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Parameter Lab 5

Ionizing radiation is a well-known human carcinogen linked to a variety of cancers including thyroid cancer, leukemia, breast cancer and soft tissue sarcomas. The molecular mechanisms of radiation-induced carcinogenesis remain poorly understood. Significant evidence has been accumulated supporting the dominant role of chromosomal rearrangements in the carcinogenesis initiated by radiation exposure. Since ionizing radiation induces double strand DNA breaks, it is conceivable that chromosomal rearrangements are formed directly by mis-rejoining of free DNA ends produced by radiation and located close to each other in the nucleus. In thyroid cancer, RET/PTC rearrangements, which usually form via an inversion in chromosome 10q, are a molecular signature of radiation-associated tumors. Recently, we have established an in vitro model of dose-dependent generation of RET/PTC in human thyroid cells after exposure to radiation. This system will serve as an important tool for further studies of radiation-induced DNA damage and chromosomal rearrangements. In this proposal, we will test the hypotheses that RET/PTC rearrangement is a direct result of mis-rejoining of double-strand DNA breaks induced by radiation exposure. Specifically, we will characterize the frequency and spectrum of radiogenic breaks and rejoining kinetics in the RET gene region, test if one or two DNA breaks are required for the rearrangement, and compare the effects of irradiation and 1-131 on the generation of RET/PTC rearrangements. We will also determine the fate of cells after they acquire the rearrangement and will characterize the earliest stages of radiation-induced carcinogenesis initiated by various RET/PTC types. Finally, we will find whether these rearrangements are predisposed by altered function of several genes playing a central role in double-strand break repair in vitro. Then, we will test the role of alterations in these genes in predisposition to radiation-induced thyroid cancer in human populations. These studies will expand our understanding of the mechanisms of radiation-induced carcinogenesis and provide important information for radiation risk assessment and protection applicable to a variety of areas such as clinical use of external radiotherapy and I-131, occupational radiation exposure, space exploration, and radioprotection from potential accidents involving nuclear power reactors or radiological terrorism.

Autism is a neurodevelopmental disorder that affects approximately 0.6% of the U.S. population. The causes of autism spectrum disorders are under intense investigation, with strong evidence for genetic substrates. Lifetime costs of caring for autistic individuals are high, both in terms of a) quality of life for the affected individuals and their families;b) financial expenses to the families, educational systems, and health care agencies. Discovery of multiple gene mutations, copy number variants, and epigenetic factors in people with autism has spurred the development of mouse models with the homologous mutation. Genetic manipulations in mice offer an optimized experimental strategy to understand the consequences of candidate gene mutations. Effective treatments for the core symptoms of autism are currently limited to early behavioral interventions. Discovery of effective pharmacological treatments requires a greater understanding of the genes, biological mechanisms, and environmental factors that contribute to the causes of autism. Animal models with robust phenotypes relevant to the diagnostic symptoms of autism offer an optimized experimental strategy to test the efficacy and safety of proposed treatments. Our Laboratory of Behavioral Neuroscience (LBN) is an international leader in behavioral assays for transgenic and knockout mice with mutations in genes expressed in brain pathways involved in neuropsychiatric disorders. We collaborate with a large number of molecular genetics laboratories that contribute mutant lines of mice with mutations in risk genes for autism to our research program. In FY2010 we tested shank1 knockout mice generated by Morgan Sheng at the Massachusetts Institute of Technology, shank3 knockout mice gnerated by Joseph Buxbaum at Mt. Sinai School of Medicine in New York, neuroligin2 and neuroligin4 knockout mice generated by Nils Brose at the Max Planck Institute, and BDNF trangenic mice in our laboratory and collaboration with Postdoctoral fellow Francesco Papaleo in Danny Weinberger's CBDB group, IRP, NIMH. A new line of engrailed2 mice recently arrived from Manny DiCicco-Bloom and Jim Millonig at RW Johnson Medical School in New Jersey. Neuroligins and shanks are families of cell adhesion proteins that regulate synapse maturation. Single mutations in several different neuroligin and shank genes have been detected in a small number of autistic individuals. During FY2010, shank1, shank3, neuroligin2, and neuroligin4 mice were evaluated on social and repetitive behavioral assays, and control measures of general health, by Research Fellow Dr. Mu Yang, Senior Research Associate Dr. Jill Silverman, Postbaccalaureates Adam Katz and Sarah Turner, and HHMI student interns Leuk Woldeyohannes and Dieynaba Diagne. Results to date indicate normal adult sociability but reductions in specific juvenile social interactions in some of these shank and neuroligin mutant mice, relevant to the first diagnostic symptom of autism. Because shank3 mutations also appear in the chromosomal deletion that defines Phelan-McDermid syndrome, Postbaccalaureate Danielle Abrams and HHMI student intern Harry Simon are testing sensory and motor functions and cognitive abilities relevant to this co-morbid neurodevelopmental disorder in the shank3 mice. Postdoctoral fellow Florence Roullet and Postbaccalaureate Roheeni Saxena analyzed social olfactory cues and responses using a novel scent marking task. Results indicate generally normal social olfactory communication in most cases in these lines of mutant mice. Postbaccalaureate Mark Harris worked with former postdoctoral fellows Maria Luisa Scattoni and Markus Wohr to analyze ultrasonic vocalizations in social settings, including pup separation, male/female interactions, and exposure to social olfactory cues. Significantly fewer social vocalizations were seen in some of these lines of mice, indicating abnormalities in social communication, relevant to the second diagnostic symptom of autism. During 2006-2008, we discovered autism-relevant phenotypes of the inbred mouse strain BTBR T+tf/J (BTBR). Lack of sociability was confirmed in multiple independently bred cohorts, on multiple social tasks including juvenile play, adult social approach, adult reciprocal social interactions, and social transmission of food preference (Yang et al.,2007a,b, 2009;McFarlane et al., 2008), relevant to the first diagnostic symptom of autism, abnormal social interactions. During 2009-2010, postdoctoral fellows Maria Luisa Scattoni, Florence Roullet,and Markus Wohr discovered that BTBR emitted fewer ultrasonic vocalizations in many social settings as compared to B6, and emitted less olfactory scent marking in some social settings (Roullet et al., 2010;Scattoni et al., 2010;Wohr et al., 2010), relevant to the second diagnostic symptom of autism. High levels of repetitive self-grooming are routinely detected in cohorts of BTBR (Yang et al., 2007a,b, 2009;McFarlane et al., 2008), relevant to the third diagnostic symptom of autism, repetitive behaviors. The absence of a corpus callosum in BTBR is unlikely to explain their phenotypes, as corpus callosum lesions did not affect social behaviors in C57BL/6J (B6), a standard control strain of mice with high sociability (Yang et al., 2009). More global underlying disruptions of white matter connectivity may contribute to the BTBR phenotypes. Genetic mechanisms responsible for the autism-like phenotypes in BTBR were pursued in FY2010. In collaboration with Dr. Elliott Sherr, University of California San Francisco, Dr. Yang and Postbaccalaureate Adam Katz completed the behavioral phenotyping for a quantitative trait loci linkage analysis (QTL) of the BTBR x B6 cross, designed to discover genes in the BTBR background that correlate with their autism-like phenotypes. 400 F2 mice were scored for social and repetitive behaviors. Tailsnips of the 400 behaviorally tested F2 mice are currently being genotyped in Dr. Sherr's laboratory. Biological mechanisms underlying the high levels of repetitive self-grooming in BTBR were evaluated by Dr. Silverman, in collaboration with Dr. Jim Koenig at the University of Maryland Psychiatric Research Center. High circulating corticosterone suggested a stress-related mechanism. However, low responses by BTBR on stressful behavioral tasks, and similar levels of corticotropin releasing factor in BTBR and B6 brain, indicate that repetitive self-grooming in BTBR is not mediated centrally by a brain response to stress. A major translational component of our Project MH-002179 is the preclinical search for potential treatments. Robust and highly replicated social deficits and repetitive self-grooming in BTBR provide a good model system for testing the ability of drugs and behavioral treatments to reverse and prevent autism-like symptoms. During FY2009, Dr. Silverman and Postbaccalaureate Sarah Turner discovered that MPEP, an mGluR5 antagonist reported to reverse phenotypes in Fragile X mice, blocked repetitive self-grooming in BTBR at doses that were not sedative. During FY2010, Sarah Turner and summer student intern Seda Tolu replicated this effect of MPEP. Their preliminary data indicate the same amelioration of repetitive behavior with a more selective mGluR5 antagonist, MTEP. Postbaccalaureate Sarah Turner tested CX546, a prototypic ampakine modulator, and detected significant improvement in sociability in BTBR in the first study. Postbaccalaureate Mike Karras is now repeating the CX546 experiments with a different drug vehicle and a different route of administration. These pharmacology studies provide evidence for the translational value of our assays in optimized mouse models of autism, for preclinical discovery of novel therapeutics to treat the diagnostic symptoms of autism spectrum disorders.

This project will evaluate the feasibility of argon and CO2 lasers for performing ablative surgery of the middle ear and semicurcular canals of experimental monkey subjects. In addition to delineating safe and appropriate output parameters (burst duration and power levels) for vaporizing or perforating hard and soft otic tissues, auditory and vestibular function will be measured electrophysiologically and behaviorally in order to determine if laser microsurgery is accompanied by harmful and sensorineural side effects. After completing all functional measurements postoperatively, the animals will be sacrificed and temporal bone sections processed for evaluation of inner ear morphology. BIBLIOGRAPHIC REFERENCE: Wilpizeski, Chester R. Otological applications of lasers. Chapter 4 in M. Wolbarsht, ed., Laser applications in medicine and biology, vol. 3, Plenum Press, 1977.

Chickens which develop idiopathic scoliosis have demonstrated several abnormalities in the mechanical and metabolic properties of collagen. How these changes affect the animal's spine causing the scoliosis is unclear. This proposal will study the mechanical properties of bone in chickens with scoliosis and relate the information with that currently known about the mechanical properties of bone in other conditions affecting collagen. In addition, the growth patterns of the scoliotic spine will be investigated with tetracyline labelling. The information gained will be related to the spinal curvature in hopes of elucidating possible causes of idiopathic scoliosis.

The hallmark of B lymphopoiesis is the sequential productive genomic rearrangement of immunoglobulin heavy (Ig) then light chain loci. Following in-frame Ig recombination, expression of the pre-BCR is associated with IL-7 dependent clonal expansion. However, pre-B cells must exit cell cycle before initiating Igk recombination. Failure to do so risks genomic instability and leukemic transformation. Downstream of the IL-7R, STAT5 drives proliferation by inducing cyclin D3 while simultaneously repressing Igk recombination. We have now elucidated three mechanisms by which STAT5 represses Igk. First, STAT5 binds the Igk intronic enhancer as a tetramer and recruits the histone methyltransferase Ezh2 that decorates the Jk and Ck regions with H3K27me3. STAT5 also represses the bromodomain containing epigenetic reader BRWD1. In the absence of BRWD1, Igk did not recombine efficiently and this was associated with diminished Jk accessibility and disordered nucleosome positioning at both Jk exons and the Igk enhancers. BRWD1 was required for clearing nucleosomes from extended GAGA motifs and for recruiting RAG1/2 to Jk. These findings suggest critical and comprehensive roles for BRWD1 in chromatin remodeling, gene transcription and in targeting recombination to Igk. Finally, STAT5 induces expression of cyclin D3, which both drives proliferation and represses Vk transcription. Confocal and super-resolution microscopy demonstrated that in pro-B cells, Vk was imbedded within a web of closely juxtaposed nuclear matrix-associated cyclin D3 and elongating-pol II (transcription centers). In these cells, Vk did not access the transcription centers. However, upon transition to the small pre-B cell stage, or when cyclin D3 was absent, one allele of Vk looped into e-pol II. In contrast, in small pre-B cells JkCk more commonly associated with e-pol II in a biallelic manner. These later findings suggest that Vk accessibility, and not JkCk accessibility, contributes to allelic exclusion. In toto, these findings suggest a comprehensive model of Igk regulation in which radically different mechanisms of repression, control of mobile Vk segments access to e-pol II and epigenetic regulation of a fixed JkCk platform, determine Igk accessibility to recombination, and ensure that proliferation and recombination remain mutually exclusive. Aim 1. Define the functional importance of BRWD1-mediated chromatin remodeling in B lymphopoiesis. Aim 2: Determine how GAGA motif recognition by BRWD1 contributes to Igk recombination. Aim 3. Determine the mechanisms regulating Vk accessibility.

7. Project Summary/Abstract More than 50 million procedures were performed with GI endoscopic devices in 2009. Since endoscopes cannot be sterilized, risk of infection from contaminated endoscopes can be tangible and needs to be urgently addressed. Infection at endoscopy has recently been recognized as a significant risk after two patient deaths from CRE infection transmitted by contaminated duodenoscopes at the UCLA Medical Center in 2015. Since 2013, more than 100 patients have been affected by CRE in Chicago, Seattle, Pittsburgh, and Los Angeles. These and many other outbreaks have been reported to CDC over the years. Endoscope contamination is directly linked to inadequate cleaning by current manual cleaning protocols and by automated endoscope reprocessors (AERs). Despite manual brushing of the wider suction/biopsy channels, narrower channels cannot be brushed, and can only be flushed with enzymatic cleaners. It is now recognized that existing cleaning methods are deficient, and this is further magnified by the difficulty of removing biofilm from endoscope internal channels. To overcome the above limitations, we have developed a highly effective technology based on the flow of special nano- and microfibers through the endoscope internal channels, which results in the unparalleled removal of organic soil, bioburden and, most importantly, biofilm. During flow, floc fibers make contact with a channel surface and generate a high hydrodynamic force which, at a close distance (<100 nm), is capable of removing contaminants, including biofilm. High-level cleaning and removal of multispecies biofilm have been demonstrated in our Preliminary Studies, including in narrow 1.6 mm air/water channels that cannot be currently brushed. The technology can be envisioned as cleaning the surface of channels at nanometer or molecular levels, and can be termed ?nano-brushing.? The proposed technology is based on rigorous and rational formulation of flow of suspensions and can be properly modeled as described in the proposal. This Phase I SBIR includes three Specific Aims to: i) Define MFC formulations and process parameters in simulated experiments; ii) Optimize process parameters to remove biofilm from endoscope internal channels; and iii) Adapt, refine and optimize the cleaning and rinsing processes in actual endoscopes.

The merozoite interacts in a receptor specific manner with the erythrocyte surface and is the stage against which immunity may work to block invasion. Thus, merozoite surface components are of interest for their role in erythrocyte recognition and as antigens for induction of protective immunity. We are identifying P. knowlesi receptors for attachment to monkey and human erythrocytes. We are studying one antigen on the merozoite surface that undergoes antigenic variation to understand the molecular basis for this variation. We are studying P. falciparum mutants that use an alternative receptor for invasions of human red cells. We are attempting to adapt P. falciparum to subprimates as an animal model for vaccine trials.

common shared clinical question of the Translational Center for Serotonin and Stimulant Addiction (TCSSA) is the role of 5-HT neurobiology in impulsivity and cue reactivity endophenotypes which associate with cocaine addiction. Project 2 will employ and refine behavioral measures of impulsivity and cue reactivity in rats, mechanistically link the status of 5-HT^R and/or 5-HT2cR expression and function to specific behavioral profiles, and test the hypothesis that treatment with M100907 (selective S-HT^R antagonist), WAY 470 (selective 5-HT2cR agonist) or their combination will normalize behavioral and molecular patterns of expression. We will initially identify the endophenotype for impulsivity based upon the degree of response inhibition in the differential reinforcement of low rates schedule (DRL-20) task and for cue reactivity based upon lever responses reinforced by drug-associated cues during forced abstinence from a well-defined cocaine self-administration paradigm. We will also investigate how basal levels of impulsivity interact with the progression of drug-taking and drug-seeking. In concert with Project 3, we propose that gains in treatment effectiveness will be possible with the selective homo- and/or heterodimeric 5-HT2R ligands. Promising compounds will be evaluated for in vivo bioavailability in a stairstep approach utilizing simple rodent assays that will also serve as dose-ranging studies for the more intensive assessment of their effectiveness in models of impulsivity and cue reactivity. Project 2 will be driven and adapted directly from the clinical neurobiology insight (Project 1) and take a molecular-level view to elaborating the role of 5-HT in targeted endophenotypes (Project 3, Core B). Careful analyses of the status of 5-HT^R and 5-HT2CR expression and function and the effects of treatment with extant and novel selective 5-HT^R and 5-HT2cR ligands or their combination, in rodent models will shape the rationale for future hypothesis-driven neurobiological studies in humans (Project 1) and clinical assessments of new selectively-targeted serotonergic drugs (Project 3). Lav Abstract. No effective, accessible medication for the treatment of stimulant addiction is currently available. We will establish the ability of existing and newly designed drugs to suppress relapse in rodent assays which model human drug-taking.

Feeding behavior is critical for animal survival, and is also a fundamental aspect of energy homeostasis. The growing incidence of eating disorders and their associated health costs have led to intensive research efforts directed to understand the mechanisms and signaling pathways that control and regulate food intake and energy homeostasis. This process appears to be regulated by a highly complex neuroendocrine system involving a multitude of neuropeptides. However, given the high chemical complexity and wide distribution of neuropeptides, the precise molecular mechanisms at the cellular and network levels remain elusive. This is, in large part, due to a lack of analytical capabilities to measure and identify these low abundance endogenous signaling molecules in a complex microenvironment. Clearly, the development of highly sensitive and selective analytical tools for neuropeptide identification and quantitation is in great demand. This project aims to fill this gap by constructing and implementing a unique analytical measurement platform and developing improved mass spectrometry (MS) - based methodologies for probing peptidergic signaling in feeding with enhanced sensitivity and selectivity. We have chosen to study the simpler and well-defined crustacean stomatogastric nervous system (STNS) and its associated neuroendocrine organs, to facilitate the technology development and validation. Furthermore, the wealth of information about the neuropeptides present in this model system and its well-defined physiology provide unique opportunities to address fundamental neuroscience problems related to the neuropeptidergic modulation of complex behaviors such as feeding. The specific aims of this project include: (1) To develop direct tissue in situ peptide profiling and quantitation via in-cell combination (QUICC) methodologies by matrix-assisted laser desorption/ionization (MALDI) Fourier transform mass spectrometry (FTMS). Major neuroendocrine organs isolated from food deprived and satiated animals will be analyzed and compared for their peptide content;(2) To develop in vivo microdialysis sampling techniques and isotopic labeling strategies coupled to nanoflow LC/MS for differential display of circulating peptides in response to feeding;(3) To develop a hybrid strategy combining the use of isotope-assisted de novo MS/MS sequencing and sequence homology searching to identify and discover novel neuropeptides, with focus on the peptides showing differential expression and secretion in response to feeding;(4) To test physiological effects of the newly discovered peptides on the feeding circuits (gastric mill and pyloric neuronal networks in the stomatogastric ganglion). Collectively, these proposed experiments will develop and illustrate improved methods and capabilities for neuropeptide analysis. This project will also discover a large number of new peptides and provide the neurochemical basis toward a detailed mechanistic understanding of the peptidergic regulation of feeding behavior. The molecular insights gained from studying such a small system can be transferred to the larger, more complex vertebrate systems and could potentially lead to the development of new therapeutic strategies for feeding disorders.

Dr. John Parker is a D.V.M. research scientist with a Ph.D. in molecular virology from Cornell University. Dr. Parker is currently a post-doctoral fellow (NRSA F32 AI10134) at Harvard Medical School in Dr. Max Nibert's lab. Dr. Nibert, the sponsor of this MCSDA, is an internationally recognized leader in the field of reovirus molecular virology and has a strong track record as a research mentor. Dr. Parker's immediate career goal is to attain advanced training in cell and molecular biological approaches to viral pathogenesis. This training will complement and significantly broaden his existing expertise in molecular virology, which together with his experience as a veterinary clinician should help him achieve his longer-term career goal of becoming an independent, nationally competitive, veterinary research scientist. These goals will be achieved over the requested three year period of this MCSDA by providing Dr. Parker the opportunity to learn advanced techniques in live-cell video imaging and protein biochemistry as applied to the study of virus-host interaction. In addition, during the course of the proposed MCSDA, Dr. Parker will assume more responsibility for decisions regarding the direction of this research project after consultations with the sponsor. This responsibility, together with further experience in written and oral presentation of research findings will give Dr. Parker the tools needed for an independent research career. The training and research will take place in the Dept. of Microbiology and Molecular Genetics at Harvard Medical School, one of the foremost biomedical research institutions in the world. The research plan will test the hypothesis that the virus inclusion bodies (vlBs) that form in reovirus-infected cells co-opt the cellular misfolded protein response in order to concentrate and sequester viral proteins and RNAs away from the general cellular milieu and by so doing promote viral replication and assembly and induce cytopathology. In particular this research plan will examine the morphogenesis of vlBs and their interaction with microtubules and intermediate filaments. Striking similarities between vlBs and inclusion bodies (aggresomes) found in neurons of patients afflicted with neurodegenerative diseases such as Parkinson's and Huntington's disease will be explored. The pathophysiologic effects associated with the development of vlBs are likely to be the same as those associated with inclusion body formation in neurodegenerative disease. This research should significantly advance our understanding of how cytopathology and disease are induced during viral and neurodegenerative disease by the formation of intracellular inclusion bodies.

