Robert Miller - US grants

DESCRIPTION (Verbatim from applicant's abstract): This research application represents a broad investigation into the mechanisms of cell communication in the vertebrate retina. Our objectives are centered on three main issues. These include (1) the mechanisms in the inner retina which are subserved by the release of glutamate and the different types of glutamate receptors, both ionotropic and metabotropic, which interact to regulate the excitability of ganglion cells; (2) the identification and biophysical characterization of voltage-gated Ca2+ channels in ganglion cells with special emphasis on T-type Ca2+ channels, their pharmacological properties and cellular distribution in the dendrites and soma. We plan to evaluate how T-type Ca2+ channels contribute to nerve impulse generation and whether these ion channels play a role in amplifying synaptic currents which are generated on the dendrites. The third broad mission of this application is dedicated to a better understanding of dendritic physiology by recording from dendrites and isolated dendrosomes to further define the physiological properties of AMPA, NMDA and KA receptors. The methods used in this study will include electrophysiology, Ca2+ imaging and the use of photolysis to introduce chemical agents quickly and apply them locally at visually targeted dendritic regions. These experiments will be carried out in the amphibian retina and are designed to enhance our understanding about the mechanisms by which cells interact with one another and contribute to the excitability of retinal ganglion cells. The health-related implications of this research are extensive and relate to the mechanisms which control cellular functions of ganglion cells and how these mechanisms may help or hinder the stability of these cells when confronted with the stress of several different disease states, including glaucoma and diabetic retinopathy.

This research proposal is an experimenal investigation into (1) the identification of synaptic transmitters, (2) the physiological basis of transmitter action at the single cell level, and (3) the morphological identification of physiologically and pharmacologically characterized cells. Inhibitory and excitatory transmitter agonists and antagonists are added to the bathing medium using perfused-retina eyecup preparations of rabbits and several types of amphibians (mudpuppy and tiger salamander). Analysis of drug action is based on intracellular electrode experiments using conventional recording techniques, single electrode voltage clamp techniques ad a lock-in amplifier for quantitative measurements of conductance changes. A similar, parallel set of experiments will be carried out using enzyme dissociated cells maintained in tissue culture conditions. Intracellular recording of single dissociated cells will be analyzed as agonists/antagonists are added to the bathing medium, iontophoretically applied, or introduced through pressure injection. The isolated retina of amphibians and rabbits will be studied for types and levels of amino acids released into the bathing medium, using reverse-phase high performance liquid chromatography. Particular emphasis will be placed on the identity of rod and cone transmitters. These experiments will also be combined with tissue culture techniques to fractionate the retina into limited neuronal populations, through the use of neuroactive ligands. The goal of this research is to identify synaptic transmitters and associate them with specific pathways and cell types. Several disease states which afflict the retina (retinitis pigmentosa; macular degeneration), show prolonged states in which photoreceptor function is compromised while leaving a more or less intact retina. An understanding of the chemicals involved in photoreceptor transmission, together with the synaptic receptors which mediate their action may help in the design of drug therapy to assist patients with limited vision.

The proposed investigations are designed to identify environmental factors that support axon growth during experimentally induced axon tract formation in the developing mammalian CNS, and to determine if those, or similar factors can be implicated in the failure of regeneration in adult animals. In earlier studies we have shown that nitrocellulose implants coated principally with astrocytes from young animals are able to support axon growth in neonates, and when transplanted, suppress scar formation and promise directed axon growth in the mature mouse forebrain. Implants coated with astrocytes from mature animals fail to support axon growth in either developing or mature animals. To identify the cellular and molecular properties of purified astrocytes important in the support of axonal growth, we will first purify astrocytes from implants of different ages, and compare the capacity of young against old astrocytes to support axonal elongation "in vitro". Second, we will assess whether this change from young to old is an intrinsic property of the astrocytes or depends on environmental factors. Third, we will determine if astrocytes preferentially support axonal growth from neurons of the same, rather than remote regions of the brain. Fourth, monoclonal antibodies will be generated that distinguish between young and old astrocytes; and used to influence axonal growth on astrocytes "in vitro", will be used to investigate the molecular basis of the axon/astrocyte interaction through biochemical characterization of their antigens. These studies will provide a better understanding of the role of astroglia in the regulation of CNS axonal growth.

health science research support; university;

Astrocytes of the vertebrate CNS have been proposed to perform a wide variety of different functions including acting as a substrate for axon growth during development, contributing to the induction of the blood brain barrier and forming glial scars following injury to the adult CNS. Our recent studies suggest that astrocytes are a heterogeneous class of cells. In the proposed studies we will examine astrocyte diversity in the rat spinal cord and test the hypothesis that distinct types of astrocytes perform distinct functions in the developing, adult and injured spinal cord. Three different approaches will be used to define astrocyte diversity in the developing spinal cord: single cell cloning, retrovirus mediated gene transfer and the generation of cell type specific monoclonal antibodies. Initial studies suggest that cultures of neonatal rat spinal cord contains five morphologically distinct types of astrocytes. We will determine the time of origin, and the factors, that regulate proliferation and differentiation of each of these different cell types. Retrovirus mediated gene transfer will be used to determine the cell lineage relationships of the different types of astrocytes, both in cultures containing all classes of spinal cord cells and in the intact developing spinal cord. Recently isolated, as well as novel cell-type specific monoclonal antibodies will be used to distinguish biochemical differences between these cell types and to localize them in the intact spinal cord. The functional potential of the different types of astrocytes will be examined by analyzing their ability to support neurite outgrowth from developing neurons, suppress glial scar formation and reestablish the blood brain barrier after injury in the adult. The extent of neurite outgrowth and its molecular basis on the different populations of astrocytes will be established using purified cell populations in vitro. Transplantation of nitrocellulose implants coated with the different populations of astrocytes in to the adult CNS will be used to assess the ability of each type of cell to suppress glial scar formation and re-establish the blood brain barrier after injury. These studies will provide new, and important information on the extent and functional significance of astrocyte diversity in the spinal cord. Since the response of astrocytes to CNS injury is important for functional recovery it is critical to define specific astrocytic cell types in the CNS and to describe their individual functions following a CNS lesion. Such information is essential if we are to understand and subsequently manipulate the complex astrocytic response to CNS injury.

This research proposal is an experimenal investigation into (1) the identification of synaptic transmitters, (2) the physiological basis of transmitter action at the single cell level, and (3) the morphological identification of physiologically and pharmacologically characterized cells. Inhibitory and excitatory transmitter agonists and antagonists are added to the bathing medium using perfused-retina eyecup preparations of rabbits and several types of amphibians (mudpuppy and tiger salamander). Analysis of drug action is based on intracellular electrode experiments using conventional recording techniques, single electrode voltage clamp techniques ad a lock-in amplifier for quantitative measurements of conductance changes. A similar, parallel set of experiments will be carried out using enzyme dissociated cells maintained in tissue culture conditions. Intracellular recording of single dissociated cells will be analyzed as agonists/antagonists are added to the bathing medium, iontophoretically applied, or introduced through pressure injection. The isolated retina of amphibians and rabbits will be studied for types and levels of amino acids released into the bathing medium, using reverse-phase high performance liquid chromatography. Particular emphasis will be placed on the identity of rod and cone transmitters. These experiments will also be combined with tissue culture techniques to fractionate the retina into limited neuronal populations, through the use of neuroactive ligands. The goal of this research is to identify synaptic transmitters and associate them with specific pathways and cell types. Several disease states which afflict the retina (retinitis pigmentosa; macular degeneration), show prolonged states in which photoreceptor function is compromised while leaving a more or less intact retina. An understanding of the chemicals involved in photoreceptor transmission, together with the synaptic receptors which mediate their action may help in the design of drug therapy to assist patients with limited vision.

DESCRIPTION (provided by applicant): Understanding the cellular and molecular mechanisms that regulate the generation of oligodendrocytes, the myelinating cells of the vertebrate central nervous system is essential both to establish a comprehensive vision of neural development and to effectively generate new therapeutic approaches towards demyelinating diseases. In the last funding period we have made substantial gains in defining the location and molecular signaling that regulates the appearance of oligodendrocyte precursors and their dispersal in the developing spinal cord. In the current proposal we outline a series of approaches that are designed to further our understanding of the cellular and molecular interactions that promote later stages of oligodendrocytes development and to relate that information to the regulation of successful myelin repair in the adult CNS. Building on new approaches and recent preliminary data we outline 3 specific aims that investigate distinct but related aspects of oligodendrocyte development. In the first aim we will test the hypothesis that mature MBP+ oligodendrocytes influence the timing and success of spinal cord myelination as the direct producer of myelin and through feedback signals to OPCs. In addition we will determine whether a local postnatal depletion of MBP+ oligodendrocytes compromises myelin repair in the same region of the adult following a second insult. In the second aim we will characterize the role of GFAP+ astrocytes in the generation of the spinal cord oligodendrocyte lineage and myelination and the third aim will address the role of both mature oligodendrocytes and astrocytes in the control of remyelination in the adult spinal cord. These studies take advantage of a novel approach developed in the laboratory during the last funding period in which we are able to selectively eliminate distinct populations of mature oligodendrocytes and astrocytes in precise locations at any point in the developing and adult CNS. To accomplish this we have generated a series of transgenic animals in which we express an inducible caspase 9 (iCP9) off cell type specific promoters. Induction of the iCP9 through cross-linking with a small molecule variant of the cell and tissue permeable FK506 stimulates apoptosis specifically in the targeted population of cells. These animal models provide us with a unique opportunity to assess the consequences of selective cell loss on myelination and myelin repair in the intact vertebrate CNS. The data generated during the course of these studies will provide new directions for the development of therapies for demyelinating diseases such as multiple sclerosis.

