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It is well recongnized that ketone bodies are excellent fuels for energy production in developing rat brain. Our on-going studies have shown that ketone bodies are also good precursors for lipid synthesis in developing brain. Since hypothyroidism and maple-syrup urine disease, an inborn error in the branched-chain amino acids are known to cause retardation of mental development and function, the effects of these two conditions on the metabolism of ketone bodies in developing rat brain will be investigated. Specifically, the effects of hypothyroidism on the following parameters will be evaluated: (1) the development of ketone body-utilizing enzymes and of several key mitochondrial enzymes, (2) the distribution of these enzymes in the free-mitochondrial and synaptosomal fractions, and (3) the synthesis in vitro of cerebral lipids from ketone bodies and glucose. Hypothyroidism will be induced in new-born rat pups by injecting 131I. In an experimental model for maple-syrup urine disease, the effects of leucine and alpha-ketoisocapruate on the metabolism both in vivo and in vitro of ketone bodies and glucose by developing rat brain will be studied.

Objectives: The aim of the present study is to increase our understanding of the development of self-concepts in children and adolescents and to add to our knowledge of the social factors that influence self-image formation. Over the years psychiatry, psycholgy, and sociology have each contributed to knowledge of the structure of the self-concept, its place in the personality system, its bearing on cognitive, affective, and behavioral processes, and its determination by interpersonal interaction, culture, and location in the social structure. Our main purpose is to add to this accumulated body of knowledge by focusing on certain important issues especially in need of further research. First, we hope to learn more about self-concept change and development. Speculation on the biological, psychological, and social factors giving rise to self-concept change is ample, but systematic data are rare. A second aim is to subject certain products of clinical experience and psychoanalytic insight--in particular, the "idealized image"--to systematic data analysis and to learn how location in the social structure may affect its formation. A third objective is to examine certain important but generally neglected global dimensions of the self-concept; the stability of the self-concept is of particular interest. A fourth aim is to learn more about how membership in certain groups, statuses, or social categories are associated with various self- concept components. Although some literature on minority group status and on sex is available, many important questions remain unanswered or, if answered, are inadequately documented. Fifth, although it is contended that the maintenance and enhancement of the self are of prime motivational importance (Lecky, 1945; McDougall, 1932; Snygg and Combs, 1949; Scwartz and Stryker, 1971; Allport, 1955), hard knowledge about the concrete manifestations of these mechanisms is still inadequate. Our special objective is to examine further the selectivity mechanism; currently, speculative observations (Rosenberg, 1967) outrun systematic data. Finally, our aim is to shed light on certain other important but neglected topics, such as ego-extensions, identification, and self- values, or those having special practical import, such as academic achievement.

Relapse to alcoholism remains a vexing clinical and national health problem. Efforts to match alcohol dependent patients to specific treatments based on their clinical characteristics have produced mixed results. Pharmacogenetics (the study of genetic influences on therapeutic response to drugs) offers a powerful new tool to match specific elements of an individual patient's complex genetic blueprint with targeted pharmacotherapies to which that individual may optimally respond. The purpose of this proposed research is to apply pharmacogenetic techniques to predict which alcohol dependent patients will respond favorably to a trial of a selective serotonin re-uptake inhibitor (SSRI) for the prevention of alcoholism relapse. Our central hypothesis is that genetic differences affecting serotonin transporter function will influence an alcohol dependent individual's treatment response to the SSRI, citalopram. To test this hypothesis, we will perform a 14-week, randomized, double blind, parallel group comparison of citalopram and placebo in treatment seeking outpatients who meet DSM-IV criteria for alcohol dependence. All subjects will receive a single Motivational Interview and 9 brief sessions of a manual-guided Compliance Enhancement Therapy designed to promote treatment adherence and enhance motivation to quit or cut down on drinking. Post-treatment follow-up assessments will be conducted at 4, 12 and 24 weeks. Subjects' DNA will be genotyped to determine allelic variants in the promoter region of the serotonin transporter gene that have been found to markedly affect serotonin re- uptake and influence treatment responsiveness to SSRIs. We predict that individuals who carry two long variant alleles (1/1 homozygotes) of this polymorphism will exhibit a significant reduction in drinking days in response to citalopram compared with patients homozygous for the short variant allele (s/s homozygotes). To our knowledge, this will be the first study conducted in alcohol dependent patients to test whether pharmacogenetic differences in the function of the serotonin transporter (the site of action of these medications) influence the treatment response to a SSRI in nondepressed women and men. The study is designed to maximize the likelihood of finding treatment efficacy for citalopram over placebo by excluding subjects with severe alcohol dependence and marked impulsive traits in which SSRIs have not been found to be effective, controlling the exposure to the concomitant psychosocial intervention to minimize a psychotherapy ceiling effect, and by controlling the potential moderating effects of sex and cigarette smoking. The successful completion of this single center study may lead to future multicenter trials in more heterogeneous populations, and to studies using serotonin receptor subtype-specific medications.

Acute thromboses of arteries are usually a medical emergency. The long-term objective of this project is to develop a pulsed fluidjet system for intravascular use to hydraulically remove clots and debris from occluded arteries via small caliber angiographic catheters. We propose a pulsed fluid irrigation system combined with aspiration through the lumen of the catheter. A specialized pump, catheter, and control system will be developed. The pump will provide variable pressure, flow, and pulse pattern. The attenation of the pressure pulse in the catheter will be analyzed to optimize the jet. The controls will allow the physician to select various operating parameters for the cutting jet. The goal of Phase I is establish the feasibility of such a system through laboratory testing of catheters and determination of cutting parameters on tissue samples. During Phase II, the system will be fabricated and refined through in vitro and in vivo animal testing. The resulting system for clearing thrombi will have immediate commercial application and could improve the health care for millions of patients.

PROJECT SUMMARY/ABSTRACT Chemoresistant metastatic disease presents the most serious threat to cancer patients despite the increased arsenal of targeted therapeutic options available to clinicians. While intense study into late-stage cancer progression has revealed a number of mechanisms that contribute to chemoresistance, little is known about the intracellular signaling mechanisms that desensitize cells to cytotoxic chemotherapy. In an effort to address this knowledge gap, we recently performed a large-scale RNA-interference (RNAi) screen intended to comprehensively identify critical kinases and phosphatases in the human genome that alter or modify tumor cell sensitivity to chemotherapeutic agents. In this RNAi screen, we identified a novel phosphatase, MK-STYX, which potently suppressed the response of tumor cells to a wide variety of chemotherapeutic drugs. Our central hypothesis is that MK-STYX specifically controls mitochondrial function by regulating phosphorylation of the machinery required for ATP synthesis, and thereby serves an essential role in the induction of chemotherapeutic-induced cell death. The objective of this project is to determine how MK-STYX regulates cellular ATP levels, and thus modulates intrinsic apoptosis. We propose the following specific aims to address this hypothesis and to understand its significance in the context of metastatic colorectal carcinoma: (1) Identify the catalytic mechanism of MK-STYX in the mitochondria; (2) Identify the mechanism whereby MK-STYX regulates chemoresistance; (3) Establish the role of MK-STYX in colorectal cancer progression and chemoresistance. Consistent with our central hypothesis, we have shown that loss of MK-STYX increases ATP production. Therefore, we predict that the elevation in cellular ATP due to loss of MK-STYX is sufficient to inhibit apoptosome formation and entry into apoptosis. We have shown that MK-STYX interacts with two additional mitochondrial proteins and we will mechanistically determine the mitochondrial function and the molecular consequences of each of these interactions. We have also shown that loss of MK-STYX expression correlates with colorectal cancer progression. To determine whether loss of MK-STYX mediates chemoresistance in vivo, we will test the efficacy of standard chemotherapies on a colorectal xenograft model using cell lines that demonstrate variable expression of MK-STYX, or have been manipulated to decrease endogenous MK-STYX levels. We will also determine the prognostic significance of MK-STYX protein levels in a cohort of patients with colorectal cancer.

The vast majority of current human vaccines function by eliciting protective antibody responses. Neutralizing antibodies are an important component of protective immunity against a wide range of viral infections in humans and in animal models. T cell help to B cells is a fundamental aspect of adaptive immunity to viruses and the generation of immunological memory. Follicular helper CD4 T cells (Tfh) are the specialized providers of help to B cells. Tfh cells depend on expression of the transcription factor Bcl6. Tfh cells are important for the formation of germinal centers. Once germinal centers are formed, Tfh cells are needed to maintain them and to regulate germinal center B cell differentiation into plasma cells and memory B cells. There is compelling evidence that Tfh cells are limiting for the magnitude of germinal centers and hence limiting for the germinal center derived high affinity antibodies, memory B cells, and long-lived plasma cells that are the basis of long term humoral immunity. Therefore, there is substantial potential for an understanding of Tfh cells to facilitate better antiviral immune responses and vaccine-elicited humoral immunity. Despite these recent advances, our understanding of Tfh cells is still in the early stages, and the transcription factor (TF) pathways that control Tfh differentiation and define Tfli functions remain poorly understood. In Project 1, we will identify, characterize, stratify, and interconnect TFs that control Tfh differentiation and function to an acute viral infection, while also determining important TF regulators of Th1 differentiation.

In this proposal we are dissecting immunologic responses in the human respiratory tract that are part of the host's normal defense appartus. Airway derived proteins and phagocytic cells comprise the immune components of these responses and are retrieved from the lungs by bronchoalveolar lavage (BAL) during fiberoptic bronchoscopy. Immunoglobulin G is a particularly important element and we are concentrating on unraveling its role: initiating lung parenchymal inflammation, enhancing alveolar macrophage phagocytosis through opsonic antibody, and causing immune complex formation. Important to this analysis of IgG was to accurately quantitate it and other immunoglobulin and protein constituents of the airway milieu and to characterize the 4 gamma heavy chain subclasses within the IgG population. Lung washings (and blood) from patients with a variety of diffuse interstitial lung disease will provide a contrast with normal values. During the past year of this project, we have made substantial progress in 1) characterizing the relative amounts of immunoglobulins 2) developing better methods to isolate IgG antibody from lung fluids, 3) identifying inflammatory mediators secreted by alveolar macrophages and in BAL fluid and 4) adapting new methods for measuring Ig secreting plasma cells and respective gamma subclasses in BAL. In the next year, we plan to quantitatively measure IgG subclasses in BAL from smokers, nonsmokers and patients with diffuse interstitial lung diseases. In addition we will pursue the function of IgG opsonins derived from patients with cystic fibrosis and will investigate the regulation of chemotactic factors secreted by alveolar macrophages.

The objective of the proposed research is to develop new types of organic molecules that can bind metal ions, forming complexes that are soluble in organic solvents and membranes. In these new molecules, pyridine and other heterocycles are oriented toward the metal-binding cavity, cleft or channel by fusion of rings, forming the most highly preorganized ligands known. Eight large-ring or helical complexing agents are proposed for study. Six are torands in which a large ring is formed by complete fusion of rings. Four of these torands have already been synthesized. Studies of the first torand will be extended to include complexes with metals throughout the periodic table. Particular emphasis will be placed on complexes of heavy metals and lanthanides do may result in applications, such as metal sensors, luminescent markers and NMR imaging agents. A new torand synthesis will be applied to formation of "tubular" discotic mesophases (liquid crystals). Two new "expanded" or "mixed-heterocycle" torands are proposed and their complexation of large metal ions and transition metal clusters will be examined. Two helical ligands of different sizes are proposed for study and their binding of alkali metals will be examined. These helical ligands and the tubular liquid crystals are potential approaches to synthetic ion channels that could be used to model biomembrane channels or to produce permselective membranes. Double-helical or even triple-helical polynuclear complexes may be formed by these novel ligands. Analogues of these complexes might be developed to interact specifically with helical biomolecules, such as proteins and nucleotides. All complexes will be studied spectroscopically and crystallographically to improve fundamental understanding of the interactions of metals with biomolecules. The new complexing agents will be tested as ionophores in ion-selective electrodes for analysis of metals in biological fluids. The polynuclear transition metal complexes formed by expanded torands may be useful as metalloenzyme models and as polyfunctional or multiple-electron catalysts.

DESCRIPTION (adapted from application abstract): Dr. Diamond received his Ph.D. in 1992 (Cell/Dev Biology) and M.D. degree from Harvard University, where he worked with Timothy Springer, who describes him as `an extraordinary level of graduate student that one may see only several times in a lifetime.` The candidate has nineteen publications, eight of these as first author, the first four research done in college and the next fifteen from his Ph.D. work in immunology, focusing on the role of CD11b/CD18 on leukocytes. After graduating from the MD/Ph.D. program he initially began a post-doc to continue his study of integrins in the Drosophila genetic system, but then decided to continue clinical training in order to be able to combine clinical work with basic investigation. Having now completed a residency and in the midst of an infectious diseases fellowship, he requests funding to obtain new training in viral pathogenesis and molecular epidemiology so that he can apply his skills in a system that is new to him. Dr. Diamond is at the very highest level of prior research training and experience that can be considered appropriate for the K08 award. The career development plan is outlined for five years, with courses in epidemiology and biostatistics during phase I, followed by the period of independent research in phase II. The proposed research addresses the role of interferon in infection by dengue virus (DV), both to investigate how IFN modulates DV infection, and how DV alters the IFN response. The candidate proposes to use this award as a vehicle for learning a new area of research that will allow him to add virology and molecular pathogenesis to his knowledge base and to apply his prior research and clinical skills in a novel system.

Thin filament-associated actin-binding proteins control both actomyosin-based muscle contraction and cytoskeletal formation. To elucidate the mechanisms required for muscle thin filaments to function, it is crucial to determine the structural interactions of the regulatory proteins involved. As a means of achieving our objective to understand the physiology of cardiac, skeletal and smooth muscle control systems, we will examine the architecture of muscle thin filaments at a fundamental structural level and characterize the changing interactions of thin filament-linked proteins that regulate muscle activity. We will use state-of- the-art electron microscopy and electron tomography coupled with image analysis and 3D reconstruction to establish the macromolecular structure of actin-binding proteins on thin filament actin. Using these techniques: (1) We aim to determine the structural basis of troponin-tropomyosin regulation of cardiac and skeletal muscle activity by analyzing interactions of tropomyosin and troponin on thin filaments, which are governed by Ca2+binding to troponin and myosin-crossbridge binding on actin. To accomplish this goal, (A) we will test our newly proposed atomic model for troponin-tropomyosin localization on thin filaments by generating single particle and electron tomographic reconstructions;(B) we will test the hypothesis that mobile domains of troponin-I latch onto actin to constrain tropomyosin in the inhibitory "blocking" state characteristic of relaxed muscle;(C) we will test both the hypothesis that tropomyosin assumes the contours of the F-actin helix as a relatively stiff coiled coiled-coil and the alternative view that tropomyosin is flexible. (2) We will test the hypothesis that mutant cardiac troponin and different tropomyosin variants perturb muscle regulation by causing an imbalance in tropomyosin's position that alters the regulatory state of thin filaments. (3) We will assess the regulatory role of thin filament-linked caldesmon and calponin in defining tropomyosin position in vascular and visceral muscle. (4) We will determine the structure of nebulin bound to actin to complete our map of thin filaments. In each study, reconstructions fitted to the atomic resolution maps of F-actin will demarcate molecular contacts of binding proteins with actin at near atomic resolution ("hybrid crystallography"). Lay summary: Studies on troponin-tropomyosin regulated filaments, with particular attention devoted to normal and mutant proteins derived from cardiac muscle, will lead to an elucidation of the molecular regulatory mechanisms governing cardiac contraction, which is essential for tracing cardiovascular disease processes. Studies on smooth muscle filaments will aid in understanding the fine-tuning of smooth muscle contraction thus revealing key controls for vascular tone and pulmonary airway resistance, determinants in, e.g., hypertension and asthma. Actin filaments and associated proteins are major participates in diverse cellular systems, underscoring the broad significance of the proposed work. Our goal is to elucidate the control mechanisms that regulate cardiovascular and skeletal muscle activity. We will examine structural changes at a molecular level that are orchestrated by regulatory proteins and which control muscle shortening and force production. Understanding the underlying molecular physiology governing contraction and relaxation in heart muscle and blood vessels is key to deciphering cardiovascular disease processes, controlling blood pressure and identifying novel targets for drug development.

DESCRIPTION: (Applicant's Abstract) Despite advancing cure rates in childhood acute lymphoblastic leukemia (ALL), 25-30% of all patients eventually succumb to their disease. The long-term objective of the proposed research is to improve clinical outcome in this subset of patients. By early identification of patients who are likely to relapse, it should be possible to instigate potentially curative therapy in a more timely manner, thus boosting the proportion of long term survivors. This goal will be pursued through three interrelated projects. In the first, overexpression of WT1 and BCL-2 genes will be assessed as markers of minimal residual disease (MRD) in childhood ALL patients. The underlying hypothesis is that these two indicators are more widely associated with leukemia than current markers, and will significantly expand capabilities for prospective identification of high risk patients. Specific Aim 2 seeks to expand results obtained during the previous period of support, suggesting that immunologic monitoring of MRD has clinical utility in the assessment of childhood ALL patients. Immunologic findings in sequential bone marrow samples from patients with B- and T-lineage ALL will be compared with event free survival, as well as presenting clinical and biologic risk features, to establish the independent predictive strength of this assay. The data will also provide opportunities for cross comparisons with results of WT1 and BCL-2 screening in Specific Aim 1. Based on encouraging preliminary results, studies in Specific Aim 3 seek to assess the clinical utility of MRD investigations using peripheral blood instead of bone marrow. Success in this endeavor will radically improve remission studies in patients with ALL, by overcoming the practical and ethical constraints posed by sequential bone marrow aspirations in children. The clinical significance of MRD has been in doubt because of the lack of prospective studies in a large group of uniformly treated patients. The studies proposed in this application should meet that need and demonstrate the feasibility of clinical management decisions based on MRD detection in children with ALL.

The inflammatory bowel diseases (IBD) are chronic inflammatory disorders of the intestine with autoimmune characteristics. Although the pathogenesis of these disorders is poorly understood, many of their features point to a dysregulated T cell response as a major, if not dominant, etiologic factor. An understanding of the inductive events that regulate development of T cells of the gut-associated lymphoid tissues (GALT) under normal and inflammatory conditions will be required to better understand the evolution of these diseases and mechanisms by which they can be managed. Recent studies indicate that commitment to different cytokine/effector phenotypes by naive T cells occurs during the initial antigenic stimulation and is controlled by APC-associated molecules and cytokines. We propose that the abnormal T cell responses in IBD may follow from aberrant phenotype differentiation in the gut. A major focus will therefore be to define where antigen presentation to naive T cells occurs in the normal and inflamed GALT and how expression of costimulator molecules and cytokines in these sites contribute to induction of alternative T cell differentiation pathways. The experimental approach will be to use an alpha-beta-TCR transgenic (Tg)mouse with specificity for ovalbumin (OVA), as a model to allow control of antigen exposure to naive Tg T cells in vitro and in vivo. Novel in situ and immunohistochemical analyses will be used to determine single-cell T cell responses. The first aim is to examine the sites of OVA antigen presentation in the normal and inflamed gut and to determine the cytokine and proliferative responses of naive T cells in these sites. The evolution of distinct T cell phenotypes in the absence of the critical regulatory cytokine IL-4 will be examined by breeding the OVA TCR transgenes into mice carrying homozygous null mutations for IL-4. The second aim is to explore the hypothesis that tolerance to enteral antigens is initiated by antigen presentation on costimulator-deficient enterocytes. Initial studies will examine the APC function of isolated enterocytes for responses of OVA-TCR naive T cells and Th1 and Th2 clones. We will then examine the effects of costimulator expression on these cells in vivo and in vitro, by generating mice in which a B7 transgene is expressed on the small intestinal epithelium under control of the intestinal fatty acid binding protein (I- FABP) promoter. This transgene will be bred into the OVA-TCR Tg mouse and oral tolerance to OVA will be examined. The third aim will be to characterize the phenotypes of proinflammatory and protective GALT T cells divided on the basis of expression of the CD45RB isoform. By using T isolated from OVA-TCR Tg mice, we will be able to control exposure of the separate populations to antigen after adoptive transfer to SCID mice. This will allow us to dissect the earliest antigenic responses of these two populations during disease progression or prevention. In parallel studies, T cell clones of distinct cytokine phenotypes will be derived from OVA TCR Tg mice in vitro and will be examined for their capacity to produce or prevent inflammatory enterocolitis upon exposure to OVA antigen after transfer into SCID hosts.

