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Dry eye is a common problem with a severe impact on the quality of life and potential vision-threatening complications. It often results from lacrimal insufficiency caused by immune-related processes, as in Sjogren's syndrome, or by hormone changes associated with aging and various physiological states. One critical hormonal influence on the lacrimal glands appears to be prolactin. Studies with human subjects, hypophysectomized rats, transgenic mice, and acinar cells in primary culture indicate that prolactin can impair lacrimal function, even at serum concentrations within the range of normal values. Moreover, the source of the prolactin that impairs lacrimal gland function may be the lacrimal glands themselves. The lacrimal glands express prolactin mRNA and protein, which may act as an autocrine or intracrine factor that in some circumstances may interfere with secretion. This project will use lacrimal acinar cells in primary culture to answer the following questions: 1. Does locally expressed prolactin act as an autocrine/intracrine factor that supports secretory functions at normal concentrations and impairs them at excessive concentrations? Neutralizing antibodies and antisense reagents will be used to minimize actions of locally expressed prolactin. Expression constructs will be used to overexpress prolactin. Acinar cell morphology, carbachol-induced protein secretion, expression of polymeric immunoglobulin receptors, and expression of ion transport proteins will be evaluated for changes related to altered prolactin expression. 2. Do altered forms of prolactin (16 kDa and phosphorylated 24 kDa) that have inhibitory effects in other cells inhibit lacrimal secretory function? The effects of overexpressed and added forms will be tested as in Specific Aim 1. 3. Does lacrimal prolactin act as a paracrine factor contributing to autoimmune activation? Antisense reagents and expression constructs will be used to suppress or enhance acinar cell prolactin expression, and the modified cells will be tested for their ability to promote proliferation of autologous lymphocytes in mixed cell reactions. This work will advance our understanding of significant mechanisms in lacrimal physiology, and it will have immediate implications for the direction of other studies now in progress. It also has the possibility of stimulating much work well beyond its present scope, including epidemiological studies aimed at identifying sub-populations of lacrimal deficiency patients with different etiologies, followed by design of appropriate, highly specific therapies.

This project will test the ability of anti-inflammatory agents to block decrease in blood flow directly measured in the skin, deep tissue and bone marrow of subjects with sickle cell anemia. We hypothesize that therapeutic interventions that interrupt adhesive interactions between sickle erythrocytes, leukocytes, platelets and vascular endothelium will lessen the vaso occlusion, hemoglobin desaturations, and release of mediators of vascular damage induced by intermittent hypoxia. While sickle cell anemia (SCA) is thought of as acute episodes of marked red blood cell (RBC) sickling, sickle-related vaso occlusion occurs continually even dudng non-crisis periods. The cumulative effect of these processes is end-organ damage. Recent data from animal models underscore the importance of leukocyte adhesion to the vascular endothelium in the genesis of sickle vasoocclusion. It is therefore likely that therapies that interfere with the formation of the adhesive interactions between leukocytes, sickle red cells and vascular endothelium will decrease end-organ damage. RBC sickling is triggered by hypoxia. Sleep studies in SCD children demonstrate that 10 to 40 episodes of desaturation occur each night with oxygen saturation dropping to 75 to 85 percent. We directly measured decreases in blood flow in response to nitrogen-induced hypoxia in subjects with sickle cell disease. These decreases are six-times greater than normal controls (p<.001). We will develop this model of vaso occlusion in humans and extend the studies to include measurement of hypoxia-induced changes in inflammatory mediators, markers of vascular damage, and measures of deep tissue blood flow. These parameters will be measured in response to nitrogen-induced hypoxia as well as hypoxic episodes naturally occurring during sleep. We will then use anti-inflammatory agents to block the changes in blood flow and increase in markers of vascular damage after detailed characterization of the N2 challenge and sleep hypoxia models. If these agents are successful, we will have direct evidence in humans that inflammation plays a role in vaso occlusion. Furthermore, this model may serve to test candidate treatments for sickle cell disease. While not within the scope of the present proposal, we anticipate that therapeutic interventions that abrogate hypoxia-induced blood flow decreases and changes in markers of vascular damage detected in these human models will decrease frequency of crisis and lessen the degree of end-organ damage if tested in larger clinical trials.

In this project, mechanisms of normal cellular immunity and tumor immunology are studied: immunological surveillance of cancer patients and the effects of chemotherapy and immunotherapy on the immune response are explored. We are characterizing acute lymphocytic leukemia according to the immunological subtypes of lymphoblasts and the capacity of lymphoblasts to express immunoregulatory helper and/or suppressor functions in vitro. The role of B cell alloantigens in lymphoblastic leukemia is also under investigation. Particular emphasis is placed on immunologic study of the monocyte-macrophage system (including development of new clinical assays of human monocyte function) in malignancies and immunodeficiency diseases, in the newborn, and in malignant histiocytosis. We are also investigating serum suppressors of cellular immunity in cancer patients, and the role of immune adjuvants and their ability to stimulate granulocyte recovery following marrow-suppressive chemotherapy.

The family of receptors involved with cellular adhesion has been termed integrins because of the predicted function of these proteins in integrating the extracellular matrix with the cytoskeletal framework. The prototypic integrin, the fibronectin receptor, mediates the interaction of cells with fibronectin. The purpose of this study is to elucidate the cytoplasmic interactions of the fibronectin receptor and ultimately to characterize the molecular basis for the fibronectin receptor-cytoskeleton association. The work proposed herein will concentrate on identifying and characterizing intracellular proteins that interact with cytoplasmic domains of each subunit of the fibronectin receptor. Each of the two cytoplasmic domains of the receptor possesses the potential to interact with intracellular proteins. Synthetic polypeptides representing the individual cytoplasmic domains will be coupled to Sepharose and used as affinity matrices for isolation of binding proteins from detergent extracts of cells or tissue. Preliminary chromatography experiments using an affinity matrix of a synthetic peptide representing the cytoplasmic domain of the fibronectin receptor beta subunit coupled to Sepharose has been used to identify a novel protein. This protein, which we have named fibulin, may mediate cytoplasmic connections of the fibronectin receptor as well as the other adhesion receptors that share the beta subunit. Such connections potentially include an interaction with the cytoskeleton. In addition, this molecule may be involved with receptor signal transduction and modulation of receptor function. A significant portion of the work proposed in this study will concentrate on characterization of the structure and biological function of fibulin. A complete amino acid sequence of fibulin will be determined from a combination of protein and cDNA sequencing. Particular emphasis will be placed on characterizing the interaction of fibulin with the fibronectin receptor, with itself, and with other cytoplasmic proteins. Using cDNA and antibody probes the expression of fibulin will be examined in both normal and tumor cells. Additional affinity chromatography experiments are proposed to identify and protein(s) that might interact with the fibronectin receptor alpha subunit cytoplasmic domain.

The proposed research is directed at understanding the molecular mechanisms by which the papovaviruses, polyoma and SV40, cause major changes in host gene expression. Research will focus primarily on understanding the viral mediated regulation of one particular viral induced enzyme, dihydrofolate reductase (DHFR). For these studies we have isolated methotrexate resistant cells in which DHFR is a major gene product, accounting for over ten percent of the soluble protein. This 200-fold increase in the level of DHFR in these cells is accounted for by an increase in the level of DHFR mRNA and the number of DHFR genes. Proposed experiments are designed to understand 1) polyoma mediated induction of DHFR synthesis, 2) the role of T-antigen in the regulation of DHFR gene expression during abortive infection by SV40, and 3) the relationship between viral mediated control of DHFR synthesis and the control by cell cycle, serum growth factors, and cyclic nucleotides. An important feature of the proposed research is the use of methotrexate resistant cell lines in which one of the viral induced enzymes, DHFR, is a major cellular protein. This feature will allow the use of currently available techniques to study changes in the regulation of DHFR gene expression that occur as a result of infection by polyoma or SV40. As a detailed investigation of the effect of papovaviruse infection on the regulation of host gene expression these studies should lead to a better understanding of how these and other DNA tumor viruses transform cultured cells to a malignant phenotype and cause tumors in susceptible animals.

The objective of this proposal is to study the local immune response in chronic periodontal disease. The research plan contains three stages: 1) Explant cultures of gingival tissue were to be used to examine de novo biosynthesis of immunoglobulins, complement components and other proteins. The immunoprecipitation of biosynthetically 14C labeled proteins. 2) Determine specificities of gingival antibodies RIA, ELISA, modified Jerne plaque or passive hemaglutination. 3) Isolate T and B cells for gingiva and characterize their cell surface receptors and blastogenic responsiveness.

DESCRIPTION (adapted from abstract): Cystic fibrosis (CF) is the most prevalent autosomal recessive lethal genetic disease in the U.S. with 5% of the population carrying a mutant CFTR gene. The long term goals of this proposal are to understand how mutant CFTRs fail to function correctly in CF patients and how these failures might be repaired. The deltaF508 mutant of CFTR is retained in the endoplasmic reticulum (ER) and thereby fails to traffic properly to the apical membrane of epithelial cells. While the mechanism of this trafficking failure may involve recognition of improperly folded CFTR by protein chaperones, preliminary data indicate that phospholipid interactions with the first nucleotide binding fold domain (NBF-1) might also play a role in CFTR trafficking. Dr. Pollard and his colleagues have therefore hypothesized that aberrant interactions between the mutant NBF-1 domain and specific phospholipids might contribute to the ER retention and degradation, and thus cause the disease. To test this hypothesis, the investigators propose: 1) to determine the specificity of phospholipid interaction with NBF-1 and the alteration of this specificity by the deltaF508 mutation by measuring lipid interaction with wild type or mutant NBF-1 using biophysical assays; and 2) to show that NBF-1 induces permeability changes in membranes, and that deltaF508 mutation changes the lipid specificity. This property will be used to develop a screening assay for identifying drugs that might affect mutant CFTR trafficking in CF patients; 3) to show that changes in the phospholipid specificity of NBF-1 have direct consequences for CFTR trafficking in vivo. This aim will be approached by analyzing wild type and deltaF508-CFTR trafficking in a cell line which is temperature sensitive for lipid biosynthesis. The significance of this proposal is that it suggests a novel approach to the mechanism of CF, involving a trafficking defect affected by phospholipid interactions with the mutant protein. Identifying this defect can provide targets for repair. If successful, this approach will set the stage for development of therapeutic means for correcting the aberrant interactions between mutant CFTR and specific phospholipids.

Project Summary/Abstract This renewal proposal supports the maintenance of Cornell?s long standing and successful Molecular Biophysics Training Grant Program. This program provides vigorous interdisciplinary training merging the fields of physical and biological sciences. The program, now in its twenty-seventh year, continues to advance the training of our most motivated and well-qualified students by focusing their graduate academic work around a core curriculum and creating an engaging, cohesive community. The trainees may have undergraduate degrees in either the physical or biological sciences and must have been admitted to the Graduate School at Cornell for training leading to the Ph.D. in a Graduate Field of one the thirty-one participating faculty members who span nine departments, seventeen fields, and four Colleges. All participating faculty are associated with Cornell?s well established and continuously growing Biophysics Program, and have well-funded quality research programs either in physics with strong biological applications or in biology with strong physical connections. The overall research interests of the faculty are broadly distributed and include: structure and function of proteins and other macromolecules using theoretical approaches, synchrotron X-ray crystallography, electron spin resonance spectroscopy, and multidimensional NMR; single molecule studies of dynamics of molecular motors; structures and molecular mechanisms of cell membranes, receptors, and neurotransmitters and associated cellular functions; materials and technology developments including nonlinear laser microscopy, steady-state and time resolved spectroscopy and imaging, single channel recording, nanophotonic optical tweezers, and nanofabrication. Through collaborations and University center facilities, Cornell offers bountiful opportunities for innovation and creative research. The program supports 11 trainees each year, with each individual supported for a maximum of three years. Trainees undertake interdisciplinary studies with advanced courses in mathematics, quantum mechanics, statistical thermodynamics, biochemistry, molecular and cell biology, computation and instrumentation, and other special topics. In addition, students participate in multiple program functions including a weekly Biophysics Colloquium, Summer Student Seminar Series, a yearly campus-wide Biophysics Symposium, and a yearly campus-wide Responsible Conduct of Research Symposium. These activities serve to provide a strong, unified program identity within the broad interdisciplinary structure of the program. Thesis research and collaborations in the laboratories of the participating faculty complete preparation for a career of teaching and research in molecular biophysics. Through these experiences, trainees gain exposure to, and experience in, interdisciplinary biomedical research.

We recently discovered a tumorigenic factor interactome connected through the tumor suppressor microRNA-198 in human pancreatic cancer (PC) patient samples and confirmed the tumor suppressive roles of miR-198 in PC animal models. We found that miR-198 is downregulated in PC and is involved in an intricate reciprocal regulatory loop with mesothelin (MSLN), which represses miR-198 through NF-kB-mediated homeobox transcription factor POU2F2 (OCT-2) induction. Furthermore, miR-198 repression leads to overexpression of pre-B-cell leukemia homeobox factor 1 (PBX-1) and valosin-containing protein (VCP). The dysregulated PBX-1/VCP axis leads to an increase in tumorigenicity. Reconstitution of miR-198 in PC cells results in reduced tumor growth, decreased metastasis, and increased survival through direct targeting of MSLN, PBX-1, and VCP. Our preliminary data strongly suggest the significant role of miR-198 and this interactome in PC pathogenesis. In addition, we found that miR-198 can sensitize PC cells for gemcitabine killing because miR-198 effectively downregulates VCP expression and inhibits autophagy maturation in PC cells. In this proposal, we hypothesize that miR-198 and this interactome could serve as a potential prognostic marker and the miR-198 replacement therapy could attack this tumorigenic network through a central vantage point and improve therapeutic efficacy in pre-clinical animal models. Three specific aims are proposed: 1). Demonstrate the significant role of miR-198 and the tumorigenic factor interactome in human PC clinical prognosis in a large cohort of PC patient samples; 2). Design and characterize MSLN-specific targeted miR-198 nanoparticles for specifically delivering miR-198 to PC cells; and 3). Demonstrate the therapeutic efficacy of MSLN-targeted miR-198 replacement in PC patient-derived xenograft (PDX) mouse models. The project will substantially contribute to PC research and will have an enormous impact on clinical practice for patients with PC.

This is a competitive renewal of an Independent Scientist Award (K02 MH01153) for Paul Worley M.D. Dr. Worley is currently an Associate Professor in Departments of Neuroscience and Neurology at The Johns Hopkins School of Medicine and he devotes full time effort to basic research. The focus of research is the molecular basis of protein synthesis-dependent neuronal plasticity. During the tenure of the previous award period, the laboratory has cloned and characterized several novel brain immediate early genes (IEGs) including Cox-2, Homer, Rheb, Narp and Arc. Analysis of the function of these genes has lead to important new insights into how neurons response to activity and this information is broadly relevant to cortex development, drug addiction and memory. The K02 Award has substantially contributed to Dr. Worley's career by assuring stable salary support and allowing him to commit full effort to research and career development. The research plan has been successful based on record of publications and current support by grants (RO-1) from NIHM, NIDA, NINDS and NIA. The Institution is supportive of continued full time research commitment by the P.I. as evidenced by expansion and renovation of laboratory and office space. Plans for future career development will extend the focus of analysis of IEGs to include studies of cellular physiology and system level function. The Research Plan describes our proposed studies of Homer that will examine its role in regulating cellular calcium dynamics and activity-dependent behaviors, including cocaine sensitization. Planned activities make full use of the outstanding opportunities for collaboration and career development at Johns Hopkins School of Medicine.

The marginal cell of the stria vascularis is associated with the production of the endocochlear potential and potassium-rich endolymph. These facilitate the mechanoelectrical transduction in the sensory hair cells. With the whole-cell recording method we previously had found that an inward current of the marginal cell is blocked by micromolar concentrations of amiloride; the dose is consistent with amiloride- sensitive sodium channels. Immuno-cytochemical study showed that immunoreactivity to antibodies raised against sodium channels from the bovine kidney, is distributed in marginal cells as well as vestibular dark cells. These observations are consistent with the importance of amiloride-sensitive channels in absorbing sodium ions from the endolymph to maintain its unique ionic composition. Preliminary studies of ion channel regulations in these cells were carried out by examining immunocytochemical localization of various GTP-binding proteins.

Although the Warburg effect has long been known as the hallmark characteristic of cancer cells, its exploitation for cancer therapy has never been successful because of failure in identifying an appropriate target protein. Recent studies suggested PFKFB3, which causes a massive increase in F-2,6-P2, the most potent activator of mammalian glycolysis, as the most causative protein of the Warburg effect. We hypothesized that a drug-like PFKFB3 inhibitor will prevent onset of the cancer-specific glycolysis by preventing the F-2,6-P2 surge and, eventually, induce apoptic death of cancer cells. To test this hypothesis, we propose to develop a drug-like PFKFB3 inhibitor. To obtain the molecular basis of the target, which is necessary for design of the desired inhibitor, we determined the crystal structure of human PFKFB3 and suggested a molecular model of its catalytic mechanism. We also performed an initial drug discovery study and obtained several promising PFKFB3 inhibitor molecules. Taking advantage of the progress we have made, we now initiate a full-scale study to develop a drug-like PFKFB3 inhibitor and test if it can cause inhibition of cancer glycolysis and, ultimately, apoptic death of cancer cells. To obtain the molecular templates for PFKFB3 inhibitors, virtual screening and subsequent functional screening of database compounds will be carried out. Hit compounds will be selected on the bases of binding affinity, selectivity, amenability to chemical modification, and structural novelty. The selected hits will be optimized to be drug-like via chemical modification. During this optimization step, new molecules will be synthesized from the hit templates and tested regarding their potency, cell- permeability, stability, and target-selectivity on cultured cancer cell lines. The compounds selected from this test would be considered as the drug-like PFKFB3 inhibitors. Finally, the biochemical and pharmacological properties of these final lead molecules will be investigated to test our hypothesis. This project is aimed to develop a novel drug-like compound with potential of anti-cancer drug. This compound will be designed to inhibit cancer glycolysis, which has never successfully exploited for cancer therapy. Thus, successful completion of this project will have an immediate impact on development of new class cancer drug and new strategies in cancer therapy. PUBLIC HEALTH RELEVANCE: This project is aimed to develop a novel drug-like compound with potential of anti-cancer drug. This compound will be designed to inhibit cancer glycolysis, which has never successfully exploited for cancer therapy. Thus, successful completion of this project will have an immediate impact on development of new class cancer drug and new strategies in cancer therapy. [unreadable] [unreadable] [unreadable]