9550649 Natvig The proposed research would involve undergraduate researchers in a collaborative effort to obtain fundamental information about genome structure in the filamentous fungus Neurospora crassa. The research aim of the Neurospora Genome Project is to sequence and map genes that encode expressed proteins. The mission of the project is to increase the number of students receiving laboratory experience in molecular genetics. Project goals include: (1) obtain partial sequences of random cDNA clones and compare sequences with known genes; (2) Identify cosmid clones corresponding to cDNA clones and (3) map cosmids to the appropriate chromosomes using RFLP analysis. Five undergraduate and two graduate students will participate in the proposed work. ***

DESCRIPTION: The purpose of this project is to characterize the role of GRO-a, an a-chemokine recently demonstrated by Dr. Miller's laboratory to be a major environmental factor that modulates the response of oligodendrocyte precursors to PDGF. In the first Specific Aim, the chemokine structural motifs required to enhance the PDGF proliferation response of oligodendrocyte precursors will be defined. In the second Specific Aim, the applicant intends to establish optimal conditions for interactions between GRO-alpha and PDGF to elicit oligodendrocyte precursor chemotaxis, and the capacity of GRO-alpha to enhance PDGF receptor signaling. In the third Specific Aim, molecular mechanisms of GRO-alpha action will be elucidated by evaluating ligand binding characteristics, as well as by determining the involvement of calcium and G proteins in signal transduction cascade. In the fourth Specific Aim, the distribution of biologically effective chemokines in the developing spinal cord will be determined.

bioimaging /biomedical imaging; computer graphics /printing; computer program /software

0073943 Paterson, Scott R
0074099 Miller, Robert B.

Exactly how plutonism, especially high volume plutons in continental magmatic arcs, fits in with active continental margin tectonics is controversial. Needed parameters include understanding magma chamber processes, how fabrics relate to the magma chamber and rates of processes during emplacement. This project will utilize excellent exposures in the Tuolumne Intrusive Complex to address several of these issues by a comprehensive integrated study of several transects across the complex. Results are expected to place greater constraints on the coupling of magmatic and host rock processes during chamber growth, on processes (mixing mingling, collapse) occurring along internal margins during emplacement of subsequent magma pulses, the connections to a volcanic feeder system and the relationship between magmatic fabrics and all the operative processes.

This research proposal is designed to improve our understanding of the physiological mechanisms which regulate the excitability of ganglion and amacrine cells in the vertebrate retina. The methods we will use in this study represent a unique combination of modeling and computer simulation analysis together with physiological and morphological experiments. One of the major objectives of this combined study will be to expand and refine a detailed, multichannel model of impulse generation in ganglion cells with special emphasis on the role which dendrites play in contributing to the impulse encoding properties of these cells. For this project we will use patch-electrode techniques directed to studies of the dendrites of single, identified, dissociated ganglion cells to identify and characterize the types of voltage-gated ion channels in dendrites. Data from these studies will be used to refine our ganglion cell model to more accurately reflect the contribution which dendrites make to impulse generation. Similar modeling studies will be carried out in amacrine cells which generate impulse activity. A second phase of this research is to examine the mechanisms of transmitter release from bipolar cell terminals with special emphasis on the ribbon synapses which appear to serve as an amplification device for exocytotic release of glutamate. This approach will also include models of AMPA and NMDA receptors connected to different cellular regions using compartmental models of realistic cell morphologies. Models of the time course of synaptic currents generated by AMPA and NMDA glutamate receptors will be based on kinetic studies we will carry out using rapid perfusion techniques. We will also generate a multiple ion channel model to simulate impulse encoding in mammalian ganglion cells, based on realistic morphologies and impulse train records from whole-cell recordings of cat ganglion cells. The purpose of this research is to formulate models which will work in synergy to guide and enhance our experimental efforts to decipher to mechanisms that are critical for function among third-order retinal neurons.

Exercise therapy in healthy sedentary adults improves limb strength by about 30%, increases aerobic capacity (VO2max) by about 15%, reduces depression and anxiety, and increases mood and self esteem. An increasing body of evidence indicates comparable benefit occurs in disease states that are relevant to ALS, such as post-polio syndrome - a disease of the lower motor neuron for which increased strength and aerobic capacity has been demonstrated - and multiple sclerosis - a disease of the upper motor neuron for which improved strength, aerobic capacity, mood, and quality of life has been shown. Neither the efficacy nor the safety of exercise therapy for ALS, or any of the other neurogenic diseases represented by the MDA, is known. The specific aims of this study are to determine if a supervised 4-month exercise training program can safely improve muscle strength, aerobic capacity, pulmonary function, mental health, and qualify of life in patients with ALS. The design is parallel, randomized, controlled, and blinded, and the sample size is 40 patients. If exercise increases muscle strength by the same mean magnitude in ALS as for healthy sedentary adults, it would represent a gain comparable to the expected mean decline in muscle strength over 9 months of the disease course. The possibility also exists that in ALS psychosocial benefits may be found from exercise which will surpass the physiological ones (strength and aerobic capacity). The recent data from multiple sclerosis (MS) is encouraging wherein exercise therapy had a significant impact in improving quality of life, strength, and endurance. Exercise therapy is an inexpensive, universally available, therapeutic modality. Improvement by exercise therapy of any of these measures without adverse effect would be expected to have a significant impact on clinical practice, not only for ALS, but also for other neurogenic diseases causing weakness.

DESCRIPTION (provided by applicant): Myelination is a crucial process in vertebrate CNS maturation. Lack of myelination during development or loss of myelin in the adult leads to severe functional deficits. CNS myelin is produced by oligodendrocytes that develop in appropriate numbers and at appropriate locations to myelinate all long projection axons. The local control of oligodendrogenesis during development or following demyelinating diseases is not well understood. We recently identified a novel regulatory mechanism that appears to play a key role in regulating the development of oligodendrocytes and in myelination. The chemokine GRO-alpha, signaling through its receptor, CXCR2, regulates the proliferation of oligodendrocyte precursors in response to platelet derived growth factor (PDGF) and provides a potent stop-signal for oligodendrocyte precursor migration. In this renewal application we will use transgenic animals that either lack CXCR2 receptors, or overexpress the ligand GRO-alpha to define the function of GRO-alpha/CXCR2 signaling during oligodendrocyte development and in response to demyelination. To unambiguously identify oligodendrocyte lineage cells we will utilize transgenic animals in which these cells are specifically labeled through expression of Enhanced Green Fluorescent Protein (EGFP) driven off the PLP promoter. We will 1) test if the GRO-alpha/CCR2 pathway regulates development of oligodendrocytes and myelination in the spinal cord, by comparing the timing and degree of myelination between normal and CXCR2 null animals; 2) identify if GRO-aplha/CXCR2 signaling inhibits the migration and dispersal of oligodendrocyte precursors by transplanting EGFP+ cells into different environments as well as direct time lapse migration analyses of EGFP-labeled cells in slice preparations; 3) determine whether GRO-alpha/CXCR2 signaling enhances or inhibits remyelination in the adult CNS by comparing recovery from lysolecithin induced demyelination or induction of EAE in wild type and CXCR2-null animals. These studies will define GRO-alpha as an important regulatory molecule in oligodendrogenesis and a potential target for therapy in diseases of myelin, such as Multiple Sclerosis, and disorders of unregulated gliogenesis such as oligodendroglioma

0003444
Harper
0087829
Miller
Accretionary and subduction complexes are commonly difficult to reconstruct due to subsequent structural complexity and lack of distinctive lithologies, fossils or stratigraphic sequences. Ophiolites, either of ocean floor or back-arc origins, provide unusually robust data that can guide structural and stratigraphic correlations and tectonic reconstructions in areas of controversial histories. This collaborative project will examine the Ingalls ophiolite complex of the north Cascades in order to help discriminate between several regionally important assembly possibilities. The work involves geochemical characterization, age dating and provenance analysis of the Ingalls complex and comparison of the results to other ophiolites that extend for 1000 km along the western United States margin. These data will help determine whether the Ingalls complex was formed near its present position, was transported long distances laterally as part of Baja-BC, or is exotic with respect to stable North America.