The a9[unreadable]1 integrin is highly expressed in airway smooth muscle. Mice we have generated lacking this integrin only in smooth muscle cells have marked in vivo airway hyperresponsiveness and lung slices from these mice have increased airway narrowing. We have previously shown that the a9 subunit cytoplasmic domain directly binds the enzyme spermine/spermidine acetyltransferase (SSAT), the rate limiting step in catabolism of higher order polyamines, and that this association and polyamine catabolism are important modulators of a9[unreadable]1 function. Pharmacologic stabilization of SSAT also augments a9[unreadable]1-dependent prevention of airway smooth muscle contraction. In the current application we will systematically evaluate the effects of a9[unreadable]1 on responses of airway smooth muscle to multiple contractile agonists and to isoproterenol-induced relaxation using 4 parallel experimental systems (in vivo AHR, tracheal ring contraction, airway narrowing in lung slices and shortening of airway smooth muscle cells). We will utilize a variety of mutant and chimeric constructs of a9 and SSAT, in vivo and in vitro studies with SSAT knockout mice, catalytically inactive SSAT mutants and genetic and pharmacologic inhibitors to thoroughly examine what contribution interaction of a9 with SSAT makes to this response. Force generation in smooth muscle depends on calcium-dependent actin-myosin cross-bridging and parallel actin polymerization, and we will systematically evaluate the effects of a9[unreadable]1 on each of these pathways. Because the two major cytosolic effects of higher order polyamines are prevention of potassium efflux through Kir channels and activation of the lipid kinase, PIP5K1?, we will pay special attention to the roles of Kir channels and PIP5K1? in this process. The proposed studies will test the overall hypothesis that ligated a9[unreadable]1 normally serves as a brake on airway narrowing by concentrating SSAT, catabolizing polyamines and thus inhibiting potassium efflux and/or PIP2 production, resulting in reduced calcium oscillations, decreased actin-myosin cross-bridging and/or impaired actin polymerization. Abnormalities in this pathway, either acquired or genetic, could contribute to diseases such as asthma that are characterized by enhanced airway narrowing. PUBLIC HEALTH RELEVANCE: Project Exaggerated airway narrowing is a central feature of asthma. We have found that mice lacking a specific protein (an integrin) only in smooth muscle have exaggerated airway narrowing. In this proposal we will determine how this integrin normally prevents airway narrowing, a process that could be perturbed in diseases such as asthma.

This proposal seeks support for the training of Dr. Supriya Saha, a medical oncologist at the Massachusetts General Hospital (MGH) Cancer Center, toward his goal of becoming an independent physician-scientist with a focused clinical practice in hepatobiliary cancers and a research laboratory dedicated to the study of cholangiocarcinoma. Dr. Saha will be mentored by Dr. Nabeel Bardeesy, a leader in mouse models of gastrointestinal cancer, and Dr. Andrew Zhu, a clinical expert in hepatobiliary cancers. Intrahepatic Cholangiocarcinoma (ICC) has an average survival of < 1 year and has been increasing in incidence for several decades. With no effective targeted therapies or methods for prevention or early detection currently available, there is an urgent need for a greater focus on basic and translational ICC research. Recent studies have identified missense mutations in the metabolic enzymes isocitrate dehydrogenase 1 or 2 (IDH1/2) as the most common genetic alterations in ICC. Mutant IDH (IDH*) has been proposed to contribute to tumorigenesis through an intriguing mechanism whereby the IDH* enzyme produces an `oncometabolite', 2-hydroxglutarate (2HG), that inhibits a family of enzymes requiring alpha-ketoglutarate as a co-factor. Unfortunately, the lack of animal models of IDH*-driven ICC or human cell lines with endogenous IDH mutations has hindered elucidation of the oncogenic mechanisms and of effective therapies against IDH* disease. Over the last 3 years, Dr. Saha has worked in Dr. Bardeesy's laboratory to overcome these hurdles by defining the mechanism by which IDH* promotes ICC and generating the first genetically engineered mouse model of IDH* ICC. Simultaneously, Dr. Saha designed and implemented a clinical protocol to obtain fresh patient samples to generate a rich panel of human ICC cell lines and patient-derived xenografts. In collaboration with Dr. Cyril Benes, these tools were subjected to a high-throughput drug screen to identify a class of kinase inhibitors with potent and specific efficacy against IDH* ICC. During the award period, Dr. Saha will complete studies required for the effective translation of his work into an early phase clinical trial. This includes evaluating the se of targeted kinase inhibition in an autochthonous mouse model of IDH* ICC, using proteomic approaches to identify the specific targets and mechanism of action of kinase inhibition in ICC and exploring the potential mechanisms of resistance that may develop in this disease. This work will benefit from the breadth of expertise at MGH, which houses leading research groups in cancer genetics, molecular biology, signal transduction and cancer therapeutics, and was the first institution to identify IDH mutations in ICC. Given his extensive background in laboratory research, promising preliminary results and the superb mentorship he will receive, Dr. Saha anticipates applying for independent funding within the third year of this award.

The Na,K-ATPase is the plasma membrane enzyme that actively transports Na + and K+ ions against their electrochemical gradients, with the hydrolysis of ATP. It is thought to be the single largest consumer of energy in the central nervous system, accounting for 30-40% of ATP hydrolysis. Three different isozymes of the Na,K-ATPase are expressed in the CNS, and in some instances are rather strikingly localized within different cell types. Their individual properties and physiological roles are not yet well known. Available evidence suggests that the isozymes may differ in their affinities for ATP and cardiac glycosides, and in their susceptibility to regulation by unidentified intracellular factors. In addition, ischemic injury has been reported to cause a selective and rapid inactivation of one of two isozymes in the heart. The hypothesis of this proposal is that the Na,K-ATPase isozymes in the CNS are expressed in different cell types, where their affinity for ATP or susceptibility to regulation or degradation may either exacerbate or ameliorate ischemic injury and edema. The properties of the Na,K-ATPases may play a role in explaining the different vulnerability to ischemia of different classes of neurons. The proposal has several objectives: to determine which cell types express which isozymes; to investigate their functional properties and predict which isozymes will remain active in marginally perfused tissue; and to investigate their inactivation secondary to a rise in intracellular Ca2+. We will utilize immunocytochemistry with specific monoclonal antibodies to determine the cellular and subcellular distribution of the Na,K-ATPases in the rat CNS. We will separate the isozymes, and determine their individual affinities for ATP, Na and K+, as well as investigate the possible use of GTP as a substrate. Finally we will investigate the mechanism of modification or inactivation of each isozyme by Ca 2+-dependent proteolysis and by calnaktin-mediated Ca 2+ inhibition. This basic research should lay the groundwork for a rigorous analysis of ion movements, energy consumption, and their role in cell death and edema in ischemic brain, and contribute to a detailed understanding of ion pump function.

During the forthcoming year the following main topics will be studied: (1) The effects of electrical acupuncture conditioning on single unit activity evoked in the spinal nucleus of the trigeminal by stimulation of the infraorbital nerve. (2) The effects of drugs which block presynaptic effects on primary afferents on the conditioning effects of electrical acupuncture stimulation. (3) Efforts to determine at a microscopic level which peripheral structure or structures are activated when the demostrated conditioning effects are obtained.

Human polymorphonuclear leukocytes (PMNs or neutrophils) are essential to the innate immune response against invading microorganisms. In contrast to the acquired immune response, which is dependent on previous interaction with specific bacteria, the ability of PMNs to kill microorganisms is immediate and non-specific. Inasmuch as PMNs produce highly toxic microbicidal components, moderation of infection-induced inflammation is critical for limiting host tissue destruction. This moderation is especially important given that PMNs are the predominant immune cell in most bacterial infections. A key aspect of our research investigates how PMNs ingest and kill bacteria, and elucidates post-phagocytosis sequelae such as apoptosis, processes crucial for the resolution phase of inflammation. Thus, one of our research objectives is to elucidate molecular processes in human PMNs that facilitate resolution of infection. To that end, we used genomics methodologies to establish a global model of host cell-pathogen interaction that provides fundamental insight into the resolution of infection in humans. A second focus of research in my laboratory investigates how bacterial pathogens such as Staphylococcus aureus and Streptococcus pyogenes (group A Streptococcus or GAS) evade human innate host defense to cause disease. Although most bacteria are killed readily by PMNs, some human pathogens have evolved mechanisms to inhibit phagocytosis and death resulting from exposure to ROS and microbicidal products. For example, strains of S. aureus which produce Panton-Valentine leukocidin cause lethal necrotizing pneumonia in non-immunocomprimised individuals, the molecular basis for which is unknown. We hypothesize staphylococcal pathogenesis includes evasion of PMN killing and undetermined host-susceptibility factors. GAS successfully evades PMN phagocytosis and killing to cause human infections such as pharyngitis and necrotizing fasciitis (flesh-eating syndrome). To date, our studies include identification of genes and proteins used by S. aureus and GAS to evade destruction by human neutrophils, hence contributing to virulence, survival and pathogenesis.

Alcohol consumption and HIV infection are frequently co-existent pathologies. Muscle wasting is a common feature of both conditions. The alterations in immune responses resulting from chronic alcohol consumption have been hypothesized to enhance the transmission and acquisition of HIV or the progression from HIV infection to acquired immunodeficiency syndrome (AIDS). Based on available information, it is possible to speculate that these are either related to the direct effects of alcohol on the immune system, or are secondary to the impact of alcohol consumption on the nutritional state of the individual. Excess alcohol consumption is associated with a approximately 50% incidence of skeletal muscle myopathy. Alcohol consumption impairs the nutritional state of the individual either as a result of decreased food consumption or as a result of decreased absorption. affecting micronutrients which in turn have been shown to modulate circulating and tissue levels of growth factors. Hence the effects of alcohol consumption on muscle wasting appear to be multifactorial. Alcohol-induced myopathy appears to be predominantly the result of decreases in muscle protein synthesis, and is characterized by decreased weight, protein, RNA and DNA contents in skeletal muscle. The general hypothesis of the present proposal is that alcohol consumption accelerates and worsens the muscle wasting associated with HIV infection, leading to increased morbidity and mortality. Infection of Rhesus monkeys with simian immunodeficiency virus (SIV) has been established as an excellent model system for studying the pathogenesis of HIV-like infection. The disease is characterized by diarrhea, weight loss, lymphopenia, thrombocytopenia, and lymphadenopathy/lymphoid hyperplasia progressing to immunosuppression with marked reduction in CD4+ cells and in the CD4+/CD8+ cell ratio, and opportunistic infections. The aim of the present proposal is to characterize the time-course and relative contribution of alterations in muscle protein synthesis and proteolysis to the progression of muscle wasting associated with chronic alcohol consumption and SIV infection individually and combined. These studies will allow for the longitudinal investigation of the progression of the alterations in muscle metabolism beginning with a healthy, non-infected animal, throughout the acute infectious period and throughout progression to full blown AIDS. These will provide the preliminary data for a more mechanistic approach to the study of the etiology of alcohol-induced muscle wasting and its impact on a chronic infection.

Our lab is interested in understanding the fundamentals of centrosome biogenesis. We aim to uncovering the mechanisms that control the centriole duplication and centrosome maturation cycles. Through our own genome-wide RNAi screen and from other published screens, we now have a large number of candidate genes that play critical roles in these two cycles. What is lacking in the field is a true understanding of these proteins functions. Our lab uses a variety of biochemical and cell biological methods, including high resolution live cell imaging, to perform structure/function studies on known and candidate centrosomal proteins. One such protein we are currently investigating is the human Pericentrin ortholog, termed Pericentrin-Like-Protein in Drosophila. Our in-vivo structure/function analysis of PLP has revealed many new functional aspects of this protein, which are most likely conserved from Drosophila to Humans. Similar experiments are being carried out on a group of proteins that have been shown to preferentially localize to either the mother or the daughter centriole. In support of this project, we have generated many molecular tools and transgenic animals that will be used to perform standard cell biological experimentation in combination with genetic loss-of-function analysis using RNAi in cell culture and allelic analysis in the animal.

Human serum enzyme assays for clinical diagnosis usually make use of measurements of catalytic activity. In many situations, this may not be ideal; e.g., isoenzymes reflecting different damaged tissues may be indistinguishable or inactive enzymes may be released. Radioimmunoassay offers a very sensitive and specific way in which to measure enzyme concentration and to distinguish isoenzymes. Creatine kinase was chosen as a model for this approach because of its clinical importance and its simple isoenzyme distribution. The enzymes from human skeletal muscle (MM) and brain (BB) have been purified, used as immunogens to raise antibodies, and labeled wth 125I. Radioimmunoassays for each of these enzymes were developed and were found to correlate well with standard enzyme assays in clinical use. An attempt is now being made to develop a specific assay for the hybrid MB species.

Approximately 10-20% of adults experience chronic abdominal symptoms (abdominal pain/discomfort and associated bowel changes [constipation and/or diarrhea] compatible with a diagnosis of irritable bowel syndrome (IBS). In the US as well as other western countries, women seek health care services disproportionately to men. IBS is defined as chronic abdominal pain/discomfort accompanied by altered bowel pattern (diarrhea, constipation or both [mixed]). The public health impact of IBS in the US is enormous with direct and indirect costs totaling approximately $6 billion/year. Studies suggest an interplay between increased gastrointestinal (GI) permeability ('leaky gut'), abnormalities in the composition of the GI microbiome (defined as bacteria, their genomes and interaction with the host), altered immune responses, autonomic dysfunction (high sympathetic tone), and psychosocial distress lead to the symptoms and the subsequent functional impact of IBS. Our long term goal is to delineate the contribution of pathobiology and psychosocial distress to better inform patient management e.g., diet versus cognitive behavioral approaches). We propose to compare GI microbiota, intestinal permeability, cytokines, and GI and psychological distress symptoms in menstruating women with IBS and without IBS. We also will derive important information about women with IBS with no evidence of abnormal GI markers. The current proposal will also allow us to define microbiome composition and pathophysiological features tied to symptoms, and likely etiology. Thus, the first specific aim is to compare GI microbiome, permeability and cytokines in women (18-45 yr. of age) with IBS (n=100) vs. Healthy Controls (HC) (n=50) without IBS. Based on pediatric data, we hypothesize that among women with IBS, those with a GI microbiome enriched with Proteobacteria/Enterobacteriaceae, increased GI permeability or increased cytokine levels will have greater abdominal pain symptoms versus those without this Proteobacteria/Enterobacteriaceae enriched microbiota composition, normal permeability, or lower cytokine levels. The second aim will be to compare abdominal pain, other IBS symptoms and psychosocial distress symptoms in those IBS subjects with abnormal versus normal GI biomarkers. In addition we will explore patterns of associations among GI biomarkers, and of GI biomarkers with abdominal pain and other symptoms. The goal is to identify possible IBS subgroups based on biomarkers. This research is innovative because we will use biomarkers (e.g., GI microbiome analyses, permeability, and serum markers applied novelty to characterize pathobiologically what heretofore has been primarily a phenotypically and arbitrarily defined condition. We will integrate psychosocial factors (e.g., anxiety, somatization, co-morbid functional conditions) that our preliminary data suggest are likely to predominate in some women with IBS.

This proposal is submitted to help organize and conduct the 1989 Gordon Research Conferences on Calcium Phosphates, to be held July 24 to 28 at Salve Regina College, Newport, Rhode Island. This meeting is the ninth in a continuous series and allows the interaction of approximately 135 international scientists from universities, industry, and government who study chemistry, biology, physics, dentistry, medicine, biomaterials, mineralogy, oceanography, geology, soil science, environmental science, and related fields and share a common interest in calcium phosphate research. The general theme of the 1989 Conference is "Calcium Phosphate Research - Linking the Basics to Applications," and topics planned for discussion include (1) the nucleation and growth of calcium phosphates; (2) interactions between calcium phosphate minerals and organic matrices in biological systems; (3) remineralization of tooth enamel and root; (4) physicochemical mechanism of dental caries; (5) synthesis, fabrication and properties of new calcium phosphate biomaterials; (6) interactions between calcium phosphate implants and tissue; (7) roles of calcium phosphates in diseases in which mineralized tissues are destructed or pathological calcified deposits are formed; (8) calcium phosphates in industrial and environmental processes; and (9) new experimental techniques in calcium phosphates research. The strength of this Conferences lies in the fact that scientists with highly diverse backgrounds bring their expertise together to exchange scientific information, and generate new approaches for research solutions for problems in all areas of calcium phosphate studies. The meeting thereby contributes to the scientific cooperation and understanding between laboratories both within and outside this country.

Studies of the biology of mucosal HIV-1 transmission are limited because the circumstances of viral exposure usually cannot be observed and can never by controlled. Nonhuman primate models are limited because massive titers of SIV are required to establish infection after mucosal challenge and sexual transmission between macaques has not be observed despite frequent sexual contact and the presence of infectious virus in genital secretions. Serological data from the mangabey colony at Yerkes were analyzed during this study period. This analysis indicated that the risk of seroconversion began at the age when mounting behavior is initiated and rates of seroconversion are higher in female mangabeys compared with males. A series of experiments were planned to test the hypothesis that SIV is sexually transmitted among mangabeys and to determine if sooty mangabeys could be a useful model for studies of viral selection during sexual transmission and for the evaluation of topical virucides for the prevention of viral transmission. These studies may greatly facilitate vaccine design and the development of topical agents for the prevention of sexual transmission of lentiviruses.

Summary: The purpose of the research is to study the cellular and molecular mechanisms of adaptation to stress with emphasis on the regulation of the various components of the hypothalamic pituitary adrenal (HPA) axis. This includes the expression of hypothalamic corticotropin releasing hormone (CRH) and vasopressin (VP), pituitary CRH and V1b VP receptors, and adrenal steroidogenesis. At the hypothalamic level, in vivo and in vitro studies suggest that cAMP can mediate positive and negative regulation of CRH transcription. Activation of CRH expression by cAMP is markedly dependent on downstream elements in the CRH gene. Stimulation of cAMP production by forskolin increased luciferase activity driven by the CRH-promoter. This was caused by increases in transcription, shown by nuclear run-on-assay, as well as increases in mRNA stability and translation, shown by experiments using tetracyclin inducible constructs. Addition of the 3 prime untranslated region downstream of luciferase in a CRH promoter driven reporter construct markedly potentiated foskolin-stimulated CRH luciferase activity due to a potentiation of cAMP-stimulated mRNA translation. Concerning negative regulation, in vivo studies showed that stress induces expression of the CREM isoform, inducible cAMP early repressor (ICER) in the hypothalamic paraventricular nucleus (PVN). Studies using the recently characterized cell lines (4B and H32) revealed that transfection of ICER markedly inhibit cAMP-induced stimulation of CRH promoter activity after cotransfection with a luciferase reporter gene driven by a CRH promoter fragment. Western blot analysis of nuclear extracts of cells incubated with forskolin revealed time dependent increases in ICER. This paralleled formation of ICER-CRH CRE complexes in the electromobility gel shift assay (EMSA) suggesting that endogenous levels of ICER can interact with the CRH CRE. EMSA using hypothalamic nuclear extracts of control and stressed rats and CRH CRE radiolabeled oligonuclotides showed several shifted bands, of which the two lower bands were supershifted with CREM antibody. Extension of studies on immune-neuroendocrine interactions showed that alterations in fluid homeostasis secondary to endotoxemia are due primarily to renal insensitivity to VP rather than defective hypothalamic VP secretion. These studies revealed marked and sustained decreases in VP V2 receptors and aquaporin 2 in kidney medulla following LPS induced endotoxemia, and that these alterations lead to impaired capacity to concentrate urine. Transcriptional and post-transcriptional mechanisms regulating the number of CRH and VP receptors in the pituitary play an important role in the control of HPA axis activity. An essential element regulating V1b receptor transcription is a GAGA box located in the proximal promoter, which binds a protein complex found in pituitary nuclear extracts. Stress causes transient increases in GAGA binding activity an effect possible mediated by VP. Experiments using the hypothalamic cell line, H32, which expresses endogenous VP receptors showed that VP caused a rapid increase in GAGA binding activity followed by a decline. This effect of VP paralleled changes in ERK phosphorylation and it was mimicked by epidermal growth factor (EGF) and blocked by inhibitors of the EGF receptor and MAPK pathway. While the early increase was blocked by intracellular calcium chelators, the declining phase was prevented by protein kinase C inhibition. Studies using receptor subtype specific analogs or different cell lines transfected with either V1a or V1b receptors showed that the pathways involved in transactivation of the MAPK pathway by VP are cell specific and independent of the V1 receptor subtype. This data suggest that VP released into the pituitary portal circulation during stress activates V1b receptor transcription through transactivation of the EGF receptor. At the translational level, studies showed that the 5 prime untranslated region (5?UTR) of the V1b receptor mRNA contains elements, which can inhibit or stimulate translation. While the presence of upstream open reading frames in the 5 prime untranslated region play a role maintaining low translational activity in basal conditions, an internal ribosome entry site (IRES) can initiate translation independently of cap. Stress conditions which upregulate V1b receptors stimulate IRES activity through protein kinase C and PI3 kinase dependent pathways, providing a mechanism for rapid stimulation of V1b receptor translation to meet physiological requirements.