We aimed to identify the reproductive targets of pollutants by studying high exposures. We entered into a collaborative agreement with the Ukrainian Institute of Pediatrics, Obstetrics and Gynecology and the University of Bristol in 1993 to conduct the research described here. The Ukraine Placental Study had the following goals: 1. To document the levels of common pollutants experienced by pregnant women in two urban areas, using environmental measurements and biological markers 2. To describe the reproductive outcomes of these women 3. To determine what relationship, if any, exists between the estimates of exposure and the outcome of the pregnancies. PAHs. Concentrations of PAHs were measured in placentas from 200 women from two cities in Ukraine, Kyiv and Dniprodzerzhinsk. The participants had no special exposures and were chosen from among subjects in an ongoing study of reproductive health. All seven of the polycyclic aromatic hydrocarbons were found in all placentas, with the sole exception of benzo(a)pyrene in one placenta. Chrysene was present at the highest concentrations, with median 1.38 ng/g dry weight. Dibenz (a,h)anthracene and benzo(g,h,i)perylene had the lowest concentrations; each had median 0.73 ng/g dry weight. Concentrations in Kyiv were slightly higher than those in Dniprodzerzhinsk, but the difference was significant only for dibenz(a,h)anthracene. Dibenz(a,h)anthracene and benzo(g,h,i)perylene increased significantly with maternal body mass index, but other PAHs showed no such pattern. Hospital admissions. We investigated the reasons for hospital admissions in a sample of deliveries from Ukraine, a country of the former eastern bloc. We enumerated all hospitalizations among 3,099 women who delivered live singletons of at least 20 weeks gestation in two urban areas of Ukraine and abstracted data from their medical records. . More than a third of the women were admitted to hospital during their pregnancy, and 91 % of the admissions were for a pregnancy complication, primarily threatened abortion or early labor. Median length of stay for all admissions was 12 days. The ratio of admissions to deliveries was 52 per 100.The Ukrainian ratio of hospitalizations to deliveries is notably higher than any that have been published in studies from the United States and Australia, reflecting patterns of care that stress hospital?based treatment. This high ratio does not necessarily mean that Ukrainian women are sicker, although that may be the case. Preterm birth. The economic, social and health problems faced by former eastern bloc countries after the demise of the Soviet Union are unique in the recent history of Europe. We conducted a study in two urban areas of Ukraine, asking if the traditional predictors of preterm delivery continue to be associated with risk under these conditions. Subjects were pregnant women with LMP between 12/25/92 and 7/23/94. Self-completed questionnaires and the medical record provided data. We compared 137 spontaneous preterin deliveries with 2886 full term births, using all established risk factors for which we had data. Maternal age was the variable most strongly related to preterm birth. Being 18 or less had an OR of 3.7; being 30+ had an OR of 2.5 relative to the reference group of age 25-29. Placental complications and pre-existing hypertension had ORs of 2.7 and 2.3 respectively but the confidence interval included 1.0. Low net pregnancy weight gain (less than 10 kg) was significantly associated with preterm birth, but the rate of net weight gain was not. Marital status and educational category were only weakly related. Comparison with England. We compared the rates of spontaneous preterm birth in Avon County, England and in two urban areas of Ukraine, using the same study methodology in both sites and accounting for differing in maternal risk factors. We used data from the European Longitudinal Study of Pregnancy and Childhood; eligibility required residence in geographically defined areas and last menstrual period in a specific time window. Data were collected by questionnaire and medical record abstraction between 1992 and 1995, using comparable instruments and methods in both countries. Preterm births were classified into those that were spontaneous, and those that involved medical intervention. Rates of spontaneous preterm birth were compared in the two sites, taking account of maternal characteristics. Placenta and birth weight. We investigated whether the difficult conditions in the former eastern bloc in the last decade might have altered the weight of the placenta or its relationship to other measures of fetal size from what has historically been reported. Placentas were obtained from 1621 singleton births of at least 28 weeks gestation in a Ukrainian city during 1993-94, using a systematic protocol. Information on maternal characteristics was obtained from two questionnaires completed by the mothers. Data on pregnancy complications were abstracted from the medical record, as were absolute measures of birth size (infant weight, infant length, crown-rump length, and head circumference). We examined the relationship of placental weight and ratio to these variables. The range of placental weight was 100 to 1000 grams, with a mean of 470 g. Mean placental ratio was 13.9%. Placental weights increased and placental ratios decreased with gestational age. Larger ratios were related to larger maternal BMI. Absolute measures of infant size and placental weight were mutually positively correlated. Placental ratio, infant length, and ponderal index were nearly uncorrelated. Ukrainian placentas were of similar absolute and relative weight as those historically reported, as was their relationship to other indicators of infant size. Alcohol and breastfeeding. We aimed to replicate a previous study of one-year olds that reported deficit in motor development associated with moderate alcohol use during lactation, using different but comparable population. The mental development of 915 18-month-old toddlers from a random sample of a longitudinal population-based study in England was measured using the Griffiths Developmental Scales. Frequent self-administered questionnaires during and after pregnancy provided maternal data. The dose of alcohol available to the lactating infant was obtained by multiplying the alcohol intake of the mother by the proportion of breast milk in the infant's diet. We compared this dose with the Griffiths scales, taking into account potential confounding variables in the sample. ACE inhibitors. We explored the association between enalapril exposure and adverse outcomes in pregnancy, taking into account other possible risk factors. We analyzed a series of all usable cases reported to the FDA between 1986 and 2000 in which enalapril was a suspect drug for the observed adverse outcomes (N=110). Parameters of exposure and reported outcomes as well as information on potentially confounding variables were systematically abstracted from this series by a single physician. Because exposure to ACE inhibitors after the first trimester of pregnancy had been associated with adverse outcomes in the existing literature, we divided the cases into those exposed in the first trimester only (considered as the baseline group) and cases exposed beyond or after this time. Frequency of reported adverse outcomes in the second group was compared with those in the baseline group; odds ratios were computed, taking account of potentially confounding variables by logistic regression where appropriate. Dr. Little has retired; Beth Gladen (DIR/BB) will assume role of PI on this Project.

Work will be carried out on two aspects of the biology of Trichinella spiralis: 1) Immunobiology of T. spiralis infection in rats and mice. The antigens responsible for induction of protective immunity are contained in secretory granules, which are localized to the stichocyte cells. These antigens will be fractionated and characterized by a variety of chemical and physical techniques. The detection system to be used for these antigens will include Ouchterlony analysis and in vitro bioassay for adult worm fecundity. The stage of the parasite most susceptible to the effects of protective immunity will also be investigated. 2) The Nurse-cell parasite relationship. The nuclear and cytoplasmic events leading to the formation of the Nurse cell (i.e. modulated infected muscle cell) will be investigated utilizing biochemical and cytological techniques.

The purpose of this project is to develop a number of organoironcarbonyl reagents to be used in the synthesis of a number of natural products which are important in the health sciences. The type of organoironcarbonyl complex on which our work has focused thus far are tetracarbonyliron complexes of alkenes. In one phase of this work we have already prepared a number of alkenes with geminal carbonyl substituent, including methylenemalonates and alpha-acylacrylates, which are capable of forming stable tetracarbonyliron complexes. These alkenes and the corresponding complexes are being developed as reagents for the construction of polycyclic fused ring natural products. A specific objective of this part of our work is the total synthesis of estrone, a centrally important steroidal hormone. In another phase of this work, a new approach to macrolide synthesis is being developed through the use of tetracarbonyliron complexes of acrylate esters which incorporate a potential internal nucleophile. Formation of the macrolide ring involves generation of the internal nucleophile with base followed by intramolecular nucleophilic addition of nucleophile to the acrylate ester. The scope of this reaction type is currently being established after which the method will be applied to the synthesis of specific naturally occurring macrolides.

The development of new female-initiated HIV prevention methods, including oral and topical microbicides, is urgently needed to stem the growing epidemic among adolescent women. Despite the disproportionate impact of HIV/AIDS on adolescent women, they have been markedly under-represented in HIV prevention trials. For example, only one phase III microbicide trial has attempted to recruit adolescents under the age of 18, even though sexual debut and high risk sexual behavior may occur years earlier. There are important scientific, ethical and programmatic reasons to consider recruitment of adolescent women. Nevertheless, challenges to recruiting and retaining adolescent women in HIV prevention trial research will be numerous. This proposed study will conduct formative community-level research and a pilot prospective clinic-based study in India and Tanzania to systematically examine the challenges and opportunities related to recruitment and retention of adolescent women aged 15-21 in clinical microbicide trials and make specific recommendations about whether and how such trials should be adapted to accommodate adolescent participation. The specific aims of this study are: Aim 1: To identify the HIV risk characteristics of adolescent women and determine whether these risks differ by younger (15-17) versus older age groups (18-21) and by country context. Risk characteristics of interest include: types and patterns of sexual partnerships;types of sexual risk-taking and risk-reduction behaviors;perceptions of risk for HIV, sexually transmitted infections (STIs), and other negative health outcomes;and psychosocial and environmental factors contributing to risks; Aim 2: To evaluate the legal, socio-cultural and service delivery factors that hinder adolescent women's participation in microbicide trials and make specific recommendations to enhance their participation; Aim 3: To examine and compare adolescent women's (aged 15-17 and 18-21) understanding of and ability to adhere to the requirements of trial participation in the context of a prospective clinic-based study;and Aim 4: To determine adolescent women's acceptability and use of a proxy gel, including circumstances in which participants apply the gel;reasons for non-use of gel;negotiations about gel use with partners;and the influences of gel use on sexual satisfaction for themselves and their partners.

Obesity and cognitive dementia may seem to have little in common other than both can have severe health consequences and both have so far been highly resistant to therapeutic interventions. Traditionally, investigators have viewed the problems of obesity and metabolic syndrome, on one hand and cognitive dementia on the other, as involving distinct etiologies, that target different underlying behavioral and biological functions, and that rely on largely separate brain structures and circuits. However, recent epidemiological findings point to a link between obesity in mid-life and the emergence of Alzheimer's disease and other serious cognitive impairments in the aged. These findings have encouraged the hypothesis that obesity may be causally-linked to cognitive impairment. Using a rodent model, this present project will explore (the perhaps counter-intuitive) idea that excessive energy intake leading to obesity and related metabolic disorders (e.g., diabetes, hypertension) is a consequence of interference with learning and memory processes. Based on the results of prior support for our work, we have developed a model that describes the learning and memory mechanisms that underlie the ability of animals, including humans, to use internal cues corresponding to "satiety" to signal when food and cues related to food will be followed by appetitive or desirable postingestive ( i.e., post oral) stimulation. Our research indicates that this type of signaling can be degraded by (1) the consumption of sweet no- or reduced-calorie foods/fluids that may weaken t he normally strong predictive relationship between the orosensory properties of food and the caloric or nutritive consequences of eating;(2) consuming diets that impair the functioning of a brain structure (i.e., the hippocampus) that underlies the ability of rats to use their satiety cues to predict when eating food will or will not produce reinforcing postingestive outcomes. A result of degrading the operation of these learning mechanisms is excess intake and body weight. The present research will examine whether learned control mechanisms develop without explicit laboratory training, whether consumption of high-fat diets interferes with these mechanisms, and whether this interference has potential to produce significant weight gain. These studies promise to yield important new information about the role of dietary factors in both obesity and cognitive decline. PUBLIC HEALTH RELEVANCE: Obesity and cognitive decline are both serious threats to the public health and to the of quality life. This project will explore the possibility that both of these disorders have common environmental origins and brain substrates. The idea that disruption in a specific learning process gives rise to obesity will also be examined.

Neurons communicate with each other in the brain through specialized junctions, called synapses. During brain development, numerous new synapses are established and new synapses continue to form throughout life. The long-term goal of the research proposed in this application is to determine the molecular basis of synapse formation in the vertebrate brain. The first proteins have now been identified that organize synapse formation and development. One such protein is SynCAM 1, a synaptic cell adhesion molecule that connects pre- and postsynaptic sides. Importantly, SynCAM 1 induces the formation of new, fully functional excitatory synapses between neurons. It is highly expressed in the developing brain during intense synaptogenesis, indicating a broad function for this molecule in synapse formation. Such synaptogenic functions have been validated in cultured neurons and in vivo. The objective of this application is to define the signaling pathways through which SynCAM 1 organizes synapses and determine how other trans-synaptic proteins act in concert with it. The central hypothesis of this application is that SynCAM signaling organizes developing synapses and regulates synaptic function at later stages. To attain the objective of this application, three specific aims will be pursued. The first aim of this application is to determine the intracellular signaling pathways through which SynCAM 1- mediated synaptic adhesion instructs synapse development, focusing on changes in the synaptic cytoskeleton. Second, it will be analyzed how trans-synaptic interactions act in concert to assemble synapses and shape their structure. Third, it will be determined to which extent SynCAM 1 functions in vivo together with other synaptic adhesion molecules to organize synapses. These experiments involve the biochemical characterization of SynCAM binding partners and their activities. Functional analyses of SynCAM interactions will be performed by quantitative immunocytochemistry, imaging of synapses in cultured hippocampal neurons, and electrophysiological recordings. In addition, the in vivo relevance of these interactions will be tested using structural and functional studies of synapses, including ultrastructural analyses, electrophysiological recordings, and behavioral analyses. Achieving these goals is important for human health, as altered synapse organization affects the wiring of neuronal circuits and synaptic plasticity. These changes are associated with alterations in human behavior, the ability to learn and remember, and addiction to drugs of abuse. Furthermore, deficits of synapse formation likely underlie neurodevelopmental disorders such as autism. In summary, this application aims to identify the molecular interactions involved in synapse formation. The progress under this application will allow testing to which extent these synapse-organizing processes are affected in disorders of the human brain and whether they represent novel points of therapeutic intervention. PUBLIC HEALTH RELEVANCE: Nerve cells communicate with each other in the brain through specialized junctions, called synapses. These junctions form in the human brain soon before birth, and changes in this process impair the wiring of the brain and can cause mental retardation. This research program is relevant to public health because it will analyze how nerve cells connect to each other in the healthy brain, allowing us to understand what steps go wrong in developmental disorders.

Influenza A viruses exhibit extreme diversity as exemplified by the multiple serotypes of the hemagglutinin (HA, H1-H18) and neuraminidase (NA, N1-N11) surface antigens. To date, only three of the possible 198 combinations found in avian and other animal reservoirs have been associated with human pandemics (H1N1, H2N2, H3N2). However, ever increasing anxiety about the potential for infections by infections of zoonotic origin have heightened concern about emergence of a human transmissible virus that could devastate the human population. The emergence of H5N1, H6N1, H7N7, H7N9, H9N2, and H10N8 viruses in the human population are constant reminders of this possibility. Influenza B viruses with its two distinct lineages further increase the health and economic burden of seasonal influenza worldwide. This proposal seeks to elucidate, at the structural level, key sites of vulnerability on influenza virus that can be utilized to develop therapeutics as well as improved vaccines. Antibody-mediated neutralization of influenza virus is a complex combinatorial problem for the human immune system as it is presented with diverse, highly variable and constantly evolving viruses. While neutralizing antibodies against human flu are traditionally regarded as being strain specific, recent advances have shown that much broader responses can be mounted that give valuable insights into conserved sites of vulnerability. We are therefore amassing compelling evidence that a sustained, cross- serotype response can be mounted against influenza and this vital information can now be harnessed for design of small molecules, peptides, and proteins to target these key sites of vulnerability, thereby blocking influenza infection. No effective drugs are currently available for preventing the entry of influenza virus. Thus, we will elucidate common features for recognition of sites of vulnerability of pandemic and emerging influenza viruses from crystal structures of diverse HAs with broadly neutralizing antibodies and sialosides that are mimics of the natural receptor. A combined biophysical, biochemical, and chemical approach employing state- of-the-art structural biology, glycan arrays, and chemical biology will be used to provide key insights into influenza virus neutralization that enable design of novel therapeutics to control and combat future influenza pandemics and seasonal epidemics.

The three areas of OCD research are: Suicide Risk in the Medical Setting Suicide is an international public health threat. In the U.S., it is the 2nd leading cause of death for youth and the 10th leading cause of death for adults. As suicide risk screening becomes a national priority for medical settings, non-mental health clinicians require valid, psychometrically-sound screening instruments for assessing suicide risk. However, there are very few such mental health assessment instruments that have been validated empirically with medically ill patients in hospital settings. Recognizing this gap early and capitalizing on clinical and research expertise within our group, we began developing a risk of suicide screen for pediatric patients in the emergency room in 2008 which resulted in a validated tool, the ASQ (Ask Suicide Screening Questions). The ASQ has been translated into Spanish, French, Dutch and Chinese and is currently being implemented in emergency departments in the U.S. and abroad. The tool is available on the NIMH website at http://www.nimh.nih.gov/news/science-news/ask-suicide-screening-questions-asq.shtml for public use. Since the development of the ASQ, the focus of our ongoing suicide screening studies has been to develop and validate brief screening instruments that can be administered by non-mental health clinicians in other medical settings and with specific patient populations. We currently have 5 IRB-approved protocols addressing suicide screening in different medical settings (NCT00623493, NCT01517126, NCT02140177, NCT02050867, NCT02830334). We are addressing areas of need, including pediatric and adult emergency department patients, and pediatric and adult medical inpatients. Our research is current with the February 2016 Joint Commission (JC) Sentinel Event Alert calling for suicide risk screening of all medical patients in a hospital setting and is consistent with NIMH's commitment to join with the National Action Alliance for Suicide Prevention to reduce the suicide rate by 20% over the next 5 years. In addition, our research team is working closely with NIH CC leadership in order to meet the JC recommendations; we are utilizing our research to inform implementation of suicide risk screening at the NIH CC and hospitals nationwide. Mental Health Aspects of Coping with Chronic Medical Illness Born out of our clinical work on the PCLS and neurocognitive assessment service for the past decade, a natural research focus has emerged from our collaborative work with other Institutes at NIH. For example, working with the Pediatric Oncology Branch we began to study distress and its correlates in medically ill children, particularly those with life-limiting illnesses, through 3 protocols over the past 5 years (NCT00824278, NCT00969579, NCT02423031). The collaboration has been highly productive leading to the development of the first advance care planning guide for adolescents and young adults with life-limiting illnesses, Voicing My CHOiCES http://www.agingwithdignity.org/forms/voicingmychoices.pdf. This planning guide is available at www.agingwithdignity.org Another study along these lines (NCT01778478) focuses on how to screen and diagnose psychiatric disorders in young adults (ages 18-25) with and without cancer. The study examines the feasibility of using the structured psychiatric diagnostic interview, Kiddie-Schedule for Affective Disorders and Schizophrenia Present and Lifetime version (K-SADS-PL), in this age group and evaluates whether the parent interview affects diagnostic findings. In addition, the study assesses psychosocial developmental milestone acquisition among young adult cancer patients compared to their healthy peers. Neuropsychiatric Aspects of Immune and Infectious Disorders (including HIV/AIDS), Genetic, and other Disorders The OCD Neuropsychology Unit is involved in a broad range of research protocols that are studying cognitive and emotional functioning in various diagnostic groups. These research protocols include genetic disorders (such as Mobius syndrome, methylmalonic academia, Cerebral Autosomal Dominant Arteriopathy with Sub-cortical Infarcts and Leukoencephalopaghy (CADASIL)), infectious and immune diseases (HIV, progressive multifocal leukoencephalopathy, Ebola, anti-NMDA receptor encephalitis), brain tumor (re-irradiation effects), and chronic fatigue syndrome. A large focus of these neuropsychiatric studies is HIV/AIDS. HIV-related research activities in the OCD fall into two categories: 1) NIH Intramural NeuroHIV Program, and 2) Collaborations with multi-site research networks, including an NIH DoD HIV/AIDS associated neurocognitive disorder protocol. In 2008 the NIH, led by NIAID, initiated a partnership with the District of Columbia (DC) government with the goal of reducing the impact of HIV on DC, which had almost a 3% prevalence rate of HIV infection, the highest of any city in the US. The program was also created to develop research infrastructure in the city to generate knowledge and change public policy as well as to develop research across NIH. In alignment with the NIMH's commitment to reduce the burden of mental illness in the HIV/AIDS population, NIMH, NINDS and NIAID have developed 2 intramural protocols (NCT01875588; NCT01692236) investigating HIV-related neurocognitive disorders, which are a clinical challenge and threat to the long-term health of people living with HIV/AIDS. In addition, we have worked to assess the mental health needs of HIV patients and build an interdisciplinary approach to HIV and mental health in DC, leading to funded projects being initiated at other DC institutions.

Data on daily climatic conditions as reported from approximately 300 weather stations have been obtained from the National Climatic Center, and linked to daily mortality data for the period 1972 through 1977. An age-specific analysis of average daily mortality at various levels of climatic variables will be performed in order to ascertain relationships between age and climatic conditions. Climatic variables will include temperature, relative humidity, rapid changes in temperature, departures from normal temperatures, and sustained heat waves and cold fronts. Levels at which hypothermia and hyperthermia are more likely will be investigated in greater detail. Concomitant information on socioeconomic status and geographic location of the cases will also be considered.

In immunologic lung disorders such as sarcoid activated alveolar macrophages are key to the pathophysiology of the alveolitis. These disorders can be successfully treated with systemic corticoids. Since therapy is often compromised by toxic side effects, localized inhalation therapy would be optimal. However, due to the poor aqueous solubility of corticoids, no inhalable dosage form currently exists which is optimally suitable for small particle nebulization to alveolar lung regions, where the inflammation occurs. We propose a novel therapeutic approach to such immunologic lung disorders: Alveolar delivery of liposome-incorporated corticoids by aerosol in order to promote corticoid uptake by alveolar macrophages, and thus modulate the inflammatory alveolitis at the macrophage level. The overall objective of this project is to initiate research that will lead to improved inhalation therapy of immunologic lung disorders, using sarcoid as an immunologically well-defined and corticoid- responsive example. The rationale is that corticoid inhalation could be more effective by targeting liposomal drug directly to alveolar lung regions and to macrophages. While optimal therapeutically effective levels could be established, systemic drug levels, thus side effects would be significantly reduced. Prolonged retention of liposome-corticoid in the lung may result in prolonged therapeutic efficacy at the cellular level. We propose to (1) design lyophilized liposome-corticoid dosage forms suitable for small particle aerosolization; (2) compare in vitro human macrophage uptake and receptor binding of liposome-corticoid and corticoid in solution; (3) determine the efficacy of liposome-corticoids on regulation of human macrophage immune functions n vitro; and (4) determine the in vivo distribution of liposome corticoids in rat lung, plasma and extrapulmonary tissues.