This Core Grant will further the research aims of 17 vision investigators at the University of Minnesota by supporting four research and service modules. The Digital Imaging module, consists of two components, the Confocal Microscope facility and the Image Analysis facility. The Confocal facility will be used by Core Grant investigators to acquire images from histological sections and from live tissues. The Image Analysis facility will be used to process, analyze and produce hard copies of images acquired on the confocal microscope and images produced in investigator laboratories. The Digital Imaging manager will supervise the Confocal and Image Analysis facilities and train investigators and staff on the use of the equipment. The Information Technology module will provide essential technical support for the Digital Imaging module as well as for individual investigators using microcomputers. The Information Technology manager will maintain the Core Grant network server, will provide networking and microcomputer support to investigators, and will create archival backups of investigator data. The Histology module will provide a fully equipped histology laboratory and the and the services of the module histologist to Core Grant investigators. Procedures offered by the facility include paraffin and frozen sections of ocular tissue, hematoxylin and eosin staining of sections, immunohistochemistry, PCR, in situ hybridization, nuclease protection assays, RNA extraction, plasmid design and construction, and sequence analysis. The Machine Shop module will provide the services of a skilled machinist to Core Grant investigators who require custom design and machining of equipment and components for their research. The modules will be linked together by a dedicated computer network that will facilitate investigator access and will enhance the capabilities of the facilities. Each module will be directed by an established NEI-funded investigator. The Core Grant will be administered by an advisory group comprised of the grant PI and module directors. The Core Grant, by providing research and service facilities, will further the research efforts of vision researchers at the University of Minnesota.

DESCRIPTION (provided by applicant) Fetal ischemic insult to the developing CNS results in irreversible and devastating functional consequences such as those observed in cerebral palsy. A hallmark of such early ischemic damage is a dramatic reduction in myelinated white matter. We propose to use a powerful model of fetal ischemia combined with biochemical and immunological tools that identify distinct stages in oligodendrocyte maturation to define the precise events affected by fetal ischemic insults. Normal oligodendrocyte development involves a number of crucial regulatory events, only some of which appear compromised by fetal ischemia. We hypothesize, based on preliminary data, that the loss of white matter after ischemic injury reflects changes in regulatory molecules that control the proliferation, migration and survival of oligodendrocyte precursors. In the first aim we will identify the specific stages at which oligodendrocyte lineage cells are lost following fetal ischemic insult and test the hypothesis that the insult alters the expression of growth factors required for oligodendrocyte development. In the second aim we will test the hypothesis that prenatal ischemic insult reduces the proliferation and migration of oligodendrocyte precursors. In the third aim we will quantify oligodendrocyte precursor cell death following ischemic insult and test the role of several different identified regulatory molecules in the induction of oligodendrocyte cell death. These studies utilize a model of fetal ischemia to identify the injury-susceptible events in oligodendrocyte maturation that eventually result in loss of white matter. Completion of this project will identify candidate molecules that will be useful for the development of novel therapeutic interventions for treating a common and devastating pediatric CNS problem.

A collaborative grant has been awarded to Drs. William Henley and Robert Miller (Oklahoma State University), Mark Buchheim (University of Tulsa) and Mark Schneegurt (Wichita State University) to establish a Microbial Observatory at the Salt Plains National Wildlife Refuge in northern Oklahoma. The briny remains of an ancient sea that once covered middle America rise to the surface and evaporate under dry conditions to leave a crust of white salt on the barren, flat, 65-km2 Salt Plains. Rainfall events dissolve the salt crust and create temporary streams and ponds, altering the landscape. Salt conditions change rapidly in time with rainfall events and in space as the plains give way to vegetated areas. The rapidly changing conditions and high surface temperatures, salt concentrations and UV exposure make this an extreme environment. The Salt Plains Microbial Observatory will use a combination of classic microbiology techniques and leading-edge genetic techniques to characterize microbial communities (bacteria and algae) and study how they survive in such a harsh environment. It is expected that many novel microbes will be discovered, and a repository of microbial biodiversity will be established, along with Internet databases of genetic information. Educational opportunities will be available for undergraduate and graduate students, including a summer course for students from groups underrepresented in the sciences.

Capturing the biodiversity of this remarkable environment will preserve unique microbes that have beneficial properties for man, as potential sources for new antibiotics, drugs, and enzymes for industrial processes and 'green' chemistry. Fundamental ecological questions will be addressed that have widespread application to the management and conservation of our natural resources. Surviving under such harsh conditions, the microbes on the Salt Plains will serve as models for organisms that may be found on other planets. Representing the first extensive study of a non-marine, terrestrial, hypersaline environment, the Salt Plains Microbial Observatory is poised to make significant new discoveries.

This research proposal has been stimulated by our discovery that CD38, an ectoenzyme originally thought to be exclusively localized to lymphocytes, is present in significant levels in the cell membranes of retinal Muller cells. To understand the functional role of CD38, we have developed a hypothesis, derived from our own experiments, in which we propose that external NAD, released from cells, activates CD38 to produce internalized second-messengers, such as cyclic ADP- ribose (cADPR)and/or nicotinic acid adenine dinucleotide phosphate (NAADP). Our working hypothesis is that cADPR and/or NAADP modulate the internal levels of calcium in Muller cells and that cADPR alters calcium through activation of ryanodine receptors. Activation of ryanodine receptors triggers calcium waves which can be blocked or modified by ryanodine, caffeine and thapsigargin, the latter of which depletes internal calcium stores. Calcium waves may alter the functional state of the Muller cells by releasing agents, such as ATP, which can affect neurons and glial cells. In addition, the changes in calcium may also trigger changes in the expression of proteins which alter the phenotype of the Muller cell. Such changes may contribute to the reactive nature of Muller cells in responding to mechanical or traumatic insults and in response to disease states. This proposal goes beyond issues related to the regulation of internal calcium through external NAD; we will thoroughly examine the mechanisms by which NAD is released from cells using both imaging and HPLC techniques (on-exchange HPLC) to assay NAD. We will determine the kinds of cells and the cellular mechanisms which permit cellular release of NAD, such as the possibility that gap junction hemi channels torn an NAD permissive pathway. We will also investigate whether externally produced cADPR could serve as a paracrine hormone to modify the behavior of nerve cells, using calcium imaging and external application of cADPR. Our general hypothesis, stimulated by our recent discoveries of CD38 and the action of NAD, is that CD38 serves as the epicenter for an external/internal communication pathway in which the afferent path is through NAD released from cells which, through CD38 is converted to internalized cADPR (and possibly NAADP)and that this pathway enhances the internal calcium levels of Muller cells and triggers calcium waves. The efferent pathway of this hypothesis is that cADPR, whether synthesized externally by CD38 or released from internal Muller cell storage circulates extracellularly and is internalized by neurons to serve as a feedback pathway to after calcium levels in retinal neurons and perhaps the pigment epithelial cells. Thus, this proposed pathway is one in which the magnitude and levels of calcium induced in Muller cells, provides a feedback, regulatory mechanism which engages and supports changes in neuronal calcium. If this hypothesis can be experimentally established, it will form the basis of an entirely new method by which neurons and glia support each other to stabilize retinal function.

The Major Histocompatibility Complex (MHC) is a set of genes that controls many aspects of immune responses including resistance and/or susceptibility to infectious diseases. The MHC has also been linked to many other biological phenomena ranging from the success or failure of pregnancy to behavior and kin recognition by smell. This project will be to study the evolution and function of the MHC genes in mammals over a larger evolutionary time-scale than has been done previously. The project will characterize the content and diversity of the MHC in a model species of marsupial, the short-tailed opossum. Marsupials, such as opossums and kangaroos, belong to a lineage that separated from placental mammals, such as humans and mice, approximately 120 million years ago. The region of the opossum genome containing the MHC will be cloned, its gene content determined, and ultimately sequenced. The genes present in the opossum MHC will be analyzed for their expression in different tissues at different stages of development to gain insight into their functional roles as well.

The MHC is the most gene rich region of the human genome. Understanding its evolution requires comparisons among distantly related species. The data on marsupial MHC generated by this project will provide the first opportunity for comparison between different subclasses of mammals. In a broader context, study of marsupial immune systems also provides unique opportunities not available using traditional mammalian models. Marsupials differ from placental mammals primarily in reproduction and development; they give birth to young that are less mature and must complete development in a pouch. This creates immunological problems for the marsupial, such as how to protect the newborn once it is in the pouch. Understanding how such problems are solved in different species requires greater knowledge of their immune systems. This research project will be the first large-scale investigation of one of the major components of the immune system in a marsupial, the MHC. This project is also part of an ongoing research program involving international collaborations with investigators in Australia. This research program has involved a large number of students, both graduate and undergraduate, in these international collaborations in the past and will continue to do so.