The objective of this continuing program is to provide veterinary students with an opportunity to engage in hypothesis-based biomedical research during the formative stages of their education. Veterinarians, and the veterinary profession in general, have much to contribute to scientific discovery in medical disciplines. An education in veterinary medicine is inherently broad-based and comparative. Veterinary students are trained to integrate medical literature from a variety of sources dealing with the full array of animal species, using problem solving and comparative approaches to evaluate disease pathogenesis and therapeutic strategies from molecular mechanisms through whole animal clinical features. As such, an education in veterinary medicine provides a solid and broad foundation upon which to develop a focused area of scientific expertise. Helping veterinary students discover this synergy and enhancing their interest in biomedical research is the goal of the current training proposal. [unreadable] [unreadable] The program will center on the trainee working full-time in a research laboratory, conducting experiments with the guidance and direct supervision of a faculty mentor. Disciplines represented by participating Cornell University faculty include infectious diseases, genetics, physiology, cancer biology, reproductive biology, toxicology, and food safety. Supporting sessions will include seven research information modules delivered in a small group discussion format with a special emphasis on animal-based experimental systems. Module topics include: 1) cell and molecular biology, 2) genomics and proteomics, 3) transgenic animal models, 4) comparative animal-based biomedical research involving non-rodent models, 5) infectious diseases and food safety, 6) experimental design and statistical analyses, and 7) laboratory animal medicine and comparative pathology. Students will also complete a graduate course focused on ethics and professional responsibilities of research scientists. Structured time outside of the laboratory, however, will not exceed two hours in any given week. [unreadable] [unreadable] [unreadable]

MacCHESS, a synchrotron radiation research resource for macromolecular crystallography, has the overall goal of advancing the frontiers of structural biology through innovate technical research and development. MacCHESS builds upon the National Science Foundation investments in the Cornell High Energy Synchrotron Source (CHESS), which maintains the synchrotron radiation laboratory, and the Laboratory for Nuclear Studies, which operates the storage ring. At MacCHESS, technical developments are driven by both core and collaborative research projects involving a broad range of macromolecules. In addition to performing technical R&D and core research, MacCHESS provides specialized instrumentation for macromolecular crystallography and a staff for user training and equipment maintenance. MacCHESS has established itself as one of the most productive facilities in the world for macromolecular crystallography with over 350 papers published during the past five years as a result of MacCHESS related activities. Of these, 65 were published in the high visibility journals, Science, Nature and Cell. Currently, CHESS stations A-1 and F-1 are used primarily for monochromatic oscillation data collection and station F-2 is used for MAD phasing experiments. A key component of this proposal is the development of two new stations for macromolecular crystallography, G-1 and G-2. Developing these stations for crystallography will be cost effective because funding for G-line will be provided by the NSF with matching funds for Cornell University. During the next five years, MacCHESS technical R&D will focus on (1) new x-ray optics, (2) CCD and pixel array x-ray detectors, (3) new instrumentation for small crystals, crystallography and phasing, (4) new software for data collection, analysis and transfer, (5) innovate uses of the world wide web to create a macromolecular crystallography "collaboratory", (6) upgrading of computing hardware and (7) instrumenting G-line. Core and collaborative research will focus on (1) novel phasing methods, (2) implementation of x- ray detectors, (3) applications in molecular medicine, (4) large macromolecular structures and (5) G-line commissioning. MacCHESS will continue to provide service in a wide variety of scientific areas. Particularly notable is the past record of productivity in the area of AIDS-related structural biology. Technical developments resulting from MacCHESS research are freely available to the scientific community and will be disseminated through meetings, workshops and other mechanisms. We will work towards increasing interactions with other synchrotron sources in an effort to maximize the benefits of our efforts.

A series of studies completed during our previous project period concentrated on describing the relative role of age, cardiovascular endurance, and the menstrual cycle in determining women's ability to tolerate work in hot environments. Our results indicated that fluctuations in hormonal levels plays a minor role and that most of the variability between individuals was due to differences in age and fitness levels. Several mechanisms were hypothesized to explain the interaction of these two variables with thermoregulatory responses. The purpose of the proposed series of studies is to investigate these mechanisms in detail. The relative contribution of central and peripheral mechanisms in effecting changes in the sensitivity of the sweating mechanism, patterns of body fluid shifts, and responses to acclimatization procedures will be quantified and evaluated for age and/or fitness related differences in response. The results of such studies will help us understand how females respond to the stress of working in hot environments as well as providing insight into some of the unanswered questions in the area of thermoregulation.

Histamine release from mast cells and blood basophils is being studied as one of the immunological mechanisms involved in inflammation. Among the histamine releasing agents employed are IgE antibody, and the Ca++ ionophore A23187. The relationships between IgE antibody crosslinking, basophil desensitization and histamine release were analysed by kinetic studies. Cultured rat basophilic leukemia cells are used as a model for the studies of the IgE receptor and changes in phospholipid methylation during cell activation.

Abstract Understanding lung development and the signals provided by the embryonic environment are of great relevance to public health. A better knowledge of the cues that guide proper lung growth and the process that forms a highly branched, specialized structure will shed light on the causes of improper development. Congenital disorders, such as pulmonary hypoplasia, or insufficient lung growth, result when embryological processes go awry. Improper development may also predispose to adult conditions such as asthma. The goal of this project is to elucidate the dynamic processes involved in lung development and determine the dependence on neural stimulation for lung growth. The quail is an amniote with similar development to mammals, and it is much more accessible for genetic labeling experiments to study embryogenesis. Confocal and two-photon imaging of transgenic quail lungs that have multiple cellular structures labeled in different colors will be used to dynamically illustrate early lung development. The importance of neural stimulation for lung growth and branching will be measured by modulating neural inputs to the lungs and assessing growth and cell fate. These studies will contribute insight into the dynamics of lung development and what regulates the process. Such knowledge may lead to interventions to rescue congenital disorders, treat asthma, guide efforts to engineer replacement tissue, or stimulate lung repair following injury in adults. PUBLIC HEALTH RELEVANCE: Project Narrative Increasing our knowledge of the factors that direct the proper embryological development of the lung has great implications for improving public health. Understanding how the lung develops will help clarify the causes that underlie congenital disorders and may lead to new interventions for infant respiratory conditions and chronic syndromes such as asthma. Better insight into how the lungs form may also facilitate tissue engineering or the ability to stimulate lung repair following injury.

DESCRIPTION (from the application): The long-term objectives of this project are to define the changes in knee cartilage mechanical function, morphology, and composition resulting from injury with the specific aims of better evaluating cartilage injury by non-invasive means such as magnetic resonance imaging (MRI), and developing therapeutic strategies such as repair and grafting. Injury to the cartilage of the knee joint is an extremely common problem that crosses all age groups and injury severity levels from high energy sports injury to low impact trauma. In particular, damage to the knee joint meniscus frequently leads to degenerative joint disease, including deterioration of the articular cartilage which is characteristic of osteoarthritis. The research design proposed in this project centers on the canine knee (stifle) joint, and two hypotheses: (1) Meniscus injury or partial excision of the meniscus will lead to diminished mechanical properties and deterioration of articular cartilage, and (2) Repair or reconstruction of a meniscus will delay or diminish degenerative changes in articular cartilage. Meniscal and articular cartilage properties will be analyzed after meniscal injury, after partial meniscal excision, after meniscal repair, and after meniscal grafting with a biologic scaffold. Specifically, analysis will include material properties of articular cartilage, and material properties of grafted, non-grafted, and repaired menisci at three months and one year after injury. The materials properties of the tissues will provide longitudinal and correlative data for interpretation of histologic changes, collagen subtyping, and analysis of synovial fluid biomarkers such as the 3B3+, 3B3-, and 5D4 proteoglycan epitopes at the same time intervals. Finally, and importantly, high resolution MRI (under 100 micron resolution) images will be obtained in the meniscal injury group at one year after injury. The MR images analyzed to create volumetric quantification, curvature analysis, and water composition of meniscus as well as cellular organization, articular cartilage thickness and volume, and collagen organization within the articular cartilage. The continuation of this research beyond this proposal will lead to further development and refinement of knee joint cartilage reconstructive protocols using modern principles of tissue engineering.

This proposal is for a five year renewal (years 6-10) of a Program Project to pursue gamma-secretase modulating compounds as Alzheimer's disease (AD) therapeutics at the University of California, San Diego, consortium with Mayo Clinic Jacksonville and Washington University, St Louis. In the first five years of support, we have shown that nonsteroidal anti-inflammatory drugs (NSAIDs) have a novel secondary action that modulates gamma-secretase processing in a cyclooxygenase (COX) independent manner. The activity of a subset of NSAIDs preferentially lowers the amyloidogenic A[unreadable]42 peptide both in vitro and in vivo, leading us to propose that this activity may be one explanation for the apparent risk reduction of AD in chronic NSAID users. As we have identified many compounds in addition to NSAIDs that shift gamma secretase cleavage, we now generically refer to these compounds as gamma-secretase modulators (GSMs). Our research efforts have contributed to the introduction of the first GSM, R-flurbiprofen ("Flurizan" renamed Tarenflurbil") into the clinic for testing in AD treatment, and led to the preclinical development of additional GSMs. The two major goals of this PPG are to further understand the mechanism of action of GSMs, and to test their activity in humans as well as determine their ability to lower disease-associated biomarkers in AD individuals. Project 1 will examine the site of action of GSMs with respect to effects on gamma- versus epsilon-cleavage and test the efficacy of combination treatments in rodents. Project 2, which has now incorporated the former Chemistry Core, will extend studies relating to the binding site of multiple GSMs, examine the relative contribution of GSMs with respect to reducing A[unreadable]42 production vs. inhibiting A[unreadable] aggregation, and conduct animal studies to better define how GSMs acutely alter A[unreadable] levels in the brain, CSF, and plasma of mouse AD models. Projects 1 and 2 will coordinately examine the biological properties of shorter A[unreadable] peptides which are elevated by these GSMs. Project 3 will determine if R-flurbiprofen lowers A[unreadable]42 in human CSF by measuring newly synthesized A[unreadable] through in-dwelling catheter and will test whether a GSM (ibuprofen) will reduce disease associated CSF biomarkers and brain volumetric changes by neuroimaging in AD subjects in a one year placebo controlled double-blind treatment study. Successful completion of these studies will provide greater insight into 1) how the GSMs shift gamma cleavage, 2) how GSMs attenuate AD-like phenotypes in rodent models, and 3) how GSMs and GSM based NSAIDs may work in humans. By providing additional preclinical and clinical information on GAMs, such studies should contribute significantly to the further development of GSMs as Alzheimer's therapeutics. PRINCIPAL INVESTIGATOR: Dr. Edward Koo is a world renowned and experienced AD research. He is well qualified to lead this program project. REVIEW OF INDIVIDUAL COMPONENTS CORE A - Administrative Core, Dr. Edward Koo DESCRIPTION (provided by applicant): The responsibilities of the Administrative Core are to coordinate and integrate the activities of the three Projects to ensure maximum cooperation and coordination of efforts among program investigators. Further, the Core will provide administrative and financial support for the Projects on an ongoing basis. The Core will organize the annual visit of the External Advisory Committee to review the scientific progress of the Projects. The Core will interface with the Data Safety and Monitoring Board who will oversee the safety aspects of the clinical studies proposed in Project 3. Further, the Core will provide fiscal and management oversight of the Alzheimer's Disease Cooperative Study (ADCS) who will be directing the biomarkers study proposed in Project 3. Another goal of the Core is to enrich aging and Alzheimer's disease research by facilitating the dissemination of results and interaction with other investigators at UCSD, Mayo Clinic Jacksonville, and other local institutions. The Administrative Core is responsible for assuring compliance with animal welfare, scientific integrity, and financial policy requirements of UCSD and NIH. Lastly, the Core will be responsible to assembling the annual progress report to NIH.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Human metapneumovirus (hMPV), a newly-recognized member of the Paramyxoviridae family, mediate serious lower respiratory tract illness in very young children, the elderly and immunocompromised patients (1-3). hMPV causes bronchiolitis, pneumonia and flu-like syndromes, as well as asthma exacerbations, and it is considered a substantial public health problem. Since its discovery in 2001, many studies have provided a better understanding of the epidemiology associated with this viral infection. However, fundamental questions regarding the pathogenesis of hMPV-induced lung disease and the host immune response are not fully understood. Furthermore, no treatments or vaccines for hMPV are currently available. The lung innate immune system represents a critical component of the host defense against viruses and is coordinated at the cellular level by activation of transcription factors that regulate the expression of inducible gene products with antiviral and/or inflammatory activity. Key to the development of novel therapies for hMPV infection is in- depth understanding of hMPV disease pathogenesis and the nature of virus-host cell interactions that lead to either infection or killing of the virus. In this respect, the study in the interaction between hMPV and innate immune cells are of particular interest. Type I Interferon (IFN) is known as key component of the innate immune system responsible not only for broad cellular antimicrobial activity in response to viral infections, but also for its role in linking innate and adaptive immune response (4). Using an experimental mouse model of hMPV infection (5,6) and primary human cells in vitro (7), I have demonstrated that type I IFN plays a fundamental role in regulating hMPV pathogenesis. More importantly, hMPV interferes with the Toll-like receptor (TLR)- induced type I IFN production in vitro (7,8) and in vivo (5). However, the mechanisms of activation and regulation of type I IFN by hMPV infection have not yet been elucidated. Our overarching hypothesis is that hMPV activates distinct innate signaling pathways in antigen presenting cells, thus triggers immune responses that lead to efficient viral clearance and elimination of the disease. This hypothesis is based on the discovery that hMPV activates type I IFN production in human plasmacytoid dendritic cells (pDC) through the endosomal compartment where TLR7/8/9 can be activated. In addition, the cytosolic protein MDA5 is necessary for the production of type I IFN in human myeloid DC (preliminary data). In this grant, we propose to identify the mechanism(s) by which hMPV activates TLR-dependent and - independent cellular signaling in vitro and in vivo. We will achieve these objectives through the following specific aims: Aim 1. To define the molecular mechanism(s) by which hMPV induces type I IFN production in primary human cells. We will investigate how hMPV activates TLR signaling pathway to induce type I IFN in pDC and which pathway is activated by hMPV in moDC and monocytes. Aim 2. To determine the role of lung macrophages and DC in the production of type I IFN in hMPV infection. We will investigate the relative contribution of macropages and DC in the production of type I IFN in vivo using an experimental mouse model of infection. Furthermore, we will determine the contribution of pulmonary macrophages and DC in hMPV pathogenesis. Aim 3. To investigate the biological relevance of TLR-dependent and independent pathways in hMPV infection in vivo. We will characterize the virulence and pathogenicity of hMPV in mouse model of infection using transgenic mice deficient in TLR7, MyD88, and MDA5 expression. We will assess viral replication, clinical disease, lung function and inflammation, and cytokine/chemokine/IFN expression.

During hypo- and hyperbaric decompression, gas-supersaturation of tissues and blood can cause bubbles to form there with serious medical consequences. The growing use of hyperbaric environments by man has made this problem of air embolism increasingly important. The objective of the present physiological study is to increase the understanding of the early etiology of air embolism, an area which has received relatively little attention in the past. Essential information is lacking as to where and how the bubbles form, and the factors which cause the dramatic lowering of liquid cavitation stability in vivo are still to be established. Several fundamental aspects of gas bubble nucleation and initial growth will be investigated by cavitation experiments on solutions, interface systems, biological fluids, microorganisms, and tissue preparations. The basic methods and procedures which have been developed in the present phase of these studies will be supplemented by high-speed cinemicrographic recordings of the cavitation events. This will allow detailed determinations of the loci for the appearance of bubbles, and their microscopic rate of growth for various pressures and decompression rates. It is of further interest to determine both the possible lower supersaturation ranges required for true nucleation in various in vivo systems and the physical parameters which govern this process. The latter will be of particular value for reaching a quantitative, theoretical formulation of the nucleation events. Our long range goal is to aid in the prevention and treatment of air embolism by contributing to the development of better hyperbaric gas mixtures and decompression procedures.

[unreadable] The proposed Northwestern University magnetic resonance imaging (MRI) training program will provide trainees opportunities to learn MRI basics and perform MRI research in diversified disciplines. The preceptors of the program come from various departments and background. Research interests include (1) Cardiac imaging; (2) Vascular Imaging; (4) MRI-guided endovascular interventions; (4) Neuro imaging; (5) Physiological imaging, including perfusion, diffusion, and oxygen consumption; (6) Breast imaging; (7) Liver imaging; (8) Targeted molecular imaging; (9) Spectroscopy; and (10) Signal and image analysis. The trainees will be graduate students pursuing PhD in Biomedical Engineering Department with basic background in an engineering discipline or a related field. The training program will last for 5 years. We will enroll 3 trainees in the first year and 5 trainees for years 2-5. The training program will include didactic instructions, laboratory research rotations, clinical rotations, industrial experience, seminar series, and mentored dissertation research. The strengths of our program include the wide range of research interests (technical developments and medical applications, basic scientific investigations and clinical studies, morphological, functional and molecular imaging, animal experiments and human studies), close interactions between MR physicists and clinicians, strong ties to industrial partners, and unsurpassed access to state-of-the-art MRI systems. [unreadable] [unreadable] [unreadable]

During the last year, the project has focused on chemokine receptors on human helper (CD4+) T cells and on cancer cells. We described the patterns of expression for eight chemokine receptors on blood CD4+ T cells from multiple healthy adults, and we studied in detail the subsets of memory CD4+ T cells that express the receptors CCR5 and CCR2. Our studies suggested a pathway of progressive effector/memory differentiation from the CCR5-CCR2- to CCR5+CCR2- to CCR5+CCR2+ cells. Sensitivity and rapidity of T cell receptor (TCR)-mediated activation, TCR signaling, and effector cytokine production by the subsets were consistent with such a pathway. The CCR5+CCR2+ cells showed the greatest responses to tetanus toxoid (the result of prior vaccination) and herpes simplex virus-1 (the result of prior and persistent (latent) infection). CCR5+CCR2+ cells also expressed the largest repertoire of chemokine receptors. By contrast, the CCR5+CCR2- cells had the greatest percentages of suppressor CD4+ T cells and activated and dividing cells, and were most susceptible to programmed cell death. Data from some mouse models have suggested that CD4+ T cells with effector capabilities, which would be important for initiating immediate protection against infection, are short-lived in vivo. Our results are important because they indicate that, in fact, increasing memory cell differentiation can be uncoupled from susceptibility to cell death and is associated with responsiveness to multiple inflammation-associated chemokines, suggesting that vaccination (or infection) can produce a stable population of effector-capable memory cells, which are highly enriched in the CCR5+CCR2+ subset and are ideally equipped for migration into tissue in order to mediate rapid recall reactions against infectious agents. Increasing the numbers of such cells in response to immunization might enhance the efficacy of vaccines. In other studies, we analyzed histone modifications at the promoters of the genes encoding CXCR3 and CCR4 in order to understand how expression of these receptors is regulated. CXCR3 and CCR4 are receptors associated with Th1 and Th2 differentiation, respectively, and we analyzed patterns of histone modifications on cord blood cells activated in vitro under Th1 or Th2 polarizing conditions as well as on CD4+ T cells isolated directly from adult blood. Our most notable observation was that the gene for CCR4 could be highly induced in cord blood cells under non-polarizing conditions in vitro without many of the activating changes in histone H3 that were found at CCR4 in Th2 polarized cells and in CXCR3-CCR4+ cells from blood. The low levels of the enabling histone H3 modifications at CCR4 suggested that the gene remained in a relatively inactive configuration in the non-polarized cells, which was supported by our finding that without continued activation, levels of CCR4 expression in the non-polarized cells fell over time. By contrast, levels of CCR4 were maintained on the cells that had been activated initially under Th2 conditions. An important conclusion was that the roles for histone H3 modifications in gene expression can depend on the overall transcriptional environment, producing significant discordance between modifications at promoter histones and expression even for a single gene. Moreover, our data demonstrated that histone modifications contribute to the mechanisms whereby gene expression can be made persistent versus transient. Our studies on chemokine receptors in cancer focused on CXCR4, which has now been reported in more than 23 types of human cancer. The data suggest that CXCR4 is a potential marker on tumors for aggressive behavior and/or poor response to therapy, as well as a possible therapeutic target. Currently, CXCR4 expression in tumors can only be evaluated in biopsy specimens, which provide data limited to the site of the biopsy and to a single time point. AMD3100 is a CXCR4 antagonist that is FDA-approved as plerixafor/MozobilTM for the mobilization of hematopoietic stem cells, and we have evaluated a radiolabeled form of AMD3100 (64CuAMD) in order to detect and quantify CXCR4 expression in cancer using positron emission tomography (PET). For preliminary studies in mice, we used Chinese hamster ovary (CHO) cells and Lewis lung carcinoma (3LL) cells that had been transfected to express CXCR4, along with the parental control cells, to produce subcutaneous and/or lung tumors. CXCR4-expressing but not CXCR4-negative tumors could be visualized by PET in both subcutaneous sites and in the lung at six hours after injection of 64CuAMD. Dosimetry experiments were also done in mice for estimating the allowable dose for administration to humans, which provided a tentative limit of 444 MBq (12 mCi), which might make imaging in patients possible.

