Datasets:

suolyer

/

pile_nih-exporter

like 0

In heterogametic species there is an imbalance in the number of sex-linked genes between males and females. Therefore, it is essential that transcription is equalized between the sexes and between the sex chromosomes and autosomes, a process called dosage compensation. Though different dosage compensation mechanisms have evolved, they all share a common initial step of distinguishing the X-chromosome from the autosomes. The model organism Drosophila melanogaster has evolved a single-step mechanism for dosage compensation, providing a simple model for studying how a single chromosome is discriminated from the rest of the genome. In Drosophila, selective two-fold upregulation of the single male X-chromosome equalizes transcription between the sexes. The selective identification of the X-chromosome involves the recruitment of a ribonucleoprotein complex, Male-Specific Lethal (MSL) that is expressed only in males. The MSL complex initially localizes to GA-rich sequences within seed sites on the X-chromosome, termed MSL Recognition Elements (MREs). However, MSL complex cannot bind directly to MREs and these sequences are distributed throughout the genome. The Larschan laboratory has recently identified a previously unstudied zinc finger protein, Chromatin-Linked Adapter for MSL Proteins (CLAMP), that links the MSL complex to MRE sequences. Furthermore, CLAMP is highly enriched on the X-chromosome independent of MSL complex and therefore is likely to be involved in the earliest step of X-identification. Therefore, the identification of CLAMP provides the first opportunity to define the X-chromosome features that promote its identification for dosage compensation. Based on strong preliminary data, I hypothesize that identification of the X-chromosome requires contributions from primary genome sequence and higher-order nuclear organization mediated by the CLAMP protein. I will test my hypothesis by defining the role of linear and three-dimensional genomic organization in enrichment of CLAMP on the X-chromosome. First, using Chromatin Immunoprecipitation- qPCR (ChIP-qPCR), I will determine whether enrichment of CLAMP on the X-chromosome occurs cooperatively or additively as the number of tandem MRE sequences increases. Second, I will determine if putative initial seed sites cluster together in three-dimensional nuclear space using the Chromatin Interaction Analysis by Paired-End Tag Sequencing (ChIA-pet) method. The proposed experiments will use state-of-the- art approaches to provide key insight into how initial identification of the X-chromosome is achieved.

Transgenic mice containing the HTLV-I tax gene develop tumors of peripheral nerve sheaths,iris, adrenal medulla and salivary gland, myopathy and localized lymph node hyperplasias. The mechanisms leading to these abnormalities remain unclear, but may involve the deregulation of cellular genes important for cell growth and differentiation. The expression of tax in these transgenic mice has been correlated with the induction of the genes for the interleukin-2 receptor (IL-2-R), the granulocyte-macrophage, colony-stimulating factor (GM-CSF), and more recently, nerve growth factor (NGF). Cell lines from the peripheral nerve and salivary tumors appear to secrete significant quantities of IL-6 and perhaps other B-cell-related growth factors, which may account for the lymphadenopathy associated with these tumors. This transgenic mouse system provides an important in vivo model for HTLV-I-induced transformation and tax activation of growth-regulating genes.

This application is submitted in response to RFA DK-06-504 to continue the efforts of the Inflammatory Bowel Disease Genetics Consortium and our role as a Genetic Research (GRC) with the central goal of identifying susceptibility genes contributing to the pathogenesis of inflammatory bowel disease (IBD). This will be accomplished through the following specific aims. Specific Aim 1: Expansion, Development and Management of Consortium Resources. Our GRC will recruit additional IBD cases, controls, and parents with a focus on ulcerative colitis and early onset cases. Extensive clinical data will be collected on recruited subjects and biospecimens (EBV-transformed lymphoblastoid cell lines, DNA, serum, whole blood) will be ascertained and stored at the NIDDK Genetics Repository for use by the Consortium, individual GRCs and outside investigators. Specific Aim 2: To employ a variety of approaches to identify genetic variation that contributes to IBD susceptibility. Our GRC will be involved in the follow-up of European ancestry genome-wide association study results. A particular initial focus of our GRC will be on a) integrating the suggestive linkage data and genome-wide association data in the IBD2 linkage region followed by additional association mapping within IBD2, and b) a resequencing, detailed haplotype and additional association study of the IL23R gene region. Specific Aim 3: To build a risk model of IBD through understanding genetic influence on variations in phenotypic expressivity, gene pathway analysis, and gene-gene (G x G) and gene-environmental (G x E) interactions. A particular initial focus of our GRC will be on gene-gene interaction analyses and pathway analyses of genes along the IL23R pathway. Our GRC may also take on other projects as genes/genomic regions advance to confirmed evidence for contribution to disease through follow-up of the Consortium's genome-wide association data. IBD is a chronic inflammatory disease of the gastrointestinal tract which primarily affects young people and is characterized by long-term illness and the need for potent medical therapy and substantial surgical therapy. The work of the IBD Genetics Consortium will enable us to identify important predisposing and disease modifying genes contributing to the pathogenesis of IBD which has the promise to: (1) identify persons at risk for disease, (2) predict disease course, (3) aid in selection of treatment, (4) understand pathophysiologic mechanisms such that novel preventive and therapeutic interventions can be developed. Advances in IBD gene identification and methodologic approaches may also be applicable to other complex genetic disorders.

ABSTRACT Two of the major problems in attaining euglycemia in subjects with type 1 diabetes (T1D) are hypoglycemia (including fear of severe hypoglycemia) and post-meal hyperglycemia. The latter commonly occurs in adolescents secondary to missed meal and snack insulin boluses. Both of these issues will be addressed over the five years of this grant. As over half of severe hypoglycemic events occur during sleep, we will continue our development of algorithms to predict and prevent nocturnal hypoglycemia using a closed loop system (CLS) in which the information from a continuous glucose monitor (CGM) is used to suspend delivery of insulin by an insulin pump. Initial studies will be done in the Clinical Translational Research Centers (CTRCs) to document safety of the Florence CLS (Abbott Diabetes Care) and we will then conduct a randomized clinical trial in the home environment in years 2 and 3 of the grant. Similarly, a fully portable CLS will be used to detect and correct hyperglycemia resulting from missed food boluses. The safety and efficacy of algorithms which utilize the signal form a CGM and provide output to an insulin pump will initially be studied in the CTRC's in year 3 of the grant. In years 4 and 5 of this grant we plan to assess the combined algorithms for prevention of hypoglycemia and hyperglycemia in a fully ambulatory system developed by Abbott Diabetes Care. We believe that prevention of hypoglycemia, particularly at night, and reduction of post prandial hyperglycemia will result in improvement in glycemic control and reduction in HbA1c levels.

Project Summary The overall objective of the Administration (Admin) Core efforts is to provide a robust and dynamic infrastructure for the Center of Excellence for Encephalitic Alphavirus Therapeutics that enables consistent and notable progress through the milestones. Led by the Program Director, Dr. Jonsson (UTHSC, University of Tennessee Health Science Center), the Core will be the main interface between the Scientific Advisory Committee (SAC), the Research Project PIs, and NIAID. To support the achievement of the aims and milestones of the Center, Dr. Jonsson will be supported by Jayne McKinnie (Admin Core Associate) to: (1) ensure coordination and dissemination of information across the research projects, (2) coordinate results required for filing intellectual property, (3) publish results of disclosed leads and provide novel resources to the scientific community, (4) coordinate input into the refinement of the product development plan outlined in the Overall section with our University partners and SAC, (4) attract commercial partners for future SBIR or similar proposal efforts, and (5) administratively support the Center in collaboration with our regulatory consultants to prepare for our meetings with the FDA at the end of year 3 and at the start of year 5. Specific aims of the Admin Core will be to: Aim 1. Organize, manage, and document Center communications and findings; Aim 2. Manage and ensure fiscal responsibility and regulatory compliance; and Aim 3. Manage lead prioritization and documentation, the refinement of the Product Development Plan and a path to Commercialization.

[unreadable] While much progress has been made to control preventable infectious diseases, infections remain a major cause of morbidity and mortality. Many of the traditional treatments for common infections are no longer effective because of the fast-growing problem of antimicrobial resistance, first associated with hospitals but increasingly widespread in the community. Antibiotic resistance is now a global problem of major concern. The ultimate goal of this proposed Center for Interdisciplinary Research on Antimicrobial Resistance (CIRAR) is to develop strategies to implement and evaluate a long term collaborative program of interdisciplinary research on reducing antimicrobial resistance. The specific aims of the CIRAR are: (1) To plan, implement, and evaluate outcomes of a core program and curriculum to prepare medical researchers to conduct interdisciplinary research; (2) To develop the expertise of CIRAR investigators in interdisciplinary research; (3) To conduct and evaluate outcomes of interdisciplinary demonstration projects related to the prevention and control of antimicrobial resistance; (4) To implement dissemination and implementation strategies that maximize the sustainability and expansion of CIRAR's interdisciplinary research; and (5) To formalize plans for a sustainable Interdisciplinary Research Consortium for Antimicrobial Resistance. The first component of the Center is the development and implementation of formal training for conducting interdisciplinary research which will be incorporated into several ongoing programs within the institution and made available by internet. In Years 2 and 3 the interdisciplinary research curriculum will be further disseminated. The second CIRAR component is the development of interdisciplinary demonstration projects designed to rationalize antimicrobial use and reduce antimicrobial resistance. In the final year, plans for a sustainable Consortium on antimicrobial resistance will be formalized. [unreadable] [unreadable]

DESCRIPTION:(provided by Applicant) Antibody Production by B cells employs 3 separate types of genetic alterations. The variable region exon of immunoglobulin (Ig) heavy (IgH) and light (IgL) chain genes is assembled from germline V, D, and J segments during early B cell development. V(D)J recombination is initiated by the RAG-1/2 endonuclease, which introduces DNA double strand breaks (DSBs) at participating V, D, and J segments, and is completed by non-homologous end-joining (NHEJ) proteins that seal the RAG-generated DSBs. In response to antigen, mature B cells change the IgH constant region (CH) expressed with a particular variable region via a distinct recombination process termed class switch recombination (CSR). At this stage, the variable region coding sequence may be further altered by somatic hypermutation (SM). While the nature of the enzymes that initiate CSR or SM remains elusive, both processes, like V(D)J recombination, may involve DSBs and thus, employ NHEJ. We have shown that defective NHEJ during attempted V(D)J recombination in a p53 tumor-suppressor deficient background reproducibly leads to murine pro-B cell lymphomas that harbor translocations between the IgH and c-myc loci. We now propose to elucidate further the role of NHEJ proteins in CSR and SM, with a particular focus on how defects in these processes, and in V(D)J recombination, may lead to the generation of chromosomal translocations that contribute to the generation and maintenance of mature B cell lymphomas. To focus potential oncogenic mutations on mature, as opposed to progenitor B cells, we will employ several strategies, including conditional gene targeted mutation to inactivate NHEJ or activate specific oncogenes in mature B cells. Also, we will insert V(D)J recombination signal sequences, CSR target sequences (S regions), expressed variable region gene sequences, or meganuclease restriction enzyme (I-Sce 1) target sequences adjacent to the c-myc, bcl-2 or bcl-6 genes within various NHEJ or checkpoint-deficient ES cells to test for ability to stimulate translocations in appropriate B lineage cell populations. To eliminate large-scale breedings and facilitate rapid assays, we will introduce certain compound mutations into ES cells and assay effects on translocations and lymphomagenesis via Rag-2 Deficient Blastocyst Complementation. Once desired mature B cell lymphoma models are developed, we will employ gene-targeted mutations to determine the role of IgH expression, IgH enhancers, and particular oncogenes in the generation and maintenance of the transformed phenotype.

The research outlined in this proposal is designed to provide a basic understanding of the role of persisting antigen in the regulation and maintenance of the immune response. In view of difficulties involved in analyzing interactions between persisting antigen, antibody and the various cell types involved in the immune response in vivo, in vitro model systems have been developed which are amenable to manipulation and yet accurately reproduce phenomena observed in vivo. The aggregated human serum albumin (HSA) antigen used in this system appears to persist in rabbits for at least a year, and in the absence of anti-HSA antibody, it is capable of inducing a secondary response. The data obtained to date suggest that circulating antibody levels are maintained and regulated in vivo by an antibody feedback system. The feedback system prevents persisting antigen from inducing antibody synthesis when circulating levels are high. However, when antibody levels fall below a critical level, the antigen is exposed and stimulates HSA reactive memory cells to synthesize antibody and to produce additional memory cells. The data to be obtained from the proposed research should provide a basis for critically evaluating this mechanism for maintaining circulating antibody levels and immunological memory. Persisting antigen appears to be retained on follicular dendritic cells in the draining lymph nodes of both mice and rabbits. We plan to compare the ability of mouse dendritic cells with typical mouse macrophages in their ability to stimulate B-cells and cooperate with mouse T-cells.

A model system has been developed to study retroviral oncogenesis, and the genetic factors regulating susceptibility to tumor induction. Avian leukosis virus (ALV) induces bursal lymphoma in chickens, after integration of viral long terminal repeat (LTR) sequences next to the c-myc proto- oncogene. Labile or short-lived proteins appear to regulate c-myc hyperexpression, because LTR-enhanced transcription is specifically decreased after inhibition of protein synthesis. This lability is observed only in pre-B cell types, suggesting that it may be important in the B cell specificity of ALV tumor induction. Moreover, lymphoma-resistant chicken strains exhibit stable LTR-enhanced transcription (unaffected by inhibition of protein synthesis), suggesting that labile regulation of LTR-enhanced c- myc transcription may be important in lymphoma susceptibility. Five LTR binding proteins from bursal lymphoma cells have been characterized using the gel shift assay. Three proteins are specifically labile in pre-B cells (decreased binding after protein synthesis inhibition) and thus may be essential for LTR enhancement. An in vivo footprinting technique will be used to determine if labile protein binding also occurs in vivo. The contribution of each protein binding sequence to LTR enhancement will be analyzed by transfection of wild type or mutant LTR-reporter gene constructs into bursal lymphoma cells. The genes encoding the major labile proteins are being cloned and sequenced, to obtain DNA and antibody probes for analysis of the role of these proteins in lymphomagenesis. Labile protein expression will be compared in different cell types, to determine why it is specifically labile in pre-B cells. Western and Northern blot analyses will determine if this lability is regulated by changes in protein half life or by post- translational modifications such as phosphorylation. Binding protein expression will be analyzed during B cell development, by Western blotting and immunofluorescent staining, to determine if the proteins are labile in ALV target pre-B cells, and stable in mature B cells no longer susceptible to lymphoma. Protein expression will be compared in lymphoma-susceptible and -resistant strains, to determine how stable expression could be involved in resistance to lymphoma. These studies will give insight to the regulation of c-myc hyperexpression during tumor induction, and the role of the labile LTR binding proteins in tumor susceptibility. An understanding of the mechanism of ALV lymphoma susceptibility can be applied to studies of oncogene activation and lymphomagenesis in humans.

Microbial calcification has proven to be a useful model for studying biologic mineral formation. The mineral species formed by Bacterionema matruchotii is crystallographically indistinguishable from biologic apatite of bones and teeth. The calcification nucleator of B. matruchotii has been identified as proteolipid: basic, hydrophobic protein complexed with acidic phospholipid. Calcifiable proteolipid has been isolated from marmoset bone and human dental calculus. A within-strain comparison of B. matruchotii and a variant exhibiting impaired calcifiability has provided insight into cellular regulation of calcification. A record of the process of mineral formation in the organism provides a base line for further studies. The purpose of the proposed research is to define the mechanism of intracellular apatite deposition in B. matruchotii through continued characterization of the nucleating entity, metabolic studies and examination of the process of apatite formation. Cellular regulation of calcification will be investigated by examining the interaction of calcifiable proteolipid with cell membrane components and through comparative studies with other mircoorganisms. Methodology developed for the microbial system can then be applied to vertebrate mineral formation.

One in two hundred children is born with limb deformities. An under- standing of the teleology of these defects, whether induced by taratogens, caused from a stochastic dysfunction of the developmental program, or the result of congenital defects, will require an under- standing of the normal process of limb development. Many of the genes that are suspected of mediating the formation of the limbs are now being identified. Particularly prominent among these genes is the set of Hox genes that may be required to specify the positional information needed for the establishment of the correct set of limb cartilages and bones. Gene targeting in mouse embryo-derived stem cells provides the means for generating mice with disruptions in each of these genes. The goal of this grant is to use gene targeting to undertake a systematic analysis of this gene complex and thereby directly establish the role of each of these genes in limb development. Not only will the phenotypic consequences of disrupting any particular gene be determined, but also through epistasis and molecular genetic analysis, it is hoped that insight will be gained as to how this set of genes functions as a network to determine the overall three-dimensional structure of the limb.

The proposal is to study the association, if any, that exists between oral contraceptive usage and increased risk of stroke among married women of childbearing age in a developing country. The study will be undertaken with the epidemiological case-control approach. Taiwan is selected as the study site because of her excellent household registration and vital statistics system and medical care services. The association between the use of hormonal contraceptives and thromboembolic diseases of the heart, lung and brain has now been fairly well established on a number of epidemiological studies conducted mainly in the United Kingdom and United States. Similar studies to confirm, or deny, such association is much needed in the developing countries where the genetic background, life style, and dietary habits of people are different. The family planning policy implication of the proposed study is self-evident. In the coming (second) year, approximately 200 stroke patients, 400 hospital controls, and 400 matched healthy neighborhood controls will be additionally interviewed to bring up the total sample size for the entire study to 300, 600, and 600 respectively in these categories.

This is a proposal to investigate the function and regulation of 20-hydroxyeicosatetraenoic acid (20- HETE) synthesis in the vasculature, more specifically, its participation in endothelium dysfunction in models of increased vascular expression of cytochrome P450 (CYP) 4A. 20-HETE is a primary eicosanoid in the microcirculation where it participates in the regulation of vascular tone. In rat renal arteries, CYP4A expression and 20-HETE production increased with decreased arterial diameter. CYP4A overexpression in small arteries and arterioles increased vascular reactivity and myogenic tone. Recent studies and preliminary results suggest that the endothelium is a target for 20-HETE bioactions. Smooth muscle-specific CYP4A1 expression via Ad-SM22-4A1 induces a marked CYP4A-dependent and 20-HETE-mediated endothelial sprouting in renal arterial microvessels. In vitro, 20-HETE is a potent angiogenic factor stimulating capillary-like tube formation of endothelial cells by a mechanism that may include MAPK activation and induction of inflammatory and angiogenic proteins (IL-8 and VEGF). In vivo, intravenous injection of Adv-CYP4A2 causes hypertension and renal arteries from these rats display endothelial dysfunction, which can be reversed by inhibition of CYP4A activity. Arteries from Adv-CYP4A2-transduced rats produce more 20-HETE and less NO;they also express higher levels of inflammatory proteins (ICAM and VCAM). These findings raise the possibility that vascular 20-HETE is an important determinant of endothelial dysfunction, a condition that is characterized by decreased NO bioavailability and enhanced endothelial activation, and are the basis for the proposal's hypothesis: Vascular overexpression of CYP4A fosters prohypertensive mechanisms via increased production of 20-HETE in a manner that may include endothelial dysfunction and activation. This hypothesis will be tested by 1) determining the relationship between hypertension, endothelial dysfunction and activation, CYP4A expression and 20-HETE synthesis;2) determining whether the functional consequences of increased vascular expression of CYP4A is associated with endothelial expression and synthesis of CYP4A and 20-HETE, respectively;and 3) exploring mechanisms underlying 20-HETE mediated endothelial dysfunction and activation.