SUMMARY/ABSTRACT Reverse-phase protein arrays (RPPAs) offer a powerful functional proteomic approach to investigate molecular mechanisms and response to therapy in cancer. MD Anderson Cancer Center has been a leader in the implementation of this antibody-based technology that can assess many protein markers across large numbers of samples in a cost-effective, sensitive and high-throughput manner. The platform currently assesses ~300 protein markers, covering all major signaling pathways and most drug targets. Its utility was demonstrated through its selection as the sole platform for characterizing >10,000 patient samples through The Cancer Genome Atlas (TCGA); and recently it has been designated as one of two NCI Genome Characterization Centers, and will characterize up to ~10,000 samples from ongoing NCI initiatives and other consortium projects. For TCGA project, the applicants built The Cancer Proteome Atlas (TCPA), a web platform for visualizing and analyzing RPPA data, which has a community of >5,000 users worldwide. The long-term goal is to promote the ability of functional proteomics to impact cancer research and the development of relevant therapeutic strategies. The current objective is to expand the scope of TCPA by adding new functionalities and datasets, and to enhance and improve its existing analytic capabilities. Working relationships have been formed to link TCPA with other widely used bioinformatic resources (e.g., cBio, UCSC Genome Browsers, Firehose and Synpase) and other ITCR projects. An experienced, multidisciplinary team has been assembled to pursue four specific aims: Aim #1. Develop an open source, all-in-one software package for processing RPPA data. This effort will standardize each informatic step for RPPA data generation including experimental design, quality control, and data normalization. The resultant program will be exported to other RPPA facilities. Aim #2. Expand and enhance our existing web platform for the analysis of patient-cohort RPPA data. The web platform will cover other patient cohorts, incorporate other types of molecular/clinical data, and provide pathway/network-based analytics. Aim #3. Build a user-friendly, interactive, open web platform for the analysis of cell line RPPA data. This effort will collect and compile RPPA data of >1,500 cell lines, and develop a web platform parallel to Aim #2. Aim #4. Promote TCPA and active interaction with the user community. This effort will provide documentation, hands-on workshops, and bug fixes, and build web APIs for interaction with other tools. The expected outcome is the first, dedicated bioinformatic resource that fully integrates RPPA data generation, analysis and user feedback, allowing for fluent exploration and analysis of high-quality proteomic data in a rich context. The project is important because it will greatly enhance the quality and reproducibility of RPPA data from important consortium projects; substantially reduce barriers biomedical researchers face in mining complex functional proteomic data; serve as a hub for integrating proteomic data into other widely used bioinformatic resources; and directly facilitate development of protein markers for precision cancer medicine.

Transcription factors in the nuclear factor ?B (NF-?B) family are evolutionarily conserved master regulators of immune and inflammatory responses. They are activated in response to ligation of many receptors including T-cell receptors, B-cell receptors, members of the tumor necrosis factor (TNF) receptor superfamily and the Toll-like receptor/interleukin-1 receptor (TLR/IL-1R) superfamily. The I?B kinase (IKK), comprising IKKa and IKK, is at the heart of NF-?B activation and mediates two NF-?B activation pathways. The canonical NF-?B pathway is triggered by microbial and viral infections and pro-inflammatory cytokines and is dependent on IKK phosphorylation and activation. The alternative pathway is triggered by certain members of the TNF cytokine family and selectively activates IKKa. Activated IKK phosphorylates I?Bs, leading to their polyubiquitination and subsequent degradation by the proteasome. The freed NF-?B dimers translocate to the nucleus to mediate transcription. Because of its importance in NF-?B activation, IKK, especially IKK, has become a potential therapeutic target for many human diseases. The regulatory protein NEMO (also known as IKK? or FIP-3) interacts with IKKa and/or IKK to form the IKKa, IKK or IKKa/ holo-complex. The intact IKK holo-complex is approximately 700-900kD in molecular mass containing multiple copies of IKK2 and NEMO. IKKa and IKK both contain the following conserved recognizable domains: a kinase domain (KD), a leucine zipper domain (LZ), a helix loop helix domain (HLH) and a C-terminal NEMO-binding domain (NBD). NEMO contains an N-terminal kinase-binding domain (KBD), a minimal oligomerization domain (MOD) that is also the ubiquitin binding domain (UBD) and a C-terminal zinc finger domain (ZF). IKK and NF-?B signaling has attracted tremendous attention with more than 30,000 papers published on the subject. Despite the biological importance, not a single successful structure determination has been reported on IKK, an indication on the difficulty of the project. To elucidate the molecular basis of IKK function and to assist the discovery of IKK inhibitors, we propose a series of structural and functional studies on IKK, in particular, IKK and its regulatory protein NEMO. Public Health Relevance: The I?B kinase (IKK) is at the heart of NF-?B activation and a potential therapeutic target for many human diseases. The proposal seeks structural studies of IKK, which will enhance our understanding on the molecular basis of IKK function. In addition, the proposed studies will provide a structural basis for discovery and optimization of IKK inhibitors in the treatment of inflammatory diseases and cancer.

Progress in healthcare research depends heavily on demonstrations of efficacy and safety via clinical trials. Bayesian methods offer a valuable alternative mode of analysis for clinical trials. By comparison with standard frequentist methods, Bayesian methods provide more interpretable outputs, use all the available evidence, and lend themselves to the more complex analyses demanded by complex healthcare research. Unfortunately, few biostatisticians have received training in Bayesian methods. We propose to produce user-friendly software to enable Bayesian analysis of data from clinical trials. This software will implement the complete functionality of Spiegelhalter s BART software, but in a much easier-to-use package. We will develop an associated web-based tutorial. In Phase II we will develop the software into a full-featured Bayesian analysis toolkit for clinical trialists. PROPOSED COMMERCIAL APPLICATION Clinical trials represent a 10 billion dollar industry. Statistical software for this industry is itself a significant business. We sell iBART both to drug company trialists and to federally sponsored clinical researchers. We expect iBART s tutorial and the Bayesian approach in general to appeal especially to the many trialists without specialized statistical qualifications.

This proposal will investigate several interrelated immunologic aspects of the basic host-parasite relationship in the mouse model of schistosomiasis mansoni. This important, wide-spread worm infection is prevalent in much of Africa, the Caribbean and South America. The long term objective of this research is to understand sufficiently the balance between host and parasite to allow manipulation of the system in favor of the host. These studies should improve the understanding of cytokine/lymphokine involvement in the production and immunoregulation of the primary pathogenic mechanism in murine schistosomiasis the schistosome egg-induced granuloma (Specific Aim #1). Specific Aim #2 seeks to define major cross-reactive, immunoregulatory idiotypes which are expressed on antibodies specific for schistosome soluble egg antigens (SEA) and elucidate their role in granuloma modulation at the antibody and T lymphocyte levels. Examination of the potential role (s) of gamma/delta (gammadelta+) T cell receptor- bearing T lymphocytes at different stages and in certain anatomical locations during schistosomiasis (Specific Aim #3) should yield useful information about the function of these newly studied cells, and may clarify previously unstudied aspects of host responsiveness in schistosomiasis in the dermis, spleen, liver and intestine. Specific Aim #4 states, and will explore the validity of, a new hypothesis regarding differential T cell responses which result in eosinophilopoiesis in schistosomiasis. The answers to questions posed in these specific aims should provide information that will contribute to our fundamental immunologic knowledge as it applies to schistosomiasis. When germane, data and concepts from these studies bay be useful in understanding other medically important conditions, such as chronic infections, autoimmune diseases, cancer and transplantation. These settings parallel schistosomiasis in the sense that in each the body's immune system must "learn" to deal with chronic antigenic exposure.

Cerebral palsy (CP) is the most prevalent physical disability originating in childhood of which spasticity and weakness are primary clinical signs. Spasticity management in this population has changed dramatically in the past two decades, first with the introduction of selective dorsal rhizotomy (SDR) and most recently with the introduction of intrathecal baclofen (ITB) pump implantation. ITB is efficacious for persons with spasticity of spinal origin; however, the clinical results in CP, while generally positive, are less well-established, reduction in spasticity is typically less marked, and functional gains are less impressive or even equivocal. Reported positive effects of ITB include a relaxation in spasms and spasticity which may reduce associated discomfort and improve ease of movement. A major unresolved question is whether muscle weakness is a direct effect of ITB or whether only an apparent weakness occurs due to elimination of spasticity, as seen after SDR. Exacerbation of weakness could prove problematic in these patients who are already significantly weak. The Specific Aims of this project are to:(1) quantify the changes in voluntary torque production, spasticity and selective control as a result of ITB; and (2) determine the interplay of these clinical changes on functional motor outcomes so as to improve clinical application of this therapy in CP. The following hypotheses will be tested: a) ITB will have a negative effect on isometric and eccentric peak torque production of eight (8) major lower extremity and two (2) representative upper extremity muscle groups in a dose-dependent manner; b) Isolated control of muscles opposing spastic agonists during movement tasks and gait will be conversely improved in a dose-dependent manner; c) Functional gains, including changes in gait temporal-spatial parameters and the Global Function Scale of the PODCI, will not be dose-related. Alternatively, these will depend on the individual's underlying motor capabilities, the amount of change in spasticity and strength produced by ITB, and how these changes interact to alter functional performance. Our long term goal is to improve spasticity management in CP by more precise patient selection and dosage adjustment, and greater consideration of adjunct therapies such as strength training post-operatively.

KS is the most common neoplasm occurring in patients with AIDS. Kaposi's sarcoma-associated herpesvirus (KSHV) is universally present in Kaposi's sarcoma tissue and in a rare subset of non-Hodgkin' lymphomas termed primary effusion lymphomas (PEL). A causal role for KSHV in the pathogenesis of KS has been suggested by epidemiological data, and is supported by the presence of KSHV gene products, which can alter the cellular activation state, activate cell cycling, and inhibit apoptosis. HIV infection greatly increases the risk of KS development in KSHV-seropositive individuals. In a PEL cell culture-based model, HIV replication stimulates lytic phase replication and transmission of KSHV. The major goal of this research proposal is to determine the mechanism underlying the stimulation of KSHV replication by HIV. A second goal is to define the potential influence of KSHV gene products upon replication of HIV in the PEL cell model. To achieve these goals, the HIV gene product(s) required for stimulation of KSHV replication will first be determined. Proviruses with knockout mutations in key gene products will be analyzed in the HIV-PEL model for their ability to induce lytic KSHV replication. The role of HIV-1 Tat, Env, Vpr, and Nef in altering the replication of KSHV in the absence of other HIV gene products will be examined by expression of these gene products or controls within cells harboring KSHV in latent phase. Although HIV- and KSHV co-infected cells undergo lytic phase replication, a soluble factor released from HIV-infected PEL cells is also capable o stimulating KSHV lytic replication. The identity of this soluble factor will be pursued through measurement o released cellular cytokines, measurement of released HIV and KSHV gene products, and by biochemical purification techniques. HIV replication may act to induce lytic replication through the activation of an. immediate-early KSHV gene, KSHV Rta. The role of HIV infection in inducing transcriptional activation of KSHV Rta and of additional KSHV transcripts will be assessed in the HIV-infected PEL-cell model. The mechanism of HIV spread in two PEL cell lines will next be investigated, with an emphasis on identifying KSHV gene products, which facilitate transmission and replication of HIV. Finally, the effect of HIV infection upon KSHV lytic replication will be investigated in a model of primary monocyte-derived macrophage infected with KSHV and HIV. These studies have relevance to the pathogenesis of AIDS-KS, and may provide insights into interactions between HIV and KSHV, which occur in co-infected individuals.

In persons with congenital nystagmus (CN), the incessant to and fro oscillations of the eyes produce continuous motion and smear of the retinal image. However, unlike many patients in whom nystagmus is acquired, individuals with CN rarely report the visual world to be moving. A robust extraretinal signal has been shown to exist for the eye movements in CN, which neurologically "cancels" much of the retinal image motion. One aim of the proposed research is to document how operation of this "cancellation" process depends upon the foveation periods of the CN waveform - brief intervals of relatively low eye velocity during which a target of regard is imaged at or near the fovea. A related aim is to compare the temporal characteristics of the "cancellation" of CN to that for smooth eye movements in normal subjects, to determine whether the "cancellation" mechanism is altered in CN. In addition to perceiving a stationary world, persons with CN typically don't perceive visual targets to be smeared. A second aim of this research is to quantify the extent to which perception of image smear is reduced in subjects with CN and evaluate the contributions of three potential mechanisms: l) masking of smeared retinal images by a clearer image that is available during the foveation periods in the CN waveform, 2) suppression of image smear by the extraretinal signals for CN, and 3) neural sharpening of the moving retinal image by "deblurring", as has been reported to occur in normal vision. The third aim of this research is to document the effects of the retinal image motion in CN on two basic visual functions. First, sensitivity to changes in target orientation will be measured in subjects with CN and compared to the variability of torsional eye position, during both the entire CN waveform and just the foveation periods. Second, thresholds for perceiving stereoscopic depth will be measured and related to parameters of CN eye movements and to visual acuity. The stereothresholds of normal subjects for targets that move to simulate the retinal image motion in CN will document how stereopsis is affected by the CN retinal image motion per se. These results will also clarify how normal subjects maintain fine stereopsis, despite substantial retinal image motion and vergence errors that accompany voluntary head movements. Over all, the results of the proposed research will define how the potentially debilitating symptoms of oscillopsia and perceived image smear are prevented in subjects with CN, and how the characteristics of CN affect visual orientation sensitivity and stereopsis. A more complete picture than currently available of the visual capabilities and limitations of individuals with CN will result.

To understand how the several mesenchymal cell types in the adult mouse mammary gland interact with mammary epithelium to regulate its development. We expect to mimic normal mammary morphogenesis and terminal differentiation, in monolayer cultures.

MINOS will develop new methods and techniques to test the hypothesis that changes in conformation and/or assembly determine biological outcomes. The staff and scientists of the SIBYLS beamline provide comprehensive expertise in the targeted areas of nucleic acid binding proteins. High Throughput (HT) Small Angle X-ray Scattering (SAXS), Macromolecular Crystallography (MX), and hybrid computational methods. MINOS builds upon our results developing and employing SAXS to define accurate conformations and assemblies in solution in combination with PSI high-resolution crystal structures for detail. Biological information involves changes in shape as well as active site chemistry. SAXS provides robust analyses of shape and conformational change in solution whereas crystallography provides precise information on structural chemistry. Leveraging our existing SAXS and molecular biology expertise, we will innovate new methods and technologies to integrate and advance PSI and community characterizations of key human proteins and their complexes (with partner proteins, DNA, and RNA). MINOS will work closely with PSI centers and individual researchers to identify promising targets and constructs, optimize solution conditions, and provide solution conformation and assembly results that complement PSI high resolution crystal structures. MINOS will provide new methods, tools, and strategies to characterize key human and higher eukaryotes proteins and their complexes for structural biology and medicine, which have been challenging for current PSI and community efforts. Technical goals include identifying and optimizing SAXS data collection strategies for human proteins and their complexes in concert with high resolution structural studies within the PSI centers, rescuing stalled protein targets, and developing hybrid methods and techniques for easing the bottlenecks that currently place real limits on overall PSI productivity. The Specific Aims will endeavor to 1) develop and apply innovative HT SAXS methods to solve solution structures of PSI:Biology defined targets, and 2) use solution scattering technologies to link PSI and community structures to biology. Structures determined by PSI and community collaborations will direct SAXS experiments, test functional implications from SAXS structures, and provide critical details for defining conformational trajectories in solution. Collectively the proposed Aims provide a clear path to leverage PSI and research community strengths and technologies for imaging human and higher eukaryote proteins and their complexes with major impacts on biological understanding.

In the last year, upgrades were performed on the spinning disk confocal system in the Laboratory of Cellular and Molecular Biology Microscopy Core. 27 researchers used the resources of the Laboratory of Cellular and Molecular Biology Microscopy Core. While most of the researchers come from the Laboratory of Cellular and Molecular Biology, the Core has been used by scientists from the Laboratory of Immune Cell Biology, the Section on Cellular and Developmental Biology at the National Institute of Child Health and Human Development, the Genetic Disease Research Branch and The University of Maryland Department of Physics. Almost all of the Principal Investigators in the Laboratory of Cellular and Molecular Biology have projects that involve the Core facility. Dr. Lawrence Samelson uses Core resources for the project Biochemical Basis of T Cell Activation. Dr. Carole Parent's projects, Signaling Events Regulating Chemotaxis and Chemotactic Signals Regulating Human Neutrophil and Breast Metastatic Migration use Core instruments. Dr. Paul Randazzo has made extensive use of the Core for the projects Regulation of focal adhesions and Turnover of invadopodia. Dr. Ying Zhang uses Core instruments for the project Molecular Mechanisms of TGF-beta Signaling Pathway. The Core has been involved with the project Cbl Proteins as Regulators of Tyrosine Kinase Signaling from Dr. Stanley Lipkowitz, who is now Chief of the Women's Malignancies Branch. In addition, the Core facility has been used by personnel working with Principal Investigators from other groups including work with Dr. Jonathan Ashwell on the role of ZAP-70 in T cell activation. This research usually involves the use of a Leica SP8 Laser Scanning Confocal Microscope or a PerkinElmer UltraView Spinning Disk Confocal Microscope, with some usage of our Total Internal Reflection Fluorescence (TIRF) microscope. Most of the users view immunofluorescent staining on fixed samples with the Leica LSCM while the spinning disk confocal is generally used for live cell imaging. We routinely use our TIRF microscope for PhotoActivation Localization Microscopy (PALM) and direct Stochastic Optical Reconstruction Microscopy (dSTORM). These high resolution techniques allow us to determine the location of single proteins clustered in signaling complexes in T cells with an localization error of around 20 nmfor PALM and 5 nm for dSTORM. We can now perform two color PALM imaging and multiplexed 3-D dSTORM imaging.

Optimizing HIV Protease Inhibitor Safety and Efficacy via Intracellular Targeting Abstract The AIDS epidemic is a global crisis with 31 million people worldwide who are living with HIV. According to the World Health Organization's 2010 revision of antiretroviral treatment therapy guidelines, creating less toxic therapies is a top priority to reduce adverse effects and improve compliance with therapeutic regimen. HIV protease inhibitors are a mainstay of highly active antiretroviral therapy (HAART) with annual sales of over $2.5 billion. All marketed HIV protease inhibitors (PI's) are substrates for cytochrome P450 and are co- administered with ritonavir, a pharmacokinetic booster. Boosting maintains therapeutic levels of compound to suppress viral replication and avoid incidence of viral mutation. Ritonavir is a strong inhibitor of the cytochrome P4503A4 isoform with a of Ki 5-70 nM which helps reduce P450 interactions of the co- administered PI. Eliminating P450 interactions of PI's would also eliminate the need for ritonavir. Toxicities attributed to ritonavir used alone or in combination with other HIV protease inhibitors (PI's) include hepatotoxicity, carotid artery thickening, hypercholesterolemia, hyperglycemia, and lipodystrophy. The elimination of P450 interactions for PI's is a high-priority research focus at several pharmaceutical companies, including Merck, Sequoia Pharmaceuticals, and Concert Pharmaceuticals. The elimination of ritonavir has been shown to reduce toxicities associated with HAART in short-term studies, and would increase compliance with therapeutic regimens. The specific aims of this proposal are: Aim 1. Design and synthesize a novel library of PI fragments linked to FKBP ligands. Aim 2. Screen and select library compounds for the ability to inhibit HIV protease employing an enzymatic assay and evaluate in vitro pk/pd. Aim 3. Re-screen the best PI candidates resulting from Aim 2 for microsomal stability, potency in cell infectivity assays, and p-glycoprotein assays to select candidates for further preclinical evaluation.