This is part of a larger endeavor whose aim is to systematically investigate executive control functions, studying their development, neural bases, and genetic & neurochemical modulation from infancy through old age. The focus here is on inhibition of attention, inhibition of action, and cognitive flexibility during early development. It is hypothesized that: (1) Errors made by children of 3-10 years reflect problems with flexibly switching. Even 3-year-olds will succeed at steady-state attentional inhibition (selective attention) and steady-state action inhibition (inhibition of old stimulus-response mappings). Lags of up to 3-6 years will be found, however, between when children can first show inhibition in steady-state and when they can switch back and forth. (2) This progression does not depend on improved memory; even the younger children will be able to state the correct rule on each trial. It does depend, though, on improvements in the ability to execute the mental computations necessary to translate abstract rules into practice. The core problems in the development of executive control functions are the abilities to flexibly switch mental settings and to flexibly manipulate information in one's mind, not inhibition or memory per se. (3) Many of the errors made by infants and preschoolers reflect difficulty in grasping that two things are conceptually connected if they are not physically connected (e.g., not part of the same entity), and the flip side, difficulty grasping that attributes of a single entity can be separated and the entity conceptually redescribed from different perspectives. Thus, while switching attentional focus and response mappings is difficult, when the stimulus dimensions are separated, children will succeed at such switching at a very young age. The proposed research will test these and other hypotheses, charting the development of inhibitory control and cognitive flexibility in the early years of life. This will provide valuable insights into why children have difficulties, conditions for optimizing their performance, and benchmarks by which to assess children's development or deficits. A more refined understanding of executive control functions will assist efforts to understand how these are differentially affected in disorders such as Attention Deficit Hyperactivity Disorder, Obsessive Compulsive Disorder, schizophrenia, addictions, and autism. Insights into the conditions under which children can succeed may yield procedures that might prove helpful to those afflicted with disorders affecting these critical functions. [unreadable] [unreadable]

The training proposed in this NRSA contains both a practical research and didactic component. The broad aim of the research component is to determine the extent to which the presence and severity of metabolic syndrome (MetS) contributes to aging-related functional decline, and to examine whether this relationship is mediated by excess inflammation. Because maintenance of physical function is a critical factor in the ability of older adults to remain independent, the research component of this project is highly relevant to the mission of the NIA. The project will be accomplished via secondary analyses of data collected in the Health ABC Study, a prospective cohort of 3,075 well-functioning white and black elders (70-79 years), well characterized for MetS, with several indices of physical function and inflammation. Specifically, this project will (1) determine whether the presence and severity (number of components) of MetS is associated with lower physical function at baseline and with a decline in physical function over time in older adults; (2) determine whether the presence and severity of MetS is associated with inflammation at baseline and with changes in inflammation over time in older adults; and (3) determine whether, and to what degree, inflammation reduces the effect of MetS on physical function. Statistical methods to be used include logistic regression and mixed effects modeling, as well as mediation analysis. This project will provide a practical opportunity for the candidate to utilize the statistical and epidemiological skills she will learn as part of the proposed coursework (described below). The research will also provide the candidate with direct, practical experience in data management and longitudinal data analysis. For the didactic component, Dr. Beavers will complete a series of courses offered through the Master's program in Clinical and Population Translational Sciences at the Division of Public Health Sciences and Translational Science Institute at Wake Forest University in statistics, epidemiology, and clinical trials, such as Foundations of Clinical and Translational Science, Epidemiology, Clinical Trial Methods, and Applied Linear Models. Completion of these courses will provide a more comprehensive understanding of statistical and epidemiological concepts and clinical trial methodology, allowing Dr. Beavers to be able to conceptualize, plan, and execute clinical research in her future career. The research findings will provide better characterization of the relationship between MetS, inflammation, and physical function, which may assist in the development of preventive and therapeutic efforts for older men and women with functional disability, while the coursework in Clinical and Population Translational Sciences offers a unique opportunity to maximize the candidate's post-doctoral training at Wake Forest University and develop competencies in conduction and translation of clinical and population research. PUBLIC HEALTH RELEVANCE: Maintenance of physical function is a critical factor in the ability of older adults to remain independent. Better characterization of the relationship between common preventable conditions, such as metabolic syndrome, and physical dysfunction is critical to efforts aimed at preventing disability. Given the growing demographic of persons aged 65 years and older, coupled with the economic and emotional burden related to age-associated loss of physical function, prompt identification of modifiable functional decline risk factors is of considerable public health concern.

This proposal is for continuation of collaborative research in the natural history, epidemiology, biology, and treatment of childhood cancer at the Children's Hospital & Medical Center (CHMC) in Seattle, Washington as a full member institution of the Childrens Cancer Group (CCG). The specific aims of this proposal are: 1) to develop better treatment of childhood cancer; 2) to develop a better understanding of the biology of childhood cancers; and 3) to develop a better understanding of the natural history, epidemiology, and late effects of treatment of childhood cancer. CHMC will pursue these aims by contributing patients to phase III randomized intergroup and CCG trials, by participating in CCG pilot therapeutic trials that will form the basis for future randomized trials, by participating in new agent trials, by contributing specimens and data for biology, epidemiology and late effects studies, and by providing leadership in the scientific and administrative efforts of CCG. During the first three years of the last grant period, 1989,1990 and 1991, two hundred-forty-eight patients were entered onto therapeutic studies, 181 onto epidemiology and late effects studies, and 36 onto biology studies. At the end of 1991, there were 838 patients in active follow-up after completing a CCG therapeutic study. In the first 8 months of 1992, following Dr. Miser's arrival at CHMC, 97 patients were entered onto CCG therapeutic protocols (12 patients per month) and 83 patients onto non- therapeutic protocols (10 patients per month). CHMC is an important contributor to the leadership of CCG: Dr. Chard, former Associate Chairman for leukemia studies and former vice chairman of CCG, is the principal investigator, a member of the nominating committee, and an important group leader. Dr. Miser is a member of the CCG Executive Committee; Associate Chairman for CCG solid tumor studies; Chairman of the Bone Tumor Strategy Group; Chairman of CCG-7881, Chairman of CCG-8605, Chairman of the CCG pilot osteosarcoma program, and a member of a number of other therapeutic and strategy committees. Dr. Geyer is a member of the Brain Tumor Strategy Group, Chairman of CCG-9921, Chairman of CCG-0896, Chairman of CCG-B921, and a member of several other brain tumor committees. Dr. Bernstein is a member of the biology committee and principal investigator of the ANLL reference laboratory. Dr. Pendergrass is Chairman of CCG-8602 and a member of a number of epidemiology committees. Drs. Milstein and Berger are members of brain tumor committees. Dr. Schaller is a member of the surgical and neuroblastoma committees. Dr. White is a member of the imaging committee. Dr. Breiger is a member of the psychology committee.

Structural studies we have conducted to date on highly purified porcine relaxin indicate that it has a molecular weight of approximately 6,700 and consists of two non-identical chains (alpha and beta) of similar size which are linked by disulfide bond(s). We propose to determine the amino acid sequence of the porcine relaxin chains and the location of the disulfide bond(s). We then propose to determine that portion of the relaxin molecule responsible for its biological properties--the so-called biologically active site. We propose to locate the tissues responsive to relaxin in the rat. We will employ a sensitive and precise radioimmunoassay for relaxin to determine factors which influence the secretion of porcine relaxin immediately preceding parturition. BIBLIOGRAPHIC REFERENCES: O.D. Sherwood, K.R. Rosentreter, and M.L. Birkhimer, "Development of a Radioimmunoassay for Porcine Relaxin Using 125I-Labeled Polytyrosyl-Relaxin," Endocrinology 96:1106, 1975.

In the proposed effort, GW Medical Technologies will asses the benefits, risks, and effectiveness of a peripheral blood lymphocyte (PBL) simulation index diagnostic test for Alzheimer's disease (AD). The company's test detects AD by evaluating a patient's stimulation index against a predetermined cutoff, where the stimulation index is defined as the ratio CD69 presentation on mitogenicly stimulated peripheral blood lymphocytes to the CD69 presentation of un-stimulated lymphocytes. In preliminary studies the company found a statistically significant difference (p<0.05) in the mean stimulation index of AD patients and cognitively intact controls and was able to identify samples from AD "probable" patients with high sensitivity and selectivity (83% and 96% respectively). However, these early results are suspect due to the limitations of the original test methodology and protocol. Early testing relied on manual processes for data collection, transfer and analysis, which could introduce significant errors. Early testing also used a small sample size (n=72) and did not include samples from patients with other forms of dementia. In the proposed effort, the company addresses all three of these limitations by developing automated routines for data collection, handling and analysis and by conducting a randomized, blind clinical study (n=300) that includes control samples from both cognitively intact individuals and patients with other forms of dementia. Should this study prove successful, a this test could provide a preclinical diagnosis of AD, improve patents' quality of life, and reduce the cost of care.

The long term goal of this project is to identify biological roles for mammalian glycans in development and cancer. Glycosyltransferase gene mutants of mice, embryonic stem (ES) cells and CHO cells will be used to investigate the basis of cell-type specific blocks to differentiation and transformation caused by alterations in specific glycans. Our recent studies show that 1) oocyte-specific knockout of complex and hybrid 7V-glycans compromises ovulation and oocyte developmental competence, but fertilization, blastogenesis and implantation proceed in the absence of these glycans; 2) the bisecting GlcNAc on complex W-glycans of non-hepatocyte glycoprotein(s) promotes hepatocarcinogenesis and liver regeneration after partial hepatectomy; and 3) deletion of mouse protein O-fucosyltransferase 1 generates a canonical Notch signaling phenotype identifying the Pofutl gene as an essential component of the Notch signaling pathway. We now propose in Specific Aim 1 to investigate the molecular basis of the requirement for complex and hybrid A/-glycans in oogenesis, and to identify roles of mucin O-glycans and Ofucose glycans in early development after oocyte-specific deletion of the core 1 p3GalT-l (Clgaltl) or Pofutl genes. In Specific Aim 2 the hypothesis that a triantennary, GlcNAc-terminating complex N- glycan is required for spermatogenesis will be investigated by deletion of a novel GlcNAc-transferase gene that is expressed almost exclusively in testis and by eliminating complex and hybrid vV-glycans from spermatocytes, spermatogonia or Sertoli cells. The Pofutl gene will also be deleted in spermatocytes and spermatogonia or Sertoli cells to identify roles for Notch signaling in spermatogenesis. In Specific Aim 3 the hypothesis that the Mgat3 gene may function as a tumor suppressor in mammary gland will be tested in the MMTV-Polyoma middle T (PyMT) mammary tumor model which metastasizes to lung and in cell signaling assays. In addition, roles for N- and 0-fucose glycans and Notch signaling will be investigated in mammary gland development and transformation by selective deletion of the Mgatl and Pofutl genes in mammary epithelia before puberty using MMTVCre, or during pregnancy using whey acidic protein (WAP) Cre recombinase.

The long-term objective of this project is better understanding of the contribution of prostaglandins, thromboxanes and other products of arachidonic acid in human airway function. This goal will be approached by in vitro studies of excised human lung tissue and supplemented by analogous studies of guinea pig lung. Studies will focus upon: (a) regulation of the tension of pulmonary smooth muscle by arachidonate metabolites; and (b) the role of prostaglandins and thromboxanes in the bronchospastic response accompanying anaphylaxis in sensitized lung tissue. Two experimental models will be employed simultaneously to pursue these questions. These are: (a) organ bath studies of the smooth muscle response of both lung parenchyma and large airways, both separately and together, under various experimental conditions; and (b) chopped lung model of anaphylaxis using passively sensitized human lung chiefly, but also actively sensitized guinea pig lung fragments. Radioimmunoassay for PGE, PGF2a, TxB2, PGD2 and the prinicpal metabolite of prostacycline (6-oxo-PGF1a) will be used to develop a profile of arachidonate products during antigenic challenge of sensitized human and guinea pig lung. Specific experimental protocols will evaluate the effect of basic physiologic parameters on PG production, the pharmacology of smooth muscle response to exogenous prostaglandins and thromboxanes, the effect of anti-inflammatory drugs (including anti-inflammatory steroids) on large and small airway sensitivity to smooth muscle agonists, and confirmation of preliminary evidence for the existence of a prostaglandin releasing factor derived from lung mast cells.

A number of studies have identified patterns of parenting that are associated with early childhood problems of offspring, including maladaptive temperament traits, aggression, and attachment problems. These early traits are harbingers of more frank mental disorder in later childhood. Recent investigations of the parenting-offspring temperament connection have suggested that these relationships come about from multiple influences, including reciprocal causation or feedback, and probably common genetic and cultural influences as well. The proposed study takes advantage of a large longitudinal data base to add information uniquely useful for making these distinctions in a three generational design. Data gathered since childhood on a large general population sample of children are to be used in models estimating the influences on parenting and child rearing practices used with their offspring, and the origins of the relationships between offspring temperament and parenting variations. The longitudinal follow-up of the parents and offspring over the transition year from infancy to early childhood (9 to 24 months) will allow the development of models in which the relative magnitude of various influences on these relationships can be estimated and tested. Comparisons of such effects in first-born nd second-born children from the same families, assessed at fixed ages of the offspring, will permit tests of hypotheses regarding the origins of within-family differences in parenting and offspring temperament. Biological markers will be examined for consistency with the theoretically proposed dimensions of temperament.

Gammaherpesviruses (GHVs) establish lifelong chronic infections that place the host at risk for numerous cancers. During chronic infection, GHVs express viral gene products that stimulate host-cell proliferation and differentiation, processes thought to facilitate long-term latent persistence and contribute to tumorigenesis. However, GHVs are not acutely transforming, and cancer is rare given the high incidence of infection among adult humans, estimated at more than 90% for Epstein-Barr virus. This suggests that host cells are equipped with an intrinsic resistance to GHV-driven proliferation and cellular immortalization, but few host molecules capable of mediating this effect are known and whether these molecules functionally restrict GHV persistence and disease in vivo has not been established. Experiments described in this proposal seek to identify and confirm the in vivo relevance of host molecules with the capacity to limit GHV chronic infection and disease in three Specific Aims. Based on preliminary studies that employed murine gammaherpesvirus-68 (MHV68) infection of mice, experiments described in Specific Aim 1 test the function of host tumor suppressor protein p53, a critical molecule for preventing neoplastic disease, as an innate barrier to MHV68 latent persistence and cellular transformation. Experiments presented in Specific Aim 2 seek to define mechanisms whereby p53 limits MHV68 latent infection. Experiments in Specific Aim 3 will provide the first in vivo test of the long- standing hypothesis that latency-associated nuclear antigen, a conserved GHV disease determinant capable of inhibiting p53 functions in tissue culture, is necessary to overcome a p53-mediated restriction to enable long- term latent infection. These experiments harness the powerful mouse and MHV68 genetic systems to address a question that is fundamental to our understanding of the GHV-host dynamic. Further, we anticipate that results of this work will inform new therapeutic approaches aimed at enhancing p53 functions to limit or prevent GHV-related cancers, especially in high-risk patients such as HIV-infected individuals or solid-organ transplant recipients.

Emerging evidence suggests that racial barriers in the patient-physician relationship may be an important source of disparities in the quality of health care. These barriers include cultural dissimilarity between doctors and patients; social discomfort or distrust when interacting with people from other racial or ethnic groups ("interracial anxiety"); and negative racial bias or stereotyping of minority patients by physicians. Training in "cultural competence" has become the primary vehicle for reducing the impact of racial barriers betweens patients and physicians. To date, however, there has been limited empirical evidence to inform the content of cultural competence training. Specifically, there has been scant research on the nature and influence of specific racial barriers in the patient-physician relationship, and on the knowledge, attitudes, and skills needed to reduce them. The objective of this developmental grant application is to develop and test tools necessary to measure racial barriers and cultural competence in the patient-physician relationship, as a first step towards assessing their role in causing and mitigating, respectively, racial disparities in health care quality and outcomes. Our specific aims are to: 1) develop a psychometrically sound instrument measuring cultural competence among physicians; 2) test the reliability and validity of scales measuring interracial anxiety among physicians; and 3) test the validity of a computerized, interactive tool measuring racial bias among physicians. We will use a multi-step approach to achieve these aims, including expert scientific review of draft survey items, cognitive interviews with practicing physicians, a national physician survey, and psychometric analysis. Developing and testing measures of cultural competence and racial barriers in the patient-physician relationship will provide essential tools necessary to advance the study of racial disparities in health care. These measures will serve at least three crucial functions. First, they will allow us to empirically assess the role of physician bias, and other racial barriers in the patient-physician relationship, in explaining disparities in the quality of health care. Second, they will allow us to determine which (if any) facets of the broad concept of cultural competence reduce racial barriers and in turn reduce health care disparities. Both of these steps will help inform the content of cultural competence training and other interventions to reduce disparities. Finally, rigorously developed measures of racial barriers and cultural competence will provide needed evaluation tools for judging the effectiveness of cultural competence programs, many of which are currently being developed under NIH sponsorship. The aims of this project are to develop and test tools needed to explore the role of racial barriers in the patient- physician relationship as a source of disparities in the quality of health care, and the role of cultural competence among physicians as a means to mitigate those barriers. These tools are needed to inform and evaluate future interventions to reduce pervasive racial disparities in health care quality and outcomes. [unreadable] [unreadable] [unreadable] [unreadable] [unreadable] [unreadable]

This P41 Biotechnology Resource Grant application seeks renewed support for our successful efforts to develop and apply innovative neuroimaging technologies within the highly integrated multimodal framework of the Center for Functional Neuroimaging Technologies (CFNT). The overall goal of this established NIBIB Biomedical Technology Resource Center (BTRC) is to provide advanced technology resources to more closely examine, and thus better understand, the human brain in health and disease. To this end, we seek to develop new techniques and advance existing technologies to acquire and analyze functional images of the working brain, with unprecedented physiological precision and spatiotemporal resolution, and to deploy these innovative tools to promote investigation of complex neuroscientific questions. Through coordinated research and development, collaborative research, service use, training and dissemination activities, our BTRC has built a standard of excellence in developing, sharing, and supporting the use of multimodal imaging tools that have consistently advanced capabilities for research that spans many basic science and clinical domains. Central to this effort are our four Technology Research and Development (TRD) projects to improve and extend techniques for non-invasive magnetic resonance image analysis (Project 1) and acquisition (Project 2), electromagnetic source imaging (Project 3), and optical neuroimaging (Project 4). Directly motivating the Aims of these TRD projects is a strong network of Collaborative Projects, which both challenge the TRDs to continue to innovate the next-generation neuroimaging tools, and reciprocally, employ the new tools we develop to drive their own research forward in new directions. Another essential element in this framework is the extensive application of our resources by a wide and diverse Service Users community. Finally, the TRDs, Collaborative and Service Projects contribute to the BTRC's Training and Dissemination mission, which additionally includes multiple dedicated Fellowships and Workshops, strong web presense, and important industrial partnerships, providing multiple channels to share the knowledge needed to apply the tools we develop with the scientific community locally, nationally, and internationally.

We have used recombinant DNA techniques to construct a series of vectors which function in both Escherichia coli and mammalian cells. These constructs fall into two categories: shuttle vectors and mammalian expression vectors. Shuttle vectors are designed to permit efficient bidirectional transfer of genes between Escherichia coli and mammalian tissue culture cells. We have applied bacteriophage lambda site-specific recombination mechanisms towards developing a new class of such vectors. Mammalian expression vectors are designed to facilitate study of eukaryotic promoters, activators, and other transcriptional regulatory elements. We have constructed expression vectors which permit the Escherichia coli enzyme chloramphenicol acetyltransferase to be used as a rapid, sensitive and specific assay for function of such elements.