[unreadable] DESCRIPTION (provided by applicant): In this application we are requesting support for a selected subset of invited speakers as well as students and postdoctoral fellows to attend the 15th biennial meeting of the International Society for Developmental Neuroscience to be held in Edinburgh 4-7th of August 2004. Recent years have seen substantial advances in our understanding of the cellular and molecular mechanisms that regulate processes fundamental to development of the nervous system. This meeting will highlight specific areas of development where our advances in the basic understanding have begun to elucidate underlying mechanisms of disease and/or illuminate novel avenues for therapeutic approaches. For example, planned sessions range from animal models of Autism, through mitochondria alterations in the developing CNS to the role of stem cells in development and repair. The program is aimed at attracting basic scientists working in neural development as well as physician scientists working in areas of neurological disease. In addition, the long-term health of the field of developmental neuroscience is dependent on fostering young investigators and 2 sessions will be dedicated to young investigators with short talks selected from submitted abstracts. Furthermore, women will chair approximately one third of the general sessions. This is an exciting time in developmental neuroscience and the types of scientific interactions afforded by this meeting will help to advance our understanding by bringing together workers in distinct but related areas. [unreadable] [unreadable]

HRD 0450375

Enhancement of Doctoral Research Capacity in Environmental Toxicology at Southern University at Baton Rouge (SUBR)

Southern University at Baton Rouge

PI- John W. Owens

With NSF support, Southern University at Baton Rouge will strengthen its science, technology, engineering and mathematics educational and research enterprise by expanding interdisciplinary research focused on the effects of environmental pollutants on health at the molecular level. Research topics include: (1) Biochemical Role(s) of Plant and Environmental Estrogens in Hormone-dependent Cancers, (2) Release of Inflammatory Mediators Following Exposure to Particulate Pollution, (3) Interaction of Fluorinated Surfactants with Natural Organic Matter (NOM), (4) Mechanisms of Butadiene-Induced p53-Mediated Apoptotic Signaling, (5) Molecular Basis of Degenerative Diseases Mediated by Oxidative Stress, and (6) Responses of Endothelial Cells to 1,3-Butadiene.

The intellectual merit of the research program in environmental toxicology at SUBR is that it makes a significant contribution to the body of knowledge of molecular environmental toxicology in general and to molecular mechanisms associated with the ill effects of 1,3-butadiene in particular. The broader impact the broader impact of the research in environmental toxicology is that it will make a significant contribution to SUBR's mission of becoming nationally competitive as a Doctoral/Research University-Intensive institution. At the same time the program is helping to address the under-representation of Blacks and other minorities in the sciences.

[unreadable] DESCRIPTION (provided by applicant): In this application we are requesting support for the seventh Gordon conference on Myelin to be held in II Ciocci, Barga, Italy from May 23-28, 2004. The field of myelin biology has seen significant advances since the last meeting including the characterization of transcription factors regulating critical steps in the development of myelinating cells, the identification of cellular mechanisms that control the behavior of remyelinating cells in diseases such as Multiple Sclerosis and the unraveling of the molecular complexes at axonal-glial junctions. The development of new animal models of demyelination promises to provide further critical insights into the normal function of myelin and its dysfunction in a variety of pathologies. The program for this 7th meeting has been developed to highlight these new advances in our understanding of the biology of myelin and myelinating cells and their role in disease. Care has been taken to minimize overlap in speakers with the previous meeting, or with related meetings in the same year and to expand the focus of the meeting beyond simply "myelin". The study of myelin and the biology of myelinating cells is; however, undergoing a period of expansion with the recruitment of scientists studying diverse aspects of membrane trafficking, cell signaling and cell migration. The program has been designed to attract scientists working both directly on myelin and its associated cells as well as in related areas of brain tumor biology and regeneration. This enhanced diversity will continue to increase the variety of myelin-associated scientist and provide for a dynamic and broad-spectrum meeting. To specifically encourage the participation of young investigators, registration and travel stipends will be offered as funds permit. All attendees will be expected to contribute to an oral presentation or present a poster. In addition to formal sessions, extensive poster sessions will encourage productive interactions between scientists with different expertise and in keeping with the goals of the Gordon Conference organization; such synergy will provide critical impetus and guidance to the field of myelin biology. [unreadable] [unreadable]

Most newborn animals are highly vulnerable to assault from pathogens and infectious diseases. In response to these threats mechanisms have evolved to provide the newborn with maternal factors for protection. This is particularly true for marsupial mammals, such as opossums and kangaroos, whose young are born less mature than that of humans or other placental mammals. In contrast to placental mammals that are born with a fairly developed immune system, marsupials do not develop their immune system until after they are born. The goal of this project is to investigate whether novel mechanisms in the marsupial immune system have evolved to protect the newborn. The project focuses on a population of cells called B lymphocytes, or B cells, which are the cells that make antibodies, and utilizes a model marsupial species, the gray short-tailed opossum, which recently had its complete genome sequenced, the first for a marsupial. In contrast to humans where antibodies from the mother cross the placenta, opossum newborns are born lacking maternal antibodies and do not develop mature B cells until they are nine days old. The goals of the project are to use cellular and molecular techniques to investigate the diversity of antibodies provided by the mother via the milk and the timing, location and diversity of antibody producing B cell development in the newborn. This project will provide a better understanding of strategies used in early immune protection in mammals. This project will also increase the available knowledge of the immune system of a marsupial species that can be used to model fetal and early newborn development and is also used as a model of cancer and infectious disease.
This project will involve both graduate and undergraduate students and provide the opportunity for a postdoctoral fellow to gain experience mentoring undergraduate students. In addition to direct research experience, students at all levels, including undergraduate, will participate in publications, presentations at national and international meetings, and the collaborations with domestic and international colleagues, the latter primarily in Australia. The work outlined in this project will be integrated with ongoing comparative genomic analyses with marsupials and, in that way, will contribute to the larger comparative genomic efforts and has significance for the NSF Tree of Life Initiative.

Oklahoma State University (OSU) proposes to adapt the MIZZOU ADVANCE mentoring plan in order to increase the retention and advancement of women in science and engineering. The long term goal of this is project is aimed at identifying and developing changes to university policies and practices to better serve and represent its changing academic workers in terms of gender and ethnicity. The goals of the program are to bring awareness of barriers to women?s and underrepresented groups? advancement across STEM fields. The data gathered in this effort will help to institutionalize positive changes at the university level and identify department-specific barriers in order to increase overall participation and advancement of women and minority faculty to senior and leadership ranks. Activities targeting the culture, practices and structures that impede advancement of women faculty can only be successful through the intervention of the faculty within STEM departments, who are best positioned to collaborate with university leadership to improve persistent organizational problems. Through its partnerships with regional institutions in Oklahoma, OSU plans to expand the network of institutions and individuals from other STEM underrepresented groups, such as African Americans and Native Americans, to equip them with knowledge in order to empower them to begin addressing the under representation of women in academic science and engineering.

Project activities will include (1) Mentoring and networking programs that span STEM departments, including a two-tier mentoring program to enhance career development for women and for faculty of color; (2) University-wide programs and workshops aimed at increasing career flexibility and culturally sensitive interventions; (3) Workshops for STEM faculty from OSU and regional institutions; and, (4) OSU ADVANCE website that includes best practices, research, electronic newsletter. Given OSU's partnership with a regional HBCU and three tribal colleges, this initiative will also contribute to the development of adapting these interventions to include underrepresented groups in removing barriers for women in STEM

The broader impacts of the OSU Advance Partnership are significant and wide ranging. In addition to addressing the under representation of women in senior faculty and leadership positions in STEM areas at OSU, we expect to enhance recruitment and retention of women in junior faculty positions as well. This program of actions and interventions will provide an important and tested model for wider applications in supporting and promoting the success and advancement of women in STEM fields. The scientific work force of the United States will be vitally enhanced by ensuring that women and people of color, two rapidly growing segments of the labor force, are positioned to assume positions of leadership in STEM fields at OSU and nationally. Students at OSU, both undergraduate and graduate, will be an important part of data collection and analysis. This will, in turn, both advance their academic experience and training, as well as providing them the opportunity to participate in transformational research and interventions. We are committed to ensuring the highest level of diversity in recruiting student researchers from traditionally under-represented groups. We would note that OSU has one of the largest Native American student populations in the United States in addition to other traditionally underrepresented student populations. The PI and co-PIs have worked closely with the Oklahoma Louis Stokes Alliance for Minority Participation Program (OK-LSAMP), NABS (Native Americans in Biological Sciences), SACNAS (Society for the Advancement of Chicanos and Native Americans in Science, and similar student and professional programs.