RTI International has proposed innovative methodological and applied research entitled Geospatial and Time Series Analysis of Food Prices and Obesity: Evidence from Sugar-Sweetened Beverage Prices to enhance scientific capabilities for conducting obesity policy research. This project is motivated by the research community's need for high-quality community-level measures of food prices and by the currently active debate among academic researchers, policy analysts, and lawmakers on the efficacy of large sugar- sweetened beverage (SSB) taxes. The proposed spatial economics research will determine the feasibility of constructing high-quality community-level food price measures using optical scanner data on supermarket sales and household purchase records collected by The Nielsen Company. The utility of the price measures in informing obesity policy will be validated by matching these measures with geocoded National Health Interview Survey (NHIS) data and examining the causal effect of SSB prices on body mass index (BMI). Price endogeneity will be controlled by using instrumental variables that are correlated with the costs of SSB and non-SSB supply but uncorrelated with SSB and non-SSB demand. The proposed research will directly benefit obesity policy researchers and policy makers through (1) the development of new food price microdata and (2) the enhanced understanding of the prospect of leveraging economic incentives such as junk food taxes and/or healthy food subsidies to reverse the obesity epidemic. Upon completion, the price microdata will be made available to the research community. The proposed 2-year project has two aims: Aim 1: Gain insights regarding the feasibility of developing high-quality community-level measures of food prices based on existing scanner data on supermarket sales and household purchases and matching these price measures with geocoded health survey data. Aim 2: Validate the utility of the food price microdata in an econometric model relating obesity to SSB prices and measures of the built environment. Use the results to shed light on whether large SSB taxes are likely to affect health outcomes.

TMD is a highly prevalent (i.e., up to 15-20% among women of reproductive age) chronic pain syndrome with profound consequences including disability, psychosocial dysfunction, and reduced quality of life. While psychosocial interventions have shown tremendous promise in reducing TMD symptomatology, standard cognitive-behavioral treatments for pain are costly and time-consuming to administer. As a consequence, significant interest has been generated in the development of interventions that minimize the logistical burdens on healthcare providers and patients (e.g., "minimal-contact", "self-help", and "brief treatments"). While these treatments are widely used in the context of certain conditions (e.g., arthritis), their application in TMD has been minimal. Moreover, none of the few existing studies of psychosocial interventions for TMD make explicit use of social support, a powerful intervention with benefits for many chronic conditions. Thus, the principal goals of the present proposal are as follows: (1) to develop a minimal contact/home-based psychological intervention based on cognitive-behavioral principles for pain management that also makes explicit use of social support, and (2) to test the efficacy of this intervention, using an appropriate control group, in reducing pain and improving functioning. [unreadable] [unreadable]

Cell differentiation, reprogramming and malignant transformation are major events characterized by remarkable changes in the epigenome and involve remodeling of DNA methylation patterns. In cancer tissues, DNA methylation patterns are drastically different from those in normal tissues. Two major events are observed, (i) global DNA hypomethylation in cancer affecting predominantly repetitive DNA sequences, and (ii) gene-specific hypermethylation of CpG islands affecting hundreds of genes. The mechanisms how these cancer-associated DNA methylation patterns arise are largely unknown. In 2009, it was reported that a sixth DNA base, 5-hydroxymethylcytosine, is present in substantial amounts in certain mammalian cell types. 5-hydroxymethylcytosine (5hmC) is created from 5-methylcytosine (5mC) by enzymatic oxidation carried out by the TET family of proteins. One model proposes that 5hmC is an intermediate in DNA demethylation. Our hypothesis is that defects in the 5mC oxidation pathway are responsible for altered DNA methylation patterns in human tumors. We have established methodology for precise quantification and genome-wide mapping of 5mC and 5hmC. Our goal is to determine the level and the genomic distribution of 5hmC in normal human tissues and in malignant tumors. These data will be compared directly with the distribution of 5mC in the same tissues. We will focus primarily on two tumor types: (1) human grade II/III astrocytomas, because these tumors frequently contain mutations in isocitrate dehydrogenases (IDH1 or IDH2), an enzymatic activity potentially impacting on the 5mC oxidation pathway; and (2) myelodysplastic syndrome (MDS), because this malignancy often is characterized by mutations in one of the TET genes, TET2. The third Aim will focus on functional studies of TET and TET-associated proteins and their aberrations in cancer. PUBLIC HEALTH RELEVANCE: DNA cytosine-5 methylation patterns in cancer are aberrant and are characterized by frequent hypermethylation of CpG islands. It is unknown how these changes in tumors are initiated. Recently, it has been shown that 5-methylcytosine can be oxidized enzymatically to 5-hydroxymethylcytosine. In this application, we propose that changes in 5-hydroxymethylcytosine patterns are hallmarks of malignant transformation and are related to the aberrant DNA cytosine methylation patterns seen in tumors. We will analyze this novel epigenetic mark as well as 5-methylcytosine in normal and malignant tissues. We will also investigate if and how mutations affecting the 5- methylcytosine oxidation pathway have an effect on genomic methylation patterns in human tumors. Functional studies of 5-methylcytosine oxidases (TET proteins) and their associated factors will support these studies so that a comprehensive picture of the importance of this pathway in human tumorigenesis can be obtained.

Fish are known conduits of metal exposure to humans and wildlife. Forty-eight states currently issue public advisories limiting fish consumption to reduce human exposure to Hg and other toxins. Yet, there is much unexplained variation in metal levels in fish from different lakes even within the same region. Project 7 studies mechanisms driving lake-to-lake variation in burdens of top priority metals (Hg, As, Cd, Zn, Pb) in aquatic organisms. We focus on the trophic transfer !of metals (i.e., movement of metal from water through the food chain to fish) in a multiple stressor context (e.g. multiple metals, pH, DOC, nutrients, low food) across a gradient of lake types. We hypothesize that much variation in fish metal burdens is driven by fundamental differences in the food web structure among lake types and in the ability of particular taxa to accumulate, magnify or dilute metals. We will predict this variation based on environmental properties that vary across lakes, such as lake productivity, adjacent land use, dominance of "key conduit" species, use of "conduit" habitats for foraging, timing and nature of metal inputs, and the toxicological responses of the conduit organisms. Our research across 60+ lakes identifies three aspects of food web structure that increase trophic transfer of metal to fish: (1) larger bodied, lower complexity zooplankton food webs, (2) dominance of key conduit zooplankton taxa (e.g., Daphnid), and (3) lower algal biomass and zooplankton abundance. We also plan to test for additional influences of factors including lake productivity, use of the littoral zone for feeding, presence of other environmental stressors. We further seek to strengthen the scientific basis for lake-specific management and public health warnings and plan to develop Daphnia as a model organism for toxicogenomic analysis to understand gene-environment interactions underlying effects of multiple metal stressors in aquatic ecosystems. This proposal has five specific aims. Aim 1 characterizes metal trophic transfer pathways in the field and tests whether the transfer to fish diminishes!in eutrophic or urbanized lakes. Aim 2 determines strength and consistency of specific taxa and specific lake habitats (littoral Vs. pelagic) as conduits of metals to fish. Aim 3 links temporal patterning in deposition of multiple metals to watersheds and inputs to lakes, and determines the source of key depositional events using multielement emission source fingerprinting, a new stable Hg isotope technique, and meteorological trajectory analysis. Aim 4 quantifies combined toxic effects of multiple environmental stressors associated with metal burdens in situ on Daphnia. Aim 5 characterizes genomic response to metals among natural populations of Daphnia, in order to further the development of Daphnia as a general toxicogenomic model species for assessing metal exposure and effects in natural field populations.

Catecholamine neurotransmitters have been implicated in the pathophysiology of several neurological and psychiatric disorders such as schizophrenia, depressive illness and Parkinson's disease. The enzyme which catalyzes the first and rate limiting step in the biosynthetic pathway of catecholamine neurotransmitters is tyrosine hydroxylase (TH) and its gene expression is limited to catecholaminergic neurons. In this proposal, human neuroblastoma cell lines which are either adrenergic (TH-expressing) or cholinergic (TH-nonexpressing) will serve as the model system. The molecular events and factors governing this differential expression of the TH gene will be investigated. In order to gain a deeper understanding of gene regulation in the nervous system, it is of critical importance to define which nuclear proteins (trans-acting elements) specifically bind to the promoter DNA sequences (cis-acting elements). Characterization of the molecular interactions between trans-acting and cis-acting elements should lead to the successful isolation of the gene(s) for the relevant trans-acting factor(s). As an extension of these studies, tissue-specific expression will further be investigated in transgenic mouse models. The results from these studies may provide insights not only into the regulation of catecholamines in general but also into the etiology of some human brain disorders that exhibit altered catecholamine expression.

The aim of the proposed study is the further delineation of B cell differentiation and characterization of B cell triggering in mice. The basic tools in these studies will be antisera specific for the two B cell differentiation markers Lyb3 and Ia.W39 which we have defined previously. Both these antigens are expressed selectively on a functionally defined B lymphocyte subset that is absent in adult mutant mice carrying the xid gene and in newborn normal mice. Since we have previously shown that Lyb3, an isogenic B cell marker, is a receptor for triggering signals, we will now analyze the nature of these signals by studying whether Lyb3 has binding capacity for T cell replacing factor(s). The role of Ia.W39, coded for by a gene in the I-A region of the H-2 complex, as an effector molecule in cell interactions will be tested. In particular, we will examine whether Ia.W39 is essential for optimal presentation of antigens, which are under immune response gene control mapping in the I-A region. Since the membrane expression of both antigens is controlled by a gene on the X-chromosome, there might be a structural or organizational relationship between the two molecules: We will analyze their chronologic appearance during ontogeny. Further we will attempt to in vivo and/or in vitro modulate their expression in order to test whether there is a mutual interdependence in the mechanism of surface expression. We will also study whether a selective loss of receptor/effector function (see above) is seen in these manipulated B cells. We will try to produce hybridomas secreting monoclonal anti-Lyb3 and anti-Ia.W39 antibodies.

The goal of this project is to demonstrate that lead CounterAct compounds atenolol (AT) and levetiracetam (LV) given after organophosphate (OP) pesticide and nerve agent induced status epilepticus (SE) are a safe and effective treatment to reduce OP SE mortality and morbidity, including behavioral abnormalities, cognitive impairment, acquired epilepsy (AE) and mossy fiber sprouting. SE is a major medical emergency seen with exposure to OPs from chemical threats from terrorist attacks or from exposure by accident or natural disasters. Advances have been made to treat the seizures associated with SE and the cholinergic crisis from OP exposure, but at present there are no therapies available to prevent mortality and the long term morbidities associated with OP SE. Our research has made a major advance in understanding how OP SE causes mortality. Our PR indicate that cardiac irritability in the first 7 days after OP SE is the major cause of mortality and this can be reduced by treatment with AT and LV. We made a breakthrough in our preliminary results (PR) indicating that AT plus LV may reduce mortality by greater than 70% and also significantly reduce morbidity by greater than 50%, including behavioral abnormalities, cognitive impairments and the development of AE. This PR also suggests that AT and LV can reduce cardiac irritability and cardiac and neuronal damage after OP SE. This study will use the OP pesticide paraoxon (POX), the OP nerve agent surrogate diisopropyl- fluorophosphate (DFP) and the nerve agent sarin to induce SE in rats. Our laboratory is ideally suited to conduct these studies and has developed the necessary skills to carry out the following specific aims: Aim 1: Determine whether AT and LV can reduce mortality following POX induced SE and conduct pharmacokinetic and pharmacodynamic analyses for CounterACT lead compounds AT and LV when administered intra- muscularly and orally. Aim 2: Determine whether AT and LV can reduce mortality following DFP and sarin induced SE and evaluate the acute and chronic effects of intramuscular injections on injection site musculature. Aim 3: Evaluate whether AT and LV can reduce cardiac irritability and cardiac pathological changes following POX, DFP and sarin SE. Aim 4: Determine whether AT plus LV can reduce the development of depression-like symptoms and provide neuroprotection following POX, DFP and sarin SE. Aim 5. Evaluate whether AT plus LV can reduce the development of cognitive impairment, the development of AE and mossy fiber sprouting following POX, DFP and sarin SE. The PR demonstrate the feasibility of these studies and underscore the potential significance of conducting this research. AT and LV have been used for many years clinically to treat hypertension and seizures, respectively, and thus their use in humans has been well established. If these preliminary findings are documented in this study, we will conduct a pre-IND meeting with the FDA for ultimately getting an IND for the use of AT and LV by intramuscular administration as an effective treatment for reducing mortality and morbidity from OP SE.

This is a response to RFA # RFA-AG-09-002. The major goals of the proposal are to: (1) Identify biomarkers that are associated with progression from normal cognitive status to mild cognitive impairment (MCI) or dementia, with a particular focus on the dementia of Alzheimer's disease (AD) - these biomarkers may potentially include measures based on cognitive testing, magnetic resonance imaging (MRI), blood, or cerebrospinal fluid (CSF); (2) Determine which cross-sectional or longitudinal biomarker measures (taken alone or in combination) are the best predictors of progression from normal cognition to varying levels of cognitive dysfunction (i.e., MCI or AD); (3) Create a publicly accessible data base containing the clinical, cognitive, imaging, blood and CSF data - raw imaging data and samples of blood and CSF will also be available to investigators in the field, as appropriate. A team of investigators has been assembled at the study site with substantial experience in the clinical evaluation of older individuals, as well as expertise in the analysis of the biomarkers in question. Several external advisory groups will be assembled in order to provide guidance concerning the analysis of the data (particularly the CSF and blood samples, which are a non-renewable resource). In order to accomplish these goals we will: (1) complete a comprehensive clinical evaluation on as many prior participants in BIOCARD as possible in order to determine their current clinical and cognitive status - this will represent an approximate 10 year follow-up of the cohort; (2) initiate annual, longitudinal, clinical and cognitive evaluations on as many of these individuals as possible in order to complete a 15 year follow-up of the cohort by the end of the funding period; (3) complete analyses of the previously collected MRI scans, CSF samples and blood, using state-of-art techniques; (4) complete analyses of the relationship of the previously collected data to the current status of the subjects; and (5) provide these data to the scientific community through a publicly accessible database.

The goal of this study is to characterize the neurophysiologic mechanisms underlying the age-related decline in human visual cognitive function. This research will delineate specific neurophysiologic measures of subcortical functions that contribute to the age-related decline in cognitive function. It is hypothesized that the impairment of the sub, cortical function of alertness contributes to the age-related decrement in visual cognitive function and is related to right hemisphere dysfunction. Alertness is experimentally modulated in a double blind placebo-controlled drug study using a CNS depressant (diphenhydramine) and CNS stimulant (methylphenidate). Alertness is operationally defined using computerized EEG frequency analysis; slow later" al eye movements; blink rate; sympathetic nervous system activity (skin blood flow based, on laser Doppler velocimetry); parasympathetic nervous system activity (heart rate, variability). Visual cognitive function is assessed by performance on serial and parallel search tasks and a directed attention task. Measures of cognitive performance include: reaction time; accuracy; variability of reaction time. The hypothesis will be tested with 120 subjects, 60 in each of 2 age groups, 25-35 years and 65-75 years. Subjects will participate on 5 separate mornings. On each morning they will be given either a placebo, 0.5 mg/Kg diphenhydramine, 1 mg/Kg diphenhydramine: 0.1 mg/Kg methylphenidate or 0.2 mg/Kg methylphenidate. They will perform the cognitive tasks before and after administration of each drug condition. The research impacts clinical management of patients with cognitive deficits. Clinicians will be provided with measurement tools to document alertness deficits in cognitively impaired patients with subcortical lesions secondary to aging, focal lesions, neurodegenerative diseases and medications. If deficits in alertness can be measured and contribute to cognitive decline in the elderly, then strategies to increase alertness could be offered by clinicians. This may be particularly helpful in problem areas for the elderly such as driving and ambulation, where intermittent lapses in alertness or attention may produce significant injury. The normative data for cognitive function does not presently account for the subject's level of alertness. Since the level of alertness may account for a large amount of the variability in cognitive function, especially in the elderly and neurologically impaired population, the definition of a measurable state of alertness will improve the sensitivity and specificity of all clinical cognitive testing.GRANT=R35AG09014 The aim of this LEAD application is to test the value of examining cellular and molecular abnormalities in extra-neural tissues from patients with Alzheimer's disease (DAT). It will test whether or not reported abnormalities relate to the presence of -the clinical syndrome of DAT or certain of its subgroups (familial vs sporadic, early vs.late onset, with vs without Parkinsonism, myoclonus, depression, or early aphasia). DAT patients and disease and intact controls of comparable age and sex will all receive detailed examination including neuropsychological testing; follow-up where possible will be to autopsy. Skin cell cultures including biopsy will be meticulously standardized to ensure that DAT and control cells are studied under identical conditions including identical biological age in culture. Parameters measured in the cultures will include two related to the materials which accumulate in DAT brain: amyloid precursor protein, and materials which react with antibodies to paired helical filaments (PHF). (Recent studies by the PI and co-workers indicate that skin cells accumulate anti-PHF reactive materials when grown under specified conditions, much more in DAT cells than in controls). Other parameters to be measured have been reported abnormal in DAT calls in at least two laboratories: isoproterenol-stimulated cyclic AMP synthesis, cellular calcium homeostasis, and [U-14C]glutamine oxidation. Data will be stored in a relational data base (SIR-software) and relations among clinical and laboratory findings analyzed in detail (SAS statistical software). Dr Ronald Black, an assistant professor of Neurology, will develop methods to quantitate anti-PHF reactive materials, compare the amounts of these materials in soluble and insoluble fractions of affected and unaffected areas of DAT and control brains, and then compare their amounts in cultured DAT and control cells. He will gain expertise in clinical as well as laboratory research in dementias by participating actively in the clinical evaluations. One pilot study will examine a possible increase in anti-APP reactive materials in DAT granulocytes, and a second possible abnormalities in phosphokinase activities in cultured DAT skin cells. Future pilots will also study other potential markers. The proposed investigations will extend the PI's ongoing mechanistic studies of abnormalities in cultured DAT cells. They will test directly whether or not the abnormalities studied relate closely to the clinical syndrome of DAT or to DAT subgroups.

Several aspects of obligate intracellular parasitism are being studied in vitro using cell culture and electro-optical systems developed with the program. Future research will be directed at (1) obtaining a clearer understanding of the mechanism of penetration of vertebrate cells by Trypanosoma cruzi trypomastigates and (2) developing in vitro systems for the continuous cultivation of malaria merozoites.

Comprehensive nutritional analysis and food management for metabolic kitchens in research settings require a different set of functions than those supplied by the general software created for nutritionists. These research nutritionists need to be able to create diets with a specific nutrient composition. The solution of this class of problem requires linear programming techniques that meet nutritional researcher's needs and are not available with existing programs. The objective of Phase II is to develop ProNutra, a research dietitian software tool for metabolic research studies, which was specified in Phase I. ProNutra contains several other innovative features, such as the ability to: automatically modify a study's diet schedule to meet the diet requirements of each of the subjects, create kitchen management reports that specifically handle the needs of a research metabolic kitchen, and store and export study, subject, and diet data to third-party statistical software packages with complete user control over the data selection, format, and order. These innovations will result in new methodologies that will: increase accuracy and efficiency of diet design and preparation, provide standardization in research methods among metabolic wards, facilitate the use of valid nutrition research methodologies, and improve the ability to monitor subject response. PROPOSED COMMERCIAL APPLICATION: The completed product in Phase III will be marketed to the general nutritionists in the industry but especially those at the clinical Research Centers, the USDA's Human Nutrition Research Centers, military, industry, hospitals, teaching institutions, and private practice. A modified version with multimedia extensions will be created for home recipe management.

Cigarette smoking remains the single most preventable cause or morbidity and mortality in the United States, accounting for approximately 440,000 deaths per year. It is thus essential that researchers continue to investigate the factors relevant to the treatment of nicotine dependence. Drug-related expectancies represent the consequences that an individual expects from the use (or non-use) of any given substance. With respect to smoking, research has focused on smokers'expectancies for the use of cigarettes. These expectancies, as measured by the standard smoking expectancy questionnaires, are robust predictors of smoking motivation and behavior. However, no prior study has examined smokers'expectancies for the non-use of cigarettes. That is, no previous study has investigated the consequences that smokers expect when they quit smoking (that is, abstinence-related expectancies). However, these expectancies likely have considerable significance to the treatment of nicotine dependence. The primary goal of the current study is to examine smokers'expectancies for abstinence via the development of an abstinence-related expectancies questionnaire: the Smoking Abstinence Questionnaire (SAQ). An initial pool of SAQ items will be developed via reference to the literature, focus groups with current smokers, and expert panel review. A draft version of the SAQ will be administered to 500 current smokers and refined with the use of established psychometric procedures, including factor analysis. The SAQ's relationship to smoking-relevant variables will be examined, including its relationship to nicotine dependence, withdrawal, motivation to quit, abstinence self-efficacy, negative affect, and smoking expectancies. We hypothesize that the SAQ will have significant relationships with each of these variables, thereby providing evidence for its construct validity. The proposed study will provide valuable information regarding the process of quitting and important treatment-related process variables. Moreover, the development of the SAQ will allow for continued, systematic, research of the role of abstinence-related expectancies in the quitting process. Finally, this investigation may help inform smoking treatment. Thus, the current study will advance the development of nicotine dependence treatments. Relevance: No prior study has investigated the consequences that smokers anticipate when they quit smoking, although these expectancies likely have significance in the treatment of nicotine dependence. The primary goal of the current study is the development of an abstinence-related expectancies questionnaire. Thus, the current study will advance the development of nicotine dependence treatments.