We are better defining the natural history of Ataxia-Telangiectasia (A-T). 1) We have developed a clinical scoring system and applied that system to a cross-sectional cohort of A-T patients. Among patients less than 18 years of age there is a clear correlation between age and cumulative A-T score. 2) We have noted that there is a lengthy delay between agerage age at which neurologic systoms appear and average age of diagnosis. We have analyzed the reasons for the delay and its consequences. 3) We are surveying all patients for the presence of opportunistic infections and complications of immunization. 4) We have noted significant nutritional deficiencies in a sub-group of patients and are looking for correlations with neurologic dysfunction. 5) We have noted little or no correlation between severity of pulmonary disease and presence of immunodeficiency. Instead, there appears to be correlation between pulmonary disease and problems with swallowing.

SUMMARY Inhibition of intestinal bile acid absorption has been shown to reduce plasma cholesterol. Although, bile acid sequestrants are clinically shown to lower cholesterol, the development of better therapeutic modalities to directly inhibit bile acid absorption is necessary. Apical Sodium Dependent Bile Acid Transporter (ASBT) absorbs majority of the bile acids in the ileum and is essential for maintaining circulating bile acid pool. Therefore, ASBT inhibition represents an attractive therapeutic target for lowering plasma cholesterol. In this regard, recent studies from our laboratory demonstrated that ASBT is inhibited by signaling intermediates including protein tyrosine phosphatases (PTPases) via membrane recycling events. Also beneficial dietary components such as green tea catechin, (-)-epigallocatechin-3-gallate, EGCG, inhibits ASBT function in a lipid-raft dependent manner. We hypothesize that phosphorylation/dephosphorylation processes, lipid raft-dependent mechanisms and membrane trafficking events play critical roles in the inhibition of ASBT function. Hence, a comprehensive understanding of these inhibitory pathways is crucial to exploit their utilization as an effective therapy for hypercholesterolemia associated with diabetes mellitus. Our preliminary studies showed that ASBT function and expression are upregulated in rat model of streptozotocin (STZ)- induced diabetes mellitus. This in vivo diabetes model will provide an exceptional tool to investigate the underlying mechanisms of ASBT upregulation in diabetes mellitus as well as determining the impact of ASBT inhibition on associated hypercholesterolemia. Our studies are designed to systematically delineate the cellular and molecular mechanisms inhibiting ASBT utilizing in vitro models and examine their dysregulation in vivo utilizing diabetes mellitus rat model. In Specific Aim 1, we will investigate the regulation of ASBT function and phosphorylation by protein phosphatases (PPase) in cell culture models. In Specific Aim 2, our studies will focus on elucidating the inhibitory mechanisms of ASBT function by membrane recycling events and lipid rafts as well as delineating the molecular basis for EGCG-mediated inhibition. Studies designed for Specific Aim 3 will focus on investigating mechanisms underlying ASBT upregulation in rat model of STZ-induced diabetes mellitus and determine the efficacy of the beneficial dietary compound EGCG and specific ASBT inhibitors (developed by Biotechnology Company Albireo) in lowering the levels of plasma cholesterol. Our proposed studies are critical for providing novel insights into the regulation of ASBT under normal and pathophysiological conditions and may provide better strategies for the management of hypercholesterolemia associated with several disorders.

The Emory Consortium for Innovative AIDS Research in Nonhuman Primates aims to understand the B and T Cell Biology of Protection from and Eradication of SIV/SHIV Infection. The consortium brings together an interdisciplinary mix of highly collaborative, and productive investigators in a range of HIV vaccine and cure disciplines to address the overarching hypothesis that a successful prophylactic HIV vaccine will require a strong and sustained systemic and mucosal immune response, comprised of functionally targeted antibody and tissue resident CD8 T cell immunity, in concert with a modulated HIV-specific CD4 T cell response that maintains low numbers of HIV targets in mucosal tissue, while providing adequate help for a strong adaptive response. Moreover, we postulate that such a potent and balanced vaccine response will, in the context of active latency reversing agents, reduce viral reservoirs and thus maintain suppression of virus replication following cessation of highly active antiretroviral therapy. The approaches in FOCUS 1, aimed at understanding the mechanisms of vaccine protection, will utilize state of the art adjuvants coupled with native trimeric Env immunogens to induce and mechanistically dissect strong durable humoral responses. In addition, we aim to fully characterize and harness a novel population of tissue resident CD8 T cells to effectively synergize with the humoral immune response in providing protection from SHIV challenge. Because recent data suggest that SIV/HIV specific mucosal CD4 T cells could enhance infection, we will also explore the potential for modulating the susceptibility of these cells to enhance protection. In FOCUS 2, aimed at defining mechanisms of reservoir eradication, we will utilize novel latency reversing agents and immunostimulants to define an optimal viral reactivation program, and then will combine these with the optimized vaccine approaches defined in FOCUS 1 to explore the potential of this combination to yield a sustained suppression of virus replication following withdrawal of highly active antiretroviral therapy. These experimental approaches will be supported by an effective Operations and Management Support component and five state of the art Scientific Research Support Cores in order to fully characterize the magnitude, function, specificity and repertoire of the humoral response. Single cell analytics and transcriptomics will also support characterization of innate and adaptive signals at the cellular level.

Newborn and juvenile eyes have an emmetropization mechanism that uses visual signals to precisely adjust the axial length of the eyes to the optical power, so that images are in sharp focus. However, nearly 30% of the U.S. population develops axial myopia, in which the eyes are elongated relative to the focal plane. Although glasses, contact lenses and refractive surgery can provide an optical correction, the increased axial length of the eye significantly raises the risk of retinal detachment, choroidal neovascularization and glaucoma, making myopia the 7th leading cause of blindness in the U.S. The emmetropization mechanism has at least three main components: 1) the retina, which responds to the sign and amount of defocus (or other visual cues); 2) a signaling cascade that originates in the retina, travels through the retinal pigment epithelium (RPE) and choroid, and reaches the sclera (the fibrous outer coat of the eye); and 3) fibroblasts in the sclera which respond to the signaling cascade by regulating the axial length. Our working hypothesis is that retinal responses to visual stimuli control the remodeling of the scleral extracellular matrix and, in particular, the slippage of the layers (lamellae) of the sclera across each other. This, in turn, controls scleral extensibility, axial elongation, and refractive state. The proposed studies will examine the retinal responses to visual stimuli and the scleral responses to the signaling cascade. In the previous project period, we identified two novel dissociation paradigms in which similar visual stimuli produce different scleral responses due to the prior history of the eye (somewhat longer or shorter than normal, an eye-size factor). Specific Aims 1 and 2 will use these dissociative paradigms to test our hypothesis that the source of this variability is localized in the patterns of gene expression in the sclera, rather than in the retina, and to identify the specific scleral signaling pathways that are involved. Specific Aim 3 will use an association paradigm in which three different methods (darkness, form deprivation, and minus-lens wear) will be used to induce myopia to identify the pattern of retinal gene expression that is common to all three and contributes to the retina go response. The proposed studies will apply proteomic tools and our Pathway-focused PCR Assay that have been developed in this laboratory to assess retinal mRNA and protein-expression patterns that characterize retinal go and retina stop responses, and scleral mRNA and protein-expression patterns that characterize the sclera go, sclera stop and sclera ignores responses in our well-characterized animal model, tree shrew. These animals are closely related to primates and, like humans, have an all-fibrous sclera. These studies will enhance our understanding the emmetropization mechanism at the level of gene expression and may lead to successful optical or pharmacological interventions to slow or prevent myopia. PUBLIC HEALTH RELEVANCE Myopia (nearsightedness) affects over 25% of the U.S. population and perhaps one billion people worldwide. In addition to the cost (over $14 billion annually) of eye exams, glasses, contact lenses, and refractive surgery, even low amounts of myopia raise the risk of developing blinding conditions. This application will identify genes and gene products that control the axial length of the eye as a step toward finding ways to prevent or minimize myopia in children.

The long term objective of this proposal is to determine the underlying pathophysiology that leads to remarkably divergent cellular events in alpha vs. Beta thalassemia. Accelerated apoptosis or programmed cell death (PCD) is most likely responsible for intramedullary hemolysis in both human and murine beta thalassemia. Integrally associated with PCD, is the very specific alteration in the asymmetry of the phospholipid bilayer with an outward movement of phosphatidylserine (PS). The mechanism by which PS is exposed and recognized on thalassemic red cells or red cell precursors, rendering them ~edible~ to macrophages, is not clear. The principal objectives of this proposal are to define the contribution of enhanced PCD to ineffective erythropoiesis in beta thalassemia and further our understanding of the mechanism(s) responsible for enhanced PCD. In particular, we will discover how and why PCD leads to alterations in the phospholipid bilayer, resulting in PS exposure at the surface of affected cells, and how this in turn acts either alone or in concert with other signaling systems to lead to the recognition and removal of the affected erythroid precursors. To pursue these goals, we propose the following three specific aims: 1. Investigate programmed cell death in murine thalassemia, 2. Investigate the mechanisms responsible for the movement of PS from the inner to outer monolayer and 3. Determine the factors that render a cell with PS on its outer surface recognizable and removable. The murine alpha and beta thalassemic models we have created provide us with mice with graded degrees of clinical severity necessary to test the proposed hypotheses. We will use fluorescently labeled annexin V to quantitate, isolate and study the biochemical and functional characteristics of subpopulations of cells with PS on their surface, define the PCD driven mechanism for PS exposure, and identify factors modulating macrophagic recognition. We will explore our hypothesis that the heme/hemichromes associated with the globin chains mediate oxidant attack and play a role in PCD and macrophagic attack on apoptotic erythroid precursors. The successful accomplishment of these objectives should enable us to develop a detailed mechanistic understanding of the pathophysiology of an import human disease. The insights generated are also likely to further our understanding of the loss of PS asymmetry and the role of oxidative damage in programmed cell death.

The goal of newborn screening is to detect potentially fatal or disabling conditions in newborns, thereby providing a window of opportunity for early treatment, often while the child is still asymptomatic. Such early detection and treatment can have a profound impact on the clinical severity of the condition in the affected child. If left undiagnosed and untreated, the consequences of the targeted disorders can be dire, many causing irreversible neurological damage, intellectual, developmental and physical disabilities, and even death. In 2006, the American College of Medical Genetics (ACMG) developed newborn screening guidelines that recommend that all newborn infants be screened for 31 core conditions and that 26 secondary conditions identified during the core evaluations be reported. These recommendations have been accepted by the HHS Secretary's Advisory Committee on Heritable Disorders in Newborns and Children (ACHDNC) (authorized by the Children's Health Act of 2000), and by the Secretary of HHS. Most states now use this or very similar panels for newborn screening. Currently, there are thousands of rare disorders that have been identified and hundreds that could potentially benefit from newborn screening. MPSI is a rare, inherited disease of metabolism in which an individual lacks an enzyme called lysosomal alpha-L-iduronidase. Without the specific enzyme individuals cannot break down long chains of sugar molecules called glycosaminoglycans. The glycosaminglycans build up and can damage organs, including the heart. The severe form of MPS1 is known as Hurler syndrome in which death occurs in early childhood. Attenuated forms (Huler/Scheie and Scheie syndromes) have a later onset in which death occurs either in the teens or twenties. Hurler syndrome is the predominant form of the disease (75-80% of patients).

Anti-neutrophil cytoplasmic autoantibodies (ANCA) were first described in 1982. We reported the association of ANCA with necrotizing glomerulonephritis (GN) in 1988 and recognized myeloperoxidase (MPO)-ANCA. Since then, substantial insight into a number of issues has been gained including: 1) separation of ANCA antigens into MPO-ANCA or proteinase 3 (PR3)-ANCA; 2) clinical and pathological associations of ANCA with necrotizing GN and small vessel vasculitis (SVV); and 3) that ANCA participate in the pathogenesis of vascular inflammation. Despite these strides by the ANCA community little is known about the derivation of the human ANCA autoimmune response or the precise antigenic epitopes important in eliciting an autoimmune response during disease onset or during disease relapse. This project has three specific aims. The first aim studies the human PR3 and MPO-ANCA immunoglobulin gene expression. We hypothesize that MPO-ANCA and PR3-ANCA expression is dependent on specific light and heavy chain usage and that both MPO and PR3 are selecting antigens in the maturation of this autoimmune response. We developed a technique to amplify message from single B cells, allowing the study of somatic mutations of the amplified immunoglobulin genes. In the second aim, we will study specific epitopes on MPO and PR3 during disease onset and during relapse of ANCA-GN. Is the fine specificity of epitope usage during disease onset and relapse the same? We have clone fragments of MPO and PR3 into expression vectors to perform these studies. The third aim is the most exciting and may provide a new general understanding of autoimmunity and of the ANCA immune response in particular. It is known that peptides translated from the anti-sense strand of DNA bind to sense proteins with substantial selectivity and affinity. This concept is formulated as the molecular recognition theory. We have developed exciting preliminary data to support the novel concept that ANCA react not only with PR3, but also with complementary peptides to PR3. Is the immune response to the complementary peptide part of the autoimmune response? Are PR3-ANCA and antibodies to complementary peptides examples of an idiotypic anti-idiotypic relationship? We will have all the reagents necessary to explore this paradigm. The project will dissect human ANCA and ANCA antigens during disease onset and during disease relapse. We will ascertain whether the molecular recognition theory pertains to the human ANCA immune response in that ANCA may react, not only to PR3 and MPO, but to peptides complementary to these ANCA antigens.

The principal aim of this project is to elucidate the molecular mechanism of active transport in bacterial organisms. In the simple fermentative organism, Streptococcus faecalis it is known that cellular accumulation of K ion and certain amino acids is coupled to ATP hydrolysis by an ATPase associated with the plasma membrane. In order to understand the coupling mechanism we are attempting to characterize the protein subunits in the ATPase and in the membrane sector with which the enzyme is associated. More particularly we are trying to isolate the protein components that are part of the "proton-channel" envisaged by the chemiosmotic theory. For this purpose we will use an isolated ATPase-membrane sector complex which is sensitive to the energy transfer inhibitor, dicyclohexylcarbodiimide. We are also trying to characterize a small peptide segment in the ATPase molecule which is involved in the attachment of the enzyme to the plasma membrane. This segment is selectively cleaved from the alpha chains by limited treatment of the isolated ATPase with chymotrypsin.

PLANNING AND EVALUATION (P&E) CORE The purpose of the Planning and Evaluation Core (P&E Core) is to monitor the progress toward the implementation of goals and objectives of the Partnership. Planning and evaluation are the ultimate responsibilities of the Pis, and Drs. Jaime Matta (PSM) and W. Jack Pledger (MCC) will lead this core and will be responsible for monitoring progress through ongoing evaluation processes, maximizing resources, identifying novel directions for the Partnership, and reporting progress to institutional leaders and the NCl. The P&E Core includes the Internal Advisory Committee (lAC, P&E Table 1) and the Program Steering Committee (PSC, P&E .Table 4). The lAC is responsible for internal planning and evaluation (P&E Core section 1) and new project selection (P&E Core section 2). The PSC provides external review and evaluation (P&E Core section 3). The decisions made by this core will be guided by data systematically accumulated and organized by the Evaluation Process Subcommittee (EPS) (membership below). This subcommittee is a unique strength of this core as it is led by an evaluation and process expert, Dr. Susan McMillan. Dr. McMillan is the Thompson Professor of Oncology Nursing at the University of South Florida, has training in evaluation and measurement, and has served as an evaluation consultant for several federal grants over the past decades. She is external to both PSM and MCC and as such will be able to objectively direct this subcommittee to produce formal systematic evaluations.

The processes within the cochlea which enable it to achieve its high sensitivity and sharp frequency selectivity are still largely unknown. The primary goal of the research proposed here is to improve our understanding of cochlear function by developing a realistic mathematical model of cochlear mechanics. This model development will involve the formulation of hypotheses concerning cochlear function which will be tested by comparing numerical solutions of the model with experimental observations published by other researchers. Among the major objectives are: 1) an investigation of the possible role that active elements within the cochlea may play in boosting the sensitivity and frequency selectivity and frequency selectivity of basilar membrane displacements; and 2) a description of the changes in model parameters that correlate with common hearing impairments or normal developmental gradients. A secondary goal of the proposed research is the development of computer algorithms which simulate the frequency analysis of the cochlea and are fast enough to be considered for use in applications such as speech spectrograms or cochlear implant prostheses.

The long term objective of this proposed research is to develop a better encapsulant which not only protects lead-pigmented paint, but also detoxifies the lead. This liquid component is intended to combat lead poisoning, the leading environmental childhood disease and provide a safer workplace for lead abatement workers. This would be the first lead abatement product that actually detoxifies lead in case of ingestion and is a safe water base formula. The proposed liquid may be used in several commercial products such as: 1) coatings which would be applied as an interim step before stripping or repairs to protect workers during removal, 2) washing solutions, 3) encapsulants, 4) treatments of lead waste and 5) fogging solutions. Preliminary testing indicates that this liquid formula detoxifies lead by 50%. Phase I research is needed to improve the detoxification levels of the initial formula, analyze the feasibility of incorporating calcium compounds into a final encapsulant product and confirm preliminary results via independent products. Phase II research is necessary to confirm long term effectiveness, simulate actual extreme environmental conditions and formulate optimal mechanical characteristics to meet proposed ASTM methods for encapsulant products. PROPOSED COMMERCIAL APPLICATION: ACT-LAC has shown the potential to detoxify lead pigmented paint and its water base formula is non- -toxic. The ACT-LAC formula may be incorporated into A) a lead safe barrier B) encapsulant C) post washing solution D) lead waste treatment and E) fogging solutions.

Research on medical decision-making makes use of insights from economics and statistics to build quantitative models of clinical decisions. These models rely on assumptions about the use of information by decision-makers, including the core assumption that people process information in a narrowly logical manner. Recently, the psychology of decision-makers, including their emotional states, is being incorporated into these models to augment the role of cognition. Research on older adults differs importantly from other populations in a number of ways that may impact medical decision-making, especially the role of emotions. Pursuing work begun with his dissertation, William Dale, MD, PhD, will enhance his interdisciplinary training in geriatrics and public policy by focusing on a deeper understanding of emotions in medical decision-making in older adults. This research agenda initially utilizes the clinical model of prostate cancer, a widely prevalent disease in older men, and one in which the role of the emotion of anxiety may influence decision-making. Although no consensus exists on the "right" clinical pathway for detecting and treating prostate cancer, there is wide agreement that patient preferences are vital for making choices. Initial investigations indicate the inclusion of anxiety in utility assessments for men with localized prostate cancer dramatically affect the cost-effectiveness ratios for treatments. Further research to assess the contribution of anxiety to utility assessments for these men is on-going. This study serves as a specific core example of the overall career goal of understanding the role of emotions in medical decision-making in older adults. Older adults care occurs in a complicated socio-medico-psychological context that demands sensitivity to the importance of issues such as functional impairments, multiple co-morbidities, polypharmacy, and care-giver burden. As a geriatrician, Dr. Dale is sensitive of including these concerns in pursuing his research. Enhancing his broad-based training in public policy studies and geriatrics with a targeted, in-depth understanding of psychology and medical decision-making under the expert guidance of his mentors, Dr. Dale will build the necessary expertise for a career researching the role of emotions in medical decisions for older adults.