DESCRIPTION (provided by applicant): The prevailing notion that mammalian spinal cord oligodendrocyte precursors arise from restricted regions of the ventral ventricular zone as a result of direct signaling by sonic hedgehog appears to require revision. Dorsal regions of the spinal cord generate oligodendrocytes, as do multiple domains in more rostral CNS regions. Perhaps more compelling, oligodendrocytes can be generated in the total absence of Shh. We propose that commitment of cells to the oligodendrocyte lineage is regulated not by global signals but by local cell-cell interactions that preferentially occur in particular regions of the ventricular zone. Consistent with this hypothesis we have found that the appearance of OPCs is discontinuous along the rostral-caudal axis in the ventral ventricular zone and that interspersed between these cells are neuronal precursors suggesting that neuronally derived signals regulate the initiation of oligodendrogenesis. Support for this hypothesis comes from our recent studies demonstrating that retinal neurons are necessary and sufficient to promote oligodendrogenesis. In this proposal we will extend these studies to develop a clearer understanding of the environmental regulation of early oligodendrogenesis. In the first aim we will examine whether the ability to induce OPCs is a characteristic shared by multiple populations of neurons including those in the PNS, and characterize the signaling pathways responsible. Our preliminary data suggest that electrical activity is critical for the ability of neurons to induce oligodendrocyte precursors and in the second aim we propose to test this hypothesis in vitro and in vivo. How the population size of oligodendrocytes and their precursors are regulated during development is unclear. We propose that cell numbers are dynamically controlled to match environmental needs. To directly test this hypothesis in vivo we will use a novel cell type specific depletion approach to locally eliminate mature oligodendrocytes in aim 3 and OPCs in aim 4. Analyses of the responses to oligodendrocyte lineage cells to stage specific deletion will identify the major regulatory steps in oligodendrocyte development and provide novel targets for therapeutic intervention in demyelinating diseases such as Multiple Sclerosis. PUBLIC HEALTH RELEVANCE: Myelination is the fatty insulation that surrounds the processes of neurons and allows them to communicate rapidly with each other. In the brain and spinal cord, myelin is made by oligodendrocytes and they are essential to allow the adult nervous system to function correctly. How oligodendrocytes are formed is not well understood and the studies in this application will identify the environmental signals that promote their initial appearance and control the number of oligodendrocyte lineage cells in the mature central nervous system. Such information will provide new targets for the development of treatments for demyelinating diseases such as Multiple Sclerosis.

DESCRIPTION (provided by applicant): The mission of this project is to bring new clarity to the importance of NMDA receptors in the retina, with a special emphasis on how their contribution to retinal function is controlled by the synthesis and release of endogenous D-serine. It is now apparent that D-serine, an amino acid whose functional visibility was off the radar screen just a few years ago, is synthesized by glial cells of the retina and released and regulated in such a way that astrocytes and Muller cells have some control over the excitability of retinal ganglion cells. It is well understood that the neurotransmitter glutamate cannot open the ion channel of NMDA receptors without the presence of D-serine which serves as a coagonist or perhaps a co-neurotransmitter. One of the most pressing problems in retina and brain research is to understand the role of D-serine and NMDA receptors for three important reasons. First the NMDA receptors are unique among glutamate receptors because of their relationship to excitotoxicity, created by their permeability to calcium and induced by excessive glutamate stimulation. This property of NMDA receptors can be additive to the ganglion cell risk factors found in conditions such as glaucoma, ischemia, injury or diabetic retinopathy. A second health-related matter of importance comes from the modern treatment of schizophrenia, where thousands of patients are being treated with high doses of oral D-serine to activate NMDA receptors which are thought to be hypoactive and possibly the cause of the disease. Since D-serine crosses the blood-brain barrier to achieve its effects, we can safely assume that it also crosses the blood-retinal barrier and, based on our own work, the addition of exogenous D-serine into the retina can enhance NMDA receptor activity, particularly since no high affinity removal system for D-serine is present in the retina, as it is in the brain;this could render cells in the retina more at risk than those in the brain. Yet a third important factor in D-serine study relates to the mounting evidence that D-serine is critical for development in the retina and brain and our more recent studies suggest that abnormally elevated D-serine levels can contribute. The studies proposed in this application will provide new information about how the intrinsic control mechanisms of D-serine synthesis, storage and release serve to regulate the excitability of retinal ganglion cells through modulation of NMDA receptor availability. We propose to study three different strains of mice, each of which has a genetic defect of the regulatory mechanisms of NMDA receptor coagonist function, including a deficiency in glycine transport, a deficiency in the degradation of D-serine (DAAO) and the absence of the synthetic machinery for D-serine synthesis (serine racemase). Our studies will include electrophysiological methods, studies of the mechanisms of D-serine release through chemical detection and immunohistochemical and EM methods to evaluate whether the mechanisms related to D-serine synthesis and NMDA receptor distribution are changed due to an interruption in the normal coagonist functions of this novel amino acid. PUBLIC HEALTH RELEVANCE: Our knowledge of the role that NMDA receptors play in retinal function and their regulation by D-serine has critical implications for issues of public health. In the retina, D-serine and its actions on NMDA receptors make an important contribution to the light sensitivity of ganglion cells but may also be involved in exacerbating degenerative phenomena of ganglion cells, such as those associated with glaucoma, ischemia, injury or disease states like diabetic retinopathy. In addition, understanding NMDA receptors and D-serine regulation has implications for patients with schizophrenia, some of whom take high doses of D-serine to enhance NMDA receptor function related to their disease;these patients may also be at higher than normal risk for retinal damage through excessive activation of retinal NMDA receptors by therapeutic doses of D-serine which readily cross the blood-retinal barrier and have access to the retina, which lacks a high affinity transport system to remove D-serine, something that is present in the brain.

DESCRIPTION (provided by applicant): Schizophrenia is a complex, debilitating mental health disorder associated with significant morbidity and mortality. The molecular- and cellular-based mechanisms that contribute to schizophrenia remain undefined. Although schizophrenia has classically been considered a neurotransmitter-based disorder, there is emerging evidence that dysregulation of oligodendrocyte function is a key contributor to the mental deficits seen in afflicted patients. Currently there is not a tractable system that allows for the direct interrogation of the functional properties of neural cells types from patients with schizophrenia. Patient-specific sources of cells that are capable of robust and reproducible differentiation into specific neural lineages do not exist. We propose to develop a cell-based system whereby neural cells from afflicted individuals can be functionally assayed to interrogate the molecular mechanisms underlying schizophrenia. To achieve this goal we have developed a cutting-edge proposal that that incorporates the skill and expertise of multiple disciplines. In Aim 1 we will derive and characterize patient-specific, induced pluripotent stem (iPS) cells from juvenile-onset schizophrenia patients and controls. Since iPS cells are pluripotent, having the ability to differentiate into all cell types of the human body, in Aim 2 we will differentiate patient-specific iPS cells line into oligodendrocyte progenitor cells (OPCs) to provide a cellular source for oligodendrocytes. In the second phase of this project will characterize, compare, and functionally assay these patient-specific, iPS cell-derived oligodendrocytes from control and juvenile-onset schizophrenia patients using both in vitro and in vivo assays (Aims 3 and 4 respectively). We will also actively procure additional samples to derive and characterize patient-specific, iPS cells from juvenile-onset schizophrenia patients and controls during this second phase (Aim 5). There is great potential for patient-specific iPS cell technology to profoundly impact our understanding of human development and disease by providing genetically distinct, functional sources of human cells. By completing the aims set forth in this proposal we expect to provide a detailed characterization of oligodendrocyte function in patients afflicted with schizophrenia and provide insight into the pathophysiology of this complex disease. We have established an interdisciplinary team that combines strengths in clinical schizophrenia research, neural differentiation and function, as well as pluripotency and iPS cells to interrogate novel questions about the cellular and molecular dysfunction that contributes to schizophrenia. We expect that results from our studies will have immediate relevance to the understanding and treatment of this human disease. PUBLIC HEALTH RELEVANCE: Schizophrenia is a serious psychiatric condition with a worldwide prevalence of approximately 1%. Individuals with schizophrenia experience very severe symptoms and are at an increased risk for suicide, unemployment, permanent disability, and homelessness. Affected adolescents experience even more severe symptoms, tend to be more chronically dysfunctional, suffer from greater cognitive impairments, and may have greater functional and social disability than those with adult-onset schizophrenia. Unfortunately, the cause of schizophrenia is currently unknown. Results of our studies will provide a detailed characterization of brain cell function in patients afflicted with schizophrenia and will offer insight into the mechanisms that contribute to this complex, devastating disease.

Collaborative Research: Tectonic links, magma fluxes, and single mineral geochemistry in plutonic systems from 5-30 km depth, Cascades core, Washington

Intellectual Merit. Continental magmatic arcs are the site of massive additions of mantle derived magmas which serve to drive melting, thickening, and reorganization of the crust. Much of what we know about Cretaceous and older continental magmatic arcs results from studies of the physics and chemistry of plutonic rocks, and the derivative data strongly influence models of crustal growth and evolution. In contrast to modern arcs where single eruptive units may be analyzed, plutons record a much richer, albeit complex, history and represent a much greater percentage of the total magmatic flux than volcanic rocks alone. Critical questions concern how and at what rate intermediate composition magmas move through the crustal column, and how the crust accommodates these potentially large fluxes? Key to answering these questions is to understand how large masses of crystallized magmas or plutons and batholiths are constructed. To address these questions, collaborative research is proposed to better understand sub-arc magmatic plumbing systems. This project focuses on the roots of a continental magmatic arc in the North Cascades that comprises three well-exposed intrusive bodies emplaced at different depths in the crust. Integrated field mapping, structural analysis, mineral geochemistry, high-precision geochronology and coupled isotopic studies will be employed to [1] determine the degree to which plutons represent single magma batches or complex mixtures of partially crystallized magmas of different origins, [2] estimate magma flux rates and timescales for melt generation, transport, and eruption of intermediate composition magmas, and [3] evaluate potential linkages between regional tectonic strain and magma formation. This multidisciplinary collaborative research is expected to contribute significantly to understanding of magma plumbing systems and the formation of plutons in continental magmatic arcs.