Mesenchymal stem/progenitor cells (MSCs) hold considerable potential for a wide range of tissue regeneration therapies. While their differentiation capacity has been demonstrated extensively, mechanisms that control their plasticity remain poorly understood. Observations by our laboratories and others have shown that proliferating MSCs express lineage-associated molecules prior to induction of differentiation. This has led to a suggested model of differentiation where commitment to a specific cell type results from the combined effects of increased expression along the induced pathway and repression of genes related to other lineages. MicroRNAs are endogenously expressed, small RNAs that may regulate this process. By acting as transcriptional repressors, they have recently emerged as regulators of cellular differentiation in cancer and stem cells. In this proposal we will investigate the role of specific microRNAs and their gene targets on MSC differentiation. We have performed microRNA profiling on MSCs undergoing osteogenic and chondrogenic differentiation and identified miR-130b, miR-432 and miR-559 as differentially expressed miRNAs among both lineages. Functional studies in MSCs suggest multilineage regulation by miR-130b; its overexpression enhanced the osteoblast phenotype and repressed smooth muscle differentiation. Differential gene expression analysis in miR-130b transfected MSCs has identified several mRNA targets that could serve as control points for osteo- and myogenic differentiation. Building on these findings, Aim 1 of this proposal will identify direct targets of miR-130b and assess their role in osteo-, chondro- and myogenic differentiation. The same approach will be followed to assess the functional effects and gene targets of miR-432 and -559. In Specific Aim 2, we will investigate whether the functional effects of these microRNAs are replicated in vivo. MicroRNA overexpression and inhibition experiments will be performed by systemic delivery of lentiviral vectors encoding mimics and hairpin inhibitors to developing mouse embryos. At specific developmental stages embryonic tissues will be evaluated for bone, cartilage and smooth muscle formation. Successful completion of this study will 1) determine how miRNA-mediated gene silencing in mesenchymal cells alters commitment to osteo- chondro- and myogenic lineages and 2) identify potential regulatory roles for these specific microRNAs during musculoskeletal development. This should improve our current understanding of mesenchymal stem cell differentiation and aid future cell-based therapies for musculoskeletal repair.

During the past two decades there has been a dramatic increase in the use of drugs by pregnant women. While the target for drugs administered during pregnancy is the mother, the fetus often becomes an unwanted recipient. Unfortunately, use of drugs during pregnancy can cause structural malformations in the fetus. We propose that many commonly used drugs also can produce more subtle biochemical, physiological and behavioral teratogenic defects in the fetus. The effects of these drug teratogens may not be apparent at birth or even in childhood, but may lie dormant or "latent" until years later. We propose that many drugs may interfere, in utero, with the normal differentiation of the liver and brain, resulting in permanent defects in drug metabolism, disposition and action in the adult. By administering commonly used drugs (i.e., barbiturates, benzodiazepines, analgesics, etc.) to pregnant and lactating rodents we plan to study in their adult offspring the delayed teratogenic effects of the drugs on in vitro hepatic microsomal drug metabolism and drug receptor levels in the brain and to correlate these biochemical findings with n vivo measures of drug action.

We propose that nutrition intervention and physical activity will decrease the amount weight gained, the amount of protein consumed, dyslipidenias, hypertension, and hyperinsulinemia and affect the incidence of progressive fibrosing and atherosclerotic lesions in the renal allograft.

This is a multi-center, open-label, safety and efficacy study to assess the use of rhFIX in the treatment and prevention of bleeding in severe and moderate hemophilia B patients who have previously received blood products. It is comprised of three segments: (1) a baseline PK segment, (2) a treatment segment, and (3) a surgical Segment (if appicable). Patients will undergo screening during a 30-day period before the first dose of rhFIX is administered in the Baseline PK Segment.

Ras family GTPases are critical components of cell regulatory systems that control proliferation, differentiation, and cell survival. Inappropriate regulation of these systems directly contributes to initiation and progression of human cancer. This proposal is directed at increasing our understanding of the composition, organization, and function of cell regulatory networks engaged by Ras family GTPases. Our focus is on the dominant effector pathways that mediate oncogenic Ras-induced cell transformation. Our specific aims are 1) revealing the molecular basis of the contribution of Ral GTPases to support of human tumor cell proliferation and survival; 2) assessing the role of two candidate Ras effectors in limiting cellular responses to mitogenic signals; and 3) defining the contribution of scaffolding proteins to the generation of signal fidelity on the ERK1/2 MAP kinase cascade. [unreadable] [unreadable] [unreadable]

Yersinia enterocolitica is a Gram-negative pathogen responsible for a range of clinical syndromes but is primarily associated with gastrointestinal disorders. A number of important paradigms of pathogenesis have emerged from the studies of Y. enterocolitica and Y. pseudotuberculosis. In addition these enteropathogens have served as important models of bacterial invasion, a process primarily encoded by inv. Virulence properties such as invasion can be characterized at the molecular level due to the ease of manipulation of these bacteria in the laboratory and the existence of an excellent murine model of infection for dissecting the host-pathogen interaction. In studies to further our knowledge of inv and its role in virulence we identified a gene, rovA, that regulates expression of inv both in the laboratory and during infection. Subsequent studies demonstrated that RovA acts as a DNA binding protein and promotes inv expression by displacing a repressor complex from the inv promoter. The rovA mutant was less virulent than either the wild type strain or the inv mutant. The rovA virulence defect is characterized by reduced systemic dissemination and an increased LD50 after oral infection. Infection using the i.p. route abrogates the rovA defect, suggesting RovA is required for events occurring either in/from the intestine/colon or in the Peyer's patch. Because the rovA mutant virulence defect was more significant than that of an inv mutant alone, this suggested RovA regulates additional virulence determinants. However, RovA did not appear to regulate the expression of previously identified virulence determinants. Using whole genome microarray analysis we identified 64 genes potentially regulated by RovA, suggesting the regulon may be quite large. The long-term goals are (i) to understand how these genes are regulated by RovA and how that is coordinated with expression of other virulence factors, and (ii) to determine which RovA regulated genes (rrg) contribute to virulence. Specifically we propose the following: (Aim 1) What is the role of the RovA regulon during infection? Recently our understanding of how enteric pathogens interact with the intestinal mucosa and spread systemically has changed significantly but questions still remain. In addition, questions regarding how these bacteria spread from host-to-host are more tractable. Given the known virulence defects of the rovA mutant, we hypothesize that the RovA regulon will play a role in these host-pathogen interactions and feel that a more detailed understanding of both wild type and the rovA mutant with respect to these aspects of infection is warranted. (Aim 2) Which rrgs are important for virulence and how do they contribute to individual RovA-associated phenotypes? (Aim 3) Are all rrgs regulated in the same way as inv? By knowing when and where these gene products are expressed combined with information regarding the phenotype of mutations in these genes we may be able to gain a better understanding of their function. [unreadable] [unreadable] PUBLIC HEALTH RELEVANCE: Gastrointestinal disease is a significant cause of morbidity and mortality in infants and of morbidity in adults. Our long-range goal is to gain a full understanding of the Yersinia enterocolitica virulence factors and their contribution to the disease process at the molecular level. This will expand our understanding and ability to intervene therapeutically not only with Yersinia but with many other gastrointestinal pathogens as well. [unreadable] [unreadable] [unreadable]

The proposed Drug Education Proect seeks to lower the number of youth in the Model Neighborhood Area who become drug dependent by reducing their predisposition toward drug use. Specific program objectives include extention of education and training efforts towards broader coverage of the MNA, strengthening of the training program and periodic review, evaluation and refinement of program methods and activities. Program activities include: an educational component which features value clarification and decision making exercises, and which is devised for neighborhood groups, skill-training centers and schools; a training component enabling particpants to develop drug education projects within neighborhoods; an evaluation component which aims at providing pre- and post program analyses, sophisticated attitude probes and feedback mechanisms.

As the applicant notes, herbal medicines are part of folk medicine in developing and developed countries and have been used extensively to reduce pain and discomfort. Unfortunately, very little controlled research has been conducted to determine whether these agents actually produce analgesia, their possible sites and mechanisms of action, and their efficacy and potency relative to known classes of analgesic agents. The major hypothesis of this proposal is that purified components of herbal medicines have potential as analgesic and antihyperalgesic agents and their efficacy and potency can be evaluated using animal models of inflammatory persistent pain. The first aim is to characterize the efficacy of systemic administration of active components of some herbal medicines in a rat model of inflammatory pain and hyperalgesia. The investigators will use their well-characterized model if inflammation to examine the effects of these active components on pain and hyperalgesia that persists for hours or days. This model will also distinguish between analgesic and antihyperalgesic activity. The second aim is to investigate the possible sites of action of agents that have analgesic efficacy by administering these agents to the site of inflammation or to localized sites in the central nervous system. In this instance the investigators will be examining whether these agents have actions in the periphery at the zone of injury, or in the control nervous system at sites of hyperexcitability and sensitization. The third aim will be to compare the efficacy and potency of active components of herbal medicines with known antihyperalgesic and analgesic agents. The principal investigator and his collaborators will compare the relative potency of active components of herbal medicines to known opioids, excitatory amino acid antagonists, neurokinin receptor antagonists and nonsteroidal antiinflammatory agents. The investigators will also administer herbal-derived agents in combination with specific antagonists of analgesia (e.g., naloxone) to reveal possible mechanisms of action.

We are investigating the mechanisms by which the virus, Q-beta, subverts the host's protein synthetic machinery into performing functions in viral RNA replication. Specifically, do the protein synthesis elongation factors Tu and Ts perform functions in Q-beta replicase related to those they perform in protein biosynthesis? We are also studying the structure and mode of action of Q-beta replicase, as well as its mechanism of template specificity. We are building Q-beta replicases with mutant elongation factors and with host polypeptides from distantly-related procaryotes. Other projects include a study of subunit relationships of RNA polymerase and a study of the roles played by Tu and Ts in protein synthesis and in other host processes.

Pre- and postnatal exposure to opioids can profoundly affect CNS development and function. Since the fastest growing population of opiate (heroin) abusers are young women of childbearing years, there is a public health interest in understanding how opioid signaling affects CNS development. Opioids given experimentally in vivo or to slice cultures during pre- or perinatal periods can alter survival and proliferation of neurons and astroglia, cause permanent changes in CNS structure and adversely affect learning and memory. Opiate exposure also reduces neurogenesis in adult hippocampus by 40 percent. Opioid effects are complex. Depending on the cell type or receptor (mu, delta or kappa) targeted, opioids can be toxic or protective and can have distinct effects on cell maturation. The survival of oligodendrocytes (OLs) and formation of myelin is critical for CNS function. Although opiate abuse can result in myelin pathology, essentially nothing is known about opioid effects on OLs either during neonatal or perinatal periods or in the adult. Our work has defined the existence of opioid signaling pathways in cultured OLs by showing that: (a) OLs express mu- and kappa-opioid receptors in a temporally specific pattern; (b) OLs have physiologic responses (survival, proliferation, myelin production) to selective manipulation of receptors; (c) OLs synthesize, process and probably secrete 2 classes of endogenous opioids (dynorphins, enkephalins). The central goals of this proposal are to determine the spatiotemporal expression patterns of opioid receptors and peptides within the developing and mature CNS, and to determine the role that opioids play in modulating the survival and function of OLs. Functional studies center on the role of dynorphin peptides since our findings show dynorphin mediates effects on the survival of OLs and neurons. A secondary goal is to determine whether manipulation of opioid signaling pathways can promote OL survival and myelination in clinical conditions with myelin pathology. Proposed studies use complementary in vivo and in vitro approaches employing mice deficient in opioid receptors and dynorphin to: (1) Identify spatiotemporal patterns of opioid receptor expression on OLs in the CNS; (2) Identify spatiotemporal patterns of dynorphin expression on OLs in the CNS and determine if dynorphin peptides are secreted; (3) Test the hypothesis that signaling through kappa-opioid receptors promotes OL survival and activates the PI3-kinase/Aktl pathway; (4) Test the hypothesis that some dynorphin peptides have glutamatergic effects on OLs. Techniques used include cell culture, immunostaining, in situ hybridization, immunoblot, confocal microscopy and adenoviral transfection.

The purpose of this application is to study the role played by van Willebrand factor (VWF) in initiating platelet deposition at sites of vascular injury, particularly in areas of the circulation characterized by rapid blood flow. The goal is to elucidate the mechanisms of adhesive interactions and cellular responses that are relevant to the arrest of bleeding from wounded tissues but also to the development of common and serious diseases caused by arterial thrombosis, such as myocardial infarction and stroke. The first aim is to define the biomechanical characteristics of the bonds formed between the VWF A1 domain and the platelet ilycoprotein (GP) Ib-alpha in relation to specific structural aspects of the A1 domain. The second aim is to define the mechanisms through which soluble VWF multimers and membrane tethers contribute to stabilizing platelet adhesion mediated by GP Ib-alpha. The third aim is based on structural evidence that alpha-thrombin may stabilize the VWF A1 domain-GP Ib-alpha interaction, and may thus play an unexpected role in modulating the initial adhesion of platelets to a thrombogenic surface through a mechanism that is independent of platelet activation. The proposed studies will clarify the biological significance and mechanism of this novel athrombin function. The fourth aim is to characterize the signaling events related to the interaction between the VWF A1 domain and GP Ib-alpha, and obtain a more definitive definition of the mechanisms through which the interaction contributes to platelet activation. The fifth aim is to define how VWF binds to extracellular matrix components, in particular to define the potential biological significance of the interactions with collagen type VI and the oligosaccharides that are present in proteoglycans. Extensive interactions with all the other projects in the program and with a core A will facilitate the development of these aims. The results of the proposed studies will have an impact on public health by providing novel mechanistic information on processes that are central to normal hemostasis and pathological arterial thrombosis.

The purpose of the proposed research is to investigate the functions of the placental members of the prolactin/growth hormone family in the mouse. Since prolactin has a broad spectrum of activities in animal physiology, these related placental hormones are likely to be important regulators of the physiological changes that occur during pregnancy. Four placental members of the prolactin/growth hormone family have been identified in the mouse. We hypothesize that the interactions of these hormones with their receptors are important in regulating maternal physiology, fetal development, and the successful completion of gestation in the mouse. We propose to prevent the synthesis of one of these placental hormones (placental lactogen II) by generating a mouse strain in which the gene encoding this protein has been disrupted. Since this protein (as well as placental lactogen I) binds to the same receptor that recognizes prolactin, we will investigate the locations, timing, and levels of prolactin receptor expression in the developing mouse fetus. We will also generate mice that have a mutated gene for the prolactin receptor; this will test if the interaction of this receptor with these two placental hormones is essential for normal fetal development. If mice that lack the prolactin receptor are able to develop, then these mice would be of value in investigating the requirement for this receptor in such processes as growth and metabolism, reproduction, osmotic balance, lung development, and immune response in the adult. Two of the placental members of the prolactin/growth hormone family in the mouse do not bind to either the prolactin receptor or the growth hormone receptor, and their physiological roles and targets have not been defined. Our recent data indicate that physiological concentrations of these two proteins regulate angiogenesis: one of these proteins (proliferin) stimulates angiogenesis, while the other (proliferin-related protein) inhibits this process. We therefore hypothesize that these proteins play important roles in initiating and limiting placental angiogenesis and that they bind to specific receptors present on capillary endothelial cells. The actions of these two placental hormones in regulating angiogenesis will be further characterized, and the receptors through which these hormones act will be identified. In addition, we hypothesize that the expression of proliferin may occur in tumors, with this hormone then acting as a paracrine angiogenesis factor that promotes tumor growth and metastasis. By introducing an expression construct for proliferin into tumor cells, we will determine if we can increase the tumorigenicity of these cells. Similarly, the effect of expression of proliferin-related protein will be tested as a possible means of inhibiting angiogenesis, and therefore the growth, of tumors.

To assess the effects of intrathecal baclofen on persons with generalized dystonia secondary to cerebral palsy or traumatic brain injury. The research design has two phases. First an open label trial of intrathecal baclofen is infused by an external pump. If the patient responds to the trial, they may continue to a second phase: a randomized, double-blind trial of baclofen versus placebo after programmable pump implant. Outcome measures for this study are based on our dystonia scale and functional assessments.

The Informatics/Data Management Core will be responsible for providing computer-based data management tools that facilitate the storage, retrieval, and analysis of the data generated in this research. The system must be accessible to each laboratory participating in the project, and should work as a communications gateway and server. It should have power and memory to quickly process large amounts of data for analytical and simulating statistical analysis. The system will build on the prototype database of FCCC and that being developed by the Human Genome Initiative for management of pedigree data and laboratory reagents and results and will have an analysis component to perform linkage segregation and risk analysis.

The primary goal of this proposal is to systematically examine the psychological and physiological responses of narcotic addicts to stimuli previously associated with drug-taking behavior. Drug addicts and non-addicted control subjects are shown stimuli reminiscent of drug-taking behavior in the form of a videotape (VT) and an assortment of drug paraphernalia. Prior to the stimulus presentation, measures of mood state and craving are taken. During the presentation, physiological measures of heart rate, skin resistance, skin temperature and respiration are monitored along with subjective level of craving. Subsequent to the VT, the psychological measures are taken and compared to the pre-stimulus scores. The same measures are also taken before, during and after viewing control films, including a non-drug-related, emotionally neutral film and non-drug-related stress film as controls for general arousal and stress factors. Goals of our study include an assessment of the stability of drug addicts' response systems. Initial results indicate that a longer period of time might be necessary for habituation to the experimental set-up, as compared with controls. In subsequent studies we will attempt to answer the following questions: (1) Are the subjects' responses specific to drug-related stimuli? (2) Are the responses limited to addicts, i.e., those with conditioning experiences - or at least, is the response topography different from those control groups? (3) Are the responses greater in subjects who have been addicted longer - thus having a longer conditioning history? and (4) Are the conditioned responses a function of drive or current levels of opiate use? This project is an attempt to expand on prior studies by the applicants demonstrating psychological and physiological conditioned responses to a VT. The present design should accomplish this in a systematic manner. The use of appropriate control subjects, precise presentation of stimuli and additional physiological measures will allow us to draw firm conclusions for our results.

GOAL: To decrease the morbidity and mortality of cancer in Wisconsin. OBJECTIVES: 1. Planning and Legislation. To develop a comprehensive plan for control of cancer in Wisconsin and a strategy for its enactment into formal legislation. 2. Epidemiology and Evaluation. To further develop and maintain a strong WCCC Biometry component to aid in the planning, implementation and evaluation of Wisconsin cancer control programs. 3. Public Information and Eduction. To develop and promote needed programs to inform the public about cancer. 4. Professional Information and Education. To conduct necessary and evaluable programs to inform and educate health professionals about cancer. 5. Prevention and Early Detection. To promote optimal practices in cancer prevention and early detection. 6. Diagnosis and Treatment. To organize regional, cooperative, site-oriented programs, networks and task forces dedicated to improving the diagnosis and treatment of cancer. 7. Rehabilitation and Continuing Care. By demonstration, to inform the public and professional communities in Wisconsin about effective rehabilitation and continuing care possibilities and practices for cancer patients. 8. New Projects Development. To catalyze, encourage and support the development of new ideas in cancer control.

