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Osteoporosis affects over 44 million Americans leading to direct annual expenditures in excess of $20 billion. Measuring the success of bone-sparing therapies requires not only sensitive tests but also convenient patient procedures since many osteoporosis sufferers are elderly. In the present study, we propose to test the utility of saliva, an easily collected biofluid, to measure changes in bone turnover in a group of peri-menopausal women who are participating in a randomized, double-blind, placebo-controlled 5-year study conducted as part of the Kronos Early Estrogen Prevention Study (KEEPS). Mayo Clinic is one of [unreadable] 8 participating centers nationally. The primary purpose of KEEPS is to study the effect of estrogen [unreadable] formulations on atherosclerosis. Fortuitously, bone turnover marker levels in serum and bone mineral density values will also be obtained meaning that KEEPS offers an extraordinary opportunity to investigate saliva. The central hypothesis of this application is that host derived bone-specific proteins present in human saliva will serve as biomarkers of systemic bone turnover and bone mineral density. The rationale for our proposal is that if levels of bone turnover markers in saliva correlate with levels in serum and/or BMD, then saliva, an easily collected biofluid, can be used to monitor systemic bone turnover. We are particularly well prepared to undertake this research because: (1) we have demonstrated that the bone turnover markers can be detected by conventional enzyme-linked immunoassays in human saliva; and (2) the KEEPS infrastructure ensures a rigorous clinical research environment. We will test our hypothesis by pursuing the [unreadable] following two specific aims: [unreadable] 1) Determine whether salivary bone turnover marker levels correlate with serum levels in a controlled clinical trial population. The working hypothesis is that levels of biomarkers in saliva are representative of levels of the same biomarkers in serum. [unreadable] 2) Determine the relationship between changes in salivary bone turnover marker levels and changes in BMD in a controlled clinical trial population. The working hypothesis is that bone turnover as measured by changes in levels of salivary biomarkers in the saliva of subjects receiving MHT is associated with increase in BMD when compared to subjects receiving placebo therapy. [unreadable] This ancillary study application addresses a key directive outlined by the NIDCR Panel on Genomics and Proteomics of Oral, Dental and Craniofacial Diseases which stressed the need to investigate saliva as a diagnostic tool. We anticipate that our results will identify how salivary biomarkers can be used to assess the osseo-status of patients with, or at risk for, osteoporosis. [unreadable] [unreadable] [unreadable] [unreadable]

The purpose of this Medication Development Research Center grant is to systematically and comprehensively test different potentially therapeutic drugs using human laboratory models of drug abuse for opiates, cocaine and cigarette smoking. This center grant will also examine how the results from human laboratory models will generalize to an outpatient clinical setting. Four individual projects and three Cores have been submitted. The first project focuses on the effects of dynorphin A (1- 13), an endogenous opiate, in opiate abusers. The proposed studies examine the effects of dynorphin on heroin withdrawal, the effects of dynorphin on the self-administration of opiates, the interaction of dynorphin and morphine in opiate-naive and opiate-dependent subjects, and the self-administration of dynorphin. The second project examines the effects of ritanserin, a 5-HT2 antagonist, and nifedipine, a calcium channel inhibitor, as potential pharmacological agents in the treatment of cocaine abusers. Studies will be conducted to determine the effects of these medications on human laboratory models of cocaine self- administration, relapse and withdrawal. The third project will examine the effects of ritanserin using an outpatient clinical setting in female cocaine abusers who do or do not have a current history of affective disorder. Studies will be conducted to examine the effects of ritanserin on cocaine self-administration and relapse to cocaine. The fourth project examines the role of a metabolite of nicotine, cotinine, in tobacco dependence. The proposed studies will examine the effects of cotinine during cigarette abstinence, physical dependence of cotinine, the self-administration of cotinine, and the effects of cotinine on cigarette smoking. The strengths of this application include 1) the testing of novel compounds as potential medications, 2) the use of parallel designs and methodologies across drugs, 3) the extension of laboratory findings to an outpatient setting, 4) the presence of ongoing clinical research programs and funded grants in these areas among the investigators, and 5) well established collaborations among the investigators, and 6) the integration of the proposed clinical studies with strong preclinical and basic research programs in drug abuse, and a drug abuse training grant, at the University of Minnesota. All four studies will use the services provided by the three proposed Administrative and Support, Analytical and Statistical Cores.

Mitochondrial generation of ATP, and the processes which control it, are crucial to nearly every function performed by cells. Uncoupling proteins 2 (UCP2) and 3 (UCP3) are homologues of UCP1 which provide an important level of control over mitochondrial generation of ATP. Accordingly, there has been much interest in discovering the biochemical and physiological function of UCP2 and UCP3. Gain-of-function, overexpression studies have suggested that UCP2 and 3 have uncoupling activity, and when greatly overexpressed in animals, regulate whole body energy metabolism. However, these high level overexpression studies do not provide insight into the function of endogenous levels of UCP2 and 3. Towards these ends, we have created loss- of-function gene knockout mice lacking either UCP2 or UCP3. In the Progress Report section, we provide strong evidence that UCP2 and UCP3 control rates of ATP production. In the case of skeletal muscle, where UCP3 is abundantly expressed, the physiological significance of this is presently being explored. In the case of pancreatic beta-cells, where ATP couples glucose sensing to insulin secretion, this has extremely important implications for regulation of insulin secretion. UCP2 is abundantly expressed in beta-cells. Of note, mice lacking UCP2 have markedly increased glucose-stimulated insulin secretion, demonstrating that UCP2 negatively regulates this process. Of pathophysiologic significance, UCP2 is significantly upregulated in islets of obese mice with inadequate beta-cell function and type 2 diabetes. Importantly, obese mice lacking UCP2 have restored first-phase insulin secretion, increased serum insulin levels and greatly decreased levels of glycemia. These results strongly suggest that UCP2 is a key component of beta-cell glucose sensing, and is a critical link between obesity beta-cell dysfunction and type 2 diabetes. These important issues will be explored in the following Specific Aims: Specific Aim 1: To define the roles of UCP2 and UCP3 in regulating mitochondrial bioenergetics. Specific Aim 2: To determine the mechanism-of-action by which UCP2 negatively regulates insulin secretion. Specific Aim 3: To determine the role of UCP2 in linking obesity to beta-cell dysfunction and type 2 diabetes. Specific Aim 4: To determine the mechanism by which the obese state induces UCP2 expression in islets. These studies should provide major insights into the physiologic and pathophysiologic functions of UCP2 and 3.

The aim of this project is to test the hypothesis that smooth muscle cells from patients with Crohn's disease and ulcerative colitis induce expression of CD1d on intestinal epithelial cells, which in turn leads to an inappropriate inflammatory response. We propose to determine if certain candidate cytokines are responsible for the upregulation of CD1d, which is an antigen-presenting MHC-like protein. Supernatants from co-cultures of patient (and control) smooth muscle cells and an intestinal epithelial cell line are sent to the immunomodulation core lab for analysis.

Oligodendrocytes (OLs) are the myelinating cells of the central nervous system and play a critical role in white matter formation. Serious clinical disorders affect central nervous system white matter during early development. These conditions include periventricular white matter injury (PWMI), which affects up to 20% of very low birth weight premature infants. PWMI is due to loss of Pre- oligodendrocytes (PreOLs), which are proliferative cells that develop into myelinating OLs. Presently, we are unaware of pharmacological approaches that specifically target PreOLs, resulting in increased proliferation of these cells. Because white matter injury is the leading cause of nervous system injury in premature infants, developing new treatments for PWMI will have a major clinical impact. Under Phase 1 of this SBIR, we proposed to use high-throughput screening to identify compounds ("hits") that would stimulate PreOL proliferation. These studies proved to be highly successful, as we identified diazoxide as a stimulator of PreOL proliferation, and we observed that this compound promotes myelination in a murine model of PWMI. Diazoxide acts by activating KATP channels, and we were able to demonstrate KATP channel component expression in PreOLs. We also found that other KATP activators stimulate PreOL proliferation. Thus, we achieved the major goal of our Phase 1 proposal. In Phase II studies, we propose to extend our study of diazoxide in preclinical, proof-of-concept studies involving cell culture and animal studies. We will: (1) Define OL-stage specific responses to diazoxide. (2) Evaluate protection against PWMI in animal models. (3) Assess long-term effects of neonatal diazoxide therapy. The long-term goal of this work is to develop novel therapeutic agents for the treatment of white matter injuries in premature infants. Support from the NIH through the SBIR program will serve to strengthen the productive academic/industrial collaboration established between scientists at Yale University (Dr. Rivkees) and JS Genetics (New Haven, CT) to discover novel drugs. Ultimately, research such as this will lead to important discoveries with significant public health benefit and commercial value. We also anticipate that these studies will lead to the development of novel approaches for treating and preventing white matter injury in the tens of thousands of premature infants born and hospitalized each year. PUBLIC HEALTH RELEVANCE: The goal of this work is to assess the utility of diazoxide as a therapeutic agent for the treatment of white matter injuries in premature infants. We anticipate that these studies will lead to the development of novel approaches for treating and preventing white matter injury in the tens of thousands of premature infants born and hospitalized each year.

The proposal deals with the mechanism of transduction in mammalian taste reception. A new method for detecting the effects of tastants at the single cell level is introduced. The method permits tastants to be applied to the normal taste-sensitive regions of the taste-bud while at the same time recording the cell electrical response. Recordings are made using patch-clamp technology. With these methods the characteristics of the cells in the anterior part of the rat tongue will be examined. Single fiber analysis of the rat chorda tympani suggests the existence of cells that will have a highly selective sodium ion conductance in the cell apical membrane. There should also be less selective cells reactive to various salts and acids. A catalog of cell types will be made to test this hypothesis. The kinetics of the changing intracellular potential will be studied under conditions of normal chemical stimulation. The possible presence of action potentials will be noted and the kinetics of cellular adaptation will be studied. Preliminary data show that the apical membranes of some taste cells have amiloride-blockade conductances. The ion specificity of these amiloride-sensitive apical membrane conductances will be examined. The possible involvement of cAMP in the sweet taste transduction will be explored. This will be done on the single cell level, and also using a live-rat model in which the chorda tympani response to sucrose will be measured with changes in the transepithelial potential or current under voltage clamp. Preliminary data indicate that a basolateral membrane conductance is blocked during stimulation by sucrose. The question of the regulation of the expression of amiloride-blockade sodium conductances will be addresses. Among sodium-deprived rats there is evidence that regulation may be effected through sodium-regulating hormones. We shall explore this possibility electrophysiologically at the cellular level and with a live-rat model. Atrial natriuretic factor, acting via cGMP, is one of the possible regulatory substances. Taste is a major factor in determining food intake. The conditions and substances that modulate its expression will have a strong impact in the areas of sodium-sensitive hypertension and feeding disorders such as obesity.

Bordetella pertussis, the microorganism which causes the disease commonly known as whooping cough, produces several toxins (i.e., pertussis toxin (PT) and heat labile or dermonecratic toxin) which appear to play important roles in pathogenesis of the organism. PT in addition is a major protective antigen which is a promising candidate for the development of a new acellular pertussis vaccine. The current project concentrates on elucidating the mechanisms by which pertussis toxin interacts with cells and elicits its diverse pharmacologic actions. The initial event in the interaction of PT with cells appears to be a rapid and essentially irreversible binding of toxin to cells. Using the interaction of PT with fetuin as a model, studies have been conducted which demonstrate that PT interacts with the carbohydrate moieties present in fetuin. Similar carbohydrate structures are present on cell surfaces and in a variety of serum glycoproteins including IgE and other immunoglobulins. The significance of these observations relative to the intoxication of cells and to specific adjuvant effects exhibited by PT are under consideration. The mechanism of action of PT is the toxin catalyzed transfer of an ADP-ribose moiety from NAD to specific acceptor protein (Ni). (Ni) is a cell membrane protein, which couples hormone receptor interaction to modulation of adenylate cyclase. Most of the effects elicited by PT appear to be associated with the covalent modification of (Ni). PT has served as an effective probe in purification of the regulatory (Ni) component and in elucidating the molecular mechanisms by which (Ni) acts in modulation of adenylate cyclase by inhibitory effectors. It has been established that (Ni), in part, functions by changing the affinity of the hormone receptor for ligand, thus when cells are treated with PT high affinity of binding of ligand is eliminated. Additional studies are in progress attempting to define more clearly the role of (Ni).

Our goal is to understand the role of scaffold proteins in the organization of signal transduction. Scaffolds determine the outcome of signal transduction by controlling the location of cell surface receptors and connecting them to downstream effectors. This proposal focuses on post-synaptic glutamate signaling, which mediates excitatory neurotransmission. Glutamate receptor signaling pathways are organized by the membrane-associated guanylate kinases (MAGuKs). In excitatory neurons there are four MAGuKs (PSD-95, PSD-93, SAP102 and SAP97/Dlg). Existing biochemical data is in conflict with functional data regarding the specific role each protein plays in synaptic plasticity. This proposal investigates the molecular basis of MAGuK structure and specificity. We have a working reconstitution of receptor-scaffold interactions in the post-synapse to provide the missing quantitative data on MAGuK affinity and selectivity for glutamate receptors. Receptor cytoplasmic domains are attached to a planar phospholipid bilayer, creating a functionalized surface that mimics the postsynaptic membrane. Aim 1 will test the hypothesis that functional differences between the four PSD-MAGuKs arises from differences in the kinetics of receptor binding. Single molecule fluorescence and simulations will solve the structure of all four, full-length MAGuKs by placing the known structures in context. We can watch individual binding events in real time to quantitate the MAGuK binding to both NMDA and AMPA receptors and also Stargazin. Aim 2 will test the hypothesis that differences in MAGuK quaternary structure give rise to differences in binding affinity for receptors and other ligands. Aim 3 is to confirm te structure of PSD-MAGuKs in vivo. Characterization of the GFP-tagged PSD-95 (and a live-cell FRET ruler) in vitro will form the basis for quantitative interpretation of live-cell FRET measurements. These studies are advancing towards a physical and kinetic description of PSD assembly. We strive to achieve a cellular structural biology by reconstituting higher-order systems. This reconstitution will eventually serve as a platform to incorporate additional post synaptic components. These results will indicate how much of the variable signaling behavior in the synapse is attributable to the scaffold itself. Describing the molecular events in excitatory signaling is a fundamental challenge in neuroscience with direct relevance to brain development, memory and learning, and many neurological and neuropsychiatric disorders.

Evidence in both humans and mice shows that immunoregulatory mechanisms are transmitted from mothers to their newborns via breast milk. However, early childhood infections by certain pathogens such as respiratory syncytial virus (RSV) compromise these protective mechanisms thereby predisposing to disease (asthma) later in life. Our preliminary data suggest a role for virus-induced Th2 responses in impairment of immunoregulatory mechanisms in newborn mice. In previous studies we have shown the importance of regulatory T cells (Tregs) expressing (mTGF-) in maintaining immune tolerance in the airways of mice. Given the important role of Tregs in suppressing unwarranted allergic responses in the airways, we investigated whether RSV infection compromises immune tolerance mechanisms in early life. Using the same model of airway tolerance previously used by us to demonstrate the importance of mTGF- in airway tolerance, it was recently shown that mothers tolerized to the antigen (Ag) ovalbumin (OVA) can transfer OVA and TGF- to their newborns via breast milk. A role for induced Tregs (iTregs) in maternally-transferred tolerance was presented in this study. If RSV infection in newborns can promote allergic disease, we asked whether Treg-mediated immune suppression is compromised by viral infection in the newborns. Indeed, our preliminary data provide evidence of RSV-mediated breach of maternally-transferred tolerance. Since RSV infection promotes Th2 responses, we reasoned that Tregs might change phenotype and function due to instability of Tregs in the context of inflammation. Our preliminary data show the presence of Tregs co- expressing Foxp3 and GATA-3 in RSV infected lungs, which is attenuated in IL-4R-deficient mice. We also show impairment of suppressive function of Tregs isolated from RSV-infected lungs. Additionally, we have observed that dendritic cells isolated from the lungs of infected mice have a more activated phenotype compared to those from uninfected lungs and when exposed to RSV are less efficient in inducing Tregs. These observations lead us to formulate the major hypothesis of this proposal, which is that RSV infection impairs maternally-acquired tolerance mechanisms in newborns. To address this hypothesis we will: Aim 1. Investigate the role of virus-induced Th2 cytokines in impairment of tolerance and Treg function in newborn mice. Aim 2. Study the contribution of the CX3CR1 pathway engaged by the viral glycoprotein G in impairment of Treg function in RSV-infected neonates. Aim 3. Examine the ability of aerosolized liposome-encapsulated ATRA to restore Treg function and airway tolerance in virus-infected lungs of mice and non-human primates.

The large majority of older patients with autoimmune Graves' hyperthyroidism and atrial fibrillation (AF) have coexisting activating autoantibodies (AA) to the beta1-adrenergic (B1AR) and M2 muscarinic receptors (M2R). Adrenergic and muscarinic agonists are known to enhance the likelihood for developing AF. HYPOTHESIS: AA in the aging thyrotoxic heart facilitates and/or causes AF. We have demonstrated AABAR) in 94% and AAM2R in 88%; and both autoantibodies in 83% in Graves' hyperthyroidism + AF compared to 10% of Graves' patients in normal sinus rhythm (P<0.001). All patients with AF had at least one of the two AA. These autoantibodies altered electrophysiological activity in pulmonary vein atrial sleeve cells such as predisposes to AF in experimental models; supporting our concept that elevated AABAR and AAM2R as well as thyroid hormone are major risk factors for AF. PROPOSAL: We will use translational and mechanistic studies to examine the electrophysiological interrelationships of thyroid hormone, age and activating autoantibodies as a cause of AF. METHODS: (Spec. Aim A) Expansion of epidemiological studies to identify the prevalence and function of autoantibodies in non-Graves hyperthyroidism and AF; (Spec. Aim Bi) Identify age and thyroid hormone dependent triggering of ectopic action potentials in the presence of target-specific autonomic AA in a canine pulmonary vein atrial sleeve tissue preparation in vitro; (Spec. Aim Bii) Study the development of AF (by ECG telemetry) in young (6 mo) and old (>5yrs) rabbits immunized to produce target-specific AAB1AR and/or AAM2R without and with thyroid hormone. We will then use sophisticated electrophysiological studies incorporating right atrial ganglia stimulation to measure the pulmonary vein atrial sleeve cell threshold for induction of AF. This model will determine the combined effects of AABAR and AAM2R on the atrial substrate for AF. METHODS also include a FRET-based micro-cAMP assay to examine the allosteric effects of AA. This assay will assist in determining whether these AA act as agonists and whether they also serve as partial antagonists to their normally operative orthosteric ligands. Our studies are NOVEL in that they will identify a spectrum of activating autoantibodies in the heart and demonstrate their role in immune-mediated mechanism(s) leading to AF in hyperthyroidism. Our data are relevant not only to hyperthyroidism but will also lead to a better understanding of these mechanisms in other and more common forms of AF which frequently coexist with AA. POTENTIAL IMPACT ON VETERANS HEALTH CARE: AF is an important risk factor for stroke and heart failure in our aging veteran population and leads to increased mortality and morbidity. The present study addresses this issue and may permit identification of future therapeutic options.

The human T-cell lymphoma virus, HTLV-I, has been found to be associated with patients with adult T-cell leukemia. Studies are underway to understand the mechanism of malignant transformation and immunologic response of individuals infected with this virus. An HLA class I epitope is expressed when cells are infected with HTLV-I but not HTLV-II. Differences in the env region of the provirus suggest that this epitope is present in the large envelope protein of HTLV-I. HTLV-I virus has been found in B-cell lines that grow spontaneously from patients with adult T-cell leukemia. The virus is expressed and infects T-cells indicating selective tropism for T-cells and not B-cells. These B-cell lines spontaneously produce acid labile and acid stable beta interferon and B-cell growth factor. T-cell clones with specific function lose their specificity when infected with HTLV-I. Immunoglobulin from B-cells from chronic lymphocytic leukemia cells from patients with serum antibodies to HTLV-I have antibody activity to HTLV-I glycoproteins suggesting an indirect effect of the virus in diseases other than adult T-cell leukemia. Antibodies were raised to HTLV-I protein produced by transfection of mouse cells with a vector containing selected portions of two retroviruses. This antibody reacts with the small envelope protein (p21) and p42 the product of the pX region of the virus.

Experiments have been performed that demonstrate an active non- specific immunosuppressive factor(s) in human cord blood and in the serum of rats transplanted with Morris hepatoma 7777. The factor appears to be concentrated in the fractions containing the highest concentration of alpha1-fetoprotein (AFP). These fractions inhibit in vitro human and rat lymphocyte cultures stimulated by phytohemagglutinin (PHA), pokeweed, and allogeneic lymphocytes. Acting on the hypothesis that AFP may be an immunoregulatory protein, in vitro and in vivo studies will be performed to determine: 1) whether the active factor is AFP or, if not, what is it?; 2) what is the mechanism(s) of action?; 3) what is the biologic significance of the material? Fractionation experiments have been designed to isolate the active factor. Dose response and kinetic studies are proposed to provide insight into the mechanism of action. Depending, in part, on the results of these studies, in vivo experiments will be performed to determine the possible biologic significance with respect to normal immunologic regulation and abnormal inhibition accompanying malignancy and other diseases.