Broader Impacts. This research will support Ph.D. students at MIT and USC, and at least 2 M.S. students at SJSU, and multiple undergraduate student assistants at all 4 institutions. Bpth SJSU PIs have supervised 2 B.S. research projects supported by their most recent grants, and Paterson teaches a formal research class at USC that has involved >20 undergraduates in NSF-supported research. Graduate students from SJSU and USC will visit Kent's lab at OSU to conduct their own analyses, and the MIT graduate student will visit SJSU and assist in SHRIMP analyses. Four M.S. students at SJSU have gone to MIT to carry out U-Pb dating under the tutelage of PI Bowring and his students, and this type of interaction is expected to continue. All of the PIs routinely incorporate their research in class lectures and use research samples in lab courses. Most importantly, The PI's are demonstrating to a large cross section of new students the power of strongly interdisciplinary and collaborative work.

Directed block copolymer self-assembly is one of the promising technologies to solve the problems of conventional lithography for advanced semiconductor technologies. In contrast to conventional lithography, block copolymer self-assembly provides sub-20 nm features with lower cost and higher throughput, though defects and precise positioning are fundamental research topics that need to be addressed. The goal of this project is to address these challenges for integrating directed block copolymer self-assembly technology into practical integrated device fabrication process at the circuit level and demonstrate the fabrication of the key features for next generation devices. Block copolymer self-assembly and guided template patterning methods are proposed as a proof of concept demonstration for fabricating ultra-high density memory arrays and sub-20 nm contact holes for high-density silicon circuitry, respectively.

The broader impacts of this project cover both fundamental research and commercial manufacturing at the university-industrial interface. This project can have direct impact to promote block copolymer self-assembly technology as an economically efficient extension to conventional lithography for next generation device fabrication, benefiting the semiconductor manufacturing industry and the broader electronics industry greatly. It also will substantially stimulate the study of polymer materials design for better functional performance. As part of a outreach to the broader society, a few video clips will be produced and posted onto public websites to help the students understand the science and technology of the self-assembly. This university-industry collaboration provides an excellent training ground for the participating students in a multidisciplinary environment at the interface of fundamental research and industrial manufacturing.

DESCRIPTION (provided by applicant): Oligodendrocyte precursors (OPCs) differentiate into oligodendrocytes that are the myelinating cells of the vertebrate CNS. Myelin sheaths wrap axons in the brain and spinal cord and maintain axonal function and promote rapid conduction of electrical impulses. Any damage to myelin sheaths, such as occurs in multiple sclerosis, results in loss of axonal conduction and ultimately axonal degeneration and irreversible neural disability leading to serious physical or mental impairments. Multiple sclerosis is a devastating disease that affects more than 300,000 individuals in the United States. Current therapies are directed towards regulating the inflammatory aspects of the disease, however long term functional recovery will depend upon successful myelin repair in the CNS. Recent studies suggest that many areas of demyelination in the brains of MS patients contain OPCs but the ability of these cells to repair damage is limited because they fail to differentiate for reasons tat are currently unknown. A detailed understanding of the mechanisms controlling OPC maturation and myelination will therefore provide new insights and novel therapeutic strategies for enhancing myelin repair in MS. The experiments described in this proposal will explore the roles of the intracellular signaling molecule cyclin dependent kinase 5 (Cdk5) and its co-activators p35/p39, in regulating the development of OPCs, myelination and remyelination. Cdk5 is known to be involved in various signaling pathways that are key for CNS development. Our preliminary data has revealed novel functions of Cdk5 in controlling the development of OPC and myelination. The proposed study will explore whether Cdk5 within cells of the oligodendrocyte lineage regulates their development and myelination in vitro and in vivo using molecular and genetic approaches. We will identify the roles p35/p39 play in mediating Cdk5 modulation of OPC maturation and myelination. To determine whether the Cdk5 pathway is a novel potential target for therapeutic development we will test whether Cdk5 is essential for remyelination in adult CNS after the induction of focal demyelinating lesions. To accomplish this we will selectively delete Cdk5 from OPCs in the adult CNS during demyelination. Successful completion of the proposed studies will provide critical insights into the signaling mechanisms regulating OPC maturation and myelination and provide novel targets to promote myelin repair. PUBLIC HEALTH RELEVANCE: Multiple sclerosis is a devastating, irreversible and progressive disease that result from failure of myelin repair and degeneration of demyelinated axons. Unfortunately, no effective therapeutics are currently available to promote myelin repair. Understanding OPC differentiation and myelination is critical for developing new strategies for promoting repair of myelin lesions in MS patients. We have identified Cdk5 as an important regulator of oligodendrocyte development and the current studies will define the role of Cdk5 and its activators, p35 and p39 in myelination and myelin repair. Results of our studies will provide critical insights into the signaling mechanisms regulating OPC maturation and myelination and provide novel targets to promote myelin repair.

Intellectual Merit:
The Santa Barbara Coastal LTER (SBC) is an interdisciplinary research and education program established in April, 2000 to investigate the role of land and ocean processes in structuring ecosystems at the land-sea margin. The main study area is the Santa Barbara Channel and the steep coastal watersheds, small estuaries and sandy beaches that drain into it. The focal ecosystem of the research is giant kelp forests, a diverse and highly productive marine ecosystem that occurs on shallow rocky reefs at the interface of the land-sea margin in the Santa Barbara Channel and other temperate regions throughout the world. The major emphasis of this project is developing a predictive understanding of the structural and functional responses of giant kelp forest ecosystems to environmental forcing from the land and the sea. The amount of nutrients and organic matter delivered to the kelp forest from land and the surrounding ocean varies in response to changes in climate, ocean conditions and land use. Variation in the supply of these commodities interacts with physical disturbance to influence the abundance and species composition of kelp forest inhabitants and the ecological services that they provide. The overarching question motivating this research is: How are the structure and function of kelp forests and their material exchange with adjacent land and ocean ecosystems altered by disturbance and climate?

To address this question LTER researchers will focus on three themes: (1) biotic and abiotic drivers of kelp forest structure and function, (2) material exchange at the land-ocean margin, and (3) movement and fluxes of inorganic and organic matter in the coastal ocean. The relevance of this research is far reaching as LTER scientists are addressing fundamental questions pertaining to biodiversity and ecosystem function, vulnerability and resilience of communities to climate change and fishing, the roles of land use and fire on landscape change and watershed hydrology, and the physics of dispersal in the little studied coastal waters of the inner continental shelf. The dynamic nature of kelp forests, including their frequent disturbance and rapid regeneration coupled with high productivity and diverse food webs make them ideal systems for investigating ecological questions that require decades to centuries to address in other ecosystems. This project will utilize a variety of approaches including: (1) coordinated long-term measurements, (2) manipulative field experiments, (3) measurement-intensive process studies, and (4) integrated synthetic analyses and modeling that allow for predictions beyond the spatial and temporal scope of our measurements, and help guide future research. SBC's information management system, which focuses on data organization, integrity, preservation and web-based public access geared for a variety of end users will facilitate these efforts.

Broader Impacts:
Education and training are tightly integrated into all aspects of this research. LTER personnel have successfully developed a multifaceted, interdisciplinary approach to education and outreach that highlights research interests of SBC investigators, students, and the general public. Programs include active links with K-12 students and teachers that target historically under-represented groups from under serving, low-achieving schools. The LTER participants are also very proactive in undergraduate and graduate student training, direct public outreach, and productive interactions with the media, government agencies and local industries. The LTER will continue these outreach and education programs and maintain efforts to attract additional funding to support them. The LTER is committed to sharing research results with resource managers, decision makers, stakeholders, and the general public who are interested in applying our findings to policy issues concerning natural resources, coastal management, and land use.

