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We propose a new UPCI Shared Resource Facility, entitled the Cancer Biomarkers Facility (CBF), which is motivated by the current and rapidly growing demand from UPCI basic and clinical investigators for integrated and cross-disciplinary biomarker investigations. This shared resource is Cancer Center managed. Recent publications and results from UPCI researchers demonstrate that biomarker research studies, particularly approaches utilizing cutting-edge molecular analysis technological platforms, offer abundant promise for molecular diagnosis and classification of human malignancies. Studies utilizing cutting-edge analytical platforms are expected to rapidly lead to translational applications for improved detection, diagnosis, and prognosis, and for determining efficacy of cancer treatments in patients. The CBF, and its constituent technical platforms, have already provided, and will continue to provide into the next funding period of the UPCI Cancer Center Support Grant (CCSG), clinical applications of state-of-theart systems level molecular profiling that make a major impact on cancer diagnosis, assessment of prognosis, and response to treatment. Accordingly, the mission of the CBF is to provide faculty expertise and collaborative consultation including integrative bioinformatics support across the spectrum of these omics technologies, instrumentation, and resources, and together with trained technical staff, to work with UPCI basic and clinical researchers in translational research studies focused on identifying, characterizing, and validating biomarkers for cancer risk prediction, eariy detection and screening, diagnosis, molecular classification/differential diagnosis, prognosis/prediction of disease progression, patient selection/stratification and treatment selection, prediction of therapeutic response, treatment response monitoring/assessment, and metastasis and recurrence prediction and monitoring. The proposed integrated CBF has been established and developed over the current CCSG funding period with sustained investment by UPCI building on the existing strong foundation of established UPCI shared facilities and resident instrumentation and capabilities, together with highly relevant ongoing NCI-funded translational research studies utilizing these resources, including the Eariy Detection Research Network Biomarkers Discovery Laboratory, and the Lung Cancer, Head and Neck Cancer, and Skin Cancer SPORE grants to UPCI faculty investigators. The systems level studies toward identification and validation of novel cancer biomarkers conducted within the CBF are enabled by the following technical platforms presently established and implemented at UPCI: Mass Spectrometry, Microarray, Luminex, and lllumina. Combined with integrated bioinformatics support from the UPCI Cancer Informatics Services (CIS), the CBF provides comprehensive omics technology and expertise in a fully integrated systems biology scientific environment to support the basic, clinical, and translational research needs of UPCI investigators.

The objectives of this study are (1) the characterization of some microsomal, mitochondrial or soluble conjugating and hydrating enzymes of extrahepatic tissues as compared to the liver enzymes, (2) to determine the effects of selected xenobiotics upon these enzymes, (3) to assess the relative importance in vivo of this extrahepatic enzyme activity as compared to the liver, following ingestion or administration of chemicals and drugs (substrates) via various routes including inhalation, (4) to investigate the perinatal development of some detoxifying enzymes in hepatic and extrahepatic tissues of a few species and to determine the protective role, if any, of these enzymes against localized tissue toxicity induced by chemically reactive electrophilic metabolites, such as epoxides, (5) to compare the metabolism of chemically reactive epoxides and their unsaturated hydrocarbon precursors in an isolated perfused rabbit lung preparation, and (6) to assess the role of the cell's nucleus (nuclear membrane and nucleoplasm) in the formation and subsequent biotransformation of chemically reactive metabolites.

This K01 application proposes career development and research activities customized to facilitate the candidate's (Dr. Noah Webster) transition from conducting basic social science research to behavioral intervention research. The candidate's long-term career goals include becoming an independent research scientist conducting research aimed at: a) improving the health and independence of older adults; and b) reducing later life health disparities. This will involve developing, testing, implementing, and disseminating a social network-based intervention to improve participation in behavioral health (e.g., physical activity) interventions within low resource settings. Career Development/Training Aims: In order to accomplish career goals the candidate is in need of targeted and interdisciplinary training in: 1) intervention development; 2) intervention evaluation; and 3) objective assessment of physical activity. This training will be facilitated through coursework and interactions with an interdisciplinary mentoring team led by Dr. Neil Alexander. Training Environment: The candidate is situated within the largest academically-based social science institute in the world. The resources available to him at the Institute for Social Research as well as the University of Michigan's Schools of Medicine and Public Health together offer an unparalleled supportive and stimulating environment for conducting research at the intersection of social science and intervention research. Research Aims: While only 16% of people age 65+ engage in recommended physical activity levels, increases may be achieved through activation of social resources. A social network-based approach that systematically identifies and involves influential agents of change in a community is proposed to facilitate physical activity-related information dissemination and behavior change. This project will leverage Go4Life, NIA's evidence-based physical activity campaign and capitalize on the strength of social ties. The intervention addresses socio- economically linked health disparities by developing the intervention for use in affordable (HUD subsidized) senior housing. The project will address three specific aims: Aim 1) Identify agents of change in an affordable senior housing community who will then be invited to form a committee to disseminate Go4Life materials through planning, publicizing, and participating in community-wide activities over 12 months. Aim 2) Evaluate intervention feasibility using a mixed methods approach. Aim 3) Establish preliminary network effect on changes in physical activity and identify influential network mechanisms. The proposed feasibility study is the first step in using locally available and low-cost resources to affect behavior change among socio-economically vulnerable senior housing residents. In the short-term, findings will provide preliminary data to conduct a multi- site efficacy trial which will implement and evaluate successful components of the intervention. In the long- term, understanding how to leverage social networks to promote and sustain increases in physical activity will provide key information to advance the science of behavior change as well as reduce health disparities.

Chronic inflammation has been associated with a variety of human cancers. Although all surgical prostate specimens contain evidence of inflammation, the causal relationship between inflammation and prostate cancer has not been established. Interleulin-17 (IL-17) has been well accepted as a critical cytokine in inflammation. Both TH17 cells (T helper cells secreting IL-17) and IL-17 cytokine are increased in prostate cancer specimens, and the IL-17 receptors, IL-17RA and IL-17RC, are expressed in prostate cancer cells. However, the fundamental question of whether IL-17 plays an active role in prostate cancer needs to be determined. Our preliminary experiments revealed that in a mouse model of prostate cancer caused by conditionally mutant for Pten in the prostatic epithelium, IL- 17RC deficient (IL-17RC-) mice displayed smaller prostates and developed a reduced number of invasive prostate cancers with decreased inflammatory infiltration, reduced cellular proliferation, and increased apoptosis, compared to mice that express IL-17RC. Further, the fibromuscular stroma surrounding the prostatic glands was significantly thicker in IL-17RC- mice, a finding that we have associated with a decreased expression of matrix metalloproteinase 7 (MMP7). Addition of a recombinant mouse IL-17 induced the expression of MMP7 in the mouse prostate. Based on these findings, we have formulated a central hypothesis that, in prostate carcinogenesis caused by a Pten mutation, IL-17 facilitates prostate cancer formation and growth through an MMP7-mediated mechanism. This concept has clinical significance because blocking IL-17 or its downstream effectors such as MMP7 has the potential to be developed into new therapeutics in the prevention and treatment of prostate cancer; further, assessing the expression of IL-17- MMP7 signaling axis can be utilized as a prognostic indicator of prostate cancer. We propose to test our central hypothesis through the following three specific aims: Aim 1: Does MMP7 mediate IL-17's function in facilitating prostate cancer formation and growth in Pten- null mice? Aim 2: Assess the efficacy of targeting IL-17-MMP7 axis in preventing prostate cancer formation and growth in Pten-null mice. Aim 3: Determine the association between the IL-17-MMP7 axis and progression of human prostate cancer. Successful completion of the proposed studies will provide new insights into the molecular mechanisms underlying IL-17-mediated prostate carcinogenesis. Further, if any or all of the tested agents show efficacy, they can potentially be developed into preventive and/or therapeutic drugs against prostate cancer and other cancer types where IL-17 plays a role. The IL-17-MMP7 axis can potentially be utilized as new biomarkers in the prognosis of prostate cancer and in distinguishing between aggressive and indolent prostate cancers.

Models of virus-induced demyelination have been of great interest to investigators concerned with Multiple Sclerosis. We have been studying the murine Theiler's model of virus-induced demyelination, in which data strongly suggest an immunopathologic mechanism of myelin degeneration. The identification of additional animal models in which similar mechanisms might be operating would be important as it would suggest that demyelination by combined viral-immunologic attack may represent a more general pattern of virus-CNS interaction. The general objective of this project is that of further characterizing the Theiler's model my morphological and immunopathologic techniques and to add two further models of virus-induced demyelination in which immunopathologic mechanisms are suggested. Specific aims are as follows: (1) Ultrastructural studies of different mouse strains with special emphasis on the C3H strain in which conspicuous Schwann cell participation is observed during remyelination. (2) Ultrastructural studies of mice infected with cell cultured adapted Theiler's viruses TO4 and WW. These viruses produce a slower disease with apparent clinical sparing of grey matter and striking Schwann cell remyelination of CNS white matter. (3) Ultrastructural and autoradiographic studies of the remyelinating phase of Theiler's infection with special emphasis on Schwann cell mechanisms of remyelination in CNS and Schwann cell origin. (4) Studies of demyelination in two new infections produced by a temperature-sensitive (ts) mutant of Vesicular Stomatitis Virus (VSV), tsG41, and a ts mutant of the human isolate Chandipura virus (CV) ts472. (5) Immunosuppression and adoptive immunization experiments with all three models to better characterize immunemediated demyelination. (6) Light and ultrastructural immunoperoxidase studies aimed at (a) visualizing viral antigen in relation to demyelinating lesions, (b) localizing antiviral and/or antimyelin antibody in diseased white matter. (7) Studies of the host-immune response both serum and cell-mediated to both viral antigens and myelin basic protein. With such studies, we hope to more clearly establish these models as representative of virus induced immune-mediated demyelination, and in addition to increase our understanding of the complex process of CNS myelin repair.

Fungal sepsis, or fungemia, is a serious public health concern afflicting over 100,000 patients every year in the US where almost 38% of those do not survive. In fungemia early targeted treatment is life-saving, and considering the rate at which the disease develops, is perhaps the most crucial factor to improving outcomes. Unfortunately, given the time-consuming diagnostic process today, the underlying cause of this disease is typically identified too late. Current diagnostics do not facilitate early initiation of a targeted antifungal intervention - as they require culturing, which requires as long as 2-5 days for accurate identification of the infecting pathogen. Hence current treatment protocols use 'best-guess' approaches which are not only less effective, but likewise increase complications, and increase the prevalence of drug resistant pathogens. Having met our and exceeded our Phase I Specific Aims, this Phase II proposal focuses on the automation of the entire diagnostic into a fully-integrated, sample-to-answer consumable enabling the automated species- level identification of the most clinically prevalent fungal pathogens directly from phlebotomy specimens, in roughly 2 hours; without human intervention. The sample-to-answer diagnostic we will develop will have profound impact, both by improving outcomes by providing a means to develop a hypothesis driven first-line intervention, and likewise by reducing the use of excessive and unnecessary antimicrobials. To succeed in this Phase II we have put together a top-notch team at HelixBind, including experts in assay development, application of synthetic nucleic acid analogues, the design and implementation of sample-to-answer automated diagnostics, and in biomedical instrumentation development. Additionally we have enlisted key strategic advisors in clinical microbiology, surface chemistry, and nucleic acid analogues as well as successful entrepreneurs with experience in the development and commercialization of medical devices. Together, we will build upon our already impressive Phase I results, having established species level capability at sub-10 CFU/ml sensitivity levels directly from spiked human samples. To establish the performance capability our automated and culture-free diagnostic, we will culminate this Phase II by assessing the clinical specificity and sensitivity of our diagnostic wit the assistance of our clinical partner. Having achieved our Phase II Specific Aims, we will move forward and develop deployable instrumentation/consumables and validate our entire diagnostic in preparation for the pivotal clinical trial.

Skin and soft tissue infections (SSTI) are one of the most common problems encountered in clinical practice and affect millions of individuals annually in the United States. These infections may range from uncomplicated superficial SSTI to life-threatening complicated deep SSTI. Treatment of SSTI has been significantly complicated by the increasing emergence of multidrug-resistant pathogenic bacteria. As a result, there is a pressing need for the development of new therapeutic approaches. The objective of this proposal is to investigate the utility of a non-antibiotic approach, antimicrobial blue light theray, for multidrug-resistant SSTI. Specifically, we propose to: Aim 1: Demonstrate the efficacy of antimicrobial blue light therapy for superficial or deep SSTI in rodents infected with Gram-positive methicillin-resistant Staphylococcus aureus (MRSA) or Gram-negative multidrug-resistant Pseudomonas aeruginosa. We will use superficial impetigo in mice and deep incisional surgical site infections in rats as the representative models of superficial and deep SSTI, respectively. Both immunocompetent and immunocompromised mice/rats will be tested. Bacterial inocula of 3x105 and 3x107 colony-forming units/rodent will be used to represent the infections caused by a low and a high inoculum, respectively. We will use the in vivo bioluminescence imaging technique to quantitatively and noninvasively monitor in real time the extent of infections in living animals. Blue light (405-415 nm wavelength) will be initiated at 30 min, 24 h, and 48 h after bacterial inoculation, respectively. The efficacy found with blue light therapy will be compared with that of clinically used antibiotics for SSTI. Aim 2: Determine the potential side effects of antimicrobial blue light therapy for SSTI. We will first compare the susceptibilities to blue light inactivation in vitro between pathogenic bacteria and host cells. We will also determine whether the pathogenic bacteria can develop resistance to blue light inactivation. The experiment will be implemented by carrying out repeated cycles of sub-lethal blue light inactivation of bacteria in vitro followed by bacterial regrowth. Finally, wewill investigate whether blue light is genotoxic to host cells by using the alkaline comet assay after irradiation of host cells in vitro and in vivo. Successful completion of these specific aims will provide the foundation required to assess the efficacy (Aim 1) as well as the potential side effects (Aim 2) of antimicrobial blue light therapy for SSTI, and will help establish protocols for the use of this prophylaxis/therapeutic option. Given the significant drawbacks in current management of multidrug-resistant SSTI, this new technology using blue light exhibits great potential.

This is an application for support for the planning and initial implementation of a program in research in surgical oncology at the Medical College of Virginia/Virginia Commonwealth University. The organizational structure in surgical oncology, and many of the resources required for such a program, alredy exist at this institution. The purpose of this specific application is to facilitate the development of a high quality, broad cancer research program that is both relevant to the National Cancer Program and effectively involves young surgical oncologists in both basic and clinical research. It is specifically designed, by effective planning, to lead to one or more collaborative program projects at its completion. The long range of this planning project is to assist in developing individuals who will strengthen academic surgical oncology as a discipline and to make this field responsive to the future needs of cancer research.

We propose to study and develop for practical use a new class of closed sequential tests of hypotheses which has recently been proposed by the principle investigator. Our research will have two goals: (1) to extend the theory to diverse distributions of data; and (2) to compare numerically the expected sample sizes and operating characteristics of the new tests with those several existing tests--for example, tests of fixed sample size, the sequential probability ratio test, and tests whose continuation regions are wedge-shaped. We expect our results to be especially pertinent to such statistical questions in medical research as testing hypotheses about the levels of radiation found in individuals over time, testing hypotheses about the time until recovery from an operation or onset of a disease, and testing hypotheses about the effectiveness of drugs and vaccines.