The goals of this program are to use biological characteristics of tumor cells as a means for improving grading of human cancer through use of more objective parameters. The object of grading tumors is to estimate the potential of the tumor for growth, invasion, and metastasis, which are important factors to clinicians in planning therapy. Tumor cell attachment to new substrates is being compared to normal cell attachment using reflection contrast microscopy. A major characteristic of cancer cells, release from density dependent growth control, is also being studied as is the role of lamellar cytoplasm in tumor cell growth. The distribution of enzyme sites on the membrane surface of human oncogenic fibroblasts has been compared to the distribution in nononcogenic cells. Some new projects characterize human cancer cell populations in archival tissues and with needle aspiration cytology. A study of endometrial epithelium and its growth dynamics in relation to endometrial hyperplasia is now underway, and a separate project on the dynamics of cellular response to cytostatic agents in head and neck cancer is in the process of activation.

Stimulus equivalence, the ability to categorize stimuli on the basis of relations shared through associative contingencies and not physical features, may be a necessary prerequisite for symbolic Functioning and language development. Monkeys, chimpanzees, and sea lions can form some of the emergent relations that determine empirically the existence of equivalence classification: reflexivity, symmetry, and transitivity. Of these criteria, transitivity is the most difficult to ascertain because it is the most behaviorally complex; it is the ability to infer a relation between two stimuli given that both are related to a third stimulus. A positive outcome on transitivity tests may indicate the potential for symbolic functioning in the absence of language, a theoretical position with profound implications for understanding how cognition relates to the development of communicative behavior. A scant number of studies have suggested that rats also are capable of transitivity; however, certain methodological issues limit interpretation of these findings. We have developed a paradigm for rats to circumvent these issues so that a practical laboratory animal model of transitivity can be established and elaborated for future research: Rats will be trained in an automated nose poke apparatus to develop two arbitrary stimulus classes. A matching-to-sample task using six olfactory Stimuli will be used to teach explicitly some of the possible stimulus pairs within a class; e.g., match to B and then B to C. If rats have developed emergent transitivity, then they should show high curacy on probe trials for matching A to C, the untaught stimulus pair. Rats are expected to display transitive behavior. If they do, some specific studies for future research will be suggested that would proceed logically from such results. Ultimately, this rat model will lead to advances in the assessment of non-language based symbolic behavior, and to applications in the fields of behavioral toxicology and animal models of developmental disabilities.

Our work over the past year has been in collaborative support of NICHD protocol 06-CH-0038, "Body Heat Content and Dissipation in Obese and Normal Weight Adults". Activities included:[unreadable] [unreadable] 1. Further refinement of a "recovery-enhanced infrared imaging" method, to gauge the reaction of human subjects to mild cold stress of the fingers. [unreadable] [unreadable] 2. Further development and implementation of algorithms to analyze localized low-frequency oscillations in skin temperature during exercise, and to reduce artifacts due to movement.[unreadable] [unreadable] 3. Further development of a method to locate perforator vessels that travel (and conduct heat) from subcutaneous tissues to the skin's surface. IR imaging was also used to guide placement of telemetric skin patches for continuous, 24/7, recording of temperatures overlying perforator vessels. Correlations were made with rest, exercise, and digestion of meals.[unreadable] Study in progress.

Glucocorticoids (GC) produced by the adrenal cortex mediate the body's adaptive response to stress. The synthesis and secretion of these steroids is regulated by the hypothalamic-anterior pituitary system. In response to stressful stimuli, the hypothalamus is induced to secrete corticotropin releasing hormone (CRH). CRH is transported via the blood to the anterior pituitary where it stimulates the secretion of the neurohormone ACTH, which then stimulates the adrenal cortex to synthesize and secrete GC. Feedback inhibition by GC on both the anterior pituitary and the hypothalamus completes the regulatory loop. CRH influences feeding and sexual behavior in rats, and hypersecretion of CRH is thought to be involved in depression. Knowledge of the regulation of CRH gene expression will provide the groundwork for understanding both normal and abnormal responses to stress. The first step will be to establish the basis for tissue-specific activation of the CRH gene in the paraventricular nucleus of the hypothalamus and other localized regions of the brain. Transgenic mice bearing the rat CRH gene and varied amounts of flanking DNA will be constructed and tissues analyzed by in situ hybridization in order to locate the cis-acting DNA sequences necessary and sufficient for appropriate expression of the transgene in adult mice. Transcription of the CRH gene is high late in gestation but drops immediately after birth in the stress nonresponsive period. The ability of transgenes to recapitulate this modulation of CRH gene expression during development will be monitored. The regulation of CRH gene expression will be further studied in transgenic mice following adrenalectomy, to identify the DNA sequences critical for feedback inhibition by GC. Immortalized cell lines producing gene products characteristic of differentiated hypothalamic cells would be invaluable for studying CRH gene regulation. To achieve this, tissue-specific elements from the CRH gene will be engineered onto a gene encoding an oncogene (SV40 T-antigen) as well as a selectable marker (neomycin) and the hybrid genes used to produce transgenic mice. Immortalized CRH producing cell lines will be derived from these animals using the selective agent. CRH transgenic mouse studies will shed light on the intricacies of cell-specific expression in the brain and hypothalamus, develop cell lines necessary to study regulation of gene expression, and thus provide the basis for understanding clinically important disorders such as depression.

Numerous studies have shown that there are endocrine abnormalities associated with endogenous depression. The major alterations include resistance of the serum cortisol rhythm to suppression by dexamethasone (dex), alterations in the circadian secretion pattern of serum cortisol, and a blunted thyrotropin stimulating hormone (TSH) response to thyrotropin releasing hormone (TRH). However, the exact relationship between these endocrine alterations and the depression remains unknown. It is possible that these endocrine abnormalities may be associated with certain non-specific features of depression rather than with the depression per se. For example, depressed subjects have altered sleep patterns including decreased slow-wave sleep (SWS), fragmentation of sleep, and early morning awakening. Because sleep is one of the basic synchronizers of not only the normal rest-activity cycle in humans but also of many endocrine and metabolic events, and since hormone secretion is altered during sleep in patients with affective disorders, we hypothesize that some hormonal alterations observed in depressed patients may be mediated primarily by the sleep disturbance. In fact, the endocrine responses to dex and TRH in depressed patients may be normal responses which are desynchronized with respect to time, as a consequence of the disturbances in sleep. We intend to examine these chronoendocrine variables in normal volunteers and endogenously depressed patients before and after sleep deprivation. The number of subjects or patients chosen for each of the experiments outlined in this proposal will be determined by statistical power analysis of already published or pilot data to minimize the chance of a type II (beta) statistical error.

Our long term objective is to understand the roles of regulatory carboxypeptidases in physiological and pathological processes. Membrane-bound carboxypeptidase (CP) M is an important regulator of the kallikrein- kinin system via its ability to generate des-Arg-kinin agonists of the G-protein coupled kinin B1 receptor (B1R). Kinins are important regulators of renal and cardiovascular function and inflammatory processes. The major objective is to investigate a novel allosteric protein-protein interaction between CPM and B1R that enhances receptor signaling. Specific Aim 1: To explore the interaction of CPM and B1R on the membrane and determine the residues important for their interaction. Hypothesis: positively charged residues on CPM's exterior bind to membrane phospholipids to orient CPM on the membrane and basal interaction with the B1R is mediated by its C-terminal domain whereas allosteric modulation is mediated by a surface near the catalytic domain. We will: (i) determine whether CPM and B1R directly interact. (ii) investigate the importance of the exterior positive charges on CPM for proper membrane orientation and binding to the B1R. (ii) determine the site(s) on the B1R that mediates its binding to CPM. (iii) determine the site(s) on CPM that interacts with the B1R. (iv) generate peptides to interfere with CPM/B1R interactions. Specific Aim 2: To elucidate the characteristics of the interaction between CPM and the B1R that results in allosteric modulation of receptor activation. Hypothesis: CPM enhances B1R function by binding to an allosteric site and substrate binding by CPM results in B1R conformational change and enhanced receptor activation. We will determine: (i) the role of CPM specificity and peptide substrate affinity on allosteric modulation of B1R activation and (ii) the effect of CPM allosteric modulation on B1R conformation. Specific Aim 3: To determine the role of CPM-mediated allosteric modulation of B1R activation in G-protein coupling, -arrestin interaction and downstream signaling and to determine the importance of CPM/B1R interaction in regulating endothelial barrier function. Our hypothesis is that CPM interaction with the B1R enhances all B1R signaling via basal interaction but further enhances Gq-coupled responses by allosteric modulation. Furthermore, disruption of CPM/B1R interaction will attenuate B1R-mediated endothelial barrier damage in response to B2R agonist. We will determine: (i) G-protein coupling mediated by CPM interaction with the B1R vs. agonist binding. (ii) the involvement of -arrestin-2 recruitment in B1R agonist activation vs. CPM mediated allosteric enhancement . (iii) the downstream signaling output from the B1R via Gq and Gi mediated pathways in response to agonist or CPM interaction. (iv) the role of CPM in allosteric modulation of B1R signaling in regulating endothelial/vascular permeability. These studies will elucidate a novel mechanism for allosteric enhancement of B1R signaling by CPM which also generates its ligand. Understanding how this process can be regulated could lead to the development of novel drugs to treat renal and cardiovascular diseases. PUBLIC HEALTH RELEVANCE: We are investigating the interaction of two proteins, carboxypeptidase M and B1 receptor, on the cell surface that results in increased production of regulatory molecules that affect cell and organ function. The B1 receptor is known to regulate kidney and cardiovascular function, especially under conditions of infection or inflammation. Understanding how the interaction of these proteins can be enhanced or blocked could lead to the development of novel drugs to treat renal and cardiovascular diseases.

Inflammation is a process essential for host defense and tissue repair. However, exuberant defense may cause pathogenic changes leading to vascular diseases such as atherosclerosis. The primary goal of this renewal application is to build from the previous granting period to characterize proinflammatory cytokines such as TNF-activated signal transduction in vascular endothelial cells (EC) to identify novel JNK activation pathways in inflammation. TNF signaling events are bimodal: survival signal via activation of NF-kappaB, inflammation/apoptosis via activation of JNK. The major hypothesis of this proposal is that inhibition of JNK without disruption of NF-kappaB activation provides a valid approach for anti-inflammatory therapy. We have identified several molecules including ASK1 and AlP1 specifically mediating JNK (but not NF-kappaB) signaling. Moreover, atheroprotective laminar flow inhibits TNF-induced ASK1-JNK and gene expression of proinflammatory molecules. We have cloned a novel Ras-GAP protein (ALP1) and demonstrated that AlP1 is critical for TNF-induced ASK1-JNK activation and is a new target of atheroprotective laminar flow. The specific hypotheses to be investigated are: AlP1 specifies TRAF2 for ASK1-JNK activation; AlP1 is a flow sensor in prevention of TNF-induced ASK1-JNK activation. Three specific aims are proposed: 1) To determine the roles of AlP1 in TNF-induced JNK pathway. 2) To define the roles of AlP1 in flow-mediated inhibition on TNF-induced ASKI/JNK activation. 3) To determine the role of AlP1 in atherosclerosis in apoE- /- mouse model. This proposal should provide candidate proteins that are specific or unique targets for TNF-mediated inflammatory events and facilitate development of new therapeutic approaches to control inflammation in various disease settings.

The three research projects will be supported by an Administrative Core that will help to coordinate the smooth exchange of compounds and data between the projects and facilitate the interface with NIH scientific and administrative personnel. The Core will be led by Dr. Kellar, the PI for the overall Program. An Executive Committee, composed of the PI, the three Project Leaders and NIH scientific personnel assigned to our program will meet twice each year to review scientific progress, exchange information about specific research plans and designs and, if deemed advisable, adjust priorities. The two Executive Committee meetings each year will be planned for Georgetown University in May and Duke University in October or November. The meeting at Duke will coincide with the annual Duke Nicotine meeting for convenience and economy. The administrative Core will make the arrangements for these meetings; therefore, we have included a modest travel budget to allow Dr. Levin to travel to Georgetown and for the PI and Project Leaders to travel to Duke (this travel budget is independent of the travel budget in each Research Project, which is for trips to attend and present data at national scientific meetings, such as the Annual Meeting of the Society for Neuroscience or for the SRNT meeting). In addition to the two annual meetings of the Executive Committee, the Administrative Core will arrange monthly telephone conferences for the Committee to discuss progress, problems, and/or new ideas. Dr. Eric Donny (U. Pittsburgh), an expert on nicotine self-administration, has agreed to serve as a consultant to Project 2. The costs associated with these consultations are included in the budget for Project 2. The Administrative Core will establish a data storage center that will encompass archives of hard copies of data and a database of electronic versions in the Department of Pharmacology, Georgetown University. All three projects will send their data to the Administrative Core, and the Georgetown University Information Security Office (Mr. David Smith, Director) will help design the necessary security arrangements to protect all confidential information. The data center will be managed by Dr. Xiao and a part-time research assistant in the Core. We plan to enter the data into a central database on a secure, password-protected web space. All key personnel of this NCDDDG will have access to the data center via password. The data maintained in this way should help greatly in the management of the program and in the preparation of papers for publication, presentations for meetings and the annual progress reports to NIH.

Sexual transmission is the primary route by which human immunodeficiency virus (HIV) is acquired. In the prevailing hypothesis about the initial events of infection, dendritic cells (DC) serve as a major vehicle for trafficking HIV across the mucosa and to secondary lymphoid organs, such as lymph nodes (LN), where robust viral replication can be initiated in T-cells. A DC-specific molecule, DC-SIGN, appears to be involved in binding and transfer of infectious HIV to CD4+ T-cells, although it does not function as an entry receptor. The SIV macaque model represents the best experimental system for examining the significance of DC-SIGN in transmission. However, lack of suitable reagents for studying DC-SIGN's role in vivo has hampered efforts in this area. In this application, we propose to develop reagents for the SIV macaque model that will allow us to test the hypothesis that virus attachment to DC mediated by DC-SIGN plays a significant role in transmission and dissemination of SIV. The following aims have been proposed for this study: Aim 1: To develop monoclonal antibodies against macaque DC-SIGN. To develop monoclonal antibodies against native SIV Env. Aim 2: To determine whether macaque DC-SIGN mediates transmission of SIV or SHIV to T-cells in vitro, and to determine if anti-DC-SIGN monoclonal antibodies or anti-Env antibodies will neutralize binding of virus to DC-SIGN and subsequent transmission of T-cells. The development of monoclonal antibodies to DC-SIGN will provide critical reagents for examining the localization of DC-SIGN+, DC populations in tissues. Furthermore, identifying monoclonal antibodies to DC-SIGN as well as the SIV envelope that neutralize virus attachment to DC-SIGN and subsequent transfer to CD4+ T-cells will serve as a basis for vaccine and passive immunotherapy strategies aimed at preventing infection.

The aim of this project is to assess muscle triglyceride content in human subjects non-invasively using 1H and 13C NMR-spectroscopy in order to evaluate whether defects in lipid metabolism might be responsible for alterations in glucose metabolism in people with insulin resistance.

Major histocompatibility (MHC) Class I gene activation occurs during embryonic development, so that at birth most of somatic cells express the antigens derived from both paternal and the maternal MHC genes. The precise timing, and the types of Class I antigens expressed during embryonic development have been so far controversial. Class I antigen expression during fetal development poses another unsolved immunological question, i.e. even though the fetus carry histoincompatible paternal Class I antigens, allograft rejection is not detected during pregnancy. To determine Class I antigen expression in embryogenesis we studied cell surface expression of Class I antigens throughout mouse gestation. Two series of monoclonal antibodies, one reacting with all types of Class I antigen (rat xeno antibodies), another reacting with classic, polymorphic H-2 antigens (mouse alloantibodies) were employed. A significant antibody binding was noted in embryos at gestation day 10 (somite stage) and after. No binding was detectable in earlier embryos. Thus, the time of the antigen expression is later than previously indicated. Only monoclonals reacting with all types of Class I antigens were positive on day 10 embryos. Studies on the onset of Class I gene transcription and that of the protein synthesis are underway. Studies of the effect of anti-paternal Class I antibodies (often produced in pregnancy) on the fetal immune development have been continued; radiolabeled monoclonal antibodies were injected into pregnant mice, radioactivity was found in various fetal tissues, much of which represented intact Ig as tested by SDS-gel electrophoresis, indicating active passage of the antibodies to the fetuses. Class I antigen expression and the immune function of the animals treated with the antibodies are being studied. To elucidate T cell activity in pregnancy, the ability of generating cytotoxic T cells in multiparous mice was examined. We found that during pregnancy T cell reactivity is markedly increased in allopregnant animals, but not in syngeneic matings indicating maternal allosensitization.

PROJECT SUMMARY Depression is a chronic and debilitating mental illness. Anhedonia, one of the two core symptoms of major depression, is particularly difficult to treat. Individuals can experience one or more types of anhedonia, defined as a loss of interest or a loss of pleasure. However, the neural circuits and molecular substrates underlying anhedonia and other key depressive symptoms remain poorly understood. The arcuate nucleus (ARC) of the hypothalamus contains two distinct neuronal populations expressing proopiomelanocortin (POMC) and agouti-related protein (AgRP) that are well known for their roles in homeostatic and hedonic control of feeding. We demonstrated that chronic unpredictable stress causes different types of anhedonia and behavioral despair and induces opposite changes in spontaneous firing rate of POMC and AgRP neurons. We hypothesize that POMC and AgRP neurons play distinct roles in chronic stress-induced depressive behaviors. The specific aims of this project are designed to determine 1) how chronic unpredictable stress regulates POMC and AgRP neuronal activity; 2) whether SIRT1 in POMC and AgRP neurons functions as a molecular mediator of chronic stress-induced depressive behaviors and neuronal dysfunction; and 3) whether selective activation or inhibition of POMC and AgRP neurons causes or reverses depressive behaviors.

The fushi tarazu (ftz) gene of Drosophila melanogaster functions in determining the formation of alternating segmental primordia during the early stages of embryogenesis. Ftz mutations affect the expression of engrailed (en), a gene required for the determination of posterior segment primordia, and Antennapedia (Antp) a gene required for determining the identity of thoracic segmental primordia. The objective of this proposal is to test the hypothesis that the homeodomain-containing protein encoded by ftz activates the transcription of en and Antp by binding to specific DNA sequences near these genes. ftz protein binding sites will be identified by DNase I footprinting using a beta- galactosidase-ftz fusion protein purified from E. coli extracts. How the deletion of ftz binding sites affects en and Antp expression will be assessed in vivo in embryos that contain transformed copies of en-lac Z or Antp-lac Z reporter gene fusions. The mechanism of ftz regulation will be probed by experimenting with the number, location and orientation of ftz binding sites at the en and Antp loci in lac Z reporter gene fusions. Results from these experiments will help explain how spatial patterns of gene expression in embryos are generated through genetic regulatory mechanisms and will provide an example of how homeodomain-containing proteins regulate gene expression.

This application is a request for a Scientist Development Award for New Minority Faculty (K01) that will enable Dr. Nyborg to continue to develop her programmatic line of research in the prevention of aggressive/violent behaviors in males of African descent. African American males are particularly at risk for negative outcomes. Fifteen percent of youth under the age of 18 are African American, but they are overrepresented in criminal and victimization statistics. Data point to a need for culturally relevant prevention programs that specifically address the psychological well-being of African American males. Dr. Nyborg's training goals are to (1) further develop her knowledge of creating culturally sensitive interventions for African American youth, (2) acquire knowledge of and experiences with public policy creation, analysis, and advocacy, (3) increase knowledge of and experiences with school based mental health interventions, specifically violence prevention and school-based mental health services, (4)advance her knowledge of scale development, (5) acquire knowledge with regard to qualitative data collection and analysis, and (6) improve her grant and manuscript writing. These training goals will be achieved through (1) the resources available at Brown University, (2) the high quality of mentorship provided through Dr. Anthony Spirito, Dr. Cynthia Garcia Coil, Dr. Marion Orr, Dr. Kenneth Dodge, and the assembled expertise of the consultant team, (3) focused coursework and clinical experiences, and (4) the proposed project. The proposed project extends the natural progression of Dr. Nyborg's research. The project will focus on three related sub-studies with African American adolescent males. First, an adolescent hypermasculinity scale will be developed. Second, the relations among experiences of racism, hypermasculinity, and aggressive behavior will be examined. Third, an intervention development trial will be conducted. The intervention will be designed to teach positive coping strategies in dealing with experiences of racism via anger management, and problem solving skills, as well as activities intended to promote feelings of empowerment (e.g., having prominent African American men speak on their experiences with racism). [unreadable] [unreadable]

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. The Bovine Viral Diarrhea Virus, similarly to HCV, is a member of Flaviviridae, and within this category, it is a Pestivirus. Matt Paulson has identified a mutation site of the HCV NS4B homologue in BVDV which resulted in an increased resistance following the infection (the cells did not die after infection). We aim to first identify the cellular interacting partners of the normal and the mutant protein. We have compared several isolation methods and using magnetic beads so far we have identified one of the expected interacting partners.