Objective To characterize the prevalence and causes of endometriosis in the aged female monkey, and to assess immune alterations associated with endometriosis. In the course of a study on the long-term effects of dioxin exposure, Drs. R. Bowman and S. Rier discovered that female monkeys exposed to either 5 or 25 ppt TCDD for 4 years were at greater risk for developing endometriosis as they aged (Rier et al., 1993, Fundamental and Applied Toxicology). 71% of females in the high dose group and 43% in the low dose developed endometriosis, in contrast to 33% of the control subjects. These females are now approximately 24 years of age, and provide a unique opportunity for assessing endocrine and immune sequelae associated with this chronic disease. We have been evaluating lymphocyte cytolytic responses and in vitro cytokine production in these subjects. In addition, we have been investigating the natural prevalence of the disorder throughout the colony by means of an in-depth analysis of necropsy records over the last 15 years. Recently, we have identified certain family lineages of monkeys at greater risk for this gynecologic condition. Future studies will focus on predictors of disease in offspring in these high- and low-risk families. Key words endometriosis, aging, inflammation, cytotoxic responses, female toxicology

Mycobacterium tuberculosis has a unique repertoire of virulence mechanisms and generates a diverse spectrum of clinical disease. M. tuberculosis has two large families of proteins, the PE and the PPE proteins, which are likely to be critical to virulence. The PE and PPE proteins are rare in nonpathogenic mycobacteria but account for four percent of the M. tuberculosis genome. Many are secreted or surface localized and thus perfectly positioned to play critical roles in host-pathogen interactions. It has been difficult to use genetics to define the function of these proteins because of the sheer number of potentially redundant gene products. However, the fact that PE and PPE proteins are secreted in the absence of a signal sequence for the general secretory pathway suggests that there is an alternative secretion system for these proteins. In Aim 1, we will identify and target this secretion pathway in M. tuberculosis. We expect that by disrupting the appropriate processing of some or all PE and PPE proteins, we will define the contribution of these proteins to the growth and virulence of M. tuberculosis. In the Aim 2, we will study the relationship between PE and PPE protein secretion, post-translational modification and the immunogenicity of these proteins. This work will refine our model of how PE and PPE proteins generate antigenic diversity in M. tuberculosis. Together, these data will dramatically increase our understanding of PE and PPE proteins and provide a foundation for future studies of their function and role in pathogenesis [unreadable] [unreadable] Despite the fact that tuberculosis kills millions of people annually, little is known about how it causes disease. Here we will develop tools to define the function of a unique family of mycobacterial proteins, the PE and PPE proteins that are likely to be critical to virulence. This work will provide a foundation for understanding the role of these proteins in the interaction between M. tuberculosis and the infected host. [unreadable] [unreadable] [unreadable] [unreadable]

The primary purpose ofthe Biostatistics Core (Core D) is to continue to provide biostatistical expertise for the design and analysis of all the protocols contained in the Program Project Grant. The Specific Aims of Core D are: Aim I. Assist in the design of experimental protocols and insure the proper sample size is selected to provide reasonable power Aim II. Facilitate data transfer and data quality assurance for all projects Aim III. Analyze and interpret the results from data gathered from the experiments Aim IV. Assist in the preparation of statistical components of presentations, reports and manuscripts Aim V. Develop and disseminate new biostatistical approaches for the analysis of the data; and Aim VI. Participate in discussions and present statistical seminars As we have done in the past, every new experimental protocol will be written in conjunction with a biostatistician from Core D who will provide design expertise and sample size justification for the animal care protocol. Members of the Core will attend the weekly Department of Hypertension and Vascular Research staff meetings. This will facilitate introductions to new PPG staff and also enable the scheduling of biostatistical seminars to cover topics of interest. We anticipate that the efforts of the Biostatistics Core staff (50%) will be divided equally with all four projects contained in this application. We reiterate that the presence of a Core provides availability of a set of biostatisticians with dedicated time and multiple years of experience with the Program Project. This availability is essential to the continuing success of the grant. Although located in a separate building the Biostatistics Core staff are a short walk from the Hypertension and Vascular Research Division, always available by e-mail, and always available for face-to-face meetings to discuss project design and contribute to the preparation of both manuscripts and grants. RELEVANCE (See instructions): The presence of biostatistical expertise is synergistic with the scientific expertise of the investigators in the Program Project. Their presence improves the scientific output from the PPG.

The proposed interdisciplinary training program at the University of Minnesota combines graduate training in the computational, chemical, physical, and engineering sciences with graduate training in neuroscience. Neuroscience is a highly interdisciplinary field that uses a variety of experimental approaches to understand the development, structure, and function of the nervous system. As neuroscience matures, the need grows for quantitative modeling, physical and chemical insights, advanced technologies, and state-of-the-art hardware and software that the computational, chemical, physical and engineering sciences can provide. Therefore, interdisciplinary graduate training is needed to take the maximum advantage of these opportunities. The proposal incorporates the strengths, resources, and administrative structures of several existing graduate programs and the University of Minnesota Supercomputing Institute with an interdisciplinary faculty with diverse research interests, to provide a new paradigm in graduate education. We propose to offer 5 three-year Fellowships each year to attract outstanding pre-doctoral students. Enrolling in existing degree programs in Biomedical Engineering, Chemistry, Computer Science, Mathematics, Neuroscience, Physics and Scientific Computation. The Fellows will be trained across disciplines using a variety of tools including special interdisciplinary coursework, research rotations, dual thesis advisors, special seminars and symposia and unique training opportunities. Each trainee's thesis work will cross the disciplines of neuroscience and the physical/computational sciences. An advisory system will help guide students through the program. Also, several mechanisms are proposed to evaluate the effectiveness of the training program. The trainees will receive instructions in the responsible conduct of research. The proposal documents the efforts that the training program and University will make to ensure that traditionally underrepresented students are recruited and included in the program. On completion the trainees will be prepared for research careers in academia, industry and government. The overall goal is to train the next generation of scientists who can bridge the gap between biology and the physical/computational sciences. Broader impacts include advancing our understanding of the brain, cross-fertilization of the disciplines, and establishing a new model for interdisciplinary graduate training.

During the past three years the applicant and his students have developed highly specific methods for the isolation of native plasminogen from human, bovine and ovine plasma. Their methods, which are now used worldwide, demonstrated for the first-time that affinity chromatography can be used to purify a proenzyme. The applicant proposes during this grand period to (1) develop affinity chromatography methods for the isolation of human and bovine plasmin, (2) to attempt to obtain crystals of human and bovine plasminogen and plasmin and (3) study further the mechanism of the activation of native plasminogen from human, bovine, and other species as time permits.

This is an application for renewal of a longstanding program for predoctoral training in Pharmacological Sciences. Eight training slots are requested to support students during their first two years. The program seeks to meet the need for well-trained scientists who can maintain rapid progress in applying advances in biology to medicine. Rigorous training in molecular biology, genetics, biochemistry, structural biology, and cell biology, as well as pharmacology, forms the foundation of the program. These many disciplines and others such as systems biology and physiology are reflected in the research activities and classes offered to the students. The training program is a specialized area of interest within the Biological and Biomedical Sciences (BBS) graduate program at Harvard Medical School. The training program draws its faculty members from various basic science and clinical departments. The central department for this program is Biological Chemistry and Molecular Pharmacology, although faculty members from other departments play important roles in the training program. The various departments and the training grant faculty are highly interactive. The research activities of the training grant faculty span a broad spectrum of pharmacological sciences with multiple areas of research strength. Students in the program are closely advised and monitored, both before and after starting dissertation research. In their first year, they take core courses covering multiple disciplines in basic biomedical sciences, and a course that stresses reading original research papers and critical thinking. They are required to take a core pharmacology course. They go on to take advanced courses in pharmacology and in areas relevant to pharmacological sciences including human biology, which also stress critical and quantitative thinking. Full time dissertation research follows course work, laboratory rotations, and qualifying examinations. Students receive training in teaching. They participate in multiple other important activities of the training program including a seminar series, a yearly Symposium, and a journal club. This training plan should ensure the strengthening of a program that aims to train students to go on to match or even exceed the accomplishments of previous trainees who now fill leadership positions in pharmacological sciences.

The goal is to investigate the regulatory role which extracellular matrix (ECM) exerts on type II pneumocytes. The extracellular components of the lung have long been known to influence the physical/mechanical properties of the tissue and thereby to play an important role in the function of normal and diseased tissue. alterations in the content and/or organization of connective tissue elements are involved in pulmonary fibrosis and emphysema, both of which are of extensive clinical significance. Recent reports of the effects of ECM on cellular proliferation and differentiation make it clear that ECM constituents cannot be regarded as an inert scaffold, but that they exert significant effects at the metabolic level. The overall goal of the proposed research is thus to investigate the hypothesis that the ECM of the lung exerts a regulatory effect on differentiated metabolism and function of type II pulmonary epithelial cells. The approach involves definition of the effects of biologically significant ECMs on type II cell morphology, as well as on retention of a typical metabolic profile. Specific metabolic steps at which these effects are exerted will be identified and these changes will be attributed to specific ECM components and/or characteristics. ECMs to be investigated range from well-defined materials, including collagens, laminin and fibronectin, to biological matrices of complex composition, including "biomatrix" isolated from whole lung and "matrix gel" elaborated by an EHS-tumor. Parameters to be monitored, which appear to provide an index of differentiated type II cell function, include cell viability, morphology, size and shape; synthesis and secretion of phospholipids and proteins associated with surfactant production; the presence of typical cytoskeletal and/or membrane characteristics, including expression of specific keratins and activit;y of Beta-adrenergic receptor-ligand interations; and the quantity, activity ratio and isozyme pattern of the cyclicAMP-dependent protein kinase system. Further investigations will extend these baseline observations to examine the effects of paraquat-induced oxidant damage on ECM function and on the interaction of type II cells with their substratum. Together, these experiments will define the biochemical mechanisms by which ECM components influence maintenance expression and physiological control of differentiated metabolic function of type II cells, as well as how these influences may be affected by oxidant damage.

Our previous research established the pattern of acquisition of cell surface receptors during normal human myeloid differentiation. These membrane markers, now being used to examine leukemic myeloid differentiation are detected on cells from only a minority of patients. The objectives of this proposed research are: (1) to induce differentiation in freshly isolated human acute nonlymphoid leukemic cells (ANLL) by incubation with known inducers of differentiation and examine cells for differentiation, viability and recovery; (2) to assess differentiation by cell surface markers for IgG and complement, morphology, and cytochemistry; (3) to investigate whether 5'-nucleotidase and microviscosity can be used as new markers of myeloid membrane maturation; (4) to induce membrane differentiation using HL-60 cells (a human acute promyelocytic leukemia cell line) and clone these cells to examine the mechanism of induced membrane marker expression; (5) to investigate whether chemotherapeutic drugs also will induce differentiation in both HL60 and freshly isolated leukemic cells; (6) to compare in vitro induction of differentiation to in vivo effects using a diffusion chamber technique in mice; and (7) to compare in vitro chemotherapeutic results with in vivo clinical response in patients receiving the same agents. The overall objective of this research is to provide a useful in vitro model to aid in the diagnosis, treatment, and prognosis of ANLL.

SUMMARY Metastases of tumors are associated with more than 90% of cancer deaths. Despite years of therapeutic development, mortality has improved only incrementally by few months at best. The preclinical development phase for modern metastatics currently misses critical quantitative information on metastatic progression. Multi-point in vivo observation of early metastasis and quantitative assessment of its development would enable unprecedented precision of longitudinal control. PhotoSound Technologies, Inc. proposes a novel platform for in vivo molecular imaging that addresses the critical barrier in quantitative preclinical imaging of metastasis via contrasted dual-modality 3D imaging approach. The proposed project promises to enable in vivo quantification of numbers and volumes of early metastatic tumors, which is critical for preclinical development of future anti-metastatics. The platform integrates a 3D photoacoustic tomography into a multi-modality imaging platform with a coregistered 3D fluorescence unit (PAFT) and a switchable optical nanoprobe targeted to the studied metastatic cells. The nanoprobe has capability to activate optical and fluorescence contrasts upon external illumination with safe levels of pulsed laser radiation, and it was designed to maximize benefits of dual-modality PAFT imaging. The fluorescence imaging component of PAFT is used to boost detection sensitivity by providing low-resolution spatial constraint for the distribution of activated nanoprobes, which are then precisely mapped in 3D by photoacoustic imaging component. The ultimate objective is to maintain the molecular sensitivity of state-of-the-art fluorescence techniques, while boosting spatial resolution of the detected metastasis 10-fold. Current trends on $1.5B market of in vivo small animal imaging favor commercial introduction of the proposed multi-modal imaging platform, which is designed for table-top application and has a 3D anatomical reference component implemented through a photoacoustic unit. PAFT could be also used as a universal instrument for 3D functional imaging of volumetric blood content and oxygenation without a need for any contrast agent, imaging of various NIR absorbing probes and bioluminescent cells. Such versatility would be attractive for Animal Research Facilities engaged in a broad spectrum of fundamental and preclinical imaging studies. Phase I project will demonstrate feasibility for the PAFT-nanoprobe imaging platform to detect and quantify early metastasis in a preclinical murine model. The focus of Phase II will be the development of a commercial imaging platform that is optimized to monitor the in vivo effects of anti-metastatic therapies. When our system is benchmarked against the current standards of optical imaging, we expect to find a 10x increase in spatial resolution enabling detection of metastases separated by only 0.5 mm, as well as quantitative assessment of individual tumor volumes.

Gene therapy using adenoviruses is limited by the inability to selectively deliver DNA to specific targets. Retargeting can be accomplished by introducing fibroblast growth factor (FGF) onto viral capsids through its conjugation to neutralizing anti-viral antibodies. As a result, vectors bind to cells not through their native receptors, but rather through FGF receptors overexpressed on tumors and proliferating endothelium. In addition, an increased transduction efficiency is accomplished, which we hypothesize results from the high affinity of FGF receptors (Kd= approximately 2 pM) and their ability to traffic to the nucleus. In this application we propose to examine the mechanisms of FGF retargeting in vitro. Specific aim one will define the roles of FGF and adenoviral receptors in cell surface binding and transduction. Specific aim two will confirm the intracellular pathways of virus internalization and gene delivery. Phase II studies would use this information to rationally develop in vivo models for examining the influence of FGF-retargeting on viral toxicity and immunogenicity, and for the development other ligand-viral conjugates. The data generated will validate the use of growth factor retargeting for gene therapy, and establish the groundwork required for its use in clinical trials. PROPOSED COMMERCIAL APPLICATION: The proposed studies are designed to develop therapeutic products for gene therapy. Initial drug candidates will target adenoviruses to growth factor receptors overexpressed on tumor cells and associated vasculature, while at the same time eliminating normal viral tropism for uninvolved organs. Targeted delivery will allow transduction of tumors with cytotoxic proteins and other anti-proliferative agents, and accomplish antitumor activity by destruction of tumor cells and/or proliferating endothelium. This approach will allow application of the growing number of adenoviral vectors to cancer gene therapy by eliminating their major limitation, namely non-targeted delivery to normal tissues.

The main objective of our application to NIH is to establish the Chicago Tri-lnstitutional Center for Chemical Methods and Library Development (CTCMLD), which is designed to address the fundamental challenges of high-throughput organic synthesis and to provide major advances that will significantly facilitate the development of small-molecule libraries for broad biological screening. Our CTCMLD application is strongly supported by the three participating institutions and the Chicago Biomedical Consortium, which provided a $2 Million Lever Award to match the NIH funding. The CTCMLD will have several major goals listed below. Center Objective 1: Conduct four highly integrated projects that are designed to significantly facilitate the generation of chemical diversity and assembly of high-quality small-molecule libraries. The four proposed projects are: (a) practical synthesis of new nitrogen-based heterocyclic libraries (leaders: Scheldt and Gevorgyan); (b) multi-component assembly of polyketide libraries (leader: Yamamoto); (c) assembly of scaffold-unbiased small-molecule libraries (leader: Kozmin); and (d) development of tailored surfaces for new reaction development, miniaturized library synthesis and small-molecule immobilization (leader: Mrksich). The scientific projects will be carried out at the highest level, resulting in the development of an arsenal of new synthetic methods, which in turn will enable the generation of approximately 20,000 new compounds of 20 mg scale in highly efficient, parallel manner. Center Objective 2: Training the next generation of scientists, who will develop and arsenal of new methods and strategies for high-throughput organic synthesis. Center Objective 3: Establish core research facility for library synthesis, characterization and cheminformatics. The Core facility will have three highly integrated components: High-Throughput Synthesis Component located at the University of Chicago, Hit-to-Lead Development Resource located on Northwestern campus and Cheminformatics Core at the University of Illinois at Chicago. Objective 4: Provide broad outreach to the biology community enabling rapid and efficient screening of new compound libraries. In addition to providing new compounds for the NIH Roadmap Molecular Libraries Initiative, we will collaborate with three major high-throughput screening centers in Illinois, and many individual biology laboratories. The increased connectivity and synergy resulting from the work of the CTCMLD will represent a major advance in the Chicago area's capabilities and a paradigm shift in how the Chicago academic community will approach and solve problems at the interface of chemistry, biology and medicine. [unreadable] [unreadable] [unreadable]

The long time goal of our studies is to delineate the molecular mechanism that regulates the immune defense against invading pathogens. The study proposed is focused on the role of the family transcription factors of interferon regulatory factors (IRF) in the innate and acquired responses to viral infection. These factors were shown to participate not only in the induction of Type I IFN genes but also in the induction of other cytokines, chemokines and genes directly involved in the antiviral and anti-inflammatory responses. Over the past years we have sequentially isolated IRF-3, IRF-7 and IRF-5 and have shown that these factors serve as direct transporters of virus induced signaling. The function of these factors is not redundant since these factors are expressed in different cell types and stimulate a profile of distinct genes. Among these three factors IRF-7 plays a limiting role in the induction of IFNa that was shown to be important both for the innate and adaptive immune responses. It is our hypothesis that these three IRFs, especially then IRF-7, are regulating both of these immune responses. The key objective of this application is to validate this hypothesis and to determine the molecular mechanism by which IRFs activates the targeted genes in response to pathogens. [unreadable] The study has three aims. [unreadable] In Aim#1 we shall examine the molecular mechanism involved in the IRF-7 mediated activation of antiviral genes. [unreadable] In Aim#2 we shall determine whether Toll 3 and Toll 9 responses to dsRNA and CpG DNA respectively results in the activation of IRF-3, IRF-5 and IRF-7. [unreadable] In Aim #3 we shall determine whether the stimulation of macrophages and dendritic cells by virus, dsRNA and CpG DNA targets similar or distinct set of antiviral genes. [unreadable] We believe that the basic understanding of the role of IRF factors in pathogen induced cellular responses will provide a new therapeutic platform for the treatment of the immune and inflammatory disease. [unreadable] [unreadable]

Physician Postdoctoral Research Training in Perioperative Medicine (PPRTPM): More than 41,000 anesthesiologists practice in the US, but only a small number are physician-scientist researchers in this field of anesthesiology/perioperative medicine. In FY13 fewer than 120 MD or MD PhD anesthesiologists were PI on an NIH grant. Nevertheless, anesthesiology is a requisite component of every medical center, hospital and outpatient surgical facility because of the 40 million surgical procedures performed annually. Advances in pharmacology, imaging, and technologies used in interventional/procedural medical care make the field of anesthesiology/perioperative medicine rich with research career development opportunities. The goal of this program is to address the as yet unmet need to train more committed physician-scientist anesthesiologists. PPRTPM program direction, aims and objectives: Program leadership will be provided by Dr. David Eckmann, serving as contact PI, with further leadership provided by additional Executive Committee members Dr. Gordon Barr, Dr. Roderic Eckenhoff and Dr. Andrew Ochroch. All are members of the University of Pennsylvania Department of Anesthesiology and Critical Care. Their responsibilities will be focused on directing four theme-based research training tracks, three of which are devoted to laboratory research trainees and one of which is devoted to clinical research trainees gaining research training in perioperative medicine. The aims of the PPRTPM are to: Identify, recruit and foster research trainees, both anesthesiology residents and clinical sub-specialty fellows willing to commit to training and career development in perioperative medicine research Match up trainees strengths and interests with mentoring teams Provide guidance for structured learning opportunities Maximize the opportunities for mentored research and career mentoring The objectives of the PPRTPM are to: Train a cadre of committed physician-scientist anesthesiology researchers to advance the field of perioperative medicine research. Provide these individuals with the skill sets and foundation for career advancement Encourage leadership and innovation PPRTPM goals-to pursue the aims and objectives through a training program consisting of: Didactic opportunities, including core requirements and courses designed to provide research skills Seminars, workshops and a journal club focusing on research and progress in the field Mentoring with a team approach, mentor training and scholarship oversight Programmatic interactions with mainstream research through local/national professional interactions.

The rapid accumulation of genome sequences and protein structures during the last decade has been paralleled by major advances in sequence database search methods. The powerful Position-Specific Iterating BLAST (PSI-BLAST) method developed at the NCBI formed the basis of our work on protein motif analysis. In addition, Hidden Markov Models (HMM) and protein structure comparison methods were applied. During the last year, we made further progress in detailed analysis of the classification, evolution, and functions of several classes of proteins. Specifically, we studied in detail the protein domains that are involved in eukaryotic RNA interference mechanisms and showed that the protein machinery of eukaryotic RNAi was pieced together from ancestral archaeal, bacterial and phage proteins that are involved in DNA repair and RNA processing. We also used computational methods to identify a novel prokaryotic toxin-antitoxin systems that are predicted to function via RNA binding or cleavage and other, diverse mechanisms. We explored the evolution of the eukaryotic phagocytosis system and demonstrated the presence of actin-related domains in a distinct set of crenarchaeota and korarchaeota, suggesting that the archaeal ancestor of eukaryotes might have already possessed a primitive form of branched cytoskeleton that facilitated engulfment of other prokaryotes. We further investigated the evolution and genomic context of prokaryotic homologs of the eukaryotic argonaute protein (the key component of the RNAi system) and showed that the genes encoding these proteins tend to be located within "genomic islands" that also contain many other genes for various defense systems. These findings suggest that the prokaryotic homologs of argonaute are components of a novel defense system that also includes a variety of other putative nucleases that we identifies using sensitive methods of domain analysis. We additionally contributed to experimental and structural analysis of a variety of domains previously identified in our computational studies, in particular, the key components of the prokaryotic CRSIPR-associated acqauired immunity system.