Work continues on the identification and characterization of circulating immune complexes and aggregates in systemic lupus erythematosus and other connective tissue diseases. Particular attention is focused on cryoprecipitates, complexes which can be precipitated by the first complement component (Clq), and complexes which can be adsorbed to membrane receptors on Raji cells. Complexes are being elevated after digestion with pronase to determine residual material such as DNA. The role of immune complexes in stimulating both normal and sensitized lymphocytes is being evaluated. The effect of rheumatoid factor in modulating the effects will be studied during the coming year. A "third population" of lymphocytes has been well defined. Extensive studies are currently being carried out to evaluate the function of these "third population" or "L" cells. Characteristics of the lymphocyte membrane are being evaluated in detail to determine if there is any special property of lupus lymphocytes. Anti-lymphocyte antibody is being used as a probe to determine if there are any specific markers on SLE lymphocytes which might indicate the presence of a virus or viral remnant.

This study aims to examine the nutritional, metabolic and growth effects of antiretroviral therapy (ART) on children in South Africa, the country with the largest number of children requiring ART in the world. Currently little is known on the effects of ART in children, and what is known comes from industrialized countries, where the malnutrition and infectious co-morbidities so prevalent in African children are often not present. The findings from this study will be important in informing both the clinical management and public health policy of the worldwide initiative to provide ART to the world's HIV-infected children. Specific aims of this study are to: 1) describe growth and body composition changes in the 2 years following initiation of ART; 2) describe the incidence of metabolic derangements and lipodystrophy in these children and temporal relationship with commencement of ART; 3) describe the relationship between pretreatment nutritional status and drug tolerability/ metabolic abnormalities in the 2 years after initiation of ART; 4) determine if body composition changes are related to changes in viral load or CD4 percentage; 5) determine predictors of ART failure. We will recruit 150 treatment naive symptomatic prepubertal HIV-infected children who are commencing triple-drug antiretroviral therapy. Study participants will be recruited during the first 2 years of the study, and will be recruited from 2 sites: the HIV Family Clinic in King Edward VIII Hospital in Durban; and at the Africa Centre/Hlabisa Health sub-District ART program in rural KwaZulu Natal. ART will be provided according to ART guidelines of the KwaZulu Natal Provincial Department of Health (KZN DOH). These guidelines recommend stavudine, lamivudine and lopinavir/ritonavir for children less than 3 years, or stavudine, lamivudine and efavirenz if older than 3. Study participants will be followed monthly initially and then 3 monthly. We will measure attained growth (height and weight), body composition (skinfold thickness and bioimpedance assessment), lipodystrophy (clinical features and skinfold ratio), markers of dyslipidemia and glucose homeostasis, evidence of drug adverse effects and evidence of treatment failure (based on changes in CD4 cell percentage and HIV viral load) at each visit throughout the study. We will use the following statistics to describe growth patterns and frequency of metabolic abnormalities following initiation of ART: means/medians and 95% confidence intervals, bivariate and multivariate logistics regression models to determine the relationship of pretreatment nutritional status, drug tolerability and metabolic abnormalities, and predictors of treatment failure. All data will be entered into Epi Info and will be analyzed using standard PC-based data analysis packages, including SAS and SPSS. [unreadable] [unreadable]

Replication competent vaccinia virus (W), the current vaccine for smallpox, can cause severe complications after vaccination, especially in immune suppressed individuals. We are seeking to develop strains of VV that are replication competent, and thus will induce a strong immune response, without the complications associated with vaccination with the current vaccines. Thus, the Aim of this proposal is to prepare conditional mutants of VV that are either dependent on an FDA-approved drug for replication, or are treatable by an FDA-approved drug. For drug-sensitive viruses, individuals who experience complications could be treated with the FDA-approved drug. For drug-dependent viruses, should complications arise, the drug could be withdrawn from vaccinated individuals as a treatment. Drug-dependent viruses have the added advantage that they would not be able to spread in a viable form from vaccinated individuals to contacts. The relative immunogenicity and safety of these two strategies will be compared with that of a current vaccine (Dryvax) in immunocompetent mice (immunogenicity and safety), and in immunodeficient SCID mice (safety only). The most promising of these strains will be engineered into a virus background suitable for use in humans, prepared under GMP conditions and tested in chimpanzees and humans for safety and immunogenicity, compared to Dryvax.

This application for a Health Services Research Enhancement project aims to strengthen and expand the capacity and infrastructural facilities at the Institute for Urban Research (IUR) and Public Health Program (PHP) at Morgan State University to conduct rigorous health services research. The proposed interdisciplinary research activities, during the three-year funding period, will focus on selected health services issues in such areas as teenage pregnancy, maternal and child health, school health programs, reproductive health, breast-feeding, and other health and demographic issues. The proposed protect activities will encompass five specific aims: (1) to strengthen and expand some on-going health services research activities at IUR and at PHP; (2) to initiate and conduct new faculty small research projects in selected health services areas; (3) to strengthen and expand on-going research assistant ships and internships for students and to increase the small pool of African American researchers trained in health services research; (4) to strengthen existing research facilities and resources at the IUR and PHP by: updating their computing facilities and by establishing a new health services and research library; and (5) to develop a leadership role in African-American health services research at the local, state, and national levels. The application describes existing research facilities, staff, gaps in research facilities, and the evolution of its health services research. The application then describes aims, objectives, and methods of three new health services research projects that will be conducted during the 3-year funding period. All these three research protects will focus on health services research issues disproportionately affecting African Americans in inner-city Baltimore and its surrounding areas by utilizing the core research facilities that will be established or strengthened at the IUR and at the PHP under the proposed Health Services Research Enhancement Program.

Approaches to understanding the molecular basis of fertilization have been severely hampered by a lack of knowledge of the macromolecules involved in this process. In particular, the materials surrounding the eggs of most animal organisms, termed egg investments or integuments, play essential roles in several aspects of fertilizaton such as sperm capacitation and the block to polyspermy. The biochemistry of the egg integuments is largely of a gross and/or elementary level, e.g. they are protein or glycoprotein in nature; their macromolecular composition is almost totally unknown. In an attempt to understand fertilization in molecular terms, it is proposed to: 1) determine the number and kinds of macromolecules in egg integuments of the South African clawed toad, Xenopus laevis; 2) elucidate the molecular structure of these macromolecules; 3) define the roles these macromolecules play in sperm capacitation and the block to polyspermy; and 4) correlate structural (chemical and physical) information from the molecular supramolecular, and cell ultrastructural levels with functional activity in sperm capacitation and the block to polyspermy to provide an integrated molecularly based understanding of these processes.

Systemic mastocytosis (SM), a myeloproliferative disorder with variable clinical manifestations, is associated in most cases with the D816V mutation in KIT. The identification of the KIT D816V mutation in patients with systemic mastocytosis has gained a major prognostic significance in the last several years, largely because of the availability of tyrosine kinase receptor inhibitors such as imatinib. However, imatinib is ineffective in patients carrying KIT D816V mutation. In collaboration with Deciphera Pharmaceuticals, we thus evaluated novel KIT switch pocket inhibitors on mast cell proliferation and activation. We found that neoplastic human mast cell lines harboring the KIT D816V mutation were significantly inhibited by the switch pocket inhibitors through the induction of apoptosis. Overall, switch pocket inhibitors thus may provide therapeutic benefit. Because eosinophils are sometimes elevated in patients with mastocytosis, we assessed surface and soluble IL-5R levels in patients with eosinophilia and/or mastocytosis in a collaboration with Dr. Klion's research group. sIL-5R was significantly elevated in patients with systemic mastocytosis without eosinophilia. sIL-5R levels correlated with serum tryptase and eosinophil activation. These data may have implications with respect to the use of novel therapeutic agents targeting IL-5 and its receptor in patients with eosinophilia and/or mastocytosis. KIT has two major variants which differ by four amino acids (GNNK- or GNNK+) at the juxta-membrane region of the extracellular domain. Using a quantitative real-time PCR assay to assess GNNK- and GNNK+ transcripts from the bone marrow mononuclear cells of patients with systemic mastoycytosis, we found that the relative expression of the GNNK- variant strongly correlated with neoplastic mast cell involvement. Using a mast cell transfection system, we have found that he GNNK- variant is associated with increased granule formation, cellular growth and resistance to the tyrosine kinase inhibitors. Studies are underway to better understand the mechanisms behind these observations.

This research is concerned with the possible relationship between protein synthesis and specialized function in the brain. The phenomena investigated include: 1) responses of brain protein synthesizing systems to environmental alterations, 2) physicochemical and metabolic bases for these responses, and 3) the regional and cellular location of these phenomena. Attention is currently being focussed principally upon the possible role of the cyclic nucleotides in the translational control of protein synthesis in the brain. Efforts will be made to delineate the actions of these substances on ribosomal protein phosphorylation and lysosomal stability in the brain and to relate these processes to variations in protein synthesis. BIBLIOGRAPHIC REFERENCES: Roberts, S. Amino acid imbalance and protein synthesis in the brain. Trans. Amer. Soc. for Neurochem. 7, 221, 1976. Roberts, S. & C. D. Ashby. Effects of cyclic AMP on ribosomal protein phosphorylation in rat cerebrum. Trans. Amer. Soc. for Neurochem. 7, 108, 1976.

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 Hol laboratory is focused on proteins from major global pathogens that are promising targets for the development of much-needed new pharmaceuticals to treat such diseases as malaria, sleeping sickness, leishmaniasis, tuberculosis and cholera. A characteristic of the laboratory is to study proteins and in particular multi-proteins assemblies that are, by themselves, extremely interesting with respect to architecture, function and properties and then aim for novel ways to obtain high affinity inhibitors. As a result we are investigating mainly multi-protein assemblies, including cholera toxin in complex with human proteins, the unique U-insertion/deletion RNA editing system in Trypanosomatids, Peroxins involved in glycosome biogenesis, the invasion machinery of the malaria parasite, and the Type 2 Secretion System which exports cholera toxin across the outer membrane of Vibrio cholerae.

The project proposes a series of simulation experiments designed to determine the power, robustness, and validity of recently developed methods of segregation and linkage analysis, and to use insights gained from these experiments to analyze pedigree data on blook pressure and other variables related to hypertension. A particular effort will be made to detect major genes in an attempt to analyze the genetic component of hypertension. The power, robustness, and validity of three genetic models will be considered: 1) the transmission probability model, 2) the mixed model which includes components for a major gene, polygenic inheritance and sibling environmental correlations, and 3) a combined model, i.e. a mixed model with the single locus component parameterized in terms of transmission probabilities. The robustness of each model will be considered with respect to the presence of environmental skewness and kurtosis, familial environmental correlations, and dominance or epistasis in the multi-locus (polygenic) component. In particular simulated data will be based on parameters obtained from actual hypertension data, so that valid and appropriate conclusions can be drawn from these experiments. Methods of segregation and linkage analysis that are powerful, and robust with respect to failures of assumptions (particularly those found in the actual hypertension data) will be applied to two sets of data provided by other investigators. The primary set of data comprises five large pedigrees ascertained through a single hypertensive proband. The second set of data will consist of approximately ten large pedigrees ascertained at random as part of the Bogalusa Heart Study. Long range objectives include 1) the dissection of the genetic component of hypertension with a view to the identification of individuals with a genetic predisposition for hypertension before those individuals become overtly hypertensive and 2) an ongoing evaluation of new methods in segregation/linkage analysis, used to illuminate the nature of genetic components.

This revised renewal proposal is designed to delineate novel gene regulatory mechanisms and downstream pathways involved in the control of muscle glucose utilization and exercise capacity relevant to the consequences of chronic caloric excess. Current dogma suggests that accumulation of skeletal myocyte lipid leads to insulin resistance caused by derangements within the insulin signaling pathway. We have shown during the current funding period that myocyte lipid accumulation, driven by chronic activation of the nuclear receptor peroxisome proliferator-activated receptor a (PPARa), is linked to reduced muscle glucose uptake and utilization, independent of derangements in the insulin signaling pathway. The metabolic phenotype of muscle-specific PPARa transgenic mice is similar to that of the obesity-related insulin resistant state including myocyte triacylglyceride (TG) accumulation, high fatty acid oxidation rates, glucose intolerance, and reduced exercise capacity. In striking contrast, transgenic lines for PPAR2, a muscle-expressed relative of PPARa, exhibit many of the metabolic benefits of exercise in the absence of training, including increased capacity for muscle glucose oxidation and an increase in slow-twitch muscle fibers. Transcriptional and metabolomic profiling results strongly suggest that whereas PPARa and PPAR2 regulate many overlapping gene targets involved in cellular fatty acid utilization, a subset is PPAR isotype-specific. For example, PPAR2 (but not PPARa) activates genes involved in muscle glucose uptake/oxidation and the slow-twitch muscle fiber program, the latter through a distinct microRNA network. We have also shown that the PPAR coactivator, PGC-1a, is necessary and sufficient for muscle mitochondrial function and biogenesis programs that are activated by exercise training. These results have led us to hypothesize that PPARa and PPAR2 control overlapping, as well as distinct, genes in skeletal muscle and that delineation of PPAR isotype-specific targets will unveil novel strategies to re-program skeletal muscle metabolism for the prevention and treatment of the consequences of obesity, such as insulin resistance. This hypothesis will be tested by: delineating the gene regulatory mechanisms involved in the re-programming of muscle glucose metabolism (Aim 1) and fiber type (Aim 2) by PPAR2 versus PPARa;conducting unbiased genomic and metabolomic interrogations to define PPAR2- and PPARa-specific target genes and metabolic pathways (Aim 3);and performing studies in vivo in mice to validate the actions and relevance of the candidate regulatory molecules and pathways downstream of PPAR2 in the control of muscle fuel metabolism, energetics, and exercise capacity under normal physiological conditions and in the context of diet-induced insulin resistance (Aim 4). In vivo validation of novel molecules, pathways, and metabolomic signatures downstream of PPAR2 will allow us to develop a prioritized list of new candidate therapeutic targets (and predictive biomarkers) for enhancing muscle fuel metabolism and function relevant to the ravages of caloric excess. PUBLIC HEALTH RELEVANCE: We are witnessing a pandemic in obesity-related diabetes. This project seeks to identify new molecules and metabolic pathways in muscle that could serve as novel therapeutic targets aimed at reducing the development of insulin resistance and progression to diabetes in the obese population. The proposed studies also show great promise for developing new therapeutic strategies to enhance skeletal muscle function in chronic debilitating disease states and with aging.

The objectives of this research program are to investigate dynamical structures, molecular motions, solvation and ligand interactions of DNA oligonucleotides in solution and crystalline solids using molecular dynamics (MD) computer simulation and other theoretical and computational methodologies available to molecular biophysics. All MD simulations will include water and counterions explicitly for run lengths extending at least into the nanosecond regime of time. Continuation studies are proposed on shorter oligonucleotide sequences for further characterization and assessment of MD modeling capabilities and limitations via comparison with observed structures obtained from crystallography and NMR, and state of the art quantum mechanical calculations on fragments. Further development and implementation of analysis techniques for elucidation of the structural chemistry and dynamics of solvent water and mobile ions will be carried out, including elaboration of a new method aimed at providing the missing link between solvent structure and functional energetics. Applications of MD modeling to the elucidation of basepair sequence effects on structure and axis bending will be pursued on both shorter (10-12 bp) and longer ( up to 40 bp) oligonucleotides. The fundamental nature of sequence effects, still unsettled at the theoretical level, will be explored via analysis of accurate MD results in the context of current hypotheses about the competing effects of van der Waals clashes, dispersion forces, electrostatic interactions, and solvent effects at the basepair level. An alternative perspective on this subject will be created based on MD studies of the effect structural perturbations such as modified backbones and bases. New research initiatives are directed at the critical use of MD in ligand binding studies, and developing links between all atom MD modeling and terms in free energy component analysis of ligand binding processes that are otherwise difficult to estimate accurately. A critical examination of the component analysis approach will be carried out with respect to estimated errors and uncertainties in the various terms and the calculated net binding energies. Analysis methodologies for the structure, motions, solvation of nucleic acid systems will be implemented in version 3.0 of "Molecular Dynamics Tool Chest" and made available for general distribution. Building on our participation in the Nucleic Acids Data Base Project, the articulation of MD results on DNA systems with the field of nucleic acid structural bioinformatics will developed into a state of broader general utility to the field. Successful completion of the proposed research will advance our understanding of the dynamical structure of nucleic acids in solution, effects of sequence on DNA structure and axis bending important in molecular recognition processes, forge improved links between dynamical structure and functional energetics, and contribute to an improved understanding of the thermodynamics of nucleic acid-ligand binding processes.