DESCRIPTION (provided by applicant): This research project is designed to provide new methods for revealing and studying patients with psychiatric diseases with special emphasis on the root causes of schizophrenia. We are proposing a new way of evaluating schizophrenic patients based on a simple ophthalmological recording technique known as the electroretinogram or ERG. The ERG is an electrical signal routinely and non-invasively recorded from the front of the eye and is a common procedure found in most ophthalmological clinics. We propose that a variant of the conventional ERG, called the pattern ERG or pERG reflects activity of retinal ganglion cells and has a signature response component that can be attributed to light-evoked activity of N-methyl-D-Aspartate or NMDA receptors (NMDARs). Abnormal regulation of NMDARs has been implicated as one cause of schizophrenia and this project will definitively evaluate this possibility. Indeed, we hav obtained preliminary data from four schizophrenic patients in which the pERG is characteristically different than that recorded from normal controls. When we compare the schizophrenic pERG with the mouse pERG, recorded from an isolated, perfused retina preparation, the mouse pERG looks very much like the control human subjects, but when the retina is perfused with an antagonist that eliminates the contribution of NMDA receptors to the pERG, the resulting waveform looks strikingly similar to the pERG observed in schizophrenic patients. These remarkable findings have stimulated the current application, with the broad objective of recording from a wider sampling of patients, including schizophrenics that have not received anti-psychotic medication to insure that we are not observing a drug effect in pERG recordings from schizophrenic patients. The strategy behind this research is to use the pERG to study human patients with schizophrenia and compare them with age- and gender-matched controls. We will evaluate cognitive functions of each human subject through testing procedures and carry out separate experiments in mice to determine how NMDARs contribute to the pERG response. In addition, we plan to carry out experiments in which mice have been maintained on antipsychotic medication for acute and chronic periods. We will also use the mouse preparation to refine our pERG stimulation parameters to optimize for the NMDA receptor contribution and modify accordingly the visual stimulus we use for human subjects. We expect that this study will definitively address the question of NMDA receptor involvement in schizophrenic patients.

DESCRIPTION (provided by applicant): Multiple sclerosis (MS) is a complex, debilitating neural disorder associated with significant morbidity and mortality. Disease etiology is a result o unknown environmental and genetic factors, while disease pathology presents as inflammatory injury to the central nervous system (CNS) causing physical incapacity. This damage is a result of autoimmune-mediated destruction of oligodendrocytes causing demyelination. Prolonged demyelination leads to axonal damage occurring either in focal lesion sites and/or widespread throughout the CNS, depending on presentation of the disease. MS is treated with disease-modifying agents that target the immune system in an attempt to reduce the frequency of relapses and delay disease progression. These drugs have had limited success in improving patient outcomes and lack the unmet need to promote immediate remyelination. Identifying drugs that induce resident oligodendrocyte progenitor cell (OPC) mediated remyelination would provide patients a vital mechanism to halt disease progression and improve function. We propose to screen drugs that promote remyelination for repurposing in MS. To achieve this goal we have developed a proposal that incorporates the skill and expertise of multiple disciplines through a proven validation pipeline to interrogate drug entities that promote remyelination. We will begin with drugs that have a history of use in human clinical trials and assess their ability o promote rodent OPC maturation and myelin gene expression in our novel high throughput in vitro system. This system takes advantage of our technology to generate scalable sources of functional OPCs from pluripotent stem cells. Validated drug hits from our high throughput in vitro screen will then be tested in a tractable and economical ex vivo myelination assay utilizing organotypic rodent brain slice cultures. Moving forward, we will take our 5 most promising drug hits (as determined from in vitro and ex vivo assays) and test them in animal model systems regularly used to interrogate applicability to MS (lysolecithin-mediated spinal cord demyelination and Experimental Autoimmune Encephalomyelitis). Top drug hits will also be tested on human OPCs. Using our strengths in preclinical MS research and stem cell differentiation, we aim to identify efficacious pharmacological agents that promote remyelination by the end of this two year project. We are prepared to progress lead drugs identified from our studies to human clinical trials.

? DESCRIPTION (provided by applicant): Multiple sclerosis (MS) is a complex, debilitating neural disorder associated with significant morbidity and mortality. Disease etiology is a result o unknown environmental and genetic factors, while disease pathology presents as inflammatory injury to the central nervous system (CNS) causing physical incapacity. This damage is a result of autoimmune-mediated destruction of oligodendrocytes causing demyelination. Prolonged demyelination leads to axonal damage occurring either in focal lesion sites and/or widespread throughout the CNS, depending on presentation of the disease. MS is treated with disease-modifying agents that target the immune system in an attempt to reduce the frequency of relapses and delay disease progression. These drugs have had limited success in improving patient outcomes and lack the unmet need to promote immediate remyelination. Identifying drugs that induce resident oligodendrocyte progenitor cell (OPC) mediated remyelination would provide patients a vital mechanism to halt disease progression and improve function. Using an innovative pluripotent stem cell-based high throughput screening platform we have discovered a class of FDA approved drugs that enhance remyelination in mouse models of MS. In this grant we will use in vivo and in vitro models to study the cellular and molecular effects of this class o drugs on mouse and human OPCs. These studies will provide the basis for potential translation of this drug, or modified derivatives, to clinical testing as a remyelinating therapeutic.

DESCRIPTION (provided by applicant): New studies have underscored the importance of D-serine and its role as a coagonist for proper gating of NMDA receptors. D-serine deficiency has been implicated in major psychiatric disorders, including schizophrenia and bipolar disorder and clinical trials have been carried out based on the idea that schizophrenia represents a deficiency in D-serine. Thus the NMDA receptor hypofunction hypothesis of schizophrenia has already been attributed to a deficiency of D-serine by some workers. Despite the emerging importance of this atypical amino acid (proteins are made from L-amino acids), we still do not understand the release mechanisms or the cells that contain the enzymes necessary for D-serine synthesis (serine racemase; SR), storage and release. Part of this problem stems from the difficulty in finding antibodies that selectively label serine racemase, an important issue that will be addressed in this study. Recently, we have discovered a light-evoked release of D-serine in the retina, the only tissue site in which natural, physiological stimulation has been associated with D-serine release. This finding has elevated the vertebrate retina as one of the ideal target sites to investigate D-serine release mechanisms. A newly developed, second generation D-serine biosensor has been fabricated in my laboratory and developed through a collaboration with Dr. Stephane Marinesco, Neuroscience Institute of Lyon, France. Armed with this improved D-serine biosensor, we are now in a position to provide the most extensive analysis of D-serine localization and mechanisms of release ever carried out in a single CNS tissue site. The objectives of this proposal are to understand the cellular sites and mechanisms of D-serine release, storage and synthesis, using our new, carbon-fiber based D-serine biosensors combined with immunostaining methods using antibodies with proven specificity (comparing antibody specificity in wt vs SRKO (knockout). We will explore all of the major release mechanisms proposed for D-serine release from neurons and glial cells. In order to carry out these experiments, we plan to examine mice during the postnatal period of development, both before and after eye opening. The reason for this strategy is to see if the sources of D-serine synthesis and release change during development, as suggested by other workers. Specifically we want to see whether D-serine release mechanisms change from neuronal (ganglion cells) to glial (M?ller cells) during the course of postnatal development and, if so, does the release mechanism change from neuronal (heteroexchange through Asc-1 neutral amino acid transporters) to a synaptic release mediated by activation of Ca2+-permeable AMPA receptors. We also want to evaluate the D-serine release mechanisms in the adult to see if more than one mechanism is involved, as our preliminary results may suggest. We will also study a mouse line in which the SR promoter expresses eGFP in the cells that express SR. We will also explore the pathway of L-serine synthesis through the enzyme 3-PGDH which synthesizes L-serine and resides exclusively in brain astrocytes.

Project Abstract Although modern therapies have dramatically improved the outlooks for people living with HIV/AIDS (PLWHA) they are unable to cure infection, leaving these individuals burdened by a lifelong commitment to expensive antiretroviral medication. It has also become clear that these treatments do not fully restore health, nor do they address the negative social issues associated with being HIV positive, including stigma and issues related to criminalization. The development of a safe and effective HIV cure would thus greatly improve the lives of PLWHA. A major obstacle to curing HIV infection is the establishment of reservoirs of hidden or `latent' virus which evade the immune system and can re-seed infection if an individual stops antiretroviral therapy. Considerable efforts are underway to attempt to purge these HIV reservoirs in the blood and in other tissues in the body where the virus is known to persist. However, an important unknown currently hangs over these efforts. HIV is known to be able to infect certain cells in the brain and it is presently not known whether or not this virus can persist for long periods of time in antiretroviral-treated individuals and re-establish spreading infection. Since many of the therapies currently being tested to eliminate HIV do not target virus in the brain, this raises the possibility that even treatments that are very effective in the blood and other tissues may not cure infection. In this study we propose using a novel mouse model of HIV infection to first determine whether or not HIV can be passed from the brain to the blood and other tissues, and then to test how long any infectious virus in the brain can persist. If the virus in the brain persists for only short period of time then it is possible that sustained antiretroviral therapy will be sufficient to eliminate this reservoir in PLWHA, however if it is more durable then other types of therapies will have to be developed. In thus study we will also test two such therapies by determining whether antibodies or killer T cells that target HIV can prevent the spread of virus from the brain, either by entering into the brain and eliminating HIV at this source, or by eliminating any `beachheads' of virus in the blood or tissues from HIV that exits the brain. Thus, our study will both lead to a better understanding of the challenge that HIV reservoirs in the brain may pose to curing HIV infection, and will test therapeutics with the potential to overcome this challenge and contribute to curing infection.