Project Summary: 518 human protein kinases modulate the activity of -30% of all proteins, and collectively control almost all complex pathways and decisions of a cell. Despite tremendous experimental analysis on some kinases, we know little of the detailed function of most members of this uniquely important family. We propose to use genome sequences to tap hundreds of millions of years of evolutionary experimentation, in order to clarify the link between kinase sequence and biological function. We will use our extensive knowledge from the discovery of the human and mouse kinomes to predict all kinase orthologs in up to 40 vertebrate genomes. We will then map the evolutionary constraints on every residue of every kinase, and predict domains, motifs, phosphorylation sites, and other functional regions of proteins, extending the kinome catalog to unprecedented resolution and generating a wealth of hypotheses for experimental testing. Finally, we will apply this knowledge to predict the functional impact of both SNPs and somatic mutations in cancer, providing a valuable preview of the proposed cancer genome atlas. By using kinases as a model family to explore the predictive power of comparative genomics, we will develop well-validated tools and parameters which can then be applied to any human gene or gene family. Relevance: Protein kinases key controllers of cell function, are one of the most important gene families in disease and development of new drug therapies. By exploring how human kinases vary from those of other vertebrates, we can predict whether any human sequence change can predispose to disease or alter drug response, and can distinguish cancer-driving mutations in tumors from background mutations.

This research aims to study mechanisms of synaptic function with emphasis on the origins and locations of individual synaptic membrane polypeptides. By reacting epsilon-amino groups of lysines which are exposed on membrane surfaces with sodium boro (H3) hydride in the presence of pyridoxal phosphate it is possible to selectively label exposed peptide areas of intact synaptosomal membranes. Combining this technique with purification and subsequent gel electrophoresis of synaptosomal sub-fractions from chick brain allows classification of membrane polypeptides into four groups: those occurring entirely within the membrane, those exposed on both the internal and external faces, and those exposed only at one surface, either external or internal. The nature of the peptides around the exposed lysine residues will be characterized by labeling polypeptides eluted from the gels with I125, cleaving with trypsin or cyanogen bromide, and performing autoradiographic peptide mapping on the resulting peptide mixtures. Whether or not synaptic vesicles fuse with the external membrane will be investigated by comparing labeled gels and peptides of these two fractions. Distances between polypeptides in the membrane will be determined with bifunctional reagents with various bridge lengths between the functional groups. When basic parameters are defined synaptosomes prepared from synapses in differing states of activity will be examined. Alterations of membrane conformation and subsequent protein exposure may reflect neural activity. Synaptosomal membranes will be prepared from electrically stimulated brain slices and chick optic lobes that are either denervated (by enucleation) or receive minimal input (by eyelid suture). Comparing such preparations with normal synaptosomal membranes may reveal a relation between synaptic membrane structure and its functional use. Such data will have important implications for theories both of normal brain function and of the etiology of neurological and psychiatric illness.

P. aeruginosa (PA) frequently infects immunocompromised individuals with HIV-1, cancer, and cystic fibrosis (CF). Since PA is increasingly resistant to antibiotics, its infection often leads to either severe states or chronic situations with a persistent inflammatory response. Better understanding of host-pathogen interaction may suggest a more effective approach to combating this pathogen. MCP-1 is a major chemokine secreted by alveolar epithelial cells type II (AECII). Recent research has illustrated an immune role of AECII in PA infection, but the underlying mechanism remains unidentified. Our long-term goal is to understand the mechanism of host immunity and develop new strategies for controlling respiratory infections. The objective of this application is to elucidate the immune function of AECII, in particular through their secretion of cytokines and activation of AM. Our central hypothesis is that AECII can secrete cytokines (MCP-1) to enhance AM's anti-bacterial immunity through a lipid raft- mediated mechanism. We have formulated this hypothesis based on our recent findings that both AECII and AM participate in innate immunity against PA. We further found that membrane lipid rafts may be instrumental for regulating cytokine secretion. Using our primary cell model, we have discovered an immune role of AECII in enhancing AM's immunity using a conditioned AECII medium. Our data also suggest that AECII play a critical role in PA infection by secreting MCP-1 and recruiting the classically activated macrophages (CAM). The rationale is that elucidating how AECII enhance AM immunity will indicate a potential strategy to bolster immunity against PA. Our laboratory is ideally suited for this research, having the relevant expertise in isolation and culture of AECII a well as in lung infection models. We propose the following three specific aims: Specific Aim 1: Define the immune role of AECII cells in secreting cytokines during PA infection. We will identify the source of MCP-1 using in situ hybridization with AECII marker SPC. We will also use primary AECII culture to show MCP-1 as a dominant cytokine. Furthermore, AM and AECII from MCP-1-/- mice will be examined for their decreased immune function against PA infection. We will determine the ability of AECII in recruiting the classically activated macrophages (CAM). Specific Aim 2: Evaluate how lipid rafts regulate MCP-1 secretion in AECII. We will study the underlying mechanism for MCP-1 secretion and hopefully identify the involvement of ceramide-rich membrane microdomains. Acid shingomyelinase will be blocked by siRNA and chemical inhibitors for analyzing sphingolipid hydrolysis during PA early infection. Specific Aim 3: Assess the potential of super-AECII over-expressing MCP-1 in enhancing anti-PA capacity of human AM. We will create super-AECII using retroviral vectors to secrete high levels of MCP-1 and test their host defense in PA infection. We will also demonstrate that human AM can be activated by AECII and that this translational research may imply the clinical value of the immune AECII. This research will be performed by graduate and undergraduate students. Our efforts are expected to substantially advance understanding of this previously unrecognized immune function of AECII in activating AM, and may provide insights into mechanisms of cytokine secretion, with indications in development of novel therapeutics for treating this infection. PUBLIC HEALTH RELEVANCE: P. aeruginosa (PA) is a bacterium that causes severe infections, particularly in immunodeficient individuals who are suffering tuberculosis, cancer, AIDS, severe burns, and cystic fibrosis. Because PA is increasingly resistant to antibiotics, its infection usually leads to a chronic state of persistent inflammatory response. We have made the surprising discovery that MCP-1, a versatile cytokine from alveolar epithelial cells, regulates host defense and inflammatory response in PA infection. We have also noted that lipid rafts may be important for regulating cytokine production. Through secretion of MCP-1, the alveolar epithelial cells may recruit a particular subset of macrophages (i.e., classically activated macrophages) to promptly respond to infection. Studying the immune role of alveolar epithelial cells may provide new insights into the development of novel treatment for PA infection.

The long-term goal of this project is to develop strategies to improve the treatment of breast cancer. The aromatase inhibitors we pioneered in the early phases of this grant are now proving to be of value in the clinic. Although AIs appear to be more effective than tamoxifen, some patients may acquire resistance or have de novo resistance to AIs. The unique preclinical model we have developed to study the effect of aromatase inhibitors (AIs) has provided accurate predictions of clinical outcome. We propose to use this model to study mechanisms of tumor resistance. We have also developed unique tumors and cell lines that are resistant to letrozole and other AIs for use in these investigations. In the proposed studies, we will focus our efforts on several significant observations made during the current period. We plan to determine the mechanisms of resistance to aromatase inhibitors and how these might be reversed so that response to well tolerated AI treatment can be restored. The Specific Aims of the proposal are: 1) to investigate the functional the role of HER2/MAPK in estrogen receptor regulation and development of resistance to AIs. We will explore whether other inhibitors of HER2, such as lapatinib, pertuzumab and HKI-272 also reverse resistance to letrozole; 2) to investigate whether down-regulation of the estrogen receptor (ER) by fulvestrant prevents crosstalk with tyrosine kinase receptors (TKR) in AI resistance. We will determine whether combining an AI and estrogen down regulator is more effective than complete estrogen suppression in controlling tumor growth. Based on mechanisms identified, the optimal efficacy of these agents in combination or sequential strategies will be determined; 3) to investigate mechanisms involved in reversing resistance of tumors to letrozole and other AIs following withdrawal of AI treatment; 4) to investigate whether histone deacetylase (HDAC) inhibitors will convert ER negative cells to hormone responsive cells that are sensitive to HDACI + AI treatment and reduce the cancer progenitor cells or side population in these and AI resistant cells and tumors. Our studies should provide information with which to plan new strategies to treat breast cancer patients. PUBLIC HEALTH RELEVANCE: This competitive renewal application is to continue studies to understand the mechanisms involved in resistance of breast cancers to aromatase inhibitor treatment. Having identified mechanisms of resistance to aromatase inhibitors, we will then apply this information to develop strategies to reverse resistance and restore sensitivity to aromatase inhibitor treatment. These strategies will be tested in our unique model to determine their anti-tumor efficacy. The results of these studies could improve treatment for breast cancer patients.

Summary We know very little about the mechanism of INO80, how it disrupts nucleosomes and the factors governing its activity. We will take detailed ?snapshots? of INO80 during nucleosome remodeling to find how INO80 and nucleosomes are moved during remodeling. A series of orthogonal approaches will be used to arrest INO80 remodeling at distinct stages and examine conformational changes in the core nucleosome and INO80. We will build on our recent observations of the motor domain being engaged at the H2A-H2B interface and persistently displacing DNA from this surface to find why displacement occurs, the factors that control displacement and whether this displacement weakens the interactions of H2A or H2A.Z dimers with the rest of the histone octamer or otherwise disrupts the nucleosome structure. Based on the proximity of Arp5 to nucleosomal DNA, we will test the premise of Arp5 as the ?gatekeeper? regulating DNA traversing through the center of nucleosomes with wild type INO80 and mutant Arp5 in which either its histone or nucleosome binding regions have been deleted or mutated. We will also test whether the Arp8 module regulates Arp5 interactions with the acidic pocket of nucleosomes or nucleosomal DNA and if communication between these two domains is mediated by the Ino80 catalytic subunit. INO80 will be arrested at different stages in remodeling by limiting DNA translocations to specified distances, arresting with non-hydrolyzable ATP analogs, limiting linker DNA length and mutation of Arp8 and Arp5. We will probe the role of DNA sequence in INO80 remodeling because we observed coupling of ATPase activity to nucleosome movement being dramatically affected by the DNA sequence of the core nucleosome. We will find as suggested in these experiments if INO80 interactions and conformation varies depending on the DNA sequence bound by nucleosomes. In order to better examine the importance of DNA sequence in a ?native? context, we will use yeast chromatin reconstituted with recombinant histones and simultaneously examine the differences of INO80 binding and remodeling with many thousands of nucleosomes, each with a different DNA sequence. We will use our expertise of mapping protein-DNA interactions in these genomic assays to sort with high precision the interactions of the INO80 subunits along with nucleosome movement, composition and structural features at ~bp resolution to provide a detailed analysis of each of these nucleosomes in a time resolved manner when remodeled. This approach will provide more insights into the DNA sequence specificity of INO80 and if there are ?hot spots? for mobilizing/ destabilizing nucleosomes or exchanging H2A.Z in the yeast genome that doesn?t require additional factors. To confirm if INO80 behaves the same in vivo as in our in vitro assays, we will transfer several of these approaches to yeast cells so that we can measure chromatin dynamics in vivo with the same resolution as in vitro. We will compare how mutations in Arp5 and Arp8 change nucleosome dynamics in vivo, the importance of genomic position, and other factors for INO80 remodeling not present in our yeast reconstituted chromatin.

Diarrhea is the most commonly reported illness in travelers from industrialized nations to the developing world. It is estimated that of the 7.6 million U.S. residents traveling to these regions annually, 30-70% contract Enterotoxigenic E. coli (ETEC) related diarrhea [1-3]. The objective of the studies proposed here is the commercial development of TravelGAMTM, an Anti-ETEC hyperimmune Milk Immunoglobulin, intended for oral prophylaxis against diarrhea caused by ETEC. In Phase I, the clinical importance of including colonization factor antigen I (CFA/I) in the bovine vaccine was determined. During that period, ImmuCell gathered preliminary efficacy data for TravelGAM in a human challenge study. In Phase II the objectives are: (1) Evaluation of improved methods of in vitro CFA expression for use in CFA screening by utilization of cfaD constructs too up-regulate pilus expression in toxin-positive diarrheal ETEC isolates which, in their native state, do not express immuno- detectable CFAs in vitro; (2)Manufacture of TravelGAMTM containing an expanded range of anti-colonization factor antigen (CFA)s) activity suitable for prospective field testing; (3) Scale-up and cGMP manufacture of enteric coated formulation of TravelGAM; (4) evaluation of the efficacy of formulated TravelGAM in a prospective field study in collaboration with the US Navel Medical Research Unit No. 3 in Cairo, Egypt under ImmuCell's Investigational New Drug Application BB-6049. In Phase III, we hope to build upon the prospective study proposed herein by conducting a pivotal effectiveness study in collaboration with a corporate marketing partner. PROPOSED COMMERCIAL APPLICATION: ImmuCell envisions manufacturing a purified-antibody product based on immune bovine milk whey, for oral administration to patients suffering rom Travelers' diarrhea caused by ETEC. Of the 16 million people who travel from industrialized to developing countries each year 6 million are Americans. Best estimates predict that at least one third, or 2.7 trillion individuals from the U.S. will experience Travelers' diarrhea per year.

The potential of proteins to form structural complexes is a most important property which determines their function in biological systems. A rheological method to resolve and characterize this potential is proposed. Dominant intermolecular interactions will be identified in concentrated solutions of globular proteins from the concentration dependence of their viscosity. Three hydrodynamic parameters are defined to analyze the factors affecting the viscosity of proteins; the interaction factor (delta) determines the maximum interaction radius, the constraint number (Nc) measures the lower limit of this interaction radius, and the compressibility factor (Fc) evaluates the capacity of proteins to compress their radius of interaction under increasing concentration. These hydrodynamic parameters depend on the geometry of molecules or aggregates and the nature of protein-protein interactions. These parameters have been evaluated for common geometrical shapes and aggregates to serve as a basis to identify dominant interactions in concentrated solutions of globular proteins. Dynamic viscoelastic properties of these protein solutions will be used to characterize the elasticity (energy storage capacity) of these interactions and to establish a basis for their later resolution from complex systems. Using a model globular protein, the predictive capability of current structural models will be evaluated. In addition, the properties of globular proteins at higher concentrations will be related to hydrodynamic properties in dilute solutions, based on their potential to self-associate and/or interact electrostatically. The proposed research represents a pioneering effort to resolve dominant interactions in concentrated solutions of globular proteins with steady and dynamic viscoelastic measurements.

Scavenger receptors (SR) are cell surface proteins that bind chemically modified lipoproteins and exhibit broad ligand binding specificities. We have identified three classes of vertebrate and invertebrate SRs: class A (SR-A), class B (SR-B) and class C (SR-C). They participate in or influence lipoprotein metabolism, development, host defense (innate immunity, protection against septic shock and viral infection), possibly asbestosis, recognition and clearance of damaged (apoptotic) cells and macromolecules, red blood cell maturation, female fertility and atherosclerosis/coronary heart disease (CHD). Many of their functions are directly related to health and disease and are consequences of their broad ligand binding specificities. One of these, SR-BI, is a physiologically relevant HDL receptor that controls the levels and fates of plasma HDL cholesterol, including delivery to the liver and steroidogenic tissues. SR-BI mediates selective uptake of HDL cholesterol, a poorly understood mechanism distinct from classic lipoprotein endocytic uptake and cholesterol effiux. The overall goals of this proposal are 1) to elucidate the biochemical and structural bases for the high affinity, broad ligand binding specificities of these receptors by determining how their ligand binding domains (e.g., collagenous and alpha-helical coiled-coil domains of SR-AI/II) recognize diverse arrays of structurally distinct ligands, 2) to provide additional insights into the novel molecular mechanism underlying selective lipid uptake and cholesterol efflux, and 3) to provide both experimental tools and a biochemical framework with which to assess further the functions of these unusual receptors. The work will rely on the generation and functional analysis of mutant receptors generated using standard and novel methods. Detailed characterization of the structures and distinctive binding properties of mammalian and invertebrate scavenger receptors will provide important tools for the analysis of scavenger receptor function and will probably suggest new approaches for the treatment and prevention of at least some of the related diseases (e.g., atherosclerosis, infectious disease, female infertility). The proposed work may lead to methods for predicting which physiologically relevant molecules are receptor ligands; this would provide additional avenues for exploring receptor function and, possibly, the design of pharmacologic reagents. In addition, clarification of the molecular bases of the broad bind specificities of scavenger receptors may provide insight into other biological systems in which broad binding specificity is important, e.g., multidrug resistance. [unreadable] [unreadable]

Annually, more than 300,000 upper extremity injuries in the U.S. require operative treatment for repair of tendons injured in their midsubstance or at their insertion sites. These injuries lead to an estimated loss of 4 million workdays. Recent improvements in the treatment of flexor tendon midsubstance injuries have been driven by advances in the scientific understanding of repair and rehabilitation variables. By contrast, there have been no significant changes in the treatment of flexor tendon insertion-site injuries in several decades, in part because there have been few scientific investigations to support such changes. As a result, many patients have a poor clinical outcome after repair of the flexor tendon insertion site, as evidenced by decreased range of motion and loss of grip strength. Our long-term objective is to identify repair and rehabilitation techniques that will consistently produce excellent clinical function for immediate and delayed treatment of flexor tendon insertion-site injuries. In this project, we will apply a canine model of flexor tendon insertion-site injury and repair to investigate several clinically relevant variables that have not been previously addressed: 1) suture technique for reattachment of tendon to bone, 2) increased tendon force and excursion applied during rehabilitation, 3) time interval from injury to repair, and 4) growth factor enhancement of tendon-bone healing. Our primary hypothesis is that the stiffness and strength of the repair site are improved by application of increased tendon force during early, passive motion rehabilitation. In addition, we hypothesize that healing of the tendon-bone repair site can be accelerated by delivery, at the time of repair, of targeted gene products that enhance expression of growth factors that are important to early tissue healing. These include: basic fibroblast growth factor (bFGF), platelet- derived growth factor (PDGF) and vascular endothelial growth factor (VEGF). A multidisciplinary approach will be utilized for evaluation of the experimental variables, with biomechanical, histological and biochemical outcomes analyzed. The final determination of practical benefit will be based on biomechanical assessment of digital range of motion and repair-site stiffness and strength during the critical first 6 weeks of healing. Identification of improved repair and rehabilitation techniques for insertion-site injuries will be an initial step toward modernizing clinical treatment and improving patient outcomes.

The long-range objectives of this laboratory are to determine the mechanisms by which enzymes catalyze reactions, and in a more general way to determine the nature of biological interactions at the molecular level. To these we have now added an attempt to gain some understanding of the energetics of the catalytic process and to learn the relationship between the energetics of individual steps and the chemical events occurring in those steps. For some years we have focused our investigations on the glutamic dehydrogenase system which offers a wide variety of phenomena with which to pursue such studies. Having already blocked out the order of occurrence of the nine major complexes formed along the reaction time course, we now turn to the study of the detailed chemical events involved in the interconversions of those complexes. In this proposal we focus our attention on two critical areas of the reaction sequence: 1) What is the role of the carbonyl group of Alpha-ketoglutarate, and at precisely which point does the required water molecule enter the reaction course? This aim will be explored through the use of an analog lacking the carbonyl group and by a variety of 0 1 8 studies. 2) What are the events and complexes preceding and accompanying the hydride transfer step? This will be explored by a study of very pertinent model reactions and by parallel studies on the early transient steps. This latter study will include cryoenzymology and alternative kinetic approach to the study of transients which may have some application to the study of enzymes in general. Finally, we will attempt to relate a recently discovered temperature-dependent poised equilibrium between two isomeric forms of the free enzyme to changes in hydration and (or) to the large negative DeltaCp's observed in many of the complexes of pyridine nucleotide dehydrogenases. These initial experimental probes are a prelude to the definition of a serious effort in this area on our part to determine whether, as Jencks has suggested, part of the substrate binding energy can be channeled into driving a later catalytic step, and if so, how it is accomplished.

The long term goal of this research is to understand the functional basis for the expression of apolipoprotein (apo) E in peripheral tissues. The proposal consists of 4 aims that are focused on activities of apoE that influence cellular cholesterol metabolism in adrenal cells and activities that are anti-atherogenic within the vascular wall. Experiments in Aim 1 will test the hypothesis that low levels of systemic apoE act within the vascular wall to block an early step in atherosclerotic lesion development. Aim 2 will explicitly test the hypothesis that low levels of apoE provide protection against atherogenesis initiated by apoB48 lipoproteins but do not protect against atherogenesis due to apoB100 lipoproteins. Aim 3 will test the hypothesis that adrenal gland apoE expression increases adrenal cholesteryl ester storage and diminishes steroid production. Aim 4 will test the physiological importance of a newly discovered apoE-stimulated pathway for the selective uptake of low density lipoprotein cholesteryl ester.