Congestive heart failure is a major public health problem affecting millions of patients in the United States. Because heart transplantation can only be offered to a minority of these patients, pharmacological therapy remains the mainstay. Survival has improved with the use of current pharmacological therapy, but nevertheless mortality is high in patients with severe congestive heart failure. If beta-blockers reduce mortality by 25% or more, this finding would have major implications in the care of the patients with this common condition. This project seeks to evaluate the efficacy of beta-blockers to improve the survival of a common clinical condition with a high mortality rate, Class III-IV congestive heart failure. Bucindolol is an investigational non-selective beta-blocker with mild vasodilator properties. It has already been used in several hundred patients with heart failure and has been extremely well tolerated. Patients are randomized between Bucindolol and plcebo, titrated upward to the maximal tolerated dose of Bucindolol and followed for 4.5 years.

This study is designed to examine the relative value of different self-reward strategies in the long-term maintenance of breast self-examination (BSE) practice among women trained to do BSE. These self-reward maintenance procedures will be implemented by breast screening educators coordinated through the Community BSP Office in a Florida community beginning Year 2 of the Program Project. The three self-reward maintenance conditions will be: (a) self-reward instructions and materials delivered at the end of the BSE training session with no monthly prompts to encourage their use; (b) self-reward suggestions delivered by the BSE Maintenance Coordinator through the mail each month contingent upon evidence of BSE performance; and (c) external, monetary rewards and self-reward suggestions delivered through the mail on an intermittent schedule contingent upon BSE practice. Subjects will be 1800 women who attend a community-based breast screening training session in the study site where community-based BSE training efforts are underway. Training groups will be randomly assigned to conditions urging a randomized block design for six months until 1800 women have been enrolled in the study. Subjects will receive their respective self-reward manipulation for one year, followed by a second year in which all external rewards and mailed monthly self-reward prompts will be withdrawn. BSE frequency and quality, and self-reward use will be assessed through a telephone survey of all women in the three conditions at one year following BSE training and again at two years following initial training. Additionally, for a sub-set of the women in each condition, proficiency will be assessed in personal interviews using silicone breast models at each of these two measurement times.

Biomedical and rehabilitation research depends increasingly on state-of-the-art imaging modalities to visualize cells, tissues, and organs. At VAPAHCS, our research program that depends on cutting edge imaging includes the study of such important diseases and disorders for the Veteran population as Alzheimer's disease, traumatic brain injury, cancer, autoimmune disorders, degenerative disorders, vascular disease, spinal cord injury, and traumatic injuries to muscle, bone, and tendon. For so many lines of research, disease modeling and experimental therapeutics have been revolutionized by the development of imaging systems that allow for the tracking of cells in vivo in living animals over time. This approach has been used extensively at VAPAHCS for the past 10 years for the analysis of such processes as non-invasive disease monitoring, stem cell therapies, cell transplantation, tumor growth, in vivo gene expression analysis, immune cell tracking, and neurogenesis, and remains a critical element of our research program. The Perkin/Elmer IVIS Spectrum In Vivo Imaging System is a top-level research instrument that combines cutting edge technology with quality and exceptional ease of operation. This instrument will replace an outdated version that is almost ten years old and failing regularly despite routine maintenance. Furthermore, the new system features includes a camera of enhanced sensitivity providing increased spatial resolution and software that provides more powerful image processing and analysis for the key functionalities of Bioluminescence Imaging (BLI), Fluorescent Light Imaging (FLI), Cherenkov light, 3D tomography, and Spectral Unmixing. The current, outdated equipment is used up to 10 hours a day, including weekends, by investigators at VAPAHCS. The ability to replace our outdated and failing system with a new and updated system would greatly facilitate and accelerate the research program at VAPAHCS. We have outlined an administrative process to oversee the use and maintenance of the new equipment, and we outline a plan for institutional support to provide the resources for equipment service over the next ten years. Overall, the purchase of a new Perkin/Elmer IVIS Spectrum In Vivo Imaging System is critically important for the ongoing biomedical and rehabilitation research at VAPAHCS in order to allow investigators to continue to be at the forefront of research in their respective fields with access to state-of-the-art equipment.

During development, sensory experiences produce synaptic modifications that specify the capabilities and limitations of brain function in adults. In adults, very similar modifications appear to be the substrates of learning and memory. Therefore, a question of great significance is how synapses in the brain are modified by sensory experience. Mechanisms for long-term synaptic depression (LTD) and potentiation (LTP) have been identified, as has a mechanism for regulating the conditions for LTP induction. However, it remains to be determined the extent to which these mechanisms contribute to naturally occurring modifications in the brain. The mouse visual cortex provides an excellent model system for addressing this question. Visual cortex is well-known to be modified by simple manipulations of experience, such as depriving one eye of vision, and the mouse can be genetically modified to test specific hypotheses about molecular mechanisms. Moreover, while this type of plasticity changes over the course of postnatal life, it persists in adult mouse visual cortex and therefore can provide insight into how age alters the qualities of synaptic plasticity in the cerebral cortex. The aims of this proposal are to determine (1) the qualities of visual cortical plasticity across the life-span, (2) the contributions of LTD mechanisms to deprivation-induced response depression in vivo, (3) the contribution of LTP mechanisms to experience-dependent response potentiation in vivo, and (4) if changes in NMDA receptor subunit composition are permissive for experience-dependent response potentiation in vivo. These aims will be accomplished by taking advantage of the special expertise of this Center in mouse genetics, chronic recording, and the molecular bases for synaptic plasticity.

Lymphedema (LE) following treatment for breast cancer is the most common form of secondary LE in the industrialized world. It occurs in 20% to 87% of patients following treatment for breast cancer and results in significant disability. At the present time, the definitive phenotypic, genotypic and epigenotypic predictors that place patients at highest risk for the development of LE are not known. Therefore, the specific aims of this study, in a sample of patients following treatment for breast cancer, are to: determine genetic predictors of LE using a candidate gene approach and evaluate for epigenetic changes, as measured by DNA methylation and subsequent gene expression, in candidate genes associated with the diagnosis of LE. The secondary aims of this study are to: evaluate for latent classes of women with distinct phenotypic predictors of LE; and evaluate for differences in symptoms, functional status, and QOL outcomes between women with and without LE and among the latent classes with LE. The results of this study will provide new information on the underlying mechanisms for LE and allow for the development and testing of novel approaches to prevent or reduce the negative effects of LE.

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 central hypothesis of the project is that deubiquitinating enzyme UCH-L1 is an essential regulator of vascular lesion formation. The proposal will uncover a novel mechanism that UCH-L1 serves as a key molecule in assembling inflammatory signaling complex thereby fine tuning vascular inflammatory responses and lesion formation. It is our view that the outcome will provide novel insight into the understanding of the complex sequelae of inflammation in vascular lesion formation. Our hypothesis will be tested by uitilzing UCH-L1 gain- and loss-of-function approaches in vitro and in vivo to address three specific aims as follows; Aim 1. Define an essential role of UCH-L1 in regulating VSMC inflammation in vitro Aim 2. Define molecular mechanism of UCH-L1-mediated inhibition of VSMC inflammation in virto Aim 3. Determine an essential role of UCHL1 in regulating VSMC inflammation and vascular lesion formation in vivo

The goal of this research is to determine the neural basis of aggressive behavior, first in cats, ultimately in monkeys, with a view to clarifying the neural basis of aggressive behavior in man. We propose to observe naturally occurring aggressive behavior in cats with a view to determining the forms its takes, and to secure experimental control over its occurrence. We use aggressive behavior elicited by electrical stimulation of the brain to help us determine the mechanisms, and anatomical and physiological techniques to identify them. This knowledge will serve as the basis for the investigation of the aggressive behavior that occurs naturally. BIBLIOGRAPHIC REFERENCES: Huang, Y. H. and Flynn, J. P. Unit activities in the hypothalamus and midbrain during stimulation of hypothalamic attack sites. Brain Research 93: 419-440, 1975. Flynn, J. P. and Brandler, R. J. Patterned reflexes during centrally, elicited attack behavior. In: The Neural Bases of Violence and Aggression, University of Texas Press, 1975.

The purpose of this application is to request NCRR matching funds for the construction of a state-of-the-art research and training facility for the Psychology Department of Tufts University. The Psychology Department has 13 researchers engaged in a variety of collaborative bio-behavioral research endeavors focused on basic and clinical research questions in biological, cognitive and social psychology. These researchers have vibrant, well funded and growing research programs addressing important mental health issues including learning disabilities, dug addiction,, nutrition affects on behavior, anxiety disorders, infant motor and social development, mental retardation and social communication. However, their efforts are severely hampered by an aged/inadequate research and administrative facility, no capacity to expand, and by being housed in three by buildings The proposed plan addresses these problems by consolidating the department into two neighboring 21st century research facilities. The new 28,800 square foot Psychology Research Building (for which funds are being requested) will contain laboratories for researchers engaging in human research, for conducting neurochemical and neuroanatomical studies as well as offices for all faculty members, graduate students, post-docs, research staff and administrative staff. The second building, Bacon Hall (for which funds are not being requested), will continued to serve as the primary laboratory facility for faculty members who use experimental animals in their research programs. The consolidation of faculty, students, research and administrative staff into a new building, and the proximity of the two Psychology Department facilities will allow for much greater efficiency as well as future expansion for current and anticipated new faculty.

The goal of this project is to develop a unique skin coating formulation for use as a combination occlusive dressing and drug delivery vehicle as a topical treatment for psoriasis, a disease which affects 1% to 3% of the world s population. Currently used occlusive dressings are associated with local side effects and have limited patient acceptability because of poor cosmetic properties. The specific aims of the project are * to optimize skin coating formulations for use as a sustained-release steroid treatment for psoriasis * to evaluate skin coating formulations with encapsulated steroids in vivo in an animal model * to evaluate skin coating formulations with encapsulated steroids in vivo in a human clinical trial on psoriasis patients. PROPOSED COMMERCIAL APPLICATION: The cost of psoriasis care in the United States is estimated to be between $1.6 and $3.2 billion annually. By combining occlusion and drug delivery in a convenient, cost effective one-step process, we seek to improve outcomes and decrease the cost of treatment.

Sensory hypersensitivity is commonly seen in FXS patients and the FXS mouse model - the Fmr1 knockout (KO). Recent data suggests that this abnormality stems from hyperexcitability in sensory circuits. We have established that cortical microcircuits are hyperexcitable in the Fmr1 KO mouse model, and that sensory responses are enhanced in Fmr1 KO mice and FXS patients. Thus, investigation of sensory sensitivities is clinically relevant, but perhaps more important is the promise of sensory system studies to advance understanding of the mechanisms and consequences of hyperexcitability in neocortical circuitry that could represent a primary pathophysiological factor impacting the development of a wide range of perceptual, cognitive, and language skills in FXS. Further, we have identified biochemical signaling mechanisms that may underlie hyperexcitability involving processes that we and others have uncovered that can be examined in detail in KO mouse models and tested in FXS patients to develop a foundation for novel therapeutic development. The striking consistency of findings across levels of investigation and species offers an unprecedented opportunity to investigate mechanisms of brain dysfunction in a mouse disease model and translate it directly to patients - a multidisciplinary mission that is ideal for a Center environment. Our Center is organized to pursue precisely this aim with a tightly integrated and highly novel scientific program of translational research. Project 1 (Huber/Gibson; UTSW; co-investigators) will determine the cellular, molecular and synaptic mechanisms of auditory neocortical dysfunction using in vitro brain slices in FXS mouse models. Project 2 (Razak/Etheii/Binder; UCR; co-investigators) will study auditory sensory processing deficits in vivo in FXS mouse models, test mechanisms, and examine developmental and structural correlates of these deficits. Project 3 (Sweeney/Byerly, UTSW, co-investigators) will investigate auditory cortical processing deficits using novel neurophysiological strategies in individuals with FXS. All Projects will examine candidate mechanisms of sensory hyperexcitability with an acute pharmacological probe strategy to test mechanisms of interest in parallel studies of mice and patients.

In the proposed program we plan to study novel human glycosyltransferases. Much emphasis will be made on the identification and characterization of enzymes present in ovarian epithelial linings and fluids. Synthesis of various oligosaccharides which can be effectively employed as acceptor-substrates for certain glycosyltransferases have already been accomplished in our laboratory. A number of these synthetic substrates have proven to be very useful and valuable acceptors for glycosyltransferases from sources other than human. Our main strategy for the characterization of the product resulting from the reaction of a glycosyltransferase and its acceptor involves the synthesis of the expected saccharide which can be used as a reference compound. The structure of the saccharide isolated from enzymatic product will be established by n.m.r., mass spectroscopy, and methylation studies. We plan to employ glycosidases for the structural studies of the new product. Adequate use of newly acquired HPLC instrumentation will be made to facilitate these investigations. The proposed studies are directed toward the characterization of the spectrum of glycosyltransfeerases which are responsible for elongation of carbohydrate units O-glycosidically linked to Serine or Threonine. We are concentrating on the study of glycosyltransferases which have not been identified but whose existence is inferred from structural studies and the "one enzyme-one linkage" concept. Our studies will further elucidate the biosynthetic pathway of glycoconjugates. It is anticipated that glycosyltransferases of defined specificity, as proposed in this program in conjunction with glycosidases, may be of general value in further understanding the roles of specific oligosaccharide structures in the complex mileu of cell-surface glycoconjugates.

The aim of this application is to develop a rapid, easy-to-use, and inexpensive multi-sample diagnostic system to identify sexually transmitted infection (STI) pathogens, such as Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG) in non-traditional healthcare settings. According to the CDC, chlamydia and gonorrhea, caused by C. trachomatis and N. gonorrhoeae, respectively, are the first and second most frequently reported STIs in the US and likely the rest of the world. These STIs are often under-diagnosed, leading to delayed treatment, continued spread and higher public healthcare costs (~$16 billion annually). As a result of their prevalence and long term health consequences, there is a need for rapid, sensitive methods of detecting STIs in order for patients to get results and treatment immediately. It is important that patients receive results and initial consultation or treatment during the first patient visit, as they rarely return for a second visit. Point-of- care (POC) tests are an important strategy to address the STI epidemic, and because many affected by STIs (young and/or poor) often have limited access to traditional healthcare, POC tests that can be performed in non-traditional settings (university clinics, community public health care clinics, jails, detention centers) could greatly reduce STIs. The aim of this application is to develop a rapid, easy-to-use, inexpensive diagnostic system for identifying STI pathogens such as CT and NG. The system will combine AI Biosciences, Inc.'s novel nucleic acid extraction cartridge and a rapid real-time thermal cycler to form a multi-sample system for use in many non-traditional healthcare settings. In Phase I, a high performance, low-cost self-contained nucleic acid extraction and purification cartridge will be developed to perform a 10 min. extraction. The extracted NA samples will be placed in an thin-walled PCR card and amplified using an innovative water based cycler to perform rapid (20 min for 40 cycles) multiplexed real-time polymerase chain reaction (PCR) for CT and NG targets. We will demonstrate that the entire assay can be completed in less than 30 min. The proposed work will be carried out in collaboration with STIs and fluidic/thermal characterization experts. Our sample preparation cartridge and the PCR technologies are highly suitable for parallel, automated sample processing. This is a substantial advantage over other integrated systems being developed as all of them can only process one sample at a time, making several units necessary to meet the minimum throughput demand. The successful development and commercialization of our technology will impact how sophisticated molecular diagnostic assays can be implemented in non-traditional healthcare settings.

Gene delivery to the liver for inherited metabolic disorders provides the opportunity for specific treatment of two forms of glycogen storage disease (GSD). Glycogen accumulation due to single enzyme defects represents the prototype of metabolic storage disease. A detailed biochemical understanding and availability of mouse models for GSD Ia, and GSD II make these conditions excellent examples for correction of the metabolic defect in the liver by gene replacement therapy. In order to demonstrate the feasibility of this approach, Dr. Byrne and his colleagues propose to investigate the AAV-mediated delivery of the gene for glucose-6-phosphatase (G6P) and, acid alpha-glucosidase (GAA) in animal models of GSD I and II. The proposed study is designed to examine the ability of AAV to direct the sustained hepatic expression of G6P and GAA in murine models GSD. The vector has been shown to yield high-level, long-term expression of a number of therapeutic proteins without eliciting a clinically significant immune reaction. The investigators have recently demonstrated high-efficacy gene transfer of GAA into embryonic tissues, cultured adult and neonatal rat cardiomyocytes, as well as, adult rat heart, and murine skeletal muscle in vivo using this approach. Substantial preliminary evidence in his laboratory and others demonstrates the utility of over-expression of therapeutic proteins in hepatic tissues. In principle, this system has the capability to deliver the therapeutic protein to all tissues via secretion from the hepatic platform. Additionally, correction of G6P deficiency examines the important question of direct correction of a microsomal enzyme defect by hepatocyte transduction. They now propose to evaluate this potential by assessing the effectiveness and biological impact following AAV-mediated reconstitution of G6P and GAA in animal model of GSD. The effectiveness of several hepatic specific promoters will be tested in vitro using immortalize cell lines from the representative mouse models. One of the important considerations of systemic delivery of corrective vectors will be evaluated by new technologies using MR imaging and MR spectroscopy. The efficiency of processing and targeting of lysosomal enzymes will be examined by strategies which allow for the augmentation of the phosphotransferase enzyme involved in lysosomal enzyme secondary processing or by anti-sense of critical proteins which control lysosomal targeting and thereby result in a preference for the secretory pathway. These studies will yield important new information in establishing a clinically relevant treatment for these fatal diseases and add new understanding to the basic pathophysiology of GSD.

Use of Engineered Nervous Tissue Constructs to Repair Extensive Nerve Injury Each year approximately 360,000 people in the United States suffer a peripheral nerve injury, which is a leading source of lifelong disability. While a primary strategy to repair major peripheral nerve injury is to bridge the damage with autologous nerve grafts, producing nerves of sufficient length and number has posed a significant challenge. The so called gold standard in peripheral nerve graft repair (i.e. the autologous nerve graft) is limited by the time consuming harvesting of donor nerves and complications arising from the harvesting surgery. In addition, most alternative methods currently used for nerve graft repair (e.g., synthetic tubes) are limited in the length that they can span to promote repair and are typically used for gaps of less than 2 or 3 cm. Here, we propose to utilize a novel tissue engineering technique to create transplantable nervous tissue constructs for major peripheral nerve repair. The key of this procedure is to use a specially designed microstepper motor system to produce continuous mechanical tension on axons spanning two populations of neurons in culture. As our central hypothesis, we propose that engineered living nervous tissue constructs will promote recovery after major peripheral nerve injury by providing a living labeled pathway to guide host axons from the proximal nerve stump across large nerve lesions to reinnervate the target tissue. In addition to providing a pathway through the injured gap, we also propose that axons from the construct will grow into both the proximal and distal nerve stumps. In the first part of this proposal, we will repair acute extensive acute nerve injury in an animal model using our novel living engineered nervous tissue constructs. In the second part of this study, we will attempt major reconstruction of brachial plexus injury in rodents, spanning the forelimb from the vertebrae to the paw using our living nervous tissue constructs. We will directly compare the outcome of animals receiving the nervous tissue construct containing the elongated dorsal root ganglion cell cultures with groups receiving conventional autologous nerve reconstruction (reverse autologous graft), repair with a synthetic tube alone (material substrate control), no repair (no treatment control), immunosuppression control (receiving daily cyclosporine A injection) and physical rehabilitation control. In the third part of this study, we will assess the immunogenicity of our nervous tissue construct. We will investigate the hypothesis that the lack of MHC I expression in our elongated DRG neurons is the main reason that immunorejection does not occur in transplanted hosts. If successful, our novel tissue engineered nerve construct could revolutionize methods for major nerve reconstruction by providing laboratory grown 'off-the-shelf' living nerves ready for transplant in patients with extensive peripheral nerve injuries. PUBLIC HEALTH RELEVANCE: In this study we propose to use a fundamentally novel tissue engineering technique to create nerve constructs for repair of extensive nerve injury in an animal model. If successful, our findings could benefit patients with devastating nerve injuries by providing laboratory grown 'off-the-shelf' living nerves ready for transplant.