The human HPRT locus will be cloned. Radioactive cDNA probe will be made on template in RNA from HPRT plus cells that does not associate with DNA from human cells that have an X-ray-induced deletion of the HPRT locus. The probe, nitrocellulose transfer techniques and autoradiography will be used to screen plaques formed by shotgun recombinant DNA phages for those with inserts complementary to probe sequences. After these clones are amplified those with functional HPRT genes will be identified by transfer of their DNA into HPRT- cells using micro-injection or calcium-phosphate precipitation techniques. If the approach outlined above is successful we will attempt to clone genes of the human major histocompatibility complex using DNA from cells having homozygous deletions in this region, which we have produced with X-rays. Microinjection experiments and hybrids produced by fusing human malignant with non-malignant cells will be used to determine if the following traits are dominantly expressed: morphological transformation, anchorage-independent growth and tumorigenicity in nude mice. For traits that are dominantly expressed a combination of DNA-mediated genetic transformation and microinjection techniques will be used to clone the responsible cancer genes.

: Adriamycin (doxorubicin) is a potent, broad-spectrum antineoplastic agent effective in treating a variety of cancers including both solid tumors and leukemias. However, the clinical value of this drug is limited by the development of a cumulative and irreversible dilated cardiomyopathy that is manifested as a progressive loss of ventricular performance in patients receiving repeated doses of the drug. Adriamycin cardiotoxicity is also characterized by a dose dependent decline in mitochondrial oxidative phosphorylation and a decrease in high-energy phosphate pools. Our hypothesis is that this results from a cumulative and irreversible modification of regulatory factors affecting the membrane permeability transition (MPT) pore by adriamycin. The enhanced sensitivity to induction of the MPT leads to a futile, energy-depleting cycling of calcium across the mitochondrial membrane, which we suggest accounts for the dose-dependent loss of respiratory efficiency and ATP synthesis in cardiac tissue from adriamycin-treated rats. We further suggest that these mitochondrial changes contribute to the progressive inability of cardiac tissue to tolerate metabolic stress, particularly those associated with induction of the MPT such as ischemia and reperfusion. Another critical element to this adriamycin-induced mitochondrial cardiomyopathy is its irreversibility, which constitutes a very serous problem clinically in treating cases of pediatric or recurrent neoplasias. The fact that the effect of adriamycin on mitochondrial bioenergetics outlasts the persistence of drug residues in tissues suggests that adriamycin, at sufficient doses, initiates an irrevocable sequence of events that continue beyond elimination of drug. Our hypothesis is that adriamycin interacts with mitochondrial membranes to initiate a series of reactions that lead to increased rates of free radical generation. Furthermore, we suggest that this is a self-perpetuating process that increases in magnitude despite the termination of drug treatment and elimination of drug residues from the tissues. Careful and rigorous testing of these jointly related hypotheses will provide valuable insight into the pathogenesis of adriamycin cardiomyopathy and is essential to the development of mechanism-based therapeutic strategies to minimize the dose-limiting effects of this clinically important anticancer chemotherapy.

The overall objective of the research proposed is to develop an understanding of the enzymatic reactions involved in the biosynthesis of terpenes through study of model reactions and investigations with biosynthetic enzymes. The synthesis of protosqualene pyrophosphate (a newly coined name for the cyclobutly isomer of presqualene) is proposed since this compound may be a new intermediate in sterol biosynthesis. Substrate specificity and mechanism in the enzymatic conversion of geranylgeranyl pyrophosphate to kaurene is to be studied through the use of structural analogues and stereospecifically labelled substrate. This project will be carried out in collaboration with Professor C.A. West at UCLA. An investigation of the carbonium ion reactions of the sesquiterpene, norcedrol is proposed as a possible model for tricyclic sesquiterpene biogenesis.

The Wisconsin Clinical Cancer Center will develop a statewide cancer control program designed to: 1. Engage health care professionals in efforts to detect cancer earlier. 2. Develop a regional network of affiliated centers providing excellent diagnosis and treatment of cancer. 3. Conduct cooperative projects in cancer detection, diagnosis, therapy and rehabilitation. 4. Serve regional units with central data management resources. 5. Inform the public of advances in cancer control, ways they can improve outcome in cancer.

The candidate seeks a K01 career development award that will encompass training in clinical research to complement his multidisciplinar training in immunology, microbiology, genetics and epidemiology and enable him to advance his scientific career by investigating the role of the gut microbiome in Crohn's diseae (CD)under the influence of major genetic risks.CD is an inflammatory bowel disease (IBD) resulting from defects in the mucosal immune response to enteric bacteria in genetically susceptible individuals. Over 70 susceptibility loci, particularly immune response genes such as NOD2/CARD15, IRGM and ATG16L1, have been associated with CD risk in individuals of European ancestry. Previous studies have established the distinct membership and abundance of the gut microbiota in CD patients compared with healthy controls, inferring a possible role of the microbiome in CD pathogenesis. Accumulating evidence supports the role of the major CD susceptibility genes, NOD2/CARD15, IRGM and ATG16L1 in the processing of microbial antigens and innate immunity. Moreover, studies showed significant differences in microbiome profiles among ethnic groups. However, it is unclear whether carriage of the major genetic risk alleles correlates with an abundance of any particular microbial species in the gut. In this study we will investigate whether bacterial profile differs between carriers and non-carriers of the major CD susceptibility alleles while focusing on a genetically homogeneous Ashkenazi Jewish (AJ) population, who has the highest prevalence of CD comparing with other ethnic/racial groups. The hypothesizes include: 1) the microbial profile of the gut is moderated by the carriage of CARD15, IRGM and ATG16L1 risk variants and 2) the CD risk in the AJ population is attributable to the unique combination of bacterial species in the gut and host genetics.To test these hypotheses, the candidate will recruit AJ CD patients enrolled in an ongoing registy of patients with IBD in the Division of Gastroenterology at The Mount Sinai School of Medicine, New York and already genotyped for the major CD risks. AJ controls without CD will come from the Mount Sinai Biobank. We will utilize 16s rRNAdeep sequencing technique followed by bioinformatics and statistical approaches to characterize the gut microbiota in study subjects with regard to disease status and genetic risks. By studying a more genetically homogeneous population (AJ), this study will attempt to gain a better understanding of the microbiome-host gene interaction associated with disease pathogenesis. This can help build comprehensive diagnostic tools to identify individuals at risk of developing CD, s well as develop novel personalized treatments for AJ CD patients. In addition, this project will help to establish a strong multidisciplinary foundation for the candidate's future career in translational research.

We have reported that beta-lapachone, a plant alkaloid, greatly increases the lethality of a variety of DNA damaging agents to mammalian dells. The drug was shown to increase the double strand breaks in DNA of the damaged cells, and to decrease the rate of disappearance of single strand breaks. It also-increased the unwinding activity of purified topoisomerase T. We hypothesized from these results that topoisomerase I is involved in repair of damaged DNA. Consistent with this, we showed that camptothecin, a specific inhibitor of topoisomerase I, also enhanced the lethality of DNA damage in cells. Our three specific aims are: (1) To test the hypothesis that topoisomerase I is involved in excision repair of damaged DNA. (2) To determine whether camptothecin or beta-lapachone enhance lethality of antineoplastic DNA damaging agents by altering topoisomerase I activity. (3) To correlate the above lethality with the increased conversion of single stranded DNA damage to double stranded breaks. The experimental approach, described in more detail below, depends upon three principle variations of established systems for studying topoisomerase I, namely (A) To use nicked gapped, and randomly damaged DNA's as substrates for the enzyme in comparison with intact DNA. (B) To determine the effects of the two drugs on both damaged cells and topoisomerase 1 activity in purified systems. (C) To make mutations of topoisomerase I and investigate the above phenomena with them as compared to the wild type enzyme, both in vivo and in purified preparations. The research proposed- in-this grant is intended to fill several gaps in our knowledge. The first concerns the role of topoisomerase I in DNA repair. How are DNA repair processes dependent upon the type of initial lesion? Is topoisomerase I involved in the repair of all kinds of DNA lesions? From a practical point of view, the combination of topoisomerase I inhibitors with DNA damaging agents may lead to enhanced antitumor activity with diminished host toxicity, and thereby improve the therapeutic indices of established antineoplastic agents.

DESCRIPTION: In this revised competing application, the Principal Investigator seeks to define the relationship of cell cycle status in hematopoietic stem cells and their ability to engraft during transplantation. Four Specific Aims are enumerated. In the first specific aim, the Principal Investigator plans to identify the cytokines which induce an engraftment defect. During the previous tenure of this grant, Dr. Quesenberry demonstrated that hematopoietic stem cells cultured in the presence of a cocktail of cytokines including IL-1, IL-3, IL6 and stem cell factor display a substantially reduced capacity to engraft in non-marrow ablated recipients. Having developed a non- myelosuppressive model of marrow transplantation in which large numbers of male donor cells are infused for several days into female recipients, the Applicant was successful in demonstrating a very high engraftment efficiency in such animals. However, when marrow cells were first cultured in the presence of a number of cytokines which act to expand putative stem and progenitor cell numbers, the ability of these cells to contribute to long-term hematopoiesis was essentially eliminated. As this non-conditioning model of stem cell transplantation would find great use in a setting of gene therapy, the Applicant proposes to determine the cytokine or cytokines responsible for the induced effect. Initial studies will be directed towards defining the amount of time cells need to be exposed to these proliferative cytokines in order to induce the engraftment defect, and refinement in the methodology will also be addressed by studying two additional systems which potentially will eliminate any barrier to transplantation (male into female might engender an immunologic intolerance). The Investigators will study Ly 5.1 into Ly 5.2 congenic transplants, and a ROSA 26 transplant model in which beta galactosidase can be tracked. These refinements should optimize the model for the subsequent specific aims, which attempt to elucidate the relationship of cell cycle status to engraftment and any experimental manipulation which might impact favorably upon this defect. In the second Specific Aim, the Investigator plans to generate both whole marrow cells and rhodaminelo/Hoechstlo fractions and using either growth factor stimulation or isoleucine deprivation attempt to develop a synchronized population of cells to test whether the transplantation defect is restricted to a specific point in the cell cycle. The Investigator will monitor cell cycle using FACS analysis and will then perform transplantations with cells throughout the cell cycle. It is hoped but not fully expected that cells that transit an entire cell cycle might provide the most useful information. In the third Specific Aim, the Investigator plans to identify a cocktail of cytokines which are capable of maintaining primitive stem cell engraftment potential. If the Investigator's hypothesis is correct, these should be cytokines which maintain viability but fail to induce cell cycle progression. To the basic cytokine cocktail of IL-11, IL-3, IL-6 and "steel" factor, modulation using different concentrations of cytokines (based on the finding that low levels of cytokines can support survival, whereas high levels support proliferation), additional cytokines which appear to act in relatively primitive cells including LIF, TGF beta, IL-1 alpha, and MIP-1 alpha, and different adherent stromal layers including Dexter cultures and the cell line TCI will also be tested for effects on cell maintenance or expansion without an associated engraftment defect. Finally, in the fourth Specific Aim, the goal will be to develop in vitro techniques to reverse cytokine-induced engraftment defects. If successful, the Investigator hopes that such manipulation will allow marrow cells to be transiently stimulated to allow retroviral integration, followed by their manipulated return to quiescence, thereby repairing the engraftment defect and thus allowing for engraftment of the cells in which retroviral gene therapy had occurred. Once again, combinations of cytokines will be assessed by following their effects on short-term clonogenic cells populations (HPP- CFC and LPP-CFC) and most importantly, on the ability to engraft in the long-term setting.

Mast cells (MCs), granulocytes, and lymphocytes are integral to the development of an allergic response. Allergic inflammation may also be generated through activation of receptors coupled to heterotrimeric G proteins (GPCRs). The purpose of this study is to understand mechanisms of G protein-mediated signal transduction in immune cells, with a focus on GPCR-mediated trafficking of leukocytes to sites of allergic inflammation. GPCRs activate a core pathway of heterotrimeric G proteins, which bind guanosine triphosphate (GTP) in exchange for guanosine diphosphate (GDP). The GTP-bound form of the G protein alpha subunit induces downstream signaling cascades, including intracellular calcium flux responsible for MC/basophil degranulation. This project focuses on a family of regulators of G protein signaling (RGS proteins), which inhibit the function of G alpha-i and G alpha-q, but not G alpha-s, proteins by increasing their GTPase activity. G alpha subunits oscillate between GDP- (inactive) and GTP- (active) bound forms based on ligand occupancy of the associated receptor. The GTPase accelerating (GAP) activity of RGS proteins limits the time of interaction of active G-alpha and its effectors, resulting in desensitization of GPCR signaling. Despite a growing body of knowledge concerning the biochemical mechanisms of RGS action, relatively little is known about the physiological role of these proteins in allergic inflammation. A major area of investigation is the recruitment of inflammatory leukocytes to sites of inflammation. Chemokines are a major class of compounds acting on leukocyte GPCRs, which orchestrate immune cell trafficking, and RGS proteins including RGS5, RGS13, and RGS16 inhibit chemokine signaling by desensitizing GPCR signals. In FY18, we continued our phenotypic characterization of patients with undefined immunodeficiencies and novel mutations in G proteins and/or RGS proteins are being characterized in collaborative studies with Drs. Orange and Su. 3 brothers in a single family with combined immunodeficiency were found to carry biallelic mutations in RGS10. We discovered that RGS10 is expressed in both human B and T cells. Patient-derived B and T cells from these patients exhibit profoundly impaired chemotaxis in response to chemokines despite having comparable RGS10 expression. Molecular studies, which are ongoing, have determined that the mutations may affect RGS10 phosphorylation and induce protein mislocalization, which could result in a gain of function. IN FY18, we defined a role for RGS5 in neutrophil trafficking. RGS5 was expressed in both human and murine neutrophils. We detected significantly more neutrophils in the airways of Rgs5-/- mice than wild-type counterparts following acute respiratory virus infection and also in the peritoneum in response to injection of thioglycollate, a biochemical proinflammatory stimulus. RGS5-deficient neutrophils responded with increased chemotaxis elicited by the chemokines C-X-C motif chemokine ligand 1 (CXCL1), CXCL2, and CXCL12, but not fMLP. Moreover, adhesion of these cells was increased in the presence of both CXCL2 and fMLP.

Project Abstract We have established the UCSD Clinical and Translational Research Institute (CTRI), with its hub located at the University of California, San Diego (UCSD). This regional center includes three universities (UCSD, San Diego State University and UC Riverside), 4 biomedical research institutes (Salk Institute, Sanford-Burnham Institute, J. Craig Venter Institute, and La Jolla Institute for Allergy and Immunology), and 3 academic hospitals (UCSD Medical Center, VA San Diego Medical Center and Rady Children's Hospital), one community health center (Palomar Health). The CTSA program encompasses 3 health sciences professional schools (Medicine, Pharmacy, and Nursing) and 2 Master's programs in Clinical Research and Drug Discovery Sciences. The overall program goals are to: 1) Develop an integrated education and training program across the translational science spectrum; 2) Empower community-based partnerships and integrate the stakeholders into translational research governance; 3) Diversify and enhance biomedical informatics infrastructure to provide a secure, confidential and inter-operative platform; 4) Provide administrative infrastructure to manage and improve clinical and translational research; and 5) Provide resources that enhance collaboration, encourage disruptive innovation and support clinical investigation. The CTRI KL2 program will provide a mentored research experience for junior faculty that ensures a diverse future workforce with the required skills for outstanding multi-disciplinary research in human health. Didactic programs will teach key skills in clinical and translational research, including Master's degrees for individuals seeking additional rigor. Mini-sabbaticals will offer experiential training to enhance the scholars' research programs. A Mentoring Core will train scholars to become mentors for the next generation of researchers. Outcomes and tracking will use new informatics tools to determine the impact of the curriculum on long term research productivity.

This proposal details a 5-year plan for the development of an academic career in Cardiovascular Medicine. The PI is a trained interventional cardiologist with a PhD, and has recently started his first faculty position at the University of California San Francisco (UCSF), Division of Cardiology. This proposal will utilize the Pi's skills in immunohistochemistry applied to the animal model of myocardial infarction (Ml) already used in the lab of the supervisor, Dr Randall Lee MD, a recognized expert in the application of new therapies for Ml. Additionally, this proposal will permit a dedicated period of time for the PI to learn the techniques and statistical analysis of high throughout microarray analysis. This will be achieved through a collaboration between the Division of Cardiology and the Gladstone Institute of Cardiovascular Disease at UCSF. Apoptosis, also called programmed cell death, of cardiomyocytes following Ml is a crucial aspect of the process of left ventricular remodeling that leads to heart failure. As the population ages and the incidence of heart failure increases, this is becoming and increasingly important clinical problem. Novel strategies to treat apoptosis following Ml are being developed, but are being tested in models of Ml in young animals. We will therefore compare the timescale of apoptosis, as well as the expression of pro- and anti-apoptotic genes, in old versus young animals. A specific inhibitor of caspases, which cause apoptosis in cardiomyocytes, will then be used to see if the anti-apoptotic effects seen in young animals are the same in older animals. This, in turn, will facilitate better planning of anti-apoptotic strategies for clinical trials in older patients. UCSF is an ideal research environment for an aspiring clinician-scientist. The Division of Cardiology and the Gladstone Institute of Cardiovascular Disease are centers of excellence in cardiac research. Specific aims 1 and 3 can be achieved in the lab of the project supervisor, Dr Lee. The microarray studies will be performed and the data analyzed at the Gladstone Institute under the supervision of Dr Bruce Conklin MD, a pioneer in microarray analysis. Additionally, an advisory committee of esteemed senior researchers will oversee the progress of this project.

Synthetic approaches to the macrolides methymycin, boromycin, and verrucarin A are described. The strategy for synthesis of the twelve-membered lactone of methymycin involves the combination of two units in chiral form, corresponding to C1-C8 and C9-C11 of the aglycone methynolide. A description of the planned synthesis of the more complex segment, using the addition of an oxoallyl cation to a furan to elaborate none of the required ten carbons at the first step, is presented. Lactonization of a fully functionalized hydroxy acid is proposed for completion of the aglycone. The plan for synthesis of the ionophore boromycin envisions construction of the two sections of the diolide by a common pathway. The "southern half" is to be assembled from five unitary segments in a specific sequence which permits a high degree of stereocontrol. The "northern half" is obtained by cyclization of the C-9 epi version of the "southern half", and these two chains are then fused around the borate core prior to completion of the macrolide ring. For verrucarin A, a cytostatic antibiotic from the fungus Myrothecium, the synthesis plan first envisions completion of a route to the sesquiterpenoid component verrucarol in stereoselective fashion. A key step in this scheme is expansion of a cyclobutyl carbinol to the bridged ring system characteristic of the trichothecenes. Attachment of the macrolide loop, assembled stepwise from three segments, to this terpenoid diol begins at the C15 hydroxyl with completion of the lactone by cyclization at the C4 hydroxyl.

Two canonical binding sites for the Ets family of transcription factors have been identified within the TCR-zeta promoter. These sites bind Elf-1, and play an important role in basal transcription of zeta. Ligand-mediated TCR degradation is inhibitable both by reagents that block vesicular acidification and by inhibitors of the ubiquitin/proteasome pathway, suggesting a complex pathway leading to TCR degradation. Our determination that the TCR is a substrate for ubiquitination has led us to evaluate ubiquitination in more detail. A family of human E2 enzymes (UbcH5A-C) have been cloned and characterized. These E2s are widely expressed in human tissues and function in conjunction with the only characterized human E3, E6-AP, in the ubiquitination of proteins. We have also been studying a novel yeast E2, UBC6, a novel type IV transmembrane protein found associated with membranes of the ER. The short transmembrane domain of UBC6 is responsible for its distribution within the cells, progressive lengthening the transmembrane segment re-targets UBC6 to the Golgi Apparatus and then to the cell surface. Two murine E3 enzymes have been characterized. One of these is the murine homolog of E6-AP, the other is a protein found to have homology to E6-AP, termed Nedd-4. Like E6-AP, Nedd-4 is an E3 enzyme, both of these function with the UbcH5 family of E2s in the ubiquitination of cellular proteins. These E3s exhibit distinct subcellular localizations, E6-AP is expressed predominantly in the nucleus, Nedd-4 is present exclusively in the cytosol. Nedd-4 has two distinct E2 bindings sites, one of which binds the amino terminus of E2s and the other showing affinity solely for the carboxyl component of E2s. These data suggest models for ubiquitination involving oligomerization of E2 and E3s.