The expected rapid increase in the prevalence of Alzheimer's disease (AD) will have a significant impact on the economic and social structure of this country and as a result, there is a critical need for research focusing on developing early intervention and prevention strategies. One approach to developing primary prevention strategies for AD is to study cognitive and neurobiological changes occurring in middle-aged individuals who do not have dementia but may be at increased risk for developing the disease. One such group is the adult children of persons with AD who are at increased risk of developing the disease due to heredity, environmental and health risk factors shared with affected parents. The University of Wisconsin is prepared to conduct such a study because of the existence of the Wisconsin Registry for Alzheimer's Prevention (WRAP). WRAP focuses specifically on adult children of persons with AD in an effort to facilitate early detection and develop strategies to reduce the risk of developing AD. To date, we have archived DMA, plasma and serum samples and conducted extensive assessments on over 825 adult children of persons with AD and control subjects without a family history. The purpose of this prospective cohort study is to conduct a second wave of laboratory and neuropsychological testing and structural and functional MRI in order to define patterns and predictors of cognitive change over a 4-year re-test interval. Our long term goal is [unreadable] [unreadable] [unreadable] [unreadable] to identify health and lifestyle variables associated with abnormal cognitive aging and the development of AD and to use this information to develop interventions that will prevent or delay the onset of the disease. Our overall hypothesis is that adult children of persons with AD will exhibit signs of abnormal cognitive aging with declines in learning, memory and executive function and volumetric and fMRI abnormalities that will be predicted by specific genetic, vascular and lifestyle risk factors for AD. The Specific Aims are: 1) determine the differential effects of family history of AD and apolipoprotein (APOE) 4 allele on the cognitive aging of participants; 2) determine the effects of vascular and lifestyle risk factors on the cognitive aging of participants; and 3) examine the differential effect of family history of AD and the APOE 4 allele on brain aging as determined by rates of longitudinal decline in brain volume and fMRI activation during episodic memory tests that are known to activate regions of the brain affected by AD. [unreadable] [unreadable] [unreadable]

The Molecular Biology Core will serve as a major resource for the Program Project, providing needed reagents and technological services. Services supported by the Core and extensively utilized will primarily be: (1) synthesis of oligonucleotides for: use as hybridization probes, DNA sequencing, polymerase chain reaction primers, generation of coding sequences in plasmid expression vectors, and (2) automated fluorescent dye-labeled DNA sequencing analysis. In addition, the following services will be available on an as needed basis: amino acid composition analysis, N-terminal microscale protein or peptide sequencing, and peptide mapping.

This study examines obese and lean individuals on two standard diets (which differ by calorie content) and the stool and urine energy losses to compare these values in lean versus obese individuals. The study is also investigating the specific macronutrient content of stool to examine differences in these parameters between lean and obese individuals. The repeatability of these measurements is also being tested in a subset of individuals. In collaboration with Dr. Jeffrey Gordons lab at Washington University in St. Louis, samples of stool will be sent for determination of the fecal bacterial flora phylotypes to examine the association between stool energy loss and enteric flora populations. [unreadable] [unreadable] Preliminary results from an initial 14 lean and 9 obese individuals failed to show any difference in stool or urine energy loss. The range of calories stool was large, varying from 2 to 9 % in both groups. Stool calorie loss on each diet was associated in each individual. On the higher calorie diet, obese individuals had greater stool calorie loss (so absorbed less food ) than lean. Further results on fecal bacterial flora phylotypes are currently pending.

The broad, long-term objectives of this research project are to develop new and more effective intervention methods to improve the health and well-being of older adults. The specific aims are to determine the effectiveness of the Transtheoretical Model of Health Behavior Change in improving the physical activity and nutritional behavior singly and in combination in an older population; determine the effects on functional ability and general health outcomes of stage-based physical activity and nutrition interventions singly and in combination; and, study how older adults change their health behaviors. Research indicates that most health promotion programs recruit only those persons who are ready to adopt the new behavior, usually only approximately 20 percent of the general population. High levels of attrition and relapse rates result in an "impact rate" of approximately 1-5 percent. This project holds the potential for developing more effective methods to promote general health of older adults. The research will utilize a 2x2 design with 4 treatment groups: nutrition intervention alone, physical activity intervention alone, combined intervention, and control. Educational materials, including a manual, newsletters, expert feedback reports, and coaching phone calls, all based on the Transtheoretical Model of Health Behavior Change, will be developed. Measures used will include general health and functional assessments; objective and subjective physical activity and nutrition assessments; and stage and process of change, decisional balance, and self- efficacy measures. The 12-month intervention will include expert system generated reports, stage- based manual and newsletters and phone coaching.

In all well-characterized epidemics there are individuals in the population that respond differently to the infectious agent. Although resistance to infection is the most common variable phenotype, variation in disease outcomes has also been observed. Epidemiologic studies have shown that inherited factors are involved in the risk of mortality to infectious agents. The HIV-1 epidemic presents a critical challenge to apply current genetic techniques to the study of host genetic variation for infection and susceptibility to infection. This problem is confounded in the studies of HIV-1 by the rapid rate of evolution of the virus. The CKR5 gene serves as a secondary receptor on CD4+ T lymphocytes for certain strains of human immunodeficiency virus. The CKR5 gene was mapped to human chromosome 3p21, and a 32 base pair deletion allele (CKR5D32) was identified that is present at a frequency of approximately 0.10 in the Caucasian population. An examination of 1955 patients included among six well-characterized AIDS cohort studies revealed that 18 deletion homozygotes occur exclusively among 612 exposed HIV-1- antibody negative individuals (2.8%) and not in 1343 HIV-1-infected individuals. CKR5 deletion heterozygotes (+/D32) were significantly elevated among patients that survive HIV-1 infection for more than 10 years, in some cases twice as frequent as their occurrence in rapid progressors to AIDS. Survival analysis clearly shows that disease progression is slower in CKR5 deletion heterozygotes. The CKR5D32 deletion may act as a recessive restriction gene against HIV-1 infection and exerts a dominant phenotype of delaying progression to AIDS among infected patients. In addition, we have identified five other alterations in the CKR5 gene, including four missense alterations in conserved residues, and an amino acid deletion. These alterations should further add to the understanding of the role of CKR5 in HIV infection and disease progression.

{\rtf1\ansi\ansicpg1252\deff0\deflang1033{\fonttbl{\f0\fswiss\fcharset0 Arial;}} {\*\generator Msftedit 5.41.15.1515;}\viewkind4\uc1\pard\f0\fs20 The Radiation Therapy Oncology Group (RTOG) is the leading multicenter and multidisciplinary research\par organization systematically testing novel radiotherapy (RT) approaches against cancer and pursuing fully\par integrated translations! research to enhance this effort. The group will build on its outstanding scientific\par accomplishments in the current funding period to conduct research based on three fundamental initiatives: 1.\par Physical Targeting: RTOG will implement and test advances in imaging and high-precision RT planning and\par delivery technologies in clinical trials. 2. Molecular Targeting: Combined with RT: RTOG will design and\par conduct hypothesis-driven trials testing the integration of novel molecular targeted anti-cancer agents with\par optimized RT or chemo-RT. 3. Translational Research: RTOG will implement powerful biostatistical and\par medical informatics approaches to its unique and inter-linked clinical, biophysical, biologic, and outcomes\par databases that will faciliate hypothesis-driven analyses of these resources,\par The RTOG's research is primarily directed toward patients with brain tumors, head and neck cancer, lung\par cancer, gastrointestinal cancer, and genitourinal cancer. More limited research strategies have been\par developed for patients with gynecologic and breast cancer and sarcoma. RTOG research is supported by\par outstanding contributions from the Advanced Technology Integration, the Translational Research Program,\par and the Health Services Research and Outcomes Committees as well as the newly relocated Biospecimen\par Resource and from the four core committees of Medical Oncology, Medical Physics, Pathology, and Surgical\par Oncology. These contributions are well coordinated by a strong administrative and scientific infrastructure\par and have resulted in increased productivity in the current as compared with the prior funding cycle in terms\par of: 1. publication record; 2. clinical trials activated and completed; 3. overall patient accrual; 4. public and\par private funding of group research; 5. collaboration with other cooperative groups and other NIH-funded\par organizations; and 6. increased participation and leadership of Canadian investigators, with 8 of 37 full\par member institutions being Canadian, Close to two-thirds of all cancer patients receive radiation therapy for\par cure or for symptom relief. Novel technology, molecular targeted drugs and advances in radiation and cancer\par biology will improve the success rate of radiation therapy through clinical research over the next six years.\par \par }

: Speech production is an integrative process in which intent is transformed in to action though a series of sensorimotor operations. For most individuals, the speech production process operates smoothly. For persons who stutter (PWS), this process intermittently breaks down. Dysfluencies in speech output, characterized by sound/syllable repetition and/or lengthening, or complete blocks of speech output, disrupt the normal spatial and temporal patterning of movement. The reason or reasons why this apparent maladaptive communicative behavior is manifest in the population, and persists into adulthood is currently unknown. A controversy in the literature is whether and how PWS differ from persons who do not stutter. One possibility is that the speech production system of PWS is not different from those of nonstuttering individuals and stuttering is the secondary characteristic of higher level impairment (cognitive, linguistic, psychological). Alternatively, the locus of the problem is in the speech motor control systems of PWS and the higher level processes interact with unstable output system causing a breakdown of fluent speech. We suggest, based on the available literature that both possibilities exists and further, that most previous attempts to resolve this issue have to limited in scope and technique as to render definitive conclusions premature. In the present proposal, we will examine the performance of stuttering individuals along a number of dimensions reflecting a sampling of cognitive, linguistic, and sensorimotor capabilities. The empirical techniques include the kinematic examination of articulator motion, electromyographic analysis of orofacial muscle activity, the examination of responses to mechanical and auditory disruptions during speaking, reaction times associated with increasing linguistic complexity and attentional demands, and functional Magnetic Resonance Imaging during language processing and production. The research focuses on both theoretical and practical issues in order to maximize the impact of the research on understanding the disorder. Many of the other investigations proposed will provide information on many of the important nervous system operations for communication and the manner in which they may be impaired in stuttering individuals. Other investigations will provide useful clinical information by describing the specific changes in speech production variables that accompany a number of clinical techniques known to reduce the frequency of stuttering. We also attempt to identify differences in movement variance functions preceding experimentally-induced stuttering episodes to determine if these episodes can be predicted from an examination of fluent speech. Once identified, such variables could be used to predict the onset of stuttering episodes and ultimately in treating the disorder.

The development and application of label-fracture cytochemistry was pursued. In label-fracture cells fixed or unfixed are labeled and then freeze-fractured. Label-fracture permits the observation in a single, coincident image the distribution of surface receptors and antigens superimposed on conventional Pt/C casts of freeze-fractured membranes. The resolution (5nm attainable) approaches the molecular level. Normal human lymphocytes were label-fractured with monoclonal anti-T3 and anti-T4 antibodies before and alter chemical fixatin by glutaraldehyde. In this work the experiments are designed to map T3 and T4 antigens on the surface, their relation to intramembrane particles revealed by freeze-fracture and their dynamics during capping. We show that while T3 and T4 glycoproteins are associated to IMP domains they do not constitute a structural subset recognizable as a specific cluster of IMPs after capping. In other work we are currently studying the lectin-binding sites in neuronal membranes. We used synaptosomal membrane preparations from rat brain and showed binding sites of concanavalin A and WGA-ovomucoid-gold on the surface associated mainly to IMP domains.

The overall goal of this project is to elucidate the factors and mechanisms that control the decision of early erythroid progenitors to undertake terminal differentiation. Much evidence suggests that a crucial aspect of differentiation decisions is a requirement for coordination with the cell proliferation program. However, our current knowledge about the connections between the cell proliferation and differentiation programs is very limited. Work from our lab and others has identified the Ets - family transcription factor PU.1 as a key regulator of the terminal differentiation decision in early erythroid progenitors. And yet, the factors that control PU.1 expression and activity in these early erythroid progenitors and the mechanisms that link the differentiation decision to the cell cycle are not known. Recently, we identified two types of factors that have a very strong potential for connecting PU.1 and the terminal differentiation decision to the cell cycle. One factor is CDK6, which we discovered inhibits erythroid differentiation, like PU.1. We also showed that: 1) CDK6 is the active G1 phase D-cyclin kinase in erythroid progenitors; 2) PU.1 controls transcription of the CDK6 gene; 3) CDK6 phosphorylates PU.1. The other factors are E2F2 and E2F4, transcriptions factors that play major roles in G1 to S phase cell cycle progression. We found that E2F2 and E2F4 occupy the promoter and upstream regulatory element (URE) of the PU.1 gene in erythroid progenitors. They also bind very close to PU.1 at many other PU.1 target genes in these cells. In Aims 1 and 2, we propose a series of experiments to determine how CDK6 and E2F2 and E2F4 influence PU.1 expression and activity and how they collaborate with PU.1 in the erythroid terminal differentiation decision. We also propose to investigate roles for CDK6 and E2F2 in stress erythropoiesis and in ex vivo self-renewal of erythroid precursors. Work from our lab and others showed that one of the principal mechanisms used by PU.1 to regulate the erythroid differentiation decision is repression of erythroid-specific gene expression Recently, we discovered that PU.1 forms a complex with the chromatin remodeling ATPase SNF2H and the maintenance DNA methyltransferase DNMT1; two enzymes that we hypothesize help PU.1 to repress erythroid gene expression. In Aim 3, we propose studies to elucidate the role of SNF2H and DNMT1 in PU.1-mediated repression of erythroid-specific genes and inhibition of erythroid terminal differentiation. The successful completion of the proposed studies will lead to new insights into the mechanisms connecting the proliferation and differentiation programs in hematopoietic cells and a much deeper understanding of how the hematopoietic system regulates red blood cell production, including during ontogeny and in stress conditions. The proposed work will also provide important information enabling the development of new approaches to managing defects in red blood cell production that occur in chronic and acute anemia.

In 1981, we will continue our study of the organization of both the main ribosomal repeat unit of N. crassa (that which codes for the 17S, 5.8S, and 26S rRNA's), and of the 5S rRNA genes. In the case of the main rDNA repeat unit, we will characterize the differences we have found in the non-coding portions of the repeat unit in about 10 different strains we have examined. Dr. Russell has putative clones of these, and we plan to do restriction mapping. Then, if the results are as we anticipate, we plan to sequence the relevant regions of several of them. In addition, we now have on hand the materials and the specific probes needed to clone the first and last repeat unit in the nucleolus organizer region. We plan to proceed with this during the first part of the calendar year. I am particularly interested in the flanking sequences of the 5S rDNA genes, of which there are many different kinds. According to one hypothesis (Selker, Driftmier, Metzenberg, Yanofsky, DeWeerd, and RajBjandary, manuscript in preparation), a degree of homogeneity of the scattered 5S rDNA genes could be maintained by rather frequent transpositions. There are some relatively straightforward ways to test this, and these will be tried. Last but not least, I hope to pursue my study of the phosphorus control genes, which have proved balky at the molecular level. I think we now have built up the skills on somewhat simpler and more tractable things to give a renewed push to this complicated set of genes.

Approximately 25% of the adult US population are current smokers, and although many try to quit, most fail. As an alternative, some smokers use potentially reduced exposure products (PREPs). However, prior harm reduction products, such as light cigarettes, have proven to be a public health disaster. Carcinogen exposure studies and epidemiologic data show that compensatory smoking behavior can make light cigarettes as harmful as regular cigarettes. Therefore, it is important to understand how people use PREPs and to assess how this relates to their harm exposure. Quest(r) cigarettes are a new PREP that uses genetically modified tobacco to provide a "step-down approach to becoming nicotine-free" by marketing cigarettes with progressively lower levels of nicotine: 0.6 mg, 0.3 mg and 0.05 mg. The primary aim of the study is to investigate the effect of progressive decreases in cigarette nicotine level on smoking topography and harm exposure measures. The study will be a within-subject, open-label design of 210 smokers not currently interested in quitting. Participants will be randomized to one of three conditions: 1) continue to smoke their own brand (control); 2) smoke Quest(r) cigarettes in the step-down order, progressively decreasing cigarette nicotine level from 0.6 mg, to 0.3 mg, to 0.05 mg (Quest(r) step-down); or 3) smoke Quest(r) cigarettes in a non-step down random order, counter balanced across subjects (Quest(r) non-step- down). The study will consist of four stages beginning with a 5-day own brand cigarette smoking phase for all participants, followed by one of the three cigarette conditions. For those smoking Quest(r) cigarettes, cigarette nicotine level will change every 10 days, either in a step-down or random fashion. The primary behavioral outcome is smoking topography; specifically total and mean puff volume, which will be assessed at each session. Carbon monoxide to assess smoke exposure, and urinary biomarkers, will also be assessed. At baseline, participants will view Quest(r) advertisements and complete a survey of product expectations to determine the impact of beliefs about Quest(r) cigarettes on subsequent smoking behavior. This study has several important public health implications. It will determine how a new PREP is actually used. It will also determine how product use relates to harm exposure. Finally, it will identify those most susceptible to changes in smoking topography that may increase harm exposure. [unreadable] [unreadable] [unreadable]

This R03 application is associated with the K08 grant entitled "Mechanisms of AIIograft Tolerance" that involves studying transplantation tolerance in a model utilizing murine islet allografts. In the first two years of in the original K08 grant period, several Specific Aims have either been completed or are well underway. The original focus of the study was to determine whether different therapies known to be effective in islet allograft prolongation worked by operationally similar or distinct immunological pathways. In the course of completing the 1st Specific Aim which examined the ability of tolerant spleen cells to coadoptively transfer tolerance (i.e., transfer tolerance despite the presence of equal numbers of naive spleen cells) to immunodeficient mice, it became clear that one therapy, in particular, developed so-called 'dominant' tolerance. Combined therapy monoclonal antibody therapy with anti-CD154 and anti-LFA-1 therapy not only led to exceptional islet allograft survival rates in 'high-responder' C57BL/6 recipients but also led to dominant transplantation tolerance in contrast to monotherapy with either anti-CD154, anti-CD154 with donor specific transfusion or anti-LFA-1. Therefore the original hypothesis that different therapies would lead to a similar form of tolerance was not bourn out with these studies. In fact, anti-CD154 and anti-LFA-1 appear to uniquely synergize and lead to a powerful form of regulatory tolerance. Given that both anti-CD154 and anti-LFA-1 are available in humanized forms, are non-lymphocyte depleting, and result in sustainable tolerance, the clinical applicability of this therapy for islet transplantation appears to be high. Because of the significance of these results, the remainder of the K08 period will include an evaluation of the dominant transplantation tolerance which results from combined anti-CD154/anti-LFA-1 therapy. The project has adapted the original studies in two other significant ways: (1) the role of NK and NK T cells will be evaluated with respect to the maintenance of dominant tolerance and (2) a new collaboration with Cytomation, Inc. will allow a more thorough assessment of graft infiltrating cells utilizing state-of-the-art flow cytometry and cell sorting techniques. The funds made available through this grant would assist the completion of Specific Aims mainly by providing for a full-time technician and by assisting with the significant animal costs associated with these studies.

Due to the anticipated significant increase in the number of potential vaccines for CDC Category A agents in the near future, it is imperative that a rapid response quality control technology be developed and validated. The key objective of this proposal is to develop and validate such a technology applicable to vaccines such as peptides, proteins and particles. The proposed technology has several distinct advantages making it particularly suitable for rapid response in distributed environments. Among its unique attributes are speed of execution, sensitivity to structural changes of importance, low cost, and inherent automation capabilities. Present quality control techniques for biologicals, including vaccines, are slow, complex, and frequently require in vivo studies to assess the level of biological activity of the product. Structural information that is directly related to activity is highly useful, but very difficult to obtain. The proposed technology focuses on development of a signature, or a fingerprint, of the structure. The signature is defined in relation to other signatures, obtained from reference samples of known structural and/or activity properties. The technology involved in the development and utilization of signatures combines physico-chemical assays with mathematical techniques. The signature is subsequently presented in an easy to interpret manner, either numerically or visually. Phase I of the proposed study would focus on a feasibility study developing signatures for various serotypes of C. botulinum toxins, as well as their fragments. Signatures will be developed that are sensitive to production and formulation aspects which result in varying levels of bioactivity. Issues such as the effects of various excipients, pH, salts, and lyophilization would be examined. The principal aim of Phase I study would be to demonstrate the ability to construct structural signatures that correlate with the effects of various treatments and conditions, and to prove its feasibility to provide unique information that could be used during rapid quality control testing. Phase II of the proposed study would concentrate on development of additional signatures for several Category A vaccines, on further analytical validation issues, and on development of automated lab-bench and portable biochip platforms to obtain the signatures in a rapid and reliable manner.

The candidate (Mark Alter) completed M.D./Ph.D. training in the Medical Scientist Training Program (MSTP) at the University of Pittsburgh in 1999 with a Ph. D. in immunology. He completed residency training in the "Triple Board" program at Brown University with combined training in pediatrics, psychiatry, and child &adolescent psychiatry in 2004. Recent research in the laboratory of Rene Hen at the Center for Neurobiology &Behavior at Columbia University has stimulated interest in the neurobiological mechanisms underlying individual differences in emotional development and their relationship to childhood emotional disorders. Dr. Alter is particularly interested in epigenetic influences on gene expression and behavior. Epigenetics refers to mechanisms outside ("epi") the actual genetic sequence that influence gene regulation. Epigenetic influences are thought to be strongest during early development and in rodents have been shown to be impacted by differences in maternal care. Thus, understanding these influences is of direct relevance to emotional disorders in children. To study the relationship of epigenetics to emotional development, Dr. Alter has used genetically identical animals to examine individual variability in emotional behavior. By studying genetically identical animals he brings focus on epigenetic influences. Using this model, he sees stable differences in emotional behavior between individual genetically identical animals. He implicates epigenetic influences on gene expression and emotion by demonstrating that by using only the pattern of gene expression in the hippocampus, a brain region important in emotional regulation, he is able to correctly predict emotional differences of individual animals. In this proposal, Dr. Alter seeks to better understand the relationship of his findings to individual variability in emotional behavior. Specifically he plans to 1) characterize individual variability in emotional behavior across the lifespan in genetically identical animals 2) relate gene expression patterns in the hippocampus to differences in emotional behavior and 3) examine epigenetic programming of genes related to emotional development. This research will likely lead to improved treatment, diagnostics, and identification of children at risk for psychopathology. Completion of his aims will require intimate knowledge of 1) mouse behavior and 2) genomics, areas in which Dr. Alter has limited background. The proposed plan provides Dr. Alter with a period o f mentored research to gain expertise in these areas. Expertise gained during this mentored period will provide Dr. Alter with the experience necessary to attain his long-term goal of developing an independent, multidisciplinary research program with an emphasis on translating increased understanding of epigenetic mechanisms related to emotional development into improved diagnostics and novel therapies for childhood emotional disorders.