There is a delicate balance between food intake and metabolic expenditures. Small mismatches lead to quite large changes in body energy content (weight). It seems likely, both on ecological and empirical grounds, that exercise constitutes an important regulator of appetite and/or body weight. The proposed experiments will examine the temporal relationship between food intake and voluntary exercise in golden hamsters and rats. It has been reported that hamsters increase their food intake during exercise, and we shall determine whether that increase reflects meal size or frequency changes. We shall examine the blood metabolites (glucose, fatty acids, ketones) associated with exercise and post-exercise periods, in relation to meals. We shall relate these findings to the failure of hamsters to increase their food intake on a variety of restricted food access schedules. Hamsters exhibit nocturnal activity bouts of several hours duration, and hence are an interesting model in which to study the relationships between metabolic expenditure and food intake. Rats are known to eat in excess of their metabolic needs during their active (night) phase, yet hamsters eat about the same by day and night and are active only at night. It is thus likely that the circadian lipolysis-lipogenesis cycles are different in these two species, and we hope to document this.

The review of our CDP plan was positive and noted that the potential candidates described were very strong. However, our reliance on a funding source for senior level post-doctoral fellows was considered not consistent with the SPORE guidelines. We have therefore revised the entire CDP to more cleariy provide research support for advanced fellows, junior faculty and established investigators who wish to develop or refocus their careers on translational breast cancer research. CDP support for research fellows through the Thomsen Family Fellowship will be used only when it is for support of research fellows transitioning to independent faculty appointments. In this submission, we have clarified our goals and our adherence to the SPORE guidelines. Our proposed plan describes in detail how promising individuals with an interest in, and expressed commitment to, translational breast cancer research will be selected. It further addresses how the CDP leadership will seek out and recruit qualified women and minorities for participation in the program. More specifically, the CDP Leadership will use program funds to recruit and nurture qualified investigators at all levels. CDP leaders will provide a system for mentoring such individuals in a broad range of disciplines and create a framework in which investigators can gain exposure to, and possibly training in, aspects of translational breast cancer research outside their areas of expertise. Ensuring that innovative ideas can develop into promising translational studies in breast cancer research, and that women, minorities and developing faculty who can make key contributions to translational breast cancer research at the FHCRC and UW, are strongly recruited and retained are also key goals ofthe current plan. In a response to the comment: Although the commitment to diversity is not questioned, a paragraph stating the gender/minority composition ofthe research programs at FHCRC/UW was not included, we have added two tables to section C.2 to address this oversight. The first presents the ethnic breakdown of faculty, postdoctoral candidates and pre-doctoral students at FHCRC as of December 31, 2008. The second provides enrollment figures by ethnicity for the UW School of Medicine (UWSOM) in 2007.

The Office of National AIDS Policy (2000) estimates that half of new HIV infections occur in youth under the age of 25. Sexual risk behaviors and substance use appear to be highly prevalent among HIV positive youth. The few studies of HIV positive youth suggest adherence rates that are dismally inadequate to manage the disease. Adherence is a multi-dimensional construct that includes taking medication, keeping appointments, and general health behaviors. Poor adherence increases transmission risk resulting from elevated viral loads. This triad of risk behaviors in HIV-infected youth (drug use, sexual behaviors, and health behaviors) must be targeted in intervention studies. We plan to pilot an empirically validated risk reduction intervention, Motivational Enhancement Therapy (MET), that can be easily disseminated and integrated into existing HIV clinics and community-based organizations addressing the needs of HIV positive youth. The proposed study is a randomized clinical trial with a wait-list control examining the utility of MET. This pilot study will use a sample of 60, ages 16-24, 30 of whom will receive MET immediately after baseline and 30 of whom will begin MET after the 9-month post-test. A repeated measures design will be used for the proposed study with a multiple baseline for the control group to examine level and trend effects. Youth will complete an initial data collection sessions (baseline), and then 30 youth will be assigned to the treatment condition. These youth will complete a three month post-test designed to coincide with treatment completion. Subsequent post-tests occur at nine months and 15 months after baseline data collection (6 and 12 months after treatment completion). At that point, the wait-list group will begin treatment. The waitlist control will receive standard care during the initial treatment period. For those in care, all four sites offer comprehensive, multidisciplinary care including social work and case management services. Those youth in the control condition who are not in care will be given referral for medical care. Youth in the control condition w ill participate in the three month and nine month post-test for the multiple baseline design. They will than enter treatment and receive additional post-tests at 12 months (post-treatment), 18 months (6months after treatment completion) and 24 (12 months after treatment completion). If successful, this intervention will provide immediate assistance to vulnerable population to prevent the spread of HIV and to minimize its negative physical and psychological effects.

The adrenergic and cholinergic components of the autonomic nervous system serve an important modulatory role in the cardiovascular system. Norepinephrine is the primary physiologic agonist in the adrenergic arm of the autonomic system. In the heart, norepinephrine is known to activate three types of receptors: alpha 1, alpha 2, and beta 1. The increase in cell calcium by norepinephrine both augments action potential triggered sarcoplasmic reticulum (SR) calcium release to cause an enhanced contractility and increases the likelihood for arrhythmogenic diastolic SR calcium release, seen in myocytes as spontaneous contractile waves. While the positive inotropic effect of alpha1-adrenergic agonists on myocardial contractility is thought to be mediated via an increase in cell inositol 1,4,5-tris phosphate (IP3), alpha1-adrenergic agonists also increase 1,2-diacylglycerol which activates protein kinase C. We examined the relative potency of alpha and beta mechanisms and the effect of phorbol ester, an activator of protein kinase C, on these neurotransmitter effects in single adult rat myocytes. Contractility was measured as the velocity of shortening during stimulation at 1 Hz. Waves were measured in a 30 sec window following 2 min of stimulation. In absence of drugs average velocity of shortening was 70 plus/minus 32 um/sec (x-SEM, n=6) and no waves occurred. Norepinephrine (1x10-5M) increased velocity of shortening to 300 plus/minus 70% control (n=6), and caused 3.6 plus/minus 1.55 waves to occur (n=6). Beta (norepinephrine plus prazocin (1x10-5M) had a similar effect: velocity of shortening increased to 310 plus/minus 93% control and 1.8 plus/minus 0.95 waves occurred (n=6). In contrast alpha (norepinephrine plus propranolol (1x10-6M) increased velocity of shortening by 37 plus/minus 28% (n=6) and no waves occurred. Thus, the increased contractility and enhanced probability for spontaneous diastolic calcium release to occur in response to neurotransmitter release in situ are essentially beta rather than alpha in nature. In Quin2 loaded myocytes, norepinephrine and beta decreased resting calcium and increased the rate of calcium uptake with KCl depolarization compared to control. Alpha had no effect on resting calcium and decreased the rate of calcium uptake with KCl depolarization.

A Pre-Clinical Model of TBI Heterogeneity. The majority of traumatic brain injuries are considered mild. Mild TBI (mTBI) is difficult to diagnose, and despite recent progress in public awareness and clinical management, mTBI (or concussion) is a persistent problem in sports, the military, and in the general population. In order to further understand injury mechanisms, identify more sensitive diagnostic tools, and begin to delve into the apparent risk for neurodegeneration, there is a need to improve pre-clinical laboratory studies. This is no easy task given patient heterogeneity and the physiological complexity of TBI. Despite decades of basic research, successful translation of potential treatments from animal to patients is extremely poor. Clinical population heterogeneity is not captured in pre-clinical studies, gravely limiting the ability to validate animal models as reliabl research surrogates. The long-term goal of this research is to improve lab-to-clinic translation using a bi- directional systems approach. The overall goal of this R21 Research Proposal is to reflect the heterogeneity of the mTBI patient population in a pre-clinical animal study and use informatics-based analysis to determine features that contribute to the injury response. The central hypothesis is that systematic institution of animal heterogeneity will result in more reproducible and robust pre- clinical TBI studies and the emergence of clinically relevant risk factors. The experimental aims are: 1) Develop a pre-clinical experimental design for mTBI that selects patient-relevant variables (gender, age, genetic variety, previous mTBI, and chronic stress) and applies them to a heterogeneous rat population using stratified randomization; and, 2) Assess acute neurological response, balance, working memory, and biomarker signature acutely following mTBI using a heterogeneous rat population. Informatics tools will be used in place of traditional multivariate statistics to extract common features of the injury response, classify them, and build predictive knowledge models. Several imbedded hypotheses will be tested to examine the response to mTBI as a function of animal sex, age, strain, previous mTBI, and chronic stress. It is expected that unanticipated relationships among the data will emerge as a result of the robust experimental design and knowledge-based model. The experimental platform presented here is novel and, if successful, can be used as a template for other studies. Addressing patient heterogeneity and clinically relevant acute outcome measures is highly significant and will increase understanding of the complexity of mTBI.

The results indicate distinct differences with maturation upon the neuronal-glial reaction of the hypoglossal nucleus to axonal injury in rats. Up to some 10 days postnatal, the neuronal membrane engulfs the presynaptic terminals in contact with its membrane and the proliferative glial phase is delayed to 1-3 weeks postoperative. In the mature neurone (21 days postnatal) the microglia proliferate earlier and lift the presynaptic terminals off the neuronal somata.

The purpose of this protocol is to determine the safety of porcine fetal neuronal cell implantation into the putamen and caudate of patients with Parkinson's diseasue, under two different methods of manipulating the human immune system to prevent rejection of the transplanted tissue.

Now that we have lowered the death rate of patients with gram negative bacteremia and septic shock by treatment with human antiserum to core lipopolysaccharide (LPS), we propose to extend these successful results by pursuing 3 specific aims: 1. To determine if J5 antiserum is an effective in preventing bacteremia and shock as it is in treating it. In these studies the antiserum would be given as prophylaxis to patients predisposed to bacteremia. In the first trial on prophylaxis with J5 antiserum we cut sharply the incidence of febrile attacks in neutropenic patients. We now propose to extent the trial by giving J5 antiserum to enough subjects to allow an evaluation of its efficacy against bacteremia as well. 2. To produce an effective, safe, human gamma globulin preparation for intravenous use in the treatment and prevention of gram negative bacteremia. Our goal during the period of this project is an IgM preparation that is free of non-specific anticomplementary activity but retains its ability to prevent and treat lethal bacteremia. After the effectiveness and safety of this preparation has been demonstrated in experimental animals, a new proposal will be submitted for a clinical trial of its safety and efficacy. 3. To produce in vitro human monoclonal antibody against core LPS and to test its ability to protect against experimental gram negative bacteremia and endotoxemia.

Interstitial cystitis (IC) is a sterile bladder condition of unknown etiology characterized by increased urinary urgency and frequency as well as suprapubic pain. Two important features of the disorder that must be explained are that 1) IC affects predominantly females, and 2) IC patients exhibit enhanced sensitivity to bladder distension. In addition, clinical observation suggests that symptoms of IC are exacerbated perimenstrually. The research proposed in this exploratory/developmental (R21) grant application is based on the overriding hypothesis that these clinical features can, at least in part, be explained by a generalized alteration in central nervous system nociceptive processing in IC, which is influenced by the hormonal fluctuations that accompany the female menstrual cycle. Based on this hypohesis, two predictions regarding IC will be investigated: first, that IC is associated with a generalized increase in pain sensitivity, which includes enhanced response to somatic as well as visceral stimuli; and second, that both clinical symptoms and responses to experimentally-evoked pain will be influenced by the menstrual cycles of the IC patients. In order to examine these predictions, the responses of IC and healthy, female controls to three, clinically-relevant laboratory pain induction procedures will be evaluated: 1) temporal summation of thermal pain, 2) ischemic arm pain, and 3) visceral pain produced by fluid distension of the urinary bladder. In addition, clinical symptoms as well as responses to the same three experimental pain procedures will be assessed aross the menstrual cycle in both IC patients and controls. It is anticipated that: 1) IC patients will demonstrate greater sensitivity to both somatic and visceral pain stimuli relative to controls, 2) clinical symptoms will be greater among IC patients during the luteal versus the follicular phase of the menstrual cycle, while menstrual cycle effects among controls will be minimal, and 3) experimental pain sensitivity for both groups will be greater during the luteal versus the follicular phase; however menstrual cycle effects will be greater for IC patients than controls. The results of this research will provide important and novel information regarding pain sensitivity and menstrual cycle effects in IC and will establish a foundation of knowledge to support more extensive investigations of hormonal influences on nociceptive processing in interstitial cystitis.

This R21 application seeks support for an investigation of the roles of marital status, social support, and self- efficacy in the effectiveness of diabetes management in a diverse population of middle-aged and older adults in the U.S. The proposed research will use structural equation modeling to analyze data collected in 2002 and 2004 from core interviews of the nationally representative Health and Retirement Study (HRS), plus data from the 2003 supplemental diabetes-specific mail survey to examine these relationships by gender, age group, and major race-ethnicity categories. The significance of the proposed research lies in the skyrocketing rates of type 2 diabetes in the U.S. over the past few decades in all segments of the population, but primarily in middle aged and older adults. Concerns about obesity and physical inactivity and their links to the onset, complications, and societal costs of diabetes have led to public health mandates at the national level-such as those included in Healthy People 2010-aimed at reversing those trends. Although prevention of diabetes is certainly an important goal, more effective management for individuals already diagnosed with diabetes is also critical for reducing the risks of future complications. Currently, less than one in eight of all adults with diabetes follow the guidelines for good management;and older adults follow them at even lower rates. Many past studies showing that self-efficacy and social relationships are key factors in diabetes-related behavioral and health outcomes are limited by their use of non-representative data sets, cross-sectional analyses, and inadequate controls on other factors that may also be important to consider. This application's goal is to examine how social relationships (and particularly marriage) and self-efficacy influence adherence to diabetes-related lifestyle behaviors and health outcomes in order to develop and test interventions that provide more effective supports for adults living with diabetes. In particular, we will: (1) examine marital status as a predictor of behavioral (diet and exercise) and health outcomes (glycemic control and diabetes-related quality of life) in middle-aged and older adults living with diabetes;(2) investigate whether self-efficacy and diabetes support mediate the link between marital status and behavioral and health outcomes of diabetes;and (3) discern whether associations among marital status, diabetes support, self-efficacy, and behavioral and health outcomes are moderated by gender, age group, or race-ethnicity. Understanding more clearly the role of marriage and self-efficacy, and particularly if and how diabetes management differs between middle-aged and older married and unmarried adults, may inform the development of more effective interventions for adults living with diabetes. This knowledge may also be all the more important given recent studies showing spouses may be at increased risk for developing diabetes themselves because of the marital context and shared environment. PUBLIC HEALTH RELEVANCE Concerns about obesity and physical inactivity and their links to diabetes have led to public health mandates- such as those included in Healthy People 2010-aimed at reversing those trends. Although prevention of diabetes is an important goal, more effective management for individuals already diagnosed with diabetes is also critical for reducing the risks of future complications. Currently, less than one in eight adults with diabetes follow the guidelines for good management;and older adults follow them at even lower rates. Understanding how social relationships (and particularly marriage) and self-efficacy influence good diabetes management in middle-aged and older adults living with diabetes may be key to developing and testing interventions that provide more effective supports, and all the more important to married couples because spouses may be at higher risk for developing diabetes themselves if their partner has diabetes.

! Project Summary Exposure to particulate matter (PM) air pollution is the fifth leading cause of premature disease and death on the planet and the number one environmental risk factor for the global burden of disease (posing a greater danger than all other environmental risk factors combined) according to the World Health Organization. Despite the growing need (and demand), the state-of-the-art for assessing personal exposure to PM is based on decades old technology that is inefficient, burdensome, and expensive. The physical burden posed by these monitors (noise, visual aesthetic, and weight) make them difficult to wear. The cost of these monitors also prevents air pollution exposure monitoring at scales relevant to epidemiologic research and occupational hazard surveillance. This Phase II proposal will develop and commercialize a novel, lightweight, and inexpensive personal sampling technology based upon ultrasonic piezoelectric pumping modules. The ultrasonic personal aerosol sampler (UPAS) has the potential to gain a wide share of the air pollution monitoring market. AST proposes developing this sampler into a commercial prototype that is inexpensive (<$200 bill-of-material cost at scale), provides both time-integrated (filter-based) and real-time measurements, is accurate (flow control and PM sampling efficiency within 4% of reference), compact, lightweight (<200g), provides better subject fitment, has no annual maintenance requirements, and virtually silent in operation. Furthermore, the technology will be versatile ? able to collect PM2.5 (along with other relevant size fractions such as PM4 and PM10, respirable, and inhalable PM) for sample durations of up to 24 hrs. Taken together, these innovative aspects suggest that the UPAS will be highly competitive with potential for rapid and substantial market penetration. Three aims are proposed: (1) Integrate a novel, real-time PM sensor into the UPAS hardware/firmware; (2) Develop a suite of different plug-and-play size-selective inlets to make the UPAS more versatile and optimize the UPAS for weight, power, performance, and usability; (3) Validate performance of the prototype through laboratory and field testing. !

Clinical Tools, Inc., in collaboration with the UPMC Health System, will create and evaluate internet-based education for physicians, residents, and medical students interested In the topic of opiate addiction. Our goal is to increase accurate recognition and treatment of opiate addiction through education of physicians and physicians-in-training. The courses will use clinical scenarios and multimedia presentations to provide a low cost distance learning opportunity that is available 2~hours a day, seven days a week. In Phase I we will create design specifications for a suite of courses related to opiate addiction based on the results from the 1997 NIH Consensus Development Conference on Effective Medical Treatment of Heroin Addiction. We will demonstrate our ability to create internet-based courses by developing a single course which is an overview of opiate addiction. We will evaluate the effect of the course on physicians' knowledge, attitude, behavior and satisfaction using assessment forms developed in Phase I. In Phase II we will create and evaluate the full suite of courses. Phase III work will involve modifying and expanding the courses to teach a full range of health professionals. PROPOSED COMMERCIAL APPLICATIONS: Quality continuing education is essential for health care providers. Providers and their organizations will request easy to use, inexpensive and engaging courses that help them detect opiate addiction and provide cost- efficient, effective treatment.

Chronic smoking is a national public health problem. Associative-based interventions (cue-exposure therapy) can be effective at reducing smoking and decreasing relapse rates, indicating that acquired associations with nicotine contribute to chronic smoking and relapse. The long-term goal of this research program is to elucidate these associative processes and their role in chronic nicotine use. Paviovian conditioned associations between a conditioned stimulus (CS) and an unconditioned stimulus (US) is one such process. In contrast to research treating nicotine as a US, little work has explored the possibility that the pharmacological effects of nicotine might serve as a CS and acquire additional excitatory properties. The present application will eliminate this deficit using a rat model. In this model, nicotine (CS) is reliably paired with the appetitive effects of sucrose (US). An association is evidenced when nicotine differentially evokes anticipatory food-seeking behavior. Specific Aim 1 will test whether changing the nature of the nicotine CS alters development or expression of conditioned responding; alterations include changes in nicotine dose and time between nicotine administration and testing. Aim 2 will test whether an associative learning or a state-dependent learning process is responsible for differential control of food seeking by nicotine. Aim 3 will assess the ability of several ligands (ABT-418, bupropion, nornicotine, cytisine, epibatidine) to prompt nicotine-like conditioned responding (stimulus substitution). Differential substitution patterns will provide insight into the neurobiological processes mediating the CS effects of nicotine. Aim 4 will assess whether extinction with a ligand that shares stimulus properties with nicotine will lead to the development of a competing association that transfers to a nicotine CS. This transfer of extinction (associative substitution) would be evidenced if rats trained with a nicotine CS, but receive extinction with another drug, show loss of conditioned responding when retested with the unextinguished nicotine CS.

This case-control epidemiological study is designed to ascertain whether there are any associations, either positive or negative, between use of exogeneous estrogenic hormones for therapy during or following menopause or for oral contraception during reproductive years, and cancer of the corpus uteri, cancer of the breast and cancer of the ovary. Previous estrogen use in women in the age group 45-74 admitted to several large Connecticut hospitals with a first diagnosis of any of these cancers will be compared to estrogen use in women in the same age group admitted to the surgical services of the same hospitals for other conditions. Information on estrogen use and other relevant variables will be obtained by means of a standardized structured questionnaire administered by trained interviewers; this will be supplemented by information provided by the patients' physicians when necessary. Diagnosis of the cancers will be confirmed by a pathologist, and particular attention will be focused on specific histological types. The search for associations will include attempts to determine whether specific estrogenic compounds are involved, how long they must be used before an effect is seen, and whether any subgroups of women are more likely to be affected than others. This study will thus involve collaboration of epidemiologists, pathologists, biometricians, and surgeons.