The purpose of the project is to develop an immunoadsorption-immunosuppression program that will effectively remove platelet alloantibodies and impair their continued production. The goal is to obtain sufficient antibody reduction to permit the survival of incompatible platelets in refractory, thrombocytopenic patients. Either intact platelets, platelet membranes or platelet antigen fractions will be used as the immunoadosorbant. These materials will be immobilized to a solid support structure and plasma obtained by apheresis techniques will be passed over the platelet immunoadsorbant to remove alloantibodies. Alternatively, intact platelets will be placed in a reservoir system and plasma obtained by membrane or hollow fiber filtration will be pumped through the platelet reservoir to achieve alloantibody adsorption. Cyclosporin A will be used as the primary immunosuppressive agent. Other agents will be added only if Cyclosporin is not effective as a single drug. Quantitative platelet antibody measurements and radiochromium labelled donor platelet survival determinations will be used pre- and post-immunoadsorption-immunosuppression to document the value of the program in reversing platelet alloimmunization. Both animal and patient studies will be conducted.

Direct methods for functional neural imaging are critical to advancements in understanding neural behavior, plasticity, connectivity and pathology. If we can directly image active neurons we will have the ability to examine neural activity more precisely than is presently the case with fMRI. We have developed an MRI-based conductivity imaging technique, Magnetic Resonance Electrical Impedance Tomography (MREIT) that can reconstruct conductivity maps with near-MRI resolution. In MREIT, small external currents are applied to an object. The MR magnetic flux density patterns created by current flow may be converted to conductivity or current density slice images. We developed this technique and have refined it to the stage of producing electrical conductivity images of animal brains in vivo, using relatively low applied currents. The large changes in membrane conductance that occur during activity cause dynamic changes in paths taken by externally applied currents. Changes in spiking activity during external current application will cause differential phase accumulation in MR data that will increase the longer current is applied. Neural activity therefore becomes visible as an increase in apparent conductivities of voxels coincident with active intracellular areas. Because the contrast controlling MREIT signals, conductivity, may only acquire positive values, phase accumulations cannot be cancelled by the presence of opposite polarity or opposingly oriented signals. This may give MREIT an advantage compared with other MRI-based methods for imaging neural currents that are based on perturbations of phase or main magnetic fields caused principally by summed axonal current flows. Thus, MREIT has the potential to detect activity in complex structures including gray matter. In this proposal, we will investigate the ability of functional MREIT (fMREIT) to detect activity-related conductivity changes in neural tissue. We will develop fMREIT techniques to image neural activity in vitro, in a several standard neural preparations, while progressively refining our methods to detect and locate active cells at high signal to noise ratio and using main magnetic field strengths conveniently used in vivo. In isolated preparations, our method has the potential to enable detailed analyses of single cell mechanisms. The method could thus be considered as a non-invasive extension of patch clamping techniques, and could stand alone for this purpose. However, ultimately we wish to image activity in vivo and our final study in this program will include a tentative exploration of fMREIT in a live animal model as a precursor to further research in this area. In summary, this study will establish the basis for functional MREIT (fMREIT) techniques. This method could ultimately be used to visualize effects of more general neural behavior and enable more fundamental analyses of neural behavior in vivo than is available with existing techniques such as fMRI.

The purpose of the Molecular Resource Core (MRC) is to provide support for the analysis of genes and gene products that are relevant to the pathogenesis of arthritis. It will support studies of immune-mediated arthritis in both humans and mice. The MRC will take a multifaceted approach in that it will develop new assays and procedures, be a source of well-characterized Collagen, provide analysis of samples under standardized conditions with stringent quality control, and be a technical and educational resource. MRC facilities will isolate and purify collagen and peptides. This protein family has many unique characteristics that make it difficult or impossible to obtain reliably from commercial sources. The MRC will use state of the art equipment for molecular analyses to aid in the development and application of the most advanced molecular techniques to the study of rheumatic disease. It will perform highly sensitive determination of mRNA levels by quantitative real time polymerase chain reaction, including assisting investigators in the design of appropriate oligonucleotide primers and probe sets an. d will provide a standardized service for sample amplification, data collection and analysis. The MRC will aid both new and established investigators in the use of DNA microarray technology to discover new factors and genetic interactions that affect pathogenesis and modulation of disease. Since many rheumatic diseases are closely linked to particular MHC genes, it will provide HLA genotype determination to identify those haplotypes relative to disease with the capacity to perform fine specificity analysis down to the DNA sequence level. The availability of these services should facilitate research in rheumatic diseases for both clinical and basic scientists at the University of Tennessee, HSC.

Maximum blood flow acceleration, beat-to-beat stroke volume and continuous external power of the left ventricle are derived from instantaneous (pulsatile) measurements of blood flow velocity and pressure in the ascending aorta of patients with ischemic and/or valvular heart disease. These parameters are regarded as giving us more information about ventricular performance than those derived from measurements of average flow and pressure. Also, the calculation of aortic input impedance from the pulsatile records of flow and pressure give us additional information about the elastic properties of the aorta as well as the location of major reflecting sites. In the group of patients studied thus far, velocity and pressure were measured using two separate catheters, but ultimately these measurements will be obtained using a single catheter with an electromagnetic velocity transducer and a miniature pressure transducer mounted at the tip. Atrial pacing will be used as transient stress to produce myocardial ischemia and sublingual nitroglycerin will be used to reduce preload and afterload in patients with and without myocardial ischemia. Pathophysiology of atrial pacing will be studied with special attention to beat-to-beat measurements of stroke volume and acceleration following the termination of pacing. These postpacing beats are of interest because they provide an opportunity to study the response of the ventricle to a sudden increase in filling volume ("auto volume load").

Understanding host-viral interactions at the organismal, cellular, and molecular levels are vital in mitigating viral pathogenesis. The goals of this project are to discover the viral microbiome and specific antiviral immunity genes in the model organism Hydra. By doing so, we will determine whether Hydra contains viruses similar to human viruses as well as being able to test Hydra specific immunity genes responsive to viral presence. Hydra provides an ideal model to test these host-viral interactions because of their simplistic composition, their ease of experimental manipulation, and their epithelial exposure to the environment without a protective barrier. Further, Hydra lack adaptive immunity features, do not contain any motile phagocytic cells, and only utilize mucous as a means to preserve its epithelium. Therefore, Hydra is uniquely suited for the study of host-viral innate immunity at the mucosal epithelium. To establish this, the project consists of two specific aims: 1.) Determine the Hydra viral composition utilizing viral metagenomics. 2.) Elucidate the Hydra antiviral innate immune response in the presence of cytosolic nucleic acids. By accomplishing these two aims we will identify viruses that cause pathogenic conditions in Hydra as well as discern viruses that similarly cause human pathogenesis. These viruses can then be tested in this innate immunity model system. Further, by identifying the Hydra antiviral genes responsive to viral infection, we will manipulate the Hydra through gene knockdown, to determine which genes are vital in limiting viral pathogenesis. PUBLIC HEALTH RELEVANCE: Establishing Hydra as an antiviral mucosal innate immunity model organism is important for human health as demonstrated by genetic variants in phylogenetically ancient innate immune genes are involved in the etiology of chronic inflammatory diseases of the epithelial barrier, such as Crohn's disease, atopic dermatitis, and asthma. These polygenic diseases are characterized by chronic relapsing inflammation of the mucosa. Such disease states and the response to them can be studied in the Hydra model system.

Liver Carcinogenesis: A small electrophoretic class of soluble proteins of target organs contains the principal protein-carcinogen conjugates of three families of carcinogens. In pursuit of the possible role in carcinogenesis of one such conjugate, and of the interaction between carcinogen and target protein from which it arises, the following are broad aims of the proposed research: 1. Characterization of the principal soluble liver azoprotein of azo dye preneoplastic liver. 2. Identification of the principal protein target of azocarcinogens in liver. Cell Transformation to Malignancy: The goal is to investigate the extracellular fibrinolytic activities that may have specifically associated with cells transformed by viruses or chemicals. Extracellular activators and inhibitors of fibrinolysin will be isolated from cultured cells and characterized. Effects of the additions of the activators and inhibitors on normal and transformed cells will be studied, in order to learn whether the fibrinolysin activating activities of transformed cells are in part responsible for some of the characteristics associated with cell transformation to malignancy.

Project Summary Core F ? Immune Function The Immune Function Core F provides broadly ranging support for immunological studies. Driven by investigator need, and a rapidly changing science environment, the Core currently offers multiplexed cytokine measurements, standard flow cytometric analysis as well as flow imaging, sorting of cell populations via flow cytometry and other methods, and ELISA, ELISPOT, and infrared imaging. The re-structuring of the Core in 2010 included a cooperative agreement with the Cancer Center Cytometry and Imaging Microscopy Facility to pool the equipment, harmonize services, and maintain formal access to CLIA-level flow cytometry. Drs. Scott Sieg and James Jacobberger have been working closely together as Co-directors and have used their complementary expertise to greatly enhance Core services. Dr. Sieg provides immunological expertise, functional assays, and biomarkers, especially in the context of HIV research; Dr. Jacobberger provides expertise on flow and image cytometry instrumentation, single-cell assays, and biomarker discovery, especially in the areas of cell cycle, apoptosis, and cell signaling. A major function of the Core is to collaborate with users to develop novel immunological assays. For example Core F, in collaboration with the Proteomics & Systems Biology Core G, is refining new flow cytometric assays for characterizing the phospho-proteome. This unique platform should open new avenues of research relevant to HIV pathogenesis, clinical evaluation, and treatment. A parallel effort is focused on developing multiple pathway signaling measurements on sub-populations of cells in unfractionated human samples (e.g., blood). The Core also interacts closely with the Clinical Core D for sample procurement. The Immune Function Core (F) is currently the one of the most heavily used of all the CWRU/UH CFAR cores and during this grant cycle the Core generated net revenues of $384,177. Program income has been steadily reinvested in the purchase of advanced instruments, such as multicolor flow cytometers, and reagents. For example in 2012, the Immune Function Core purchased a BD Fortessa ? a next generation analytical instrument ? with blue, green, red and violet lasers and filters optimized to measure fluorescent proteins. This instrument was subsequently upgraded to add a high throughput 96 well plate feed system thanks to a CFAR supplementary award. As part of the recruitment of Dr. Skaly, the CFAR will purchase a BD Biosciences FACSAria with 4 or 5 lasers and 18-color capability. The FACSAria will be maintained under BSLII conditions to permit sorting of HIV-infected cells. This state-of-the-art instrument will bring sorting capacity in line with our analytical capacities and create further opportunities for Core-Core interactions since it will be used to prepare samples for sequencing and proteomic analyses by Cores E and G.. The specifically aims of the Core follow: ? To provide access to instruments and assays to support CFAR programs. ? To develop validated assays and platforms for CFAR investigators. ? To provide consultation and training at all levels. The development of innovative new platforms, such as phospho-protein analyses, spearheaded by the Immune Function Core creates new opportunities for scientific investigation and competitive advantages by CFAR investigators. The Core will continue to promote interactions among CFAR investigators, meeting the needs of the CFAR Working Groups, and cooperating with the other CFAR Cores to provide access to cutting edge technologies.

This project examines factors affecting the incidence of racial harassment and discrimination, and the multiple consequences of the stress of racial harassment and discrimination. Research suggests that Hispanic employees who experienced racial discrimination, and their White coworkers who were aware of it, showed higher levels Of stress. Further, African-Americans and Hispanics who reported racial discrimination exhibited poorer health conditions and psychological well-being. The project tests a model which distinguishes between racial harassment (i.e., unfavorable race-related comments or gestures) and racial discrimination (i.e., unfair employment- related treatment of employees of one race over another). Further, the model predicts that a negative organizational climate for racial/ethnic minorities affects the occurrence of racial harassment and discrimination, and that those who are racially harassed or discriminated against experience increased stress, and negative job-related (e.g., job satisfaction, organizational withdrawal), health (e.g., health conditions, health satisfaction), and psychological outcomes (e.g., self-esteem, satisfaction with life). In addition, the project explores how the stress of racial harassment and discrimination differentially affects those who experience such events, and their coworkers who are aware of, or witness such events. Finally, it also examines how employees of different racial/ethnic backgrounds are differentially affected by the stress of experiencing or being aware of racial harassment and discrimination. Participants are 1500 graduate students at an ethnically diverse (approximately 50% Hispanic), South-Western, university. Participants will complete a survey which assesses their perceptions of university climate, experiences of racial harassment and discrimination and job-related, health and psychological outcomes. Data will be analyzed by the LISREL VIII approach to structural equations modeling. Results can be used to design interventions and will contribute to the understanding of the role of culture in responses to the stress of racial harassment and discrimination.

Our laboratory has in the past year conducted runs at CHESS A1, F1 and F2, NSLS X4A, X12 and X25, as well as APS to extend the resolution and improve the quality of diffraction data for crystalline complexes involved in chaperonin mediated protein folding, signal transduction, transcriptional and translational regulation. During those trips we have obtained numerous complete high resolution data sets resulting directly in the determination of the following new structures: (1) progesterone ligand binding domain complexed with progesterone; (2) human estrogen receptor ligand binding domain complexed with estradiol; (3) the chaperonin GroEL/GroES/ADP complex; (4) the T. thermophilus EF-Tu/EF-Ts complex; and (5) a P. woesei ternary complex of the TATA-binding protein, TFIIB, and a DNA sequence containing a consensus TATA box at 2.1[unreadable] (see publications section). Data recently collected at CHESS has been instrumental in solving all of the above structures. In addition a paper on the molecular mechanism for the role of phosphorylation in the regulation of hetereotrimeric G-Proteins by phosducin is also in preparation from data collected at CHESS on the phosphorylated form of phosducin. Other projects which are still in progress which have been significantly influenced by CHESS synchrotron data collection include the following: Chaperonin-Medicated Protein Folding: To understand the capacity of ATP to drive the GroEL-GroES reaction cycle, we wish to study relevant intermediates in the ATPase cycle. GroEL/GroES/(ADP)7/(AlFx)7 crystals diffract to 4[unreadable] at our home source but only initial tests were done at the CHESS F1 line this year. Searching for optimal freezing conditions is in progress. Bovine Visual Arrestin: The arrestin crystals diffract to 3.0[unreadable] resolution using synchrotron radiation (NSLS X-25 beamline and MacCHESS A-1 beamline). We have collected complete native data sets to 3.1[unreadable] (C2221; a = 169[unreadable], b = c = 191[unreadable]; crystal size: 0.2 x 0.2 x 0.03 mm3). Useful MAD, heavy atom and anomalous data sets for phasing have been collected at CHESS, X-25 and APS. Quaternary Complex of VP16 Acidic Activation Domain Complexed with hTBPc, hTFIIB on Promoter DNA. To date no high resolution structural information exists on a transcriptional activator domain bound to the preinitiation complex. Hence, this crystal structure will reveal the essential contacts made between a strong acidic activation domain and the basal machinery, resulting in transcriptional activation. The crystals diffract to about 4.0 [unreadable] on a home source (at 100 K). Several 2.6[unreadable] native data sets (P21; a = 118.7[unreadable], b = 122.2[unreadable], c = 140.8[unreadable], b = 113.0 ; crystal size: 0.4 x 0.1 x 0.05 mm3) were collected recently at CHESS F2 and F1 lines.

The title of this project has been changed to more accurately reflect the thrust of the science. The retina and pigment epithelium are neuroepitheli tissues that work in close cooperation. Specific growth and differentiatin factors are found in the eye that guide development and interactions of individual ocular tissues to form a functional visual system. Studies on t s project are focused on an understanding of the molecular biology and molecular genetics of the retina and discovering new genes that are candidates for hereditary retinal degenerations. For example, ocular tissu synthesize a number of growth factors. There now appear to be several systems present that could self-regulate growth and metabolic activity in the retina-pigment epithelium complex and be involved in eye diseases. In this regard, we have cloned and characterized a unique protein secreted from fetal human pigment epithelial cells, called pigment-epithelial derive factor (PEDF), that is "neurotrophic" to cultured human retinoblastoma cell and may affect neural retinal development in vivo. This protein also is a potent "neuronotrophic" agent in that it promotes neuronal cell survival of cultured cerebellar granule cells. Finally, PEDF is "gliastatic" in that i markedly retards glial cell growth. Along with being a candidate gene in retinal degenerations, the uses of PEDF in neuronal transplant in retina an other CNS areas are obvious.

MAP kinases are key regulatory components acting on many cellular processes such as embryogenesis, differentiation, proliferation, cell death, and acute hormonal responses. MAP kinases are activated by a protein kinase cascade, consisting of a MAP/ERK kinase kinase (MEKK), which activates a MEK which activates a MAP kinase. ERK1 and ERK2, the first mammalian MAP kinases identified, serve as prototypes for the behavior of the many related enzymes. The functions of these MAP kinases depend on their activation, their subcellular localizations, their protein-protein interactions, and their substrate specificities. We propose to continue to investigate mechanisms controlling MAP kinase pathways as follows. The nuclear uptake of ERK2 is essential for some of its most important functions. We will define mechanisms of subcellular localization of ERKI/2 by examining import and export by reconstitution in permeabilized cells, and microinjection and transfection in intact cells. We will define mechanisms determining functional complexes of MAP kinases. To do this we will use a cDNA library of mutant ERK2 molecules from which we will isolate ERK2 mutants that lack high affinity interactions with proteins including substrates, activators, and phosphatases. We will study the functions of the loss-of-function ERK2 mutants to determine the roles of individual interactions in inducing ERK2-dependent phenotypes. The ERK2 activator MEK1 binds to molecules other than ERK1/2 and Raf that are essential for signal transmission through the cascade. We will attempt to identify proteins that bind MEK1 through its proline-rich insert and determine their mechanisms of action. We will study ERK5, another member of the MAP kinase family. ERK5 is most similar to ERK2, and cooperates with ERK2 to transform cells. We will examine the mechanisms of ERK5 regulation, and determine how its actions relate to the functions of ERK1/2. We will also study a putative ERK5 ortholog in C. elegans to use genetic studies to complement work in mammals.