This project focuses on a protein phosphatase that interacts with the phosphorylated form of RLK5, a receptor-like protein kinase from Arabidopsis thaliana. RLK5 has features characteristic of the growth factor receptor kinases, including an extracellular domain of tandem leucine-rich repeats, a transmembrane domain, and a C-terminal serine/threonine protein kinase catalytic domain. Numerous serine/threonine receptor kinases have been identified in higher eukaryotes including the TGF-?/activin receptor superfamily in animals and the receptor-like kinases from higher plants. However, the mechanisms by which these receptors transduce a signal is unknown. Interaction cloning was used to identify an Arabidopsis protein which associates with the catalytic domain of RLK5. This protein, which has been termed KAPP for kinase associated protein phosphatase, consists of three functional domains: an N-terminal type one signal anchor, a central kinase interaction (KI) domain responsible for interaction with RLK5, and a C-terminal region with homology with type 2C ser/thr protein phosphatases. KI domain association requires phosphorylation of RLK5 as shown by in vitro binding assays. Interaction of a protein with the phosphorylated form of a receptor protein kinase is reminiscent of the interaction of SH2 domain-containing proteins with tyrosine kinases. SH2 domains bind with high affinity to specific phosphorylated tyrosine residues of active tyrosine kinases and are key components of many intracellular signal transduction cascades. Interaction of the KI domain of KAPP with phosphorylated RLK5 suggests that this may be a general mechanism for receptor protein kinase-mediated cellular signaling common to both receptor tyrosine and receptor serine/threonine protein kinases. The autophosphoryl-ation sites in the catalytic domain of RLK5 will be mapped with MALDI-MS.

Candida albicans is the most common fungal pathogen of humans. Its clinical importance has increased in recent years as the number of immunocompromised patients (AIDS, transplant recipients, cancer, corticosteroid treated, etc.) has expanded and the incidence of opportunistic infections has escalated. The long-term objectives of this grant are to understand the fundamental mechanisms by which pathogenic fungi communicate with each other and with their environment within human hosts, and how host hormones may regulate critical fungal processing during infection. We have described the presence of steroid binding proteins (SBPs) in C. albicans and other pathogenic fungi. These SBPs exhibit high affinity for selected mammalian steroid hormones, particularly estrogens and corticosteroids. We have also demonstrated functional responses in the fungi subsequent to hormone treatment by steroids that bind to the SBPs. These findings have led us to raise 4 hypotheses: (1) the SBPs represent hormone receptors in the fungi; (2) the fungi have endogenous hormones to match each SBP; (3) since the host hormones bind to the SBPs, the fungus responds to the hormonal milieu of the host, thus mediating physiological changes in the invading pathogen; and (4) the SBPs are evolutionarily related to the steroid hormone receptors of higher organisms. We postulate that the host-pathogen interaction contributes to differences in susceptibility to infection depending on the hormonal status of the host (e.g. gender, menstrual stage, pregnancy, treatment with birth control pills or corticosteroids, etc.). The grant will focus on the corticosteroid binding protein (CBP) and the estrogen binding protein (EBP) found in c. albicans. The aims of the grant will be: (1) to clone the genes encoding the SBPs and raise antibodies to the proteins; (2) using these molecular probes, to define the location and function of the SBPs in C. albicans; (3) among other actions, to evaluate the role of SBPs in regulating cyclic AMP, dimorphism and cellular adhesion, actions that would likely affect fungal pathogenicity; and (4) to explore the evolutionary relationship of the Candida SBPs to homologs in other pathogens, both fungal and protozoan, as well as to other species up the evolutionary tree including human. Our long-range goal is to understand better the biology of pathogenic fungi so that advances can be made in the elucidation of the molecular basis of the epidemiology of human infection and improved methods can be developed for the diagnosis and treatment of fungal disease.

Cognitive memory and habit formation are two qualitatively different learning processes based on separate neural systems, a cortico-limbic and a cortico-nonlimbic system, respectively. To see how emotional and social behavior develop in animals whose infantile global amnesia might persist from infancy through adulthood, we have prepared monkeys with neonatal limbic lesions and followed their behavioral development. Animals with neonatal removal of cortical area TE, a higher-order visual station linked to both learning systems, serve as controls. The results indicate that neonatal TE lesions leads to a transient impairment of habit formation three months later (compared to permanent impairment seen with the same lesion in adults), whereas both neonatal and adult limbic lesions leave habit formation intact. Interestingly, data on both normal and operated infants suggest that development of the nonlimbic habit system is sexually dimorphic, and that this is due to the high testosterone levels present in male infants before and shortly after birth. At ten months of age, the infants with limbic lesions show impairment in memory formation, whereas the operated controls show significant functional sparing (compared to those that received the same lesions as adults). These findings point to greater compensatory potential after neonatal cortical than after neonatal limbic removals, indicating that association areas of the cortex are immature at birth, and may thus possess greater plasticity than limbic structures. Direct evidence of neocortical immaturity in the macaque has been provided by our neurobiological studies on opiatergic and cholinergic receptor distribution and on metabolic activity. Finally, early damage to the limbic memory system produces later socio-emotional abnormalities that are similar in many respects to the behavioral syndrome seen in autistic children.

This is an open-label multiple dose study in which patients with advanced solid tumors will receive a single dose of losoxantrone as 14C-labeled compound during their first course of treatment. Blood, urine, and feces will be collected for analysis. Following enrollment, patients will receive up to 8 courses of losoxantrone, once every 3 weeks, depending on response to treatment and tolerability.

This R13 application proposes to support a 2-day annual meeting to enhance the collaborative network, and maximize the long-term national impact of the National Consortium for Multicultural Education for Health Professionals (Consortium). Funded in 2004 by NHLBI as a 5-year competitive training award program (K07) for U.S. medical schools, the Consortium uses the approach of addressing disparities by integrating cross- cultural education into health professional training. We propose to build on our success by implementing a meeting focused on the role of reflective practice to support health professional education in cultural competency and health literacy. To reduce costs, increase participation, and maximize collaboration, the meeting will be scheduled to coincide with the American Public Health Association annual meeting to be held November 2014 in New Orleans, Lousiana. The goal of this conference is to provide an opportunity for experts in cultural competency and health literacy curriculum development to network with leaders in public health education, and researchers on the impact of unconscious bias on health outcomes to further build upon the exchange of ideas which began recently among research and practice professionals in cultural competency and health literacy. Specific aims include: 1) create a network of experts in cultural competency and health literacy curriculum development and research, 2) identify common competencies, approaches and assessment methods that can be applied to education and research in cultural competency for multiple health professions; and 3) advance research and publication potential of the combined fields of cultural competency and health literacy. The meeting comprises a: 1) scientific panel session (n=30-100) on use of reflective pedagogy to teach about and assess unconscious bias that impacts patient health outcomes, (2) Learning Institute (skills- building workshop) on preparing submissions to MedEdPortal with the Association of American Medical Colleges (n=20), and 3) working session (n=30) to bring together experts from different health professions to design curriculum addressing unconscious bias for an interprofessional setting.

Abstract Phasing, defined as the estimation of haplotypes from diploid genotype data, is a fundamental problem in medical and population genetics. Phasing is a key preprocessing step for genotype imputation algorithms employed in genome-wide association studies of diseases and complex traits, and is also important for mapping molecular QTL using allele-specific reads, detecting clonal mosaicism, inferring population structure, and detecting natural selection. Considerable resources have been invested into developing accurate phasing algorithms, but currently, unsolved challenges include: (i) incorporating large reference panels, such as the Haplotype Reference Consortium, to improve phasing accuracy (reference-based phasing), and (ii) phasing extremely large cohorts using within-cohort data (cohort-based phasing). Here, we propose an exploratory two-year research program, in which we will develop methods and software for both reference-based phasing, and cohort-based phasing, using a new data structure based on the Positional Burrows-Wheeler Transform (PBWT). We aim to make fast and accurate phasing methods and software freely available to all researchers via public phasing servers. We will also explore the early and conceptual stages of developing PBWT-based methods for reference-based imputation as well. Our team has multiple strengths: our statistical and computational expertise; our track record of producing practical, publicly-available software packages for a broad range of applications in statistical genetics that are widely used by the community, and our data-driven approach to methods research. We will guide our methods development using data from 500,000 samples from the UK Biobank, and will work closely with the Haplotype Reference Consortium (see letters of support).

Our aim is to understand chemotactic migration in cells from a biochemical standpoint. To this end we have investigated this process in both phagocytic cells and connective tissue-type cells. Studies on the regulation of phagocyte chemotaxis, using formylated peptides attractants, have indicated that a tumor-derived peptide inhibits migration by reducing receptor availability, inhibiting hydrolysis of the attractant, and decreasing membrane lipid turnover. Receptor mediated internalization of attractant may be required for chemotaxis since inhibitors of translutaminase affect both processes in a well-correlated manner. Glucocorticoids, anti-inflammatory agents, appear to exert their effect by inducing the synthesis of a phospholipase A2 inhibitor. Actual locomotion involving microtubules may depend upon attractant-stimulated addition of C-terminal tyrosine to tubulin. Studies on chemotaxis in connective tissue-type cells have shown that fibroblasts respond to the cell binding domain of intact fibronectin and to platelet-derived growth factor; that angiogenesis factors induce endothelial cell migration, and that transformed endothelial cells respond to angiogenesis factors, platelet-derived growth factor, and fibronectin.

The objectives of this research is to study the chemical and structural properties of the ACTH receptor system in adrenal membranes. ACTH analogs containing fluorescent probes will be used for spectroscopic measurements of the binding interactions. Other analogs will be synthesized for use as ACTH inhibitors. The structural relationships between the hormone receptor and adenylate cyclase will be studied in attempts to solubilization and fractionation of membrane components. Ectopic receptors for hormones in a rat adrenal carcinoma will be characterized by measurement of their binding affinities for peptide hormones and catecholamines.

PROJECT SUMMARY The secretory pathway employs vesicle transport to provide a linear pathway for export of cellular products and distribution of membrane and organelle components throughout the cytoplasm. Many diseases, including neurodegeneration, involve disruption of the biosynthetic secretory pathway and unresolved secretory stress--making it essential to understand how secretion is up- and down-regulated under different physiological conditions. While the basic engines of vesicle budding, docking and fusion have been identified, little is known of how they are tuned to respond to physiological conditions and stresses. In mammals, ER-to-Golgi transport, which represents the rate-limiting step in the secretory pathway and the step most relevant to transport-related diseases, has been extensively characterized in vivo and reconstituted in vitro. In broad terms, ER-to-Golgi transport has been shown to comprise: 1) cargo sorting and vesicle budding mediated by the COPII vesicle coat; 2) homotypic COPII vesicle tethering and fusion mediated by tethers and SNAREs to form pre-Golgi organelles called vesicular tubular clusters (VTCs); and 3) VTC-mediated cargo sorting and transport along microtubules leading to fusion with the Golgi. Little is known about how these processes are adjusted dynamically to match secretory output rates with the needs to enforce secretory quality control, avoid ER stress, and keep pace with secretory protein biogenesis and cell growth. One key aspect to regulation of ER-to-Golgi transport that has become apparent in recent years is the role of ER luminal calcium. Calcium, when released from the ER by channel proteins appears to interact with penta-EF hand proteins (PEFs) in the cytoplasm that bind to the COPII coat and modulate its assembly and the rate of cargo egress from the ER. However, the mechanisms of these proteins to produce different secretory outcomes and how they are integrated with ER calcium homeostasis and calcium channels are not understood. This project will employ kinetic assays of ER-to-Golgi transport in intact mammalian cell lines, live-cell calcium measurements, and in vitro studies of PEF protein interactions with vesicle machinery to elucidate how how calcium channels, PEF proteins, and COPII components are integrated to dynamically regulate secretion rate. These studies will have wide significance because proper regulation of secretion is fundamental to key cell survival and stress pathways.

Neurons communicate by establishing connections, or synapses, with specific partners. Mechanisms that control the fidelity of these choices are poorly defined but are likely to depend on specific gene regulation. In the nematode, C. elegans, we have shown that the UNC-4 homeoprotein and transcriptional co-repressor, UNC-37/Groucho, function in VA motor neurons to block inputs normally restricted to sister VB motor neurons. We developed cell specific microarray profiling methods to identify UNC-4 regulated genes in this pathway. One of these targets, CEH-12, is homologous to the conserved homeodomain transcription factor, HB9, a known determinant of motor neuron fate in mammals. We used a powerful new strategy for visualizing motor neuron inputs to confirm that CEH-12/HB9 is in fact a VB gene normally repressed by UNC-4 to prevent the adoption VB-type inputs. These experiments also showed that this CEH-12 function is restricted to VA motor neurons in the posterior nerve cord and therefore suggest that UNC-4 must regulate other downstream genes to control synaptic inputs to anterior VAs. RNAi and genetic tests of additional candidate UNC-4 target genes from our microarray data sets have revealed that UNC-4 also negatively regulates the Frizzled protein and Wnt receptor, MOM-5. We hypothesize that a posterior source of Wnt signal acts through MOM-5 to drive ectopic expression of CEH-12 and VA miswiring in unc-4 mutants. A major goal of this project is to test this model by defining the molecular components of this Wnt signaling cascade and its mechanism of action. The existance of A/P gradients of Wnt signaling in the vertebrate spinal cord could be indicative of a similar Wnt dependent mechanism for specifying A/P differences in motor circuit connectivity. To identify unc-4 target genes that function in anterior VA motor neurons, we used high throughput genetic screens to isolate new mutations that "suppress" the Unc-4 movement defect. VA inputs in these mutants will be assessed with GFP labeled gap junction and synaptic proteins to confirm unc-4 pathway function. The DNA sequences of these UNC- 4 targets could be used in the future to identify similar genes in mammals where their roles in synaptic specificity can be explored. Thus, our work with a simple model organism is likely to provide important clues to fundamental processes governing the development of complex neural circuits in the the human spinal cord. PUBLIC HEALTH RELEVANCE Nerve cells (neurons) project elongated processes (axons) from the brain into the spinal cord to make connections or synapses with motor neurons that drive body movements. To facilitate the identification of genes that control the specificity of these connections, we are using the nematode, C. elegans, a model organism with a simple, well-defined nervous system. The results of this work should reveal similar human genes with crucial roles in the creation spinal cord circuits and therefore potentially contribute to the development of treatments for spinal cord injury and dysfunction.

Hepatitis C virus (HCV) is a major cause of community-acquired viral hepatitis. Prototype strains of the various genotypes of HCV, including some of those discovered in this laboratory, are being biologically amplified in chimpanzees, packaged and distributed for use as challenge inocula in studies of passive and active immunoprophylaxis, etc. Full-length cDNA clones of HCV (genotypes 1a, 1b and 2a) have been constructed and transcribed RNA used to transmit hepatitis C to chimpanzees by in vivo hepatic transfection. Chimpanzees, transfected with infectious cDNA clones of HCV, are being followed to determine the natural history of infection. Infectivity pools have been prepared from chimpanzees infected with monoclonal HCV (derived by in vivo transfection with RNA transcripts of infectious cDNA); these have been titered for infectivity in other chimpanzees. In addition, the availability of infectious cDNA clones of HCV has permitted for the first time a mutational analysis of genomic regions. For example, individual portions of the 3' NCR have been deleted from the full-length clone and the resultant deletion mutant clones inoculated into chimpanzees by intrahapatic transfection. Certain regions of the NCR have been identified as critical for in vivo replication of HCV. We have constructed an infectious cDNA clone of GB virus-B (GBV-B), a monkey virus that is the closest relative to HCV. In addition, we have prepared challenge pools of GBV-B and have determined the infectivity titer of these in tamarins. We plan to use the GBV-B tamarin system to study characteristics of the virus that it shares with HCV, which must be studied in chimpanzees. In other studies, we have constructed chimeric genomes from infectious cDNA clones of HCV and bovine viral diarrhea virus. These genomes can replicate in transfected cells but the resultant viral products cannot assemble into infectious virus in the absence of helper virus.We have determined the genetic heterogeneity of HCV isolates that were recovered from patients who were infected following transfusion. The sequence of the hypervariable region and adjacent portions of envelope proteins 1 and 2 were determined for multiple clones obtained from patients who had fulminant hepatitis, from patients who convalesced following acute hepatitis and from patients who progressed to chronic hepatitis C. Distinctive patterns of dynamic change in sequence of clones during the first several weeks of infection were observed. Patients with fulminant or resolving hepatitis had few changes in the sequences of clones, whereas there were many changes in the sequences of clones from patients who progressed to chronic hepatitis. Thus, the outcome of an HCV infection could be predicted in the first few weeks of the infection

Glucose 6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, isocitrate dehydrogenase and malic enzyme share a common pyridine nucleotide (NADP) as coenzyme. Activities of these enzymes are coordinated to generate adequate pools of NADPH for lipid biosyntheses. Biochemical comparisons of the parameters of pyridine-linked dehydrogenases suggest a common evolutionary origin of these enzymes. In Drosophila melangaster there appears to be two forms of NADP-isocitrate dehyrogenase and there may be two genes controlling the activity of this enzyme. A molecular characterization of this gene(s) and the transcript(s) made from it is proposed as the initial step in understanding regulation of this gene-enzyme system. Expression libraries of Drosophila cDNA will be screened for sequences specific for IDH. The identity of these cloned sequences will be verified by in situ hybridization to salivary gland polythene chromosomes and immunoprecipitation of in vitro translated hybrid selected mRNA. Clones which are identified by these techniques will be used to isolate corresponding sequences of genomic DNA. Restriction analyses will compare cDNA and genomic sequences. The developmental expression of these sequences will be determined by Southern analysis. Hybridization of strand specific transcripts of cDNA to "Northern" blots of appropriately staged pupal mRNA will allow determiniation of the orientation of transcription. Future experiments would include S1 nuclease mapping and primer extension analysis.

Most of the human population world-wide has been infected by herpes simplex viruses. Following the initial lytic infection, HSVs establish permanent latent infections within sensory neurons. Reactivation of latent virus not only results in viral disease (new infections, blindness, and encephalitis) but also contributes to HIV infection, diabetes, cardiovascular and neurodegenerative diseases. No effective vaccine is available and no therapy eliminates latency or prevents reactivation. The long-term goal of this project is to find interventions for recurrent HSV episodes by defining mechanisms that control establishment and reactivation of HSV-1 latency. The gene expression cascade during HSV-1 lytic infection begins with activation of immediate-early (IE) gene transcription by the virion protein VP16 with host factors Oct-1 and HCF-1. In contrast, the initial events in the reactivation from latency are still poorly defined. The central hypothesis of this proposal is that regulation o both VP16 expression and activity underlie the establishment of latency and reactivation from latency. These two levels of control involve multiple positive and negative inputs to allow or inhibit viral replication in the sensory neuron in vivo. Aim 1. This project will determine the mechanism of de novo VP16 gene activation and silencing in sensory neurons in vivo. The working hypothesis is that the VP16 gene in the HSV-1 genome can be regulated by action of neuron-specific and stress-responsive promoter elements and corresponding transcription factors either to allow or inhibit lytic replication upon initial infection or exit from latency. Uing recombinant viruses in a mouse model of infection and latency, we found that expression of VP16 is both necessary and sufficient to trigger the exit from latency and we identified a neuron-specific promoter for the VP16 gene. We will test whether this promoter controls the entry into lytic phase infection during acute infection and during reactivation. We will determine whether the predicted transcription factors bind to the various elements of this promoter to positively or negatively regulate VP16 gene expression, whether singly, in combination, or in competition. Aim 2. This project will define the VP16 coactivator interactions essential for VP16-dependent exit from latency and identify mechanisms regulating these interactions in vivo. Our data strongly suggest that the exit from latency by HSV is regulated by CK2 mediated phosphorylation and that this phosphorylation may be also competitively regulated by O-GlcNAcylation (a PTM that regulates signaling in response to nutrients and stress). Our goal is to elucidate the functions of each of these PTMs in viral latency and to define the roles of their crosstalk in regulating immediate early gene expression of viral proteins through VP16 transactivation. The outcomes of this work will identify transcription factors or protein modifying enzymes that could be targets for future development of therapeutic interventions for HSV reactivation.

Since its inception in 1984, the goal of the CSHL FACS Shared Resource has been to give Cancer Center members access to state-of-the-art flow cytometry equipment and support. The Resource has evolved with emerging technologies and changing research needs. In 1984, the primary use was separation of cells by DNA content or by expressed cell-surface antigens. The advent of fluorescent markers (such as the green fluorescent protein GFP) ushered in a new phase in FACS usage, as Cancer Center members used GFP to track transfected cells, and to isolate particular populations of cells from complex mixtures. Increases in the speed and efficiency of sorting by FACS have enabled completion of experiments that once would have been impossible or impractical[unreadable]such as the isolation of large quantities of tumor or stem cells for genomic and genetic analyses. The recent introduction of the new generation of proteins that fluoresce in various colors and are less toxic than their predecessors, changed the requirements for flow cytometers and increased the demand for the Resource. In response to these demands, the FACS Shared Resource has undergone an extensive series of changes[unreadable]in location, instrumentation, and personnel[unreadable]designed to expand FACS capabilities at CSHL, and improve the efficiency and reliability of flow cytometry for Cancer Center members.