This is the renewal of the Neurobiology of Adolescent Drinking in Adulthood (NADIA) Administrative Core (NADIA Administrative Core-U24, RFA-AA-15-005). The Administrative Core provides administrative and scientific leadership to achieve the goal of our Consortium: to elucidate persistent changes in complex brain function-behavior relationships following adolescent alcohol exposure. In the previous funding period, the NADIA discovered that adolescent intermittent ethanol (AIE) led to multiple pathologies in adulthood, including altered anxiety, social behavior, cognitive flexibility, conflict behavior, sleep, impulsivity, decision making, incentive salience, increased adult alcohol drinking and decreased response to alcohol, as well as altered neuroimmune gene expression, neurogenesis, epigenetic histone-methylation changes, and decreased choline acetyltransferase expression. To promote and facilitate this progress, the NADIA Administrative Core is the main organizational unit of the Consortium and serves as the liaison between the eight Research Components, the two Scientific Cores, the NIAAA, the NADIA Steering Committee, and the External Advisory Board. This Core organizes all Consortium activities including retreats, progress reports and External Advisory Board evaluations of cores and components. The Administrative Core ensures consistent and forward progress by facilitating communication and promoting integration of data among the components and cores. The Core develops scientific and conceptual themes as well as standard operating procedures that cross components. The Core provides a repository for all NADIA publications (at the time of submission, 119 publications were published or in press from the previous funded period) and data generated by the Consortium. Finally, the Core facilitates dissemination and translation of NADIA-generated data. Understanding the impact of underage drinking on adult neurobiology is important to guide public health initiatives, and the NADIA Administrative Core creates synergies for such discoveries across the Consortium.

A successful virus infection involves entry into the cell; uncoating, expression and replication of the genome; assembly and release of infectious virus particles; and defense against specific and non- specific host immune mechanisms. Combined genetic, biochemical, electron microscopic, and immunological approaches are being used to investigate these complex processes. Two genetic approaches have been developed and applied to the study of vaccinia virus assembly. For essential genes, the elements of the E coli lac operon were used to construct inducer-dependent conditional lethal mutants of vaccinia virus. Repression of the D13L gene, which encodes a 65,000 Dalton protein, prevented the formation of the immature spicule-coated viral envelope and mimiced the action of the drug rifampicin. Repression of the F18R gene allowed virion morphogenesis to proceed up to the stage of differentiation of the virion core. The second approach, gene knock-out, was used to investigate the function of the outer viral envelope and the release of virions from cells. Deletion of the viral gene encoding the VP37 envelope protein did not affect the formation of intracellular infectious virus but prevented the membrane wrapping of virions and their fusion with the plasma membrane. Evidence was obtained for an additional post-budding step in virion release. With some strains of vaccinia virus, enveloped virions remain attached to the surface of cells but can be released with trypsin treatment. To evade the host immune system, vaccinia virus encodes a variety of proteins. We demonstrated that the complement control protein (VCP), the secreted product of the C2IL gene, is able to prevent complement- enhanced antibody-mediated neutralization of infectious virus. These data demonstrated that VCP blocks the alternative, as well as the classical, pathway of complement activation. A mutant virus containing a knock-out deletion of the C2IL gene was highly attenuated when injected into the skin of animals. The basis for the interferon- resistance of vaccinia virus was investigated. The product of the K3L gene was found to inhibit the double-stranded RNA activated protein kinase and to thereby prevent the interferon induced phosphorylation of the alpha subunit of eukaryotic initiation factor 2. Evidence against the possibility that vaccinia inhibits host protein synthesis by degrading or causing the underphosphorylation of components of the cap- binding translation initiation factor complex eIF-4F was obtained.

Project Summary Sleep deprivation presents an increasing threat to individual health and public safety as well as an economic burden due to lost productivity, traffic accidents, occupational accidents, and skyrocketing healthcare costs. There has been an astonishing rise in the number of individuals affected by sleep deprivation with approximately 35% of U.S. adults and a staggering 70% of teenagers reporting insufficient sleep. Sleep deprivation induces significant impairments in memory and performance, aggravates psychiatric and neurological disorders and increases disease risk, especially neurodegenerative disorders such as Alzheimer?s disease. Given the significant number of people affected by sleep deprivation, and the potentially devastating consequences of sleep loss in terms of disease and dementia, it is essential to identify the cellular consequences of sleep deprivation and to define the specific molecular targets and processes impacted. Recent research suggests that the influence of acute sleep deprivation on memory occurs at the cellular and synaptic level, although the specific mechanisms through which sleep deprivation exerts these effects remain poorly understood. The hippocampus, a critical brain region for memory, is particularly susceptible to the effects of acute sleep deprivation. Previously, we found that sleep deprivation decreases protein synthesis in the hippocampus leading to impairments and deficits in synaptic plasticity. We hypothesize that sleep deprivation targets multiple processes that affect the regulation of gene expression, which is comprised of changes in transcription, RNA processing and localization, and protein synthesis. The objectives of this proposal are to define the molecular and cellular mechanisms through which sleep deprivation impacts gene regulation and to define the affected subregions and cell types within the hippocampus. In Specific Aim 1, we focus on the signaling pathways through which sleep deprivation affects protein synthesis to adversely impact long-term memory and synaptic plasticity with a focus on identifying mechanisms of resilience to sleep loss. In Specific Aim 2, we investigate the effects of sleep deprivation on RNA fate at the subcellular level and explicitly detail the effects of sleep deprivation on the pool of mRNA available for translation. In Specific Aim 3, we employ state of the art techniques to define the impact of sleep deprivation across subregions within the hippocampus and within individual cell types providing a detailed spatial map and cellular signature of the effects of sleep deprivation. The results from our comprehensive proposal integrating in vivo behavioral manipulations to mitigate the effects of sleep deprivation on memory, the subcellular analysis of the effects of sleep deprivation on RNA fate and protein synthesis, and the identification of cell specific signatures of sleep deprivation, will provide significant insights into the negative impacts of sleep deprivation on memory, potentially leading to the development of therapeutics to counteract the consequences of sleep loss on cognition and neurodegenerative disorders.

Articular cartilage damage is one of the most expensive of the debilitating non-life threatening diseases in the US (Jackson et al., 2001c). Non-arthroplasty treatments for cartilage defects-such as lavage, debridment, and microfracture-appear to provide short term pain relief and the long-term outcome of these procedures remains unclear (Gilbert 1998). The current6 standard-of-care treatment for focal cartilage defects is the transplantation of autogenous osteochondral plugs from non- weight bearing areas; an approach which has had reasonable short-term success (Sanders et al., Fitzpatrick and Morgan, 1998; Horas et al., 2000), but also faces many unresolved technical challenges including matching donor and host site profiles and tissue material properties. The objective of this study is to explore the feasibility of stabilizing focal cartilage defects by inserting a non-degradable synthetic plug into the defect. Our hypothesis is that the structural integrity of a permanent plug and its surrounding tissue can be maintained by insertion of a plug that is more compliant compared with the surrounding cartilage. The plugs intended for use in this study are degradable synthetic plugs made of poly(vinyl alcohol) PVA, the properties of which can be altered through compositional and structural changes. The first requirement of this study is to choose two hydrogel plug groups with different instantaneous mechanical property profiles. The second phase is to insert both plug types into a distal femoral rabbit osteochondral defect model. Our hypotheses will be tested by quantifying the stability of plug fixation to the surrounding bone, assessing how adequately the plug shares load with the surrounding and opposing cartilage, and how effectively it transfers load to the underlying bone. The specific aims can be summarized as follows: Specific Aim 1: Specify two groups of hydrogel plugs so that their structure and composition results in; a) material properties that are within the range or published values for rabbit temporal condyle articular cartilage, and b) material properties with a 50% increase in compliance compared to rabbit femoral condyle articular cartilage. Specific Aim 2: Quantify the structural, compositional, and mechanical incorporation of hydrogel plugs into rabbit femoral condyle focal osteochondral defects over time periods extending to 12 months, as a function of time and of hydrogel material properties. The goal of our approach is to stabilize the cascade of events that accompanies the presence of focal cartilage defects, a cascade that left untreated usually results in osteoarthritis. It is our contention that drilling through subchondral bone and inserting a hydrogel plug with specifically chosen material properties will provide an acceptable pain free, functional clinical outcome.

Reaching for objects is a natural and common behavior. A great deal of behavioral and physiological effort has been devoted to understanding reaching behavior. This project is the continuation of a unique attempt to understand how humans are able to reach accurately despite the highly variable dynamics of their own movement patterns. Specifically, this project attempts to understand reaches involving simultaneous extension of the arm and torso rotation. The project was originally based on the observation that when we turn and reach for an object, the Coriolis forces generated between the torso and arm are orders of magnitude greater than the interaction forces generated between brachial segments during simple reaches not involving trunk rotation. If dynamic perturbations of this type and magnitude are applied to the arm by a robotic manipulandum, large movement errors result. Subjects can learn to move accurately again through repetition but the adaptation does not generalize far beyond the workspace region in which the mechanical perturbation was experienced. In the first five years of this project, we showed that 1) humans can create vastly different Coriolis forces during different reaches, 2) these force variations have no discernible effect on the accuracy of turn and reach movements, 3) this accuracy is achieved through both feedforward compensation for anticipated Coriolis forces and on-line compensations, 4) adaptive self re-calibration of feedforward mechanisms is possible, and 5) signals about foot-centric torso rotation are implicated in re-calibration. Our new aims include 1) understanding the control of posture and gaze during turn and reach movements and 2) discriminating feed forward and feedback mechanisms in reaching movements made during passive body rotation. The experimental findings and analyses will provide a comprehensive evaluation of the role of feedforward predictive mechanisms in the control of reaching movements involving body rotation.

Orofacial clefts, specifically cleft lip and cleft palate, are common and costly congenital anomalies whose etiologies remain largely unknown. One of the most promising clues to the causes of orofacial clefts is that women who use vitamins containing folic acid in early pregnancy are at much lower risk for cleft-affected pregnancies. Although the underlying mechanisms by which folic acid contributes to these reduced risks are unknown, the evidence suggests that folate intake prevents clefts by compensating for susceptibilities in folate/one-carbon metabolism. However, clear identification of genetic determinants in this pathway through association studies has proven elusive. The approach we propose to further define the molecular genetic mechanisms behind orofacial clefts is based on two previous observations from our group. First, deep sequencing reveals a substantial number of novel, nonsynonymous variants in folate pathway genes (frequencies <=1%) that adversely affect enzyme function, yet are remediable by folate supplementation. Second, a discovery-sequencing study of all folate pathway genes in the context of spina bifida, an anomaly with many similar attributes to isolated clefts, revealed compelling risk signatures only by analyzing biologically relevant allelic combinations. These data suggested that combinations of alleles, both common and rare, are integrated into metabolic function, which ultimately underlies disease risk. Thus, we hypothesize that genetic susceptibilities in one-carbon metabolism may also be etiological for clefts and that these susceptibilities can be conferred by both low-frequency and common alleles, and possibly by synergy between relevant combinations of pathway variants. To test this hypothesis, we will sequence the coding regions in all folate/homocysteine pathway genes (N=32) from a population of ~375 cleft-affected infants and ~375 controls. We will test enzyme variants, individually and in combinations, for their functional impact and nutritional remediation based on quantitative cell-based assays in the yeast S. cerevisiae, and correlate allele distribution and functional studies with clinical phenotype. In addition, we will evaluate the relevance of murine models of orofacial clefts to human etiologies in two ways. First, we will sequence the coding regions of human orthologs of those mouse genes with convincing contributions to lip/palate closure (N=20) to identify the full spectrum of mutation to test whether such genes/variants play a role in human cleft development. Second, we will investigate the hypothesis, based on experiments in mice, that folate exerts its preventive effect through its control of methyl donor flux and subsequent epigenetic changes. Thus, we will explore global DNA methylation in cleft-affected and control newborns, which could lead to logical extensions at specific genomic loci. These studies will better define the causality of orofacial clefts as well as uncover the remedial mechanism of nutritional supplementation. This research plan capitalizes on an ongoing and successful collaboration that unites a unique combination of expertise for its execution.

This application is a renewal of the application titled "Trial of a Cognitive Intervention for Older Adults." This application is for the Field Site at Johns Hopkins University. Phase I of ACTIVE (Advanced Cognitive Training for Independent and Vital Elderly) was a randomized controlled trial of three cognitive intervention arms, addressing the question of whether improving basic cognition aided in maintaining functional independence in elders. As to be reported in JAMA (11112102), Phase I found strong, broad, and durable cognitive ability-specific training effects. The effect sizes were comparable to or greater than the amount of cognitive decline observed in other longitudinal studies, suggesting that the interventions have the potential to reverse age-related decline. There was minimal transfer of training effects to everyday activities (i.e., functional competence). However, it should be noted that through the two-year followup, there was no evidence of a significant decline in ADL and IADL status. Therefore, to adequately understand the cognitive transfer effects of the training interventions, a longer followup period is required, particularly to see whether there is a separation of the change trajectories for everyday activities of trained and untrained participants over time. Phase II of ACTIVE is proposed as a followup study focused on measuring the long-term impact of training effects on cognitive function and cognitively demanding everyday activities. The Phase II followup will consist of one assessment to include the Phase I post-test battery and a clinical assessment. The ACTIVE cohort (n = 2832) is a special sample, containing substantial oversampling of African American, socioeconomically poor, and very old adults. The Specific Aims of Phase II of ACTIVE are: 1) to determine whether the cognitive interventions (as initial treatment or as a consequence of repeated boosters) have long-term protective effects on functional outcomes; 2) to document any delayed transfer of the cognitive training to secondary outcomes; and 3) to identify individual factors that affect response to intervention. As in Phase I, the primary analytical approach to detecting treatment effects on both cognitive and functional abilities will be a repeated-measures, mixed-effects model incorporating all design features as fixed effects and individual-level variability as random effects. Other multivariate analyses including lagged and cross-lagged analyses of change using latent change analysis, structural equation modeling, and growth curve analyses will also be used as appropriate to characterize relationships between individual difference factors and change in functional competence. Retention is projected conservatively at 72% with 65% of the cohort providing full data and another 7% providing partial data at year 5. Power analysis shows that extending the study will make it possible to observe effect sizes on the order of 0.05-0.10 with excellent power, in the range of at least 80-90%.