The aim of this research is to determine the effects of deprivation on visual development by studying children treated for dense, central cataracts. A large sample of these children is being treated at The Hospital for Sick Children by surgically removing the cataract and fitting the resulting aphakie eye with a contact lens. Our objectives are to determine the influence on the visual outcome of: (a) the timing of deprivation, by comparing children treated for congenital, developmental, and traumatic cataracts; (b) the duration of deprivation by comparing children who were first treated at various times after diagnosis of this type of deprivation, by comparing children treated for unilateral versus bilateral cataracts; (c) the extent to which the normal eye was patched (in unilateral cases); and (d) the presence of the additional common problems of persistent hyperplastic primary vitreous microcornea, microphthalmus, nystagmus, secondary glaucoma, and/or strabismus. Because the animal literature suggests that the answers may vary for different visual functions, seven aspects of vision will be measured: (a) grating acuity, (b) better acuity, (c) contrast sensitivity, (d) vernier acuity, (e) peripheral vision, (f) the symmetry of optokinetic nystagmus, and (g) color vision. Standard clinical tests, modified for aphakic eyes, will be used to assess letter acuity, peripheral vision, and color vision. Grating acuity will be measured with preferential looking and optokinetic nystagmus. For contrast sensitivity, an oscilloscope and the method of limits will be used to determine the minimum contrast at which stripes of different spatial frequency are visible. For vernier acuity, a computer-controlled interactive video display and the method of limits will be used to determine the minimum detectable offset between parallel lines. For the symmetry of optokinetic nystagmus (OKN) a large display of moving stripes will be used to determine whether OKN can be elicited easily both when striped move from the temporal field toward the nasal field and when they move in the opposite direction. The results should elucidate the effects of deprivation on visual development, and indicate the optimal treatment for children afflicted with cataracts. The methods refined for this study also will be useful for evaluating the consequences of other visual defects and their treatment, and for screening children for possible eye disorders.

Project Summary Since enzymes are involved in most life processes, it is crucial to gain a detailed understanding of their action. The importance of such an understanding is highlighted by the fact that many diseases can be controlled by developing drugs that block the action of enzymes in crucial biological pathways of pathogens that cause these diseases. Quantifying the analysis of enzyme catalysis can be greatly advanced by computer simulation approaches that correlate enzyme structures with their activity. Here the progress should involve both advances in quantifying the fundamental basis of enzyme catalysis and in developing practical predictive power in studies of diverse classes of enzymes. Thus we propose to continue in advancing the frontiers of this field, while shifting a significant part of our focus to the the emerging field of enzyme design. The advances on this front should provide a deeper understanding of catalysis, help in controlling what a given enzyme is doing as well as the use of specialized enzymes. We note, however, that the progress in enzyme design has not yet led to designer enzymes that rival native enzymes. Thus, it is clear that the potential of this field can be greatly enhanced by computational approaches that evaluate the activation barriers of the reactions that are being catalyzed. In order to progress in both quantifying enzyme design and in enhancing the general understanding of enzyme catalysis, we propose parallel advances in the following directions: (i) We will validate the reliability of our EVB-based computer-aided enzyme design by reproducing the observed catalytic effects of key designer enzymes. (ii) We will continue to develop more quantitative design concepts as well as fast screening methods. After exploring the predictive power of these approaches, we will use them in collaboration with research groups that are involved in actual enzyme design experiments. (iii) We will continue to advance quantitative computational methods including the paradynamics QM(ai)/MM-FEP approach that should help us to obtain activation free energies of enzymatic reactions by ab initio methods. (iv) We will quantify the relationship between folding and stability and explore the relationship between thermostability and catalysis. (v) We will explore the catalytic effect of directed evolution, trying to elucidate its relationship to natural evolution. (vi) We will conduct studies of several important classes of enzymatic reactions. (vii) We will continue with the systematic examination of different nonelectrostatic catalytic proposals.

During the past year we have continued to examine the role of polyglutamate formation in the cytotoxic action of methotrexate. We have examined various aspects of the formation, retention, and binding of polyglutamates in human breast cancer cell lines. The major findings have been 1) that higher polyglutamates, particularly those with 4 or 5 glutamyl groups, are preferentially retained by human breast cancer cell lines, 2) that these longer polyglutamates have slower rates of dissociation from the enzyme as determined in whole cells, 3) studies with isolate dihydrofolate reductase have shown that methotrexate and its polyglutamates have roughly equivalent dissociation constants, implying that unidentified factors, such as association of DHFR with thymidylate synthetase in intact cells, may account for the slower off rate seen in the whole cell, and 4) methotrexate polyglutamates have additional sites of inhibitory action, including potent inhibition of thymidylate synthetase and AICAR transformylase. We have also identified and described gene amplification in a human small-cell carcinoma line derived from a patient treated with high-dose methotrexate, and have shown that the amplification is unstable in culture, likely because of the failure of the double-minute chromosomes to segregate equally during cell division. The loss of double-minute chromosomes in this cell line during continuous passages in tissue culture was associated with a fall in intracellular DHFR and a return to sensitive state. We have initiated studies on pleiotropic drug resistance in human and murine tumor cell lines. Through continuous exposure to colchicine in tissue culture we have derived 10- to 20-fold resistant cell lines which display cross-resistance to adriamycin and vincristine. In murine cell lines this resistance is reversible with verapamil, while in human breast cancer cell lines the resistance is only minimally reversible. In addition, the human breast lines show a cytotoxic response to verapamil alone. We plan to define the calcium channels present on these cells and will study the relationship of channels to drug resistance. We have also initiated studies attempting to transfer drug resistance through DNA-mediated transfection to sensitive receptor cell lines.

Type 2 diabetes (T2D) and cardiovascular disease (CVD) are among the leading causes of morbidity and mortality in US Veterans, as well as the US population at large. T2D is a widely-recognized risk factor for CVD, and T2D leads to worse CVD outcomes. However, there remains considerable clinical heterogeneity among individuals with T2D. Even among individuals with apparently similar glycemic control, there is significant variability with respect to who will develop CVD. To develop more effective strategies to prevent CVD in this high-risk population, better approaches for quantifying CVD risk are needed. Using novel computational approaches, we will consider dense phenotype and genotype data to identify the subpopulations of individuals with T2D who are at the highest risk of heart and vascular disease. In Aim 1, the relationship between traditional CVD risk factors, such as cholesterol, blood pressure, and smoking, and three heart and vascular disease phenotypes: peripheral artery disease (PAD), coronary heart disease (CHD), and cerebrovascular disease, will be tested. To account for the fact that these outcomes frequently occur in the same individuals, statistical models that treat the traits as correlated-within person outcomes will be used. To determine if the addition of genetic information improves the prediction of CVD outcomes, the impact of genetic risk scores, based on preliminary studies from the VA Million Veteran Program and other published work, on the models will be assed. In Aim 2 dense phenotype data will be extracted from the electronic health record and novel artificial intelligence based biclustering algorithms will be used to identify hidden subtypes of T2D. The association of these subtypes with CVD outcomes will then be assessed. In Aim 3, a similar approach will be taken to elaborate T2D subtypes based on DNA variants known to associate with T2D, CVD, and their risk factors. Finally, the genetic and phenotypic data will be jointly considered. These approaches will be applied across data from both US Veterans, using the Veterans Aging Cohort Study and the VA population at large (via the Corporate Data Warehouse), and non-Veterans, using data from the PennMedicine BioBank, Penn Data Store, and UK Biobank. Successful completion of this project will help to elucidate the phenotype structure of T2D and identify individuals at the highest risk of T2D. These results will lay the ground work for developing tailored strategizes for CVD prevention in T2D and help realize the promise of precision medicine for heart and vascular disease.

The ATP-dependent Chromatin-remodeling complex, Rsc, was originally identified in yeast. It has a subunit composition similar to the well-characterized SWI/SNF: 2 subunits are shared between the two complexes and at least 4 others are homologues of each other. However, the function of Rsc is distinct from SWI/SNF. Rsc is essential for the mitotic growth of yeast, whereas SWI/SNF is not. Rsc mutants are arrested at G2/M transition during the cell cycle, and the arrest is dependent on spindle-checkpoint genes. The mutants are also more sensitive to microtubule- destabilizing drugs. These data suggest that Rsc may play a role in mitosis, perhaps by stabilizing mitotic spindle formation or have a function at kinetochores. We have previously purified several ATP-dependent chromatin remodeling complexes from human that are closely related to either yeast SWI/SNF or Rsc. By microsequencing and cloning, we now identified the BAF180 subunit of human SWI/SNF-B complex as a human homologue of yeast Rsc subunits, Rsc1, Rsc2 and Rsc4. It has the same set of biochemical motifs as the three Rsc subunits: 6 bromodomains, 2 BAH regions, and a minor-groove binding domain. Database search of the completed genome database of human, Drosophila and C.elegans showed that BAF180 is the closest homolog of the three Rsc subunits, suggesting human SWI/SNF-B as a human version of Rsc. We renamed this complex as PBAF (for Polybromo-associated BAF complex). We subsequently identified many other subunits of human PBAF complex and found a number of them to be identical to those in human SWI/SNF. We also demonstrated that BAF180 localizes at kinetochores of mitotic chromosomes, consistent with a function for PBAF during mitosis. This work has been published in PNAS. As a continuation of this project, we have identified three possible new components of this complex. We have obtained antibody against one of them, BAF200. With this antibody, we were able to show that BAF200 is indeed an intrinsic component of this complex. BAF200 contains a domain found in a component of yeast RSC complex, implying that BAF200 may be a conserved functional subunit within RSC family of complexes. We are currently performing siRNA knockdown experiments to identify genes regulated by BAF200 and its associated complex.

In aged cats a series of interdisciplinary studies will be carried out with the objective of defining changes in sheep and wakefulness due to the aging process as revealed by correlated behavioral, electrophysiological and neuroanatomical techniques. The circadian distribution of a sleep and waking states and the attendent patterns of electroencephalographic, electroocular, electromyographic, cardiac and respiratory activity will be examined. These data will be individually analyzed and cross-correlated to generate a comprehensive behavioral and electrophysiological profile of state-dependent patterns of neural activity. Combined light (Golgi) and electron microscopic analysis of the neural substrates for the behavioral and electrophysiological data obtained during sleep and wakefulness will be carried out to provide a comprehensive description of the regulation of state-dependent processes in the aging feline brain. We have chosen to study the influence of age on central neural processes in cats in order to pursue a controlled, interdisciplinary experimental program that could not be realistically performed in humans. Additionally, in our preliminary studies, we found dramatic changes in sleep and wakefulness in aged cats. Since all neural and behavioral patterns of activity are carried out either during sleep or wakefulness, a detailed description of aging changes in these fundamental states, combined with functionally oriented neuroanatomical studies, was deemed an appropriate avenue to pursue in our initial interdisciplinary explorations of the aging process in the central nervous system.

Insulin resistance is a major factor in the pathogenesis of type 2 diabetes and recent studies have established a strong relationship between intramyocellular triglyceride accumulation and insulin resistance in skeletal muscle. In this proposal the mechanism of fatty acid induced insulin resistance in skeletal muscle will be examined in awake rats and unique transgenic and knockout mouse models using state-of-the-art methodology including nuclear magnetic resonance spectroscopy (NMR), gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-tandem mass spectrometry (LC/MS/MS) in combination with radioactive isotope techniques. Recent studies by our group have demonstrated that fatty acids induce insulin resistance in skeletal muscle by inhibiting insulin activation of IRS-1 associated phosphatidylinositol 3-kinase (PI 3-kinase) which we hypothesized was mediated by activation of a serine kinase cascade involving protein kinase C? (PKC?). This grant will further examine this hypothesis by examining the sequence of events leading to activation of PKC? in skeletal muscle. Specifically, increases in intramuscular content of fatty acyl CoA, ceramides, diacylglycerol, and triglyceride in relation to activation of PKC?, IRS-1 Ser307 phosphorylation and insulin simulated: 1) insulin receptor tyrosine phosphorylation, 2) IRS-1 tyrosine phosphorylation and 3) IRS-1 associated PI 3-kinase activity will be examined in awake rats during a lipid infusion designed to raise plasma fatty acid concentrations. In order to examine the putative roles of PKC? and Jun kinase 1 in mediating fatty acid induced insulin resistance, the effect of lipid infusion and high-fat feeding on insulin action and signaling in skeletal muscle will be examined in PKC? and Jun kinase 1 knockout mice. To test the hypothesis that accumulation of intramyocellular fatty acid metabolites are responsible for mediating insulin resistance in skeletal muscle, insulin signaling and action will be examined in UCP3 overexpressing mice (to promote increased muscle fatty acid oxidation) and fatty acid transport 1 (FATP1) gene knockout mice (to block fatty acid entry into skeletal muscle) following lipid infusion and high-fat feeding. Finally, in order to test the hypothesis that n-3 fatty acids protect against fat induced insulin resistance by serving as natural ligands for PPAR[unreadable] resulting in peroxisome proliferation and increased hepatic fatty acid oxidation, the effect of feeding a diet enriched with n-3 fatty acids versus isocaloric control and safflower (% fat matched) diets on insulin signaling and action in PPAR[unreadable] knockout and wild type littermates will be examined. The results from these studies should yield important new insights into the mechanism of fatty acid induced insulin resistance in skeletal muscle leading to the identification of potential novel targets for treatment of type 2 diabetes.

SUMMARY Eukaryotic cells contain within them a myriad of spatially distinct sites that serve a variety of functions. To facilitate this organization, eukaryotic gene expression is routinely spatially regulated through the trafficking and sequestration of thousands of different RNA molecules to distinct cellular locations. Misregulation of this process leads to detrimental phenotypes in a wide range of systems, from developmental defects in Drosophila to neurological disease in humans. Despite this importance, our knowledge of the regulation of RNA localization is quite limited. For other modes of post-transcriptional regulation like splicing, our understanding of how the interactions of RNA binding proteins (RBPs) and RNA motifs lead to specific outcomes is much more mature. This relies on many years of work by many groups that have defined the regulatory language of splicing and allows us to make predictive and combinatorial models about how splicing is regulated across conditions and cellular environments. We lack such an ability with regards to RNA localization, in large part because we lack the analogous ?parts list? that defines the language of localization regulation. Generally, the effect of RBP/RNA binding on post-transcriptional regulatory processes like splicing or RNA decay is consistent across cell types. For example, if an RBP promotes the splicing of an exon in one cell type, it often exerts a similar effect on that exon in another cell type. However, because RNA localization is inherently tied to cell morphology, the generality of localization regulation across cell types is unknown. Combinations of RNA motifs and RBPs that result in RNA localization to projections in neurons are also broadly present in non-neuronal cell types. Are these RNAs trafficked in non-neuronal cells? If so, to where? The answers to these questions first require a better knowledge of the underlying regulatory language of localization. The experiments proposed here are the beginnings of our efforts to define this language and test its generality. We have developed methods to isolate and profile subcellular transcriptomes from the projections of neurons and the apical and basal regions of epithelial cells. We will use these techniques to take a biochemical and transcriptome-wide approach to defining RBP/RNA interactions that regulate localization in two mammalian cell types: neurons and intestinal epithelial cells. By identifying transcripts that are mislocalized in RBP-null cells, we will identify functional RBP/RNA interactions. Using a massively parallel reporter assay, we will take an unbiased approach to finding RNA sequences that regulate localization. By comparing the activities of identified functional RBP/RNA interactions across cell types, we will for the first time be able to directly assess the generality of RNA localization. This methodical and innovative approach is the first step in our efforts to shed light on this fundamental but poorly understood cellular process.

This research is designed to investigate nutritionally-induced alterations in hormones and the energy needs of the body. It is specifically designed to study the effects of over- and undernutrition on thyroid hormone and catecholamine metabolism, and the possible effects these changes have on thermogenesis and the body economy. Nutritionally-induced changes in the metabolism of thyroid hormones and catecholamines will be correlated with changes in thermogenesis to determine whether these changes represent adaptive responses with important survival value.

Significance Gut-associated lymphoid tissue (GALT) is an important site for early viral replication and dissemination and severe CD4+ T cell depletion. These changes are not adequately reflected in peripheral blood or lymph nodes. GALT will be valuable in determining mechanisms of SIV-associated CD4+ T cell depletion. Objectives The present study was performed to examine the kinetics of viral replication and CD4+ T cell depletion in GALT in comparison with peripheral blood and lymph nodes during the course of SIV infection in rhesus macaques. Results Viral replication and severe CD4+ T cell depletion occurred in GALT during primary SIV infection in rhesus macaques. This was accompanied by an increase in intestinal CD8+ T cell percentages and increased potential of expression of Th1 type cytokines. These changes could reflect altered T cell homeostasis in GALT and play an important role in SIV-associated enteropathy and viral pathogenesis. Future Directions Future studies will focus on determining the mechanisms of SIV-associated CD4+ T cell depletion in GALT. Studies of anti-retroviral therapy of SIV-infected macaques will be performed to determine the mechanisms of CD4+ T cell repopulation and viral suppression in GALT. KEY WORDS gut-associated, lymphoid tissue, viral replication, SIV FUNDING NIH Grant AI043274 PUBLICATIONS Smit-McBride, Z., Mattapallil, J., McChesney, M., Ferrick, D. and Dandekar, S. Journal of Virology 72:6646-6656, 1998. Mattapallil, J, Smit-McBride Z, McChesney, M, and Dandekar, S. Journal of Virology 72:6421-6429, 1998. Smit-McBride, Z, Mattapallil, J., Villinger, F., Ansari A, Dandekar, S. Jounal of Medical Primatology 27:129-140, 1998.

Cellular and molecular mechanisms involved in T-cell-mediated autoimmunity affecting the retina are being studied in animal models of experimental autoimmune uveoretinitis (EAU). The questions are aimed at elucidating the mechanisms governing normal development and maintenance, as well as the pathological breakdown, of self-tolerance to retinal antigens. To address these questions, rats and mice are immunized with retina-derived antigens, or with synthetic peptides, representing fragments of these antigens, to induce EAU. Susceptibility to disease induction is being evaluated in various rodent strains of known genetic characteristics, in the hope of delineating the hereditary mechanisms that predispose to uveitis. Genetically altered (transgenic or "knockout") mice are used to evaluate the role of various components of the immune system in the immunopathogenesis of uveitis. In vivo functional T-cell lines and clones are developed from lymphoid organs of rats and mice immunized with uveitogenic ocular antigens. The functional properties and specific receptors of these cells are being studied with the aim of developing strategies for in vivo targeting of the autoimmune cells. Lastly, EAU in rats and mice serves as a template for the preclinical evaluation of new drugs and compounds as to their effects on the various stages of immunopathogenesis and final disease expression. The goals of these studies are to identify the immunogenetic factors predisposing to uveitic disease, learn about the pathogenic mechanisms involved, characterize the immunoreactive cells and their mediators, and use this knowledge of design novel and rational approaches to immunotherapy.

Our aim is to understand the mechanisms of some of the functions of the excitable membrane and cilia by genetic dissection. This is done by producing molecular lesions with mutagenesis and analyzing the resultant effects on behavior, electrophysiology and the ultrastructure. Our choice of material greatly simplified such a study. Paramecia are ciliated unicells with excitable membranes capable of generating action potentials. Using the relatively sterotyped locomotor behavior as the indicator of the state of membrane and ciliary activity, and taking advantage of the presence of autogamy in Paramecium aurelia, mutants can be selected which carry molecular lesions affecting membrane activity or ciliary structure. Membrane mutants have been isolated and studied behaviorally, genetically, and electrophysiologically. The first case of genic deletion of action potential in a known cell has been demonstrated in our system in the case of the mutant "Pawn". We aim to continue this fruitful interdisciplinary approach. Specifically, we propose to screen for other behavioral mutants with membrane or ciliary defects using a proven method and two newly devised methods based on galvanotaxis and the use of chemicals with known effects on membrane and ciliary activity. We also plan to continue the genetic and bioelectric analysis of the mutants on hand, especially the Na-specific mutants, "Fast-2" and "Paranoiac". A small part of our effort will be directed to the analysis of the ultrastructure of ciliary mutants. The search for the defective gene products which are correlated with the modified electrical properties and behavior will be conducted in collaboration with other laboratories so as to make maximum use of the experience of the principal investigator in genetics and electrophysiology.

In this new grant proposal, investigators with expertise in cellular immunology, molecular biology, purification of mycobacterial antigens and diagnostic techniques propose to use human T-cell lines and clones to screen a lambda gtll genomic DNA expression library for M. tuberculosis The goal of this screening will be to identify recombinant antigens of potential use in protection against tuberculosis and in the diagnosis of infection and disease due to the tubercle bacillus. Since tuberculosis is a leading cause of morbidity and mortality worldwide, this is a health related project. Direct T-cell screening of recombinant mycobacterial antigens is proposed, a novel approach consonant with the importance of cell-mediated immunity in protection against and delayed-type hypersensitivity skin testing in diagnosis of tuberculous infection. Different bacteriophage will be amplified in E. coli Y1090 and used to prepare lysogens in E. coli Y1089. phage multiplication and production of fusion proteins will be induced. Then, fusion proteins will be purified from bacterial lysates by immunoabsorbent and molecular sieve chromatography. Initial screening will be for recombinant mycobacterial antigens capable of inducing 3H-thymidine incorporation in antigen-specific T-cell lines from healthy tuberculin reactors. Bacteriophage encoding positive fusion proteins will be screened in smaller batches by diluting the E. coli lysogens and rescreening until relevant clones of recombinant bacteriophage are identified. Screening for possible protective antigens will involve testing individual recombinant antigens for induction of interleukin-2 and interferon-gamma production in antigen-specific T-helper cell clones. Screening for antigens useful in skin testing will consist of demonstrating species restriction. Such antigens will induce DNA synthesis in antigen specific T-cell lines and may be capable of activating T-cell clones specific for M. tuberculosis or reacting with antibody to species-specific epitopes. They must not, however, activate T-cells or react with antibodies which reflect hypersensitivity to nontuberculous mycobacteria. Candidate diagnostic antigens will be evaluated for specificity in elicitation of delayed-type hypersensitivity skin test responses in sensitized guinea pigs.