Oral pre-exposure prophylaxis (PrEP) against HIV infection has demonstrated efficacy in men who have sex with men, serodiscordant couples, and young heterosexuals; however, efficacy was not shown in two studies of women. Lack of adherence is the leading hypothesis for these discrepant findings. Adherence outside of rigorous clinical trials is unknown and is essential to know in order to determine whether PrEP will be an effective public health strategy. The proposed study will determine if HIV-uninfected people in serodiscordant couples outside a clinical trial will adhere adequately enough to PrEP to be protected against HIV transmission. Approach: This study builds on our experience conducting the Adherence Sub-study within the Partners PrEP Study (a phase III clinical trial of tenofovir and emtricitabine/tenofovir PrEP) and will be integrated into a recently funded study of a public health delivery model of antiretroviral therapy-based HIV prevention strategies involving 500 serodiscordant couples in Uganda and Kenya (R01MH095507, PI: Baeten). Our Specific Aims are a follows: 1) Assess the level, trajectory, and correlates of PrEP adherence in research-naive discordant couples receiving PrEP in a public health delivery model with adherence counseling practical for scale-up; 2) Determine the relationship between patterns of adherence and risk exposure outside of a clinical trial; and 3) Determine if the level and patterns of real world adherence and risk exposure determined in Aims 1 and 2 substantially alter current estimates of PrEP effectiveness and cost-effectiveness. Innovation: We will: 1) characterize PrEP adherence in serodiscordant couples recruited into a demonstration project of PrEP delivery involving limited adherence counseling that is practical for scale-up settings; 2) use a novel conceptual framework to understand PrEP adherence behavior; 3) use text message reports of sexual behavior combined with electronic adherence monitoring to characterize temporal patterns of sexual exposure and PrEP adherence; and 4) develop models of the tenofovir drug concentration-time relationship, using prospectively collected samples, which can be used to determine which patterns of adherence are likely adequate for protection against HIV infection. Investigators: Dr. Jessica Haberer is a new investigator with significant experience studying adherence to antiretroviral therapy and PrEP in sub-Saharan Africa. Her team includes expertise in clinical epidemiology (Drs. Jared Baeten and Connie Celum), adherence measurement (Dr. David Bangsberg), qualitative analysis (Dr. Norma Ware), tenofovir pharmacokinetic modeling (Drs. Craig Hendrix and Ayyappa Chaturvedula), statistical analysis (Dr. Deborah Donnell), cost-effective modeling (Dr. Tim Hallett), and sub-Saharan African research experience (Drs. Nelly Mugo and Elly Katabira).

DESCRIPTION (based on applicant's abstract): The research contained in this application will focus on host factors that modulate HIV infection. Specifically, the application will concern the role of the human fetal thymus and placenta in in utero HIV infection and the unexpected consequence of early spontaneous abortions suggested by evidence gathered on Baylor grant R01 AI32466-04, "Role of the Placenta in Transmission of HIV." The long-term objective of this research application is directed at answering critical questions of the importance of host factors in the early HIV immunopathogenesis in vertically-acquired HIV infection acquired in utero. The specific aims are to: 1) explore the role of the thymus in early rejection of the HIV-infected fetus in first and second trimester spontaneous abortions in HIV-infected women, 2) determine the role that cytokines in the placenta play in fetal loss in HIV infection, and 3) compare the phenotypic/genotypic characteristics of HIV in the early trimester aborted HIV-infected fetus to those in the mother. The applicant proposes to explore the immune mechanisms of rejection of the fetus, possibly mediated by factors (inflammatory cytokines, interleukin-1 beta, interleukin-6 and tumor necrosis factor-alpha) derived from the placenta or by the fetus as a consequence of HIV infection. The project will utilize an existing model of HIV infection, a fetal thymus organ culture (FTOC) system. This model system of HIV infection will allow the careful interpretation of the importance of timing of HIV infection and viral burden/viral strain in early fetal death. The methods to be used include: 1) recruitment of HIV-infected pregnant women in a Pediatric/Obstetrical Research Center; 2) pathological examination of fetal and maternal products of conception, including in situ hybridization studies; 3) denaturing gradient gel electrophoresis, single strand conformation polymorphism, gene sequencing, and T cell and macrophage/monocyte tropism studies of HIV strains recovered from fetus and mother; 4) measurement of mRNA and protein product of interleukin-1 beta, interleukin-6, and tumor necrosis factor-alpha; and 5) HIV infection (and pathological consequences) in an FTOC system in vitro.

Although there is an important genetic contribution to the normal range of variation seen in human behaviors as well as human behavioral disorders, very little is known about the genes that influence behavior in any organism. In particular, there are currently few model vertebrate systems in which the study of behavioral variation in a natural, outbred population can be genetically dissected. The recent development of genetic and genomic tools for the threespine stickleback (Gasterosteus aculeatus) presents an opportunity to study the genetic basis of behavioral variation in a well-defined ecological and evolutionary context. The mating behaviors of the threespine stickleback are perhaps the best-studied of any animal system. Previous work has shown that divergence in male and female mating behaviors leads to reproductive isolation between stickleback populations that have adapted to living in two different habitats ('benthic'and 'limnetic') in a single lake. There are multiple lakes in British Columbia in which these benthic and limnetic 'species pairs'have evolved, and similar changes in mating behavior have occurred independently in each lake. The aim of this study is to investigate the genetic underpinnings of behaviors involved in reproductive isolation between benthic and limnetic sticklebacks from two lakes (Priest and Paxton) using quantitative trait locus (QTL) mapping. These experimental populations will be raised and behaviorally evaluated under fully natural conditions in outdoor ponds. By investigating the genetic basis of behavioral phenotypes involved in reproductive isolation (e.g., male nesting behavior and female mate preferences), this work will test whether parallel species pairs of sticklebacks in isolated lakes share similar genetic architectures for convergent behaviors involved in reproductive isolation. This project will also test whether QTL influencing behaviors involved in reproductive isolation map disproportionately to genomic regions affecting traits known to be important for mate selection, such as body size and shape. Importantly, the QTL mapping experiments proposed here will serve as a first step in the identification of genes that underlie behavioral differences in natural stickleback populations. Results in sticklebacks are likely to have important implications for understanding the genetic basis of behavioral variation and behavioral disorders in humans because many of the genetic and neural pathways that underlie behaviors are likely to be conserved between fish and humans. In addition, humans and sticklebacks have migrated and adapted to new environments over a similar evolutionary timeframe, suggesting that studying the genetic architecture of behavioral variation in sticklebacks has direct relevance to similar studies in humans.

The main purpose of this project is to test the efficacy of epsilon-aminocaproic acid administered during the 14 day period immediately after the bleeding episode. The activities of the last year have been mostly those of data collection; the randomized portion was suspended because EACA was shown to be superior at the time. Supporting data are included.

Cerebral malaria (CM) is a severe and potentially fatal neurological manifestation of disease caused by infection with Plasmodium falciparum. Despite effective anti-malarial therapy, approximately 25% of CM survivors develop long-term neurological deficits, such as memory loss and motor coordination impediments. The mechanisms that mediate this neurocognitive impairment are not well understood. Neuronal injury has been associated with the neurological deficits in several neurodegenerative diseases and may contribute to the impairment seen in CM. In this regard, damage to neuronal axons has been observed in both human and murine experimental CM (ECM). Furthermore, improper regulation of tau protein, an axonal protein important for microtubule stability and cytoskeletal organization, has been demonstrated in mouse and human disease. The neuronal injury and neurocognitive impairment observed in CM may result, in part, from abnormalities in tau. We hypothesize that pathological tau is one of the main mediators of neuronal damage and subsequent neurocognitive decline in CM. To address this hypothesis, we propose the following specific aims: AIM1: To characterize the role of tau in ECM. Aim1a: To examine the localization and secretion of hyperphosphorylated tau in the brains of mice with ECM and compare our findings to mice with malaria-associated severe anemia and uninfected mice. Aim1b: To characterize the contribution of tau to the neuronal damage and the subsequent neurocognitive deficits which occur during ECM using both a monoclonal antibody against phosphorylated tau and tau-knock-out (KO) mouse experiments. AIM2: To determine the role of tau in an in vitro human CM model. We will challenge cultured immortalized human neurons with supernatants obtained from blood-brain barrier co-cultures of astrocytes and endothelial cells (with and without P. falciparum exposure) and determine the effects on neuronal morphology and function as well as tau protein. We will also quantify secreted and intracellular tau levels and examine whether targeting the protein immunotherapeutically with PHF-1 antibody reverses neuronal damage. Our goal for this project is to further establish tau as a significant contributor to the pathogenesis of CM. Targeting this protein therapeutically may prevent both damage to neurons as well as the subsequent neurocognitive impairment which occurs during disease.

The goal of the proposed program is to address several technical feasibility questions (in Phase I), and then demonstrate (in a subsequent Phase II) incorporation of Sonetics'groundbreaking CMUT-in-CMOS ultrasound transducer technology into a novel, high-performance 2D ultrasound array suitable for commercial applications. Phase I specific aims will investigate bandwidth improvement for 2D array elements, scaling and enhancement of on-chip readout circuits, and reduction of dielectric charging effects related to device membrane sealing layers. This phase will culminate with the fabrication and characterization of a 64-element 2D array prototype, which will be used to evaluate the imaging potential of a larger scale array. Full implementation into a prototype scanhead (including a 128x128 element CMUT array) suitable for real-time 3D imaging would be demonstrated in Phase II, with the long-term goal of commercializing the technology (as well as other CMUT-in-CMOS-based products) for the ultrasound equipment market, forecast to be $3.75B globally by 2010. The academic segment of this market would benefit from the practical realization of fully-populated 2D ultrasound arrays, which will improve the quality and speed of 3D imaging for disease diagnosis. As well, the potential of CMUT-based scanheads to reduce the largest cost element of many state-of-the-art ultrasound systems, while simultaneously exhibiting improved imaging capability, will provide increased opportunity for researchers and students to undertake real-time, in vivo studies of living systems-something 3D ultrasound is uniquely positioned to do from a cost, availability, and convenience perspective (vs. MRI, PET, or CT, for example, all of which require expensive, dedicated facilities and technicians). The clinical market stands to gain similarly, as 3D ultrasound provides safe, convenient, real-time imaging technology-enabling improved triage and better patient outcomes, lower-cost imaging, and reduced staffing needs, all of which will serve the U.S. healthcare industry well as it seeks to control skyrocketing costs. PUBLIC HEALTH RELEVANCE: Potential benefits to public health from the successful development of Sonetics'novel ultrasound transducer technology include: improved availability and affordability of high-quality 3D medical imaging for disease diagnosis;improved medical training as low-cost 3D ultrasound enters the classroom;and lowered health-care costs for society as a whole, as better ultrasound improves clinical triage and patient outcomes. Furthermore, ultrasound is the only technology with the potential to become a convenient, safe, real-time imaging tool for use in limited-budget facilities such as lowincome health clinics.

Anxiety and affective disorders constitute a group of heterogeneous illnesses that are common and show significant heritability. Considerable work has focused on identifying the genes involved in anxiety and affective disorders providing interesting leads, but no definitive answers. One of the most prominent childhood risk factors for the development of these illnesses is behavioral inhibition (BI), a temperamental disposition characterized by extreme shyness and inhibition in response to novel situations or strangers. Using young rhesus monkeys, we have developed a model that is analogous to childhood BI and have demonstrated that individual differences in monkey BI are significantly heritable. In addition, we have used functional imaging to identify the brain regions associated with BI. In this project we will measure BI, associated physiological parameters, and functional brain activity in a large multi-generation pedigree of rhesus monkeys. These phenotypes will be used in whole genome linkage analyses to investigate the genetic basis of brain mechanisms underlying anxiety and depression. By combining proven approaches to the study of the genetics of complex disease with simultaneous analysis of intermediate brain reactivity phenotypes, our novel strategy using young rhesus monkeys will: 1) identify novel genes that influence BI, 2) quantitate the influence of genetic variation on individual differences in reactivity of the neurocircuitry underlying emotion, and 3) determine specific genes that are involved in mediating both individual differences in BI and increased reactivity of emotion-related brain circuits. These studies are not feasible in rodent species or humans. The proposed experiments provide an invaluable opportunity to identify novel genetic factors that play a major role in the development of human anxiety and affective disorders, results that will be immediately relevant to children at risk for the development of psychopathology. PUBLIC HEALTH RELEVANCE Anxiety and affective disorders constitute a group of mental illnesses that can be inherited. Using young rhesus monkeys as subjects, this study will: 1) identify genes that influence behavioral inhibition, 2) quantitate the influence of genetic variation on individual differences in reactivity of emotion-related brain circuits, and 3) determine specific genes that are involved in mediating both individual differences in behavioral inhibition and increased reactivity of emotion-related brain circuits. The proposed experiments provide an invaluable opportunity to identify genetic factors that play a major role in the development of human anxiety and affective disorders, results that will be immediately relevant to children at risk for the development of psychopathology.

This proposed project intends to address challenges identified by and realize the full potential of two earlier SEPA-funded projects: a project completed in 2012, Education for Community Genomic Awareness (Grant Code: R25RR022703); and our currently project (2014- 2019), A New Genomic Framework for Schools and Communities (Grant Code: 8 R25 GM129186-05). These projects focused on addressing the critical need for modern genomics curriculum for middle and high school students by connecting classroom instruction with the community to give both students and community members opportunities to understand, explain and apply ideas about health-related phenomena to their lives tied to gene-environment interactions, natural selection, and evolution. The implementation of these projects has produced evidence of success in student learning and interest, it has also produced three challenges to the realization of their full potential: 1) Aligned with the Next Generation Science Standards, these units are vastly different than those teachers are familiar with and making community connections are also difficult to orchestrate; 2) The potential for maintaining and enhancing student engagement was limited by short periods during the school year and lack of continuity from year to year; 3) The extent of support provided by the project to schools suggests the need to develop a model for sustained support from internal sources once the grant period is over. This research educational program plan outlines a novel design for science education that combines three components: 1) high quality curriculum materials, 2) innovative professional learning, and 3) a community health/science education consortium. These components work together to support and sustain genomic and environmental health learning and inspire interest in careers for underrepresented middle and high school students both in and out of school. The result will be a new, innovative, sustainable educational program with the following characteristics: 1) ?Health in Our Hands?, a coherent set of curricula for secondary grades that maximizes deepening of student understanding; 2) A ?Community of Practice? linking teachers with informal educators, learning new teaching practices, making seamless connections between the classroom and community, and enabling students to apply what they have learned to real-world health issues; 3) ?Greater Flint Health in Our Hands? consortium of health-related organizations, providing the mentors and the community-based activities, and support for future growth of the learning program after the expiration of this grant.

Recent genetic evidence indicates that the integrin class of fibronectin-binding adhesion receptors (5E1 and others) can regulate both the form and function of the heart. Integrin ligation drives recruitment of a number of structural and signaling molecules to the ventral plasma membrane collectively termed a "focal adhesion" which serves to link the force-generating actin cytoskeleton inside the cell to the extracellular matrix (ECM), and to coordinate activation of downstream signaling pathways. The non-receptor tyrosine kinase, Focal Adhesion Kinase (FAK) is strongly activated by both integrins and growth factors, and is a likely candidate to integrate downstream signals from these diverse pathways during growth and development. Indeed, germline deletion of FAK results in mesodermal defects and embryonic lethality between E7.5-10 similar to the phenotype observed in both fibronectin-, and D5-null mice. Although a direct role for FAK in cardiac development has yet to be examined, hearts from FAK-null embryos revealed a lack of separate mesocardial and endocardial layers, indicative of a defect in cardiomyocyte maturation. Interestingly, recent work by our group and others clearly indicate that FAK is activated in cultured cardiomyocytes by a variety of hypertrophic stimuli including, phenylephrine (PE), endothelin I (ET-1), angiotensin II (AII), and hypo-osmotic stress, and that increased cardiac FAK activity is observed in vivo in hypertrophic hearts. The idea that FAK activation plays a direct role in the development of cardiomyocyte hypertrophy is evident from our seminal findings that the activation of FAK is required for PE-stimulated hypertrophy of cultured cells and similar findings from others that FAK is required for maximal ET-1 and stretch-induced hypertrophy in vitro. The experimental goals of this proposal are to test the hypothesis that FAK regulates cardiac development and pathological hypertrophy in vivo and to identify the FAK-dependent signaling pathways involved in these processes. We will generate genetically modified mice in which FAK will be deleted in a temporal and cardiac-restricted fashion using Cre/LoxP technology to examine a functional role for FAK in cardiac growth. We will also establish a cardiac cell culture model to identify FAK-dependent signals and target genes that are differentially regulated by hypertrophic stimuli. [unreadable] [unreadable] [unreadable]

The goals of this research as stated in the grant title are the optimization of hygroscopic aerosol delivery to the human bronchial tree. We are achieving these goals by a combination of experimental studies on physical models of the bronchial airways, theoretical calculations of aerosol drop size and size distributions effected by evaporation, development of aerosol evaporators, and ultimately testing of the delivery system on human lungs and on patients.

Excessive alcohol consumption remains a leading cause of preventable death in the United States. Alcoholic liver disease (ALD) is a major cause of morbidity and mortality from heavy alcohol drinking and is the most prevalent cause of liver disease-related death. Despite increased understanding of the many cellular and molecular events occurring in ALD, the causal mechanisms remain elusive, especially with regards to the factors responsible for disease progression and severity. To advance mechanistic understanding of this serious liver disease, the current proposal will investigate the importance of circadian clock disruption and `time-of-day' as critical risk factors for ALD. These views stem from the growing recognition that circadian clock disruption is linked to numerous pathologies and diseases. One essential function of the molecular circadian clock is to provide a selective advantage of anticipation, allowing for rapid and temporally appropriate adaptation to metabolic stress, immune challenges, and environmental insults. When these processes fail, disease ensues. We propose that perturbation of circadian clocks underlie many of the metabolic and inflammatory events contributing to alcohol-induced liver injury. As such, we will determine the role of the hepatocyte clock and the monocyte clock in alcohol-induced liver injury. In support of this concept, we have found that the circadian clock is significantly altered in livers of chronic alcohol-fed mice, genetic disruption of the liver clock increases levels of hepatic inflammatory mediators, and alcohol-induced liver injury and steatosis are elevated in mice with a disrupted liver clock. Furthermore, studies show that disruptions in normal time-of-day patterns in alcohol drinking increases risk for ALD and binge alcohol drinking is now recognized to be harmful to health. Collectively, these observations have led us to hypothesize that circadian clock disruption exacerbates alcohol-induced liver injury and inflammation. Similarly, we propose that the magnitude of alcohol toxicity is dependent on the time of day of alcohol ingestion. We will test these hypotheses through two specific aims. In Aim 1, we will mechanistically show the critical role of cell autonomous clocks in chronic alcohol- induced liver injury by using two genetic mouse models, the hepatocyte-specific BMAL1 knockout mouse and the monocyte (myeloid cell)-specific BMAL1 knockout mouse. In Aim 2, we will use the `chronic + binge' alcohol model and determine the `window' or time of day that the liver is most sensitive to alcohol binge toxicity. Successful completion of this project will reveal the importance of cell-specific circadian clocks in alcohol-mediated tissue injury and inflammation and show that `time-of-day' is a significant risk factor for liver injury from binge alcohol drinking. Our long-term goal is that the scientific knowledge gained from these pre- clinical animal studies will lead to future translational investigations using various chronobiology-based therapeutic approaches for treatment of ALD and other related liver diseases.