Triple negative breast cancer (TNBC) is one subtype of breast cancer that frequently relapses and leads to worse outcome than patients with hormone receptor-positive subtypes. PARP is currently the most promising drug target for TNBC, and multiple PARP inhibitors (PARPi) have been developed and tested in clinical trials. Although PARPi show higher response rate in patients carrying BRCA mutations, there are still high percentage of BRCA mutations carried patients does not respond to PARPi. Thus, developing strategies to make PARPi treatment more effective and to identify biomarkers to stratify patients is critical. Our recent publication in Nature Medicine (2016) showed that in response to reactive oxygen species, c-Met interacts with and phosphorylates PARP1 at Y907. Moreover, we demonstrated that c-Met-mediated phosphorylation is critical for PARPi resistance, and that c-Met inhibitors sensitize TNBC cells to PARPi. The long-term goal of our research is to develop the effective therapeutic strategies for TNBC. To this end, we will seek the novel biomarkers and treatment strategies to improve the efficacy of PARPi. We hypothesized that PARP1 protein is regulated by its phosphorylation, and that phosphorylation status of PARP1 and the expression of the corresponding kinases that phosphorylate PARP1 will serve as appropriate biomarkers for combinational treatment. Multiple kinases can phosphorylate the same substrates, resulting in signal crosstalk. Also, TNBC is a heterogeneous disease, and the distinct kinases play an important role in different TNBC. Therefore, we also hypothesize that similar to c-Met, other protein kinases also have functions to regulate PARP1 activity through phosphorylation in TNBC. Potential molecules we will study are EGFR, which also directly interacts with PARP1 and phosphorylate it. We will investigate the role of these kinases in PARP1 regulation and PARPi resistance. Thus, we propose the following three aims; Aim 1. To systematically validate the significance of c-Met?mediated phosphorylation in PARPi resistance in mouse models and TNBC patient samples; Aim 2. To determine the role of EGFR-mediated PARP phosphorylation in PARPi resistance in TNBC; Aim 3. To determine the role of c-Met and EGFR interplay in PARPi resistance in TNBC. If our proposal is successful, several phosphorylation sites in PARP1 can be used as biomarkers to guide the combinational treatment of PARPi and correlated kinase inhibitors. We will particularly focus on c-Met and EGFR because inhibitors of these kinases are currently used in the clinic or in clinical trials, allowing for faster progression of our biomarker-guided rationale combination therapy into clinical trials.

Ultimately the primary function of all somatic cells in the testis is to ensure a microenvironment that provides adequate androgen and nutritive support for spermatogenesis and confers immunologic tolerance for the mitotic spermatogonia located outside the blood-testis barrier. It is well known that the pituitary hormones LH and PRL affect androgen secretion by Leydig cells. What is less well understood is the identity and function of the local peptidergic factors that make up the non- steroidal microenvironment of the interstitium. LACAF is an interstitial cell protein stimulated by HCG and growth factor but inhibited by prolactin. LACAF activates adenylyl cyclase in Leydig cells but this activation is not functionally directed towards steroidogenesis. Such differential regulation is by known Leydig cell trophic factors suggests that LACAF is an autocrine regulatory factor. This protein shows some sequence homology with the testicular protein of the VIP family of peptides that shares this capacity to activate cyclase without concomitant effects on steroidogenesis. We will study the function and regulation of these two proteins in parallel. Prolactin preferentially enhances the secretion of two other interstitial proteins, one of which is a known immunomodulator, suggesting the prolactin affects testicular immune elements. The second protein, PIL, may be a member of the transforming growth factor Beta family of peptides. The physiological significance of this regulated pattern of interstitial proteins is presently unknown. Our proposed studies will focus on the unique features of the regulation of LACAF, PIL and complement components by prolactin. The ability to determine the various forms of the prolactin receptor MRNAS affords the opportunity to determine the cell-specificity of these prolactin-induced changes in interstitial protein secretion. LACAF and PIL proteins will be further characterized. Cloned CDNAS for these interstitial proteins will be isolated and their structure characterized. Moreover, specific probes will be generated for use in determining both physiological and aberrant expression of these proteins in vivo and in vitro studies. Regulation of these interstitial proteins will be studied by analysis of their MRNA levels as a function of endocrine status using hybridization in situ and immunohistochemistry. The mechanism(s) of action of these peptides will be investigated by assessment of their effects on both seminiferous tubule and interstitial cell function. This research plan will lead to new, significant data on the mediators of intratesticular signalling and their physiological relevance in the testis.

Intimal hyperplasia in arterialized vein bypass grafts is a significant cause of vein graft stenosis and delayed graft failure. Injury at the time of implantation or as a consequence of transplantation into the high pressure arterial system may contribute to these delayed events. Alterations in gene expression accompany implantation and arterialization injury. These alterations lead to intimal hyperplasia, including transformation of endothelial cells to an inflammatory state and initiating migration and transformation of smooth muscle cells from the contractile to secretory states thus creating the lesion of intimal hyperplasia. It is our hypothesis that silencing of genes upregulated by injury to the vein wall will diminish intimal hyperplasia. Furthermore, gene silencing can be accomplished within the constraints of operating room conditions. Our preliminary data demonstrates our ability to identify candidate genes associated with intimal hyperplasia and our ability to knockdown gene expression with siRNA. Using laser capture microscopy we have separated genetic events in the endothelium from those in smooth muscle. In the proposed study we will apply these technologies to (1) systematically identify silencing targets, (2) to silence target genes in vitro, (3) to silence target genes under surgical conditions, (4) to demonstrate inhibition of intimal hyperplasia in vein grafts in vivo. Both human tissue and canine models will be used as well as all cultures, microarrays, laser capture, RT-PCR, Western Blot, and related technology. This study will greatly strengthen the application of gene silencing to clinical problems in vascular surgery, and broaden our understanding of vascular wall biology. Public Lay Summary: Scar tissue formation due to surgical injury is a major cause for failure of heart bypass grafts and bypass grafts for peripheral vascular disease. The investigators propose to prevent formation of this scar tissue by controlling expression of the genes that cause it, using techniques that can be applied in the operating room. [unreadable] [unreadable] [unreadable]

About 10% of all intracranial tumors are schwannomas, benign tumors comprised of Schwann cells with biallelelic NF2 mutations. NF2 mutation or loss is believed to cause schwannoma formation. Several lines of evidence implicate merlin, the NF2 protein, in small G-protein signaling. Biochemical evidence and our analysis of primary schwannoma cells suggest that merlin functions in Rac signaling. While normal Schwann cells are polarized, with one surface attached to axons and the other to basal lamina, schwannoma cells are unassociated with axons. This loss of polarity is likely to arise secondary to altered Rac signaling. Because the relevance of the Rac signaling pathway to Schwann cells is not known, we propose to evaluate a merlin-Rac cascade in Schwann cell growth and tumorigenesis. Our specific hypothesis is that merlin limits the duration of Rac signaling in Schwann cells. We further postulate that deregulated Rac signaling accounts for the failed axon-glial interactions characteristic of schwannomas. We plan to use a combination of gain-of-function and loss-of-function mouse models and primary cells from human schwannomas to test these hypotheses. We believe that analysis of primary schwannoma cells offers a rare opportunity to study a pure population of early stage human cancer cells. The proposed studies are expected to provide insight into regulation of normal axon-glial interactions, and to lead to strategies to understand and ultimately treat human schwannomas. [unreadable] [unreadable] [unreadable]

Hermann Lotze was an agent in the development of philosophy and medicine into independent specialized subdisciplines. The advantages of specialization when held in check by general philosophy and general medicine turned to disadvantages in the subsequent generation. The fruitful interaction of science and philosophy in the period of the rise of the German mandarins gave way to isolation in the later period of their separation. The project deals with the contribution of Hermann Lotze (1817-1881) to the German movement, a broad reform of German medical science on the foundation of philosophical theory construction. His "mechanical" program for medicine was one of many attempts to break loose from vague speculative medical theories such as contagion, humors, crises, etc. He conceived the body as an equilibrium, and disease as a disturbances of this process. His "theory of value" for both medical and philosophical disciplines was based on Kantian critical teleology. This became a foundation stone for the New Psychology in its many ramifications, including pragmatism, phenomenology, analytic philosophy, social behaviorism, and genetic epistemology.

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. Substance abuse continues to be a persistent epidemic that evades effective treatment. Multiple dependencies present a further challenge because of the need to treat different psychological and physiological effects of the different substances. This proposal has the potential to distinguish the biological basis of individual effects of METH and heroin abuse and reveal how they act in tandem to produce a speedball effect. Understanding the pharmacology of speedball reward will lead to the precise brain targets for which medications can be developed to counteract the rewarding effects of speedballs and consequently reduce the prevalence of speedball dependence.

Carnitine acetyltransferase (CrAT) is a freely reversible mitochondrial matrix enzyme that catalyzes the exchange of short-chain acyl groups between CoA and carnitine. Unlike their acyl-CoA counterparts, acylcarnitines can traverse cellular membranes. Accordingly, the interconversion between these molecules can potentially impact cellular, and perhaps inter-tissue carbon trafficking. The primary substrate of CrAT, acetyl-CoA, holds a prominent position in intermediary metabolism as the two-carbon universal end product of fatty acid, glucose and amino acid oxidation. As its major metabolic fate, acetyl-CoA typically enters the tricarboxylic acid (TCA) cycle where it drives production of reducing equivalents that in turn fuel the electron transport chain. CrAT purportedly acts to export excess carbon fuels from the mitochondria during conditions wherein the production of short-chain acyl-CoAs exceeds TCA cycle flux. By doing so, this reaction is thought to play a key role in regenerating free CoA, modulating mitochondrial acetyl-CoA/CoA balance and relieving acetyl-CoA-mediated inhibition of pyruvate dehydrogenase (PDH), the committed step in glucose oxidation. Mounting evidence from our laboratory suggest that CrAT plays a critical role in regulating whole body substrate selection and glucose tolerance;and that CrAT inactivity might contribute to obesity- and age-related metabolic dysfunction. This NRSA application proposes to elucidate the role of this enzyme in regulating whole body and mitochondrial energy homeostasis utilizing immunoaffinity purification techniques and physiological characterizations of genetic mouse models. First, we will test the hypothesis that one of the mechanisms through which CrAT controls energy metabolism is by regulating nuclear and/or non-nuclear protein acetylation. This is an important and timely line of investigation in light of the growing number of metabolic pathways that appear to be modulated by reversible acetylation. Second, we will use an inducible gene knockout approach to determine whether total body loss of CrAT negates the antidiabetic actions of carnitine therapy. These studies are expected to shed clinically relevant insights on the role of this enzyme in combating nutrient stress and metabolic disease. PUBLIC HEALTH RELEVANCE: The overarching goal of this project is to elucidate mechanisms through which supplemental L- carnitine improves glucose tolerance in obese and diabetic rodents. Results from the proposed studies are likely to yield new insights regarding the therapeutic properties of L-carnitine, a conditionally essential nutrient, while also advancing our understanding of why obesity increases risk of metabolic disorders such as type 2 diabetes. These are clinically relevant topics of intense scientific interest and controversy.

The Data Management and Statistical Analysis Core performs data management and analysis for the Clinical Core, the Neuroimaging Core, for the Neuropathology Core and for individual research projects affiliated with of the ADCC, and maintains all data collected by the ADCC in a centralized database. This facility also provides consultation in the design of experiments and statistical analysis to all collaborating investigators in the ADCC. Investigators have the option of running their own data analyses in consultation with Data Management and Statistical Analysis Core personnel, or having the core personnel provide consultation concerning interpretation of results. The centralization of these functions makes it possible for each of the research projects to benefit from more sophisticated and flexible statistical and computer tools than would normally be available for any one project and fosters sharing of data among collaborating investigators in the ADCC. The centralized database incorporates all the data on subjects who have been evaluated for participation in the ADC and is updated continuously with the results of evaluations of new subjects and longitudinal information about all subjects. Al changes in subjects' mental and physical status, as well as key outcome variables of drug trials, other clinical studies, neurobiological and postmortem studies are recorded. The core has fully achieved and exceeding its original specific aims. A new local area network (LAN) computer system using all new equipment has been installed this year, and is used to implement centralized data management. The database now encompasses not only data gathered since the inception of the ADC but also all data collected prior to establishment of the ADCC in a uniform format with consistent labeling and data codes. A data resource has been achieved that is unique in both size and diversity. Individual investigators at NYU or elsewhere are able to use with the database for exchange and sharing of data, and procedures for sharing data with the NIA-Alzheimer's Disease Data Coordinating Center (ADCC) have been established. Integrating the formation about the behavioral, physiological and psychosocial components of the disorders being studied at the ADCC in one database has made it possible to develop a rich resource for study the normal and abnormal concomitants of aging.

The proposed research has its long-term goal of the development of a quantitative theory of discrimination learning and choice behavior in pigeons. The experiments are done in a choice situation in which one of a pair of stimuli is presented on each trial, and one response is rewarded if it occurs in the presence of one of these stimuli, and a second response is rewarded if it occurs in the presence of the other stimulus. A large number of the parameters that are known to affect learning in other situations will be systematically studied in this one. The proposed theoretical analysis is based on statistical decision theory.

CMV is a major cause of death for patients with AIDS. CMV pneumonia is often found at autopsy, but is rarely found antemortem for reasons that are not understood. As antipneumocystis prophylaxis is used more commonly, it is likely that CMV will become an increasingly important cause of morbidity and mortality. Effective anti-CMV drugs are now available. It is thus important to learn how to diagnose this entity invasively and noninvasively. This study will assess sputum, bronchoalveolar lavage, and transbronchial biopsy by a variety of conventional and novel approaches to CMV. Patients will be followed, but these data will not be used to determine therapy since their relevance is currently unknown. To date 24 asymptomatic patients with HIV infection have undergone bronchoscopy. Over 80% were culture positive for CMV. The importance of this study is that it is a comprehensive effort to determine how CMV pneumonia can be diagnosed antemortem: CMV is a major cause of death in AIDS patients.

This minimally invasive study is designed to explore various biologic effects of radiation on the pediatric CNS in an attempt to 1) obtain information on the pathophysiology of radiation-induced damage, 2) explore the association of neuropsychological deficits with biologic markers and neuroimaging abnormalities, 3) document changes in neurobehavioral functioning through longitudinal comprehensive neuropsychological assessments with comparison of various radiation therapy techniques, 4) describe changes in quality of life in pediatric patients who have received radiation therapy, and 5) attempt to identify children at increased risk of radiation-induced neurotoxicity. We are specifically evaluating effects on angiogenesis by measurement of angiogenesis related cytokines, proteins and cells including VEGF, bFGF, thrombospondin, TNF-alpha, IL-12, IL-8, circulating endothelial cells (CECs) and matrix metalloproteinases in blood and urine specimens. We are evaluating the changes in angiogenesis and vascularity by imaging, including dynamic susceptibility contrast perfusion and dynamic enhanced MRI scans. Neurotoxicity will be characterized by measuring biomarkers associated with neurotoxicity and documentation of changes in neurobehavioral functioning through longitudinal comprehensive assessments. In addition, we will describe changes in quality of life (QOL), assess changes in memory, define changes in ophthalmologic studies associated with radiation and detect changes in audiometry associated with radiation. Patients under 22 yrs of age who are referred to NCI for radiation therapy of a primary CNS tumor are eligible. Approximately 55 patients have enrolled and are followed longitudinally by a multidisciplinary team of specialists, including those from pediatric oncology, ophthalmology, radiology, audiology and neuropsychology.

This study will be conducted among a diverse group of urban and rural women between the ages of 21 and 44 years residing in eastern North Carolina. Women are studied as they are primarily shopping for and preparing family members'food, and thus have a major influence on family member's dietary practices. We propose to use a unique participant recruiting opportunity through a CDC-funded study in the local health department family planning clinic, to enroll a subset of participants from the larger study into a primary qualitative data collection effort to examine neighborhood context and "food activity spaces" as viewed by women of diverse demographic groups (Aim 1). In Aim 2, we will develop and implement a standard method for mapping farmers'markets. We will then use data collected in Aims 1 and 2 to conduct a secondary data analysis using a larger sample of women (n = 1000) recruited through the CDC-funded study in the local health department (Aim 3). The successful completion of this project will move the field of public health nutrition forward in the following three ways: (1) Determine the geographic boundaries and features of neighborhood and food activity spaces among diverse women by using participants to quantify and describe their activity spaces. This will allow future researchers to more accurately estimate buffer size in Geographic Information Systems. (2) Standardize the methodology for identifying farmers'markets, which will allow others to accurately quantify and disseminate information about markets to consumers and public health practitioners. (3) Provide greater clarity regarding disparities in access to farmers'markets and regarding potential associations between access to farmers'markets and obesity. PUBLIC HEALTH RELEVANCE: Obesity is an overwhelming public health problem necessitating community-level solutions. Here we propose to combine data from qualitative interviews, windshield tours, and a geographic information system to: (a) define and examine neighborhood context and "food activity spaces" as viewed by diverse women;(b) develop a high-quality measure of the food environment specifically related to farmers'markets;and (c) examine potential access disparities, as well as the association between access to farmers'markets and weight status among 1000 diverse women. Results will inform future research and promising environmental and policy changes to decrease obesity.

Project Summary: Small regulatory RNAs regulate gene expression in most eukaryotes. By regulating gene expression, small regulatory RNAs, play key roles in many biological processes that include development, genome defense, oncogenesis, and antiviral immunity. Small regulatory RNAs act by seeking out and binding homologous (target) RNAs in cells. By recruiting accessory proteins to target RNAs, small RNAs are able to control gene expression at many levels that include; translation, mRNA stability, and transcription. The mechanistic underpinnings of small RNA biology are widely conserved in most eukaryotes. In particular, small RNAs play an important role in regulating gene expression within most eukaryote nuclei. My lab has established systems that are allowing us to study how and why small RNAs regulate genes in animal nuclei. We are using the model organism C. elegans to understand how small RNAs regulate gene expression in animal nuclei. We are using C. elegans to address this question because of the excellent genetic tools that are available, and because of the robust and facile nature of conducting RNAi experiments in this system. Using genetic approaches in C. elegans, we have identified a molecular pathway that uses small RNAs to recognize and mark nascent transcripts (and the genes that encode these transcripts) for silencing. We have identified accessory proteins (termed the nuclear RNAi defective (NRDE) factors), which are recruited by small RNAs to nascent transcripts emanating from RNAP Polymerase II. Finally, we have shown that the association of the NRDE factors with RNA transcripts allows that NRDE factors to inhibit RNA Polymerase II during the elongation phase of transcription. Some of the NRDE factors that we have identified are conserved in mammals. In summary, our work is helping us understand how small RNAs regulate gene expression in animal nuclei, and may lead to insights into how small RNAs regulate gene expression in mammals. We are also interested in understanding why small RNAs regulate gene expression in animal nuclei. Small regulatory RNAs direct the covalent modification of DNA and histones proteins in most eukaryotic cells. These small RNA-mediated chromatin modifications are epigenetic in nature: they alter gene expression without changing the underlying in DNA sequence. We have shown that endogenous nuclear small RNAs, and the nuclear RNAi pathway, regulate the epigenetic landscape at ~1000 genes during the normal course of reproduction. In animals that lack the nuclear RNAi machinery, germ cells loose their immortal character. Thus, C. elegans uses endogenous small RNAs to regulate epigenetic states at many genes during the normal course of reproduction and this gene-silencing process is required to mediate important biological processes. Many other biological processes such as development, imprinting, X-chromosome inactivation, and paramutation are directed by epigenetic modifications on DNA and histones. Interestingly, non-coding RNAs also contribute to many, if not all, of these processes. Given the widespread connections that exist between small RNAs, non-coding RNAs, and epigenetic processes in eukaryotes, we believe that our research exploring how small non-coding RNAs regulate epigenetic landscapes in C. elegans may prove to be globally applicable to diverse epigenetic processes in animals. We do not yet understand 1) how the recruitment of NRDE factors to pre-mRNA inhibits RNAP II elongation to direct nuclear RNAi, 2) how RNAi-guided chromatin modifications contribute to nuclear RNAi in animals, 3) if/how nuclear RNAi is regulated, or 4) if the NRDE nuclear RNAi pathway is functionally conserved in mammals. Our proposed experiments are designed to answer these questions. .