The survival of effector T cells to the memory state is more effective for those T cells undergoing mitochondrial oxidative phosphorylation (OxPhos) rather than glycolysis. IL-15 drives OxPhos in T cells and has been associated with several autoimmune disorders, such as rheumatoid arthritis, where the inflamed synovium is rich in IL-15. It is currently unknown how IL-15 promotes OxPhos in T cells or how this promotes T cell survival. Dr. Budd's and Rincon's groups have made two key preliminary observations that offer an explanation. First, Dr. Rincon has shown that a newly defined protein known as MCJ (Methylation- Controlled J protein) negatively regulates mitochondrial Complex I activity and OxPhos (6), Second, Dr. Budd has shown that IL-15 downregulates MCJ expression, allowing increased Complex I activity, elevated OxPhos and reactive radicals compared to IL-2-cultured T cells. This results in S-nitrosylation and S- glutathionylation and inactivation of caspase-3 at critical Cys163, which resides in the enzymatic pocket. This R21 application thus posits a novel hypothesis that IL-15 promotes effector T cell survival through downregulation of MCJ, resulting in enhanced mitochondrial OxPhos, reactive radicals, and inactivation of caspase-3 through S-nitrosylation. We then apply our murine findings to an actual human inflammatory condition rich in IL-15, rheumatoid synovium. Specific Aim 1 will determine whether retroviral re-expression of MCJ in IL-15-cultured T cells inhibits mitochondrial Complex I activity and OxPhos, and increases caspase-3 activity. We will then determine whether IL-15 also promotes supercomplex formation, as observed in MCJ-/- T cells. Specific Aim 2 will define whether IL-15 present in human rheumatoid synovium promotes OxPhos, ROS/RNS, reduced caspase-3 activity and resistance to cell death through MCJ downregulation. Rheumatoid synovial tissue or synovial fluid T cells will be assessed for their metabolic state by Seahorse flux analysis, ROS/RNS generation, MCJ levels, caspase-3 activity, and resistance to cell death. This novel study combines the expertise of the PI, a research rheumatologist, in regulation of caspases by cytokines in T cells, with those of Dr. Rincon who first described MCJ in T cells and its role in regulating mitochondrial respiration at Complex I. The findings would provide the framework for future expanded studies on the role of IL-15 and MCJ in immune responses and autoimmune disorders, such as rheumatoid arthritis. All the tools are in place to conduct these studies, including MCJ-/- mice and rheumatoid synovial tissue cultures. The experiments also represent a focused exploratory study with the potential for high yield for human inflammatory conditions and suggestions for therapeutic intervention through modulation of MCJ, IL-15, and metabolism.

Although glaucoma is a major cause of blindness in the U.S. population, there is no single screening test that has been shown to be adequately sensitive and specific to detect very early glaucomatous optic atrophy on a population wide basis. Preliminary evidence suggests that changes in the pupillary light response may be observed in the early stages of glaucomatous optic nerve damage. Specifically, there may be an increase in latency of optic nerve constriction as an early sign of optic atrophy. In this application we propose to develop a pupillometer based upon a production microcomputer and utilizing commercially available digital imaging hardware. This instrument will be specifically oriented towards measuring latency and amplitude of the pupillary light response and comparing this data to standard values as we determine them. Our aim is to develop an instrument that can be assembled for under $5,000 in hardware costs and operated by minimally skilled technicians. Using this device we plan to study the patient population available to us in order to determine the relationship between changes in pupillary light response and glaucomatous damage as measured by computerized perimetry, computerized ophthalmoscopy and clinical examination. For this pilot study, we will assemble three groups of patients: 1. Those with documented glaucoma on the basis of intraoculr pressure, visual field changes, and optic disc examination. 2. Those considered glaucoma suspects because of suspicious discs, intraocular pressure over 21, or suspicious fields, but not a combination. 3. Those considered normal on the basis of intraocular pressure, disc exam and fields. We will perform pupillometric analysis on each patient in addition to sequential measurements of intraocular pressure, examination of the optic disc by computerized ophthalmoscoy, and Octopus computerized perimetry. These factors will be compared in order to determine the potential sensitivity and specificity of pupillometry in glaucoma screening.

The development of antibody forming cells (AFC) is being studied in tissue culture with a new system. A low density subpopulation of primed mouse spleen cells is boosted in vitro with specific antigen in the presence of isologous serum. Large and highly enriched populations of antigen dependent AFC develop. The AFC precursors can be radiolabeled initially and are found to undergo several divisions in vitro during their development into AFC. Moreover, the proliferating cells associate physically with a novel dendritic cell whose function is still unclear. This system is being used to study, more directly than previously possible, the effects of specific antigen and various accessory cells on the proliferation and maturation of AFC.

The objective of the porposed research is to examine the influence of patterns of mother-daughter communication about sex roles and sexuality on daughter's sexual and contraceptive behavior. It is hypothesized that family structural variables will influence attitudinal congruence between mother and daughter which will in turn have an impact on their communication pattern. The latter is expected to influence directly the effectiveness of the daughter's sexual and contraceptive knowledge and behavior. The research design calls for collection of demographic, attitudinal, and behavioral data from 400 black and white 15-year-olds and their mothers currently living in the Detroit, Michigan SMSA. Half of the sample will be from female-headed-households. Multivariate techniques will be used to test the major explanatory model and secondary hypotheses. The project will indicate whether and under what conditions the mother-daughter nexus is an appropriate target for population policy interventions.

Survivors of childhood Bone Marrow Transplant (BMT) are growing in numbers. The intensity of this therapy puts them at risk for long-term complications. The musculoskeletal system is one area where these long term complications may occur. Diminished bone mineral density (BMD) and muscle strength are potential morbidities of BMT. Studies looking at the musculoskeletal system and health related quality of life (HRQOL) in this population have not been conducted. The specific aims of this study include 1. Describe the relationships among BMD, muscle strength and HRQOL as they relate to age, sex, type of BMT, years post BMT, endocrine function and steroid use, in survivors who are at least two years post childhood BMT. 2. Compare the BMD of survivors who are at least two years post childhood BMT to age and sex matched established norms from healthy children. 3. Compare the muscle strength of survivors who are at least two years post childhood BMT to age, sex and size matched established norms from healthy children. 4. Compare the HRQOL of survivors who are at least two years post childhood BMT to established norms from healthy children. [unreadable] [unreadable] [unreadable]

Homopolymers of L-isomers of alanine, arginine, histidine, lysine, and proline have been oxidized in the presence of ferrous iron and a chelator, generally citrate. Oxidation of the polymers introduces carbonyls which may be detected by reaction with 2,4-dinitrophenylhydrazine or with p-aminobenzoic acid in the presence of sodium cyanoborohydride. Oxidation results in loss of acid precipitability of these homopolymers. When similar oxidations were conducted with insulin beta chain, similar oxidative changes are seen: increased carbonyl content and decreased precipitation with acid. Amino acid analysis of the insulin beta chain suggests that the attack on the peptide's residues must be random.

The proposed research will utilize tissue culture preparations to study the signals involved in controlling Schwann cell function. Experiments are proposed to study the axonal influences on Schwann cell secretion and the influences of the axon-Schwann cell unit on fibroblast function. Experiments are also proposed to study the axonal changes that occur when a neuron becomes competent to induce myelin formation. Studies in tissue culture will be directed at the problem of identifying products of Schwann cell secretion. The effects of alterations in Schwann cell secretion will be studied by utilizing agents known to either suppress or alter the secretory activity of Schwann cells. We also propose to study the ability of Schwann cells to provide trophic support for neurons. Additional studies are directed at delineating the requirement of Schwann cells on contact with extracellular matrix for the expression of normal Schwann cell function. We plan to continue studies on the mouse mutant dystrophic to test our hypothesis that an abnormality of an extracellular matrix component may explain the peripheral nerve abnormalities in this animal.

During avian vocal learning, birds memorize conspecific songs and use auditory feedback to shape their vocalizations to these stored models. Often song learning is restricted to species-specific sensitive periods, and this proposal is aimed at identifying neural changes that may encourage and temporally constrain this learning. The general approach is to describe how relevant brain regions normally change during vocal learning, and then to assess the functional significance of these cellular changes by examining how they are influenced by auditory inputs essential to song learning. The studies concern zebra finches (Poephilla guttata), a species whose song development is associated with major anatomical and neurochemical changes within brain pathways controlling song. The studies proposed examine two specific neural changes that accompany song learning; a massive addition of new neurons to the vocal motor path way, and a decline in the N- methyl -D- aspartate class of excitatory amino acid receptor within a region implicated in song development. In the first set of studies, thymidine autoradiography and retrograde tracing will be combined to assess how auditory inputs during either song memorization or vocal practice influence the survival of new neurons inserted into the vocal motor pathway. Also, long-lasting retrograde tracers will be used to determine if neuronal replacement or addition occurs within this same neural pathway in adulthood, when song patterns are stable. In the second experimental series, neurotransmitter receptor autoradiography will be used to determine if: i) the developmental regulation of NMDA receptor binding correlates with age-related differences in the propensity for vocal learning and, 2) if auditory manipulations that delay completion of either song memorization or vocal practice similarly affect the timecourse of NMDA receptor decline. This receptor subtype has been linked to numerous other instances of experience-dependent developmental plasticity and learning. Sensitive periods are characteristic of a wide variety of behavioral phenomenon (e.g. language acquisition, social attachment, imprinting) and these studies will help define neural mechanisms that encourage learning during these periods of unique susceptibility.

Selenium is an essential micronutrient in the diet of humans and other mammals. Many health benefits have been attributed to selenium that include preventing various forms of cancer (e.g., colon, prostate, lung and liver cancers), heart disease and other cardiovascular and muscle disorders. Numerous human clinical trials have been undertaken in recent years to assess the role of this element in cancer prevention, delaying the progression of AIDS, etc., at a cost of hundreds of millions of dollars, but little was known about the mechanism of how selenium acts at the metabolic level in mammals to incorporate selenium into protein. We proposed several years ago that the health benefits of selenium are due largely to the presence of this element in selenoproteins as the selenium-containing amino acid, Sec. In the ensuing years, we established the biosynthetic pathway of Sec in eukaryotes and Archaea and focused on the two Sec tRNA isoforms that we demonstrated were responsible for the synthesis of the two subclasses of selenoproteins, housekeeping and stress-related selenoproteins; and pursued studying the methylase, designated Um34 methylase, that synthesizes the methyl group at the 2'-O-postion on the ribosyl moiety at nucleotide 34 of Sec tRNA. We provided strong evidence that addition of Um34 to the isoform, 5-methylcarobxymethyl-uridine (mcmU), to form 5-methylcarboxymethyl-2'-O-methyluridine (mcmUm) requires that mcmU is aminoacylated with Sec, i.e., that the substrate for the methylase (designated Um34 methylase) which carries out this reaction is selenocysteyl-tRNA. In addition, our program focused on developing mouse models to assess the role of all selenium-containing proteins within the two subclasses, housekeeping and stress-related selenoproteins, and on individual selenoproteins in preventing and promoting cancer and in mammalian development. In the past year, we have completed and published the following projects: 1) analysis of the first selenocysteine (Sec) tRNA mutation found in a family, wherein the parents were heterozygous for a G-C base change at position 65 and the proband was homozygous. The patient, an eight year old male, suffers from abdominal pain, muscle weakness and fatigue. The level of mutant Sec tRNA was reduced substantially compared to normal tRNA and the proband synthesized stress-related selenoproteins poorly; 2) dietary selenium regulates the Sec content in selenoprotein P, the only selenium-containing protein in mammals that contains multiple Sec residues, and the replacement of Sec by cysteine; 3) loss of the glutathione peroxidase 4, a selenoprotein, was far more detrimental than the loss of the entire selenoprotein population in mouse liver, and vitamin E could compensate for its loss; 4) an examination of the antioxidant proteins in human liver and lung tumors and comparing them to antioxidant proteins in the corresponding surrounding normal tissues and to each other revealed a greater dependence of liver on either the thioredoxin or glutathione system to drive the malignancy, while lung cancer appeared to depend primarily on the thioredoxin system; 5) an analysis of the role of selenophosphate synthetase 1, an essential protein, in regulating redox homeostasis in mammals using a conditional knockout mouse model; 6) the observation that the loss of thioredoxin reductase 1 or the 15kDa selenoprotein (Sep15) in a mouse colon cancer cell line reversed several malignant properties of the cancerous properties, but surprisingly the simultaneous loss of both of these antioxidant selenoproteins reversed the anticancer effects; 7) knockout of SECp43 (function unknown) in mouse liver was found to be embryonic lethal, but had no apparent role in selenoprotein expression in liver even though it was known to form a complex with Sec synthase and Sec tRNA; 8) observation that the effects of dietary selenium levels on immune function are not limited to liver and further identify the IL-6 and interferon-gamma pathways as being responsive to dietary selenium intake; and 9) two studies involving loss of Sep15 in Chang liver cells inhibiting cell proliferation and motility at the G1 phase and cytoskeleton remodeling and membrane blebbing. In addition, a manuscript on an analysis of extensive ribosomal frameshifting in Euplotes is under revision at Nature Struct. Biol. and the 4th edition of our book entitled Selenium: Its Molecular Biology and Role in Human Health, containing 50 chapters, was completed and will be published in 2016.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Net1 sequesters the Cdc14 protein phosphatase and other proteins in the yeast nucleolus through much of the cell cycle. The Cdc5 and Cdc28 protein kinases regulate release of Cdc14 from the Net1 complex. We would like to use mass spectrometry to identify other proteins that are released from the Net1 complex upon phosphorylation by additional kinases. This study should provide invaluable information on how multiple kinases regulate the sequestration of proteins in the nucleolus.

A chemical information system is being assembled at NIH. The system permits searching through large data bases of mass, CMR and X-ray diffraction spectra and also through sub-sets of chemical abstracts with a view to identification of compounds from their physical data and also retrieval of literature information regarding such compounds. The system is more than 50% complete and sections of it are now being made available to the International Scientific Community via computer networks.

The morphological and physiological characteristics of the intersegmental interneurons and accessible intrasegmental interneurons in the lobster abdominal nerve cord will be determined using extracellular and intracellular recording, stimulation and dye-filling techniques. Iontophoretic differential dye-filling will be used to map the locations of all intersegmental interneurons with somata in the anterior five abdominal ganglia. Once the locations of the somata are known, the intersegmental interneurons and any accessible intrasegmental interneurons will be further characterized by intracellular analysis. The results will provide detailed information concerning numbers, location and possible functional relationships of these interneurons and thereby provide a framework for future analysis of interneuronal function in the lobster abdominal nerve cord.

Minimal access surgery is widely practiced, however, few prospective studies have rigorously analyzed this technique. Uncontrolled studies have reported benefits of minimal access surgery for staging, and for treatment of various GI and thoracic malignancies. Pancreatic cancers are usually advanced at presentation, accurate preoperative staging of pancreatic cancer may amongst other things: (1) prevent unnecessary open surgical procedures for unresectable disease, (2) provide better preoperative staging for patients in clinical trials, particularly in trials with neoadjuvant chemoradiation, (3) accurate staging is essential if palliative surgical procedures are considered by laparoscopically assisted surgery. The first major long-term goal of this proposal would be to establish whether staging pancreatic cancers by laparoscopy will correlate with findings at open surgery and improve resectability rates of patients coming to open surgery. Anecdotal reports have documented the feasibility of palliative procedures for pancreatic cancers by minimal access surgical techniques. The second long term goal of this proposal is to assess in a controlled Phase II trial whether comprehensive surgical management of unresectable pancreatic cancer is feasible and safe using laparoscopically assisted surgery. If our results are positive then in the second half of the study period we would be interested in conducting Phase III trials. The research methodology will incorporate standard surgical techniques of minimal access surgery. A new laparoscopic ultrasound probe will be evaluated for staging the tumors. Findings of laparoscopic staging procedures will be correlated with those at open surgery. Palliative laparoscopically assisted procedures will be compared to our retrospective experience with similar open procedures. The findings from the present proposal may substantially change the way pancreatic cancers are investigated preoperatively and managed surgically.

The broad objective of the proposed research is to investigate the metabolism and toxicity of hydrofluorocarbons and hydrochlorofluorocarbons (H(C)FCs). H(C)FCs are being developed as replacements for ozone- depleting chlorofluorocarbons. To prevent migration to the stratosphere and depletion of ozone, H(C)FCs were developed because the presence of C-H bonds in H(C)FCs was expected to enhance tropospheric lability and thereby reduce migration to the stratosphere. The C-H bonds that impart tropospheric lability may also be expected to impart metabolic lability, and this hypothesis has been confirmed. Studies in past years of support and in other laboratories demonstrate that H(C)FCs undergo cytochrome P- 450-catalyzed oxygenation reactions to afford a range of products. These findings will be extended and expanded in the competitive renewal application. There are three Specific Aims: Specific Aim 1 includes investigations of the metabolism and toxicity of H(C)FCs, including in vivo metabolic fate and gas-uptake pharmacokinetic experiments, in vitro biotransformation studies, and studies on the toxicity and cellular interactions of H(C)FCs and H(C)FC metabolites. Specific Aim 2 includes a comparison of mammalian cytochrome P-450 2E1 and bacterial cytochrome P- 450 101 (P-450cam) as catalysts for the biotransformation of H(C)FCs and development of computational predictions of H(C)FC reactivity. Specific Aim 3 will focus on synthetic, stereochemical, and analytical aspects of H(C)FCs. A major focus of this aim is to develop synthetic routes for the preparation of chiral H(C)FCs of high enantiomeric excess, which will allow, for the first time, investigation of the role of chirality in the metabolism and toxicity of H(C)FCs. The proposed research is significant because the commercial use of H(C)FCs will likely be accompanied by occupational, consumer, and general-population exposure to H(C)FCs. Although exposure concentrations may be low, the populations exposed may be large. Hence it is important to understand thoroughly the metabolism and toxicity of H(C)FCs. Moreover, some H(C)FCs (HCFC-123 and HFC-134a) induce benign tumor formation in experimental animals, indicating that H(C)FCs are not inert chemicals and that the interaction of H(C)FCs or H(C)FCs metabolites with cellular constituents may lead to toxicity.

Oral mucosal defects are secondary to oncologic resection, traumatic events or congenital craniofacial malformations such as cleft palate. Current repair treatments such as autologous grafts are restricted by donor site morbidity, tissue shortage, and retention of the original characteristics of the donor tissue. AlloDerm, a native acellular dermal matrix, with an intact basement membrane, is currently used as a dermal oral soft tissue graft, however, a second procedure for epithelial grafting is required. Keratinocyte populated acellular dermal substrates in ex vivo produced oral mucosal equivalents have been proposed, however, the low cell number obtained from harvested tissue results in graft failure. The proposed research studies will address the capacity of bone marrow-derived mesenchymal stem cells (MSC) to regenerate the oral mucosa epithelium by mimicking an environment in vitro similar to that seen in the human body. Bone marrow- derived mesenchymal stem cells (MSC) are capable of self-renewal, differentiation into cell phenotypes of mesodermal lineage, and transdifferentiation into other cell lineages including epithelial cells. Their local microenvironment composed of growth factors and extracellular matrix (ECM) is known to play a critical role in controlling the proliferation and differentiation fate of stem cells. Our hypothess is that tissue-specific extracellular matrix provides a native microenvironment that governs the ability of multipotent stem cells seeded on Alloderm to both self-renew and differentiate into functional full-thickness oral mucosa epithelium. The following specific aims will be proposed to test this hypothesis: 1) Determine the capacity of MSCs maintained on tissue-specific microenvironment to differentiate into an epithelial cell lineage and form oral mucosa epithelium in vitro; 2) Demonstrate the capacity of predifferentiated MSCs maintained on AlloDerm for oral mucosa epithelium regeneration in vivo using an athymic nude mouse model. These studies will provide training in stem cell maintenance and differentiation, quantitative gene expression, immunochemistry, flow cytometry, FACS sorting, animal modeling, and cell culture. This study is innovative because human bone marrow-derived MSC capability to transdifferentiate into epithelial cells for engineering oral mucosa epithelium has not been explored. The proposed research is significant because it will facilitate the optimization of in vitro oral mucosa equivalnt models for studies of oral mucosa biology and pathology as well as a model alternative to animals for testing of consumer products. These findings will have an impact by initiating human bone marrow-derived MSC research exploration of the field of craniofacial regenerative medicine and lay the foundation to further study the molecular complexity and dynamics of native and tissue-specific ECM in the regulation of MSC proliferation, survival, and differentiation. A better understanding of these mechanisms will move the field closer to bone marrow-derived MSC based therapeutic application by utilizing bone marrow- derived MSCs as an unlimited source of stem cells and treatment of craniofacial defects. Through these studies, I will acquire state-of-the-art knowledge and skills toward my goal to become an established dentist- scientist.