Parkinson's disease (PD) is the second most common neurodegenerative disorder after Alzheimer's. Heavily implicated in its molecular pathology is the abundantly-expressed and intrinsically disordered protein (IDP) a- synuclein (a -syn). While the precise role played by a -syn in PD is unknown, its aggregation into filamentous inclusion bodies within the brain is the defining hallmark of this and other synucleinopathies. Understanding the aggregation process of a -syn and its connection to PD is thus of great interest to the both academic and medical research communities as such information is anticipated to contribute greatly to development of effective therapies, of which there are currently none. Despite much effort however, such molecular-level information remains elusive. IDPs are defined in part by their lack of well-defined structure in vitro, apparently relying on functionally relevant binding partners to modulate and affect conformations in vivo. This characteristic structural plasticity makes IDPs inherently difficult to study as the effects of conformational averaging of conventional structural studies often lead to a dramatic loss of information about sub-populations and conformational heterogeneity critical to understanding folding in these anomalous proteins. In the case of a-syn, this problem is further complicated by an expansive range of known binding partners (small molecules, metal ions, and other proteins) of unknown biological relevance, and presently unknown cellular function(s). To overcome these challenges and reveal otherwise hidden information about the structure of a-syn critical to understanding its role and function(s), single-molecule fluorescence (SMF) methods such as Forster Resonance Energy Transfer (smFRET) in combination with Fluorescence Correlation Spectroscopy (FCS, auto- and cross-correlation) are proposed herein to probe this challenging target. The Deniz lab is a world leader in the application of these highly sensitive techniques, which report directly on even complex populations through observation structural features of individual molecules. These SMF methods will be applied to comprehensively profile and model the structural properties of a-syn by (i) examining in detail the behavior of monomeric a-syn under both non-aggregating and aggregation-promoting conditions, including a focus on the C-terminal "tail" region, hypothesized to mitigate aggregation but which has been largely neglected due to a high level of disorder in solution (presents a significant challenge for non-SMF studies), and (ii) characterizing the global structural rearrangements that a-syn undergoes in the presence of several known binding partners of suspected biological relevance. It is anticipated that such detailed information about molecular conformation and dynamics will allow for the development of a detailed structural model of a-syn function in normal and diseased states, and ultimately for a better understanding IDPs and their broad role in human health. PUBLIC HEALTH RELEVANCE: Parkinson's disease (PD) is the second most common neurodegenerative disorder after Alzheimer's disease, and has devastating effects on the muscular and cognitive abilities of those who suffer from it. This research proposal is aimed specifically at providing the broad biomedical research community with the molecular information needed to better understand and more effectively treat PD.

Interstitial cystitis (IC) is a chronic lower urinary tract disorder characterized by urinary frequency, urgency, suprapubic pressure and pain. Although various pathological mechanisms have been proposed to account for IC, the etiology of the disorder is unknown. Nevertheless, it seems reasonable to speculate that IC is an abnormality of visceral sensation and that the IC patient is either more sensitive to relatively normal afferent activity generated in the bladder or that the bladder generates abnormal afferent signals in response to an unidentified peripheral pathology. The experiments in this proposal are designed to analyze the neural pathways transmitting nociceptive information from the bladder in rats and to evaluate the influence of noxious bladder stimuli on the properties of peripheral afferent neurons and spinal neurons that process sensory input from the lower urinary tract. Several hypotheses will be tested: (1) bladder pain is triggered by normally silent (sleeping) C-fiber afferents that are sensitized by chemical mediators released at sites of inflammation or tissue injury, (2) silent C-fibers are quiescent or insensitive to mechanical stimuli such as bladder distension due to a high threshold for activation of sodium currents as well as a low threshold for activation of a potassium current which tends to decrease cell excitability, (3) inflammatory and pain producing substances sensitize bladder afferents by blocking potassium channels, (4) chronic inflammation in the bladder can alter the properties of bladder afferents to increase their excitability and alter their chemistry, (5) interactions between sympathetic efferent pathways and afferent pathways can modulate sensory input from the bladder and may be a mechanism for sensitization of silent c-fibers, (6) allodynia, defined as a painful response to a normally nonpainful stimulus, can occur in the inflamed bladder as in other organs and may be mediated by both central and peripheral mechanisms. The long term objectives of this research program are to understand the mechanisms by which irritating or tissue injuring stimuli in the lower urinary tract are detected and processed by the nervous system and in turn modulate urinary tract function. The study will utilize a multidisciplinary approach encompassing neurochemistry, electrophysiology and pharmacology.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Ex vivo imaging of myocardial necrosis, using micro-CT, using the drug Isoproterenol, up to 56 rats.

Clinical trials for neurodegenerative diseases are hampered by the lack of quantitative and objective biomarkers that reflect treatment effects in the brain. Magnetic resonance spectroscopy (MRS) has potential to directly assess disease-modifying effects of therapeutic interventions in the brain. However, MRS has not made the transition to the clinical setting, largely due to lack of standardization of data acquisition and analysis methods and compromised data quality obtained with standard clinical packages, which result in poor reproducibility of neurochemical concentrations. The primary objective of this application is to facilitate translation of advanced MRS technology to the clinical setting ina strategic alliance between MR physicists, software engineers and physician scientists. This Partnership for MRS Biomarker Development is comprised of 3 phases and incorporates a gradual shift from MRI/MRS to clinical expertise at the sites involved: Phase I will establish an MR-technologist ready advanced MRS protocol on two widely-used clinical 3T platforms. An optimized semi-LASER (sLASER) sequence was chosen for this implementation because it is considered a top candidate for recommendation for high fields by the MRS Consensus Group. Phase II will assess the performance of the protocol under ideal conditions (efficacy), namely at sites where MRI/S and clinical trial expertise overlap. We focus on hereditary spinocerebellar ataxias (SCA) because the patient cohorts are well-characterized and they present the greatest need for multi-center investigations to sufficiently sample the patient population in trials. Four sites of the Clinical Research Consortium for Spinocerebellar Ataxias (CRC-SCA), which was formed to provide infrastructure for clinical trials in the common SCAs, will participate in this phase. Finally, Phase III will assess the performance of the protocol under ordinary conditions (effectiveness), i.e. in a clinical setting with rotating MR technologists. This phase will focus o Alzheimer disease (AD), the most common cause of age associated cognitive decline and dementia, and take advantage of large ongoing neuroimaging investigations. This application has a translational focus. The bioengineering focus areas of this project include advanced high field MRI technology for neuroimaging applications, validation and reproducibility assessment of spectral acquisition and analysis methods and non-invasive technology to assist monitoring treatment response in brain disorders. The partnership sites are the University of Minnesota (leading institution), Johns Hopkins University [Phases I and II], Duke University [Phase I], University of Florida [Phase II], Harvard University [Phase II], Mayo Clinic [Phase III] and University of Michigan [Phase III]. The deliverable of this project will be a turn-key advanced MRS data acquisition and analysis protocol that has been comprehensively evaluated in multiple patient cohorts, brain regions, platforms, and institutions, including the clinical settin. A high impact is expected in early diagnosis and treatment of neurodegenerative diseases.

It is particularly important that donor and recipient be well matched for tooth transplantation because immunosuppressive drugs cannot be used to prolong their survival. At the present time it is not known to what degree humans be matched in order to assure the long-term survival of tooth allografts. The purpose of this research is to define the minimum criteria that must be met to insure a favorable outcome and to determine what immunological parameters should be monitored to detect in vivo sensitization after transplantation. For this purpose teeth will be transplanted among well matched strains of genetically-defined mice and between rhesus monkeys who have been typed for serologically-defined and lymphocyte-defined transplantation antigens. Post-transplantation analyses will permit correlations between in vitro immune assays and clinical evaluations to establish the probability of successful transplantation in humans.

The liver is the largest glandular organ in the body and is responsible for diverse functions including regulation of glucose and lipid metabolism, generation of bile, and detoxification of ingested substances. The primary cell type that is responsible for these functions is the hepatocyte. However, in order to generate a working liver the hepatocyes must function as part of community of liver cells. For these cells to properly interact they must be arranged in a specific format that produces the discreet of architecture of the liver that is so crucial for normal hepatic activity. Indeed, when the architecture of the liver is disrupted the liver cannot function resulting in hepatic failure. Hepatic architecture is produced during embryonic development when individual liver cells are generated and organize to produce tissues. The aim of this proposal is to define the basic molecular processes that control development of the liver as an organ. This will achieved by studying mouse embryos and cells that lack a specific transcription factor, HNF4a, that is required for expression of over 600 hepatic genes. HNF4a is also an essential regulator of the epithelial transformation of the hepatic parenchyma during liver development. We porpose that HNF4a regulates liver development by both direct and indirect mechanisms. The first aim will test whether HNF4a controls expression of such a large array of liver genes by regulating expression of a cascade of downstream transcription factors. In the second aim we will test whether HNF4 coordinates expression of genes encoding adhesion and cell junction proteins to elicit the epithelial transformation of the liver. Finally we will test whether it is neccessary for hepatic cells to form cell adhesions and junctions in order to control hepatocyte differentiation. Success in the proposed experiments will elucidate the fundamental mechanisms tha govern formation of the liver as an organ and will advance our ability to rationally generate and culture hepatocytes that have the potential to be used therapeutically.

The reactivities of various reagents in proton transfer and nucleophilic substitution reactions in both aqueous and non-aqueous solvents will be studied. The comparative behavior of neutral and charged acids and bases acting in the Lewis, and in the Bronsted sense will be determined from the results. The goal of the research is an understanding of solvents and structural factors which determine rates of reactions.

Thyroid hormone has major effects on the development, regeneration and function of skeletal muscle. Thyroid hormone receptor ?1 is the predominant mediator of muscle T3 action, and transcriptionally regulates a number of important genes including SERCA A1 and A2, UCP 3 and GLUT 4, as well as the muscle differentiation factors myogenin and MyoD1. Circulating T3 enters myocytes via specific transporters such as MCT8 and 10, but we have shown in the last cycle of this grant that a significant fraction of cellular T3 is generated by the intracellular monodeiodination of T4 catalyzed by the type 2 iodothyronine deiodinase (D2). While mature myocytes are stable, skeletal muscle has a robust regenerative mechanism that is markedly accelerated following injury. This involves the concerted actions of satellite cells, the muscle stem cell equivalent, an a number of different cell types localized to the muscle stem cell niche including resident stromal fibro/adipogenic progenitors (FAPs), T-regulatory cells, and macrophages. These cells produce a number of pro-myogenic cytokines and growth factors that that are necessary to coordinate the regeneration process including Notch, Wnt/?-catenin, sonic hedgehog, IL-4 and IL-6, and a number of TGF-??family members among others. During the last grant period, we showed that changes in thyroid hormone mediated by local thyroid hormone metabolism play an important role in these signaling cascades. We found that after muscle injury acute inactivation of thyroid hormone by the type 3 iodothyronine deiodinase (D3) is necessary to facilitate satellite cell proliferation and block premature differentiation that otherwise would lead to apoptosis. This is followed by increased type 2 deiodinase (D2)-mediated T4 activation that is essential for the satellite cell to differentiate into mature myoblasts and then myofibers to complete the normal muscle regeneration program. In studies employing gene knockout mouse models, we have found that a loss of either D3 of D2 dramatically impedes muscle repair after injury. Using a murine model of satellite cell transplantation therapy, we further showed that local thyroid hormone activation by D2 plays a crucial role in the engraftment of transplanted muscle stem cells. In this renewal, we propose to combine studies using our unique mouse knockout models and established FACS-sorting isolation of specific cell types. We will investigate why D2 is required for engrafted satellite cell survival and successful myotube formation, how D3 and D2 change after injury in satellite cells and other components of the muscle stem cell niche, whether deiodinases are required for the coordinated responses of other cell types in the muscle stem cell niche, and how dysregulation of this process may lead to fibrosis. This will expand our knowledge of the contributions of thyroid hormone to muscle regeneration and form the basis for potential therapeutic strategies for patients with skeletal muscle injuries.

The proposed research project involves the study of the molecular and cellular mechanism of epilepsy. Epilepsy is a family of neurological disorders characterized by the unpredictable but recurrent occurrence of seizures, which are uncontrolled sudden attacks triggered by unusually intense neuronal firing. The model system that will be used in this proposed project is a murine model of status epilepticus (SE). In our SE model, systemically administered pilocarpine induces prolonged SE which results in hyperexcitability in the hippocampus and selective neuronal degeneration as observed in human epilepsy. Seizures also rapidly induce the expression of the multifunctional neuronal modulator, cyclooxygenase-2 (COX2), in principle neurons of the hippocampus. Our preliminary data demonstrate that pharmacologic inhibition of COX2, when initiated after SE, largely prevents the loss of synaptic inhibition onto dentate granule cells, suggesting that a signaling pathway critically involving COX2 mediates loss of GABAergic inhibition after SE. My first objective in this project is to test two alternative hypotheses that could explain the COX2-mediated loss of inhibition of granule neurons after SE. First, the possibility of COX2 mediating degeneration of somatostatin labeled GABAergic interneurons in CA1 area and dentate hilus and/or second the enhanced frequency dependent presynaptic inhibition of granule cell afferents to the dentate interneuron population after SE. To test both hypotheses we will compare the number of injured neurons within the CA1 pyramidal cell layer and dentate hilar interneurons using an immunohistochemical approach, in addition to measuring intrinsic synaptic properties of the dentate interneurons and granule cells using an electrophysiological approach. As a second objective I will determine whether the principle neurons of the hippocampus are the source of COX2 involved in loss of inhibition after SE, exploring the beneficial effects of blocking neuronal and nonneuronal COX2 induction after seizure using both a pharmacological and a genetic approach. At the end of the proposed project we will be able to understand the role of COX2 induction and its signaling pathways in the synaptic plasticity observed in the hippocampal network after SE. These experiments should provide us with new information regarding inhibitory synaptic plasticity modulation in the normal and epileptic hippocampus. As an advantage, the pharmacology of COX2 and its downstream signaling are well studied and understood. Demonstrating that SE-induced COX2 signaling is involved in the synaptic plasticity and/or selective neurodegeneration that occurs in the hippocampus after SE could lead to a strategy for interrupting epileptogenesis and be used as an efficacious therapeutic treatment for epilepsy prevention and impact the lives of those with this neurological disorder.

Tissue functional properties are governed by physical and chemical processes occurring at cellular and subcellular length scales. Specifically, the physical state of tissues (e.g., osmotic and mechanical properties, state of hydration, charge density) must be characterized on distance scales below 100 nm. Understanding the interaction of polyelectrolytes with ions is critical to clarifying the basic physics of ion binding as well as physical mechanisms affecting a large number of biological processes. To determine the effect of ions on the structure and dynamic properties of synthetic and biopolymers we developed a multi-scale experimental framework by combining high-resolution techniques, such as small-angle X-ray scattering (SAXS), small-angle neutron scattering (SANS), and static light scattering (SLS), with macroscopic methods (osmotic swelling pressure and mechanical measurements). We also use dynamic methods e.g., neutron spin echo (NSE), dynamic light scattering (DLS) to observe the relaxation response as a function of length scale. Additionally, we developed a method to determine the distribution of counterions in the ionic atmosphere surrounding charged macromolecules using anomalous small-angle X-ray scattering (ASAXS). In biology, osmotic pressure is particularly important in regulating and mediating physiological processes. Swelling pressure measurements probe the system in the large length scale range, thus providing information on the overall thermodynamic response. SANS and SAXS allow us to investigate biopolymer molecules and assemblies in their natural environment and to correlate the changes in the environmental conditions (e.g., ion concentration, ion valance, pH, temperature) with physical properties such as molecular conformation and osmotic pressure. These techniques simultaneously provide information about the size of different structural elements and their respective contribution to the osmotic properties. Combining these measurements allows us to determine the length scales governing the macroscopic thermodynamic properties. It is important to emphasize that this information cannot be obtained by other techniques. We have studied the effect of multivalent cations, particularly calcium ions, on the structure of various model systems mimicking soft tissues. Divalent cations are ubiquitous in the biological milieu, yet existing theories do not adequately explain their effect on and interactions with charged macromolecules. Moreover, experiments to study these interactions are difficult to perform, particularly in solution, because above a low ion concentration threshold multivalent cations generally cause phase separation or precipitation of charged macromolecules. Since macroscopic phase separation does not occur in cross-linked gels, we have overcome this limitation by cross-linking our biopolymers, greatly extending the range of ion concentrations over which the system remains stable. In previous studies, this new non-destructive procedure has been used to investigate cross-linked gels of a model synthetic polymer, polyacrylic acid, and different biopolymers such as DNA, hyaluronic acid and chondroitin sulfate (important components of extracellular matrix) to determine the size of the structural elements that contribute to the osmotic concentration fluctuations. We have combined SANS and SAXS to estimate the osmotic modulus of hyaluronic acid solutions in the presence of monovalent and divalent counterions. We studied the collective diffusion processes in these solutions by dynamic light scattering and determined the osmotic modulus from the relaxation response. We determined the distribution of counterions around charged biopolymer molecules using anomalous small-angle X-ray scattering measurements. We applied our approach to understand the binding mechanism in glucose sensors made from smart zwitterionic hydrogels containing boronic acid moieties. Based on systematic SANS and osmotic pressure measurements we provided a thermodynamic explanation for the enhanced selectivity of these gels for glucose relative to fructose. This class of material has a great potential in the development of implantable continuous glucose sensors for use in diabetes. We developed a procedure to control the size, compactness and stability of DNA nanoparticles by mediating the interaction between ions and DNA. We quantified the effects of salt, pH and temperature on their stability and biological activity. These polyplexes are pathogen-like particles having a size (70 300 nm) and shape resembling spherical viruses that naturally evolved to deliver nucleic acids to the cells. They contain the pDNA in the interior surrounded by synthetic polymer bearing sugar residues on the surface recognized by the M cells and dendritic cells as pathogens. Because we can control the stability and biological activity of DNA nanoparticles, we believe that this knowledge can provide a solid foundation for developing new DNA-based vaccines for the treatment of various diseases. We studied the effect of calcium ions on the larger scale structure of DNA solutions and gels in near-physiological salt conditions by SANS. Analysis of the SANS response revealed two characteristic length scales, the mesh size of the transient network, and the cross-sectional radius of the DNA double helix. In gels the mesh size is greater than in the corresponding solutions by approximately 50%, reflecting the increased heterogeneity of the crosslinked system. The cross-sectional radius of the DNA chain is practically independent of the polymer concentration and the calcium ion content, and is close to the value of the DNA double helix (10 A). This finding implies that bundle formation is negligible in the present DNA gels. The results of this study show that changes in the ionic environment offer a molecular level control of the interactions between DNA strands and allow us to tune the morphology of DNA-based assemblies. In recent studies we have investigated the mechanism of fiber formation from small hydrogelator molecules in biological cells. Fluorescent imaging revealed that self-assembly directly affects the distribution of these small peptidic molecules in a cellular environment. Cell viability tests suggested that the states and the spatial distribution of the molecular assemblies control the phenotypes of the cells. We demonstrated that enzyme instructed self-assembly makes it possible to modulate the spatiotemporal profiles of small molecules in a cellular environment.

High temporal resolution ECG-gated scintigraphic angiocardiography (radionuclide cineangiography) is a computer-based, radiotracer imaging procedure that allows non-invasive visualization of the chambers of the working heart, at rest, during bicycle exercise, during therapeutic intervention, etc. The procedure yields quantitative measures of left ventricular function such as ejection fraction, peak ejection rate, etc. Work is continuing to assess the accuracy of these data in man and to establish findings for the method selected disease categories. Variations of this technique are being explored and will result in an inexpensive, portable device capable of continous real-time measurement of left ventricular function.

Over half of all U.S. citizens will be exposed to a potentially traumatic event (PTE) during their lifetime. The recent, unprecedented man-made and natural disasters of 9/11 and Hurricane Katrina catastrophically illustrate that lifetime trauma exposure rates are rising. The psychiatric response associated with traumatic exposure involves post-traumatic stress disorder (PTSD), substance-use disorder (SUD), or co-occurring psychopathology (i.e., PTSD-SUD). However, not all individuals will develop psychiatric disorders in response to PTE exposure, underscoring the question: What are the factors that lead some individuals who are exposed to traumatic events down the path to resilience and other individuals down the path to risk for these disorders? This question can be examined effectively in a sample of firefighter (FF) recruits who will experience life-threatening events on a day-to-day basis as a result of their profession. FFs exposed to PTEs may follow a number of pathways towards pathology or wellness. Unfortunately, the variables and parameters that predict risk and resilience for PTSD, SUD, and PTSD-SUD symptoms and diagnostic caseness, as well as the patterns of recovery and relapse related to these disorders, are not well understood. In the proposed study, 400 urban, professional FF recruits will be followed from selection and training, through their early careers as FFs in a prospective, longitudinal evaluation. Resilience to, or development of, PTSD, SUD, or PTSD-SUD will be examined, as well as temporal patterns of symptom expression, recovery, and relapse. Biopsychosocial variables that influence post-exposure pathways and increase or decrease the likelihood of developing PTSD, SUD, or PTSD-SUD symptoms and/or caseness will be evaluated. Following a multi-dimensional pre-employment baseline assessment (i.e., genetic markers, family history, personality factors, prior PTE exposure and substance use), PTSD and SUD symptomatology plus empirically-derived predictor variables (i.e., trauma characteristics, acute peritraumatic responses, coping, and social support) will be measured at 3-month intervals for 3 successive years. Latent growth curve analyses, structural equation modeling, multigroup structural equation modeling, and cross-lag panel analytic approaches will test temporal dependency as well as postulated relationships between risk and resilience factors. This application is the next step in our research team's programmatic investigation of trauma-related disorders among FFs, with an emphasis on factors that may inform prevention and treatment guidelines for both FFs and other trauma-exposed populations. The long-term goal of this study is to build a FF database from recruitment through retirement in order to identify patterns of risk and resilience in this highly-exposed, essential public safety population.