E. coli p-aminobenzoate synthetase and anthranilate synthetase are structurally and genetically related enzymes that catalyze similar, but slightly different, reactions. Each enzyme is composed of two dissimilar subunits, one responsible for glutamine amidotransferase (GAT) activity, and the other responsible for the aromatization of chorismate. The difference in the products is the disposition of amino- and carboxy- substituents on a benzene ring. The subunits of PABS are encoded by pabA and pabB and the subunits of anthranilate synthetase are encoded by trpE and trpG. The nucleotide and amino acid sequences of these four genes have been determined, and clearly indicate divergence from a common ancestor. Similarity is approximately 30% between the large subunits and 45% between the small subunits of the enzymes. The GAT subunits perform identical functions, and differ only in the specific interactions with the appropriate large subunit. The nucleotide sequence of a GAT subunit that functions in both anthranilate and PABA synthesis in A. calcoaceticus has also been determined, and is very similar to the E. coli pabA sequence. The experiments in this proposal focus on the determination of specific differences between the pab and trp genes that are responsible for divergence of function. The experimental approaches include construction of hybrid genes by exploiting in vivo recombination and in vitro DNA replication techniques, in vitro inter- and intrageneric subunit exchange, in vitro mutagenesis, and analysis of second site reversions. The results of the proposed studies will include information on the ways in which genes evolve from a common ancestor after a gene duplication event. The extent of total nucleotide and amino acid sequence divergence has been documented, and my aim is to determine the fraction of the divergence that is responsible for alteration of function. We will also gain insight into a mechanism in which biochemical specificity has been maintained while function has diverged. The approach and methodology are unique in that a functional determination of the consequences of sequence divergence will accompany a descriptive approach on the extent of sequence divergence.

The objective of this project is to develop a peptide sequencing procedure based in part on novel reactions first noted in this laboratory. By this procedure peptides generated by enzymatic and/or chemical cleavages will then be converted by the novel reactions to volatile components separable by gas chromatography. The novel reactions are catalyzed by pyridine - 2 -carboxaldehyde Schiff base(s). Gas chromatography/mass spectrometry provide the information requisite for sequence assignments. A dedicated computer and software facilitate data work up and sequence assignment.

Over half of all deaths in non-ischemic heart failure (HF) are due to ventricular arrhythmias, primarily ventricular tachycardia (VT) degenerating to ventricular fibrillation (VF). In non-ischemic HF, VT can initiate by non- reentrant (focal) mechanisms likely due to delayed after depolarizations (DADs). The heterogeneous HF substate may then allow focal activity to convert to reentrant VT/VF. Thus, lethal arrhythmias in HF likely require a focal triggr for initiation and a vulnerable substrate that promotes reentry. HF electrophysiological remodeling can promote DADs and cause pro-arrhythmic changes to the substrate. -adrenergic receptor (-AR) stimulation can exacerbate DADs and further perturb the abnormal ionic currents and Ca2+ transients (CaT) found in HF. Moreover, cardiac sympathetic nerve remodeling in HF may lead to localized -AR stimulation and spatially heterogeneous effects. Electrophysiological and sympathetic remodeling has been individually linked to arrhythmia in HF. However, the interplay between local -AR stimulation and altered HF electrophysiology in arrhythmogenesis has not been explored. The overall objective of this proposal is to systematically determine the role of local -AR stimulation in producing the trigger and substrate for ventricular arrhythmias and how electrophysiological remodeling in non-ischemic HF exacerbates these effects. Our over-arching hypothesis is that local -AR stimulation causes 1) spatiotemporal synchronization of DADs across many cells to provide focal triggers; and 2) local electrophysiological heterogeneity to produce the substrate for reentrant VT/VF. We further hypothesize that electrophysiological remodeling in HF exacerbates the pro-arrhythmic effects of local -AR stimulation, both in generating triggers and in modifying the substrate. To address these hypotheses, dual optical mapping of Vm and Ca2+ will be performed on isolated rabbit hearts while administering local norepinephrine (NE). Aim 1 will focus on the mechanisms by which local -AR stimulation triggers focal arrhythmia in healthy rabbit hearts. We will then systematically test the effects of HF- associated electrophysiological remodeling on the propensity to focal activity by pharmacologically mimicking key HF phenotypes. Aim 2 focuses on the role of local -AR stimulation in contributing to the substrate for reentry via dispersion o action potential and CaT properties and development of alternans. We will also test the effects of key HF-associated electrophysiological mechanisms in contributing to reentry during local -AR stimulation. In Aim 3, we will determine the arrhythmogenic role of localized -AR stimulation in a rabbit model of non-ischemic HF by applying exogenous as well as invoking endogenous sympathetic stimulation followed by a quantitative assessment of neurochemistry in HF. We will then assess the therapeutic potential of reversing key HF phenotype(s) with newly proposed anti-arrhythmic strategies. Overall, the results of this project will define the firt mechanistic link between sympathetic dysfunction and ventricular arrhythmias in non-ischemic HF and will greatly advance our long-term goal of predicting and preventing sudden cardiac death in HF. PUBLIC HEALTH RELEVANCE: Heart failure predisposes patients to deadly cardiac arhythmias (disturbances of the normal heart rhythm). Increased activity of the cardiac sympathetic nerves is known to be associated with arrhythmias. But how exactly sympathetic nerve activity leads to arrhythmias and the interaction between nerve activity and other changes that occur in the heart during heart failure are unknown. The goal of this project is to determine the role of local sympathetic nerve activity in leading to arrhythmias in heart failure. The result of this study will shed new light on proper therapeutic treatments for heart failure patients.

PROJECT SUMMARY Pre-exposure prophylaxis (PrEP), formulated as a once-daily pill, represents an effective form of biomedical HIV prevention for men who have sex with men (MSM), but its use among African American MSM remains suboptimal and low relative to White MSM. African American MSM are more likely to report suboptimal adherence and subsequently seroconvert while using PrEP. As such, the use of long-acting injectable (LAI) antiretroviral drug formulations as PrEP may be an attractive alternative for MSM interested in biomedical HIV prevention but who may have difficulties with daily pill-taking. However, little is known about what factors may influence willingness to use LAI-PrEP among African American MSM. The proposed research will leverage existing data from the N2 Study, a population-based longitudinal cohort of young HIV-negative African American MSM residing on the South Side of Chicago (n = 350). Using detailed information on the social and sexual networks of young African American MSM, we will assess whether one?s willingness to use LAI-PrEP is impacted by their position within their networks. Using a complex systems approach known as agent-based modeling, we will simulate HIV transmission in the dynamic networks of young African American MSM, allowing us to assess the potential effects of a network-based intervention in improving LAI-PrEP use and reducing HIV incidence. Identifying the social dynamics associated with future use of LAI-PrEP may make a significant impact on the future success of this prevention method and the speed at which an effective LAI-PrEP formulation is taken up among young African American MSM. In achieving our aims, the findings from the proposed research may be used to optimize the uptake of LAI-PrEP through innovative network-based interventions, potentially maximizing its ability to make a sustained impact on the HIV epidemic in the United States.

Perhaps few questions in development have been so intractable as the means by which different cell fates are produced in response to a gradient of signals. These analogue-to-digital switches underlie the determination of cell fates in many organisms. T Lymphocyte development may be one of the best systems to understand this general problem because the critical signals are given at a stage where biochemical, genetic and cell biologic methods can all be brought to play. In lymphocytes, weak or transient signals are thought to produce positive selection (differentiation and proliferation) of T cells capable of reacting to self-MHC on thymic stromal cells. On the other hand, strong signals produced by self-antigen lead to death of cells responding to self-antigen. The pro-apoptotic protein Bim is required for negative selection but is not necessary for positive selection. Conversely, we have recently found that calcineurin is essential in T cells for positive selection, but dispensable for negative selection. Surprisingly calcineurin specifically controls the activation of ERK but not other MAP kinases or IkB, suggesting a revision of the accepted signaling pathways of thymocyte selection. These observations set the stage for a biochemical march from Bim and calcineurin to the molecule(s) that divert signals from positive to negative selection with increasing signal intensity. Current studies support several possible mechanisms by which signals of different intensity could control selection. To avoid the difficulties encountered with forward analysis of biochemical pathways we will work backward from Bim and calcineurin to define the biochemical pathways that control their activity in CD4+, CD8+ thymocytes. Our goal in these studies will be to define the lowest common mediator necessary for activation of both Bim and calcineurin and hence positive and negative selection. We will then determine the mechanism by which this molecule is induced to channel high intensity signals to Bim and low intensity signals to calcineurin. We will also define the processes downstream of calcineurin that mediate positive selection including the mechanism of NFATc nuclear import and export, the set of genes that are dependent on calcineurin activity in positive selection and how these genes give rise to a population of immunologically competent peripheral lymphocytes. Defining these mechanisms should lead to a more complete understanding of immune defense and provide useful information for development of new therapies. [unreadable] [unreadable]

This application requests partial funding for the Reproductive Tract Biology Gordon Conference to be held July 2-7, 2000. Please note that due to the scientific content of the meeting we request the National Institute of Child Health and Human Development as the primary sponsor of the meeting. We also request joint sponsorship by the Office of Women's Health, the National Cancer Institute, National Institute of Diabetes, Digestive and Kidney Diseases, the National Institute for Environmental Health Sciences, and the National Institute of General Medical Sciences. The number of requested sponsors reflects the unusual nature of this conference which focuses on the biology and the pathology of the organs and organ systems of the male and female reproductive tracts, rather than on specific molecules or processes. Therefore, this meeting attracts participants from a wide variety of fields and backgrounds ranging from basic science to clinical practice. Accordingly, the principal goal of this conference is to stimulate cross- disciplinary exchange and integration of information concerning the reproductive tract. Three specific aims will allow us to accomplish this goal: First, the scientific content of the meeting will be at the highest level. To achieve this standard of excellence, we are inviting speakers who are internationally recognized experts at the forefront of their fields. There is an interesting blend of established investigators who have been leaders in their respective areas for many years and equally renowned researchers in other fields whose phenotypic analyses of transgenic mice have uncovered unexpected and novel actions of these molecules in the reproductive tract. Second, there is a strong emphasis on research that is highly relevant to important clinical questions and has potential for translation into medical practice. For example, one entire session focuses on mechanisms of hormonal signaling in the context of the mammary gland with a special emphasis on aberrations that lead to breast cancer. Third, there is a strong emphasis on the inclusion of students and junior colleagues because the future of any field depends on the next generation of investigators. In summary, we have planned an exciting meeting that is in accord with the rich traditions of the Reproductive Tract Biology Gordon Conference, which has been in existence for nearly 30 years. We envision that the new site (Connecticut College, New London Connecticut) and the intent to meet on a permanent basis with the Mammalian Gametogenesis and Embryogenesis Conference will enhance the quality of this meeting, which has consistently been rated extremely high by the attendees.

Inhibitors of the enzyme sarco/endoplasmic reticulum Ca2+ATPase (SERCA) are valuable research tools for the study of the enzyme's role in physiological processes and they also have the potential of being developed into new anti- cancer agents. A series of 2,5-disubstitued hydroquinones will be synthesized and their ability to inhibit SERCA will be assessed in bioassays. Based on the results, computational techniques such as structure-activity relationship modeling and ligand docking will be used to develop models capable of predicting the activities of yet untested, hydroquinone-based compounds. With the aid of these models, compound libraries will be screened virtually for novel SERCA inhibitors that then will be obtained and tested in bioassays. PUBLIC HEALTH RELEVANCE: The research described in this application is aimed at the development of novel, hydroquinone-based inhibitors of the enzyme sarco/endoplasmic reticulum Ca2+ ATPase (SERCA). As documented by an impressively large number of publications on the naturally occurring inhibitor thapsigargin, SERCA inhibitors are of tremendous value for the study of the enzyme's role in physiological processes. In addition, it has been shown that SERCA inhibitors can be developed into prodrugs for the treatment of prostate cancer. Since hydroquinone-based compounds are structurally simple and quite different from other SERCA inhibitors, their synthesis from inexpensive starting materials is relatively straightforward. Thus, they have the potential of being developed into valuable alternatives to currently available agents with unique physicochemical and pharmacodynamic properties.

Recent work in this laboratory has shown that a variety of cancer cells secrete nerve growth factor. In particular, mouse L cells secrete large quantities of the factor in culture. Further, the chemical properties of L-cell NGF are different from any form of NGF that has so far been studied. Our study is: (1)\to grow large quantities of L cells; (2)\to isolate and to completely purify the NGF secreted by these cells; (3)\to determine the molecular properties and structure of this protein; and (4)\to ascertain whether this naturally occurring form of NGF has biological actions that have heretofore not been recognized, both within the nervous system as well as outside of it. Further work will completely purify human nerve growth factor from serum to characterize it chemically and to develop specific immunoassays for it. Such assays then will permit the specific measurement of NGF in man. More recent studies have shown that NGF can significantly promote the healing of experimentally induced wounds in animals, that it can substitute specifically for the first component of human complement, and that it is chemotactic for leukocytes. These findings indicate that NGF may play an important role in systems other than neural tissue. The chemical and biological meaning of this new information is under study at present. (J)

The incidence of obesity worldwide continues to escalate with the spread of the Western diet and with it there has been a corresponding increase in cardiovascular disease, diabetes, end-stage renal disease and other obesity-related disorders. Of all the causes for obesity, the predominant one seems to be choice - the choice to eat more and exercise less. One of the choices that has been linked with obesity has been the selection of a high fat, calorically dense diet. Despite much research on the link between fat intake and obesity, relatively little is known about the chemosensory mechanisms that underlie the taste of fat and how these mechanisms might contribute to dietary fat intake. Our previous research has identified differences in how the taste systems in obesity-prone and -resistant rodents respond to fatty acids, a cue for dietary fat, and how this was correlated to differences in gene expression and receptor function. Moreover, we have demonstrated that these chemosensory mechanisms are modulated by diet and the development of obesity. The revised proposal focuses on pursuing a more focused, single remaining specific aim that seek to explore how the gustatory response to fat is modulated by dietary experience. There is new and emerging data that argues to the plasticity of the peripheral taste system, yet our understanding of how the system changes in relation to experience is poor at best. Specifically, we will use an approach including a multidisciplinary approach to answer the following question: 1. Is the fatty acid transduction pathway modulated by diet? Our multidisciplinary approach analyzing genes through behavior will be used to test the hypothesis that fat receptor expression in taste cells is altered during high fat feeding in a manner that results in an increased responsiveness to fatty acids. Following high fat dietary regimens, molecular, cell-based and behavioral assays will be performed to measure changes in four primary receptive elements in the fatty acid signaling pathway, including CD36, GPR 120, GPR 84 and fatty acid-sensitive DRK channels. Our extensive preliminary data generated in the previous period would argue that these are the primary components of the pathway that underlies the ability of the gustatory system to recognize and respond to free fatty acids.

There has been little systematic study of the function of tissues within the oral cavity during aging, either describing normal processes of alterations resulting from specific diseases and therapeutic procedures. The purpose of this project is to focus on 3 oral health problem areas for the elderly (salivary secretion, oral motor function and cervical caries) and examine the status of certain biological factors which would likely influence the course of such problems. Major effort has been directed at evaluating electrolyte secretions from the stimulated parotid glands (reflecting ion fluxes in various gland components) and assessing several oral motor functions (postural, masticatory, speech, swallowing).

The objective of this proposal is to investigate the use of MYXV as potential oncolytic virotherapy agent against pancreatic cancer in preclinical animal models. The proposal focuses on MYXV, and is based on our previously published reports and supporting preliminary studies. MYXV is a rabbit-specific poxvirus with oncolytic activity against many types of human cancer models in vivo, including brain tumors, melanoma and rhabdoid tumors. In addition, we reported recently that MYXV is able to infect and kill pancreatic cancer cells in vitro. Our preliminary studies provided in the main body of the proposal show that MYXV exhibits potent oncolytic activity in both immunodeficient and immunocompetent animal models of pancreatic cancer. Based on these results, we seek to evaluate MYXV in combination with current chemotherapy regimens, especially gemcitabine, for pancreatic cancer. We hypothesize that MYXV will have potent oncolytic activity against pancreatic cancer in vivo and may be combined and/or engineered to enhance current chemotherapy treatments. Wildtype MYXV and recombinant MYXV "armed" with chemosensitizing gene(s) will be evaluated as single agent therapies and in combination with current chemotherapy drugs approved for the treatment of pancreatic cancer. For the purposes of this grant, we propose to: 1) Evaluate the efficacy of wildtype MYXV oncolysis in murine models of pancreatic carcinoma. MYXV will be evaluated: a) as a single agent therapy compared to standard chemotherapies, b) in combination with gemcitabine and/or erlotinib, and as c) second line treatment therapy for chemotherapy resistant tumors. These experiments involve the use of the most common first line chemotherapies for pancreatic cancer and will therefore evaluate MYXV in the context of a clinically relevant scenario. Immunodeficient and immunocompetent murine models of pancreatic cancer will be established intraperitoneally (IP) and virus will be administered locally by the IP route. Tumor burden and survival curves will be compared between treatment groups in the presence or absence of gemcitabine and in gemcitabine refractory tumors to determine if MYXV virotherapy under the three regimes mentioned above results in an enhancement of therapeutic benefits as measured by tumor burden and survival. 2) Generate recombinant MYXV armed with chemosensitizing genes that will enhance gemcitabine-based chemotherapy. Recombinant "armed" MYXVs that express deoxycytidine kinase (CDK) or the human equilibrative nucleoside transporter 1(hENT-1) will be engineered. These viruses will be characterized in vivo to determine if the expression of the transgenes enhances gemcitabine-based oncolysis of pancreatic cancer cells in the two models described above in Specific Aim 1. Tumor burden and survival curves will be compared between armed and wildtype MYXVs treated groups to determine if the use of an armed MYXV provides a therapeutic advantage over wildtype MYXV by enhancing gemcitabine-based chemotherapy in vivo. This proposal will be the first study to evaluate the oncolytic potential of MYXV in preclinical animal models of pancreatic cancer as single agent therapy and in combination with chemotherapy drugs, as well as the characterization of armed MYXV capable of sensitizing cells to gemcitabine chemotherapy. Thus, if successful, these pilot experiments will identify MYXV as an effective oncolytic virus that can be further developed as a novel, safe virotherapy for the treatment of pancreatic cancer. In particular, if the results are as positive as we anticipate, and given the excellent safety profile of MYXV, we will pursue the production of clinical grade stocks of MYXV and the filing for an investigational new drug application (IND) at the completion of the proposed study.

Project Summary/Abstract The Department of Occupational and Environmental Health (OEH) at the University of Oklahoma Health Sciences Center (OUHSC) Hudson College of Public Health provides graduate education leading to the Master of Science (MS) degree in Industrial Hygiene and Environmental Health Sciences (IH/EHS) and the Doctor of Philosophy (PhD) degree in Occupational and Environmental Health. The MS in IH/EHS at OUHSC is one of only four ABET-accredited masters level industrial hygiene programs in the south central United States. The goal of the MS in IH/EHS is to prepare professional practitioners to apply scientific knowledge to the anticipation, recognition, evaluation, and control of environmental hazards or stresses affecting human health. The MS curriculum, which emphasizes both quantitative skills and effective communication, consists of 48 semester hours of coursework, including masters thesis research and a field practice experience, and can be completed in 22-24 months of full-time study. The goal of the PhD degree in OEH is to prepare graduates to impart and/or add to knowledge in occupational health through careers in academia or research. The PhD curriculum includes advanced didactic coursework in environmental sciences, epidemiology, biostatistics, and research methods related to occupational health, and a doctoral dissertation within the broad field of occupational and environmental health. The specific aims of the proposed renewal of the NIOSH training project grant (TPG) are: (1) to attract highly qualified and motivated students, including individuals with cultural or linguistic fluency related to underserved populations, into the industrial hygiene profession; (2) to recruit students with diverse technical backgrounds who are interested in entering the industrial hygiene field; and (3) to recruit and train very promising research-oriented students in industrial hygiene research. TPG funds will be used to support six traineeship slots per year, to be filled by full-time masters-level trainees and up to 2 full-time doctoral-level trainees. Trainees in the MS program may be supported on the TPG for up to 24 months. Trainees in the PhD program may be supported for up to 5 years. Oklahoma colleges have relatively high American Indian enrollments reflecting the state's unique ethnic mix, presenting an excellent pool of well qualified prospective trainees from under-represented minorities. Recruitment for the MS program will also target individuals with work experience in the health professions, engineering, chemistry, and environmental science who wish to make the transition into industrial hygiene practice. Recruitment for the PhD will target highly qualified individuals with BS or MS degrees in science or engineering that provide a solid foundation for industrial hygiene research.