The overall objective of this SBIR proposal is to develop and commercialize a human 3D or mini- liver bioassay system suitable for real-time analysis of metabolic functions and long-term drug toxicity profiles. At present, liver functions are mostly studied using HepG2 cell line and primary hepatocytes cultured onto collagen or polymer matrices. However, these 2D monolayer models allow only partial or short-term toxicity analysis due to rapid loss of metabolic functions. Interestingly, spheroid formation in 3D matrix/scaffold cultures is shown to improve host cell functions. In Phase I studies, we successfully demonstrated the feasibility of a new human bioscaffold- 3D HuBiogel (high-density gel) for supporting long-term survival and maintenance of primary liver cells. Moreover, integration of rotary bioreactor system allowed efficient production/analysis of viable mini-liver spheroids. We now propose to complete the development of new 3D-liver bioassay platform that exhibits improved metabolic functions, and thus will enable precise drug toxicity testing in vitro. Phase II specific goals are: 1. Establish an efficient hepatic cells cultivation protocol by optimizing 3D HuBiogel scaffolds and rotary bioreactor or perfusion culture strategies; 2. Test functionality of 3D-liver culture model by evaluating key biochemical and metabolism endpoints; and 3. Validate the practical utility of mini-liver assay system via real- time analysis of CYP450 induction and chemical/drug toxicity responses. Comparative control studies will include 3D culture scaffolds of Type-I collagen or Matrigel as well as fresh human liver-slice cultures. High throughput adaptability of 3D-liver bioassay will also be examined for toxicogenomics via CYP-microarrays. Thus, biologic relevance of new marketable 3D-liver bioassay system will be confirmed for improved drug toxicity analysis/prediction in humans. We are confident this advanced in vitro hepatotoxicity model will positively impact basic, preclinical and biomedical research arenas. Commercial Importance & Significance: No acceptable commercial liver toxicity assay currently exists which utilizes a human bio-scaffold culture system. VBI will develop a market-ready 3D-liver assay platform adaptable to HTS applications. Sale of 3D HuBiogel culture kits or in-house bioassay services would have significant world-wide market, as a research & diagnostic tool.

Candida albicans is by far the most frequently isolated human mycotic agent. In the oral cavity, oropharyngeal candidiasis (OPC) is a significant cause of morbidity in patients with HIV or AIDS. Other forms of mucosal candidiasis are also frequent in different patient populations such as infants, denture wearers, the elderly, and following antibiotic therapy. Azole derivatives, in particular fluconazole, are generally effective in the treatment of mucosal candidiasis. However, resistance has emerged as an important clinical problem. Large-scale DNA sequencing has provided an important sequence infrastructure for protein analysis. The term "Proteomics" refers to large-scale characterization of the proteins present in a cell, tissue or organism (the proteome) and involves the combined application of techniques to resolve, identify, quantitate and characterize proteins, as well as bioinformatics tools to store, communicate and interlink the resulting information. The experimental design of this proposal takes advantage of the recently completed NIDCR-funded Candida albicans genome sequencing project. The post-genomic era offers unprecedented opportunities to study host-fungal interactions. The specific aims of this proposal include: i) a pilot feasibility study of the analysis of the C. albicans proteome under a wide variety of conditions and development of a searchable proteomic map and database as a resource for the fungal community, ii) analysis of C. albicans azole resistance by proteomics and identification of proteins implicated in the regulatory networks of multidrug resistance. We will expect that these projects will establish the foundations for creating a fundamental tool for the C. albicans research community and for providing a detailed large-scale study of a biological phenomenon (drug resistance) with important clinical repercussions.

The long-term goal of this research is to provide a better understanding of metabolic and physical performance of the heart as affected by diabetes-mellitus. Specifically, I will study the regulation of cyclic AMP and glycogen metabolism along with mechanical performance in hearts from normal and diabetic rats to determine if and how these parameters are altered by diabetes. Cardiac metabolism and performance will be studied using the isolated perfused rat heart (non-working and working) which provides the advantage of allowing a study of direct effects on the heart without having to consider secondary interventions. Carbohydrate utilization and metabolism will be studied by looking at the effects of diabetes on regulation of glycogen metabolism by glucose, fatty acids, catecholamines, insulin and glucagon. Metabolic effects will be determined by measuring tissue levels of glucose-6-phosphate, glucose-1-phosphate, and uridine diphosphoglucose, and by measuring incorporation of labeled substrate into glycogen. Tissue enzymatic activities will be determined for glycogen synthase, protein kinase, synthase phosphatase, glycogen phosphorylase, phosphorylase kinase, phosphorylase phosphatase, adenyl cyclase, and phosphodiesterase. We will also examine the effects of diabetes on regulation of cyclic AMP metabolsim. The physical parameters to be monitored include coronary flow, cardiac output, heart rate and aortic pressures. Metabolic and physical parameters will be determined under normal, anoxic and ischemic conditions. This study will provide new information regarding effects of diabetes on cardiac metabolism and function under normal and simulated infarct (ischemic) conditions. A study of this nature could eventually determine if increased risk of cardiac disease is a direct or secondary effect of diabetes and may result in improved treatment of diabetics.

Estimates of the hip joint centers (HJC) are usually based on predictions from anatomical landmarks and anthropometrical measures such as pelvic breadth. There is concern regarding the relevance of this approach in patients characterized by obesity or pelvic deformity. Recent investigations have explored estimating HJCs using the functional method. Current literature often fails to disclose the form of the algorithm used in these tests, although three distinct forms exist. In addition, previous research has ignored the interaction between the algorithms, the portion of sphere surface represented by the data, the nature and magnitude of noise contained in the data, and the effect of different marker placement strategies. The goal of this project is to understand these interactions in order to know when clinical implementation of the functional method is appropriate and when it is likely to produce erroneous results. The specific aims of his project are: 1) to model the area of motion and radial perturbation values as a function of the error associated with the functional algorithms, 2) to determine the effect of area of motion on HJC estimates measured under non weight-bearing conditions, 3) to determine the effect of marker placement on radial perturbations and HJC estimates under non weight-bearing conditions, 4) to compare the functional algorithms to estimates from prediction methods and with known HJC locations based on ultrasound and DEXA imaging, and 5) to determine the characteristics of functional algorithms and marker placement strategies on HJC estimates measured during weight-bearing. In the computer modeling component of this study, the range of hip flex/ext, abd/add, and random radial noise will be systematically altered in order to produce maps of the centroid displacement. The maps calculated from these simulations will be used to establish theoretical constraints for use of the algorithms in clinic. In the clinical component of the study, the standard for HJC location will be determined with DEXA and ultrasound. Motion analysis data collected during standing and walking trials will be used to reconstruct HJCs from 1) different algorithms, 2) different virtual knee marker reconstruction methods, and 3) delimited amounts of sphere surface. The characteristics of marker noise associated with different placement strategies will be analyzed. Resulting HJCs will be compared to the standard to determine the interactions of each variable, and the acceptable range of values from each variable that will produce HJCs within 1 cm of the standard.

by the Chemistry Core. These compounds will be designed as opioid agonists and bradykinin/dynorphin antagonists in order to produce potent and efficacious antinociception targeting the pathology of neuropathic pain while eliminating antinocicpetive tolerance. The Biochemical Core contains 7 aims that will test such novel compounds. The in vitro pharmacological data in particular will provide timely feedback to the Chemistry Core on the structure-activity relationship (SAR) to further inform chemistry design. The initial binding and functional characterization of all novel compounds (est. 20 to 50 compounds/year) is necessary and essential for target-based drug discovery. To identify lead compounds, we must evaluate their affinity at multiple opioid and bradykinin receptors, and their apparent biological activity at each of these receptors. Studies will include in vitro tissue assaysto determine agonist and antagonist activity as well as novel compound activity at calcium channles using calcium fluorimetric analysis in transfectd cells. We will use a number of in vivo animal models to identify whether such novel bi-functional compounds produce antinociception as well as antihyperalgesia in inflammatory and chronic pain states. Finally, in vivo studies will be performed to detemine whether such compounds will result in antinocicpetive tolerance. Overall, Studies will be performed to identify molecules with agonist activity at one receptor and concurrent antagonist actions at a second receptor. The biochemical core provides dedicated equipment, personnel and expertise in data analysis for the entire project. It serves to centralize the use and maintenance of shared equipment and the technical training of personnel to use these equipment, management and oversight of animal protocols required by IACUC and Radiation Control, as well as to ensure data and information sharing with the Chemistry Core and the other projects. The biochemical core will synergize with projects A, C and D by providing lead compounds to directly test their hypotheses. RELEVANCE (Seeinstructions): Inflammatory and chronic neuropathic pains are growing areas of unmet medical need. Clinically, chronic pain remains poorly controlled by available therapies and thus adversely impacts quality of life (Arner & Meyerson, 1988). One reason for this lack of effect is the absence of compounds that specifically target the pathology of neuropathic pain. The goal of the chemistry &biochemical core of this PPG are to synthesize and test compounds that result in potent and efficacious antinociception while eliminating tolerance. PROJECT/

DESCRIPTION, OVERALL (provided by applicant): This application is for continued support of the Digestive Diseases Research Core Center (DDRCC) at Washington University St. Louis, which is the central institutional resource for investigators interested in the regulatory mechanisms and pathophysiology of the gastrointestinal tract and liver. The central focus of investigation is on inflammatory bowel diseases and its complications. However, we take a broad view of relevant research and will continue to support research on basic biological processes relevant to inflammatory bowel diseases, even if the systems being studied are not immediately applicable to inflammatory bowel diseases. The DDRCC supports an outstanding Research Base consisting of 62 Investigators in 10 departments with research grants totaling $17.5 million dollars (47% NIDDK funding). The DDRCC sustains its strong Research Base by actively supporting career development of new investigators through its Pilot/Feasibility Program. Nine former and four current Pilot/Feasibility awardees currently contribute to the Research Base. Four research core facilities (Murine Models/Gnotobiotic Core, Morphology Core, Functional Genomics Core and Proteomics Core) play a central role in promoting collaboration and synergy between individuals. They pool resources to provide services and expertise that would be cost and time- prohibitive to develop de novo, and maintain. To facilitate the conduct of clinical and translational research in inflammatory bowel diseases, the Clinical Component of the DDRCC consists of a Tissue Procurement Facility archives clinical samples linked to longitudinal clinical information ( >1,100 patients with inflammatory bowel diseases recruited in three years) and promotes new collaborative and synergistic interactions between basic researchers and clinicians. The DDRCC Clinical Component and Research Core facilities are integrated within the framework of the Clinical Translational Science Award - sponsored biomedical informatics infrastructure. By collecting clinical samples and linking them to integrated clinical information collected in focused longitudinal studies and research data generated from these samples by the DDRCC Research Core Facilities, the DDRCC is building a comprehensive multidimensional discovery platform from which investigators can launch further hypothesis-driven studies on inflammatory bowel disease pathogenesis.

The proposed work combines mutant and biochemical analysis to localize the genes for ribosomal proteins in the eucaryote Drosophila melanogaster. The evolution of ribosomal proteins within the genus Drosophila will be investigated by 1 and 2 dimensional polyacrylamide gel electrophoresis. Tryptic peptide analysis, on one or two of the invariant ribosomal proteins from three species will be carried out to measure the sensitivity of our electrophoretic system in detecting mobility variation. Studies on the coordinate regulation of ribosomal RNA and protein synthesis will be carried out using drugs (5-Fluorouridine and alpha-amanatin) and mutants (temperature sensitive assembly mutants) which differentially effect either the synthesis or maturation of ribosomal RNA or protein. These studies will be carried out in embryonic Drosophila tissue culture cells. An experiment is described which utilizes the specificity of alpha-amanatin in inhibiting the mRNA like polymerase, and autoradiography of H3-uridine labelled chromosomes, to localize the map position of transfer-RNA genes.

To characterize the cognitive dysfunction,behavioral disorders, type & degree of dyslexia Klinefelter's Syndrome and other sex chromosome aneuploidy.To identify the similarities/differences in the dyslexic phenotype of individuals with XXY, XYY, XXX and isochromosome X Turner's Syndrome subjects. They will be compared to primary hypogonadal XY men,normal gender and age group controls, and subjects with the same karyotype but with minimal or absent dyslexic features.

The bacterium Bartonella henselae causes a variety of disease syndromes including severe systemic life-threatening infections in AIDS patients. The NIH Plan for HIV-Related Research (FY2002) lists objective 2F as "elucidate the pathogenic mechanism of HIV-related opportunistic infections in adults and children." Despite the recognition of B. henselae as an emerging pathogen, very little is known about the novel mechanisms by which this bacterium causes disease. This project targets the most unique aspect of Bartonella pathogenesis (angiogenesis), that is observed primarily in AIDS patients with bacillary angiomatosis (BA) and bacillary peliosis hepatis (BPH). The central hypothesis of this proposal is based on a new model of B. henselae -induced angiogenesis that we have advanced requiring a major role for surface proteins and structures of B. henselae in this process. The following specific aims are proposed to test the hypothesis: 1) determine the role each of the four major surface structures of B. henselae play in interaction with host endothelial cells, 2) examine the ability of B. henselae and each of the four major surface structures of B. henselae to induce production of vascular endothelial growth factor (VEGF), 3) determine if VEGF produced by interaction of B. henselae with other cell types is able to function in a paracrine manner to induce proliferation of human microvascula endothelial cells. In vitro cell culture systems will be utilized for both the induction of VEGF (macrophages, fibroblast! epithelial cells) as well as the proliferation of endothelial cells leading to angiogenesis. The definition of the pathogenic processes utilized by B. henselae will help us better understand why this bacterium causes a mild disease in healthy individuals and severe diseases including BA and BPH in AIDS patients.

The fluid-mosaic model articulated by Singer and Nicholson provides a unifying hypothesis for the structure of biological membranes. A crucial aspect of this hypothesis is the interaction between proteins and lipids, but for nearly every case relatively little is known about such interactions. The coat protein (B-protein) from the filamentous phages (fd, M-13, fl, etc.) can be isolated in pure form in abundance, is extraordinarily hydrophobic, has a known sequence of just 50 amino acids, and forms tight associations with purified lipids. We (Dunker and Williams, J. Biol. Chem., 252, 6253, 1977) and others have shown that the B-protein can assume two distinct conformational states when associated with lipid. This is the only well-characterized example in which a protein in two distinct conformations associates with lipid. The core of this grant application is to compare protein/lipid interactions of the two conformationally different protein states using laser Raman spectroscopy, differential scanning calorimetry, and electron microscopy. We have already carried out studies of the beta-sheet-rich, c-state B-protein complexed with dipalmatoyl lecithin using laser Raman spectroscopy. In this grant application we seek support to extend these studies to other lipids with differing head and tail groups and to repeat all of these studies with the B-protein in the vastly different, alpha-helix-rich b-state.

Summary: The liver has an enormous capacity to regenerate, as demonstrated by the 2/3 partial hepatectomy model in rodents. In addition, the liver has a stem cell compartment acting as a backup regenerative system. Activation of the stem cell compartment occurs when the hepatocytes are functionally compromised, are unable to divide, or both. In stem cell-aided liver regeneration, progeny of the stem cells multiply in an amplification compartment composed of the so-called oval cells. Recent studies have also suggested that bone marrow cells can differentiate down the hepatic lineage, while other studies show they do not. Regardless of their origins, oval cells express the phenotype usually referred to as the transitional cell between hepatocytes and biliary cells. The overarching question is, which systemic signals are responsible for determining the magnitude and efficiency of oval cell activation within this type of hepatic repair? Two additional questions in oval cell biology are; how do oval cells become activated; and what role do extracellular factors play in their engraftment and differentiation into the hepatic architecture? The proposed research will explore three specific molecular mechanisms involved in these processes. In preliminary experiments, we have shown that Connective Tissue Growth Factor (CTGF), Notch-1 and Somatostatin all participate in oval cell-aided liver regeneration. This suggests that we can use these factors as starting points for deciphering the underlying mechanisms of how and why oval cells participate in liver regeneration. The studies proposed in this application will determine the extent each of these molecules play in the oval cell aided regenerative process, thus leading to a better understanding of the liver regeneration process as a whole. We will pursue the following specific aims: Specific Aim I. To test the hypothesis that CTGF and stellate cells are necessary components needed to facilitate oval cell proliferation in liver. Specific Aim II. To determine the role of Notch-1 and its receptor Jagged-1 in oval cell aided liver regeneration. Specific Aim III.To determine if somatostatin (SST) has a role in oval cell activation and can enhance the efficiency with which bone-marrow stem/precursor cells engraft in an injured liver. It is anticipated, with confidence, that the proposed studies will yield new and significant data about the mechanisms of governing oval cell activation, proliferation and differentiation.