: The National Human Genome Research Institute (NHGRI) has launched a series of research training activities in Genomics and Ethical, Legal, and Social Issues (ELSI), comprising the Research Training Consortium (RTC), with a focus on increasing the successful participation of individuals from Under Represented Minorities (URM). While the various grantees are involved in evaluating and tracking progress towards their goals for their own programs, this has not been done systematically, making it difficult to compare and pool data across programs. The major goal of RFA-HG-08-006 is to establish a Data Analysis and Coordinating Center (DACC) to facilitate and streamline the evaluation and tracking component for the entire MAP initiative. An interdisciplinary team of investigators with considerable strengths and experience in areas of direct relevance to the RFA from Washington University in St. Louis is submitting this application in response to the RFA. With a strong sense of commitment to accomplish the goals of the RFA, we propose 4 specific aims: First, we will develop complementary evaluation and tracking tools for the entire RTC after reviewing the evaluation and tracking instruments currently implemented within each of the training centers. Second, we will create a web-based Data Entry System (DES). a prototype of which has already been created for one of our Summer Institutes, for establishing a centralized database for deposition of the RTC data from grantees that will become the source of all data analysis. Third, we will periodically perform rigorous analyses of the data using standard methods and generate reports that would assess progress and outcomes, report the findings at annual meetings of the grantees and training coordinators and bi-annual meetings of the National Advisory Council of the NHGRI, and provide written reports periodically. Fourth, we will provide leadership as necessary in other areas as maybe customarily expected of coordinating centers and work closely with the RTC and the NHGRI in achieving the goals of the RFA. We believe that we have the strengths and experience as well as the people skills and familiarity with the RFA goals and yet we bring a sense of freshness, objectivity, and flexibility to the table by virtue of not being directly affiliated with any of the research training centers.

We seek support for the development of technologies that will help elucidate the three-dimensional (3-D) fine structure of cells and tissues. Two approaches to imaging will be employed: (1) thick samples of biological material will be studied in a JEM-1000 high voltage electron microscope (HVEM), and serial tilted views will be imaged to provide information about the 3-D structure of the specimen; (2) serial sections of various thickness will be imaged either in the HVEM or by conventional electron microscopy and assembled into 3-D models by a combination of image processing and computer graphics. The major thrust of our efforts will be to develop specimen preparation technologies for these imaging approaches. (1) We will continue our work on structural neurobiology, improving existing methods for the selective staining of specific cells and molecules. Tomographic methods will be combined with stereo modeling and serial section reconstruction to extract quantitative information about neuronal geometry. (2) We will investigate the utility of high pressure rapid freezing for the study of specimens too large to be well preserved by conventional freezing methods. Cryofixed material will then be treated by several secondary methods designed to permit reliable visualization of fine structure and of the location of specific antigens. (3) We will explore the utility of cryoHVEM for the study of frozen hydrated material, using flagella and basal bodies as test systems. (4) We will continue our development of methods for in situ hybridization at the EM level to reveal the location of nucleic acids with biotinylated probes.

Platelets play a central role in cardiovascular, stroke, and bleeding disease through their quantity, activation, and interaction with the inflammatory and vascular systems. The Wiskott -Aldrich syndrome (WAS) is an X- linked disorder characterized by thrombocytopenia, immunodeficiency, eczema and an increased risk of lymphoma and autoimmunity. The affected gene encodes the cytoskeletal protein, WASP. Through the RhoGTPase, Cdc42, WASp undergoes conformational change that leads to actin polymerization. How WASp results in thrombocytopenia, the most common manifestation of WAS, remains unknown. Cdc42, Src kinases, membrane phospholipids, and SH3 domains control WASP function. Our lab has discovered a new regulator of WASP, CIP4 (Cdc42 interacting protein 4), in a yeast two hybrid screen with the Src kinase Lyn as bait. CIP4 is a member of the F-BAR family of proteins, which remodel the plasma membrane and promote actin polymerization. To determine CIP4's function, we created a CIP4-null mouse. We found that CIP4-/- mice display thrombocytopenia, similar to that observed in WASP-/- mice. The mechanism for thrombocytopenia in WAS is not known. Some studies demonstrate an autoimmune basis; others a defect in proplatelet production. Because our mice do not display signs of autoimmune disease, we favor the hypothesis (defective proplatelet formation hypothesis) that CIP4-WASP forms a signaling pathway that promotes platelet production and that a deficiency of either CIP4 or WASP perturbs actin polymerization in megakaryocytes, which results in defective proplatelet formation. An alternative immune destruction hypothesis states that a defect in this CIP4-WASP pathway promotes autoimmunity and immune-mediated destruction of platelets. We propose to address these hypotheses through the following two specific aims: 1) the defective proplatelet hypothesis by culturing megakaryocyte precursors and megakaryocytes from wild-type, CIP4-/-, WASP-/-, and CIP4-/-WASP-/- mice and analyzing their proplatelet formation. We will use these unique mice strains to analyze proplatelet formation, the interaction of CIP4 with microtubules in megakaryocytes, and ultrastructural features of platelets; and 2) Test the immune destruction hypothesis by labeling platelets and measuring their clearance rates in wild-type, CIP4-/-, WASP-/-, and CIP4-/-WASP-/- mice and measuring their T, B, and NK cell functions. We will use these unique mice strains to measure platelet survival and to correlate with altered immune function.

Existing mRNA quantification methods As it is becoming increasingly apparent that gene expression in individuai cells deviates substantially from the average behavior of cell populations (Raj and van Oudenaarden, 2008), new methods that provide accurate integer counts of mRNA copy numbers in individual cells are needed. Ideally, such methods should also reveal the intracellular locations of the mRNAs, as mRNA localization is often used by cells to spatially restrict the activity of RNA binding proteins (St Johnston, 2005). One of the methods sensitive enough to detect single mRNA molecules is the MS2 mRNA detection technique developed simultaneously by the Bloom laboratory (Beach et al., 1999) and Robert Singer and colleagues (Bertrand et al., 1998). In this method, a gene is engineered to transcribe an mRNA containing many copies of a specific RNA hairpin in its untranslated region, each of which binds tightly to the coat protein of the bacteriophage MS2. This gene is then expressed in a cell that already expresses the MS2 coat protein fused to GFP. When many of the MS2-GFP proteins bind to an individual mRNA, enough fluorescent signal is generated that the individual mRNAs are detectable as diffraction limited spots using conventional fluorescence microscopy, allowing one to count the number of mRNAs in single cells. Although this technique provides quantitative and spatial information about mRNAs, its use is hindered by two substantial limitations: first, the target organism needs to be genetically modified;and second, detection of more than one mRNA species is impossible. Additionally, adding the tandem MS2 binding sites signiflcantly changes the RNA stability and the MS2-GFP fusion proteins tends to aggregate in large clusters hindering the detection of single molecules.

This proposal seeks to renew an Asthma and Allergic Diseases Cooperative Research Center that has been focusing on the mechanisms of initiation and persistence of allergic asthma. During the course of these studies and earlier work done in our laboratories and multiple other labs, it has become clear that two cytokines, IL-13 and IL-17, make important contributions to the most critical functional endpoints in asthma (airway hyperresponsiveness and mucus metaplasia) through spatially and temporally restricted effects on airway epithelial cells and airway smooth muscle. In the current proposal, individual projects will focus on how each cytokine regulates airway epithelial cell differentiation and mucous metaplasia, how these cytokines work alone and in combination to regulate contractility of airway smooth muscle, and the dynamic behavior of the cells that generate these cytokines in the airway wall. Each of these projects will have a major focus on human asthma and will rely heavily on a Clinical Subject and Biospecimen Core that will provide basic characterization of airway physiology, evidence for IL-13 and IL-17 bioactivity, and samples of bronchoalveolar lavage fluid, epithelial brushings and airway biopsies for use in each of the 3 projects. These samples and clinical information will be obtained from subjects in 3 cohorts, ACE, RITA and SARP, that will enroll subjects for segmental allergen challenge, subjects with mild-to-moderate asthma undergoing a trial of inhaled corticosteroids and patients with severe asthma, respectively. Through the proposed studies we hope to gain new insights into the dynamic effects of IL-13 and IL-17 in asthma, to develop better tools to characterize subsets of patients with asthma and to improve the prospects for targeted therapy of this disease.

Project 3: Continuous Wave Electron Paramagnetic Resonance Imaging. This technique provides non-invasive assessment of tissue/tumor redox status using redox sensitive paramagnetic contrast agents called nitroxides. We have now examining new molecules as reporters of oxidative stress, which are precursors of nitroxides and are converted to the corresponding nitroxide by oxidation with reactive oxygen species. Such oxidation should provide information on tissue oxidative stress. Instrumentation is also being developed to provide detection at higher frequencies to give enhanced sensitivity and spatial resolution.

PROJECT SUMMARY As a computational biologist, my long-term goal is to develop methods and tools to discover new or better therapeutics for cancers. In the past few years, I have identified drug-repositioning candidates for a number of primary cancers using Big Data approaches. These candidates have been validated successfully in preclinical mouse models. To maximize the utility of Big Data, I plan to translate the findings into therapeutics; therefore, I propose to develop methods to utilize transcriptomic, proteomic and pharmacogenomic data to inform individualized therapy in cancers. Current preclinical and clinical approaches including the NCI MATCH trial select therapies primarily based on actionable mutations, yet patients may have no actionable mutations or multiple actionable mutations that are hard to prioritize, suggesting the need for other different types of molecular biomarkers. The recent efforts have enabled the large-scale identification of various types of molecular biomarkers through correlating drug sensitivity with molecular profiles of pre-treatment cancer cell lines. Computational methods to match these biomarkers to individual patients to inform therapy in the clinic are thus in high demand. The objective of this award is therefore to develop computational approaches to identify therapeutics for individual patients by leveraging large-scale biomarkers identified from cancer cell lines. Through conducing this research, I expect to expand my knowledge in cancer clinical trials, cancer genomics, cancer biology, and statistics. To achieve the goal, I have gathered seven renowned experts from different fields related to Big Data Science as mentors/advisors/collaborators: Primary Mentor Dr. Atul Butte in translational bioinformatics from UCSF, Co-mentor Dr. Samuel So in cancer biology from Stanford University, Co-mentor Dr. Mark Segal in statistics from UCSF, Advisor Dr. Andrei Goga in cancer biology from UCSF, Advisor Dr. Laura Esserman in breast cancer trials from UCSF, Collaborator Dr. John Gordan in liver cancer trials from UCSF and Collaborator Dr. Xin Chen in cancer biology from UCSF. With the support from my world- class mentors, advisors and collaborators, this award will prepare me to be a leader in developing big data methods that are broadly impactful.

Publication of the revision of the DSM-III sets the occasion for a series of additional studies on the phenomenology, reliability, validity, and nature of anxiety disorder categories. Building on a series of studies on these topics conducted over the past several years we propose to examine a number of these issues. The Anxiety Disorders Interview Schedule, recently revised to be fully compatible with DSM-III-R will be utilized to determine the reliability of DSM-III-R categories using stringent test - retest methodology. Data will be collected from psychological test, physiological assessment, and periodic self-monitoring of relevant cognitive and behavioral events to ascertain similarities and differences among the DSM-III-R anxiety disorder categories as well as normals and two well-defined stress disorders, irritable bowel syndrome and hypertension. Response to CO2 challenges, and response and habituation to unexpected tones will comprise an important part of the physiological assessment. Patterns of co- morbidity will be determined for the anxiety disorder categories using hierarchy free methods and considering the functional relationships among disorders. In addition, key features of anxiety disorders based on a priori, clinical, or theoretical specification will be identified in terms of their presence or absence, as well as severity across the anxiety disorders. These key features will include panic, social anxiety, anxiety sensitivity, depression, worry, obsessions and compulsions and specific fears. All key features will be measured by questionnaire, interview, and possibly physiological responding. For example, responses to CO2 inhalations may be an index of anxiety sensitivity. Preliminary evidence from the first phase of this project indicates that many of these key features are present in other anxiety disorders (as well as the normal population) and that these features can be dimensionalized to provide profiles within each of the anxiety disorders with substantial implications for classification. Finally, patients in each of the reliably diagnosed DSM-III-R anxiety disorder categories will be followed for a period of two years to determine the course of these disorders and the stability of diagnostic categories and/or dimensional profiles.

Eukaryotic chromosomal replication is an intricate process that requires the coordinated and tightly regulated action of numerous molecular machines. Failure to ensure once only replication initiation per cell cycle can result in uncontrolled proliferation and genomic instability, two hallmarks of tumor genesis. The origin recognition complex (ORC), first discovered in yeast, is a six-subunit protein machine conserved in all eukaryotes. Yeast ORC constitutively binds to and marks the replication origin throughout the cell cycle. Licensing of the DNA replication origin starts when the critical cell division cycle protein Cdc6p binds to ORC. Recent biochemical studies with purified components indicate that Cdc6p and specific origin DNA sequence activate an ATPase switch in ORC. This induces an extended pre-replication complex (pre-RC)-like nuclease protection footprint on origin DNA that was previously observed only in vivo. Our preliminary EM work reveals a ring-like structural feature in the ORC-Cdc6p complex that is similar in size to the presumptive replicative hexameric MCM helicase. This result supports the emerging concept that the helicase is loaded by replication initiators in a mechanism similar to the loading of the DNA polymerase clamp PCNA by the RF-C clamp loader complex. The formation of the extended pre-RC-like footprint by ORC and Cdc6p, a crucial event in replication origin licensing, is ATP-binding and -hydrolysis dependent. We are interested in revealing the structural basis of this ATPase switch in ORC by studying the structures of ORC-Cdc6p-DNA in the ATP-bound form where the pre-RC footprint is formed, and in a non-hydrolyzable ATP (ATPgammaS)-bound form where the extended footprint is not formed. We hypothesize that the extended pre-RC footprint is a result of Cdc6p-induced origin DNA bending around ORC. We plan to test this hypothesis by determining the approximate binding sites of the three critical elements (A, B1 and B2) of the yeast ARS1 origin DNA. The origin DNA fragments will be biotinylated and labeled with streptavidin before binding with ORC and Cdc6p for EM analysis. These detailed structural studies will provide a long-awaited molecular mechanism for the origin licensing stage of the intricate replication initiation process. [unreadable] [unreadable] [unreadable]

The proposed project will develop a set of three interrelated research-based products: (1) an archive of exemplary behavioral and social research data on HIV/AIDS, (2) HIV/AIDS teaching modules for the university classroom, and (3) a Web-based learning center for promoting best practices in HIV/AIDS prevention program development and evaluation. These resources should facilitate progress toward the HIV/AIDS-related prevention and control objectives of Healthy People 2010 by: (1) fostering efficient dissemination of high-quality research data and conclusions on behavioral and social factors related to HIV/AIDS; (2) empowering students and educators from a variety of disciplines at the undergraduate and graduate levels to understand, communicate, and utilize research data on social aspects of HIV/AIDS; and (3) enabling prevention practitioners to perform program development as well as process and outcome evaluation tasks critical to achieving best practices standards, thus helping to bridge the gap between research and practice. The three research-based products are interrelated, allowing them to be developed more efficiently and cost-effectively through combined project work. Because each product has a different target audience, the overall impact of this project will be felt across a broad range of HIV/AIDS-related fields. Input from a Scientist Expert Panel and from professors in a variety of disciplines will help ensure the scientific merit and substantive utility of the data archive and teaching modules. Input from practitioners, local government officials, and staff members of community-based organizations will help ensure that the Web-based learning center is relevant to the needs of prevention practitioners. Phase I investigated the feasibility of the proposed products by creating product prototypes and conducting appropriate needs assessment, feasibility, and usability studies to assure the products' user-friendliness and commercial appeal. Phase II will complete the development of all three products, conduct a second round of usability tests, field test the products in university (Products 1 and 2) and HIV/AIDS prevention program (Product 3) settings, and revise them to be maximally useful to their intended audiences.

Cardiac arrhythmias are a major clinical health problem. We have developed a significant and unique solution. During the current award period, we have designed, constructed and successfully tested a novel prototype system which integrates, in real-time, all image and associated physiological variables, device signals and computer generated data derived there from required for accurate navigation and precise targeting in catheter- based ablation of cardiac arrhythmias. The system renders obsolete or unnecessary current approaches to such treatment, including open chest surgery, pacemakers, medications that don't work, and present ablation systems. Our system design is advanced, open and flexible, permitting ready integration of new imaging modalities, interventional devices and physiologic signals as they become available. This system has achieved, for the first time, real-time performance in fusing dynamic cardiac anatomy with physiologic signals. Based on this promising, but preliminary progress, we now can specify and complete the requirements for seamless clinical implementation and application. These requirements include enhancement, optimization and addition of novel algorithms and technology. We will achieve real-time segmentation of pre- and intra- operative image data, including dyna-CT and 3D ultrasound. The segmented multimodality data will be fused in real-time in the procedure room. Using tissue characterization and catheter tracking, we will develop and validate an innovative approach to physiologic visualization of intra-operative ablation (burn) sites. This capability will provide accurate visualization of the burn targets on anatomy to guide and verify the desired ablation pattern. These high-performance segmentation, fusion and visualization capabilities will be the basis for the final real-time platform for cardiac ablation guidance, which will be fully validated in detailed animal experiments. The system will be demonstrated efficacious both in the standard clinical set-up using catheter tracking and x-ray fluoroscopy (and/or pre-operative CT) and in an advanced set-up using a robotic manipulator for precise and stable catheter control. The system will be fully documented to facilitate ready reproduction. This project is poised to deliver a new generation of technology for effective treatment of cardiac arrhythmias that will result in significant clinical benefits, including dramatically improved outcomes with reduced morbidity, mortality, procedure time, x-ray exposure and cost. PUBLIC HEALTH RELEVANCE: Cardiac arrhythmias are a major public health problem with limited effective treatment options. The project will deliver a new generation of technology based upon multi-dimensional image fusion and guidance that will provide effective treatment of cardiac arrhythmias, resulting in significant clinical benefits which include dramatically improved outcomes (cases) while reducing morbidity, mortality, procedure time, x-ray exposure and cost.

Neuroblastoma (NB), a disease of trunk neural crest cells (NCC), represents the most common extracranial solid tumor in childhood. While amplification of MYCN is the strongest marker of poor prognosis in NB patients, no clinical inhibitors exist to directly target MYCN. Instead, targeting a cooperating genetic partner of MYCN represents an alternative strategy to treat these patients. Sequencing NB patient samples revealed that although recurrent single nucleotide variations is rare in NB, chromosomal aberrations are robust, particularly deletions of chromosomes 1p. Since loss of heterozygosity of chromosome 1p correlates with amplification of MYCN (strongest marker of poor prognosis) in NB, I hypothesize that amplification of MYCN cooperates with loss of tumor suppressors within chromosome 1p to transform human NCC to NB. As a postdoc in the William Weiss laboratory, I spent the majority of my time developing the necessary protocols (NCC differentiation) and tools (cloning overexpression vectors and genome editing plasmids) to demonstrate a proof of concept that I can transform human NCC to NB. I have, thus far, been successful at producing tumors driven by MYCN (10% penetrance) and MYCN/ALK F1174L (60% penetrance). For the K99 portion, I will examine other candidate NB drivers in cooperating with MYCN in accelerating NB tumorigenesis. I will also compare the resulting human stem cell (hSC)-derived NB tumors with genetically engineered mouse models of NB via whole genome sequencing and RNA-seq analysis. Thus, part of my training during the K99 will focus on bioinformatics analysis, which will be accomplished with the guidance of Hanlee Ji (Stanford) and a bioinformatics workshop at Cold Spring Harbor. After publication of the hSC-derived model of NB, my goal in the R00 phase is to identify tumor suppressors within chromosome 1p that, when deleted, cooperate with amplified-MYCN to accelerate tumorigenesis. Using the CRISPR/Cas9 system, I can narrow down the region within chromosome 1p that harbors the critical tumor suppressors. Subsequently, I can screen for individual candidate tumor suppressors by performing a CRISPR interference (CRISPRi) knockdown for each gene in that region. To bring CRISPRi to my independent laboratory for the R00 phase, I will work with Jonathan Weissman's (UCSF) group, which has pioneered CRISPRi technology, during the K99 phase. Successful completion of these studies will establish and characterize the first hSC-based model of NB, which can be used to rapidly test candidate driver genes and chromosome copy number alterations in NB tumorigenesis, as well as provide a preclinical model to evaluate the efficacy of various therapeutic strategies. My long term goal and vision for my independent lab is to investigate the significance of different chromosomal aberrations in NB and to utilize CRISPRi to not only identify key drivers of NB tumorigenesis, but also potential therapeutic vulnerabilities in a synthetic lethal screen.

We propose to establish an Advanced Center to Improve Pediatric Mental Health Care (ACIPMHC) that will be located within the Child and Adolescent Services Research Center (CASRC) at Rady Children's Hospital-San Diego. The ACIPMHC will have strong linkage to the intervention development program at the Center for Research to Practice (CR2P) at the Oregon Social Learning Center (OSLC) and to other intervention and services research programs and networks around the country. The research agenda has a strategic focus on improvement of public pediatric mental health care, primarily in the mental health and child welfare service sectors, through improved integration of evidence-based practice and usual care. The center's program of practice research spans clinical epidemiology studies linked to evidence-based practice, effectiveness, and implementation studies. The research agenda will have a primary focus on effectiveness and equity as two of the six aims for improving health care from the "Crossing the Quality Chasm" report. An overarching center perspective of cultural exchange will guide active partnership with major stakeholders in youth mental health care, including youth and family consumers, providers, program and funding managers, system administrators, policy makers, and intervention and services researchers. The Principal Research Core (PRC) will focus on practice research through two developmental studies addressing implementation of two established evidence-based practices. These studies and others to be designed by the center network of investigators will be carried out in community-based mental health and child welfare service settings, building on already developed research-practice relationships. The Research Methods Core (RMC) will advance methods for effectiveness and implementation research through four workgroups focusing on (a) design and analysis, (b) measurement, (c) mixed methods, and (d) economic analyses, as well as one developmental study addressing incorporation of research-based measurement in usual care child welfare practice. The Community Network Core (CNC) will strengthen a multiple sector infrastructure in San Diego County and link research activities to an emerging research network in the State of California that partners with the California Institute of Mental Health, a technical assistance arm of the county mental health directors in California. The Operations Core (OC) will provide leadership and will support activities in the other three cores by extending an effective infrastructure developed through two prior services research center grants and participating as one of six sites in an R-25 training consortium. All cores will advance practice research to inform improvements in mental health care that decrease racial-ethnic disparities.