Material exchange between ecosystems is being increasingly recognized as an important determinant of many ecological patterns and processes. Nowhere is this more evident than in the highly productive coastal zone, which receives large amounts of terrestrial particulate organic (POM) matter through stream and river discharge. In semi-arid regions such as southern California, the delivery of terrestrial POM to the nearshore is largely restricted to storm events that are intensified during El Nino years of above average rainfall. The processing and fate of this material is poorly known, yet there is growing evidence that it could contribute significantly to nearshore productivity. This research project will help to fill a critical knowledge gap pertaining to the origin, distribution, processing of terrestrial POM and its potential to serve as a reservoir of nitrogen storage for nearshore primary production during periods of the year when marine sources of dissolved inorganic nitrogen are low. Research on this award will be done in close collaboration with the Santa Barbara Coastal Long Term Ecological Research program (SBC LTER). As such it will augment SBC's strong contribution to student training and mentoring in interdisciplinary research at the undergraduate, graduate, and post-doctoral levels. Outcomes will be incorporated into SBC's ongoing Schoolyard LTER program, which is organized around a theme of kelp forest ecology and land-ocean exchanges and aimed at long-term connections with underserved, low-achieving schools that include year-round on and off campus activities. The LTER Investigators have developed formal partnerships with local cities and Santa Barbara County to develop vulnerability assessments of the regions coastal ecosystems and the LTER will incorporate the findings from this study into those assessments

The unprecedented drought currently in its fourth year in California, coupled with the ongoing conditions of anomalously low ocean productivity and the prospect of one the strongest El Ninos on record provide an unparalleled opportunity for researchers at the Santa Barbara Coastal Long Term Ecological Research program (SBC LTER) to test specific hypotheses pertaining to the origin, distribution, processing and bioavailability of terrestrial organic matter in coastal marine sediments and their potential for serving as a reservoir of nitrogen storage to fuel nearshore primary production during periods when nitrate concentrations are low. NSF RAPID Response award funds will be used to: (1) measure bulk properties and biomarker tracers of particulate organic matter (POM) in stream water and in coastal marine sediments at SBC sites differing in exposure to terrestrial runoff prior to and following large storm events, and (2) determine the bioavailability of dissolved organic matter (DOM) released from POM in marine sediments following large runoff events. The research will complement and inform SBC's ongoing efforts to investigate the availability and utilization of recycled forms of nitrogen in supporting the primary production of nearshore macrophytes and phytoplankton during non-upwelling periods when nitrate levels ar typically low.

DESCRIPTION (provided by applicant): New studies have underscored the importance of D-serine and its role as a coagonist for proper gating of NMDA receptors. D-serine deficiency has been implicated in major psychiatric disorders, including schizophrenia and bipolar disorder and clinical trials have been carried out based on the idea that schizophrenia represents a deficiency in D-serine. Thus the NMDA receptor hypofunction hypothesis of schizophrenia has already been attributed to a deficiency of D-serine by some workers. Despite the emerging importance of this atypical amino acid (proteins are made from L-amino acids), we still do not understand the release mechanisms or the cells that contain the enzymes necessary for D-serine synthesis (serine racemase; SR), storage and release. Part of this problem stems from the difficulty in finding antibodies that selectively label serine racemase, an important issue that will be addressed in this study. Recently, we have discovered a light-evoked release of D-serine in the retina, the only tissue site in which natural, physiological stimulation has been associated with D-serine release. This finding has elevated the vertebrate retina as one of the ideal target sites to investigate D-serine release mechanisms. A newly developed, second generation D-serine biosensor has been fabricated in my laboratory and developed through a collaboration with Dr. Stephane Marinesco, Neuroscience Institute of Lyon, France. Armed with this improved D-serine biosensor, we are now in a position to provide the most extensive analysis of D-serine localization and mechanisms of release ever carried out in a single CNS tissue site. The objectives of this proposal are to understand the cellular sites and mechanisms of D-serine release, storage and synthesis, using our new, carbon-fiber based D-serine biosensors combined with immunostaining methods using antibodies with proven specificity (comparing antibody specificity in wt vs SRKO (knockout). We will explore all of the major release mechanisms proposed for D-serine release from neurons and glial cells. In order to carry out these experiments, we plan to examine mice during the postnatal period of development, both before and after eye opening. The reason for this strategy is to see if the sources of D-serine synthesis and release change during development, as suggested by other workers. Specifically we want to see whether D-serine release mechanisms change from neuronal (ganglion cells) to glial (M?ller cells) during the course of postnatal development and, if so, does the release mechanism change from neuronal (heteroexchange through Asc-1 neutral amino acid transporters) to a synaptic release mediated by activation of Ca2+-permeable AMPA receptors. We also want to evaluate the D-serine release mechanisms in the adult to see if more than one mechanism is involved, as our preliminary results may suggest. We will also study a mouse line in which the SR promoter expresses eGFP in the cells that express SR. We will also explore the pathway of L-serine synthesis through the enzyme 3-PGDH which synthesizes L-serine and resides exclusively in brain astrocytes.

Scientific imaging is ubiquitous: From materials science, biology, neuroscience and brain connectomics, marine science and remote sensing, to medicine, much of the big data science is image centric. Currently, interpretation of images is usually performed within isolated research groups either manually or as workflows over narrowly defined conditions with specific datasets. This LIMPID (Large-scale IMage Processing Infrastructure Development) project will have a transformative impact on such discipline-centric workflows through the creation of an extensive and unique resource for the curation, distribution and sharing of scientific image analysis methods. The project will create an image processing marketplace for use by a diverse community of researchers, enabling them to discover, test, verify and refine image analysis methods within a shared infrastructure. As a freely available, cloud-based resource, LIMPID will facilitate participation of underrepresented groups and minority-serving institutions, as well as international scientists, allowing them to address questions that would otherwise require expensive software. The potential impacts of the project are significant: from wide dissemination of novel processing methods, to development of automatic methods that can leverage data and human feedback from large datasets for software training and validation. For the broader scientific community, this immediately provides a resource for joint data and methods publication, with provenance control and security. This in turn will facilitate faster development and deployment of tools and foster new collaborations between computer scientists developing methods and scientific users. The project will prepare a diverse cadre of students and researchers, including women and members of under-represented groups, to tackle complex problems in an interdisciplinary environment. Through workshops, participation at scientific meetings, and summer undergraduate research internships, a broad community of users will be engaged to actively contribute to all aspects of research, development, and training during the course of this project. 


The primary goal is to create a large scale distributed image processing infrastructure, the LIMPID, though a broad,  interdisciplinary collaboration of researchers in databases, image analysis, and sciences.  In order to create a resource of broad appeal, the focus will be on three types of image processing: simple detection and labelling of objects based on detection of significant features and leveraging recent advances in deep learning, semi-custom pipelines and workflows based on popular image processing tools, and finally fully customizable analysis routines.  Popular image processing pipeline tools will be leveraged to allow users to create or customize existing pipeline workflows and easily test these on large-scale cloud infrastructure from their desktop or mobile devices. In addition, a core cloud-based platform will be created where custom image processing can be created, shared, modified, and executed on large-scale datasets and apply novel methods to minimize data movement. Usage test cases will be created for three specific user communities: materials science, marine science and neuroscience. An industry supported consortium will be established at the beginning of the project towards achieving long-term sustainability of the LIMPID infrastructure.

This project is supported by the Office of Advanced Cyberinfrastructure in the Directorate for Computer & Information Science and Engineering and the Division of Materials Research in the Directorate for Mathematical and Physical Sciences.

Project Summary This resubmission proposal aims to elucidate the role of a histone deacetylase, HDAC11, in diseases such as multiple sclerosis (MS), and to establish HDAC11 inhibition as a potentially effective new treatment strategy for diseases including MS. MS is a chronic, immune-mediated demyelinating disease of the central nervous system. Like many autoimmune disorders, it presently has no known cure, and current drugs available for managing this disease are only effective early on and are accompanied by many adverse effects. The disease mechanism of MS remains unclear, and no effective targeted therapy is available for chronic progressive MS. Our preliminary studies show that deletion of HDAC11 ameliorates clinical symptoms in a mouse model of MS. In parallel, we discovered a novel HDAC11 enzymatic activity that is >10,000-fold more efficient than its deacetylase activity. This novel activity allows us to begin to uncover physiologic substrates of HDAC11, which in turn will help to uncover the biological mechanisms of HDAC11?s actions. One of the goals of this research is to investigate how this newly discovered enzymatic activity underlies the immune-regulatory function of HDAC11 in MS. Knowledge gained from these studies will help to further understand the disease mechanism of MS and to develop better therapeutics. Because the discovery of a novel HDAC11 activity has enabled us to develop, for the first time, HDAC11-specific inhibitors, the chief objective is to further improve these inhibitors and test whether they can be used to treat diseases such as MS in our established mouse models. Our multidisciplinary team has expertise in all aspects needed to make this project successful. Overall, the proposed studies in this application will not only yield a better understanding of HDAC11?s function in health and diseases, but may also result in a first prototype targeted therapy for the treatment of chronic progressive MS, and possibly other diseases as well.