This proposal represents a continuing effort by our laboratory to understand how certain cells stimulate sugar transport in response to increased cellular demand for glycolytic ATP. Skeletal muscle sugar transport and glycolysis, for example, are stimulated massively during exercise and anoxia. We will show that transport stimulation results from the controlled reversal of two, insulin-independent mechanisms that depress the activity of the sugar transport protein GLUT1. These inhibitory mechanisms (which can be studied in isolation in specific tissues) are RESTRAINT and REPRESSION. Restraint reduces whereas repression abolishes the catalytic activity of plasma membrane GLUT1. The broad goals of this proposal are to characterize the molecular basis of restraint, repression and their reversal and to understand how certain cells achieve coordinate regulation of transport and glycolysis. These studies will assist in our long term goal of defining the molecular basis of protein-mediated sugar transport and may ultimately be of value in the management of disordered states such as diabetes. Our studies show that GLUT1 restraint and its reversal and allosteric regulation of phosphofructokinase (the pacemaker of glycolysis, PFK) are functionally analogous. This could account for coordinate regulation of glycolysis and transport. SPECIFIC AIM 1 addresses the mechanism of restraint and therefore asks: "Does GLUT1 contain multiple regulatory domains for direct interaction with glycolytic species that also regulate PFK?" We propose biochemical reconstitutions using purified GLUT1 and allosteric activators of PFK to test this thesis. Thus far, we have discovered two GLUT1 regulatory domains. We will identify these domains by an affinity labeling/proteolysis/sequencing strategy. Finally, we will verify and extend these details and assess their significance to GLUT1 regulation by analysis of the effects of molecular mutagenesis of putative GLUT1 regulatory domains on GLUT1 regulation. SPECIFIC AIM 2 addresses the mechanism of GLUT1 repression / derepression. This mechanism is only poorly resolved although we do know that cytosolic ATP is required for both transport repression and derepression. We will test the hypothesis that GLUT 1-repression is simply a more efficient form of restraint. If repression is a unique regulatory mechanism, we will determine: 1) the role of ATP in GLUT1 regulation (allosteric or covalent) and, 2) whether this regulation is intrinsic to GLUT1 or is mediated by other cellular proteins. If other proteins are involved, we ask: What are their identities, what are their roles in regulation, are these proteins expressed uniquely in cells where transport is regulated and if so, can we reconstitute transport regulation by appropriate transfections of non- regulated cells? If successful, these studies will establish whether restraint and repression are identical or dissimilar regulatory mechanisms and will significantly extend our understanding of the molecular basis of metabolic regulation of sugar transport.

Dioxins, generated both commercially and naturally, are chlorinated polycyclic aromatic hydrocarbons that are highly toxic environmental contaminants. These agents are known to be potent rodent carcinogens and suspected human carcinogens. The best-known prototype of this group of agents is 3,4,7,8-tetrachlorodibenzo-p-dioxin (TCDD). It has been well documented that most, if not all, of the TCDD effects are mediated through the Ah receptor (AhR). Thus, in an effort to better understand the mechanism of dioxins action, I propose to investigate the molecular mechanism of the AhR signaling pathway using the overexpressed AhR and Arnt proteins. My working hypothesis is as follows: Upon ligand binding, the AhR undergoes conformational changes resulting in alteration of a number of AhR-protein interactions, which subsequently leads to a cascade of biologic events to occur. In addition to AhR-protein interactions, other interactions involving Arnt and other proteins also contribute to the action of dioxins mediated by the AhR. This proposal contains specific aims investigating the mechanism for protein factors involvement in the AhR signaling. Not only do these studies provide important information and reagents to further the study of the PAH action mediated by the AhR, but they also provide mechanistic insights on how the AhR regulates its target genes by understanding what other proteins are involved in the AhR signaling pathway. Five specific aims have been proposed: Aim 1) Identify the protein factors in rabbit reticulocyte lysate (RRL) and the Sf9 soluble extract which are responsible for the AhR/Arnt/DRE complex formation; Aim 2) Identity interacting proteins of the AhR and Arnt and examine their effect on the AhR/Arnt/DRE complex formation; Aim 3) Determine whether different forms of the AhR/Arnt heterodimer are present in cell lines which contribute to the different levels of ligand dependent transactivation; Aim 4) Identify ARA9-interacting proteins which are responsible for the ARA9-mediated AhR/Arnt/DRE complex formation and Aim 5) Determine how CyP4O enhances the AhR/Arnt/DRE complex formation.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. De-scalping the brain is a very critical step in MRI data post-processing and analyzing. Most common areas of application are visualization, surface rendering, image registration, DTI and perfusion MRI data post-processing, and decreasing the complexity of subsequent processing algorithms. Many applications related to brain MRI either require, or benefits from the ability to accurately segment brain from the non-brain tissue. De-scalping is one of the interesting and challenging problems in MRI and a number of techniques have been developed for accomplishing it. A recent study published by Fennema-Notestine et al. (2006) concluded that existing algorithms had both strengths and weaknesses, but that no single algorithm was robust enough across different sequences. There is still a need for simple and robust methods for accurate de-scalping. Here we present a new simple technique for brain de-scalping. This technique is based upon lipid ratio map computed using with and without lipid saturation images, which are routinely acquired on most of the clinical scanners.

Agents that target tumor vasculature have tremendous potential as anti- cancer therapies because they do not appear to induce drug resistance. However, these agents are cytostatic rather than cytotoxic, which poses a unique set of problems for the evaluation of drug efficacy because classical measures of tumor (response" may not be valid. It will be especially important to identify their biological mechanisms of action and to develop new methods to detect these effects in primary patient tissue specimens. To this end, we have developed a technique that allows for the detection of dying tumor endothelial cells, and our preliminary results strongly suggest that anti-angiogenic agents induce apoptosis in tumor endothelial cells. The overall goal of the present studies is to determine whether endothelial cell apoptosis is a sensitive marker of efficacy in tumors treated with anti-angiogenic therapies can distinguish patients who respond to these drugs from those who do not. To this end, we propose to: (1) Define the role of specific "survival" pathways in the maintenance of endothelial cell viability in vitro. Cells will be exposed to growth factor withdrawal or VEGF receptor antagonists, and effects on signaling pathways previously implicated in cell survival (i.e. AKT) will be evaluated. We will also isolate mRNA from these cells and analyze changes in apoptosis-associated gene expression following VEGF withdrawal. (2) Determine the role of endothelial cell apoptosis in orthotopic tumor models. Nude mice bearing human pancreatic, colon, or prostate tumors will be treated with investigational agents, and effects on tumor endothelial cell apoptosis will be measured CD31/TUNEL staining. (3) Characterize the role of endothelial cell apoptosis in the effects of anti-angiogenic therapies in patients. Levels of endothelial cell apoptosis in patients treated with anti-angiogenic agents will be correlated with tumor blood flow changes and radiographic measurements of response. These studies will allow us to rapidly determine whether tumor endothelial cell apoptosis can be used as a surrogate for clinical response in patients treated with this class of novel anti-cancer agents.

The anthrax toxin lethal factor (LF) enzyme directly enables the Bacillus anthracis bacterium to evade host immunological mechanisms, leading to circulatory shock and death. Few, if any, therapeutic options are available to counteract LF-mediated cytotoxicity at any stage of anthrax infection. The long-term goal is to better understand the mechanisms by which anthrax toxins act, and, based upon that knowledge, to develop novel and relevant biochemical tools that can be utilized to define mechanisms of LF-related cell death and tissue damage. The objective in this particular application is to evaluate new, promising molecular scaffolds for efficacy against LF-mediated toxicity. The central hypothesis is that small molecules structurally related to a new glutamic acid-based scaffold will effectively inhibit LF protease activity in vitro and in cell-based assays. The rationale for the proposed research is that an improved understanding of key structural features that contribute to LF inhibition will, in turn, lead to an improved understanding of toxin involvement in anthrax pathogenesis, and will potentially provide a promising strategy for treating postexposure anthrax. Thus, the proposed research is relevant to that part of NIH's mission that pertains to developing fundamental knowledge that can be applied to advance significantly the Nation's capacity to protect and improve health. Guided by strong preliminary data, the central hypothesis will be tested by pursuing four specific aims: 1) Design, synthesize and evaluate new glutamic acid-based libraries targeting the anthrax toxin lethal factor active site; 2) Identify new scaffolds for LF probe design using high-throughput screening; 3) Identify probe compounds with capacity to protect macrophages against LF-mediated cytotoxicity; and 4) Identify key binding modes to the LF enzyme active site by means of X-ray crystallography. Under the first aim, libraries around candidate scaffolds will be generated in silico; compounds will be prioritized using molecular modeling; and selected compounds will be synthesized and evaluated for activity against LF in vitro. In the second aim, a large-scale high-throughput screen with novel triage and hit-to-probe techniques will be conducted to identify additional potential scaffolds for probe design; refinement from hit scaffolds will follow an iterative, systematic cycle of design, synthesis, purification, and screening. In the third aim, probes will be tested to assess their relative protective efficacy in inhibiting cell death of a murine macrophage cell line in response to anthrax toxin; and in the fourth aim, prioritized compounds will be co-crystallized with LF in order to experimentally identify key LF ligand-receptor interactions. The approach is innovative, because it targets specific LF structural features that have not been fully investigated, and incorporates recently developed, highly accurate computational and experimental techniques not yet applied to the LF system. The proposed research is significant, because it is expected to provide further molecular insights into the pathways leading to LF-related cell death, and in so doing, to advance and expand understanding of the complex mechanisms involved in anthrax toxemia. PUBLIC HEALTH RELEVANCE: The proposed work addresses important and under-investigated molecular structures and mechanisms that contribute to anthrax pathogenesis. The proposed research has relevance to public health, because although weaponized anthrax continues to pose a threat to society, there is currently no effective therapeutic on the market that can counteract LF-mediated cell death, and the findings from this work are ultimately expected to guide the design of effective therapeutics that can aid persons who have been, or suspect they may have been, exposed to anthrax spores in an emergency situation.

As a noninvasive method for measuring concentration and distribution of chemicals in the living brain MRS is an important tool for studying brain function and disorders. However, robust measurement of MRS signals requires highly sophisticaed design, implementation and maintenance of various MRS techniques. MRS technology has been a very active research area, attracting major effort from top magnetic resonance research centers around the world. Clinical magnetic resonance imaging scanners optimized for performing structural and functional imaging studies also present daunting obtacles for MRS technical development. Most of the MRS protocols at NIH were developed and implemented by the MRS core. 1) Automatic correction of magnetic field inhomogeneity. It is essential to optimize the homogeneity of magnetic field for all MRS experiments because field inhomogeneity can easily destroy the critcal separation of different chemicals. More importantly, an inhomogeneous field makes effective suppression of tissue water signal difficult, therefore making reliable detection of more dilute chemicals impossible. This is particularly the case for anatomical regions of interest to psychiatric research. We have recently implemented an automatic shimming method called FASTMAP which is optimal for localized MRS studies. This method consistently out performs the automatic shimming method provided by the manufacturer. It has already greatly improved the quality of proton glutamate editing and 13C MRS experiments. 2) N-acetylaspartate mapping. The MRS core maintains a chemical shift imaging techique for mapping distribution of the neuronal marker N-acetylaspartate on 1.5 and 3 Tesla General Electric scanners. This method simultaneously generates images of N-acetylaspartate, creatine, and choline-containing compounds. Patient movements can cause artifacts in N-acetylaspartate imaging. The patient movements can be compensated for by using the signal of the partially suppressed water. Unsuppressed water would be too strong to be separated from the metabolite signals, so the N-acetylaspartate mapping sequence was revised to use residual unsuppressed water as a navigator to track and correct for patient motion. 3) Glutathione detection. Glutathione is a marker for oxidative stress. Many psychiatric and neurological disoders (such as schizophrenia, Alzheimer's disease and stroke) are associated with abnormal glutathione concentration. In collaboration with Li An and Steven Warach (NINDS) a glutathione editing method was developed on the Philip 3 Tesla scanner at Suburban Hospital for studying stroke patients. This method uses a selective editing pulse placed on the cysteinyl alpha proton of glutathione to remove the overlapping signals from creatine and GABA. Dr. Warach's group is currently using this method to study stroke patients. 4) Carbon-13 MRS. By using carbon-13 labeled glucose or the glial-specific substrate acetate, brain energetics and glutamate and glutamine cycling flux can be measured. We have succeeded in implementing and optimizing the hardware for carbon-13 MRS on a single-channel 3 Tesla General Electric scanner at NIH. Carbon-13 MRS requires two channels. An add-on stand alone second channel was used to implement a stochastic decoupling scheme. Previously we invented a method for carbon-13 MRS by combining low power stochastic decoupling and intravenous infusion of glucose with a carbon-13 label at the C2 position. This strategy makes it possible to perform viable carbon-13 MRS within the hardware contraints at the NIH. Using this strategy, we recently acquired high quality carbon-13 MRS data from the occipital lobe of healthy subjects. Initial tests of volume coil for decoupling were also successful. We are currently developing methods for acquiring carbon-13 MRS data from the frontal lobe. 5) Proton glutamate editing. Previously we implmented a single-voxel glutamate editing method with correction of eddy current effects for measuring glutamate concentration at 3 Tesla. The method requires several dozen echo time averages, therefore making it incompatible with the robust conventional chemical shift imaging. We are developing a new glutamate editing method which needs a single echo time to isolate the glutamate H4 signal. 6) GABA editing. Previously we developed a method for measuring GABA. Similar to N-acetylaspartate imaging, patient movements can lead to difficulty in accurate determination of GABA. A navigator strategy based on residual water was used to track and correct for patient movement. Special data processing software was also written to correct for phase changes because of patient motion. The improvements of the corrections have been quantified. 7) NAAG editing. We are currently developing a MRS method for measuring the peptide neurotransmitter N-acetylaspartylglutamate (NAAG) which plays an important role in glutamate signaling. Our method uses regularized lineshape deconvolution combined with echo time averaging to separate NAAG from NAA.

The primary aim is to test differences in the effectiveness of three treatments for dyspnea: Education about dyspnea; nurse-coached exposure to dyspnea with education; and a model of pulmonary rehabilitation with high-intensity exercise and education. All subjects will receive a home exercise prescription. Ninety dyspnea-limited COPD patients will be assigned randomly to one of three treatment groups for one year. The GCRC will be involved in measurement of demographic data, and outcomes, e.g., 6 minute walk distance and dyspnea with activities.

Environmental exposures, socio-economic (SE) and behavioral variables all contribute to morbidity and mortality in the United States and in other developed nations. As part of our ongoing studies on pollution and health, we have created the Hopkins Integrated National Tools (HINT) comprising national data bases on health, pollution, weather, and socio-economic and behavioral variables for the entire population of US elderly. HINT provides the unique opportunity for investigating important questions in social and behavioral research (Aim 1) and for enhancing the methodological and substantive contributions of our ongoing studies on pollution and health (Aim 2). The specific aims of this proposal are to develop and apply multilevel statistical models for: 1) determining SE and behavioral factors that influence health when these variables are collected at different levels of spatial aggregation (Aim 1); and 2) for jointly estimating short and long term effects of air pollution on disease outcomes by taking into account cross-level interactions between air pollution exposure, SE and behavioral variables. The work will contribute to establishing an integrated statistical approach for social and environmental epidemiology and therefore will enhance the research goals of the parent grant (Statistical Methods for Environmental Epidemiology R01 ES012054-01). We anticipate that findings of these studies will impact air quality regulations and will better inform public policy. Our analytical framework, which will include development of multilevel models and of computationally fast algorithms for their implementation, will find application in a broad array of policy studies. [unreadable] [unreadable]

DESCRIPTION (Investigator's Abstract): (They) propose to characterize conantokin peptides for treatment of epilepsy. Conantokins, a family of small peptides (17-27 amino acids) isolated from the venom of fish-hunting cone snails, are specific antagonists of NMDA receptors in the mammalian central nervous system. Conantokins inhibit NMDA-elicited currents and NMDA- stimulated cyclic GMP formation. Their laboratories performed the first in vivo studies showing that conantokins exhibit a potent antiseizure activity in animals. Conantokins were shown to be some of the most potent compounds ever tested in the Frings audiogenic seizure-susceptible mouse model. In addition, conantokins showed low neurotoxicity, as determined by relating the toxic dose (rotorod performance) to the effective dose. Therefore, conantokins represent a unique, highly potent and novel class of compounds for treatment of CNS disorders. In Phase I, we propose to: test conantokins for antiseizure activity using three different experimental animal models; compare the in vivo antiseizure activity of conantokins with their in vitro receptor binding activity; identify and evaluate new natural conantokins; and synthesize chimeras of conantokins to initiate structure function studies. In Phase II, more extensive in vivo characterization of conantokins for treatment of seizure disorders will be carried out. Moreover, they will establish a program for synthesis of conantokin peptidomimetics, which should increase the bioavailability of these molecules.

DESCRIPTION: The broad goal of the project is to elucidate the molecular mechanisms of initiation and control of protein synthesis in eukaryotic cells. Special focus is on the structure/function of mammalian and yeast initiation factors involved in Met-tRNAi and mRNA binding to ribosomes and on the phosphorylation of initiation factors as a general method to regulate translation rates. A major aim is to determine the subunit composition and structure of human and yeast eIF3, a complex of 10 or more protein subunits that plays a central role in initiation. Capitalizing on recent cloning of cDNAs and genes encoding these subunits, investigators will express fusion proteins tagged with his6, then purify the eIF3 complex by Ni or glutathione affinity chromatography and analyze the subunit composition. The structure of these complexes will be elucidated by determining subunit-subunit binding interactions by Far-western blotting, the yeast 2-hybrid system, affinity chromatography and/or chemical cross-linking methods. Mutant and suppressor analyses with yeast genes will provide evidence for interactions and help elucidate functional roles. Higher order structures involving eIF3 subunit interactions with other initiation factors, mRNA and/or ribosomes will be probed by similar methods. In addition, tapping mode atomic force microscopy will be applied to elucidate the structures of initiation complexes and a fluorescent-labeled mRNA will be employed to measure the rate of ribosome scanning. Studies of the role of phosphorylation will focus on mammalian eIF4B and eIF3, and on yeast eIF1A. Following the determination of the site of eIF4B phosphorylation by the p70 S6 kinase which is regulated by the rapamycin-sensitive signal transduction pathway, investigators will evaluate the importance of this phosphorylation event for regulating the factor's activity. They also will identify other sites of eIF4B phosphorylation and the kinases involved. eIF3 is known to be phosphorylated on the p116 subunit, although other subunits may be modified as well. They will exploit their knowledge of the primary structures of these proteins to determine sites of phosphorylation and their kinases. Identified sites will be mutated to alanine and aspartate and the mutant forms expressed in transfected cells to elucidate the effects of preventing or mimicking phosphorylation. Identification of the site of eIF1A phosphorylation and expressing appropriate mutant forms as the sole source of this essential protein will be carried out. The studies will extend the understanding of how phosphorylation regulates translation rates.

Atherosclerotic vascular disease remains one of the most common causes of death in the United States. The broad goal of the proposed studies is to generate a better understanding of the mechanisms of this disease process. Atherosclerosis is an inflammatory disease. It is mediated in part by monocytes that are recruited into the vessel wall by adhesion molecules, including E- and P-selectin. Two 1(1,3)-fucosyltransferases, FucT-IV and FucT-VII, participate in the synthesis of active E- and P- selectin ligands on leukocytes. Thus, expression of these fucosyltransferases, and therefore selectin ligand activity, may regulate monocyte trafficking into the vessel wall and development of atherosclerosis. The experimental goal of this proposal is to test the hypothesis that monocyte-specific FucT-dependent selectin ligand activity modulates the development of atherosclerosis. FucT- dependent changes in atherosclerotic lesion pathology will be assessed in the low density lipoprotein receptor (LDL-R)-deficient mouse model of atherosclerosis. FucT activity in LDL-R(-/-) mice will be deleted globally and in a monocyte specific manner using gene targeting strategies, as well as reconstituted in a monocyte-specific manner using transgenesis. FucT-dependent changes in atherosclerosis in vivo will be correlated with FucT-dependent monocyte selectin ligand activity measured in a flow chamber in vitro. The specific aims of this proposal are to: 1) Quantify atherosclerotic lesion size, and determine the qualitative alterations of the lesion in LDL-R(-/-) mice in which FucT-IV, or FucT-VII, or both FucT-IV and FucT-VII are deleted. 2) Determine the cellular adhesion parameters that enable FucT-IV- and/or FucT-VII-dependent monocyte tethering and rolling on E- or P-selectin under shear. 3) Define the selectin ligand-dependent monocyte/macrophage- specific contribution to atherogenesis by quantifying lesion size and determining the qualitative alterations of the lesion in LDL-R(-/-) mice with (a) monocyte-specific deletion of selectin ligand activity, and in (b) mice in which monocyte-specific expression of selectin ligand activity has been reconstituted by transgenesis. Together, the proposed experiments will determine the functional role of FucT-IV and/or FucT-VII in generating the selectin ligand activity necessary for atherogenesis. The experiments will also determine the quantitative and qualitative monocyte-specific selectin-dependent contributions to atherogenesis. This knowledge will contribute to the development of improved or novel preventive strategies, diagnostic procedures, and therapeutic options for atherosclerosis, which will ultimately reduce the morbidity and mortality associated with the disease and its sequelae.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. To obtain important reproductive data on endangered primates under field conditions.[unreadable] [unreadable] For the muriqui (Brachyteles hypoxanthus), Strier and Ziegler examined the timing of the breeding season with [unreadable] respect to the onset of ovarian cycling. We found that early conception was related to the amount of androgen and [unreadable] estradiol levels. Early conception was associated with lower androgen levels and higher estradiol levels then in [unreadable] females who did not conceive. Fedigan and Ziegler found for the white-faced capuchin (Cebus capucinus), there is [unreadable] a lack of overt signs of ovulation. A means to determine the timing of ovulation is limited. Behavioral observations [unreadable] on the males in the groups indicated that the best indicator of ovarian state was the two-fold increase in male [unreadable] affiliative and sexual behaviors during the conceptive phase. Gould and Ziegler found that for the prosimian, Lemur [unreadable] catta, or the ringtailed lemur that males who live in groups with fewer males show lower cortisol levels and [unreadable] aggression than males who live with a higher density. However, during breeding season competition, male ring [unreadable] tailed lemurs do not show significantly higher levels of cortisol. These studies indicate the type of endocrine data [unreadable] that can be obtained from nonhuman primates under totally free conditions. This research used WNPRC Assay [unreadable] Services. Note: Brachyteles hypoxanthus is not included on NCRR APR system species list. The investigators [unreadable] studied muriquis in addition to white-faced, or white-throated capuchins. This work used private foundation [unreadable] support, mentioned in previous progress reports, and also relied on WNRPC Assay Services.