Multiple sclerosis (MS) is a neurological disease of major socioeconomic importance with a suspected virus etiology. Theiler's murine encephalomyelitis virus (TMEV) provides one of the most relevant of the few available experimental animal models of virus-induced demyelination for the following reasons: a) chronic pathological involvement is limited to the white matter of the central nervous system (CNS); b) myelin breakdown leads to clinical disease; c) TMEV persists in the CNS for the life of the mouse; and, d) demyelination appears to be immune-mediated. Our studies during the initial grant period have defined genetic loci contributing to disease susceptibility, established an immune-mediated basis for the development of TMEV-induced demyelinating disease, and defined a critical role for MHC class II-restricted, virus-specific DTH responses in the demyelinating process. We have also demonstrated that chronic CNS demyelination can occur in the apparent absence of neuroantigen-specific autoimmune responses against the major myelin antigens. In addition, our studies have laid the groundwork for future studies on nonspecific (monoclonal antibody therapy) and specific (virus-specific tolerance induction) means of regulating virus and neuroantigen-specific CMI responses. Using in vivo-derived T cells and in vitro-propagated T cell clones/hybrids isolated from both peripheral lymphoid organs and CNS lesions, we propose to continue our research on defining the effector phenotype, epitope-specificity, T cell receptor usage and lymphokine-producing profile of the TMEV-specific T cell repertoire involved in the demyelinating process. We will also continue our studies examining the effects of and mechanisms responsible for both: nonspecific regulation of disease induction via therapy with monoclonal antibodies directed against MHC class I- and class II-restricted T cell subsets, T cell activation antigens, and T cell lymphokines; and, specific regulation of virus-specific immunity (both T cell and antibody levels) and disease induction via TMEV-specific tolerance/suppressor T cell induction. These studies should lead to a definitive understanding of the role of T cell- mediated immune responses in persistent virus-induced demyelinating processes and may be applicable to the understanding of the etiology and treatment of MS.

There are approximately 1.3 million US child care workers, most of who are hourly employees and classified as low-wage earners. In North Carolina, half of these workers are African American or Hispanic. Many have no health insurance, putting them at increased risk for many serious and chronic health conditions such as obesity, diabetes, and hypertension. Regular physical activity (PA) is known to decrease risk of many chronic diseases; unfortunately, most Americans, especially low-income individuals and minorities, fail to meet PA recommendations. A multi-level worksite-based PA intervention offered in child care is an innovative strategy to reach these low wage workers and address health disparities among this high-risk population. While the primary target of our intervention is worker PA, we expect that healthier and more physically active workers will be better role models and promoters of healthy lifestyles for children in their care. This two-arm, cluster RCT (n=104 centers, with 4 workers/center) will test the efficacy of a 6-month Care2bWell (CARE) intervention in increasing workers' PA compared to a Financial Empowerment (FE) control arm. The CARE intervention will include a kick-off workshop, where participants get tailored feedback about their health risks and set behavioral goals, followed by three 2-month long wellness campaigns, during which workers monitor PA, receive tailored feedback, and compete in teams for prizes. Center directors will take part in monthly webinars focused on improving the work environment to support PA and wellness. Within a social ecological framework, Perceptual Control, Social Support, and Diffusion of Innovation theories guide intrapersonal, interpersonal, and center- level intervention components. Participants in the FE arm will receive a parallel intervention to enhance financial health at the individual and center levels. The primary outcome will be accelerometer-measured minutes of moderate to vigorous PA. Primary and secondary outcomes (diet, anthropometrics, psychosocial variables, & center wellness environments) will be assessed at baseline, follow-up (6 mo), and maintenance (18 mo). Detailed process evaluation, cost-effectiveness, and meditational analysis will be used to assess mechanisms of behavior change at the individual and center levels.

Fluid secreted by the lacrimal gland is an essential component of tears and is estimated to contribute approximately 50% of the liquid volume bathing the cornea (Walcott, 1998). Recently in situ measurements of fluid production from the lacrimal glands of mice have demonstrated stimulated flow rates of 0.2-0.6 uL/minute (Walcott et al., 2002; Paranyuk et al., 2001; Moore et al, 2000). Our preliminary data indicates that the NKCC1 Na+, K+, 2CI- co-transporter and Bkca channels play important roles in fluid production of the lacrimal gland. We propose the use of knockout mice along with NZB mice (compromised fluid flow, Paranyuk et al., 2001) to study their respective roles in fluid production by the lacrimal gland. Further we propose the use of activators and inhibitors of PKC to delineate its role in the regulation of the NKCC1 co-transporter and Bkca channel. Both systems have been shown to be influenced/moduate/regulate by PKC (Standen and Quayle, 1998;Zhou et al., 2001; Clerice etal., 1995). Our proposal is focused on addressing the following hypotheses: Aim 1 Hypothesis: Basolateral blockade of the salt co-transporter (NKCC1) will significantly reduce stimulated fluid flow in controls but will be similar to NKCC1 knockout flow rates. We will also compare and contrast control and knockouts with NZB. We propose the use of NZB because our preliminary results indicates that the NZB acinar cells have significantly less amounts of NKCC1 than controls. Aim 2: Hypothesis: Apical membrane BKca channels contribute to normal fluid production of the lacrimal gland. We will test control (C57) and Bkca Beta 1 knockout mice. Aim 3: Hypothesis: PKC activity affects fluid production via regulation of K channels and/or NKCC1 transporters. We will test controls, NZB and the two knockouts (Beta1 and NKCC1).

A high-performance liquid chromatography (HPLC) method with electrochemical (EC) detection was developed for improved quantitation of carmustine (BCNU) in patients with brain tumor. Using this method, the plasma BCNU elimination (concentration-time) profile was characterised for a male patient who received 293mg of BCNU as an intra-arterial infusion over 45 minutes. The elimination of BCNU in this patient was found to be biphasic with an initial distribution phase occurring between 0 and 30 minutes after the end of the infusion. The new method is 50 fold more sensitive than the HPLC-UV method and permits direct on-column injection of plasma samples without extraction. Continued clinical studies of BCNU pharmacokinetics in brain tumor patients are in progress.

Gap junctions serve an essential role in the passage of molecules from the cytoplasm of one cell to its neighbor in both functional and homeostatic capabilities. They are defined as clusters of closely packed intercellular membrane channels embedded in the plasma membranes of two adjoining cells. The channels are composed of two hexamers of a protein (connexon) from a family of integral membrane proteins known as eonnexins. Here, we focus on the structure and function of connexin26 (Cx26), the smallest of the family. Mutations in the DNA sequence can result in hereditary sensorineural deafness and account for between one third to one half of the cases of prelingual inherited deafness in Caucasian populations. We have isolated preparations of Cx26 gap junctions in pure and sufficient amounts for biochemical and structural studies. These 2D crystals are amenable to electron microscopy (EM) structure determination and conformational dynamics as revealed with atomic force microscopy (AFM) done under hydorated conditions. In SPECIFIC AIM 1, we will determine the structure of the Cx26 hemichannel beyond 10 A using state of the art cryo-EM and improvements on image processing procedures. This involves improving specimen preparation, imaging at either liquid nitrogen or liquid helium temperature and implementation of a combined single particle/ 2D crystallographic approach to circumvent imperfect crystal lattices. In SPECIFIC AIM 2, we will construct Cx26 wild type and mutant cell lines with a tetracysteine domain genetic tag to improve isolation with FlAsH ligand affinity bead purification, stably express these in HeLa cells or in baculovirus-infected Sf9 insect cells and isolate the gap junctions or connexons for structural analysis using the methods developed in Specific Aim 1. We will construct two Cx26 mutants (P97L and T135A), each containing a single point mutation in one of the transmembrane helices that changes the effective pore properties. These mutations should be reflected in conformational changes in the 3D structure. In SPECIFIC AIM 3, we will expand coordinated AFM/EM experiments for visualizing conformational changes due to treatments known to close or alter gap junction mediated communication. Preliminary AFM images have visualized conformational changes at submolecular resolution. We have chosen five treatments that known to induce closure of Cx26 channels or hemichannels and are physiologically relevant. Conformational changes identified by AFM imaging will be further imaged using EM. Each of these goals is intended to complement the others and lead to structural and ph),siolo_ical models of Cx26 _ermane to the entire connexin family. _ERFORMANCE SITE(S) (organization, city, state) University of California San Diego La Jolla, California KEY PERSONNEL. See instructions on Page 11. Use Name SOSINSKY, GINA E. GAIETTA, GUIDO M. OSHIMA, ATSUNORI ADAMS, STEPHEN R. DOWNING, KENNETH H. FUJIYOSHI, YOSHINORI MOLLER, DANIEL J. NICHOLSON, BRUCE J. continuation pages as neededto provide the required information Organization UC SAN DIEGO UC SAN DIEGO UC SAN DIEGO UC SAN DIEGO LAWRENCE BERKEI.EY LAB KYOTO UNIVERSITY MAX-PLANCK INSTITUTE STATE UNIV. OF NEW YORK AT BUFFALO PHS 398 (Rev. 4/98) Page 2 Number pages consecutivelyat the bottom throughout the application. Do not use suffixessuch as 3a, 3b. in the format shown below. Role on Project PRINCIPAL INVEST CO-INVESTIGATOR POSTDOC (PROPOSED) CONSULTANT CONSULTANT CONSULTANT CONSULTANT CONSULTANT Principal Investigator/Program Director (Last, first, middle): The name of the principal investigator/program director must be provided at the top of each printed page and each continuation page. RESEARCH GRANT TABLE OF CONTENTS Page Numbers Face Page ......................................................................................................................................................... 1 Description,

The three-dimensional structure of DNA is quite dependent on sequence. This should be intuitively obvious from the sequence specificity required by many proteins for recognition. The few available x-ray crystal structures and NMR solution structures attest to this structural flexibility as well. The location and orientation of potential ligand binding functions on the DNA such as charges, hydrogen bonding and hydrophobic sites can be modified substantially from that which one might expect on the basis of assuming a canonical B-DNA structure. Consequently, we intend to continue development of the methodology for determination of high-resolution nucleic acid structures in solution and apply this methodology to some oligonucleotides and oligonucleotide complexes. This entails methods designed to improve the accuracy and resolution of the structures determined. Improved structures can be obtained with more accurate and more numerous experimental distance and torsion angle constraints, as well as improvements in calculating structure from these constraints. Enhancements will result from improvements in our iterative complete relaxation matrix program MARDIGRAS, development of a more encompassing density matrix approach for analysis of spectra derived from any pulse sequences (even those not yet invented), development of tailored excitation pulses, inclusion of experimental molecular motion information, and development of alternative methods of reducing experimental structural constraint data to structures. The latter includes (a) for restrained molecular dynamics simulations, use of improved force fields, empirical development of improved force fields, and use of constraint terms permitting a more realistic picture of conformational flexibility, and (b) development of an alternative restrained Monte Carlo method in torsion angle and helical parameter space, which is quite promising especially for structure refinement directly against NOE intensities. Applications will include oligonucleotides of interest, in particular sequences recognized by transcription factors or regulators, genome targets, antisense oligonucleotides, and a DNA microcircle duplex. Structures of proteins (including nucleic acid complexes) which are important for initiation or regulation of transcription will be determined. In particular, the 72-residue protein GerE which is a regulatory protein that binds specifically to a target site in promoter DNA will be the subject of study. Other proteins will be evaluated as possible candidates for study.

The primary objective of this proposal is to characterize an unusual bactericidal system in normal human serum. This system requires only early-acting complement components (C1, C4 and C2) and certain serum euglobulins unrelated to known components of the conventional or alternative pathways. The essential euglobulin components will be purified and characterized by means of various column chromatographic procedures, gel electrophoresis, isoelectric focussing and immunologic techniques. The purified euglobulins will be utilized to reconstitute the bactericidal system in the presence of the early complement components + antibody. An additional objective will be to determine the range of sensitivity to this "early complement pathway" of representative species from several medically important genera of enteric bacteria.

Johnson C. Smith University recognizes the tremendous benefits of an active research program to our overall educational atmosphere; therefore, released time is requested for those faculty who conduct supported research. The training of students in research techniques and the methodology of research is also a major goal of the program. This proposal consists of three projects which will involve ten students yearly and utilize the expertise of five faculty. Two of the proposed projects relate to opiate addiction including: (1) study of the role of brain enkephalins and adrenergic amines in the addiction process (Cain), (2) the development of new opiate-like compounds, followed by determination of their analgesic properties (Rowe-Anderson). Also proposed is (3) a biostatistical analysis of changing antimicrobial susceptibility patterns in a large community teaching medical center (Wright and Chopra).

DESCRIPTION: (Applicant's Description) Neuroblastoma is the most common extra-cranial solid tumor of childhood and only 40 percent of children with high-risk features at diagnosis survive despite intensive therapy. Goal. New approaches are needed to treat high-risk neuroblastoma since maximum dose intensity alone, or without hematopoietic rescue, has only modestly improved survival. Hypothesis. We hypothesize those new therapies that are not affected by resistance of tumor cells to chemotherapy, irradiation, or 13-cis retinoic acid will significantly improve treatment of these patients. Specific Aims. (1) To establish the safety, and within the confines of phase I studies, the efficacy (a) of cytotoxic agents with new mechanisms of activity or increased tumor specificity and (b) of biologic agents which may act on resistant tumors without overlapping toxicities with cytotoxic drugs. (2) To target in vivo levels of these agents using pharmacokinetic and dosimetric studies. Research Design. Cytotoxic agents to be studied include buthionine sulfoximine (BSO) with melphalan, a synergistic combination in which BSO sensitizes tumor cells by glutathione depletion. Melphalan will be tested in both non-ablative and marrow ablative doses. A tumor targeted cytotoxic therapy to be tested is 131 I-MIBG for specific concentrated radiotherapy delivery with ablative doses of carboplatin, etoposide, and melphalan and autologous stem cell transplantation (ASCT). Fenretinide, which is cytotoxic in vitro against drug and retinoic acid resistant cell lines, and which we have shown acts by increasing tumor cell ceramide levels, will be tested in combination with tamoxifen and possibly other agents that modulate ceramide levels. A humanized antibody/GM-CSF fusion protein directed against the GD2 antigen and capable of inducing high-level neutrophil cytotoxicity against neuroblastoma cells in vitro will be evaluated. Methods. These phase I studies will be performed by a consortium of nine institutions and will include (a) patients failing front-line phase ill trials, because of poor response to induction chemotherapy; (b) patients with progressive disease; and (c) those with minimal residual disease after myeloablative therapy. These studies will provide new approaches to improving outcome in children with high-risk neuroblastoma which can be tested in larger Children's Oncology Group phase III trials.

Fetal alcohol syndrome (FAS) is a major cause of learning and sensory deficits in people. There is growing evidence that abnormalities of neocortical function and plasticity underlie these deficits. Animals exposed to alcohol during the third trimester equivalent of human gestation and examined following a prolonged alcohol- free period were characterized by disruption of neocortical function and plasticity. However, the mechanisms by which prenatal alcohol exposure disrupts neocortical development and plasticity remain elusive. Neural plasticity in the neocortex is especially interesting in the context of the learning deficits that characterize FAS since it shares basic mechanisms with learning and memory, including a requirement for activation of the N- methyl-D-aspartate (NMDAR) receptor and the transcription factor cAMP/calcium-dependent response element binding protein (CREB), which regulates expression of genes required for cortical plasticity. Chronic alcohol exposure has important effects on NMDA receptor function, CREB activation and intracortical inhibition, all of which are crucial for cortical function and plasticity. The central hypothesis of this proposal is that these effects result in abnormal transmission of synaptic signals to the nucleus, disrupting activation of transcription factors that regulate expression of plasticity genes. The primary goal of this proposal is to rescue cortical plasticity in an animal model of FAS. The proposed studies will use molecular-genetic and pharmacological approaches to enhance transmission of synaptic signals to the nucleus of cortical neurons. The second major goal is to prevent developmental problems in the neocortex. The proposed studies will restore cortical plasticity and inhibition to normal level during and after the period when the animal is exposed to alcohol. Collectively, these studies should provide a new and exciting opportunity to elucidate how early alcohol exposure impairs cortical function and plasticity. The results of these studies may one day contribute to devise therapeutic interventions that will prevent or alleviate morbidity in FAS. [unreadable] [unreadable]

Our goal is to establish an innovative, world-class GEOHealth Hub in the Alto Mayo region of Northern Peru, a heavily deforested jungle region poised on the eastern edge of the Andes that exemplifies the dynamic and dangerous interface formed between remote traditional villages and emerging agricultural and population centers. New and old environmental issues are compounded in such regions, and the populace is not yet cognizant of the risks; lessons learned in the Alto Mayo will be readily extrapolated to similar interface situations in Peru and around the globe. Multiple environmental health risks of staggering magnitude are apparent there, but have received little objective scrutiny. Strong local traditional medicine remains pervasive, dating back millennia; modern medical care is developing. Infant and maternal mortality are alarmingly high. Through international collaboration, a GEOHealth Hub in this region would provide critical support needed by Peruvian health authorities to develop well-targeted, effective public health policies and educational strategies to address bona fide, quantified environmental/occupational health risks. We will build upon an on-going, uniquely advantageous partnership that has developed between the Peruvian Institute Nacional de Salud, the Medical College of Wisconsin (MCW) Division of Pediatric Pathology, the Milwaukee-based NIH/NIEHS-sponsored Children's Environmental Health Science Core Center (CEHSCC), the Yantalo Peru Foundation, and most recently the Oregon State University Superfund Research Program. Inherent strengths of this multidisciplinary group are magnified by new availability of a sophisticated clinical and scientific facility in Yantalo, Peru, located i the heart of the Alto Mayo, designed and operated by partnership members. Our research will focus on indoor/outdoor air quality, water/soil quality, and quality and safety of traditional medicinal plant preparations. Although health concerns of the broad populace will be addressed, the critical problems of infant/maternal mortality will be emphasized. PUBLIC HEALTH RELEVANCE: Through international collaboration, this partnership will identify needs and opportunities in environmental health in the remote, but heavily deforested Alto Mayo rainforest of Northern Peru. Perceived environmental hazards in this region are staggering, but poorly understood. Through innovative pilot studies and thoughtful planning for future studies and educational approaches we will help alleviate this alarming situation.

Cystic fibrosis (CF) is characterized as a defect of electrolyte transport of epithelial cells of exocrine tissues. Central to the pathophysiology of CF is the absence of cAMP- stimulated Cl- secretion and an enhanced rate of Na+ absorption. The cloning of the CF gene and characterization of the gene product, the cystic fibrosis transmembrane conductance regulator (CFTR), has revealed that CFTR functions as a cAMP-stimulated Cl- channel. Cloning of the CF gene led to the creation of an animal model for CF, the CFTR (-/-) knockout mouse. Interestingly, the CFTR (-/-) mouse did not exhibit a severe CF phenotype in many epithelial tissues, including the airways. Importantly, the absence of a CF phenotype led to the formal identification of the Ca2+-regulated, or "alternative" Cl- conduction pathway (Cl-A) that is molecularly distinct from CFTR and plays a protective role in preventing CF pathogenesis in the airways of the CFTR (-/-) mouse. This proposal focuses on Cl-A and hypothesizes that Cl-A is an important airway epithelial ion channel resident in the apical membrane and regulated by elevation of intracellular Ca2+. Cl-A will be investigated at three levels, 1) characterization of transepithelial Cl- currents, 2) regulation of Cl-A by Ca2+-mediated signal transduction pathways, 3) patch clamp identification of the Cl-A single channel properties. Confluent polarized epithelial preparations will be used to characterize the Ca2+-stimulated Cl- current and to identify the agonists and signal transducers that regulate this conductance. Patch Cl-Amp technique will be used to identify the Cl-A single channel properties that correlate with Ca2+-stimulated currents from CF murine airway epithelial cells. Importantly all single channel studies will be performed on cells grown on a permeablized support and only channels resident in the apical membrane will be studied. Finally, comparisons between the endogenous Cl-A and heterologously expressed candidate clones will permit the unequivocal identification of the Ca2+-activated airway Cl- channel. A complete characterization of Cl-A will lead to the genesis of new therapies for CF disease that would circumvent a defective CFTR.