Mycobacterium tuberculosis (Mtb) is adapted to grow in alveolar macrophages that provide an essential niche to establish infection in the host. After phagocytosis, Mtb virulence mechanisms block phagosome maturation permitting intracellular bacillary replication. We previously discovered that attenuated Mtb H37Ra and M. bovis BCG, at low multiplicity of infection (MOI), induce tumor necrosis factor (TNF)-alpha production and prime infected Macrophages for TNF-mediated apoptosis. In contrast, virulent Mtb strains suppress Macrophage apoptosis by interfering with TNF-alpha signals and by upregulating expression of the anti-apoptotic factor MCL- 1. Apoptosis creates conditions unfavorable for Mtb viability and enhances antigen presentation. We postulate that Macrophage apoptosis is an innate host defense against tuberculosis (TB), analogous to the role established for apoptosis of cells infected by viruses. New preliminary studies highlight the complexity of Mtb-induced Macrophage apoptosis. We find that at high MOI, virulent Mtb are equally or even more potent than attenuated strains at inducing Macrophage cell death by an entirely different pathway than occurs after low-MOl challenge. High-MOl Macrophage apoptosis involves a novel TNF- and caspase-independent mechanism triggered by cytosolic translocation of lysosomal proteases. We postulate that high-MOl apoptosis reflects a mechanism for Mtb to exit host Macrophage, analogous to cell death triggered by lytic viruses. In support of this model we found that high-MOl apoptosis progresses rapidly to necrosis, releasing Mtb to the extracellular space without loss of bacillary viability. We propose to investigate the mechanism and regulation of high-MOl apoptosis, to identify Mtb genes that trigger this response, and to explore the implications of high-MOl apoptosis for TB defense. The interaction between Mtb and alveolar Macrophage leading to death of the infected host cell and/or the infecting microbe is a critical issue in TB pathogenesis. A better understanding of the body's defenses against TB is needed to help develop new approaches for the prevention and treatment of this important disease. Our project will produce new knowledge about the fundamental relationship between the microbe that causes TB and the human cell which serves both as its target for infection and as the body's first line of defense against TB disease.

Patients with diabetes mellitus (DM) are at increased risk for major depressive disorder (MDD), which is reliably associated with poor DM outcomes. Compared to Caucasians, African Americans are more likely to develop DM and to have poor DM outcomes. They are also more likely to have untreated MDD, and to hold beliefs about the nature and care of MDD that are not well-accommodated by current mental health care delivery systems. Despite these clear disparities, there is a dire shortage of data to guide the development of culturally-appropriate interventions for MDD comorbidity in DM, because African Americans have been tremendously under-represented in research on this topic. The significance of this empirical gap is highlighted by our preliminary data, which suggest that depressive symptoms may be more disruptive to DM control for African Americans than for Caucasians, even after adjusting for potential confounders. Specific Aim 1 is to test our conceptual model, in which the association between MDD and DM outcomes (glycemic control and DM quality of life) is both moderated by race and mediated by DM self-care behavior. We plan to study 262 Detroit-region Type 2 DM patients (50% African American, 50% Caucasian) in a 10-week assessment protocol. At Weeks 1 and 10; assessments will be made of MDD presence and severity, DM self-care, glycemic control, and DM quality of life; with an additional assessment of self-care and MDD made at mid-study. Data on self-care and MDD will be aggregated across time, and the study hypotheses will be tested using OLS multiple regression path analysis. Specific Aim 2 is to gain an understanding of depression-related behavior and beliefs that will inform the development of culturally-sensitive interventions for DM patients who have comorbid MDD. To meet Aim 2, we will develop a race-stratified subsample of 30 subjects with comorbid MDD. Using a semi-structured interview tool based on the Commonsense Model of Illness Representations, we will collect qualitative data on beliefs about the nature and care of MDD. We will perform qualitative analysis to identify and compare themes by race. Finally, we will integrate our quantitative (Aim 1) and qualitative results using triangulation and other mixed-methods approaches, to characterize cultural differences in MDD representations, treatment-seeking, and mental health barriers among DM patients. The project outcomes will advance the science of illness self-management. Findings will also enable the design of trials to develop and test innovations that improve mental and physical health for many thousands of African Americans. [unreadable] [unreadable]

Project Summary. Background: Insomnia is a highly prevalent and interfering comorbidity to HIV infection. Nearly 70% of PLWHA experience significant sleep difficulties characteristic of insomnia. Efforts have been made to identify factors that contribute to the development and maintenance of insomnia in PLWHA. Extant studies have studied sleep problems, but not clinical insomnia. The purpose of the current study is to specifically study insomnia (by including insomnia specific measures) as a comorbidity of HIV. Further, we will test a behavioral and biological pathway that could explain the relationship between sleep, HIV health, and quality of life. If significant, findings may suggest that treatment of insomnia (either behavioral or pharmacological) could improve HIV health outcomes and quality of life. Research Strategy: Linked to my career development plans to become an independent investigator in the area of HIV/AIDS and sleep, the proposed project seeks to examine whether insomnia (assessed via wrist actigraphy, diary, and interviews) is associated with HIV-related health and quality of life in PLWHA (Aim 1). Secondly, this grant seeks to examine whether a behavioral and/or biological model may explain the relationship between insomnia and HIV health (Aim 2). To achieve these aims, PLWHA (N=100) recruited from the Miami Jackson HIV clinic (N=3,000; 33% Hispanic, 60% African American/Black Caribbean), will complete self-report and biological measures of sleep (e.g.1-week actigraphy); mental and behavioral health (e.g. depression, medication adherence); and immunity (e.g. as measured by biomarkers IL-6 and CRP). We will use existing medical records to identify coexisting conditions, medications, viral load, and CD4. Next Steps: Using similar methodology for assessment of sleep, the next step in my anticipated research program is to design and test evidence-based interventions for insomnia in PLWHA to help improve sleep quality and quantity, and also overall HIV-related health and quality of life. Training Plan: As an NRSA proposal, this study will lay the groundwork for my career goals of studying HIV/AIDS and sleep. The short-term goal of the enclosed training plan is to develop the skills necessary to study the impact of insomnia on HIV-related health and quality of life in PLWHA, using state of the art assessment methods. The next step (ideally, for a post-doctoral F32 and/or early career K award) is to build on these findings to develop an area of research in sleep and quality of life for PLWHA. Improved sleep (and reduced insomnia symptoms) in other chronically ill populations with several comorbidities has improved not only sleep itself, but also, other health outcomes and quality of life. Ultimately, my goal is to improve not only HIV-related health by reducing insomnia, but also quality of life.

The long-term objective of the proposed research is to elucidate the mechanisms and role of genetic toxicity in chemical carcinogenesis. In multidisciplinary integrated studies, various complementary markers of genetic toxicity will be investigated: carcinogen-DNA and protein adducts using immunological methods, sister chromatid exchange (SCE) and micronuclei in lymphocytes (MNL), and products of activated oncogenes. The overall approach will be to validate these markers in parallel experimental animal and molecular epidemiological studies by assaying biological samples following in vivo exposure to a number of carcinogens. In the first phase, quantifiable and well-characterized model compounds (chemotherapy agents, ethylene oxide) will be studied. In the second phase, following development of antibodies to the specific DNA and protein adducts of interest, the same battery of immunological and cytogenetic assays will be applied to two environmentally relevant chemicals (vinyl chloride and styrene). The feasibility of extending this approach to benzene and butadiene will also be evaluated. This approach will provide greater understanding of the relationship between DNA binding and measurable chromosomal damage and will permit assessment of the relative usefulness of each as a marker of biologically effective dose of carcinogen in future biomonitoring and molecular epidemiology studies. The proposed research will be carried out by developing new antibodies to carcinogen-DNA and protein adducts, modifying existing immunological methods to increase their sensitivity, implementing the MNL assay, and applying innovative methods (cytofluorography, fluorescence line narrowing, and image intensification microscopy) to quantification of DNA adducts.

Increased development and use of minimally invasive surgery (MIS) procedures demand improvements in endoscopic imaging to provide a more comprehensive view of the operating field. Current trends in enhancing endoscopic imaging are toward stereoscopic viewing and true or pseudo 3D perception. However, such systems currently being introduced are based on the use of standard endoscopes with a limited field of view (approximately +60 degrees about the optical axis, at best). With ongoing support from NIH, InterScience, Inc. is currently developing a direct viewing rigid endoscope based on our patented and patent pending C-Viewa technology that can capture a field of view greater than a hemisphere (>2p steradian solid angle) onto a single image plane. In anticipation of the increased use of MIS that demands more comprehensive view of the operating field, we propose to utilize our C-View technology to present the clinician with a field of view with depth perception and true spatial configuration. It is the purpose of the proposed effort to develop a projection and display technology compatible with our C-View Endoscope technology that provides depth and spatial information to the clinician and can be expanded to stereoscopic 3D field visualization. [unreadable] [unreadable] [unreadable]

The long term objective of this proposal is to understand the function of the human retinoblastoma (Rb) gene(s). It is postulated that the loss of function in both alleles of this gene located in chromosomal region 13q14 is responsible for the development of both Rb and secondary malignancies in patients carrying the mutant Rb allele. The mechanism of tumor development may also be similar in patients with osteosarcomas without any hereditary susceptibility to retinoblastoma. In addition the same mechanism of tumor formation has been implied for the development of Wilms' tumor. Specific aims include determining changes in human Rb and second primary tumors following the addition of a 13 chromosome containing the normal wild type Rb allele. Parameters tested in such hybrid cells will include changes in ability to produce tumors in nude mice, differences in the capacity to form colonies in semisolid medium, changes in specific protein markers and alterations in the expression of oncogenes which are known to be amplified and/or highly expressed in the original parental cells. In addition fibroblasts from patients with the hereditary deletion and non-deletion forms of Rb will be transformed with various chemical and physical agents to determine if the loss of the normal wild type Rb allele at 13q14 results in the transformation of these cells. Such studies will provide further evidence that the retinoblastoma gene functions normally to suppress tumorigenicity and that loss of function within both alleles is responsible for tumor formation. Expression of oncogenes in these transformed cells as well as the specific role of the N-myc gene in the development of retinoblastoma and secondary malignancies will be examined. Methodologies will include microcell fusion, 2-dimensional gel electrophoresis, restriction fragment length polymorphism analysis, esterase D assays, karyotypic analysis and standard recombinant DNA technology. These studies should lead to a much better understanding of a class of human cancer susceptibility genes which are recessive and appear to normally function to "suppress" tumor development. Information gained from these investigations should not only further our understanding of human cancer development but also could suggest clues to possible ways of preventing malignant progression.

Human melanocytes are neural crest derived cells that perform a unique function in the skin. While they are a minority of the population of the epidermis, they are responsible for the majority of photoprotection of the skin from ultraviolet radiation (UVR) through the production and transfer of melanin to keratinocytes [1]. Melanocytes, or their stem cells, are also progenitor cells for the most deadly of skin cancers, melanoma, which arises as a step wise progression from benign, to minimally invasive, to metastatic tumor [2]. Paracrine factors, produced primarily by keratinocytes, but also by fibroblasts, control multiple melanocyte functions, including dendricity, migration, growth and pigmentation, and also regulate normal homeostasis of melanocytes in the skin. Disordered of these growth factors, their receptors, or signaling pathways, are implicated in melanoma progression, as well as in disordered pigmentation [3]. Therefore, discovery of new or novel paracrine factors, and defining receptors and signaling pathways stimulated by these factors, are important to understanding melanocyte regulation in the skin, and melanoma tumor progression. Semaphorins are a large family of proteins that were originally identified in the nervous system, and are critical regulators of axon guidance. During the last 5 years, numerous reports show that semaphorins and their receptors (Plexins and Neuropilins) are widely distributed and regulate multiple biologic processes, including cell adhesion and migration, neurite extension, immune function, organogenesis, and tumor progression. Our published and preliminary data show a previously unrecognized role for semaphorins in human melanocyte function. Semaphorin 4D and semaphorin 7A regulate multiple cellular processes in other cell types, including neurite outgrowth and retraction, tumor progression and immune modulation [4]. We are the first to show that semaphorin 4D (Sema4D) and semaphorin 7A (Sema7A) control melanocyte attachment, spreading and dendricity, which are critically important for normal skin pigmentation. Further, our data suggest a potential role for Sema4D and Sema7A in melanoma progression, because their cognate receptors (Plexin B1 and Plexin C1 respectively) are either reduced or absent in melanoma. Finally, our preliminary data indicate a novel role for Sema4D in downregulation of c-Met receptor activation, a receptor critically important for melanocyte cell growth, migration, differentiation, and melanoma progression. We hypothesize that Sema4D and Sema7A regulate melanocyte migration and dendrite formation through stimulation of intracellular signaling pathways that include the GTP binding proteins Ras, Rho, Rac, integrins and LIM kinase- cofilin pathways. We predict that effects of Sema7A are mediated by a balance between 1- integrin and cofilin activation. We predict that effects of Sema4D are mediated by a balance between c-Met receptor inhibition and Plexin B1 activation. Finally, we predict that loss of Plexin B1 and Plexin C1 expression contribute to melanoma progression through enhanced melanoma cell growth, migration, and survival due to loss of c-Met receptor inhibition and coflin activation, respectively. PUBLIC HEALTH RELEVANCE. Identification of new growth factors that regulate human melanocyte function is important for understanding pigmentation of the skin, and in the transformation of benign melanocytes to malignant melanoma. We have identified two proteins, Semaphorin 4D and Semaphorin 7A, which stimulate human melanocyte function through the receptors Plexin B1 and Plexin C1 respectively. Further, we show that both of these receptors are reduced or absent in melanoma cell lines, and, in the case of Plexin C1, in melanoma in vivo. The goal of this project is to define how Semaphorins signal through Plexin receptors, and to understand the mechanisms by which loss of Plexin receptor expression contributes to melanoma metastasis.

Despite numerous studies that have been carried out on plaque, saliva and tooth mineral, very little is known about events taking place in the model which permits the determination of many of the relevant physicochemical parameters on the ultramicro scale within "white spots" and sound enamel under a wide variety of conditions outside the tooth. Enamel sections 200 to 750 Mum thick are sealed on one side and cemented to a thin plastic sheet on the other. Microscopic wells, 50 to 100 Mum in diameter, are drilled through the plastic sheet into sound or caries regions in he enamel section, depending upon the nature of the experiment. Test solutions are deposited into these wells and the change in composition of these solutions is followed as functions of time and the composition of the external medium. The composition of the solution in the microwell is determined through the use of specific ion electrodes or through microcolorimetric analysis. Galvini potentials are simultaneously determined with microreference electrodes. The goals of this research are: (1) to obtain data defining the time scale of events occuring within the lesion during caries information; (2) to develop a valid mechanism of the caries process; (3) to obtain iformation about the physical properties of enamel relevantn to mineralization and demineralization; (4) to discover which part of the caries process is most amenable to interruption or reversal; and (5) to devise new micro and ultramicro techniques for the analysis of oral tissues.

Objective 1: Develop a FV-based gene therapy approach for the treatment of subjects with LAD-1. 1.1 Pre-clinical studies to evaluate the efficacy of FV in human LAD-1 CD34+ cells. We have conducted extensive pre-clinical studies to investigate the efficacy of clinical grade FV expressing the human CD18 cDNA (FV-hCD18) in HSPCs collected from subjects with a molecularly confirmed diagnosis of LAD-1. Cells were transduced ex vivo with FV-hCD18 for 16 hours. Flow cytometry of CD34+ cells cultured for 3 days after transduction demonstrated CD18+ cell surface expression in 39-42% of cells. Genetic correction of HSPCs from LAD-1 patients restored the chemotactic function of neutrophils differentiated from these progenitor cells in vitro. Transplantation of FV-hCD18-transduced LAD-1 HSPCs into immuno-deficient (NSG) mice resulted in high-level, clinically relevant gene marking levels in vivo. The average percentages of human cells expressing CD18 in the murine BM 5 months after transplantation were 36.0 3.9%. Quantitative PCR analysis of vector integrants within engrafted human cells indicated a single integration event occurred in most of long-term repopulating HSPCs. Flow cytometry-based lineage analysis of BM from mice transplanted with CD34+ cells transduced with FV-hCD18 revealed human CD18+ cells in both CD13+ myeloid and CD20+ lymphoid compartments. Using next-generation sequencing technology, a total of 101 unique integration sites were recovered in repopulating cells and revealed a polyclonal pattern of integration with no evidence of insertional mutagenesis or tumorigenicity five months after transplantation. 1.2 First-in-human clinical trial testing safety/efficacy of FV for gene therapy of patients with LAD-1. Based on pre-clinical evidence of safety and efficacy discussed above, we have designed a first-in-human phase I/II gene therapy clinical trial using FV for the gene therapy of LAD-1. A pre-IND type B meeting was held with members of the Center for Biologics Evaluation and Research (CBER) within the FDA. Regulatory review and accrual are currently on hold due to delays in production of GMP grade FV. Objective 2: Develop and evaluate safety of CRISPR/Cas9-based strategies for permanent site-specific delivery of a therapeutic gene in human HSPCs. 2.1 Evaluate off-target Cas9 activity in human HSPCs We performed CRISPR-Cas9-based genome editing in human HSPCs and assessed the acquisition of de novo somatic mutations in an unbiased, genome-wide manner via high-throughput, whole-genome sequencing (WGS) of single-cell-derived HSPC clones. Importantly, to distinguish between naturally occurring spontaneous somatic mutation, potential cell culture-induced mutagenesis, and bona fide Cas9-mediated genetic alterations, the experimental design included a parallel set of control treatments applied to samples of the same bulk HSPC population. HSPC samples were either i) mock electroporated, ii) electroporated in the absence of effector molecules, iii) electroporated with recombinant Cas9 alone, or iv) electroporated with ribonucleoprotein complexes (RNPs) consisting of Cas9 bound to single guide RNAs (sgRNAs) targeting either the cxcr4 gene or the safe harbor locus AAVS1. Experimentally manipulated cells were cloned by limiting dilution and single-cell isolates were expanded in vitro to obtain sufficient genomic DNA for WGS library preparation and high-throughput sequencing. We employed somatic variant calling algorithms to identify de novo insertions/deletions (indel) mutations, single nucleotide variants (SNVs) and structural variants (SVs) captured within the WGS datasets for each treatment group relative to the un-treated, bulk HSPC reference sequence. Consistent with previous studies investigating CRISPR-Cas9 off-target activity in mouse embryos and human iPSCs, our WGS-based analysis of potential Cas9 RNP-associated off-target mutational events in primary human CD34+ HSPCs suggests that genome-editing utilizing electroporated Cas9 RNP complexes does not result in a significant, Cas9-specific mutational burden within recipient cells. This observation has relevance to current Cas9-based ex vivo genome-editing strategies. 2.2 Homology-independent targeted integration (HITI). We have utilized a novel NHEJ-based approach to Cas9-mediated transgene knock-in, known as HITI, to achieve robust site-specific transgene integration within human CD34+ HSPCs. As proof-of-concept, a copGFP expression cassette was targeted to the genetic locus ITGB2 encoding the beta-2 integrin subunit CD18. First, a HITI donor template bearing a CMV promoter-driven copGFP reporter gene flanked by 20-nt ITGB2-specific sgRNA target sequences (designated ITGB2-ts) was constructed and packaged within recombinant adeno-associated virus serotype 6 (rAAV6) capsids. Mobilized human CD34+ HSPCs isolated from healthy donors were transduced with rAAV6-copGFP and electroporated with pre-formed, ITGB2-targeted Cas9/sgRNA ribonucleoprotein (RNP) complexes. Upon nuclear entry, the ITGB2-ts-flanked reporter cassette and the endogenous ITGB2 target gene are concomitantly cleaved by Cas9, thus promoting NHEJ-mediated transgene insertion at the site of the Cas9-induced chromosomal double-strand break. To determine the optimal time of rAAV transduction, HSPCs were transduced with rAAV6-copGFP at either 48 hr or 36 hr pre- or 0.5 hr post-RNP electroporation. Gene edited cells were cultured for up to 28 days post-electroporation and periodically sampled for flow cytometry and genomic DNA (gDNA) extraction. Transduction of HSPCs prior to electroporation resulted in enhanced cellular viability compared to post-electroporation transduction. Flow cytometry revealed efficient rAAV transduction at 4 days post-transduction (range 18-46% copGFP+ cells). The percentage of cells expressing copGFP slowly decreased over the extended culture period, stabilizing at approximately 5-10 percent of the bulk cell population at >2 weeks post-electroporation. Site-specific transgene integration was confirmed by PCR analysis of bulk cell gDNA using reporter- and flanking gene-specific primer pairs. To estimate the frequency of transgene integration, CD34+ cells were plated in a CFU assay and transplanted into NSG mice. Approximately 12% of colonies and similar percentages of human cells at 5 months after transplantation demonstrated copGFP expression. Integration junction-specific PCR analysis of gDNA derived from GFP+ colonies and post-transplant human CD45+ cells confirmed that reporter gene expression was attributable to integrated donor template sequences, as opposed to rAAV-copGFP episomes or random transgene integration events. In summary, HITI-based transgene knock-in provides an effective alternative to HDR-mediated donor template recombination in human CD34+ HPSCs. Objective 3: Develop a targeted, non-genotoxic conditioning regimen based on anti-c-MPL antibodies conjugated to immunotoxin. In collaboration with Dr. Zhirui Wang and Dr. Diogo Magnani, proof-of-concept experiments have been initiated in non-human primates (NHP). Construction of anti-c-MPL monomeric and dimeric single-chain variable fragment (scFv) fused with the diphtheria toxin fragment 390 (DT390) is underway. This recombinant toxin fragment is safer because it lacks the native toxin-binding domain, therefore, preventing internalization of toxin in off-target cells. The resulting scFv-DT390 (69.55 kDa) and svFv2-DT390 (96.5 KDa) will be produced using the yeast Pichia pastoris expression system, as previously described for anti-CD3-DT390. Evaluation of in vitro and in vivo rhesus macaque HSPC target cytotoxicity and pharmacokinetic studies will be performed in FY20. Pending success of these proof-of-concept experiments, options will be explored to develop this concept clinically.