The Kinesin-2 family of molecular motors transport cargo along cilia and flagella in a process termed intraflagellar transport. Disruption of intraflagellar transport results in truncated cilia and flagella at the cellular level, and at the organismal level leads to defects in body plan development and organ failure. There is a concerted effort underway to define the molecular machinery underlying intraflagellar transport, but characterizing this process at the molecular scale is made difficult by the virtually complete lack of understanding of the molecular mechanism of the Kinesin-2 motor. To answer cellular questions such as how the motor activity is regulated and how many motors are required for transport, it is essential to first characterize Kinesin-2 motors at the single-molecule level, and define the key biochemical transitions that underlie their mechanism. A unique and puzzling feature of Kinesin-2 motors is that instead of containing two identical motor domains like most kinesins, they are made up of two different motor domains. Domain swapping experiments carried out in the PI's lab have now shown that these two heads move at different speeds, leading to the hypothesis that their activities are tuned to optimize transport characteristics of the intact heterodimer. [unreadable] [unreadable] The goal of the proposed work is to define the molecular mechanism of Kinesin-2 function by characterizing KIF3A/B, the mouse Kinesin-2 ortholog. Single-molecule fluorescence and optical tweezer experiments will measure the performance characteristics of these motors (speed, force production and microtubule affinity) and will uncover the inner workings of these motors to reveal the structural basis of movement and regulation. The specific aims are as follows: 1) Test whether the two heads step along microtubules at different rates. 2) Determine whether kinetic differences arise from structural differences in the motor domains or from coordination between the motor domains. 3) Measure the number of sequential steps motors take during each encounter with a microtubule and the dependence of this processivity on external load. 4) Identify the biochemical transitions in the Kinesin-2 kinetic cycle that control motor speed and processivity. Experimental data will be incorporated into computational models of Kinesin-2 motility to test hypotheses regarding the cellular behavior of these motors. [unreadable] [unreadable] Understanding the molecular mechanism of Kinesin-2 motility is important for uncovering the molecular basis of transport-based diseases such as polycystic kidney disease, defects in sperm motility, and retinal degeneration. Furthermore, insights into the mechanochemistry of kinesin motors will aid in developing anti- tumor therapies targeting mitotic kinesins. [unreadable] [unreadable]

This is a resubmission of a competitive renewal application for a Cardiovascular Research Training Program (CRTP) that was established twenty years ago at the University of New Mexico (UNM). It has served to provide opportunities for predoctoral and postdoctoral trainees from underrepresented backgrounds to gain research training in cardiovascular biology. This grant has also been a catalyst for the development of an integrated program of research and training in cardiovascular sciences at UNM. The CRTP has bridged the basic and clinical sciences and has fostered new productive collaborations between disciplines. This area of research and training emphasis has particular relevance to the state of New Mexico where the incidence of cardiovascular-related disease is high, especially in the Hispanic and Native American populations. During the tenure of the grant, several new programs have evolved that unify research in the cardiovascular sciences at the UNM Health Sciences Center (HSC), including the formation of the Vascular Physiology Group with researchers from the School of Medicine and the College of Pharmacy. In addition, since the initial funding of the award, the UNM HSC has identified signature research programs including one focused on Cardiovascular and Metabolic Disease (CVMD). This area of research receives special funds from the Office of Research to support pilot studies and to enhance collaborative projects between its members. All of the mentors on this grant are members of this signature program with a key member of the T32 serving as the leader of the CVMD program. Collaborative research is also facilitated by the structure of the Biomedical Sciences Graduate Program that offers a unified training environment within the UNM HSC. The graduate program is non-departmental and provides interdisciplinary training over a broad base of biomedical science in the first year, followed by in-depth training in the chosen discipline. The goals of th current application are to build upon this framework and to improve our success in recruiting and training scientists from underrepresented backgrounds. To foster this latter objective, we have designed recruitment strategies that seek to improve integration of our program with existing minority-based undergraduate and master's programs at UNM and partner institutions in the region. We will also promote collaborations with clinician scientists in the recently funded Clinical and Translational Sciences Center (CTSC) to provide new opportunities for training in translational research. Leaders of the CTSC are mentors on this T32 and will help to coordinate research opportunities in the CTSC for both pre- and post-doctoral trainees. Twenty productive and collaborative faculty members from both basic science and clinical departments will serve as faculty mentors. The CRTP will continue to support eight predoctoral and two postdoctoral positions per year. Planned appointment durations are 2-3 years for predoctoral students and 2 years for postdoctoral trainees, with subsequent support provided by individual training fellowships or mentored career awards. (End of Abstract)

Allelic loss and loss of function of tumor suppressor genes (TSGs) are the most frequent genetic abnormalities in lung cancer. Replacement of TSG function alone is therapeutic and often leads to lung cancer cells undergoing apoptosis or cell cycle arrest in vitro. Currently it appears that such replacement therapy must be done with genes rather than small-molecule mimics of TSGs. We recently demonstrated that restoration of function of 3p21.3 TSG FUS1, a proapoptotic protein, with the use of systemic nanoparticle delivery successfully cured mice with large human lung cancer orthotopic xenografts. In a phase I clinical trial systemic nanoparticle therapy delivered the FUS1 TSG to distant sites in stage IV nonsmall cell lung cancer (NSCLC) patients after intravenous injection. The FUS1 gene is inactivated in primary tumors due to 3p21.3 allele haploinsufficiency and defective post-translational modification of the remaining gene product. Enforced expression of the wild-type FUS1 in 3p21.3-deficient NSCLC cells significantly suppressed tumor cell growth by induction of apoptosis, functioning as a TSG in vitro and in vivo. However, FUS1 overexpression in human bronchial epithelial cells and other normal cells does not affect their viability. We also observed that exogenous expression of wild-type FUS1 protein in NSCLC and SCLC cells deficient in FUS1 had inhibitory effects on several oncogenic protein tyrosine kinases (PTKs), including EGFR, PDGFR, c-abl, and c-kit in NSCLC and small cell lung cancer cell lines. Associated with this PTK inhibition, there was a markedly enhanced cell response to the clinically available tyrosine kinase inhibitors (TKIs) imatinib and gefitinib. Thus, combined treatment with FUS1 and TKIs led to a significant growth inhibitory effect on lung cancer cells that were resistant to TKIs given alone. We hypothesize that treatment with the FUS1 gene delivered by nanoparticles combined with TKI therapy will have additive or supra-additive growth inhibitory and pro-apoptotic effects on lung cancer cells overcoming TKI-induced or intrinsic resistance. Our long-term goal is to develop personalized, pathway-targeted treatments which are more effective and less toxic than current treatments. The specific aims in this proposed study are 1) to determine whether FUS1-induced apoptosis and growth arrest are potentiated in various lung cancer cells /n vitro and in vivo in tumor xenograft models by TKIs that are currently being used in the clinic, 2) to identify sensitivity and resistance phenotypes associated with FUS1 expression and molecular signatures associated with these phenotypes in human lung cancer cell lines and tumor specimens and validate candidate signature molecules in a larger population of lung cancer cell lines, tumor xenografts, and clinical specimens from lung cancer patients, and 3) to conduct a Phase l/ll clinical trial combining Fl/S7-nanoparticles and erlotinib in stage IV lung cancer patients who have progressed following treatment with platinum-containing regimens.

Group A streptococci remain an important cause of morbidity and mortality as a result of infections and as a consequence of the postinfectious sequela of acute rheumatic fever. Rheumatic fever and chronic rheumatic heart disease are leading causes of cardiovascular death and disability in developing countries, and have recently increased in incidence in the U.S. Among the potential virulence factors contributing to the pathogenesis of streptococcal diseases, the role of the hyaluronic acid capsule is perhaps the least well understood. The objective of this proposal is to elucidate the role of the capsule both in virulence and in the immune response to streptococcal infection. Both microencapsulated and unencapsulated isogenic mutant strains will be derived from a mucoid (i.e., highly encapsulated) strain of group A Streptococcus by transposon mutagenesis. These mutant strains will be used to examine the role of the capsule in virulence. Specifically, the ability of strains with altered capsule expression to produce lethal infection in mice will be compared with that of the wild type mucoid strain. The effect of partial or complete loss of capsule expression on resistance to phagocytosis will be examined using an in vitro assay of opsonophagocytosis. The mutant strains will also be studied in a mucosal colonization model to determine the effect of degree of encapsulation on the establishment and persistence of mucosal colonization, and on the humoral immune response to both colonization and infection. These studies will lead to a better understanding of the pathogenesis of streptococcal infection and of the immune responses of the host which may play a role in acute rheumatic fever.

Amplification of the genes coding for rRNA (rDNA) occurs in the oocytes of various species of organisms. Recent work indicates that rDNA is replicated during amplification by a rolling circle mechanism. In organisms where it has been studied, the genes coding for 5S RNA (5S DNA) are not amplified. The main objectives of this proposal have been and still are to determine whether amplification represents a generalized phenomenon associated with oogenesis; to determine the extent of diversity which exists in regard to the degree of amplification and the nature of amplified DNA; and to correlate this diversity with differences in the organization of amplified DNA. Emphasis will be placed on the last objective. To this end, molecular mapping techniques will be applied to somatic and ovarian rDNA of the house cricket, Acheta domesticus. We wish to determine the molecular size and organization of DNA sequences coding for rRNA, spacer sequences, and the entire rDNA repeating unit. Moreover, we would like to determine whether amplification in A. domesticus occurs via a rolling circle intermediate. Other workers have reported amplification of DNA in chromomeres of A. domesticus which apparently do not contain rDNA. Do these contain 5S DNA and is 5S DNA amplified? Finally, do vertebrates other than fish and amphibians amplify their rDNA? Is rDNA amplified in the uninucleolate oocytes of mammals? An answer to the last question is relevant to man because of the similarities between oogenesis in man and in other mammals.

The Contractor will attempt to determine vital status as of March 1, 1981, of all Coronary Drug Project (CDP) patients still alive at the termination of follow-up in February, 1975. For all patients who have died, an attempt will be made to determine cause of death.

The central theme of this program involves studies of the mechanisms of action of cytochrome P450 enzymes and nitric oxide synthase. The principal approach is the synthesis and characterization of reactive iron porphyrin species that are thought to be intermediates in the catalytic cycles and to relate the interconversions of these species toward a molecular understanding of these proteins. Cytochrome P450 is the central protein involved in drug detoxification and hormone metabolism while nitric oxide and peroxynitrite. Synthetic metalloporphyrins can be employed as probes to intervene in these processes in diagnostic ways. Thus, these agents may prove to be significant tools for elaborating the biology of superoxide, peroxynitrite and NO. These same metalloporphyrins have shown impressive activity in animals suggesting their application as pharmaceutical agents. Our effort seeks to provide a foundation of mechanistic and kinetic information which can be applied to in vitro models, cell culture studies and whole animal models of specific disease states such as ischemia-reperfusion, sepsis and autoimmune diseases. Experiments are aimed at determining what reactive intermediates are formed and what their biological targets are likely to be. The elaboration of these will processes will facilitate the design of metal complexes for the catalytic decomposition of peroxynitrite and these other species. The studies of nitric oxide synthase aim to illuminate the range of mechanisms of NOS and to seek out new oxidation processes that may help with the rational development of NOS inhibitors. Peroxoiron(III) porphyrin species are now readily accessible in aqueous solution at ambient temperature. Rapid kinetic techniques have been developed to study the reactivity of these species. A central question is how to modulate the chemistry of these complexes between nucleophilic and electrophilic pathways that are essential to understand the variety of P450 mediated processes. Synthetic and semi-synthetic phospholipid assemblies are used to model and understand the larger scale events in P450 and NOS action and to probe the permeability of membranes to reactive oxygen species. Membrane binding by amphiphilic metalloporphyrins is extraordinarily sensitive to pH. This is an unprecedented observation that may have immediate application to the design of metallopharmaceuticals.

This report briefly describes our findings as mentioned in our objectives. Interesting MRI findings emerged related to the identification of a new intermediate phenotype and cortical circuitry. We tested whether altered dorso-lateral-prefrontal cortex (DLPFC) connectivity is heritable, independent of DLPFC activation, and is affected by a genetic variation in the ZNF804A gene. Siblings of patients with SZ showed increased DLPFC inefficiency vs. controls. Our connectivity analyses showed abnormal DLPFC functional coupling with the hippocampus formation (HF) and to a lesser degree the rest of the PFC, in patients and siblings but not controls. Correlation between PFC activation and connectivity in the siblings was not detected, concluding that these events may be independent. The ZNF804A gene affects distributed network-based neurointegrative deficits that reflect genetic risk mechanisms for SZ. These deficits are mildly present in siblings and normal controls with the risk-associated genotype. This suggests that altered PFC activation and coupling are independent intermediate phenotypes, one of which is modulated by susceptibility genes. A SNP in ZNF804A appears to modulate PFC-HF coupling but not PFC activation. DLPFC, which communicates with HF, inferior parietal lobules and ventrolateral PFC during working memory (WM), is probably susceptible to genetic modulation and our data suggest that genes related to increased SZ susceptibility effect in this modulation. Imaging Genetics has allowed us to expand our knowledge of genetic interactions that support biologic phenomena. We studied interactions between NRG1 and NMDA-glutamate pathway partners. NRG1 plays a role in brain development, neuronal function and is a SZ risk gene. We show an interaction between NRG1 and SZ which we validated using BOLD fMRI on WM in controls. Carriers of risk alleles showed inefficiency in processing in DLPFC. We showed epistasis between SNPs inNRG1 and ErbB4 and a 3-way interaction between NRG1 and SNPs in ErbB4 and AKT1. Each of these interactions (NRG1 x ErbB4 x AKT1) was validated using fMRI in controls carrying the risk SNPs and all were found to be inefficient in DLPFC processing. Our data suggest complex epistatic effects implicating a NRG1 molecular pathway in cognitive function and pathogenesis of SZ. Another study, explored the impact of an AKT1 functional variation on human medial temporal lobe (MTL) development and plasticity. We found another 3-way interaction of AKT1 with BDNF and COMT, adding to convergent evidence identifying effects of AKT1 variation in memory-dependent neuroplasticity and structural brain processes in development and SZ and epistatic interactions with neurotrophic and dopaminergic processes that modulate AKT1 effects. AKT1 modulation via pharmaceuticals effected cognition and PF-MTL brain structure in SZ. Therefore, AKT1 and related neuroplasticity and developmental pathways may influence cognition and risk for SZ through effects that may be pharmacologically modifiable. Other allelic variations we found in DAOA and COMT genes interact to increase risk for SZ and impaired DLPFC information processing. This again, demonstrated a functional yet statistically significant interaction between risk alleles in DAOA and COMT which may play a role in glutaminergic and dopaminergic dysregulation. Lastly a variant of the FGF20 gene, which modulates hippocampal biology, mRNA expression, verbal episodic memory, and morphology, was shown in T allele carriers to increase HP mRNA expression, had relatively larger HP volume, reduction in verbal episodic memory, and a sharp decline in HP volume with normal aging. From mRNA expression to brain morphology to cognition and interaction with aging, we confirm a role for the Parkinsons disease-associated FGF20 gene in brain structure and function during development and aging. Another study used healthy controls to explore the neural mechanism through which selective updating of information is stored in WM. A novel wm task was designed that parsed WM maintenance, overwriting, and selective updating. A functionally coupled network consisting of midbrain regions were selectively engaged during wm updating when compared to both overwriting and maintenance. In both behavioral and neurophysiological terms, we revealed that selective updating was clearly parsed from maintenance and nonselective updating of information. Analysis also showed differential midbrain-dorsolateral prefrontal interactions during selective updating between subjects rated as low and high-performers. The midbrain engaged the DLPFC during updating to a greater degree in low performers vs. high performers. These findings of meso-cortico-striatal network engagement during updating supports the notion of dopaminergic modulation of striatal gating of DLPFC during the selective updating of wm content, which was predicted in computational models of wm function. Future studies using genetic assays and pharmacological manipulations to investigate this network will be needed to further support these claims. The group also investigated neurophysiological correlates of altered brain function associated with aging. Our study analyzed age-related brain morphometric changes using cortical thickness, cortical gray matter volume, and surface area as morphological indices. It has been previously reported that there are global and regional changes in gray matter volume as we age. Using a surface-based automated reconstruction technique, we analyzed healthy subjects and found with age, there are global and regional changes in cortical volume, thickness and surface area. Age-related atrophy, in most cortical regions was indicated by a reduction in thickness however, most striking was that the prefrontal cortex showed a reduced thickness, surface area, in addition to volume which confirmed previously published reports. Interestingly, the average global surface area reduction was greater than in any one cortical region alone. The area that appeared preserved with age was the MTL, but this may have been due to limitations in our method. Future studies will be needed to assess brain histological changes that occur during normal aging as related to these morphometric measures. We will continue to explore the effect of aging on brain structures and systems, and the role of genetics on these changes. Finally, we explored changes in GABA levels in SZ in vivo and examined the genetic association with the ErbB4 gene. We found significant effects in A carriers as increased ErbB4 expression and higher cortical GABA levels. To our knowledge, this is the first study to measure GABA in living human brain. It is unclear whether this association reflects functional differences in enzyme activity or is a neuroanatomical decrease in the total number or distribution of interneurons or synapses. Another study examined a method to reproduce quantification of brain metabolites and parse gray/white matter differences that impact interpretation of imaging results in clinical populations. There have been inconsistencies in the reproducibility of MRS studies that quantify GABA levels in vivo, such as field strength, scanning techniques, acquisition and processing which resulted in varied measures. GABA is a major inhibitory neurotransmitter in human brain and its dysfunction has been implicated in many neuropsychiatric disorders. We used a J-edited, single voxel-based spectroscopy method to measure GABA + macromolecules in the anterior cingulate cortex and right frontal white matter. In fact, our method allowed us to quantify metabolite differences between gray and white matter. Reproducibility of estimations of brain metabolites may lead to a clearer interpretation of results where voxels have both gray and white matter and may be applied to studies of clinical population.