Cell and tissue imaging core (Core C). This program project focuses on the effects of locoregional expression ofcytokines, and their effect on the tumor microenvironment. This demands study of the efficiency of delivery of tranduced cells, and multiple different analyses of the effects of these cells on the tumor. Central questions in projects are: 1) how effectively are cells delivered to the tumor? 2) Is the appropriate message and the protein product expressed by these cells? 3) is any therapeutic effect seen? This latter component includes study of cellular infiltration, angiogcnesis, tumor senescence, and cellular recruitment to the tumor environment. In each case these studies will rely critically on quantitative optical methodologies. The Center for Biolo_c lmaging, in which this core service will be performed, is designed with this function in mind. It is equipped to perform a continuum of optical methods including all types of light and electron microscopy essential to this program project. Within the scope of this project the principal goals of the core will be various. For example we will quantify and characterize cellular infiltrates into the tumor micro-environment. We will determine whether exogenously transfected cells reach tumor targets, quantify their abundance, determine whether these cells are expressing the transduced cytokines, and examine structural changes resulting from the expression of these cytokines. To perform these functions we will use a diverse array of microscopy technologies. At the light microscopic level these include: histological, immuno-histological, in situ hybridization, laser confocal, 2 photon and live cell technologies. Furthermore, our considerable experience in computerized image processing and morphometry will allow quantitative analysis of observed phenomena to corroborate earlier, possibly quite subtle qualitative changes. This core will be used extensively by all projects, though the imaging tools used will vary from project to project. In previous submissions of the proposal, this core was rated as superior, well integrated into the program and offering essential and integrated services to the program. As such the changes in this application are few, and are described in the introduction.

The Coronary Drug Project is a national co-operative research study testing efficacy of five therapeutic regimens in preventing death from recurrent myocardial infarction in a group of men who have suffered one or more documented myocardial infarctions prior to entry into the study. The five drug regimens under study are Dextrothyroxine, Nicotinic Acid, Clofibrate and conjugated Equine Estrogens in high and low dosage. In addition, there is a placebo group. Mortality serves as the primary end point with recurrent myocardial infarction and coronary insufficiency as some of the secondary and tertiary end points. The participants are being followed in a routine and systematic manner according to a common protocol. There is continuous safety and data monitoring of the entire research effort. The study design includes a minimum of five years of follow-up for each participant. All laboratory procedures relevant to drug efficacy and possible toxic end side effects are performed in a central laboratory. All data acquistion is submitted on standard forms to a coordinating laboratory for computer and statistical analyse. The Coronary Drug Project Aspirin Study initiated in 1972 is a double- blind clinical trial testing the efficacy of acetylsalicylate acid in reducing mortality and morbidity from recurrent coronary heart disease. Patients are followed in the same manner as the other Coronary Drug Project patients, with careful monitoring of possible toxic end side effects.

Our aim is to develop novel antibody conjugates for use in the radioimmuno-detection of tumors. In response to the problems associated with antibody-DTPA-radiometal imaging methodologies with regard to liver deposition of the metal, a number of more specific chelators will be investigated. Based on the capability to tightly bind the metal, and because of their greater lipophilic character and lack of excess functionality, enhancement of membrane transport of the ligand-metal complex is to be expected. The overall synthetic strategy will allow a large number of alternatives to be considered in the future, while the compounds close correlation with the ubiquitous natural products would not indicate any future cytotoxic problems. Antibody conjugation will follow protocols established in these laboratories and elsewhere. Successful completion of Phase I followed by fulfillment of the compounds potential in the Phase II in vivo studies; will lead to a new generation of tumor imaging agents.

Clinical observations and research data from animal models suggest that a delay of ovulation of the preovulatory folicle can result in defective embryonic development, abortion and birth defects. Recent data indicate that alterations in hormonal concentrations due to maternal age are responsible for the detrimental changes in the oocyte that lead to subsequent abnormal development. Estadiol rises early in relation to time of ovulation during an induced delay of ovulation or in old rats during a spontaneous delay of ovulation. Absorption of this early rise in estradiol with an antiserum during prolonged estrous cycles prevented the detrimental effects of delayed ovulation. Thus it appears that age associated changes in hormonal production can cause alterations in the preovulatory oocyte. The primary site of aging is not known and could be the ovary or the hypothalamus. The present project will use orthotopic ovarian transplants between young rats and old rats before they become acyclic to differentiate aging of the ovary from aging of the hypothalamus-pituitary unit in the alteration of those hormones responsible for defects of embryonic development. Also, pregnancis resulting from ovulations from reciprocal orthotopic transplants will be used to study the role of the aging ovary on developmental anomalies.

Strabismus is a misalignment of the visual axes which is relatively common in the general population with estimates of 5-6%. When this disorder occurs in infants, it can lead to severe deficiencies such as loss of central vision from one eye, known as amblyopia. Current therapies for restoration of visual alignment are based on weakening the relatively too strong antagonistic eye muscle, either by surgical recession or pharmacological denervation with botulinum toxin and muscle tightening by surgical resection. There is currently no therapeutic approach that strengthens the weaker muscle. The proposed research is based on recent evidence showing that the strength of muscles is regulated by trophic factors. Experiments were designed to identify trophic factors that either directly affect muscle fibers or provide feedback signals between eye muscles and innervating motor neurons. In the long-term, this project seeks to supplement surgical treatment of strabismus with a pharmacological treatment targeted at trophic interactions. Using an advantageous chicken animal model, we will test the hypothesis that an experimentally weakened developing eye muscle can be strengthened with trophic factors. Injections of trophic factors into selected eye muscles during a critical period of development may restore balanced eye movements by mimicking intrinsic trophic mechanisms. The effectiveness and duration of acute and chronic application of trophic factors will be explored. Additional studies will characterize adaptive responses (molecular, physiological and morphological) that are induced by denervation with botulinum toxin, mechanical denervation, or muscle lesion. Trafficking of trophic factors in mutant mouse models with motor neuron degeneration will be analyzed to identify underlying causes of motor degeneration. Weak and strong human eye muscles of different ages will be examined to compare expression of trophic factors and their receptors. Our studies will focus on trophic factors with established effects in the oculomotor system: glial cell line-derived neurotrophic factor (GDNF), insulin-like growth factors (IGFs), and cardiotrophin-1 (CT-1). A combined pharmacological, molecular, physiological and morphological approach, including the ultra-structural level, will provide a meaningful assessment of the prospects for a trophic, pharmacological treatment of strabismus and other eye muscle disorders as an alternative or supplement to current surgical and denervation procedures.

Our main research interests reside in the intracellular events -changes in signaling and in gene expression- which direct T-lymphocyte differentiation and survival, both during intrathymic T-cell development and in mature T-cells. Our first experimental approach focuses on T-cell positive selection, an intrathymic process which ensures the generation of an efficient T-cell repertoire by rescuing 'useful' thymocytes from programmed cell death and by inducing their differentiation into mature T-cells. While it has been shown that T-cell Receptor (TCR) engagement on thymocytes is necessary to trigger positive selection, it has remained unclear whether attributes of TCR signals (including their strength or duration) also dictate subsequent differentiation or survival events required for complete intrathymic maturation. To address these issues, we have generated a recombinant mouse model in which expression of Zap70, a tyrosine kinase required for TCR signal transduction, decreases in thymocytes that have been signaled to undergo positive selection. Thus, thymocytes in such mice can only receive transient, but not persistent, TCR signals. Using this model, we have shown that interruption of intrathymic TCR signaling in vivo (i) prevents thymocytes from completing their intrathymic maturation, despite the fact that they undergo initial differentiation events that characterize positive selection and (ii) precludes the differentiation of CD4-lineage T-cells. By breeding these mice with mouse lines carrying defined genetic modifications, we are currently investigating the intracellular signaling pathways which transduce differentiation and survival signals during intrathymic selection. As a second approach, we are developing conditional versions of intracellular signaling molecules that can be turned 'on' or 'off' by small-size ligands, to interfere with T-cell signal transduction in vivo and to monitor signaling and gene expression during the course of an immune response or during intrathymic development.

This work was aimed towards determining the biochemical function of calretinin. The initial results describe some of the basic characteristics of calretinin and will serve as the basis of further studies. These include the following: (1) Calretinin was identified in membrane fractions of rat cerebellum by both radioimmunoassay and immunoblot. Calcium produced an increase in the amount of calretinin associated with the synaptic membrane fraction and decreased the calretinin in cytosol relative to that found in the presence of EGTA; (2) Several approaches were used to search for calretinin target proteins. Iodinated calretinin was found to bind to proteins in several subcellular fractions on nitrocellulose blots of one dimensional SDS gels; (3) A purification procedure was developed for obtaining large amounts of homogeneous recombinant calretinin from plasmids. No differences were found in the physicochemical properties of native and recombinant calretinin (molecular weight, isoelectric point, calcium binding kinetics and behavior on ion exchange: gel filtration and hydrophobic columns); (4) A series of experiments used specific resins to study the hydrophobic properties of calretinin and the resins. A partial calcium dependence of calretinin binding observed for some resins (octylsepharose) was dependent on ionic strength and pH.

Approximately 70% of patients with Chronic Obstructive Pulmonary Disease (COPD) are obese or overweight. Tobacco smoking that causes COPD also is associated with other poor health habits that can lead to obesity and cardiovascular disease. Symptoms of COPD are often non-specific and include dyspnea and exercise intolerance. COPD treatment guidelines recommend stepped escalation of inhaled medications to improve these symptoms, but make little mention of the effect of co-existing obesity or weight loss interventions because of insufficient evidence. Cardiovascular disease is a leading cause of mortality among patients with COPD, and obesity is associated with important risk factors for cardiovascular disease including dyslipidemia, hypertension, and diabetes. Comprehensive lifestyle interventions that include calorie-controlled healthy eating, increased physical activity, and behavioral self-management strategies consistently result in modest, clinically significant weight loss and associated reductions in cardiovascular risk factors. That overweight and obese patients with COPD would not only reap similar clinical benefits from modest weight loss is an intuitive - but untested - concept. Therefore, we propose to conduct a highly valid, multicenter, patient-level randomized, pragmatic clinical trial to produce first-ever data on the effectiveness of a self-directed 12-month evidence-based lifestyle intervention targeting modest weight loss and increased physical activity among overweight and obese patients with COPD. We aim to serve as a Protocol Leadership Group (PLG) for the National Heart, Lung, and Blood Institute's Multi-Site Clinical Trials for the Pulmonary Trials Cooperative (PTC) in order to test if intervention participants have better outcomes through 24 months of follow-up compared to usual care control patients in terms of weight loss, exercise tolerance using the 6-Minute Walk Test, and dyspnea using the Borg dyspnea score. Secondary outcomes include, generic health-related quality of life using the SF-12, and major cardiovascular risk factors using Framingham risk score (including lipids), central obesity by waist circumference and blood pressure. We will oversee enrollment of 1000 patients at multiple clinical sites eventually chosen and contracted by the PTC's Network Management Core (NEMO). As the PLG for our proposed HOPE trial, we will cooperate with NEMO and will provide trial oversight, data management and reporting, and safety monitoring.

This research will examine the role of prolactin, estradiol and brain catecholaminergic and LHRH neurons in the age-related transition to irregular estrous cyclicity and eventual complete acyclicity. Experiments, using the 8-16 month old female rat as an expermental model, have been designed to determine which of the following working hypotheses is (are) the most likely explanations(s) for the onset of irregular cyclicity and acyclicity: (1) Prolactin and/or estradiol may become elevated in aging females and feedback to alter the cyclic profile of catecholamine-LHRH dynamics and cyclic gonadotropin release. (2) Age-related changes at the hypothalamic level may change the synthesis, metabolism and/or release of catecholamines and/or LHRH. The spontaneous neural changes result in an acyclic pattern of gonadotropin release. (3) Alterations in the responsiveness of the preoptic area to the positive feedback of estradiol may be the primary age-related change such that steroids become incapable of stimulating cyclic gonadotropin surges. (4) Changes in pituitary lactotroph sensitivity to dopamine and/or estradiol may occur during the aging process and lead to changes in prolactin secretion and ultimately to acyclicity. It is possible to test these hypotheses because of several recent methodological advances. A microdissection technique used in conjunction with sensitive radioenzymatic and radioimmunoassays for norepinephine dopamine and LHRH permit analysis of changes in these parameters in specific brain structures which are known to be involved in the basal secretion and surge of gonadotropin and prolactin. Catecholamine turnover rates (alpha methylpara tyrosine method) will be used as an index of neurona activity. New receptor assays for estradiol and prolactin allow examination of changes in receptor binding in the brain and pituitary with minimal pooling animals for tissue samples. These technique allow for neuroendocrine analyses to proceed at a greater resolution than previously possible such that the questions posed in this proposal may be approached.

ABSTRACT The human Brainspan data was created to identify all transcripts involved in neural development and to help understand of how specific risk genes affect human brain development. In addition, these data will have important clinical relevance for translational medicine; these data can help discern which risk alleles associated with psychiatric and neurological disorders influence transcription and alternative splicing across different regions and developmental stages. Also, most Brainspan samples were processing for whole-genome sequencing (WGS) and/or DNA methylation analysis, which enables direct comparisons of single basepair changes, copy number variation, and RNA editing events in the developing human brain. As such, Brainspan data holds biologically and clinically important data on the genetic and molecular mechanisms underlying the development and increased disease susceptibility of the human brain. To expand upon this resource, we aim to create a matched profile of the human brains RNA modification landscape (epitranscriptome), for both methyl-6-adenosine (m6A) and 5-methyl-cytosine (5mC). We will profile the developmental trajectory of the RNA modifications and their activity in non-coding regions and impact on splicing, RNA editing, AU-rich regulation of transcripts, and association with DNA methylation changes (epigenetics). Finally, we will also test the impact of these modifications from patient-derived iPS cells that will be grown and assayed over five time points. This will be accomplished over five years, and across 1,075 samples, across the Mason and Sestan labs, with collaborators at the Broad institute available to help with assays and access to GTEx data from adult brains with m6A profiles. We will achieve these goals across three main aims. (1) Create a neuro-developmental map for epitranscriptome sites and levels, with an emphasis on m6A and m5C, for 35 brains from four time periods, and five regions of the brain, chosen based on their large differences seen in the BrainSpan data and prior implication in neurological development. (2) Detail the inter-individual variation in epitranscriptome levels and their epigenetic regulation using m6A variation with the changes in expression levels, and then link epigenetic changes to altered gene expression and m6A regulation. (3) We will delineate the epitranscriptome changes in autism brains and manifestation in patient-derived iPS cells, including an examination of epitranscriptome variation across 30 banked Autistic brain samples and testing of the impact on disruption of the readers and writers of RNA regulation (on induced pluripotent stem cells). These will represent the first-ever epitranscriptome maps from primary tissue of Autism brains and help guide future studies that examine the dysregulation of Autism gene expression networks and epitranscriptome states.

Macular Edema contributes to many of the leading causes of blindness in America including diabetes, age related macular degeneration and uveitis. Currently, a wide number of studies reveal that altered expression of cytokines, including vascular endothelial growth factor and tumor necrosis factor act to increase blood vessel permeability. Further, research from our laboratory, as well as others, reveals activation of atypical protein kinase C isoforms are required for the permeability response for these and other permeabilizing factors. By screening a commercially available library, we have already identified a class of inhibitors for this target and in this grant we propose to combine medicinal chemists, structural biologists and cellular and molecular physiologists to develop compounds that control retinal blood vessel permeability in multiple models of eye disease. Importantly, measures of retinal function will mimic clinical assessments. The successful completion of these studies will provide a robust chemical pharmacophore, pharmacokinetic analysis, mechanism of action and in vivo effectiveness for atypical protein kinase C inhibitors to treat macular edema with specific leads available for clinical trials.

Core C: Data Management and Statistical Core Project Summary/Abstract Efficient, clean, and organized data, together with valid, flexible, and reproducible statistical design and analysis techniques, are an integral component to achieving the scientific mission of the UCI ADRC: to discover, quantify, and validate factors that influence the risk of AD across the lifespan. The overarching goal of the Data Management and Statistical Core (DMSC) is to provide logistical and intellectual support to all ADRC investigators at all phases of scientific projects. Data management support includes comprehensive data management and dissemination of data arising from the Uniform Data Set (UDS) and all ADRC projects. DMSC personnel are responsible for the development of National Alzheimer's Coordinating Center (NACC) approved collection forms, maintenance of the ADRC database, and the timely transfer of accurate data to the NACC. DMSC faculty and staff will also be intimately involved in the conception, design, implementation, analysis, and reporting of research conducted by members of the ADRC. The DMSC emphasizes regular communication with ADRC investigators and proactive involvement in ADRC-sponsored projects. The DMSC also promotes the development of independent research programs among its members. During the proposed research period, projects will include the development and validation of novel data collection mechanisms for harmonizing heterogenous Big Data streams, as well as the development of novel statistical methodology for efficient biomarker discovery and validation. These DMSC-specific research endeavors will increase the intellectual contribution of the DMSC to the scientific community at large, and will also lead to improved methods for collecting, entering, and analyzing complex data obtained through local ADRC projects. !

Translational research, bringing new laboratory findings quickly to improve prevention, treatment, quality of life, and survival for breast cancer patients, has been the focus of the team now forming the Baylor Breast Center for 25 years. During the first ten years of our SPORE, our tumor bank which made much of this rapid translation possible became a national resource, while basic cell and molecular biology research suggested new clinical implications for endocrine and chemo- therapy resistance, breast cancer prevention, metastasis, development of premalignant lesions, and roles for tumor suppressor genes BRCA-1 and -2 in DNA repair. Developmental projects ranged even further in seeking new translational possibilities. In this SPORE renewal, we build on the results developed in our earlier work and on new findings and new technologies, in several directions. (l) Compelling new data indicates that cross-talk from growth factor and stress pathways to the estrogen receptor and its coactivators may lie at the root of clinical resistance to tamoxifen, and suggests feasible clinical interventions. We will lay the preclinical groundwork for definitive trials and examine in clinical specimens the prognostic and predictive value of key intermediates from these pathways. (2) Our gone expression array studies offer the promise of a rational risk classification of DCIS. We will assess key markers indicated by these studies on a unique series of DCIS specimens, to evaluate their potential to predict progression to invasive cancer. (3) Tamoxifen can prevent ER-positive breast cancer in many high-risk women, but does nothing against the emergence of ER-negative disease. Based on ongoing treatment studies plus our own laboratory data, we now propose preclinical studies and an early clinical trial of the receptor tyrosine kinase inhibitor Iressa in chemoprevention of breast cancer in women at risk. (4) Our discovery that a single hypersensitive estrogen receptor mutant is found in many hyperplasias and in most node-positive breast cancers, but not in normal tissue, suggests that this mutant could be not only a powerful risk indicator but a uniquely specific treatment target. We will explore both possibilities. (5) Our preliminary expression array data in responders vs. nonresponders to neoadjuvant Taxotere implies that the gone expression profile might strongly predict clinical sensitivity or resistance. We will test in a randomized trial the ability of expression profiles to distinguish Taxotere from AC sensitivity, and confirm markers from these profiles in sets of retrospective clinical specimens. (6) Although important parts of our unique breast Tissue Resource were lost in last year's floods, much remains, and we propose important new accessions to further enhance this critical resource. (7) Pathology, Biostatistics, and Administrative Cores will also support key aspects of the proposed work. (8) Our highly successful Developmental Projects Program and Career Development Program will continue to encourage new ideas and new investigators in translational breast cancer research.