Despite recent advances, development of nucleic acid (NA) therapies continues to be impeded by the challenges of effective delivery. GeneSegues, Inc. (GSI) has developed a nonviral delivery technology, sub-50 nanometer (s50) capsules, which co-opt the raft/caveolar uptake path for efficient intracellular delivery of NA drugs. The raft/caveolar path is an endogenous, intracellular transport mechanism that can efficiently deliver internalized complexes into the cell, avoiding the inflammation and drug degradation mechanisms that have consistently thwarted effective delivery of NA drugs. Exploiting this receptor-mediated, size-sensitive path is achieved with a novel capsule design comprised of a single-layer crystallized polymer shell which shields the NA drug, sterically stabilizes the shell, and targets the capsule. In liver, GSI has demonstrated initial proof-of-concept for liver-targeted, cell-subtype delivery of functional plasmid DNA. Hepatocyte-specific delivery in mice was demonstrated with s50 capsules employing asialoorosomucoid (ASOR) ligands targeting the asialoglycoprotein receptor (ASGPr), while delivery to LSEC's was achieved using hyaluronan (HA)-coated s50 capsules targeting the HA receptor for endocytosis (HARE). We now propose to initiate preclinical development of these liver-targeted, plasmid-bearing s50 capsules for therapeutic application. Additionally, in view of the promising therapeutic potential of RNAi, we propose to also synergistically assess the utility of s50 capsules for systemic in vivo delivery of therapeutic RNAi oligonucleotides to defined liver target sites. Therefore, our proposed specific aims are to (1) determine utility and safety of s50 hepatocyte-targeted capsules for systemic delivery of double and single stranded RNAi oligonucleotides, using a published siRNA sequence (siApoB-1) encapsulated in s50 ASOR-ligand capsules and (2) assess safety of LSEC-targeted s50 capsules for systemic delivery of plasmid DNA, using an epitope-tagged reporter gene encapsulated in s50 HA-ligand capsules. Biodistribution will be assessed by radiolabel for NA's delivered via HA-coated as well as ASOR-coated s50 capsules, and any evidence of early inflammatory responses will be assessed for plasmid and RNAi cargos. If successful, we expect this body of work will provide a solid framework for partnering with NIH and/or drug developers to move forward encapsulated NA therapies for liver disease. PUBLIC HEALTH RELEVANCE: Nucleic acid drugs offer much promise for treating diseases of the liver. However, delivering nucleic acids into disease cells of the liver is very problematic because (1) the liver is a heterogeneous organ with many different cell types, and (2) nucleic acids can trigger drug degradation and toxic reactions once inside the cell. We propose to further develop nucleic acid delivery technology capable of accessing and penetrating specific cell types within the liver, and of avoiding intracellular defenses, for effective treatment of liver disease.

Benzene is an important industrial chemical (greater than 2 billion pounds produced annually in U.S.) And component of gasoline. It is also an established human leukemogen. The shape of the dose-response curve for benzene-induced leukemia at exposures below 10 ppm is highly controversial and a key public policy and risk assessment issue involving potentially billions of dollars. Conventional epidemiology and toxicology studies are unlikely to resolve this controversy. Work performed to date on this grant has led to the development of a mechanistically- relevant biomarker that should be sufficiently sensitive to shed light on the dose-response curve in the low dose region. This biomarker is the simultaneous detection of leukemia-specific aberrations in chromosomes 5, 7, 8 and 21 by fluorescence in situ hybridization in metaphase cells. We propose to examine this and other biomarkers in the blood of 300 workers exposed to a wide range of benzene concentrations (0.5 to greater than 10 ppm) and 150 unexposed matched controls. The first year will be devoted to the collection of these biological samples with detailed exposure information and sophisticated sample processing such that many new endpoints can be examined. Prior work on this grant has also shown that specific chromosomal translocations related to leukemogenesis can be detected by the reverse transcriptase-polymerase chain reaction (RT-PCR) in benzene- exposed workers. Recent advances in kinetic real-time PCR now make it possible to quantitate accurately the level of various chromosome translocations. We propose to use quantitative real- time PCR to measure levels of the translocations t(8;21), t(9;22), t(11q23) and t(14;18) in the peripheral blood lymphocytes and granulocytes of workers exposed to benzene and unexposed controls. Our studies to date also suggest that individuals differ in their susceptibility to benzene toxicity in relation to their folate status and genetic make-up. We therefore propose to further examine the role folate, vitamin B12, and various genetic polymorphisms play in individual susceptibility to benzene-induced chromosome damage. These studies will contribute to our understanding of the mechanism of benzene-induced leukemia, the risk benzene poses at low doses, and factors associated with susceptibility to this compound.

Carnitine is important in normal oxidation of fatty acids for energy. It is provided to mammals from animal protein foods and by de novo synthesis from lysine incorporated into tissue proteins that then is trimethylated. Such proteins are degraded so that tissues including liver, heart, kidney and especially skeletal muscle have free intracellular trimethyllysine that can be converted to carnitine in several enzymatic steps including two hydroxylations catalyzed by Alpha-ketoglutarate-dependent, ascorbate-requiring dioxygenases. The liver is the major organ for final synthesis of carnitine by hydroxylation of Gamma-butyrobetaine. Carnitine levels appear to increase in various ketotic and incipient ketotic states such as diabetes and starvation; this remains to be firmly established, especially whether the increase is due to increased synthesis. We are engaged in a long-term study of metabolism and functions of carnitine, including synthesis, regulation of its synthesis and use, and degradation by intestinal bacteria. Here we propose, by studies using isolated perfused livers of rats, to learn whether increased synthesis occurs when the donor animals have been made diabetic by alloxan or streptozotocin, have been starved, or have received glucagon or the hypolipidemic drug, clofibrate. We then shall study capacities of homogenates of various tissues of normal and above animals to convert trimethyllysine and subsequent intermediates to Gamma-butyrobetaine or, in the liver to carnitine, we shall infer inter-organ dependencies in carnitine biosynthesis. Using isolated perfused livers from ascorbate-deficient guinea pigs, we shall determine whether ascorbate deficiency affects one or both hydroxylases in synthesis of carnitine. Then we shall study the mechanism of hydroxylation of Gamma-butyrobetaine to carnitine at a molecular level, selecting from amoung four proposed mechanisms. For that purpose we shall determine whether the reaction is stereospecific or steroeselective, and measure kinetic isotope effects. Finally, we outline methods to establish pathways of degradation of carnitine to trimethylamine and other products to be identified; the bacteria Ps. putida and Ac. calcoaceticus will be studied. We shall purify the enzyme(s) involved and study mechanisms of action. These organisms may serve as models for further study of truley enteric organisms of the human that could metabolize dietary carnitine and Gamma-butyrobetaine.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. My laboratory is interested in structural aspects of cell-surface receptor/ligand interactions with relevance to human health and disease. Through structural analysis we hope to answer basic mechanistic questions about transmembrane signaling, as well as produce structural data that can be used as a template for drug design. We are engaged in expression and crystallization studies of numerous glycoprotein receptors both alone and in complex with their ligands. We are expressing both soluble receptor domains as well as full-length integral membrane proteins. The receptor systems we study are primarily in the immune and nervous systems, with a special emphasis on understanding cross-reactivity in molecular recognition. Most of our projects we are critically dependent on the ability to collect x-ray data at a synchrotron facility due to the generally small and moderate quality of the crystals which we can grow of large and hetero-geneous protein complexes.

The overall goal of this continuing project is to develop efficient algorithms which will permit the construction of realistic and comprehensive mathematical models that relate normal and pathological renal function to the underlying membrane transport and flow processes in the renal tubules and their associated vasculature. The primary thrust of our research during the next period will be: 1. To use our present inner medullary models to develop fairly detailed architectural models of the inner stripe of the outer medulla and then integrate these new models with our present central core and vasa recta n- nephron inner medullary models. 2. To include additional solutes and osmolytes and investigate the role of osmolyte production in the inner medulla. 3. To incorporate a more detailed representation of transmural movement of water and solutes that takes into account both cellular and paracellular pathways and also the exchange of electrolytes and water between red blood cells and plasma in the vasa recta. 4. Adapt current algorithms for serial computers, - if necessary, develop new ones based on our split system solvers - to fully exploit the parallel and vector processing capabilities of supercomputers. This is necessary to handle the size and complexity of our current and future n-nephron models. 5. If necessary - for parallel and/or vector algorithms - (a) improve stability and accuracy of numerical methods, (b) develop hierarchal solution strategies, and (c) incorporate continuation and smoothing methods. 6. To make the non-linear Schur Complement type methods developed by us readily available to other biomedical modelers, for use on minicomputers and/or workstations. These include: (a) reduced models of the whole kidney and medulla, (b) models of epithelia and isolated perfused tubules, (c) tubuloglomerular feedback response models, and (d) neural network models.

Several recent studies indicate that tissue-derived stem cells can not only differentiate into cells of the tissue from which they are obtained, but also those from other tissues. We have been interested in the range of differentiative capacity (which we term plasticity) of CNS derived stem cells and hypothesized that one way to assay this would be to implant these cells into blastocyts and assess incorporation into the resulting mice later in development. Our results indicate a robust incorporation of these cells into extraneural tissues including bone marrow, where they express lineage markers of differentiated hematopoietic cells, bone muscle and GI tract. We believe this model provides a unique insight into stem cell plasticity because of the high efficiency of implantation and production of apparently normal pups. We propose to follow-up our studies by addressing the following specific aims: 1) We will determine whether incorporation of stem cells into blastocysts varies as a function of stem cell subpopulation or type, we will create a retorviral library of neural stem cells to assess whether specific subpopulations exist as well as comparing neural stem cells with marrow stromal cells in their ability to incorporate into blastocysts; 2) We will determine whether incorporation of neural stem cells into other tissues renders them functional by performing bone marrow transplantation of cells obtained from chimeric pups into lethally irradiated mice and assessing whether neural stem cells can repair a embryonically lethal genetic defect in bone; and 3) We will assess whether neural stem cells retain their functional capacity to develop into olfactory interneurons after blastocyst transplantation and determine whether their implantation at the blastocyst stage can repair mice with an absence of myelin.

The primary goal of the Coordinating Center (CC) for the State and Community Tobacco Control Policy and Media Research initiative is to provide support to the National Cancer Institute (NCI) and its funded projects as they shape national priorities and fill important gaps in the evidence and practice base of tobacco control and ultimately reduce the health and economic burden of tobacco in the United States and elsewhere. The CC will accomplish this goal by fostering productive collaboration and communication among the research projects (RPs), NCI, and the broader public health community. The CC will provide comprehensive project coordination; facilitate collaborations with NCI and the individual sites to support funded RPs and identify and implement complementary cross-site RPs; offer state-of-the-science analytic, statistical, and data management support; utilize methods and mechanisms that will facilitate the translation of study findings and accelerate the implementation of new tobacco control strategies; and implement measures and methods to evaluate the success of individual RPs and the initiative as a whole. To encourage communication and collaboration, the CC will provide logistical support for all meetings and develop a project Web portal. The project Web portal will provide a secure, centralized collaboration space to store the initiative's history and knowledge and unite the human interactions and connections of the group. The Web portal will also be used to disseminate information about the initiative, individual RPs, research findings, news and press releases, and publicly available data and publications. The CC will foster productive research collaboration by pairing each RP with teams that have tobacco control expertise that aligns with the aims of the RPs, facilitating the use of common methods and measures, and making unique data resources available to NCI and individual sites. In addition, the CC will convene a working group of thought and practice leaders in tobacco control who will represent the perspective of the broader tobacco control communities, including state tobacco control programs, advocacy organizations, and minority networks. This working group will participate from the outset of this initiative to help ensure that the research conducted meets the needs of practitioners and fills the most important gaps in research. This group can also assist with the translation and dissemination of findings to diverse audiences. The CC will use traditional dissemination formats and channels, such as newsletters disseminated through mail and e-mail, and social media (e.g., Twitter, Facebook) to ensure broad dissemination of findings with the goal of quickly influencing tobacco control practice.

Specific Aims: The principal objective of this study is to validate a new measure of patient outcome. The proposed instrument is a scale of cognitive functional status which is readily applicable to clinical practice and research in the elderly. This study will test the reproducibility, validity, and responsiveness of the cognitive function scale. A second objective is to integrate the results of cognitive function evaluation with those obtained using the Medical Outcomes Study instrument, which was designed to assess multiple aspects of functional status and well being, but does not include a measure of cognitive status. Integration of the cognitive function scale with the Medical Outcomes Study instrument (SF-36) will allow sensitive assessment of the negative and positive effects of medical interventions or conditions on elderly patients' mental and overall functioning in their daily lives. Experimental design and methods: This longitudinal prospective study will enroll 300 patients over the age of 65 for a baseline assessment and a second assessment, using both single-state and transition measures after six months. The patient population will consist of three groups: 100 medical clinical patients with a low comorbidity index, 100 medical clinic patients with a moderate to high comorbidity index, and 100 patients pre- and post-coronary artery bypass surgery. At both interviews, patients will complete the Cognitive Function Scale, a directed psychometric battery, the Medical Outcomes Study 36 item scale, the Geriatric Depression Scale, and the Comorbidity Questionnaire. The methodology of scale development requires that the proposed cognitive function scale satisfy the criteria of reproducibility, internal consistency, item convergent validity, variance, item discriminant validity, and construct validity. In addition, the responsiveness of the cognitive function scale will be measured by comparison with change on psychometric testing and change reported by an independent patient observer. Medical patients with low comorbidity indices are expected to show less change in cognitive functional status than medical patients with high comorbidity indices, who in turn are expected to show less change in cognitive functional status than the group of cardiac surgery patients. The interrelationship between patient- reported change in cognitive function and change in other aspects of health status, including physical, affective, social and role function and overall perception of health, will also be assessed. Long term objective: The development of a brief, easily administered, responsive, multi-item scale of cognitive function which can be used in practice and research to evaluate change in cognitive function in the elderly, and which can be integrated with other patient-centered measurements of functional status and well-being.

Using a combination of organic chemistry, molecular biology, enzymology, and NMR spectroscopy the details of the biosynthesis of uroporphyrinogen III (the precursor of heme and chlorophyll) and its subsequent transformation to vitamin B12, the anti-perinicious anemia factor, will be elucidated. Knowledge of the pathway including control mechanisms and genetic mapping will define intermediates important in diseases such as B12 deficiency and acute intermittent porphyria. All of the biosynthetic enzymes necessary for the formation of cobyrinic acid, the simplest B12 analog, will be over-expressed using the sequenced cbi genes of Salmonella typhimurium and the cob genes of Pseudomonas denitrificans and their mechanisms studies by NMR spectroscopy ia 13C- labeling. Finally, the multi-enzyme synthesis of advanced intermediates and of B12 itself will be addressed.

Altering the course of the diabetes epidemic requires a diverse array of effective changes in policies and the environments in which people live, work, and play. While prevention of type 2 diabetes (T2D) is a critical public health goal, little is known about the effectiveness of large-scale policies and programs that could help achieve this goal at the population level, or any differential effects on sub-populations. This renewal provides a platform and framework for the rigorous empirical observational study of the health impact of new and imminently available population-targeted diabetes-related programs and policy innovations by federal, state, local governments, healthcare organizations, community organizations, or businesses. New activities will construct a multi-sectoral approach to preventing T2D, related disparities and risk factors and reduce diabetes-related disparities and complications. Renewal funds will support the following: Component A: Natural Experiment Research Centers to support a 5-year multi-center network of independent research centers to evaluate innovative, health system and non-health system-based natural experimental approaches that alter the diabetogenic characteristics of US communities. Applicants must select only one of the following two tracks: ? Track 1 Evaluation of population-level programs or policies that affect population-level risk factors for type 2 diabetes (such as diet or physical activity, as well as other health behaviors; glucose; prediabetes), or ? Track 2 Evaluation of programs or policies aimed at improving care and management of diabetes, and the risk for diabetes complications. Component B: Coordinating Center to provide organizational, logistic and communication support to enhance the efficiency, productivity, and public health impact of the Natural Experiments research centers that are funded as part of Component A. Funded projects are expected to include a focus on the social determinants of health.

The major objective of the project is to identify the host factors associated with resistance to dental caries. Adults with no history of dental caries, despite environmental challenge, will serve as the focus for comparative studies with matched subjects with high caries activity. Separately collected salivary secretions, pellicle and plaque will be examined in the two groups for factors that can modify: (1) bacterial adherence, growth, and acid production and (2) the ability of the generated acid to produce demineralization. Parameters to be examined will include: (a) direct antibacterial factors, such as SigA antibody levels against specific components of cariogenic bacteria, lysozyme, lactoferrin and lactoperoxidase (amount and antibacterial activity), (b) aggregation potential using laser light to measure rate, (c) amount and nature of acids and bases produced in plaque, employing a recently modified system of liquid column partition chromatography. Salivary factors already identified as differing in the two groups (albumin binding fraction and "extra" precipitin bands) will be characterized and quantitated in saliva, pellicle and plaque. The concentration of specific phosphoproteins and cationic proteins will be compared and their protective properties assayed in an in vitro system (in collaborative studies).

The gastrointestinal epithelium functions as a dynamic barrier that serves as an interface between luminal contents and underlying tissue compartments, and is thus vital in maintaining mucosal homeostasis. Mucosal wounds have been observed following enteric infection, inflammatory bowel disease and ischemic insults. Disruption of the critical epithelial barrier allows access of luminal contents to immunologically privileged compartments thereby contributing to disease pathogenesis. In response to injury, intestinal epithelial cells (IEC) migrate and proliferate to rapidly cover denuded surfaces and re-establish the epithelia barrier. We have recently identified expression of the N-formyl peptide receptors (FPR1 and FPR2) in the intestinal epithelium. Our studies determined that a bacterial derived N-formyl peptide, fMLF and the endogenous Annexin 1 protein, which are agonists for FPR family members, promote intestinal epithelial cell migration and facilitate wound closure. Additionally, it is becoming evident that a healthy optimized intestinal microflora mediates important roles in normal gut homeostasis and recovery from mucosal insults. Recent experimental results in our laboratory revealed that epithelial cells exposed to fMLF and intact bacteria also rapidly initiate cytoplasmic signaling events, Rac and Cdc2 activation, reactive oxygen species (ROS) production, epithelial cell migration and wound closure. Thus, we believe that FPRs represent important novel type of pattern recognition receptors (PRR) in the intestinal epithelium that transmit homeostatic signaling and facilitate epithelial barrier recovery following pathologic insults. Thus, our overall objectives are to define the pathobiologic function of epithelial FPRs and microbiota in regulating intestinal homeostasis, barrier recovery and resolution of inflammation. PUBLIC HEALTH RELEVANCE: Intestinal epithelial injury occurs in a wide spectrum of clinical conditions that include inflammatory bowel diseases, enteric infection, ischemia and following surgical procedures. The loss of epithelial barrier function associated with injury contributes to mucosal inflammation. Numerous physiological processes including those influenced by the normal prokaryotic microbiota have been implicated in regulating intestinal epithelial homeostasis. Thus, given the pathologic ramifications of epithelial injury, it is vital to understand mechanisms of intestinal epithelial barrier maintenance/recovery and in defining the role of microbiota in these processes. We propose the formyl peptide receptors (FPRs) as novel pattern recognition receptors that regulate intestinal epithelial growth, motility and restitution post injury, as well as the positive influence of the microbiota in these events. Realization of the role of FPRs and their ligands on epithelial barrier regulation and wound recovery will facilitate in development of therapeutic agents to promote regeneration of the intestinal mucosa.