Wyoming Research Innovations, Inc. will develop in situ cross-linkable hydrogels, called synthetic extracellular matrix (sECM), for soft tissue repair. The ability to fill a traumatic, surgical, or chronic defect with a self-molding cell-interactive sECM prepared with a fast-curing mixture of biocompatible macromonomers would have significant clinical utility. These materials will be prepared from chemically modified hyaluronic acid and chondroitin sulfate, which are two major glycosaminoglycan (GAG) components of the ECM. Biomaterials from cross-linked GAG derivatives produce versatile new bioinspired materials. The sECM concept incorporates ECM proteins as well as GAGs, and we propose to demonstrate that crosslinking can occur in the presence of seeded cells and in a tissue defect in vivo. Two crosslinking chemistries will be investigated and the resulting materials will be evaluated for compatibility with cell growth. In situ cross-linked materials will be evaluated for in vivo efficacy in an animal model. The proposed materials address an unmet need in surgical care and wound repair. Many surgical defects, traumatic wounds and deep chronic wounds fail to heal without augmentation using grafted natural or synthetic materials. A biocompatible hydrogel that mimics ECM and is conducive to cell seeding and growth would have many important clinical applications.

The Rh antigens of human erythrocytes have been known for over 30 years. Their importance in iso-immune disease of the newborn and adult hemolytic anemia has become established. On the other hand, the chemical nature of these antigens remains very much in doubt. The reason for this is that these antigens have, thus far, not been obtained in free solution. The present evidence indicates that the antigenic structure is a combination of lipid and protein intimately linked with some membrane structure. This investigation is concerned with methods of attempting to solubilize these antigens and determine their chemical structure. The methods that are used in this investigation are the methods that have been devised to attempt to solubilize biological activity, such as enzymes, from membranes and other insoluble biological components. These include detergents, and manipulations of pH, and ionic strength, etc. Evidence of antigen activity will be looked for in the solubilized supernatants by hemagglutination inhibition and other appropriate techniques. If evidence of activity is found, further characterization will be done. BIBLIOGRAPHIC REFERENCES: In press. Solubilized Human Erythrocyte Membranes and the Rh Antigen System. Lorusso, Binette and Green. Proceedings of the Fifth International Convocation on Immunology. S. Karger, 1976.

PROJECT SUMMARY Malaria affects over half the world's population and causes nearly 450,000 deaths every year, mainly in sub- Saharan African children. Rapid development of parasite resistance to frontline antimalarials and lack of an effective, durable vaccine are the most formidable barriers to achieving malaria control and elimination. Host cell invasion by Plasmodium is essential to establish an infection and cause disease. Therefore neutralizing parasites before they enter host cells can have an enormous impact in reducing morbidity and mortality. For this reason proteins on the surface of merozoites (responsible for initiating the disease process) and sporozoites (infectious stage) are being evaluated as potential vaccine targets. However, even the most clinically advanced vaccine RTS,S, targeting the sporozoites, showed limited efficacy. The overall goal of this proposal is to develop a novel vectored gene therapy approach for malaria prophylaxis by targeting a universal pathway required for host cell invasion. This builds on our studies demonstrating a crucial role for the interaction of two Plasmodium proteins, apical membrane antigen 1 (AMA1) and rhoptry neck protein 2 (RON2), in junction formation between the merozoite and red blood cell (RBC), providing an anchor to facilitate parasite entry. Interestingly, RON2 is secreted onto the host cell and acts as a receptor for AMA1 present on the parasite surface. This is a unique example in host-pathogen interactions where both ligand and receptor are provided by the parasite to mediate successful host cell entry. A 47-amino acid conserved region of RON2 (termed RON2L) is necessary and sufficient to bind a similarly conserved hydrophobic pocket in AMA1. This points to an evolutionarily preserved ?lock-and-key? mechanism designed to prevent changes. Importantly, we demonstrated that RON2L competes for binding to AMA1 and efficiently neutralizes Plasmodium merozoites before they can enter RBCs and recent data suggests that this pathway is also used by P. falciparum sporozoites to enter hepatocytes. We reasoned that targeting this interaction can be an effective antimalarial strategy as it would make it harder for the parasites to develop resistance. To do this, we will develop an adeno-associated virus (AAV)-mediated gene therapy to endogenously express the entry inhibitor RON2L. Preliminary data demonstrated that the entry inhibitor expressed by AAV transduction is produced as a correctly folded, stable fusion protein and potently neutralized P. falciparum merozoites in vitro. We will use an in vivo rodent malaria model and a P. falciparum humanized mouse model to evaluate protection against infection and disease. Successful outcomes of our aims will provide a novel malaria prophylaxis approach by self-targeting a key step used by malaria parasites to enter host cells.

Core A is essential to the success of the SPORE and provides oversight and support of all SPORE projects and activities; ensures compliance with all general, governmental, and National Cancer Institute (NCI)/National Institutes of Health (NIH) regulations and requirements; coordinates communications and consultations with the NCI project officer and other NCI staff in preparing all required reports and publications; coordinates all SPORE-related meetings and communications of the Executive Committee, Internal and External Advisory Boards, the Biostatistics and Data Management Core (Core B), and the Tissue Procurement, Pathology and Biomarker Core (Core C); and other key functions. Over the previous grant period, Core A oversaw the annual budget/rebudget development, distribution, and oversight of grant funds; supported the Executive Committee and advisory boards in scheduling and coordinating regularly scheduled meetings and ad hoc consultations; coordinated the annual solicitation, review, and award process for the Career Development and the Developmental Research Programs; served as the primary liaison in developing potential InterSPORE collaborations and with the NCI SPORE program representatives; and interacted on a regular basis with Core Directors and Project Leaders to review progress, identify problems, and resolve issues hampering successful achievement of their aims. The past trial activation and accrual record of this HN SPORE was the appropriate target of criticism in our previous review; thus, the SPORE leadership team has implemented several mechanisms to enhance the oversight, and thus activation and accrual, of our SPORE clinical trials, including 1) quarterly accrual report submissions to the Executive Committee (and more frequently should concerns arise); quarterly presentations by the Pis of the SPORE trials to the Executive Committee on accrual progress; annual review of all trials by the Internal and External Advisory Boards; and a requirement to address accrual deficiencies within six-months or risk study closure or replacement. Major SPORE and Core A leadership changes occurred in the fall of 2006, when Dr. Scott M. Lippman succeeded Dr. Waun Ki Hong as SPORE PI and Core A Director and Dr. Rakesh Kumar succeeded Dr. Reuben Lotan as a SPORE Co-Pi and Core A Co-Director (sharing these roles with Dr. Gary L. dayman, who continues a Co-Pi and Co-Director from the previous grant period). The experienced leadership of the HN SPORE will ensure full compliance with all policies and procedures required of NCIand NIH-funded research programs to successfully achieve the SPORE's overall aims.

The assembly of triadyglycerol-rich lipoproteins (TAG-LP) involves the initial formation of a small HDL sized primordial particle with a neutral lipid core and a later process which adds TAG and phospholipid (PL) to create nascent VLDL. The N-terminal 20.5% of apoB(B0.5) has homology to lipovitellin (LV) that consists of an N-terminal beta barrel, a region of 17 amphipathic alpha helices (AaH) and 2 beta sheet domains, the A and C sheets. Proper folding of the N-terminal is necessary for secretion since certain point mutations and failure to form disulfide bonds target it to degradation. Limited proteolysis of 617 and B20.5 indicates that apoB homology to LV is approximate but not exact. Structural determination of isolated proteolytic and expressed domains using CD, NMR and X-ray diffraction will define B20.5 domains at the molecular level. The assembly with lipids as driven by the primary sequence of apoB is studied in MTP-deficient C127 cells transfected with C-terminally truncated forms. The role of molecular chaperones in folding, lipidation and in quality control is studied both in C127 and hepatoma-derived cells. The region from B20.5 to B29 recruits surface molecules, mainly PL to the nascent particle. As the peptide lengthens from B20.5 to B29 the number of surface molecules increases from approximately 5 to about 70 per particle. The sequence between B32 and B41 recruits almost 200 TAG (about 1 TAG/2.3 aa) which form nascent particles with a cryo-EM discernable core which is not seen in B32.5. Biophysical analysis shows that B37 and B41 peptides must interact directly with the core. This region contains 24 amphipathic beta strands (ApS) 12-15 aa long. Consensus 12 aa ApS and 27 aa p sheet peptides bind to oil/water (O/W) interfaces, are elastic and are not displaced at high pressure. ApoB also contains 2 major regions a2 and a3 of AaH. Consensus AaH peptides bind to O/W and air/water interfaces, but are ejected at a critical pressure into the aqueous phase. Native apoB also binds to the TAG/W interface and cannot be fully displaced by high pressure, but parts of the apoB come off when compressed and snap back on rapidly when the surfaces are again expanded. Thus, we suggest the flexible parts of apoB are AaH and the non-exchangeable part is AbetaS. This project dissects the complex pathway by which "bad cholesterol" (apoB associated cholesterol, VLDL, LDL) is made and secreted by the liver. The long term goal is to find procedures or drugs to reduce bad cholesterol, heart attack and stroke.

The effect of ethnicity on outcome in systemic sclerosis (SSc) is not well understood. The hypothesis to be tested in this proposal is that SSc is a more aggressive disease in non-Caucasians, who manifest a higher occurrence of critical organ involvement and a worse prognosis, and that reasons for this may include genetic factors as well as sociodemographic or behavioral determinants. Therefore, the investigators will: 1) select a well-defined cohort of Caucasians, Hispanics and African Americans (approximately 80 Caucasians, 40-50 African Americans and 40-50 Hispanics) with SSc of five years or less in duration from SSc patients seen at 3 southern Texas medical centers in order to ascertain their initial status and follow their course and early outcome at regular intervals for a period of at least three years; 2) determine the HLA class II genotypes as well as disease-associated alleles of other candidate genes found to be associated with SSc (eg fibrillin, Interleukin 1 receptor antagonist, DNA topoisomerase I and others); 3) determine the sociodemographic parameters of these patients; 4) determine pertinent clinical and laboratory paramenters, including disease manifestations, laboratory features and selected SSc-associated autoantibodies; 5) follow disease progression to outcomes as manifested by: a) development of end-stage pulmonary fibrosis, b) end-stage scleroderma-related renal disease, c) scleroderma heart disease, d) scleroderma-related gastrointestinal involvement, e) MD global assessment of disease severity, f) functional disability, or g) death; 6) examine the relative contributions and interactions of genetic, demographic, socioeconomic, cultural, and initial clinical and laboratory features on the course and outcome of early SSc. Elucidation of those factors affecting outcome in SSc will not only permit modification thereof, but also the identification of high-risk patients to whom earlier and more aggressive therapy could be rendered, which may ultimately improve the prognosis of this disease.

Alzheimer's disease (AD) is the most common form of dementia in the elderly, affecting about 10% of those aged 65 or over, but no treatment to delay or halt the progression of the disease is as yet available. Under the present situation where the proportion of the elderly population is progressively increasing, AD has become a significant problem for the elderly citizens including Veterans. Therefore, the study of this disease is relevant to the mission of the Veterans Administration. Recently, new evidences indicate cerebrovascular complications and impaired regional cerebral blood flow as the causative factors in AD along with its associated memory loss and behavioral changes. In turn, this evidence provides the rationale for early treatment of cerebrovascular dysfunction which is critical for delaying, slowing down or preventing the development of AD. Nitric oxide (NO) plays an essential role in the maintenance of vascular tone and hemodynamics. Currently, free NO donors, such as sodium nitroprusside and nitroglycerine, are being used in various clinical settings. However, these NO donors, releasing free NO, may not be an effective treatment for AD because the concomitant chronic inflammation and oxidative stress scavenge free NO from these donors to generate deleterious peroxynitrite. Here, we propose to evaluate the therapeutic potential of GSNO, a major transport form of NO in biological systems, for the treatment of AD. GSNO is able to induce several cell signal transduction pathways that are essential for biological action of NO via S-transnitrosylation without generation of free NO. In addition, GSNO is implicated in anti-inflammatory, anti-oxidant, and vaso-protective effects under various disease conditions. Therefore, GSNO is a promising candidate as a stand-alone therapy for cerebrovascular complications implicated in AD. In support, we have recently observed that GSNO treatment inhibited the inflammatory response and also reduced A accumulation and improved impaired learning and memory performance in aged rats with chronic cerebral hypoperfusion, an animal model for vascular dysfunction associated with late-onset AD, and in transgenic mice over-expressing human A. On the basis of these data, the following specific aims are designed to improve therapeutic reliability of GSNO in the treatment of AD, to optimize pharmacological parameters, and to investigate mechanisms underlying the drug action(s). Specific Aim 1: To establish therapeutic efficacy and pharmacological optimal dose of GSNO therapy for cerebrovascular complications associated with Alzheimer's disease: Under this aim, we propose to optimize the efficacy of GSNO treatment against cerebrovascular complications associated with development and progression of AD pathology. Two animal models will be employed for these studies: transgenic mice producing high levels of A for studies of early-onset AD and aged rats received bilateral common carotid artery occlusion (BCCAO), a clinically relevant model for human mild cognitive impairment and associated late-onset AD. Specific Aim 2: To investigate the mechanism(s) underlying GSNO therapy: Implication of anti-inflammatory and vaso-protective activities of GSNO: Under this aim, we propose to investigate the mechanism(s) underlying GSNO mediated anti-inflammatory and vaso-protective signaling cascades in brain micro-vessel endothelial cells. Focus of this aim will be to study the S-nitrosylation of cell signaling mediators regulating gene expression for inflammatory mediators and A clearance across the blood brain barrier (BBB). The results from studies described here should provide a better understanding of pharmacological, biochemical and cell biological events underlying vascular pathology of AD, and the possible utility of GSNO as therapeutics for the management of AD.