The goal of the MENTORS (Model Education Networks to Optimize Rural Science) Project is to actualize an exportable model of development and dissemination of culturally relevant and innovative programs that stimulate interest in and enhance preparation for careers in science, technology, engineering and medicine. Integrated components that will provide mentoring and career preparation include: summer research internships, science exploration camp, Field Experiences (FEs), and innovative classroom activities for students, and a rigorous professional development (PD) program for K-12 teachers. Our project will build strong partnerships between biomedical, engineering, public health and education researchers at Texas A&M University and K-12 teachers, school/district administrators, students and parents. MENTORS focuses on school districts that are rural and have a high proportion of students who are underserved and/or underrepresented in science and medicine. Such districts often lack programs to optimally prepare students for STEM careers. The multi-disciplinary Project team, which includes experts in biomedical, health disparities and biomedical engineering research as well as K-12 educational theory, curriculum development and evaluation, will work with classroom teachers to develop innovative, career based, educational modules focused on 21st century skills. The Specific Aims of the MENTORS Project are: 1. To provide authentic research and field experiences (FEs) for elementary, middle and high school students, designed to enhance STEM education and stimulate interest in and pursuit of a broad variety of science, technology, engineering and health-related careers. 2. To engage a network of K-12 teachers and biomedical, public health, biomedical engineering and educational researchers to develop learner-centered, career focused educational modules that will attract and motivate students, particularly those who are traditionally underrepresented in science, medicine and technology. 3. To provide rigorous professional development for K-12 teachers in TX, through an established and impactful Summer Institute (SI). All activities and products will be extensively evaluated using a mixed methods approach, with a variety of instruments designed to collect and analyze the most pertinent data. These will assess the effectiveness of the model as a whole, and of the components, in achieving the objectives of enhancing student interest and developing the skills needed for educational and career success.

The long term aim of the proposed research is to define the molecular structure and function of the midkine signal transduction pathway which regulates cellular proliferation and differentiation Special emphasis in this effort is placed on the molecular cloning of the cell surface Midkine signal transduction receptor which we have recently identify from a Wilms~ tumor cell line using biochemical techniques. Recent studies have established the importance of Midkine expression in Wilms~ tumor, renal cell carcinoma and lung, colon and hepatocellular carcinoma. Our studies have also shown Midkine expression in autosomal dominant polycystic kidney disease (ADPKD) and its role as an important mediator in epithelial differentiation during kidney organogenesis. Our hypothesis is that Midkine functions as an autocrine or paracrine which regulates several biological processes including the growth regulation of epithelial cells in ADPKD as well as in oncogenic transformation in Wilms~ tumor and in differential mechanisms which regulate epithelial conversion of renal progenitor cells during renal development. We plan to clone Midkine receptor gene using either an expression cloning approach or ligand affinity chromatography to purify the MK receptor protein. Additionally, we also plan to examine Midkine signal transduction pathway, then define the basic developmental pattern in the kidney of expression of all identified components of the midkine signal transduction pathway including expression of the Midkine receptor in relation to its ligand.

Bleomycin is the name which has been given to a family of antitumor antibiotics, all of which are closely related structurally and can be derived synthetically from bleomycinic acid. The bleomycins are of interest because of their clinically useful activity against, e.g. skin, lung and testicular cancer. We propose a total synthesis of bleomycin B2, MW 1342, having 19 asymmetric centers.

Aromatic hydrocarbons and their chlorinated derivatives are environmental pollutants that are, in many cases, toxic to most forms of life. Microorganisms that degrade toluene and naphthalene to carbon dioxide and water initiate metabolism by the enzymatic incorporation of molecular oxygen into the aromatic nucleus. These enzymes are called dioxygenases and are known to be multienzyme systems. The components of the toluene and naphthalene dioxygenases will be purified and characterized and studies on the mechanism of enzymatic oxygen fixation will be initiated. The products formed by the action of these dioxygenases on a variety of aromatic hydrocarbons and their substituted derivatives will be isolated and characterized by conventional chemical techniques.

Trichotillomania (TTM) is a chronic impulse control disorder characterized by pulling out one's own hair, resulting in noticeable hair loss. Although the disorder typically begins in early adolescence, there are few data available to guide treatment choice for youth with TTM. Our research group developed and empirically evaluated a manualized BT program for pediatric TTM (R21 MH61457 - M. E. Franklin, PI), completing what is to our knowledge the only randomized controlled trial (RCT) of any treatment for pediatric TTM. Results indicated a clear advantage for BT over a Minimal Attention Control (MAC) condition at post-treatment, with gains well maintained up to six months for those randomized to BT. With this encouraging preliminary work as our starting point, we now seek to replicate the findings in a larger sample and extend them by employing a more scientifically rigorous comparison condition, Supportive Counseling (SC), to allow us to rule out non-specific factors such as psychoeducation and therapist contact time as the reasons for TTM symptom reduction. Our primary specific aims are to: 1) compare the efficacy of BT to SC for treating TTM at post-treatment (week 8); and 2) examine maintenance of BT gains through an eight week maintenance phase (week 16). We also plan to: 3) examine maintenance of BT gains through a naturalistic follow-up phase (weeks 28, 40); and 4) explore predictors of acute (week 8) and long-term (weeks 16, 28, & 40) response to BT, including comorbidity, initial severity, and family psychopathology. Public Health Relevance: Development of an efficacious and transportable treatment for youth with TTM may provide front-line clinicians with specific strategies for helping these youngsters now. Moreover, effective treatment delivered closer in time to the onset of the disorder may serve as a form of prevention for the psychiatric sequelae that appear to develop in the wake of TTM in adults. The proposed study constitutes a logical and necessary next step in developing an adequate evidence base for BT in pediatric TTM, and lays the groundwork for future transportability studies where its effectiveness can be examined in the settings where most families can access pediatric mental health services. Development of an efficacious and transportable treatment for youth with TTM may provide front-line clinicians with specific strategies for helping these youngsters now. Moreover, effective treatment delivered closer in time to the onset of the disorder may serve as a form of prevention for the psychiatric sequelae that appear to develop in the wake of TTM in adults. The proposed study constitutes a logical and necessary next step in developing an adequate evidence base for BT in pediatric TTM, and lays the groundwork for future transportability studies where its effectiveness can be examined in the settings where most families can access pediatric mental health services.

Concurrent with the increased prevalence of overweight and obesity in the United States, a significant portion of older adults are increasingly at risk for developing metabolic dysfunction. Metabolic abnormalities can include insulin resistance or impaired glucose tolerance, dyslipidemia, hypertension, and abdominal obesity. These metabolic abnormalities tend to cluster together, forming what is known as metabolic syndrome (MetSy). MetSy is widely considered a "pre-disease" state associated with development of Type II Diabetes as well as cardiovascular and cerebrovascular disease. Several studies have proposed links between components of MetSy and neurocognitive and brain dysfunction;however, few studies have examined the additive effect of the cluster of risk factors that comprise the syndrome! The long-term objective of this study is to uncover evidence that individuals with MetSy, who by definition do not exhibit any overt disease process, will demonstrate subtle differences in performance and abnormal FMRI brain response on cognitive tasks. The specific aims of the study are to demonstrate subtle deficits in verbal working memory (VWM) performance and abnormalities in task-related BOLD FMRI brain response in individuals with MetSy. It is hypothesized that in comparison to a well-matched control group, individuals with MetSy: (a) will be less accurate and slower in their reaction times on the 2-back VWM test;(b) will exhibit differences in 2-back performance that will increase in relation to increased task difficulty;(c) will have reduced BOLD response in brain systems involved in VWM including the dorsolateral prefrontal cortex, supplemental motor area, and the posterior parietal cortex;(d) will exhibit compensatory responses in areas outside the primary VWM system. Additionally, it is hypothesized that increases in brain response in relation to task demand will be higher in individuals with MetSy. Relevance to Public Health In the United States, 24% of adults and nearly 44% of adults over the age of 50 years meet the clinical criteria for metabolic syndrome. Thus, this project will yield significant clinical application for millions of Americans with metabolic syndrome by providing evidence that cerebrovascular and cognitive function are affected by subtle vascular and non-vascular abnormalities before clinical signs of major disease exist.

Fragile X syndrome is the most common genetic cause of autism, occurring in 1 out of 6000 births. Affected patients also suffer from mental retardation and in some cases seizures. Current treatments involve the use of drugs to ameliorate mood and attention symptoms and to prevent seizures, but are not able to restore normal learning and emotional function. A molecular-level understanding of the neuronal defects in Fragile X syndrome will be necessary for the rational design of therapies to address the underlying cause of the disease. The protein mutated in the disease, the Fragile X mental retardation protein (FMRP), is required for regulating protein synthesis at activated synapses, the communication points between neurons. A large body of evidence suggests that the normal strengthening and weakening of synapses that underlies learning requires the careful regulation of protein synthesis by synaptic activity. Experiments have also suggested a role for FMRP in regulating both synaptic strengthening and weakening. However, the precise relationship between synaptic strengthening and weakening, protein synthesis, and FMRP is poorly understood. For instance, which proteins are synthesized during, utilized in, or required for synaptic strengthening and weakening, and which of these events are affected by FMRP loss, is not known. Research on the function of FMRP in activity-dependent local protein synthesis has been limited by the low sensitivity and resolution of methods for assessing and controlling protein synthesis in living neurons. We have developed new molecular tools that allow the real-time tracking and control of new protein synthesis and the visualization of kinase pathways involved in activity-induced protein synthesis. We propose to use these tools to examine the specificity of protein synthesis responses in synaptic strengthening versus weakening, and to study the effect of FMRP loss on these responses. We will also determine which new proteins are normally required for long-lasting synaptic plasticity, and how FMRP loss might alter those requirements. These studies will provide insight into the regulation and function of synaptic protein synthesis in persistent synaptic plasticity, identify potential molecular targets for therapeutic intervention, and produce new technologies that can benefit the larger neuroscience community.

Vitellogenin, the yolk protein precursor, serves as an excellent model protein to study the hormonal regulation of transcriptional, translational and posttranslational events. This proposal focuses on one such posttranslational event, the phosphorylation of over 90 serine hydroxyl groups in each vitellogenin polypeptide during its translocation through the secretary apparatus of the hepatocyte. Xenopus vitellogenin microheterogeneity will first be examined by resolving vitellogenin and yolk protein polypeptides and testing whether this heterogeneity arises from differential modification of a common polypeptide or rather from slightly different primary sequences. A variety of useful substrates, both in phosphorylated and unphosphorylated form, should be concomitantly obtained during this effort. Direct evidence will be sought that vitellogenin represents the principal endogenous substrate in liver microsomes from estrogen-treated animals, and optimum procedures will be defined for the extraction and purification of vitellogenin kinase activity from such liver microsomes. During kinase purification, the existence of multiple kinases will also be explored. Purified kinase(s) will be characterized, assigned to microsomal subfractions, and related to cytosolic "phosvitin kinase." Male liver microsomes will also be examined for vitellogenin kinase activity, and vitellogenin phosphorylation and processing will be examined in Xenopus oocytes microinjected with vitellogenin mRNA. These studies should accomplish several goals, including (a) elucidation of the relationship among the vitellogenin and yolk protein polypeptides and the recently described 4 vitellogenin genes in Xenopus, (b) identification of the mechanism by which vitellogenin is phosphorylated during its secretory translocation, (c) indication of the relationship, if any, between the vitellogenin kinase(s) and the ubiquitous "phosvitin kinases" described by numerous laboratories and for which there is as yet no known biological function, and (d) documentation of whether vitellogenin kinase is coordinately induced in liver by estrogen or rather is a constituent enzyme of the endoplasmic reticulum/Golgi elements of all xeropus cells. In addition, these studies will allow for the first time a direct comparison between vitellogenin phosphorylation and an analogous (perhaps even homologous) system: the phosphorylation of milk protein (caseins).

The Mouse Production and Ventricular Function Core (Unit C) has two major aims as dictated by Projects 1-3: 1) production of mutant mouse models of familial hypertrophic cardiomyopathy (FHC) and dilated cardiomyopathy (DCM) and 2) characterization of the phenotypes of these animals using both in vivo and in vitro methods. Production of mice will be accomplished in the laboratory of our co-investigators Drs. Jeffrey Robbins and James Gulick (Univ. of Cincinnati) using overexpression of transgenes linked to a cardiac specific myosin heavy chain promoter. Our collaborators Drs. Jon Seidman (Harvard Medical School) and James Lessard (Univ. of Cincinati) will provide additional mice with both myosin and actin mutations. In vivo phenotypic characterization will take place in the laboratory of Dr. David Kass (Johns Hopkins School of Medicine) by use of a miniaturized conductance/micromanometer catheter system for simultaneous measurement of left ventricular (LV) pressure and volume under near physiologic conditions. These measurements allow estimation of a variety of sophisticated parameters of systolic and diastolic function that have been previously characterized in larger mammals, including man. In vitro phenotypic characterization will be accomplished in the laboratory of Dr. Martin LeWinter (University of Vermont), using a buffer-perfused, isovolumically contracting, isolated heart preparation. The latter preparation provides estimates of a number of functional parameters under less physiologic but more controlled experimental conditions than are possible in vivo. It also provides a mechanoenergetic analysis of the efficiency and economy of the contractile machinery by relating its mechanical output (pressure-volume area or force-time integral) to its chemical energy input (oxygen consumption). In vivo and in vitro LV chamber properties delineated in the Core will be used to better understand how specific mutations result in a FHC versus a DCM phenotype in two ways: 1) they will be correlated with muscle strip (myofilament) and myofibril properties determined in Project 3 as a component of systematic integration of the results of all of the Projects and the Core at increasing levels of system complexity;2) by studying the temporal evolution of phenotypes in selected FHC and DCM models, we will determine which chamber properties are primary and which are secondary manifestations of specific mutations. The latter studies should lead to a better mechanistic understanding of the adaptations and maladaptations that occur in response to sarcomeric protein mutations.

The goal of the administrative core of the University of Colorado Lung Cancer SPORE is to provide outstanding administrative and fiscal support for the entire program effort and to provide the scientific leadership for the program. The administrative core will oversee all administrative and scientific activities of the SPORE program, review and regulate financial expenditures, develop and prepare reports. The Administrative core consists of the two SPORE principal investigators, Drs. Bunn and Miller; the SPORE executive director. Donna Berrier, a 40% grants manager/administrative assistant and 10% financial manager. This core will also develop and circulate research conference schedules, coordinate scientific review, schedule the monthly scientific meetings and aid project investigators in the preparation and publication of manuscripts as well as maintain a record of all publications emanating from this grant. It will oversee the planning and evaluation efforts including the scheduling of visits by the external advisors, the planning and coordinating of the yearly internal retreats and yearly NCI SPORE meetings, the scheduling of meetings and scientists, Executive Committee, Developmental Research Committee and Career Development Committee and the SPORE advocacy program.. The Administrative Core works with the SPORE investigators and NCI program staff to insure compliance with all federal regulations and reporting requirements. It will coordinate activities with the Cancer Center and with other SPORES to ensure that there is no redundancy, and to ensure joint projects are conducted in the most economical way. The Administrative Core will assist in community outreach efforts particularly with respect to public relations and community activities through the established Cancer Center mechanisms. The core provides support for the development and career development programs as well as Visiting scientist program. The Administrative core oversees the functioning of the other four core resources.

Monocyte-derived dendritic cells (DC) are currently used in clinical trials as carders of anti-cancer vaccines. As it has been shown that DC developing and maturing in different conditions show strong differences in their abilities to produce cytokines and to induce Th1, Th2, and CTL responses, it is likely that DC will also differ in their ability to exert antitumor therapeutic effect. Despite extensive in vitro characterization of distinct functional subsets of DC, their ability to induce immune responses of different character and magnitude has not been tested in vivo. We propose to develop a model system for evaluating and optimizing myeloid and plasmacytoid DC function in rhesus macaques. Based on previous results, we hypothesize that polarized myeloid DC1, grown in GM-CSF and IL-4 and which in vitro produce high levels of IL-12 and preferentially induce Th1 and CTL responses, will prove to be the most potent DC for stimulating Th1 and CTL responses in vivo. However, plasmacytoid DC have also been shown to induce Th1 and CTL responses, as have DC cultured in GM-CSF and IL-15. We propose to test the efficacy of DC generated in different protocols and at different stages of maturation, polarized by different sets of cytokines, as well as exposed to multiple Forms of antigen, to induce different classes of immune responses in vitro and in vivo. We will first develop protocols for generating rhesus DC, loading them with antigens, and inducing polarized phenotypes in vitro. The phenotype and functional capacities of these cells, including cytokines produced, will be characterized extensively. We will next test the different types of DC for efficient localization in T cell areas of lymph nodes after intranodal injection. Finally, we will test in vivo the ability of different DC types to stimulate polarized CD4 T cell responses to antigens and to stimulate CD8 T cells responses. Immunization strategies that provoke strong Th1-type responses will potentially be used for clinical trials being performed in other projects within this P01, and in future studies.

Since parent training as compared to direct clinic treatment is potentially a more economical and effective means of providing treatment, the field in general is moving in this direction as a method of improving the treatment of autistic children. Both the review of the literature and our pilot data provide compelling evidence that parent training is a plausible means of increasing the availability, intensiveness and effectiveness of the treatment as compared to direct clinic intervention. However, many of these results were based on post hoc analyses or case studies. The necessary systematic comparison has not been conducted. Such a comparison will hopefully allow us to assess the relative strengths and weaknesses of each of these approaches. This is important because it is necessary for the development of a treatment program encompassing the strengths of both approaches. Also, no one has yet analyzed the effect of either of these approaches on family members other than the autistic child. Without such an analysis, it is impossible to know, from the point of view of the family, whether or not the effect of the treatment is worthwhile. To evaluate the relative strengths and weaknesses of these two approaches (parent training and direct clinic treatment), and to assess the impact on the family, we propose to compare two groups. One group will receive the parent-training program, and no clinic treatment. A control group will receive direct clinic treatment for the child and no training for the parents. The measures which will be analyzed include: (1) direct measures of the child's behavior; (2) measures of parent/child interactions in both home and laboratory settings; and (3) measures of parental psychological and marital adjustment, and attitudes toward their child's potential.

The present proposal examines the principles governing language comprehension in reading. The proposed studies examine the principles underlying the syntactic analysis of sentences, principles determining the semantic interpretation of sentences, and the relation between them. The experiments all involve recording subjects' eye movements as they read sentences or short texts. Average reading time per letter in various critical regions of the sentence is used as a measure of processing complexity, along with the gaze durations associated with each word, total sentence reading time, and the probability of making a regressive eye movement which returns to portions of the text which have already been read.

The Stanford Proteomic Shared Resource is a state-of-the-art proteomics facility dedicated to providing rapid and low-cost support for proteomic methods to Stanford Cancer Center investigators. Services include protein purification, peptide and protein identification, and analytical characterization of modifications and other small compounds. The Shared Resource has the necessary scientific and technical foundation, as well as a clean physical plant to conduct high accuracy mass spectrometry. Substantial cost savings, efficiency, and scientific advancement is provided by this type of centralized facility. An advisory board provides recommendations on facility policies and activities, ensuring that this Shared Resource is aligned with the Cancer Center's strategic goals. In fiscal year 2005, the Proteomics Shared Resource has performed close to 600 proteomic analyses for 30 different laboratories, including 18 members of the Cancer Center, representing 6 of the 10 Cancer Center Programs. Use in fiscal year 2005 reflected an increase of 60% as compared to 2004. Future plans for the Shared Resource include expansion of capacity and capabilities with new mass spectrometers, an ongoing effort in improved and novel analysis software, and development of chromatographic strategies.