In the rat pituitary tumor cell line GH3, the expression of the prolactin gene is stimulated by the sex steroid 17 Beta-estradiol and the hypothalamic factor thyrotropin releasing hormone (TRH). The expression of the growth hormone gene in this tumor line is stimulated by corticosteroids and thyroid hormones. At present, the quantitative changes in mRNA production coupled to hormonal induction are most readily explained by supposing that primary control lies with transcription. We will study the primary effect of hormones on gene regulation, in these tumor cells by making a careful analysis of transcriptional units, their instantaneous rates of transcription, the formation of mRNAs from nuclear precursors and the metabolic fate of these mRNAs. These experiments utilize cDNA clones specific for growth hormone and prolactin RNA sequences. The chromosomal segments of these genes will also be isolated and cloned to facilitate a detailed analysis of transcriptional unit organization. The transcriptional units will be related to the sequences at the capped 5' end and poly-adenylated 3' end of nuclear and cytoplasmic RNA. These studies will provide criteria for the development of an authentic in vitro system of transcription, and will contribute to an understanding of regulation of gene expression in a hormone-responsive tumor cell line.

Research is divided into studies concerning the regulation of i) humoral immune responses to HIV-1 envelope glycoproteins, ii) virus replication in murine models of AIDS and iii) B cell activation in retrovirus- induced and autoimmune diseases. i) Serum and cells were obtained from mice and humans immunized with purified recombinant envelope glycoproteins from a variety of HIV-1 isolates in order to determine the number, specificity and cross- reactivity of B cells producing anti- bodies against gp120. Results indicate that the preferential expansion of B cells which cross- reactively recognized multiple HIV strains can be achieved by sequen- tially immunizing mice with different gp120 isolates. Glycosylated gp120 was found to act as a more efficient immunogen than non- glycosylated envelope glycoprotein or V3 look-region specific peptides. ii) Two murine models of HIV infection are being examined. In the murine AIDS model, levels of T cell activation and lymphokine production are being studied at the single cell level. In transgenic mice expressing gp120 proteins from HIV, the effect of immunization on tolerance and disease progression has been investigated. We are also studying SCID mice reconstituted with human PBL from gp160 vaccinated normal volunteers to analyze the degree of protection conferred by various immuni- zation regimens. An outgrowth of this work has been our finding that anti-HIV reactive cells are present initially in the peripheral cirulation and then home to the bone marrow of immunized donors. iii) We have been studying the number and antigenic specificity of lymphocytes secreting specific antibodies and lymphokines in patients and mice with SLE and AIDS. Results indicate that two processes - polyclonal activation and (auto) antigen specific immune stimulation - combine to induce the humoral abnormalities found in disease states. Murine models of these diseases are being used to analyze the specificity and cross-reactivity of activated B cells. This work is also providing considerable insight into the degree to which HIV-1 specific B cells are activated in patients with ARC and AIDS.

The proposed study addresses critical gaps in research that have impeded efforts to improve intervention and address mental health disparities for diverse, urban, low-SES infants and their families. Compelling longitudinal data have shown that insecure infant attachment is linked to later psychopathology (e.g., Sroufe et al., 2005); thus, it is crucially important to understand precursors of attachment that can be targeted in preventive intervention. Meta-analysis, however, shows that this goal has proven elusive, particularly in low-SES families (De Wolff & van IJzendoorn, 1997). There is a great public health need for effective intervention for infants in low-SES families because low-SES infants tend to face multiple risk factors (McLoyd, 1998) and insecure attachment is thought to be a risk factor for psychopathology in the context of other risk factors (DeKlyen & Greenberg, 2008). The proposed study has three interrelated aims: First, the proposed research challenges current paradigms for studying precursors of attachment by examining a novel conceptualization of parental caregiving: Secure Base Provision (SBP; Cassidy, Woodhouse, et al., 2005; Woodhouse & Cassidy, 2009)2 the degree to which a parent is able to ultimately meet an infant6s attachment needs, even when demonstrating high levels of insensitive parenting behavior. The construct of SBP is rooted in Bowlby6s (1988) emphasis on the attachment figure6s role in providing a secure base for exploration to which the child can return when needed. SBP avoids emphasizing the importance of certain culturally bound parenting practices found in white, middle class samples (e.g., sweet tone of voice, moment-to-moment affective attunement), and does not pathologize other parenting practices (e.g., 3no-nonsense parenting4). The proposed study will test the relative predictive utility of SBP and maternal sensitivity2the degree to which a mother is able to accurately interpret infant needs and respond appropriately and promptly (Ainsworth, et al., 1971; NICHD, 1999)2in predicting later attachment. Additionally, the proposed research will break new ground by using a longitudinal design with a racially/ethnically diverse, urban, low-SES sample to examine the relative predictive utility of SBP vs. sensitivity in predicting infant stress reactivity (as indexed by cortisol and alpha-amylase), physiological indicators of emotion regulation (i.e., RSA), and psychosocial adjustment. Second, we will examine the role of maternal stress reactivity and maternal physiological regulation in caregiving. The notion that parental emotion regulation plays an important role in responsive parenting (Dix, 1991) has received surprisingly little empirical attention (Lorber & O'Leary, 2005), yet could be useful in understanding how to best intervene with mothers. Third, we will examine an overarching conceptual model proposing that maternal caregiving mediates the links between maternal physiology and infant outcomes. In sum, successful completion of the three aims will inform the development of interventions for diverse, low-SES families by extending current theory and research on parental caregiving, infant attachment, infant emotion regulation, and infant psychosocial adjustment. PUBLIC HEALTH RELEVANCE: There is a tremendous public mental health need for culturally appropriate and effective interventions to serve ethnically/racially diverse, low-income infants and their parents. However, because such families have received limited attention, we know very little about which parenting behaviors best predict positive outcomes for these children or about what helps parents provide more positive caregiving. Results of this study will have important implications for the development of culturally appropriate interventions that can be used to address mental health care disparities faced by diverse, low-income infants and their families.

The importance of Th1 and Th2 cells in the normal physiology and pathology of the immune system is well established. Th2-derived cytokines play a central role in the pathophysiology of allergy and asthma. Past studies have documented differences in T cell receptor (TCR)-initiated early signaling between Th1 and Th2 cells. To date, however, no Th2-specific signaling pathways have been identified. The unerlying working hypothesis is that unique TCR-proximal signaling pathways exist in Th2 cells, and that such molecular events represent potential drug targets for selectively interfering with Th2 function. This project will focus on a detailed comparative analysis of early signaling events in Th2 vs. Th1 cells with emphasis on antigen-dependent activation systems. We will: 1) Assess early signaling events during Th2 cell activation and the effects of pharmacological inhibitors/stimulators on the differentiation of naive CD4+ T cells using established clones and T cells from TCR-transgenic mice. These studies will be combined with the use of pharmacological agents which modulate defined T cell signaling events. Effects of these drugs on naive T cell differentiation into Th1 or Th2 cells will be analyzed. 2) Analyze the role of protein tyrosine kinases (PTKs) of the Src. Syk and Tec families in Th1 vs. Th2 cells, and the effects of transient/stable overexpression of wild type or catalytically inactive kinases on activation of the two subsets will be analyzed. 3) Determine the Molecular basis of Ca2+ signaling in Th2 cells, Ca2+ signals in Th2 cells have been reported to differ quantitatively and/or qualitatively from those in Th1 cells. Studies will address the potential role of three alternative Ca2+ signaling pathways mediated by: a) Gq-coupled phospholipase Cbeta: b) sphingosine kinase; or c) CD38. The latter two enzymes generate novel Ca2+-mobilizing second messengers, sphingosine-1-phosphate and cADP ribose, respectively. 4) Analyze the roles of the Ras and MAP kinase signaling pathways in Th2 cells. The function of these pathways (which are critical for Th1 activation) in Th2 cells has not been addressed. We will measure activation of Ras and its downstream Ser/Thr kinases and transcriptional activators, examine the effects of dominant-negative Ras, and assess the role of stress-activated protein kinases in Th2 activation. The information generated by this project is critical for our ability to rationally devise pharmacological treatments designed to selectively inhibit Th2 activation and, thus, interfere with allergic responses.

In order to account for the remarkable catalytic power of enzymes, it is generally considered that the activation free energy is contributed both by binding of the substrate to the enzyme (step 1) and by chemical manipulation of the bound substrate (bond-making and breaking, step 2). Popular opinion holds that most of the activation energy is supplied in step 2. We have proposed, however, that the overall catalytic process can be explained more reasonably if it is assumed that the first step (binding) contributes a more significant, and sometimes major, share of the activation energy. To support this theory, we have synthesized a large variety of test-tube models which simulate the bound substrate by being frozen into a single, favorable conformation and by having the interacting groups brought into the closest possible juxtaposition (stereopopulation control). These compounds undergo intramolecular reactions at rates comparable to those catalyzed by enzymes, sometimes even too fast to measure. The protein raises both the entropic and enthalpic components of the substrate by binding it in a single, rigid conformation. Our original theory proposed that the principal sources of free energy increase during binding were conformational freezing, desolvation, electronic deformation, etc. Our new studies with tryptophan analogs have provided yet another factor which we had not considered originally: in those cases in which a substrate is capable of tautomeric equilibrium, the enzyme may be able to stabilize (by binding alone) the thermodynamically unfavorable tautomer (Tenutautomer). This simple event would necessarily increase the free energy content of the bound substrate and serve as "activation." We have already proven the reality of this phenomenon for tryptophan by demonstrating the potent inhibitory and stereospecific properties of TENUTAUTOMER ANALOGS. We are presently involved in the design, synthesis and testing of other stable tenutautomer analogs.

Project Summary: Metastatic prostate cancer will claim the lives of over 30,000 men this year alone. There are no effective treatments that prevents disease progression. In prostate cancer, lipid production is known to drive tumor growth throughout the early and late stages. In addition to providing building blocks for tumor cell replication, lipids produced by tumors have been shown to repress immune activity, which allows tumors to grow unchecked. Clinically used immunotherapies which deactivate built-in anti- inflammatory pathways called immune checkpoints, have been highly effective at harnessing the tumor- killing abilities of immune cells. Unfortunately checkpoint blockade inhibitors are notoriously ineffective at treating metastatic prostate cancer. Studies have shown that tumors produce lipid metabolites, endogenous ligands of Liver-X-Receptor (LXR) that activate and suppress the function of immune cells. In line with this shutting down production of lipid metabolites in tumor cells reverses tumor suppression resulting in tumor destruction. As prostate cancer cells rely heavily on lipid synthesis, we postulated that prostate tumors produce LXR ligands to suppress immune function. Our studies have highlighted that prostate tumors produce lipids that activate LXR and suppress the activity of helper and effector T-cells as well as dendritic cells, all key cell types that direct a robust anti-tumor immune response. We theorized that prostate tumors specifically produce high activity LXR ligands and by suppressing LXR activity in immune cells we could stimulate tumor destruction. In this proposal, we will explore this hypothesis using three specific aims. We propose firstly to identify if prostate tumors produce unique lipids, LXR ligands, that are especially effective at suppressing immune cell activity. We also intend to determine if the concentrations of specific tumor lipids correlate with disease severity or responsiveness to treatment. Secondly, we will test if drugs which suppress LXR activation, can stimulate destruction of prostate tumors when used alone and probe whether these drugs can sensitize prostate tumors to checkpoint immunotherapies. Lastly, we will determine how LXR signaling regulates immune cell metabolism and the manner in which immune cells respond to prostate tumor growth. These studies should lead to the development of a novel dual activity immunotherapy for prostate cancer that has intrinsic cytotoxicity and immune stimulatory activity.

Respiratory syncytial virus (RSV) is the single most important viral pathogen causing acute lower respiratory- tract infections (bronchiolitis) in children. No vaccine or specific therapy is currently licensed to prevent and treat RSV infections. Exposure to environmental tobacco smoke (ETS) has been identified as a risk factor for the development of severe RSV infections, yet the mechanisms that determine ETS and infection co-morbidity are largely unknown. Recent studies support the notion that severity of clinical disease is driven by higher level of RSV replication in the airways and studies by our group have shown that expression and activity of superoxide dismutase (SOD) antioxidant enzymes are significantly decreased in the airways of infants with severe episodes of RSV bronchiolitis, particularly if infants had been exposed to ETS. Surprisingly, our new preliminary data have shown that SOD enzymes are able to inhibit RSV replication in cultured epithelial cells and in experimentally-infected mice. We propose the novel hypothesis that lung SOD enzymes have a previously unrecognized function as host antiviral gene products by limiting RSV replication and shortening viral shedding. Our secondary hypothesis is that expression and antiviral function of such enzymes are reduced/impaired in RSV-infected subjects who are exposed to ETS. In this exploratory project we propose to test these hypotheses by two Specific Aims. In Aim 1, the profile, relative abundance, and activity of SOD proteins will be analyzed in nasopharyngeal secretions (NPS) of infants with RSV infections of different clinical severity and with or without exposure to ETS. SOD levels and activity will be correlated with the concentration (i.e. viral titer/load) of RSV in NPS. In Aim 2, we will test the specific hypothesis that increasing of SOD activity in the lungs will blunt RSV replication and improve airway disease. For that, we will increase SOD expression in the lung by adeno-associated virus (AAV)-mediated gene transfer or by the use of salen-based EUK SOD mimetics, and measure RSV titer (as the main endpoint) in an experimental mouse model of RSV infection and co-exposure to ETS. The protective effect of SOD will be evaluated by examining clinical disease, lung inflammation/pathology, and viral-mediated airway hyperresponsiveness. This project may have important translational implications by suggesting new and more aggressive strategies to treat primary respiratory viral infections in high risk infants (i.e. those exposed to ETS). These therapeutic opportunities will b explored in a future R01 application based on the results of this exploratory project.

DESCRIPTION: (Applicant's Abstract) The exposure of an opioid receptor to opioid peptides or opiate alkaloids initiates a biological response. A long-term or repeated exposure to opioids causes a decreased sensitivity to the drug, leading to a reduced cellular response; this desensitization is regulated by multiple mechanisms which have been implicated in the generalized development of tolerance and addiction. Acute opioid treatments result in a rapid desensitization by functional uncoupling of the receptor from the effector system. In addition, this treatment results in a rapid internalization of the receptor into intracellular compartments. Chronic opioid treatment results in longer desensitization due to receptor down-regulation with a net loss of binding sites from the cell. Relatively little is known about the molecular mechanisms underlying these events. The objective of the studies proposed here is to explore the agonist-mediated events that lead to receptor internalization and degradation. These studies propose to explore the molecular mechanisms of opioid receptor internalization and dimerization in order to understand the functional significance of these events. The specific aims are: (i) to characterize kappa opioid receptor trafficking in neuronal cells, and (ii) to explore the role of dimerization in kappa opioid receptor function. This work will provide critical information about the early agonist-mediated events that modulate opioid receptor function. A thorough understanding of the cellular mechanisms involved in the modulation of receptor desensitization is crucial for the development of a therapeutic basis for drug addiction.

A. Mouse Homeobox And POU-Domain Genes. A novel mouse homeobox gene NKx-1, was cloned and approximately 8 kb was sequenced. The deduced amino acid sequence of the Nkx-1 homeodomain differs from the Drosophila NK-1 homeodomain by only 3 of 60 amino acid residues. Both Nkx-1 and NK-1 proteins contain an acidic region before the homeodomain. Northern analysis revealed one major band of Nkx-1 poly A+ RNA in brain and trace bands in testes and spleen. The abundance of Nkx-1 poly A+ RNA is highest in 10 day mouse embryos and progressively decreases thereafter. In situ hybridization with sections of 14-day mouse embryos revealed Nkx-1 RNA in the mesencephalon, myelencephalon, spinal cord, vertebrae, and ribs. Five additional novel mouse homeobox genes were cloned and partially sequenced. A novel mouse POU-domain gene related to Brain-3 POU-domain CDNA was cloned and about 3 kb was sequenced. The expression of Brain-1, Brain-2, Brain-4 and Skip POU-domain genes in the mouse nervous system was determined by in situ hybridization as a function of developmental age. B. Regulation of a Calcium Channel a-1 Subunit Gene. A nucleotide sequence was found in the 5 -upstream regulatory region of a voltage-sensitive calcium channel alpha-1 subunit gene that is a powerful activator of an enhancerless chloramphenicol acetyltransferase reporter gene. A CDNA expression library was screened for recombinants that direct the synthesis of proteins that bind to this nucleotide sequence. Seven kinds of clones were found that encode proteins that bind to oligonucleotides with appropriate sequence specificity. C. Promotor And Enhancer Selection. CDNA clones were obtained that encode proteins that specifically bind to oligonucleotide sequences by a method that selects for gene regulatory sequences. Partial sequences of the CDNA clones were obtained.

Introduction The Jackson Laboratory (J/\X) founded in 1929, is an independent, nonprofit institute dedicated to research and education in mammalian genetics and its application to precision medicine, as well as to empowering others in their scientific enterprise through access to our genetic resources. The JAX Cancer Center (JAXCC) comprises the cancer research and education activities at the Laboratory, as well as institutional shared resources that are used in cancer research. The large-scale distribution colonies that supply mice to the external research community are managed independently from the JAXCC by an administrative arm called JAX Mice & Services. The distribution colonies are financially self-supporting and, as a not-for-profit organization, generate surpluses that support fundamental research. The JAXCC occupies facilities on three JAX campuses, described below. The main JAX campus is an ~ 750,000 ft[2] facility in Bar Harbor Maine. We have launched a major expansion with the opening of a campus adjacent to the University of Connecticut Health Center (UCHC) in Farmington, Connecticut. This new campus, called The Jackson Laboratory for Genomic Medicine (JAX Genomic Medicine), will be a 189,000 ft[2] facility, to be completed in 2014. JAXCC members on this campus focus on human cancer genomics, computational biology and their application for precision medicine. The third campus, the JAX-West facility in Sacramento, California?has expanded to include a large, collaborative resource of patient-derived xenografted (PDX) primary human cancers, significantly enhancing the cancer resources ofthe JAXCC. The original campus in Bar Harbor has been renamed The Jackson Laboratory for Mammalian Genetics (JAX Mammalian Genetics) to reflect its role within the expanded three-campus framework. In total, we have an institution that has remarkable infrastructure to innovate and conduct science in mammalian genetics. The geographically distributed nature ofthe campuses is to our advantage in being able to capitalize on local (state) resources and talent, but unified by leadership, organization, and our focus on genetics and genomics.

This project focuses on the effects of fatty acid oxidation on recovery from myocardial ischemia. Exposure of the myocardium to long chain fatty acids has long been known to increase myocardial oxygen consumption and to impair functional recovery from ischemia. For example, perhexiline, a putative inhibitor of fatty acid oxidation, is used in Europe to treat angina, and its benefits are observed in patients maximally treated with b-adrenergic blocking agents, nitrates, and calcium channel blockers. We examined the effects of perhexiline in normoxic and post-ischemic isolated rat hearts exposed to a mixture of pyruvate, lactate, _-hydroxybutyrate, acetoacetate, glucose, and long chain fatty acids. All substrates except glucose are 13C enriched. Perhexiline significantly inhibited fatty acid oxidation and stimulated the oxidation of lactate in normoxic hearts, but the effect was not sufficient to substantially change myocardial oxygen demand. In a separate study, the effects of glutamate and aspartate on metabolic pathways were examined under conditions of cardioplegia. Again, hearts were supplied with physiological substrates, but aspartate and glutamate in the presence of cardioplegic arrest did not further alter substrate utilization patterns substantially, although acetoacetate utilization was somewhat lower compared to simple cardioplegic arrest.

The proposed studies examine the morphology and metabolism of adipose tissue from nonobese and obese patients and correlate the in vitro tissue findings with various metabolic functions of the whole individual as measured in vivo. Morphologic studies of the adipose tissue include determination of adipose cell size and number in individuals of widely different body weight. Metabolic studies in these adipose tissues investigate the glucose and lipid metabolism, and the hormone (insulin, epinephrine) responsiveness of adipose tissue as a function of adipose cell size, dietary composition and antecedent changes in body weight. Specific emphasis is placed on dissociating the effects of these three factors on the metabolic character of the adipose tissue and the overal metabolism of the individual as measured in the forearm tissues and assayed in the plasma. Additional studies will examine the relationship between obesity and pancreatic dysfunction through investigation of insulin secretion from pancreatic islets isolated from rats varying widely in body weight, adiposity and dietary intake. BIBLIOGRAPHIC REFERENCES: Salans, L.B., Reactive Hypoglycemia, in: Current Therapy 1975. Eds. Howard F. Conn, M.D., Publ. W.B. Saunders, 1975, pp. 400-402. Salans, L.B. and S.W. Cushman. Hormones and the Adipocyte: Factors Influencing the Metabolic Effects of Insulin and Adrenaline. In: Obesity. Eds. W.L. Burland, P.D. Samuel, and J. Yudkin. Churchill Livingston 1975, p. 204.

This is an application for an Academic Research Enhancement Award to investigate growth factor mediated differentiation of granulosa cells. Specifically, the growth factors, transforming growth factor B (TGFb), hepatocyte growth factor (HGF), and leptin will be investigated with respect to their respective signaling pathways. The hypothesis will be investigated that positive and negative modulation of FSH-directed estradiol synthesis by these growth factors is coupled to the Janus kinases (JAK)-signal transducer and activator of transcription (STAT) proteins and to mitogen-activated protein kinases (MAPK).

The NAEPP Guidelines were released in 1991 to foster evidence-based clinical management of asthma to improve patient outcomes. Despite widespread dissemination and regular updates, most recently in 2007, uptake of evidence-based management strategies among healthcare providers and patients with asthma remains suboptimal. National data on the content of health care visits (2003 National Health Interview Survey for Healthy People 2010) show that only 12% reported receiving patient education on asthma; 35% reported receiving written asthma plans from their health care providers; 68% reported receiving education about early signs, symptoms, and/or responses; and 42% reported receiving advice to change their home or work environment to improve asthma. Data from 2008 show little improvement. It remains unclear where major barriers to implementation of Guideline-promoted management strategies occur on the pathway from health care delivery to acceptance and practice by clinicians and patients. Furthermore, overall knowledge and acceptance of the Guidelines by health care practitioners are unknown.