Previous application of cooperative game theory to household decision making resulted in few testable or refutable hypotheses concerning the actual behavior of the household. Those applications did point to new data requirements, namely, the need to measure the separate unearned incomes of individuals in a coalition and one could test hypotheses on household demand concerning these separate incomes. In addition, they indicated that the all-important substitution effects in a bargaining household need not behave in the same way as those effects in a neoclassical household. Hence, a bargaining household could be observed to be a neoclassical one so that it would be difficult to distinguish empirically between a household that behaves neoclassically and one that operates according to the Nash axioms (Pareto optimality, solutions that are invariants to positive linear transformations, symmetry, etc.) Moreover, those applications yielded no way to distinguish empirically between a symmetrical and a dictatorial household. Acceptance of the bargaining approach thus necessitated the derivation of hypotheses that could be refuted by observed data; in short, "to give some examples of empirical propositions that might emerge," as stated in the NIH Critique. That is what the enclosed paper begins to do.

There are over three million needle biopsies performed every year in the USA, typically for the diagnosis of breast, prostate, thyroid, lung, liver, and pancreatic cancers. Because needle biopsies are less invasive than surgical biopsies, they are much more cost-effective. However, a major limitation for all needle biopsies are that the pathologist or cytologist views the tissue or cells in only two dimensions, while the important structures for determining cancer and invasiveness of the disease is more clearly seen in threes dimensions (3D) versus two dimensions. What is true in radiology is expected in pathology, 3D imaging will give a quantum increase in the information content, expected to increase sensitivity and specificity of early cancer diagnosis. For many cases such as suspected pancreatic cancer, the small needle biopsy is all the tissue that the clinical team has to test before making a lifesaving decision. In this proposal, the preferred specimen for pancreatic cancer diagnosis is a thin needle core biopsy, which the entire 1-2cm long specimen is visualized in 3D using advanced optical imaging. After needle procurement from ex vivo pancreas, all handling and preparation for highest quality 3D imaging will be prototyped and validated within a novel microfluidics device. Every tissue preparatory step of fixing, staining, clearing, and transporting to 3D imaging is conducted within the microfluidics channel, which can be automated. The goal of this project is to reduce the time for biopsy tissue preparation to less than half the standard time in a pathology lab. Furthermore, the 3D architecture of the tissue will be preserved for the first opto-mechanical measures of biopsy intactness. The diagnostic value of the needle biopsy specimens after automated preparation will be determined by experienced pathologists. The ability to visualize 3D morphology within tissue biopsies will also allow the pathologists to bette compare ex vivo diagnosis with new in vivo 3D imaging. These ex vivo technologies that rely on optically cleared tissue and traditional bright-field imaging (Z-stack imaging and optical projection tomographic microscopy) can become the pathologists' bridge for better understanding in vivo 3D imaging technologies, such as confocal, optical coherence tomography, and photo-acoustic imaging. This project is expected to lead to a transformation in pathology from 2D to 3D, and in the ability to provide less invasive, low-cost and rapid cancer diagnosis, directly affecting several millions of US citizens per year.

Dopaminergic neurons control aspects of mood, cognition and movement, and are involved in the pathogenesis of a variety of brain disorders including drug addiction. A central regulator of dopaminergic neurotransmission is the dopamine (DA) transporter (DAT) that is expressed exclusively in dopaminergic neurons and is responsible for clearance of extracellular DA. Psychostimulants such as amphetamines and cocaine elicit dramatic behavioral phenotypes primarily by targeting DAT directly and increasing extracellular DA concentration. DA neurons are functionally heterogeneous and morphologically complex, with spatially separated somatodendritic and axonal domains, and dense axonal arborizations. Proper DAT distribution in the DA neuron is critical for the function of the dopaminergic system in its normal and diseased states. The principal goal of this proposal is to elucidate mechanisms by which the DAT-mediated substrate re- uptake is regulated in DA neurons by endocytosis, long-distance intraneuronal trafficking and membrane diffusion. We have developed and characterized a knock-in mouse that expresses an extracellular epitope (HA)-tagged DAT (HA-DAT). In this mice we used a combination of quantitative electron microscopy of intact brain and acute sagittal brain slices, a novel physiological ex vivo model of the intact DA neuron, to demonstrate distinct patterns of DAT trafficking in different regions of DA neurons. Using mechanistic analysis in cultured cells, we have identified molecular determinants in the DAT that are involved in its targeting to filopodia and its mobility in the plasma membrane, and novel sequence motifs that are important for constitutive and signal-induced DAT endocytosis. We propose that since intraneuronal distribution of DAT is regulated by the balance of vesicular traffic, cell surface retention and membrane mobility of DAT, these processes must be controlled differently by intracellular signaling in somatodendritic and axonal domains of neurons and in different areas of the striatum and midbrain. To test these hypotheses, we will: 1) Define kinetics and mechanisms of long-distance DAT trafficking between spatially separated axonal and somatodendritic domains of DA neurons; 2) Define routes, kinetics, and mechanisms of constitutive and signal- induced endocytosis of DAT in DA neurons; 3) Test whether brain region- and signal-specific DAT trafficking affects DAT substrate transport kinetics. Defining mechanisms of DAT regulation by trafficking in intact DA neurons will significantly advance our understanding of the cell biology and physiology of the DA system, and may suggest potential therapeutic options for treatment of DA pathologies such as drug addiction.

These experiments propose to deal specifically with the question of chemical mediation in one resorption stimulated by the application of force. Pilot data have supported the hypothesis that chemical factors in the local microenvironment of bones exposed to forces can stimulate increased release of calcium from fetal bones. It will be determined if this stimulation calcium release is also reflected by an incrase in osteoclast numbers. If this correlation can be made, calcium release will be used as a bioassay for bone resorption in further studies of this phenomenon. Other major objectives will include a description of the effect of duration and degree of force application on the stimulation of such bone resorption mediator(s). Preliminary investigations will also be made into the chemical nature of this/these mediators(s). Correlations between the degree of root resorption in the test animals and amount of calcium release in the bioassay will also be made. Using simple orthodontic appliances on rats, tipping forces of known magnitude will be applied. The tissue immediately surrounding the point of pressure application and similar areas from control animals will be excised, homogenized and cleared of cellular debris. After normalizing these homogenates for wet weight of sample, their ability to stimulate resorptive activity will be assessed by bioassay. The bone resorption bioassay to be used is based on the release of pre-incorporated 45Ca from the fetal long bones into a tissue culture medium which has been treated with a test homogenate. The specific objectives will be carried out by generating and testing homogenates from alveolar bone exposed to forces for differing periods of time and of differing magnitudes. The chemical nature of this/these bone resorption mediator(s) will be investigated by perturbing the test homogenates and assessing alterations in their activities using the bioassy. The degree of root resorption will be assessed by quantitative SEM procedures.

The overall aim of this project is to increase the number of New York City Technical College (City Tech) students who continue their education and pursue a baccalaureate degree in science. The Research Double Bridge to the Baccalaureate program is a student research transfer oriented collaborative involving City Tech, which grants the Associate in Science degree, and two science baccalaureate- granting institutions, Brooklyn College (BC) and City College of New York (CCNY); all are part of the City University of New York (CUNY). The major aims of this project are: to increase the number from a significant pool of City Tech students who transfer to science baccalaureate degree programs at the partner institutions, to increase City Tech faculty research capability, and to enrich the science/research skills of City Tech students. These aims will be achieved by the implementation of specific activities involving City Tech and the partner colleges. The complementary activities include an enrichment program for students. The enrichment program includes a pre- research course and summer research work, access to financial aid (including MARC) as well as a home base at the senior colleges upon transfer, and a transition program to facilitate transfer. A faculty research program will not only grow a cadre of faculty researchers but will increase student research opportunities. The three institutional collaborators will build on their NSF-funded efforts at SMET institutional reform to design a transition course based on their similar core reform agendas.

The purpose of this grant is to support the scientific and clinical activities of Cancer and Leukemia Group B (CALGB) by providing necessary infrastructure to the CALGB program at The Ohio State University (OSU). The multidisciplinary collaborative research approach at OSU has a tract record of providing more effective methods of prevention, detection and treatment of adult cancer, with a particular focus on breast cancer, gastrointestinal cancers, genitourinary malignancies, hematologic malignancies, lung cancer and melanoma. This research focuses the efforts of medical and hematologic oncologists, surgeons, radiotherapists, transplanters, psychiatrists, pathologists, cytogeneticists, translational laboratory scientists, basic scientists, statisticians, epidemiologists, nurses, pharmacists, and clinical research associates on well designed and conducted studies asking interrelated clinical and basic science questions whose answers contribute significantly to patient care and to reduction of cancer within populations at increased risk. Included in this project are: 1) the exploration of new therapeutic agents, and their associated toxicities, in Phase I, II and III clinical trials; 2) the evaluation of efficacy and toxicity of new regimens including combinations of new and old agents in an effort to exploit synergistic combinations more effectively; 3) the development of multi-modal approaches to specific tumor problems using surgical, immunological and radiotherapeutic measures in optimal combinations; 4) the involvement of pertinent basic science disciplines such as molecular genetics, biochemistry, pharmacology, immunology and biostatistics in the formulation and execution of specific therapy protocols; 5) the improvement of cancer care in the community by using these protocols to educate pre- and post-doctoral students, nurses, allied medical personnel and physicians, 6) the evaluation of biologic studies in correlation with clinical endpoint to develop more rationally based cancer management, 7) the evaluation of cancer controls efforts such as early detection, and 8) the study of the psycho-social aspects of cancer. Under the overall coordination of Clara D. Bloomfield, M.D., OSU contributes to CALGB activities through patient accrual to protocols, development and leadership of research protocols, leadership and participation in the scientific and administrative committees of CALGB, housing of multiple CALGB core laboratories and facilities, CALGB meeting participation and authorship on Group publications. In addition, OSU performs institutional pilots and provides lab data that lead to new CALGB studies. Support of this program should increase our ability to prevent, detect, treat and cure adult cancer and improve the quality of life of cancer survivors. [unreadable] [unreadable] [unreadable]

Much of science, including biomedical science, consists of discovering and modeling causal relationships. Increasingly, biomedical scientists have available multiple complex data types and a very large number of samples, each of which has an enormous number of measurements recorded.. There is a pressing need for algorithms that can efficiently discover causal relationships from large and diverse types of biomedical data and background knowledge. In the past 25 years, tremendous progress has been made in developing general computational methods for representing and discovering causal knowledge from data. However, these methods are not readily available, nor easy to use by biomedical scientists, and they have not been designed to exploit the increasingly Big Data available for analysis. The proposed Center will create a computer ecosystem through which to implement and apply an integrated set of tools, new and repurposed, that support the representation and discovery of causal knowledge from large and complex biomedical data. These computational approaches will be accessible to a wide variety of biomedical researchers, data analysts, and data scientists who might not otherwise take advantage of them. Three very different biomedical problems will drive the development of the methods, tools, and interactive system architecture. While we anticipate that new biomedical discoveries will be made in each of these problem areas using the methods developed by the Center, the longer-term impact will result from the development of the computational technology itself, which will be generalizable to the full spectrum of biomedical research. The Center will be very active in the sharing of these knowledge, methods, and tools through a rich offering of training activities and through engagement with other Centers in the consortium.

RESEARCH ACCOMPLISHMENTS NOMID STUDIES: CLINICAL: 1. We analyzed 5 year outcome data in 20 patients and 3 year outcome data in 26 patients with NOMID who have been receiving escalating doses of anakinra. The initially observed good clinical response to anakinra persists, the drug is overall well tolerated. In most patients hearing and vision was persevered over 3 and 5 years, however patients who experienced hearing loss on anakinra had already significant hearing loss at baseline and more persistent enhancement on MRI. Low grade leptomeningeal inflammation was seen in up to 50% of patients at 3 years but dropped further at 5years. Although no new bone lesions occurred with anakinra treatment, preexisting bony lesions continued to expand on treatment. These data suggest that treatment with anakinra not only controls disease symptoms and inflammatory blood markers but can also prevent the progression of organ damage. Studies to assess the prevention of any organ damage in very young children are ongoing. 2. Disability on treatment with anakinra significantly improvement and persisted up to 3 and 5 years. 3. Chronic inflammation leads to growth retardation, osteoporosis. We showed that bone mineral density increases above the age-expected increase which correlate with a change in bone markers suggesting an increase in bone production and catch-up height and weight gains are seen in treated patients. 4. An ongoing study in patients with NOMID using the long acting IL-1 inhibitor canakinumab with the goal of finding an optimal dose regimen and monitoring long term outcome is currently ongoing. LABORATORY: 1. We have characterized the inflammatory skin lesions in collaboration with Dr. Lowes at Rockefeller by comparing lesional and nonlesional pretreatment skin biopsy samples to post treatment skin biopsies and normal control tissue. 2. We studied a chondrocyte primary cell lines from a bone lesions from one patients with NOMID and in collaboration with Dr. Stratakis laboratory, their group identified that the cyclic adenosine-monophosphate (cAMP) signaling pathway is activated in patients with NOMID which is similar to bone lesions of other non-inflammatory conditions including fibrous dysplasia, osteochondromyxomas, and chondro- and osteo-sarcomas. These pathways all result in activation of the wnt signaling pathways and are dependent on active caspase-1 but not on IL-1. These findings explain our clinical findings that bone lesions once they have developed are non responsive to IL-1 blocking lessons. 4. In collaboration with Dr Nishikomoris group in Kyoto a number of our mutation negative patients analyzed by conventional methods are found to have somatic mosaicism. DIRA STUDIES: 1. Last year we published on a novel autoinflammatory disease DIRA, or deficiency of the IL-1 receptor antagonist and reported 4 founder mutations in the Newfoundland population, the Dutch, the Northern part of Puerto Rica around the city of Arecibo and Lebanon. Patients present with neonatal-onset multifocal osteomyelitis, periosteitis and pustulosis and systemic inflammation. The disease mimics infectious osteomyelitis and sepsis and has a high mortality if untreated. In collaboration with Dr. Jesus from Brazil and Dr. El-Shanti from Qatar a novel 15 basepair deletion in 2 unrelated Brazilian patients was found, suggesting a novel founder mutation in Brazil. Functional analyses indicated that the protein is expressed, but has no affinity to the IL-1 receptor and stimulation assay abnormalities are identical to those of the other DIRA patients As expected and similar to all other live children with DIRA, both patients had complete resolution of skin and bone lesions and a complete laboratory response. Similarities of the bone lesions in DIRA and patients with CRMO have spurred interest in the evaluation of the IL-1 pathway in patients with CRMO as well. ADULT ONSET STILL'S STUDIES We are closing out a pilot study of 5 patients with adult-onset Still's disease (AOSD) who were treated with the long acting IL-1 inhibitor rilonacept. We are assessing long term efficacy and safety. However, 3 patients had a satisfactory response to IL-1 TRAP and continue to be on treatment since the study stopped. A less uniform response to rilonacept was observed in 2 patients, one of them was subsequently switched to the commercially available IL-6 blocking agent tocilizumab. BEHCETS DISEASE A cohort of patients with Behcets disease has been recruited by Dr. Henderson and the clinical and laboratory manifestations of the diseases are being assessed. A treatment protocol to assess the role of a novel long acting IL-1 inhibitor XOMA 052 has been approved by the IRB and is expected to start recruitment soon. UNDIFFERENTIATED DISEASE We have accumulated a number of patients who have been referred with autoinflammatory disorders presenting early in life, who have clinical similarities with NOMID and DIRA patients but can clinically not be classified. We are using a genetic approach of whole genome sequencing in collaboration with Dr. Kastners group, an immunologic approach to characterize the immune deviation and a treatment approach using approved therapeutics in the treatment of these children to obtain answers to the underlying immune dysregulatory defect. CONCLUSIONS AND SIGNIFICANCE 1. In now 2 severe autoinflammatory syndromes, cryopyrin associated periodic syndromes, CAPS (which includes FACS, MWS and NOMID), and deficiency of the IL-1 receptor antagonist syndrome, DIRA, the crucial role for IL-1 in the pathophysiology of these illnesses with multiorgan involvement has been demonstrated. 2. A novel founder mutation in 2 Brazil patients with DIRA has been identified and may raise the awareness of that disease in Brazil. 3. Somatic mosaicism in NLRP3 accounts for a large number of mutations in mutation negative patients assessed by conventional methods. 4. Long-term treatment effect of IL-1 blockade in patients with NOMID indicates that treatment of this devastating illness is safe and effective at least up to 5 years, improves disability and retards/stops progression of hearing loss and vision loss. 5. The phenotypic difference between NOMID and DIRA (no CNS, eye and ear inflammation in DIRA, compared to NOMID) and difference in skin manifestations, urticaria-like versus pustulosis, allow us to study the organ- specific immune responses downstream of IL-1 and may shed light on processes that determine specific organ manifestations. 6. The development of long acting IL-1 inhibitors may allow for better disease control in those patients who are not in complete remission and offer more convenient treatment options particularly in children in whom daily injections can be quite traumatic. A study evaluating the long term efficacy of the long acting IL-1 inhibitor canakinumab in patients with NOMID is ongoing. 7. The heterogeneous response to IL-1 blockade in patients with AOSD suggests a more complex cause for the inflammatory response in patients with AOSD. 8. The role of IL-1 in Behcets disease will be investigated with the newly approved protocol.