Huntington's disease (HD) is a dominantly inherited, incurable neurodegenerative disease affecting primarily the striatum and corticostriatal pathway. Sex differences in mice and humans with HD suggest that gonadal steroid hormones may play a role in the onset and progression of HD. Neuroprotective effects of 17--3-estradiol (E2) suggest that it may contribute to these sex differences. The goal of the proposed research is to characterize the effects of E2 on neural pathophysiology and the behavioral phenotype of HD in female mice. Because HD results in damage and altered neural activity of the corticostriatal pathway, it is likely that cortical dysfunction contributes to excessive neuronal activity and a deficit of the antioxidant vitamin, ascorbate (vitamin C; AA) in the striatum. E2 effects on glutamate (GLU) transmission may normalize cortical neuronal activity in female HD mice preventing striatal AA loss and resulting in later onset and/or slower progression of HD in females. Electrophysiology and voltammetry will be used to record neural activity in the prefrontal cortex (PFC) and monitor AA release in the striatum of behaving HD mice and wild-type controls (WT) receiving either ovariectomy (OVX) + .placebo, OVX + E2, or sham surgery + placebo. During cortical recordings, mice will be engaged in a reversal learning task as this is a cortico- striatal dependent task involving PFC. One mechanism of E2 neuroprotection may be via upregulation of glial GLU transporters such as GLT1. This may prevent the GLU excitotoxicity believed to occur in HD and increase GLU clearance in female HD mice. Immunohistochemistry and behavioral assessments will be used to determine whether E2 upregulates GLT1 in the cortex and striatum, improving the behavioral phenotype of HD in female mice. Results of this study will indicate whether E2 may protect female HD mice from striatal AA loss, normalize cortical neuronal activity and improve the behavioral phenotype associated with HD. Furthermore, results will suggest whether E2 effects on GLU transmission contribute to sex differences observed in HD mice. HD affects -30,000 people in the United States and -150,000 more are at risk for inheriting the gene. If E2 is neuroprotective in HD models, it may be a suitable therapy, particularly for women with HD. Furthermore, evaluation of the role of E2 in regulating motor circuits in the brain will provide a better understanding of how this hormone regulates normal and pathological motor control. This is particularly important because more than 40 million U.S. citizens are affected by movement disorders. E2 likely plays a critical role in motor function and neuroprotection and may represent a therapy for movement disorders and neurodegenerative conditions that is currently not well investigated. [unreadable] [unreadable] [unreadable] [unreadable]

Comparison of potency tests of recombinant FVIII manufactured by Miles and by Baxter in the one and two stage assays and the chromogenic assay indicates an extraordinary degree of both interassay and intro-assay variability. Differences between individual runs of same assays can deviate up to 100% from the labelled potency. Predilution of both products in either albumin or factor VIII deficient plasma also affects the potency, with those samples prediluted in deficient plasma having overall higher values. It is planned to do in vivo half life and recovery studies.

Control of the primary tumor is a major goal of radiotherapy for cancer. Escalating the dose delivered to the tumor provides a method to improve local control. For lung cancer patients in particular, respiratory-induced organ motion has impeded safe dose escalation. Methods to compensate for this motion or to immobilize the tumor have been developed in recent years. However, these new technologies have not been applied in concert with reduced margins that would enable dose escalation due to the lack of data characterizing uncertainty in respiratory-induced organ motion. It is critical to characterize the uncertainties associated with tumor immobilization to enable the use of appropriate margins. It is our hypothesis that the combination of image guidance techniques and integrated active breath hold radiotherapy will enable characterization and reduction of the geometric uncertainties due to respiratory-induced organ motion. Active breathing control (ABC) has been shown to be a safe and effective means of tumor immobilization for breast cancer patients which allows for the reduction of the dose to normal tissue structures such as the heart and lungs. For lung cancer patients, however, the ABC technique must be adapted to increase compliance for patients with pulmonary compliance issues. Furthermore, the integration of image guidance techniques with ABC radiotherapy enables the characterization and reduction of daily setup variation and immobilization uncertainty. The specific aims of this project are to (1) Measure the random and systematic uncertainties of tumor immobilization for integrated active breath hold radiotherapy. (2) Evaluate a model image-guidance strategy with respect to the presence of these uncertainties and to design a treatment margin to compensate for these uncertainties. (3) Quantify residual setup error in a small population of patients. [unreadable] [unreadable] [unreadable]

ABSTRACT This proposal is for a phase I/II fast track project for the STTR program with the main goal to develop a test for high sensitive detection of tau oligomers in biological fluids, which could be used for the biochemical diagnosis of Alzheimer's disease (AD) and related tauopathies. AD is the most common dementia in the elderly population and one of the leading causes of death in the developed world. One of the main problems in AD is the lack of an early, sensitive and objective laboratory diagnosis to identify individuals that will develop the disease before substantial brain damage. Compelling evidences point that the hallmark event in AD is the misfolding, aggregation and brain accumulation of amyloid-beta (A?) and tau proteins. Recent evidences suggest that A? pathology is the primary driving force of the disease initiation, but this is accomplished by induction of tau hyperphosphorylation, misfolding and aggregation, leading to the neurodegenerative cascade. Tau aggregation follows a seeding-nucleation mechanism and involves several intermediates, including soluble oligomers and protofibrils. Recent evidence has shown that tau oligomers are circulating in biological fluids and these structures appear to be key for inducing brain degeneration in AD. Our working hypothesis is that detection of misfolded tau oligomers circulating in blood may be the basis for an early biochemical diagnosis for AD. Our approach is to use the functional property of misfolded oligomers to seed the aggregation of the monomeric protein as a way to detect them. For this purpose, we have developed the protein misfolding cyclic amplification (PMCA), which represent a platform technology to detect very small quantities of seeding-competent misfolded oligomeric proteins associated with various protein misfolding diseases. Currently, PMCA has been adapted to detect misfolded prion protein implicated in prion diseases in various biological fluids, including blood and urine and more recently soluble oligomers composed of A? and ?- synuclein in cerebrospinal fluid (CSF) of patients affected by AD and Parkinson's disease, respectively. The major goal of this project is to adapt the PMCA technology for specific and highly sensitive detection of misfolded tau oligomers in human CSF and blood plasma, perform studies of specificity and sensitivity using large number of samples coming from patients affected by AD and other tauopathies as well as to evaluate the utility of tau-PMCA for monitoring disease progression. The results generated in this project may lead to the first biochemical test for diagnosis of AD. The studies included in this project will constitute the basis for regulatory approval of the test that Amprion will commercialize.

The main intention of this grant is to increase our understanding of the effect of ginseng as an anti-bacterial agent against Pseudomonas aeruginosa pathogenesis in chronic lung infection the most common cause of morbidity and mortality among patients with cystic fibrosis (CF). Although antibiotic therapy, mostly with a mixture of aminoglycoside and Beta-lactam antibiotics, is used to combat the infection, treatment often fails, owing to the emergence of alginate-producing P. aeruginosa isolates. The genetic and molecular mechanism of alginate overproduction has been elucidated in great detail. Yet, a possible therapeutic intervention has been elusive. Recent studies using animal models (described in detail later) have shown that ginseng might play a role in enhancing immune response and bacterial clearance. This research proposes to use genetic and molecular biological techniques to systematically analyze the effect of ginseng against Pseudomonas aeruginosa. Specifically this research will investigate the following: I. Analysis of the ginseng extracts. II. Determine whether ginseng modulates (down- or up-regulates) the alginate genes in vitro. III. Identify novel genes that are modulated by ginseng. Understanding the mechanism of ginseng action would be a key factor if we want to promote this alternative or supplementary therapy. This is vital to devising efficient antipseudomonal therapy and eradicating complications resulting from P. aeruginosa infections among patients with CF.

This proposal requests support for a Keystone Symposia meeting entitled Neurodegenerative Diseases: The Molecular and Cellular Basis for Neurodegeneration, organized by Ted M. Dawson, Virginia M.-Y. Lee and Stephen M. Strittmatter, which will be held in Taos, New Mexico from February 21 - 26, 2011. Neurodegenerative diseases are chronic age-dependent progressive disorders that are substantial and growing health problems, which exert a tremendous toll on the patient, family, health system and society as a whole. Accordingly, there is an urgent need to identify therapies that slow and/or reverse the progression of these disorders. Research in neurodegenerative disorders is providing tremendous advances in the molecular understanding of these disorders. New insights in cell biology, biochemistry, genomics and proteomics into these illnesses are leading to mechanism-based therapies and new tools and biomarkers to study disease progression and therapeutic efficacy. In this meeting an emphasis will be placed on understanding new molecular and common mechanisms of disease in Alzheimer's Disease (AD), Parkinson's Disease (PD), triple repeat diseases, frontotemporal lobar dementia and others. This meeting will provide a forum to bring together basic and translational scientists from both academia and industry to share data, identify key areas to focus future research, and to create new collaborations in the translational neuroscience of neurodegenerative disease. PUBLIC HEALTH RELEVANCE: According to the World Health Organization, neurologic disorders are the leading cause of death, disability and loss of quality of life worldwide. In the US, neurological disorders affect approximately 50 million Americans and cost an estimated $400 billion annually in medical and related expenses. Neurodegenerative diseases are projected to triple over the next several decades as the percentage of elderly increases dramatically.

This overall program project in trauma is designed to quantitatively evaluate various biochemical and physiological parameters in the critically ill patient. The pathophysiology of trauma has been expressed by a number of overt clinical changes manifested by the patient which have served to emphasize the severity and seriousness of trauma, "the forgotten disease". This program has been conceived to explore in depth the clinical changes with the hope of providing a better rationale for the treatment and care of such patients. The core program includes a Metabolic Facility which provides the basis for the constant clinical care of the patient and for research by the basic scientist. The program encompasses study areas to evaluate in critically ill patients the following: (1) biochemical indices related to nutritional status, (2) trace element requirements, (3) immunological function related to trauma and nutrition, (4) metabolism of carbohydrate using 14C-glucose, (5) gluconeogenesis using 14C-L-alanine and 14C-glycerol, and (6) whole body protein metabolism using 15N-L-alanine and 14C-L-leucine.

Patient falls and pressure ulcers are designated as "nursing-sensitive quality indicators" by national groups such as the American Nurses Association and the National Quality Forum. Yet research linking the occurrence of falls and pressure ulcers to differences in nurse staffing has yielded equivocal findings. Unreliable measurement of adverse events and inadequate measures of nurse staffing could account for the inconclusive findings. Prior studies have analyzed these effects at hospital and nursing unit levels, but never simultaneously. Differences in the nursing practice environment may moderate the link between staffing and adverse events, although this has not been tested. The proposed study builds on evidence developed by this research team that nurse staffing and the practice environment influence patient outcomes. This study advances our understanding of the relationship between staffing and patient adverse events by 1) calculating adverse events using secondary data from observational prevalence studies and incident reports, 2) using a comprehensive set of nurse staffing measures that account for qualitative as well as quantitative differences among RNs, 3) examining these relationships at both the nursing unit and hospital levels, and 4) exploring the influence of a formally-recognized favorable nursing practice environment on these relationships. Multi-level models will be used to predict the effects of staffing on rate of falls at the nursing unit level and pressure ulcers at both the patient and the nursing unit levels using data from 4,300 nursing units in 650 community hospitals in the ANA-sponsored National Database of Nursing Quality Indicators. American Nurses Credentialing Center Magnet Designation status will be used to differentiate the nursing practice environment across hospitals and explored as a hospital-level factor moderating the associations between staffing and outcomes. This study will strengthen the evidence base on how nurse staffing patterns and practice environments support the quality and safety of patient care.

The CAPRISA Clinical Trials Unit for AIDS/Tuberculosis Prevention and Treatment (CAPRISA CTU), strategically located in KwaZulu-Natal, South Africa, at the epicenter of one of the world's most severe HIV and tuberculosis (TB) epidemics, has recently made significant scientific contributions on tenofovir gel and pre-exposure prophylaxis, HIV-TB treatment, prevention of breastfeeding transmission, and immunity with broadly neutralizing antibodies. The scientific team includes investigators who have served at the highest levels in Network leadership. During the current funding cycle, 2,854 participants have been enrolled in 25 protocols to date, with retention rates usually exceeding 90% and data quality scores exceeding 95%. The CAPRISA CTU comprises 4 components; firstly, experienced leadership, senior investigators and CTU coordinator; secondly, an administrative and governance component; thirdly, 10 research support cores; and fourthly, 4 Clinical Research Sites (CRSs). The eThekwini CRS for vaccine, microbicide and adult HIV treatment studies, is located in central Durban adjoining a local clinic that annually treats 40,000 patients with sexually transmitted infections (HIV prevalence of 59.3% (CI: 56.5-62.0) and HIV incidence rate of 6.4 per 100 person-years (CI: 2.6-13.2)) as well as 3,500 new TB cases, two thirds of whom are co-infected with HIV. At the rural Vulindlela CRS for integrated prevention, vaccine and microbicide studies, the HIV prevalence among young women is 35.7% (CI: 32.7-38.8) and the HIV incidence rate is 6.5 per 100 personyears (CI: 4.4-9.2). In this community, HIV incidence is 10.2 per 100 person-years (CI: 4.1-20.9) in women under 20 years. The Springfield CRS for adult HIV/TB treatment studies is located at a regional referral hospital that treated 2,359 MDR-TB and 206 XDR-TB patients (71% co-infected with HIV) in 2010. The Umlazi CRS for maternal/pediatric HIV studies is at a 1,200 bed hospital that provides pre-natal services to about 12,000 women annually (HIV prevalence: 39% (CI: 36.7-41.5)). The vertical transmission rate is 2.4% (CI 1.1-4.5) at birth but increases to 6.6% (CI 5.3-8.2) at 6-8 weeks post-partum due to breastfeeding. The CAPRISA CTU headquarters is located at the Nelson R Mandela School of Medicine and houses the Administration as well as the laboratory, pharmacy, data management and IT, community engagement, evaluation and quality assurance, financial management, bioethics, communication, regulatory compliance, and training Cores. The CTU's organizational structures (Leadership Group, Executive Committee and Community Advisory Board) and communication tools (regular meetings, video conferences, monthly newsletters and website) enable effective communication, management and governance in the unit. Overall, the CAPRISA CTU has diverse well-characterized high-risk populations, well established clinical facilities, accredited laboratories, pharmacies, and data management systems, strong community linkages, and extensive experience in conducting clinical trials, together with a track record of scientific innovation available to support the 5 Networks in developing new approaches to HIV and TB prevention and treatment. RELEVANCE: The overall goal of the Centre for the AIDS Program of Research in South Africa (CAPRISA) Clinical Trials Unit for AIDS/Tuberculosis Prevention and Treatment (CAPRISA CTU) is to advance the scientific agendas and research contributions of each of the five NIAID Clinical Research Networks (CRNs) focused on 1) adult HIV therapeutic strategies, 2) strategies to address HIV in pediatric and maternal populations, 3) integrated HIV prevention strategies, 4) microbicides. and 5) vaccines

Osteoporosis has become a major health problem as the life expectancy of the general population has risen rapidly in recent years. Post-menopausal women are at greater risk because of accelerated bone loss induced by estrogen deficiency superimposed on age related bone loss. One of the important etiological factors in postmenopausal osteoporosis is the interaction between estrogen and PTH. PTH is known to be an important initiator of bone remodeling and persistent elevation of PTH, such as in hyperparathyroidism, presents as a risk factor for the development of osteoporosis. However, intermittent administration of PTH has been shown to be a promising regimen for improve bone mass in both the animals and humans. The dichotomy of this issue is not fully understood, especially in the estrogen deficient population. This project attempts to understand further the dualistic role of PTH as a catabolic and anabolic hormone in animal models of estrogen deficiency. The proposed study will have the following specific aims. Specific Aim 1. To differentiate the anabolic action of continuous elevation of PTH from its catabolic action. Specific Aim 2. To demonstrate the interactions of dietary calcium intakes with continuous and intermittent PTH administration under estrogen deficiency state, and Specific Aim 3. To study the interactions of PTH, cytokines and anti-resorptive agents at both tissue and subcellular levels in a mouse model of estrogen deficiency.

Tumor specific therapeutic antibodies direct the immune system to eliminate or prevent growth of tumor cells. Passive and active humoral immune responses are mediated via interactions between the IgG Fc domain and specific Fc receptors (FcR) present on the surfaces of effector cells. Proof of principal experiments show that it is the interaction between tumor specific antibodies and the FcR activating receptors on the surface of immune system effector cells that mediate tumor cell depletion. Modulation of the binding activity of the Fc domain to satisfy specific Fc receptor binding parameters will enable genetically engineered antibodies to act as efficient antitumor drugs. Novel Fc mutations that enhance the affinity of the Fc domain to FcgammaR3A and decrease the affinity to FcgammaR2B may play an important role in development of new therapeutics. These novel scaffolds could be ligated to Fabs that target tumor specific antigens and ultimately used as therapeutics to mount a successful cytotoxic response against tumor cells. Recent advances in proteomics and cDNA microarray technologies have aided in the identification of a number of tumor specific antigens. These peptides have been used for the development of antibodies that target specific cancer cells. To enhance the immune response against tumor cells we have developed a novel genetic screen to identify Fc mutants that preferentially bind to the activation receptor FcR3A. Further research and development of these Fc mutants should lead to therapeutic antibodies with increased capacity to trigger antibody dependent cytotoxic reactions against tumor cells. This proposal describes an experimental paradigm to identify Fc mutations, characterize the mutations in functional assays, and ultimately test therapeutic antibodies harboring the best Fc mutations in animal model studies.

DESCRIPTION: Abstract Traumatic brain injury (TBI) represents a signature injury of the Iraq and Afghanistan conflicts. TBI causes severe persistent disability including cognitive impairment and mental health problems, resulting in loss of productivity and quality of life for the young Veterans returning from the wars. The long-term health care costs of combat-related TBI have created a huge financial burden and generated serious public and personal crises in the United States. To date, evidence-based treatment for TBI recovery is not yet available. Considering the fact that TBI Veterans are currently in the chronic phase of the disease, there is a critical need to develop neurorestorative strategies for brain repair in the chronic phase of TBI. Our recent studies have revealed that a stroke-damaged brain is repairable in the chronic phase by the combination of two essential hematopoietic growth factors, stem cell factor (SCF) and granulocyte-colony stimulating factor (G-CSF) (SCF+G-CSF). Using the same approach, our preliminary studies have demonstrated the neurorestorative efficacy of SCF+G-CSF in brain repair in the chronic phase of TBI. However, it remains unclear how SCF+G-CSF repairs the brain in the chronic phase of TBI and whether SCF+G-CSF treatment could repair a TBI-damaged brain in a delayed chronic phase. The objective of this research proposal is to address these unanswered questions. Based on our recent publications and preliminary data, we hypothesize that SCF+G-CSF repairs a brain in the chronic phase of TBI through neural network rewiring, which is accomplished by its direct regulation of neurons and its indirect effects via bone marrow-derived monocytes/macrophages (BMDM). Using the approaches of live brain imaging, transgenic mice and targeted knockout mice, neurobehavioral assessments, and cell signaling, the central hypothesis will be tested and the objective of this application wil be achieved by accomplishing the following 3 specific aims: Aim 1 will determine the contribution of SCF+G-CSF on brain repair in the delayed chronic phase of TBI, Aim 2 will define the role of neural network rewiring in SCF+G-CSF-induced brain repair in chronic TBI, and Aim 3 will identify the involvement of BMDM in SCF+G-CSF-induced brain repair in chronic TBI. This project is innovative in the unique approach of using hematopoietic growth factors to repair the brain in chronic TBI, which is originally created by our group. This study will significantly advance current knowledge in rehabilitation research for TBI. This research is highly compatible with the objective and scope of the RR&D priority areas for supporting the development of therapeutic strategies on neural plasticity in chronic TBI repair. The contribution of this research is in keeping with the VA mission to ensure that Veterans achieve maximal recovery from combat-related neurotrauma.

Flow Cytometric immunophenotyping is a sensitive technique for analysis of benign and malignant tumors. We are studying the refinement of this technique and its application to diagnosis and measurement of prognostic markers in different systems. We are studying the flow cytometric immunophenotype of CLL and correlating the expression of specific antigens with morphology, cytogenetics and clinical course. Data from this study may provide prognostic markers for this disease. We have refined methods for flow cytometric monitoring of patient specimens for apoptosis induced by chemotherapy and have been able to detect apoptosis in specific cell populations. This has allowed us to demonstrate different sensitivities of specific cell lineages to chemotherapeutic agents. We have initiated a study of clonal cytotoxic T-cell populations that arise in patients with B-cell neoplasia to determine prognostic importance as well as resulting difficulties in minimal residual disease detection. As a result, we have found that clonal cytotoxic T-cell proliferations occur with surprising frequency in hairy cell leukemia. The laboratory has an ongoing interest in detection of minimal and residual lymphoma. We are using multiparametric approaches to improve the sensitivity of detection of monoclonal B-cell populations. By targeting abnormal patterns of antigen expression (eg CD10, CD5, CD23, FMC7, or abnormal intensity of antigen expression) by neoplastic B-cells in light chain detection, we are attempting to detect very small numbers of neoplastic B-cells among admixed polyclonal B-cells. The laboratory is studying specific Flow Cytometric markers of various lymphoma sub-groups to improve diagnostic accuracy. The Flow Cytometry Laboratory is developing methods for Flow Cytometric analysis to detect EBV gene expression in the research and clinical setting.