The overall objective of this research proposal is to determine if prior stimulation of the hypothalamo-pituitary-adrenal axis by hypoglycemia or exercise blunts subsequent neuroendocrine, autonomic nervous system (ANS), and metabolic counterregulatory responses to these stresses. We are nearing completion of this study, therefore final data analysis is ongoing.

The main objectives of these studies are (1) to identify and characterize all proteins that are specified by the defective human parvovirus (AAV) and to determine similarities and differences with autonomous parvovirus proteins, (2) to define the mechanism(s) by which the AAV proteins arise, and (3) to define specific functions of the AAV proteins. We have identified at least four AAV non-structural proteins. At least one of these proteins is necessary for viral DNA replication. Post-translational processing does not account for production of any AAV structural proteins although they share large segments of sequences-in-common. It is now clear, however, that these proteins originate from independent in-frame initiations. Mechanism that regulate expression of AAV proteins are of fundamental interest, and we have shown that one AAV structural protein is initiated by a codon (ACG) not known previously to act as an initiation codon in higher eukaryotes. Furthermore, our current findings (i) support a "scanning mechanism" in the translational expression of polycistronic eukaryotic mRNAs, and (ii) demonstrate that alternative mRNA splicing is required for effective translational expression of the largest AAV capsid protein. Among methods used are site-directed mutagenesis, affinity chromatography, gel electrophoresis, in vitro translation of viral RNA, DNA transfection, immunoprecipitation, and Western blotting.

The mechanisms underlying the profound modulation of parasite antigen-specific human T cell responses in lymphatic filariasis have been addressed by demonstrating the multiple pathways involved. In terms of APC dysfunction, human monocytes from patients with patent filarial infections are studded with filarial antigen and express markers associated with alternative activation of macrophages (M&#934;). To explore the role of filaria-derived parasite antigen in differentiation of human monocytes, cells were exposed to microfilariae (mf) of Brugia malayi, and their phenotypic and functional characteristics were compared with those of monocytes exposed to factors known to generate either alternatively (interleukin-4 IL-4) or classically (macrophage colony-stimulating factor MCSF) activated M&#934;. IL-4 upregulated mRNA expression of CCL13, CCL15, CCL17, CCL18, CCL22, CLEC10A, MRC1, CADH1, CD274, and CD273 associated with alternative activation of M&#934; but not arginase 1. IL-4-cultured monocytes had a diminished ability to promote proliferation of both CD4(+) and CD8(+) T cells compared to that of unexposed monocytes. Similar to results with IL-4, exposure of monocytes to live mf induced upregulation of CCL15, CCL17, CCL18, CCL22, CD274, and CD273 and downregulation of Toll-like receptor 3 (TLR3), TLR5, and TLR7. Exposure of monocytes to mf resulted in significant inhibition of the phagocytic ability of these cells to the same degree as that seen with IL-4. Our data suggest that short exposure of human monocytes to IL-4 induces a phenotypic characteristic of alternative activation and that secreted filarial products skew monocytes similarly. The mechanisms underlying the profound modulation of parasite antigen-specific human T cell responses in lymphatic filariasis have been addressed by demonstrating the multiple pathways involved. While several mechanisms have been implicated in mediating this T cell specific downregulation, the role for alterations in the homeostasis of T effector and memory cell populations was explored using multiparameter flow cytometry. Compared to filarial-uninfected endemic normals (EN), microfilaria (mf) positive infected patients (Inf) had a reduced CD4 central memory (T(CM)) compartment. In addition, Inf patients tended to have more effector memory cells (T(EM)) and fewer effector cells (T(EFF)) than did ENs giving significantly smaller T(EFF):T(EM) ratios. These contracted T(CM) and T(EFF) populations were still evident in patients previously mf+ who had cleared their infection (CLInf). These data indicate that filarial-infected patients have contracted T(CM) compartments and a defect in effector cell development, defects that persist even following clearance of infection. The fact that these global changes in memory and effector cell compartments do not yet occur in infected children makes early treatment of LF even more crucial. Beyond the APC dysfunction, T cells from patients with patent infection have induced pathways that in concert prevent Th1-type T cell activation. We have recently shown that the filarial infection at homeostasis is associated with an expansion of IL-10 producing adaptive Tregs as well as nTregs and that these are associated with the suppression of parasite-antigen induced pro-inflammatory Th1 cells. Because downregulatory mechanims are induced in chronic helminth infection, we have attempted to study the spillover effect of the downregulation on responses and diseases that are non-parasitic. To this end, we have both clinical trials underway and in vitro models that have demonstrated the influence of pre-existing chronic helminth infection on susceptibility to mycobacteria, on modulating the response to aeroallergens, and potentially to HIV and malaria. Specifically, we have recently demonstrated that coincident filarial infections profoundly alter the pro-inflammatory and Th1/Th17 responses to malarial antigens (in filarial/malarial coinfections) and to mycobacterial antigens (in filarial/latent tuberculosis coinfections). Lymphatic filarial disease manifested by overt, morbid clinical pathology, characterized by swelling of the scrotal area and limb edema (hydrocele, elephantiasis, lymphedema) has been shown to be related to immunologic and pro-inflammatory factors. Circulating microbial products such as LPS and markers associated with microbial translocation have been shown to play an important role in disease pathogenesis of certain infections like HIV. Similarly, proteins associated with the acute phase response and related cytokines also play an important role in pathogenesis. We have attempted to elucidate the role of the above mentioned factors in disease pathogenesis by comparing the plasma levels of the various markers in four groups of individuals: chronic pathology individuals with or without active filarial infection, asymptomatic, filarial infected individuals and uninfected, endemic normal individuals. We show that circulating levels of LPS, acute phase proteins and certain cytokines are significantly elevated in filarial disease with active infection but not in the other groups indicating that filarial infection induced increased production of these factors correlated with the development of filarial lymphatic pathology. Tissue fibrosis is also a hallmark of lymphatic filarial disease. Matrix metalloproteinases are a family of circulating and tissue proteins that influence the development of tissue fibrosis. They are regulated by another family of proteins called tissue inhibitors of metalloproteinases. The interplay between these proteins governs tissue fibrosis in a variety of conditions. In addition, certain cytokines are known to promote pro-fibrotic events. We have attempted to elucidate the role of the above-mentioned factors in disease pathogenesis by comparing the plasma levels of the various markers in four groups of individuals: chronic pathology individuals with or without active filarial infection; asymptomatic, filaria-infected individuals; and uninfected, endemic normal individuals. We show that altered ratios of the metalloproteinases and their inhibitorsas well as elevated levels of pro-fibrotic cytokinescharacterize filarial infection-induced lymphatic pathology. Filarial-induced CD4+ and CD8+ responses have been characterized fully (using microarray/quantitative RT-PCR) in both the generally more-responsive expatriate patients and the less responsive indigenous (with lifelong exposure) filarial-infected patients. These data provide clues to the pathways induced by infection and those systemic alterations seen in chronic helminth infection.

Deductive reasoning is central to human intelligence and plays a fundamental role in the learning and understanding of fundamental concepts during childhood. Deductions also contribute significantly to mathematical and language learning. Given the central role of deductive reasoning in education, it is critical to advance our understanding of the cognitive and neural underpinnings of its development. Neuroimaging research in adults suggests that the brain regions that are engaged in deduction partially depend upon the type of task. For example, posterior parietal regions that have been linked to visuo-spatial processing have been implicated in linear relational reasoning (e.g., Tom is taller than Bill, Bill is taller than John, therefore Tom is taller than John), whereas left inferior fronal regions associated with verbal processing have been implicated in categorical reasoning (e.g., All Tulips are Flowers, All Flowers are Plants, therefore All Tulips are Plants). However, neuroimaging studies in adults also show that the left rostro-lateral prefrontal cortex is involved in both relational and categorical reasoning, suggesting a more general role for this region in the manipulation and integration of the premises of all types of reasoning problems. Although there have been several neuroimaging studies in adults and behavioral studies in children, nothing is known about the neural basis of the development of deductive reasoning in children. This project will use functional magnetic resonance imaging (fMRI) to investigate the neural development of relational and categorical reasoning in typically developing children (8-9 versus 12-13 year olds). We will examine differences in signal intensity using conventional fMRI analyses, but we will also examine differences in effective connectivity using dynamic causal modeling (DCM). The results of this project will indicate how deductive reasoning emerges and interacts with spatial and verbal systems during childhood and early adolescence. By identifying the neural development of deductive reasoning in typically developing children, our study will also provide the necessary groundwork for future studies that will investigate how this developmental trajectory differs in children with specific language impairment and dyscalculia.

This study is concerned with the ways certain physiological and behavioral signs of arousal or irritability (e.g., heart rate variability, vagal tone, colic, sleeplessness, crying) in the first five months of life are related to measures of the child's temperament, emotional expressiveness, and physiology at later ages. By observing patterns of infant-mother interaction both at home and in laboratory settings, we further expect to determine whether individual differences in maternal behavior interact with early tendencies (as indexed by the signs listed above) in determining patterns of psycho-physiological functioning, emotional expressiveness, attachment behavior, and behavioral inhibition in toddlerhood.

We have continued our study of the insulin-like growth factor, rat IGF-II. During the past year, we have demonstrated that: (1) multiple IGF-II RNAs (1.2 to 5 kb) arise from a single gene through the use of 2 promoters and alternate polyA addition sites; (2) the IGF-II gene is transcribed from both promoters in 11 fetal rat tissues with different efficiencies. Transcription from both promoters is high in the fetus and early neonate and negligible in adult tissues; (3) 1.2 kb IGF-II RNA is translated into Mr 22,000 pre-pro-rIGF-II, whereas 4 and 5 kb IGF-II RNAs are not translationally competent; (4) IGF-II RNA is translated in different tissues, and pre-pro-rIGF-II processed to Mr 7484 biologically active IGF-II; (5) the fetal/neonatal form of the IGF carrier protein is synthesized as a Mr 35,000 precursor in cell-free translation, and cotranslationally processed to the stable, secreted Mr 33,000 form; (6) translatable RNA encoding the Mr 35,000 carrier protein precursor is present in fetal and neonatal rat liver, but not in adult liver, suggesting that developmental regulation occurs at the level of transcription; (7) polyclonal antibodies to purified type II receptor do not stimulate or inhibit IGF actions in L6 rat myoblasts, suggesting that these effects are not mediated by the type II receptor; (8) nearly full-size type II IGF receptors circulate in fetal and neonatal rat serum, and that levels of circulating receptor decrease markedly in older rats; (9) IGF-II potently stimulates neurite outgrowth in sympathetic and sensory neurons cultured from chick embryos; (10) activation of human T lymphocytes results in increased expression of type I and type II IGF receptors, suggesting that the IGFs may participate in the activation cascade; (11) two-chain insulin-IGF hybrid molecules containing the B-domain of IGF-I have increased mitogenic activity and binding to type I IGF receptors but do not bind to IGF carrier proteins.

This proposal describes our plans to build a set of software tools and an online capability for generating high-resolution, study-specific atlases from the ADNI database of images and patient data. The proposed tools will rely on the technology for large deformation diffeomorphic metric mappings (LDDMMs), to generate high-quality, multimodal brain atlases. The goal of this project is not merely a single atlas nor is it simply a set of new algorithms. Rather, we will provide an online service through which users can request custom atlases that they specify through a web interface. A computational infrastructure will deliver these atlases to the user. [unreadable] [unreadable] We will also pursue fundamental and technological improvements in the atlas construction framework as well as the validation of these technologies. In order to provide a clinical context for these developments, we will use these tools to conduct an atlas-based study on metabolic asymmetry in dementia with coinvestigators in the Department of Neurology at the University of Utah. [unreadable] [unreadable] PUBLIC HEALTH RELEVANCE: The goals of this project are multifold. We will be developing open-source software for generating LDDMMs and a web interface open to researchers around the world for automatically generating and delivering high-quality, on-demand atlases from ADNI database queries. The proposed Brain Atlas Portal will produce, as specified by the user: (i) multimodal imagery, with transformations between subjects and the mean, (ii) reliability index for the atlas, (iii) quantification of structural and metabolic variability of the cohort, and (iv) a set of longitudinal multimodal atlases for a cohort. [unreadable] [unreadable] [unreadable]

Tissue factor pathway inhibitor (TFPI) produces factor Xa (FXa)-dependent feedback inhibition of factor VIIa/tissue factor (FVIIa/TF)-induced coagulation. In humans, TFPI is expressed as two isoforms. TFPI1 contains three tandem Kunitz-type protease inhibitor domains, Kunitz-1 binds FVIIa in the FVIIa/TF complex and the second binds FXa. Kunitz-3 does not possess protease inhibitor activity. TFPI2 lacks the Kunitz-3 and the C-terminus of TFPI1. The latter is required for TFPI1's optimal inhibition of FXa and its anticoagulant activity in one-stage coagulation assays. TFPI1 interacts with protein S (PS), which enhances its anti-FXa activity, and with factor V (FV), which may prolong its clearance from plasma. Both forms of TFPI associate with the surface of cells in a glycosylphosphatidylinositol (GPI)-anchor dependent fashion, but through different mechanisms. TFPI1, present in plasma and secreted by activated platelets, appears to bind to another GPI-anchored protein(s), whereas TFPI2 contains an intrinsic GPI-anchor. We plan to determine the structures within TFPI1 responsible for its binding to PS and FV and further explore its interactions with cell surfaces. The form(s) of TFPI, which circulate bound to lipoproteins, will be identified and the relevance of a potential additional isoform of TFPI, TFPI4, will be investigated. TFPI gene-disrupted mice (TFPI KO) die intrautero of widespread thrombosis and a consumptive coagulopathy, but are rescued by a low level of either of two transgenes with FVIIa/TF inhibitory activity. One, Tie2-hTFPI1, directs expression in endothelial cells, the other, Tf-mXK1-hAlb directs expression into plasma. Transgene-rescued TFPI KO mice and appropriate bone marrow transplantation studies will be used to assess the role of platelet- associated TFPI1 and to determine the effect of low levels of endogenous TFPI on thrombosis, inflammation, and atherosclerosis using mouse models. Public Health Relevance: TFPI is a plasma and cell-associated protein, which exists as two isoforms (TFPI1 and TFPI2) and plays an important role in regulating the initiation of coagulation. The goals of this proposal are to determine the mechanism and relevance of its interactions with circulation lipoproteins, cell surfaces, and two other coagulation-related proteins, Protein S and Factor V, and to determine the effects of TFPI deficiency on thrombosis, inflammation, and atherosclerosis using mouse models.

The inheritance of variability in the behavioral and physiologic effects of psychopharmacologic agents in clinical use will be studied. The initial project will be on pharmacogenetics of d-amphetamine. Heritability and intra-individual stability of pharmacokinetic data and of the following parameters of response will be determined: 1) behavioral ratings 2) adrenergic receptor response 3) neuroendocrine response and 4) cortical evoked response.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The purpose of this research study is 1) to evaluate the safety and dose tolerance of the chemotherapy drug, vinorelbine in patients who have cancer for which there is no standard therapy or for which standard therapy would need to be modified due to impaired liver function. Vinorelbine is a chemotherapeutic agent that has been used against a variety of cancers, including cancers of the breast, lung, esophagus, and lymphnode. After it is administered, the body eliminates it. To a large extent this is done by the liver. In patients with decreased function of the liver, vinorelbine is eliminated more slowly, which could lead to an increased incidence of side effects. As a result, decreased doses of vinorelbine are recommended in patients that have evidence of decreased liver function. The amount of decrease necessary, however, can only be estimated. It would be desirable to be able to determine more accurately the best dose of vinorelbine to allow safer, and potentially more effective administration of the drug. Indocyanine green (a dye) and lidocaine (an anesthetic) are drugs that have been used to estimate how well the liver is working. This study utilizes indocyanine green and lidocaine to predict the rate of removal of vinorelbine and to guide the dosing.

Elderly people (age 65 years and older) are the most rapidly growing segment of the US population and the incidence of epilepsy is greatest in this age group. Little information is available about the use or outcomes associated with antiepileptic drugs (AEDs) by these vulnerable persons. We will examine pattems of AED use and associated outcomes among nursing home (NH) and community based elderly. For the proposed research, we have available two large electronic data sets: (1) the Beverly Enterprises Inc. centralized records (the Minimum Data Set and physicians' orders) for two calendar years (1999, 2000) for all residents (N~140,000 for two years) living in 501 NHs in 31 states, and (2) the Health Care Financing Administration's (HCFA) integration of the Medicare Current Beneficiaries Survey with Medicare claims files (N~12,000 elderly per year) for 7 calendar years. Both of these data sets have been used in part during the current project (1997-2002) by the same multidisciplinary team that will be working in the proposed project. We have a thorough working knowledge of the complexities of these data sets and the richness of information they contain. In our current research, we found a prevalence of 7.7% for AED use by NH admissions. Significant covadates with AED use at admission were epilepsy/seizure, bipolar depression, age group, and cognitive performance. During the first 3 months following admission, an additional 2.7% people were initiated on AEDs. Epilepsy/seizure indication was present in 5.8% of all admissions and an additional 1.47% of the follow-up cohort during the first 3 months. Specific aims of the proposed project are: (1) outcomes associated with any AED use by eldedy, (2) pharmacoepidemiology, characteristics, and outcomes associated with CBZ, PHT and VPA, and (3) the pharmacoepidemiology of other specific AEDs. Patient outcomes will be captured through incidence events, while AED use can be tracked continuously over time. Multivariate-modeling techniques will be used, controlling for demographics, co-morbidities, and regional effects. Other P50 teams are studying the elimination kinetics of AEDs, and we will use this knowledge to develop age, gender and co-medication specific estimated total and unbound concentrations for NH residents.

Many patients take their medications with meals to minimize side effects and/or improve adherence to the administration schedule. This conventional strategy may be deleterious if an interaction occurs between the drug(s) and dietary substance(s). Currently, such interactions are largely unrecognized. Cranberry juice (CBJ), widely promoted for its health benefits, including prevention of urinary tract infections, is a common component of many popular juice-containing beverages. CBJ recently was reported to significantly increase the systemic exposure of the CYP3A substrate nifedipine in vivo in rats. CBJ inhibited the enteric (but not hepatic) CYP3A-mediated first-pass metabolism of nifedipine, an effect that mirrored that of grapefruit juice (GFJ). Preliminary data detailed in this application indicate that CBJ contains potent enteric CYP3A4 inhibitors. Clinically significant drug-GFJ interactions result from multiple furanocoumarins that inactivate enteric CYP3A4 permanently to elicit a prolonged inhibitory effect (~3 days). Cranberry is not a citrus fruit and is not known to contain furanocoumarins. Thus, CBJ likely contains unique CYP3A4 inhibitors with unknown mechanisms of action. Multiple independent laboratories have invested considerable time and resources in identifying the active ingredients responsible for drug-GFJ interactions. This slow, costly process was due in part to the lack of a multidisciplinary approach, combined with the lack of application of relevant in vitro systems. We hypothesize that a multidisciplinary translational research approach, which capitalizes on the combined expertise of drug metabolism scientists and natural products chemists, can rapidly identify the active CYP3A4 inhibitors in CBJ using well-characterized human intestine-derived in vitro systems. Identification of the major CYP3A4 inhibitors in CBJ would allow standardization of CBJs for patients at risk for drug-CBJ interactions, as well as provide marker compounds to identify other foods with interaction potential. Specifically, following (1) an evaluation of the effects of pretreatment of CBJ on the extent of enteric CYP3A4 inhibition in human volunteers, we will use human intestinal microsomes and CYP3A4-expressing Caco-2 cells to (2) identify potent CYP3A4 inhibitors in cranberry fractions and (3) define the concentration- and time-dependent inhibitory properties of the identified CYP3A4 inhibitors. Importantly, this novel experimental paradigm could be applied widely to identify additional natural compounds that alter drug disposition in humans in vivo. The long-term goal of the proposed translational research program is to further the mechanistic understanding of the effects of dietary substances on drug disposition. The knowledge gained will provide critical information to both clinicians and the lay public as to whether specific dietary substances can be taken safely with certain medications. [unreadable] [unreadable] [unreadable] [unreadable]

HBV X protein has been shown to have a transactivating effect on the expression of a variety of viral and cellular genes. The expression of tumor suppressor gene RB was studied in a transient expression system by transfecting HBV X gene into Hep G2 cells. The HBV X open reading frame was generated by PCR from plasmid containing wild type HBV DNA and was inserted into expression vector. The plasmid pHBV-X was used to transfect Hep G2, a human hepatoblastoma cell line. Protein was extracted 48 hours after DNA transfection. The level of endogenous RB protein was analyzed by western immunoblotting using RB monoclonal antibody. RB protein untransfected Hep G2 cells and Hep G2 transfected with vector alone were also analyzed as controls. The level of RB protein in pHBV-X transfected Hep G2 cells increased nearly 10-fold compared to the control cells. Preliminary in vitro co-immunoprecipitation studies between RB protein and HBV X protein do not indicate direct interaction between RB and HBV X proteins. The finding that HBV derived X gene can alter the expression of RB tumor suppressor gene suggest a possible role for the X gene in HBV mediated hepatocarcinogenesis. Work continues in an effort to determine the mechanism of transactivation by X protein. Host mediated transcriptional factor(s) involved in X-gene activities will be analyzed by X-protein transactivation effect in different hepatic and non hepatic cell lines.