This project examines a new but poorly understood finding from functional magnetic resonance imaging (fMRI) that is providing new insights into fundamental brain architecture. Functional imaging studies show that similar sets of non-contiguous brain regions are co-activated across a wide range of tasks. Remarkably, many of these co-activations are recapitulated when subjects are at rest. Thus, resting state fMRI (rs-fMRI) is thought to reflect task-related functional networks. It is of great interest to know what principles are responsible for the maintenance of these intrinsic co-activations. Most functional connectivity studies have focused on the cortex. Deep brain structures, including the thalamus, have been implicated in these networks, but little is known regarding exactly what role they might play. Most input to the cerebral cortex passes through the thalamus, including many sensory signals, cerebelar and basal ganglia inputs. As a result, the thalamus is in an ideal anatomical position to regulate or maintain functional connectivity. Focal lesions can reveal much about the structure and maintenance of functional connectivity. Progress in this area has focused on naturally occurring lesions. Experimental lesions in non-human primates can be precisely placed, and repeated to obtain statistical confidence in their effects. Recently, rs-fMRI in non-human primates has revealed cortical networks similar to those observed in humans. Muscimol, a GABAA agonist, can be injected into tissue to inhibit local activity, and the sites of inactivation can be precisely monitored. We propose to use reversible inactivation in the macaque monkey to examine the roles of the thalamus and cortex in maintaining resting state network functional connectivity, and to characterize the effects of inactivation on the networks using novel graph theoretic approaches. This work will shed light on the neuronal underpinnings of cortical connectivity and more generally, may inform rehabilitation approaches for traumatic brain injury and other disorders.

The plasma membrane of epithelial cells is divided by tight junctions into two distinct domains, apical and basolateral, that differ markedly in their protein and lipid composition. A central question in cell biology is to determine how this asymmetry is generated and maintained. Viruses that cause influenza, measles, and other diseases depend on intracellular protein sorting pathways for their propagation. Understanding how intracellular sorting operates may provide new approaches to combating these diseases. Plasma membrane proteins and some membrane lipids are sorted in the trans Golgi network in certain cultured epithelial cells, and are packaged into transport vesicles destined for the apical or basolateral surface. Proteins anchored in membranes by glycosyl phosphatidyl inositol (GPI) are transported apically. GPI anchorage can cause this targeting. Glycosphingolipids are also delivered primarily to the apical surface. It has been proposed that GPI-anchored proteins may associate with glycosphingolipids inside the cell, and that the two may be sorted co-ordinatedly into the apical transport pathway. Our goal is to test this theory, and to isolate and characterize the lipid-protein complexes. Membrane complexes that contain both glycolipids and GPI-anchored proteins have been isolated, using their surprising insolubility in non-ionic detergents. The aim of this application is to learn whether these complexes contain the domains of associated GPIanchored proteins and glycolipids from the Golgi that were proposed above. These domains would be likely to contain sorting proteins, since they form in the sorting compartment and contain two classes of molecules that are targeted to the same surface. Isolation of these sorting proteins is the long-range goal of this work. Biochemical and ultrastructural techniques will be used to characterize the complexes. The effects of altering the cellular lipid composition and the conditions of detergent lysis on complex formation will be determined, as will the behavior of GPI-anchored proteins reconstituted into vesicles of defined lipid composition. The intracellular site(s) of origin of the complexes will be determined by subcellular fractionation and by membrane labelling prior to isolation of complexes. The next aim is to determine the protein profile of the membrane complexes, to identify candidate proteins that may be important in sorting. Finally, the possible inter-relationship of glycolipid and GPI-anchored protein sorting will be determined using specific inhibitors.

The long-term goals of this project are to identify and elucidate the physiological and pathophysiological signaling mechanisms involved in the regulation of pulmonary vascular function mediated by modulation of cGMP generation by poorly understood processes that control the soluble form of guanylate cyclase (sGC). Our studies focus on integrating our understanding in isolated bovine pulmonary arteries of how heme metabolism and various redox-related processes interact with the metabolism of reactive oxygen species (ROS) and nitric oxide (NO) in the control of pulmonary arterial smooth muscle force through mechanisms involving the regulation of sGC. Aim 1 investigates defining how the modulation of specific pathways regulating Nox oxidases by mediators promoting pulmonary hypertension (PH) control pulmonary arterial smooth muscle force through ROS mechanisms that regulate the activity of sGC. Studies in Aim 2 examine how cytosolic NADH, NADPH and glutathione redox regulate sGC through modulating oxidation of its thiol and heme sites, with an emphasis on identifying how these mechanisms of sGC regulation contribute to the actions of PH mediators and inhibit vascular responses involving nitric oxide (NO) and ROS. Aim 3 studies examine how the control of heme biosynthesis by aminolevulinic acid (ALA) generating protoporphyrin IX, and heme metabolism by heme oxygenase (HO) regulate the activity of sGC and its control of pulmonary vascular force by cGMP. Endothelium-removed bovine pulmonary arteries are studied with a combination of physiological function, molecular signaling, organ culture, transfection and metabolic modulation approaches to investigate mechanisms controlling sGC and its influence on vascular force through cGMP. Arteries from mice deficient in Nox-2 and p47phox Nox oxidase subunits, glucose-6-phopshate dehydrogenase needed to maintain cytosolic NADPH, and HO-2 are used to support mechanistic studies by defining their role in regulating sGC. Pulmonary arteries from monocrotaline-induced PH rats are examined to identify alterations in the regulation of sGC by the Nox oxidase, ROS and redox systems investigated. The regulation of sGC by ALA-elicited protoporphyrin IX generation is examined in a manner that determines if it stimulates sGC in a manner that has potential for therapeutic development as a pulmonary vasodilator which is resistant to oxidant conditions that are likely to be inhibiting sGC in PH.

Although much is known about the pathophysiological mechanisms underiying traumatic brain injury (TBI), few therapeutic strategies have been found to promote recovery after TBI. The purpose of this study will be to investigate novel therapies that promote recovery of function when initiated in an early or delayed fashion after TBI. In preliminary studies, we found that the transplantation of neural progenitor cells (NPCs) transduced to release a multi-neurotrophin in the injured brain leads to substantial histopathological protection and improvements in cognition even when initiated one week after the traumatic insult. The proposed experiments will extend these exciting findings and provide information regarding the underlying mechanisms for these improvements. This strategy is novel in that few studies have utilized transduced NPCs releasing a multi-neurotrophin that activates all of the neurotrophin receptors to optimally activate cell survival and synaptic plasticity pathways within neurons. We propose three specific aims to test the central hypothesis that transplantation of NPCs expressing a multi-neurotrophin improves functional recovery after TBI. The first aim will identify the optimal conditions for transplantation of the NPCs. We will identify the optimal multi-neurotrophin, cell dosage, therapeutic time window, and rehabilitative strategy that results in the greatest level of behavioral recovery. Our preliminary data indicate that co-transplantation of the NPCs with a soluble, clustered ephrin BS ligand, which promotes cell survival pathways in stem cells in culture and in vivo, may be an innovative strategy to foster survival of the transplanted cells in the injured brain. In the second aim, we will determine which of these transplantation strategies improves histopathological outcome, increases survival of the transplanted NPCs and possibly promotes neurogenesis. The final aim will determine whether the transplantation of the NPCs improves synaptic plasticity in the hippocampus. Thus, the scope of this grant will assess how this cell therapy improves behavioral, morphological, and electrophysiological mechanisms. To thoroughly test the effectiveness of these therapies, potential detrimental side effects will also be assessed, including posttraumatic seizure threshold changes and aberrant axonal sprouting. This project is supported by an established group of investigators who provide the wealth of expertise necessary to conduct this multidisciplinary program in TBI

DESCRIPTION: The broad objective of this application is to explore and define the mechanisms by which the autonomic nervous system regulates the circulation to support tissue perfusion, particularly in the brain, during adaptation to microgravity and readaptation to earth's gravity. The primary hypothesis is that adaptation to the unique environment of microgravity causes alterations in the autonomic nervous system that interact with microgravity-induced changes in body fluid distribution, that result in orthostatic intolerance upon return to earth. The unique characteristics of microgravity may modify neural afferent traffic and produce conflicting information. Microgravity minimizes the dynamic demands on the cardiovascular neural control. The level of physical activity is decreased, and no postural adjustments are required. This regulatory environment is likely to degrade important control mechanisms. The proposed experimental design represents and integrated approach to the testing of this primary hypothesis. The following working hypotheses will address: (1) whether efferent sympathetic nerve activity increases appropriately in response to baroreflex and non-baroreflex- mediated stimuli after space flight; (2) whether integrated clinical tests of autonomic function can detect functional impairment for use to characterize the time course of adaptation to microgravity; (3) whether regulation of the cerebral circulation changes are parallel to or independent of the regulation of the systemic circulation; and (4) whether advanced mathematical models of neural control, including both linear and non-linear dynamics, can be developed to gain insight into the integration among neurocirculatory variables and control mechanisms.

Test approximately one hundred coded chemical samples per year for four years for their ability to induce mutations in Salmonella typhimurium strains TA97, TA98, TA100, and TA135.

Attention deficit hyperactivity disorder (ADHD) is one of the most common childhood-onset psychiatric disorders afflicting 5-10 percent of children and adolescents, and 2 percent or more of all adults. Twin, family, and adoption studies support a significant role for genetic influences in ADHD. The heritability of ADHD is 70-80 percent and patterns in families suggest that a major autosomal gene may contribute to the genetic effect. The purpose of the present study is to identify susceptibility genes underlying ADHD through a genome search and linkage disequilibrium studies in 300 Affected Sibling Pair (ASP) families. The long-term goal of this research is to understand the genetic etiology of ADHD. Understanding the genetic basis of ADHD will enable more accurate diagnosis and treatment of preventive measures, tailored to the specific genetic susceptibility of an individual, to be developed. Based upon a year of extensive pilot research, and in collaboration with the Wellcome Trust Centre for Human Genetics at Oxford University, the present study is proposed to: 1. Identify susceptibility gene locations in ADHD through a systematic genome scan in 300 Affected Sibling Pair (ASP) families - specifically to test the hypothesis that a gene or genes of major effect underlie ADHD. 2. Identify susceptibility genes underlying the clinical and cognitive manifestations of ADHD through the linkage disequilibrium studies of candidate genes involved in dopamine transmission, for example, to test the hypothesis that the dopamine transporter gene influences behavioral symptoms of impulsivity and hyperactivity and not behavioral symptoms of inattention. 3. Identify the modifying role sex has in the expression of ADHD susceptibility genes -specifically to test the hypotheses that a gene located on the X chromosome modifies ADHD expression.

Neuroimaging techniques have provided effective tools for investigating the influence of pain on CNS function. Human imaging studies have identified a pain matrix composed of brain regions that are activated by nociceptive stimuli. Activation is observed in the secondary somatosensory and insular regions, the anterior cingulate as well as the primary somatosensory area and thalamus. More limited evidence indicates decreased activity in a network that includes the inferior parietal lobule, and the medial prefrontal cortex. These regions are thought to subserve discriminative sensory pain transmission and to process affective-motivational components of pain. MRI investigations in chronic neuropathic pain indicate brain neurodegeneration and decreased gray matter volume and density in patients with chronic back pain, phantom pain, or fibromyalgia, although degree and regional distribution varies. Chronic back pain patients show activation of the prefrontal cortex during spontaneous pain, the same area that shows a reduction in gray matter density, as well as disruption in resting functional connectivity of widespread cortical areas. The nature of structural changes remains to be determined, whether neurodegeneration is causal and if analgesics prevent these changes. A difficulty in human research is assembling a homogenous patient cohort with matched symptoms, disease duration, medication history and age distribution. Since subjects are not tested prior to pain onset, individual differences that may predispose pain vulnerability can not be assessed. Importantly, the response to nerve injury and to different treatments varies among patients with comparable pain syndromes and not all patients exhibit neuropathic pain behavior after a demonstrable nerve injury. For these reasons, human functional imaging has not yet provided reproducible findings specific to the disease or a pathophysiological basis for symptomology. Prior to the advent of small animal imaging, preclinical studies could not directly measure spontaneous pain, a limitation in their utility as models of neuropathic pain. However, procedural advances now allow quantification of alterations in resting state connectivity and stimulus-evoked activity. The coupling of imaging to animal models of neuropathic pain allows longitudinal study of the development of pain and its expression. Changes in brain function that occur during spontaneous and stimulus-evoked pain can be assessed as a function of pain duration: grey matter volume and white matter integrity can be assessed over time. Using MRI and MRS techniques and the spared nerve injury (SNI) model of neuropathic pain in rats, ongoing studies seek to: i) identify changes in brain structure, function and metabolism that occur in the pain matrix as a function of the duration of neuropathic pain;ii) determine whether individuals differ in vulnerability to SNI-evoked pain and whether these differences are associated with differences in brain function and iii) determine whether a manipulation that we have shown to prevent the development of or reverse the expression of SNI-evoked allodynia and hyperalgesia (see below) reduces SNI-evoked alterations in spontaneous and evoked brain activity, functional connectivity, concentrations of biochemical compounds, grey matter volume and white matter integrity. Using advanced MRI and MRS techniques developed within the IRP, we expect to identify maladaptive brain plasticity at a systems level in the SNI rats, and to correlate brain plasticity with behavioral pain measures. The proposed translational studies will enable identification of a disease-modifying strategy aimed at both preventing maladaptive plasticity and identifying intrinsic risk individuals The rostral ventromedial medulla (RVM) constitutes the efferent component of pain-control systems that descend from the brain to the spinal cord. Considerable evidence has emerged regarding participation of this system in persistent pain conditions such as inflammation and neuropathy. The RVM normally exerts an inhibitory influence on dorsal horn neurons However, persistent noxious stimulation triggers time-related alterations in RVM synaptic activity. In inflammatory pain models, descending facilitation transiently increases reducing the net effect of inhibition. Over time, descending inhibition increases resulting in decreased nocifensive behavior. After nerve injury, RVM plasticity leads to facilitation of spinal cord nociceptive output, exacerbation of primary hyperalgesia and enhanced sensory input from adjacent regions (secondary hyperalgesia). AMPA receptor activation in the RVM has been shown to inhibit spinal nociceptive transmission and nocifensive behavior. Increased AMPA receptor function in the RVM is implicated in the activity-dependent plasticity that occurs in response to persistent pain produced by tissue inflammation. Targeting and synaptic clustering of AMPA receptors is essential for efficient excitatory transmission. Neuronal pentraxin 1 (NP1) is a member of the pentraxin family of proteins that is expressed exclusively in neurons and facilitates AMPA receptor clustering. Given the postulated role of NP1 in excitatory synaptic transmission and AMPA receptor systems in pain processing, we have used gene deletion and viral-mediated transfer techniques to examine whether manipulations that target this protein can affect the expression of persistent pain. To determine the contribution of NP1 to nerve-injury evoked pain, we assessed mechanical hypersensitivity in wild type and NP1 knock out mice following spared nerve injury. Nerve injury led to marked mechanical hypersensitivity of the injured limb. This enhanced nociception is significantly inhibited in NP1 knockout mice. In order to probe the specific involvement of RVM NP1 in mediating the attenuated response of NP1 knockout mice, we infused a lentiviral vector which drives expression of functional NP1 protein directly into the RVM. Selective rescue of RVM NP1 expression in knockout mice restores allodynia produced by nerve injury. Consistent with the data obtained in NP1 knockout mice, silencing NP1 expression in the RVM of rats prior to nerve injury inhibits allodynia. Furthermore, it decreases mechanical hyperalgesia. These findings are consistent with the observation that descending facilitatory systems arising in the RVM are necessary for the maintenance of neuropathic pain and identify NP1 in the RVM as a critical element in the descending facilitation of nerve-injury evoked pain. Together, these data suggest that targeting NP1 may be a novel therapeutic strategy for reversing persistent pain of diverse etiologies. On-going studies examining the role of NP1 in other conditions including those associated with AIDS antiretroviral therapy and peripheral inflammation indicate a key role of this protein in the development of persistent pain. Preliminary studies examining the contribution of another pentraxin, pentraxin 3 suggest a global role of the pentraxin family of proteins in the pathogenesis of nerve injury, neuropathic and inflammatory pain.

The Investigator, Daniel K. Benjamin, Jr., MD, PhD, MPH, will prepare pediatricians for a career in clinical research that uses rigorous quantitative methods. Historically, 75% of drug products have insufficient labeling information needed for pediatric dosing, safety or efficacy. Dr. Benjamin's long-term research goal is to improve pediatric drug development by establishing proper dosing, safety, and efficacy of drugs used in adolescents, children, toddlers, infants, and neonates. The specific aims are to prepare trainees fora career in acadmic medicine in one of: 1) clinical pharmacology; 2) epidemiology and translational research; or 3) biostatistics and clinical trials. In order to fulfill these aims, fellows in the quantitative research methods program obtain formal training in biostatistics, epidemiology, or pharmacology and complete a Masters or PhD degree during their fellowship and first 24 months as faculty. Dr. Benjamin's research plan includes the multisite NICHD-sponsored protocol Meropenem Pediatric Off-Label Drug Study and the proposed Antimicrobial Pharmacokinetics in High Risk Infants II study. Dr. Benjamin also serves as Protocol Chair for 5 protocols sponsored through public-private partnerships between the National Institutes of Health and pharmaceutical industry. Strengths of his program include ongoing funding and active participation in several areas of neonatal pharmacology and therapeutics, including his role as Principal Investigator of the NICHD-sponsored North Carolina Collaborative Pediatric Pharmacology Research Unit (PPRU) site. The North Carolina PPRU ts a collaboration between Duke Pediatrics (Benjamin: PI) and the University of North Carolina School of Pharmacy (Thakker: co-Pi), which serves as a platform for training pediatricians in clinical pharmacology. Dr. Benjamin has formal training and a proven publication record in epidemiology and biostatistics. His expertise in these fields is further demonstrated by his leadership role directing pediatric quantitative analyses within the NCRR-sponsored Duke Clinical and Translational Science Award (CTSA). Feasibility of this program is shown by a proven track record in publications (58 peer-reviewed articles and abstracts) for which trainees are first author, and by the 16 trainees who are enrolled in or have completed masters or PhD degrees in health sciences, epidemiology, biostatics, or pharmacology. These research projects and institutional initiatives made possible by the Duke CTSA and Dr. Benjamin's collaboration with University of North Carolina combine perfectly with his role as Chief of the Division of Quantitative Sciences in the Department of Pediatrics at Duke University, and Fellowship Director of the Duke Clinical Research Institute

Childhood obesity is a major public health concern, particularly in low-income and minority youth. Sedentary behavior (SB) has been associated with obesity cross-sectionally and longitudinally and also has demonstrated negative effects on increased metabolic risk and mortality independent of weight. In addition, greater health disparities in sedentary behavior versus diet and physical activity have been shown in minority youth compared to non-minority youth. However, the sedentary behavior intervention literature has several limitations. First, all SB intervention studies used self-reports of SB. As a result, te entire intervention literature on reducing SB is based on self-reported outcomes, which are known to be unreliable. Second, all studies have limited their assessment of SB to TV viewing only, making it unclear how existing interventions affect the broad range of SBs (e,g., texting, video game playing, inactive transportation) or the degree to which time previously spent in SB is reallocated to light, moderate, or vigorous physical activity after an intervention. Third, only three studies focused on minority youth and show mixed effects on weight despite large SB disparities. Interventions also typically target increasing moderate-to-vigorous physical activity as the alternative to sedentary behavior. However, minority youth are more likely to have multiple barriers to MVPA including limited safety, access, and equipment. A novel approach is the promotion of light physical activity to displace SB. This switch represents an easier target and a potentially more appealing alternative than MVPA. Light physical activity may be done simultaneously with television watching or achieved through other popular screen activities including game consoles, avoids common complaints of getting hurt or sweaty, and acknowledges that underserved families are more likely to have safety concerns and a lack of acceptable alternatives. Despite these potential advantages, no interventions to date have promoted light physical activity as a SB displacement target. The proposed study will be conducted in 2 phases. During Phase 1, a parent-based intervention for decreasing accelerometer measured sedentary behaviors through targeting light physical activity will be developed and piloted in low-income youth. During Phase 2, the intervention will be evaluated in a randomized controlled trial (n=40) to determine its effect on decreasing sedentary behavior and relative weight as well as light, moderate, and vigorous physical activity.

Light, immunofluorescent microscopy and transmission and scanning electron microscopy will be carried out on human renal biopsies and nephrectomy specimens and of renal lesions of dogs and rats. Particular emphasis will be placed on new methods of preparation of these tissues for scanning electron microscopy.

Are maternal child-related behavioral deficits characteristic of women with a history of depression, or are difficulties present only when mothers are experiencing an acute episode of depression? Are there certain patterns of aberrant child-related behaviors that wax and wane with depressive episodes, and others that reflect more or less enduring behavioral characteristics of depressed mothers? In this longitudinal study it is possible to compare maternal characteristics of two groups of mothers with a history of affective disorder: those who are in an episode and those who are between episodes of depression at the time of their participation in the study when they are observed with their children. Prelim- inary analyses indicate that depressed mothers who are currently in episode are not more likely than those who are not in episode to report behavior problems in their children. Both groups, however, report significantly more child behavior problems than mothers without a history of depression. Nor do depressed mothers who are in a self-reported depressed mood report higher levels of child behavior problems than depressed mothers who report non- depressed moods. These data argue against the likelihood that mothers' depressed moods or depressive states (episodes) lead to systematically distorted reports of problems in their children. Depressed mothers experiencing an episode of depression, however, do report more distress in their marriages than depressed mothers who are not experiencing an episode of depression. Mothers' behaviors with and around their children while playing with a peer (an experimental laboratory situation) were compared. Depressed women in an episode of depression did not differ significantly from depressed women who were not in episode with respect to appropriateness of their interactions with their children.

Health literacy is a critically important skill that helps people to become active participants in their health care. The 2003 National Assessment of Adult Literacy showed that more than 75 million Americans had basic health literacy skills, indicating that as many as 1 in 4 Americans can have difficulty understanding information about their healthcare. Persons in racial and ethnic minorities are likely to have even lower levels of health literacy. Twenty-four percent of blacks (9.5 million persons) and 41% of Hispanics (21 million persons) have below basic levels of health literacy. These persons have lower levels of health literacy and compelling evidence, including our own findings (see below), link race and ethnicity to disparities in health via health literacy. Members of minority groups and older adults are more frequently affected by chronic diseases such as cancer, high blood pressure, heart attack, stroke, diabetes, elevated cholesterol, asthma, hepatitis C, HIV infection, mental health disorders and many others. The twin burdens of chronic disease and low levels of health literacy thus fall disproportionately on those most in need - members of minorities and older adults, all of whom likely to experience one or more chronic conditions while often not having the health literacy skills to help them cope. Chronic disease self-management (CDSM) is a logical target for a general health literacy intervention. In an approach that cuts across specific diseases. CDSM targets problems and skills needed to cope with issues such as fatigue, pain, stress, depression, sleep disturbance and treatment adherence. Studies show that in- person CDSM classes improve patients' functioning and reduce healthcare utilization, but their availability is limited due to the lack of qualified personnel and cost. Similarly, while interventions have been developed to improve health literacy, they are difficult to scale to levels needed to meet the challenge of low health literacy (for more than 40 million persons) due to their cost. Effective interventions with the potential for wider dissemination at reasonable costs are urgently needed. In a previous study, we showed that a computer-delivered tailored information intervention targeting health literacy that can deployed either as an information kiosk in a clinical office or n the Internet could be cost- effective in improving patients' health literacy and adherence. It is nt clear, however, whether the same sort of computer-delivered, multimedia and interactive approach will be effective in improving CDSM skills in persons with low baseline levels of health literacy, and if it is, whether its effects will extend beyond health literacy to general health, slf-efficacy, activation, and treatment adherence. In a second project period we will evaluate this possibility by creating a personally relevant computer-delivered intervention targeting CDSM and health literacy among African-Americans, Hispanics, and white non-Hispanics:

A phase I study of cabozantinib (Cabo) plus docetaxel (D) and prednisone (P) in metastatic castrate resistant prostate cancer In mCRPC, two randomized trials demonstrated an overall survival (OS) benefit with the chemotherapeutic agent D. However, the survival improvement is modest and new strategies are needed to enhance clinical response. D-based combinations have been evaluated as one alternative strategy. Cabo targets multiple tyrosine kinases including c-Met, vascular endothelial growth factor receptor 2 (VEGFR2) and RET. Cabo has shown activity in mCRPC, with resolution of bone lesions on bone scan, regression of soft tissue/visceral disease, and reductions in circulating tumor cells and bone biomarkers. We hypothesize the addition of Cabo to D and P, in patients (pts) with mCRPC, will have an acceptable toxicity profile and could lead to improved survival by targeting different cellular pathways simultaneously. This combination therapy may represent a safe and effective strategy to improve the outcome of mCRPC pts treated with D-based chemotherapy. This is a phase I trial to determine the safety profile and the recommended phase II dose of Cabo in combination with D and P. Pts receive a fixed dose of D (75 mg/m2 IV day 1 of each 21 day cycle) and P (5 mg po q12 hours) in combination with Cabo at three escalating doses: dose level 1 is 20 mg, level 2 is 40 mg, and level 3 is 60 mg (all po qdaily). Using a standard 3 + 3 design, three patients will initially be treated at each dose level until the maximum tolerated dose (MTD) has been defined. An expansion cohort will then be enrolled at the MTD. The accrual ceiling for the study, including both the dose escalation and the expansion phases, is set at 24 pts. Secondary objectives include assessments of pharmacokinetics of each agent, evaluation of antitumor activity of the combination therapy, and assessment of changes in molecular biomarkers for receptor tyrosine kinase and angiogenesis pathways, as well as biomarkers for bone metabolism. Restaging with bone and CT scan will be undertaken every 3 cycles. A phase II study of trebananib (AMG 386) and abiraterone in metastatic castration resistant prostate cancer. Preclinical studies support the use of an antiangiogenic approach in the treatment of prostate cancer. Trebananib is a novel peptide-Fc fusion protein that sequesters angiopoeitin 1 and angiopoeitin 2, thereby preventing their interaction with their common receptor Tie2, and inhibiting tumor endothelial cell proliferation and tumor growth. Trebananib is currently in Phase 3 trials for the treatment of ovarian carcinoma and has been shown to have clinical activity in multiple tumor types. A phase II study of trebananib (AMG 386) and abiraterone in metastatic castration resistant prostate cancer. Preclinical studies support the use of an antiangiogenic approach in the treatment of prostate cancer. Trebananib is a novel peptide-Fc fusion protein that sequesters angiopoeitin 1 and angiopoeitin 2, thereby preventing their interaction with their common receptor Tie2, and inhibiting tumor endothelial cell proliferation and tumor growth. Trebananib is currently in Phase 3 trials for the treatment of ovarian carcinoma and has been shown to have clinical activity in multiple tumor types.

Stigma is a risk factor for adverse mental health outcomes among several groups, including gays, lesbians and bisexuals (LGB), African-Americans, and the overweight/obese. The mechanisms conferring this risk, however, remain inadequately understood, hindering the development of effective interventions for these vulnerable populations. Through integrating research, theory and methods from clinical and social psychology, the present investigation seeks to understand how stigma contributes to psychopathology. The author proposes a theoretical model hypothesizing that experiences of stigma render individuals more vulnerable to established psychological and social risk factors that confer risk for psychopathology. The predictive utility and generalizability of this model will be examined in a longitudinal, prospective study among a community-based cohort of approximately 2,100 adolescents (grades 5-12). Stigma-related stressors are particularly pervasive among LGB, African-American and obese/overweight youth, but research with this age group has been lacking. Thus, the present proposal will advance our understanding of the ways in which stigma leads to psychopathology during this significant developmental period. Specific aims of the proposed work are to: (1) document stigma-related stress as a risk factor for the development of internalizing symptoms in a longitudinal sample of adolescents; (2) establish specific cognitive, emotion regulation, and social processes as sequelae of stigma-related stress; (3) determine whether these processes mediate the relationship between stigma and internalizing symptoms; and (4) evaluate the generalizability of the proposed etiologic model through the examination of three stigmatized groups. The results of the study have the potential to provide new insights into an important public health problem. PUBLIC HEALTH RELEVANCE: Because efforts to improve attitudes towards stigmatized individuals involve protracted changes over time, clinical interventions are needed for those currently experiencing mental health problems that result from stigma. This study highlights several processes including cognitive, emotion regulation and social sequelae of stigma that are modifiable through existing treatments. The results will therefore set the stage for the development of theory-based preventive interventions that seek to reduce the prevalence of psychiatric morbidity among members of various stigmatized groups. [unreadable] [unreadable] [unreadable]

Project Summary: The long-term objective of Dr. Sinn is to pursue an academic career at a major research university studying the biology and development of lentiviral vectors. This will be achieved with the aid of Drs. McCray and Voytas and the support of the Dept. of Pediatrics at the University of Iowa. The immediate interest of Dr. Sinn is the general safety and utility of lentiviral vectors for use in a broad range of gene therapy applications, such as cystic fibrosis. Gene therapy for cystic fibrosis, a disease that has pulmonary and digestive manifestations, is directly applicable to the mission of the NIDDK. To persistently expressa therapeutic transgene, a retroviral vector must integrate into a host cell chromosome. This critical process makes lentiviral vectors an attractive tool to achieve life-long gene delivery; however, the nonspecific nature of retroviral integration presents inherent hazards and variations in gene expression. If integration could be restricted to preferred genomic loci, the safety and utility of lentiviral vectors would be vastly improved. In this proposal a novel strategy is investigated to modify the integrase (IN) gene of a feline immunodeficiency virus (FIV)-based lentiviral vector to achieve restricted integration. A "tethering" model is proposed in which an engineered hybrid protein that contains a protein binding domain and a DNA binding domain will direct the lentiviral vector integration complex to preferred sites on chromosomal DNA. Three aims are proposed: 1) determine the capacity to which FIV IN can be modified and still retain its function, as determined by titering assays as well as in vitro catalytic and integrase assays; 2) confirm high affinity protein-protein interactions of modified FIV IN to the tethering protein by yeast two-hybrid assays and demonstrate that these complexes can mediate restricted integration in a cell-system. These studies will focus the ultimate goal to, 3) map integration sites of the modified vector into genomic DNA and demonstrate a restricted pattern of integration in those cells in which the tethering protein is co-expressed. Relevance: Lentiviral vectors have the potential to persistently correct genetic diseases. However, such vectors integrate nonspecifically into the host genome and therefore present a risk of disrupting normal gene function at the site of insertion. Successful site-restricted lentiviral vector integration into human genomic DNA would have exciting and broad applications in the gene therapy field.

One strategy for treating methamphetamine and similar compound addictions is the use of immunopharmacotherapeutics to elicit production of antibodies, which will bind the drugs of abuse and alter their pharmacokinetic properties in a manner that reduces or eliminates drug use. An added advantage is that immunopharmacotherapies can be used in combination with other medications to obtain an addictive or synergenic effect. This supplement to R37DA005477 involves designing, synthesizing, and coupling methamphetamine haptens to appropriate proteins (BSA, OVA, others) and to produce conjugates for use in developing monoclonal antibodies as pharmacotherapies to treat methamphetamine and other stimulant addictions.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. This project is an extension of our previous one dimension CW SRLS fitting program with dynamic exchange. It will enable a more accurate determination of the dynamic parameters related to the dynamic exchange processes, as 2D ELDOR ESR contains more spectral resolution and information than its 1D CW counterpart. We started from the MOMD version and will proceed to the SRLS version.

The long term objective of this research is to develop analogs of mitomycin C that are less toxic and more efficaceous in the treatment of cancer. Our earlier studies have emphasized the synthesis of large series of analogs that might be useful for defining structure-activity relationships. Newer directions include computer-assisted design of analogs and the use of bioactivation mechanisms to define new prodrugs. Based on a recently derived computer model for the binding of mitomycins to B-DNA, analogs will be proposed and evaluated for non-covalent binding using the computer model and the AMBER molecular mechanics program. Promising compounds will be synthesized and submitted for testing. The resulting activities will be used for linear free energy based correlations, with calculated binding energy, log P, and quinone-reduction potential as the independent variables. Nucleotide derivatives of 2,7-diaminomitosene, in which the nucleotide phosphate is substituted at C-1, will be synthesized. The nucleotides will be FdUMP, ara-CMP, 6-mercaptopurine RP, and 6-thioguanine RP. The derivatives will be evaluated for liberation of the nucleotides and DNA alkylation by the mitosene moiety on catalytic reduction. If they pass this test and they are shown to be taken into cancer cells in culture, they will be screened widely against mouse tumors, and against human tumors in cell culture. The corresponding 7-methoxy analogs also will be prepared to ensure bioreductive activation. 2,7-Diaminomitosene, which has no alkylating functionality at C- 1, will be prepared and tested by computer modeling and an experimental binding study for its ability alkylate DNA through the loss of the carbamoyloxy substituent at C-10. This theoretically important process has never been demonstrated. A new set of compounds that represent hybrid structures between mitomycins and Anderson's bis(hydroxymethylpyrrole) biscarbamates will be synthesized and tested against tumors. They are designed to make a good fit to the computer-based mitomycin binding site. Antitumor testing will receive increased emphasis in colon cancer. New analogs will be submitted to a panel of six human colon cancers in cell culture. Derivation of structure-activity relationships in this panel will be attempted.

The aim of this study is to directly measure de novo lipogenesis in lean an obese individuals using a new isotopic technique and to examine the effects f obesity and dietary macronutrient composition and caloric content on the ra of lipogenesis.

Childhood mood disorders represent a significant public health concern, with 6 to 11% of the population suffering from symptomology. Among those affected, 70% will have a recurrence during childhood and/or adolescence, and 50% will continue to experience symptoms in adulthood. These patterns of chronicity suggest that mental health problems observed later in life often have identifiable markers during early childhood. Therefore, early identification and intervention is critical. The NIH has put forth the Research Domain Criteria (RDoC), which acts as a heuristic to examine mechanisms of mental illness by suggesting the investigation of several behaviorally and biologically based processes. Although RDoC provides a valuable theoretical framework, the applicability of RDoC for developmental psychopathology research has been limited, primarily due to a lack of validated, feasible, standardized assessment batteries to examine RDoC domains in young children. Our aim is to create and validate behavioral and neurobiological assessments for young children in order to examine the relevance of RDoC domains, Positive and Negative Valence, for childhood depression and anxiety that often precedes or commingles with it. Specifically, we will study the constructs of reward valuation (Positive Valence) and acute threat (Negative Valence) using behavioral and neurophysiologic correlates of these constructs and test for associations with clinically assessed depression and anxiety in 4-6 year old children. Recruitment through the Infant and Early Childhood Clinic at the University of Michigan's Department of Psychiatry, as well as general pediatrics clinics in our health system, will ensure that a broad spectrum of anxiety and depressive symptoms, across the clinical to non-clinical range are represented in the children in our study. We seek NIH pilot funding to investigate the feasibility of behavioral and neurophysiology tasks that will long-term 1) inform assessment procedures, blending basic science advances with accessibility for patients and their families; 2) improve our characterization of underlying mechanisms of child anxiety and depression; and 3) inform subsequent interventions to address personalized child-level deficits. Findings will prove high impact as the NIMH considers the study and future application of RDoC criteria a high priority, and, yet, there are few empirical data exploring such application among young children. This proposal is responsive to the urgent need for validated, feasible, and standardized assessment batteries to examine RDoC domains in young children, and represents a critical step in a long-term plan to achieve effective and mechanism-based identification and treatment strategies for preschool-age children with depression and anxiety.

DESCRIPTION (provided by investigator): Fetal alcohol syndrome (FAS) is a leading cause of developmental disability in the Western world, and is entirely preventable. Still, 20% of women who drink continue to do so during pregnancy. Efforts at prevention have failed to access many high-risk women in time to intervene effectively, A venue frequently visited by drinking gravidas (pregnant women) is the public drinking establishment. Much damage can be prevented by intervention in the second and third trimesters when pregnancy is ordinarily visible to alcohol service personnel. Ongoing social relationships between bar staff and customers also provide opportunities for transmission of prevention information to women early in pregnancy, before pregnancy is visible. This study will design and evaluate an interactive, computer-based alcohol server-training program for the prevention of FAS and other fetal alcohol effects. The training program, on a CD-ROM, will incorporate components of demonstrated effectiveness in promoting server intervention with pregnant drinkers from a current NIAAA study, and will incorporate training issues salient to alcohol serving establishments. The program will be evaluated with 140 bar owners, managers and alcohol servers using pre- post- design to assess the impact of the program and collect both process and outcome data.

My research in the lab of Prof. Clayton will be directed toward the molecular mechanisms of mitochondrial DNA replication. To address this question, I will use the in vitro approach developed by Prof. Clayton and his collaborators. This approach is based on the fractionation of mitochondrial extracts and the development of adequate in vitro assays. Its aims are to identify the proteins required to reconstitute in vitro the various stages of mtDNA replication, as well as to characterize the cis-acting elements involved in controlling this process. The relevance of these research goals to my career goals is multiple. First, the results of my research will contribute to a better comprehension of a problem whose study I plan to pursue in Belgium. Most importantly, this research will allow me to develop a strong experience in protein purification and biochemistry of DNA replication. This gained expertise will be combined with my experience in genetics and molecular biology. Together, these will constitute very complementary, and therefore powerful approaches to address future issues in molecular biology and in particular to tackle the mechanisms of DNA replication. In addition, this new expertise will prove useful for my future teaching experience. Finally, this postdoctoral research will provide me with the opportunity to establish new contacts in my field of research, as well as to set the basis for future collaborations.

Socioeconomic disparities in cardiovascular (CV) disease are evidenced by a 2.5 times higher risk of cardiovascular mortality for low-income Americans, when compared to those in the highest income bracket. Despite well-documented awareness that social environments can influence CV disease risk, and the increasing attention given to disparities research, there is relatively little knowledge of the underlying social pathways contributing to socioeconomic disparities, and socioeconomic disparities in CV disease have only widened in the last three decades. Socioeconomic disparities exist for many CV risk behaviors, including smoking, lack of physical activity and insufficient fruit and vegetable intake, and these disparities contribute to socioeconomic gradients in CV disease. These behaviors are shaped by social environmental resource factors, including social support, loneliness and neighborhood social cohesion. These three social environmental resource measures are independently associated with CV morbidity, but their role in CV risk behaviors is not well understood. Also, interaction between social resources is likely to have a multiplier effect, where the net effect is greater than the sum of the individual effects. However, few studies utilize an ecological framework to identify the magnitude of these effects simultaneously. Furthermore, these social resource measures are often correlated with socioeconomic position (SEP), indicating that the resources may represent a fundamental social inequality. The purpose of this study is to examine the relationships among SEP and three distinct, but related, measures of social resources - social support (both emotional and instrumental), loneliness and neighborhood social cohesion - with CV risk behavior outcomes (smoking, lack of physical activity, and insufficient fruit and vegetable intake). The aims of this study are: 1) To examine associations of social support, loneliness and neighborhood social cohesion with CV risk behaviors after controlling for confounders. 2) To explore the interactions of social support, loneliness and neighborhood social cohesion as they relate to CV risk behaviors. 3) To determine the contributions of social support, loneliness and neighborhood social cohesion to SEP gradients in CV risk behaviors. This study will use cross-sectional data obtained by the Multi-Ethnic Study of Atherosclerosis, a prospective cohort study of a diverse, population-based sample of 6814 adults without apparent CVD from six US sites. Multi-level statistical modeling techniques will be used to test the proposed inter-relationships of SEP and the three social resource measures, based on an ecological framework. PUBLIC HEALTH RELEVANCE: Results of this study will elucidate the pathways that contribute to socioeconomic CV risk behavior disparities, responding to a NINR research priority area, and this knowledge may inform future research into other health disparities. This knowledge also is important for the development of socially and contextually based behavioral interventions for clinicians and researchers, meeting a critical need in health behavior research. Moreover, these findings may be used to improve patient advocacy and shape policies to address health disparities at a societal level.

Stroke is the third leading cause of death and leading single cause of disability in the United States. The estimated direct and indirect costs of stroke for 2008 are about $65 billion. It is known from prior research that early recogniton and treatment reduces the morbidity and costs associated with stroke. Thrombolysis with intravenous tissue plasminogen activator (IV t-PA) remains the only proven treatment for acute ischemic stroke who present within three hours of symptom onset with no established contraindication. However, it is also known that the rate of t-PA use in ischemic stroke patients is only 2-3%. Large scale studies have shown that poor rates of thrombolysis are due to lack of patient recognition of stroke symptoms, delay in accessing Emergency Medical Services (EMS) and in-hospital delays like lack of expedited triage.Therefore, it is well known scientifically that a multi-disciplinary approach is necessary to improve the rate of t-PA use in eligible acute stroke patients. In addition to improving knowledge on early stroke recognition in the community, the Brain Attack Coalition (BAG) determined that two levels of stroke care i.e primary and comprehensive stroke centers be created to improve emergency and other types of clinical care for stroke. With establishment of certified primary stroke centers and evidence for improved quality of care at primary stroke centers, county authorities and EMS agencies created policies to bypass the nearest emergency department and transport stroke patients to primary stroke centers. Although multiple counties are adopting the change, the impact of the policy change on regional patient outcomes has not yet been established through scientific evidence. In addition, studies on accuracy of stroke recognition by prehospital providers have shown that there is considerable scope for improvement in stroke recognition. To address these issues, I propose to conduct a study with the primary goal of studying comparative patient outcomes in regionalized and non-regionalized stroke systems. The secondary goals will be to assess the cost-effectiveness of regionalized care and accuracy of prehospital stroke recognition before and after regionalization of systems. RELEVANCE (See instructions): The study results will help to determine 1) regional differences in the rate of intravenous t-PA use and mortality among acute ischemic stroke patients 2) the impact of increasing volume in primary stroke centers and longer transport times within a regionalized system on patient outcomes.By providing evidence for patient outcomes in a regionalized stroke system.our research will provide scientific knowledge to promote health policy changes of great public health importance at a national level.