DESCRIPTION: (Applicant's abstract). The differentiation of cell types and the formation of synaptic contacts within the neural retina are guided, in large part, by molecules in the extracellular environment. Trophic factors represent an important class of extrinsic signals and have been shown to regulate certain aspects of the differentiation, as well as the survival, of discrete neuronal populations elsewhere in the nervous system. Their actions in the developing retina are poorly understood at present. Progress in the cloning and identification of trophic factor families has begun to provide the tools with which to define their role in retinal cell differentiation. The proposed research focuses on the role of nerve growth factor (NGF) and related trophic factors in the formation of the chicken neural retina. Our recent work has shown that the embryonic and adult chicken retina synthesize NGF. We have proposed that it may act in a "classical" manner as a target-derived factor for extrinsic neurons in the isthmo-optic nucleus (ION) that innervate the retina. However the embryonic retina also expresses a high level of NGF receptors, raising the possibility that NGF may act locally to regulate such early events as retina cell migration, differentiation and process outgrowth. A second factor structurally related to NGF, brain-derived trophic factor (BDNF), appears to act as a target-derived factor for retinal ganglion cells. This suggests that additional members of the NGF trophic factor family are likely to be important for guiding the normal development of the retina. The goals of this study are to first establish the relationship between the cell types that express NGF receptors and that synthesize NGF in the embryonic retina using presently available peptide antibody and nucleic acid probes. The role of NGF in retinal development will then be determined using antibodies to eliminate NGF in the embryonic eye at important development stages. The results will be interpreted with respect to two possible actions of NGF - 1) a target-derived factor regulating the survival of ION neurons and 2) a local regulator of retinal cell differentiation. Finally, chicken BDNF will be cloned and information about conserved sequences will be used to screen for additional members of the NGF/BDNF trophic factor family. The expression of BDNF and related trophic factors in the embryonic retina and optic tectum will be determined to assess their role in guiding retinal development. Defining the roles of NGF and related factors in the normal development of the retina will aid in the understanding of developmental and degenerative eye disorders and contribute to strategies and aimed at restoring retinal function following injury.

PROJECT SUMMARY Our goal is to explain the neurophysiology of breathing. Breathing refers to periodic movements of the chest and airways that ventilate the lungs. To breathe, the brain must: ? Generate a rhythm, ? Produce a spatiotemporal pattern for breathing muscles, ? Integrate sensory feedback. Here we focus on jobs 1 and 2, discovering the ionic mechanisms underlying the rhythm and motor pattern. The brainstem preBtzinger complex (preBtC) generates the inspiratory breathing rhythm. Its core rhythmogenic interneurons are derived from Dbx1-expressing progenitors (i.e., Dbx1 neurons). Because we know the site (preBtC) and the canonical cell-class (Dbx1) at the point of origin for breathing, we are well equipped to discover which ion channels influence normal inspiration (eupnea), ?sighs?, and gasps in hypoxia. We manipulate ion channels using genetic technologies. We assess breathing phenotypes in intact adult mice. We explain the biophysics of these phenotypes via patch-clamp recordings from adult Dbx1 preBtC neurons. Three types of ion channels are implicated in preBtC function: (1) Na+ channels that engender persistent Na+ current (INaP), (2) Transient receptor potential (Trp) channels that mediate Ca2+-activated nonspecific cationic current (ICAN), and (3) K+ channels that give rise to transient outward K+ current (i.e., A-current, IA). Specific aim 1 evaluates the role of INaP in Dbx1 preBtC neurons. A rhythmogenic role for INaP has been suspected for 27 years. We use intersectional mouse genetics and short-hairpin RNA (shRNA) to knockout or knock-down Na+ channel genes that give rise to INaP and evaluate its role in eupnea, sighing, and gasping. Specific aim 2 evaluates the role of ICAN in Dbx1 preBtC neurons. ICAN is a major charge carrier for inspiratory bursts. Next-generation RNA Seq technology provides us with a suite of Trp channel targets that we will acutely attenuate using shRNA to test the role of ICAN in eupnea, sighing, and gasping. Specific aim 3 evaluates the role of IA in Dbx1 preBtC neurons. IA-expressing preBtC neurons feature other key rhythmogenic properties so they may be specialized. We use intersectional mouse genetics and shRNA to knockout or knock-down K+ channel genes for IA to evaluate its role in eupnea, sighing, and gasping. Then we selectively kill the 56% of IA-expressing Dbx1 preBtC neurons to test whether they serve a specialized rhythmogenic role. The success of this project would be a watershed in terms of understanding the ion channel-level origins of real behavior. The new knowledge could be applicable to treatment and prophylaxis of respiratory pathologies with a central etiology. The new knowledge may provide general insights regarding the neural origins of motor rhythms.

The primary purpose of this grant is to conduct clinical research studies in patients being treated on protocol therapeutic efforts of the Western Cancer Study Group. These studies involve assessment of laboratory and clinical response rates to various chemotherapeutic treatment programs. Major attention will be directed to developing and effecting clinical protocols of study for enteric cancers and structuring a linear array or clinical systems approach to delineate the data needed for a more effective attack on the enteric cancers; the activities are recognized as part of the Principal Investigator's role as current Chairman of the Group's Enteric Committee. In parallel with some of these treatments where adriamycin or high-dose methotrexate are employed, laboratory assessment of plasma levels of parent drug will be carried out in an effort to relate such pharmacokinetic data to therapeutic response and clinical toxicity. The clinical activities of the program at Harbor General Hospital with respect to the Western Cancer Study Group are integrally related to joint efforts in Radiotherapy. Ancillary laboratory research studies to the clinical efforts will center in part on the influence of heat on tumor uptake of anticancer drugs. For in vitro studies clinical (surgical) material will be used; for in vivo work microwave heating of transplanted animal tumors will be used.

Project Summary/Abstract Acute myeloid leukemia (AML) is maintained by a small minority of self-renewing leukemic stem cells (LSCs). Defining and targeting the key molecules that specifically regulate LSCs might eradicate AML. Heat shock transcription factor 1 (HSF1) is known to regulate the expression of heat shock proteins (HSPs) to protect cells from misfolded protein-induced proteotoxic stress. Although HSF1 is not an oncogene, it enables cancer cells to accommodate imbalances in signaling and alterations in DNA, protein, and energy metabolism, a phenomenon called ?non-oncogene addiction?. Targeting HSPs in AML is being explored with promising results. However, targeting HSPs induces feedback that increases HSF1 activity, which may compromise treatment effects. Therefore, targeting HSF1 directly may be a more attractive alternative. However, given the critical roles of HSF1 in the maintenance of normal cellular homeostasis, targeting HSF1 could adversely affect normal hematopoiesis. Unexpectedly, our preliminary data show that HSF1 is dispensable for normal hematopoiesis, but specifically required for the self-renewal of LSCs. Mechanistically, deletion of HSF1 disrupts amino acid metabolism and mitochondrial oxidative phosphorylation (OXPHOS), which plays a critical role in regulating LSC function, and dysregulates multifaceted genes involved in LSC stemness. In addition, we identified that hepatocyte nuclear factor 4a (HNF4a) as a direct HSF1 target. Overexpression of HNF4a largely reconstitutes deletion of HSF1-induced impaired LSC function. Based on these observations, we hypothesize that HSF1 is specifically required for LSC self-renewal through regulating leukemic energy metabolism (mainly OXPHOS) and is a potential therapeutic target in AML. We propose the following two specific aims to test our hypothesis. Aim 1: To determine the underlying mechanism whereby HSF1 is specifically required for LSC self-renewal. We will determine 1) the impact of HSF1 ablation on LSC frequencies, proliferation and OXPHOS; 2) the HSF1 transcriptional targets in LSCs by comprehensive analysis of transcriptomic gene expression, chromatin accessibility and chromatin immunoprecipitation (ChIP) sequencing in the presence or absence of HSF1; 3) how HNF4a reconstitutes HSF1 ablation-induced LSC defects, especially the effect of HNF4a on OXPHOS; and 4) if deletion of HSF1 impairs mechanistically different types of AML. Aim 2: To determine if targeting HSF1 effectively eliminate human LSCs. We will determine 1) the impact of HSF1 inhibition on human LSC proliferation, apoptosis, self-renewal and oxidative phosphorylation; 2) the HSF1- dependent regulatory circuitry in human LSCs; and 3) if the expression of HSF1 protein correlates with AML therapeutic response and relapse. The expected outcomes of this comprehensive analysis are identification of the mechanism whereby HSF1 specifically regulates LSC self-renewal, providing the rationale for targeting HSF1 and using HSF1 to monitor AML progression.

The proposed research is an extension and expansion of a currently funded study [Adolescent Social Behavior. Predictors of Adjustment] that was designed to identify social interactional risk and protective factors what would moderate the deleterious influence of known stressors on social and psychological adjustment during adolescence. The study was imbedded in a larger study of the epidemiology of adolescent depression and other disorders. The overlay of the two projects provides a unique opportunity to bring to bear longitudinal data with a wide array of social, contextual, environmental, personal, and cognitive variables, including both clinical and nonclinical for tracing the course of adjustment during the transition from adolescence into adulthood. The primary goals of the proposed research are: (a) to continue and complete analyses originally planned for the current study, specifically the analyses of the T1-T2 design, and the group social interaction component, and (b) to. expand and extend the current studies. to: (i) explore the social,psychological, and other processes during the transition from late adolescence to young adulthood, and (ii) identify specific risk and protective factors that are predictive of adjustment during this critical period. The two noteworthy components of the study are (a) the prospective design that will allow us to untangle cause/effect relations, and (b) at least two indicators from different sources will be obtained for each relevant construct assessed. As such, the study is not only multi-method/multi-trait but also multi-respondent.

The specific objective of this proposed study is to quantify the mechanisms responsible for blood flow modification in normal and tumor tissues due to hyperthermia and hyperglycemia. Mature granulation tissue and VX2 carcinoma grown in a rabbit ear chamber preparation will be used as models for normal and neoplastic tissues, respectively. Using this preparation and the microcirculation experimental setup in our laboratory, modifications in blood flow in individual vessels following hyperthermia and hyperglycemia have been determined. In the proposed study, the role of red and white blood cell rheology in this flow modification will be determined. A micropipette aspiration technique available in our laboratory will be used to study cell deformability during hyperthermia and hyperglycemia. An intravital fluorescent microscopy technique will be used to study cell adhesion to vessels. This study will contribute to basic pathophysiology of tumors and aid in improving present approaches to hyperthermia treatment of tumors.

The administration of chemotherapy at the earliest time (neoadjuvant or induction chemotherapy) following diagnosis in an effort to reduce the risk of disease recurrence in patients with non-small cell lung cancer (NSCLC) is under investigation at multiple institutions in the U.S., Europe, and Japan. This approach has resulted in radiographic response rates of 56-70% with complete remission rates of 1-8% and partial remission rates of 55-64% in patients with stage IB-IIIA with 2-3 cycles of platinum plus gemcitabine, paclitaxel, or docetaxel. Patients with pathologically proven N2 disease prior to chemotherapy had negative N2 nodes on resection in 50% of cases. A complete pathologic remission has been documented in 6-16% of patients (95% Cl, 0-23%). Disease progression during treatment has been observed in 2-10% of patients, and a complete surgical resection was achieved in 70-92% of patients. It is important to devise less toxic, yet efficacious regimens that are well tolerated and can be given expeditiously so as to allow surgery to be done in a timely fashion. Neoadjuvant therapy allows for investigations of molecular parameters that may affect response to chemotherapy and patients' survival. It is our hypothesis that the expression of genes associated with activation, inactivation, and efficacy of the drugs gemcitabine and pemetrexed will predict response to therapy and prognosis. We further hypothesize that the expression of these genes will be altered during chemotherapy. We propose a phase II study of neoadjuvant chemotherapy with gemcitabine and pemetrexed in patients with resectable NSCLC, specifically correlating molecular parameters to the primary clinical study endpoint disease response (radiographic CR+PR) and the secondary endpoints complete pathological response at surgery, disease-free survival, and overall survival. [unreadable] [unreadable]

Neuronal differentiation is characterized by changes in both the extent of intercellular coupling and the expression patterns of connexin genes that encode gap junction proteins. Using a neuroblastoma culture system, we have found that stable exogenous expression of different gap junction genes results in either retarded or accelerated response to differentiating agents. Comparing gene expression using locally produced cDNA microarrays indicates candidate genes whose expression is enhanced or depressed in the transfected neuroblastoma cells. However, the preliminary studies raise important questions regarding the technique, including threshold of detection, reliability for each of the spots in the array and detection of clusters representing gene expression patterns. The Specific Aims of this application are to apply mathematical algorithms and modify experimental techniques so as to: 1) minimize the spot ratio errors; 2) determine expected values and controllable fluctuations of gene expression; 3) build the pre-Hilbert space of standard gene expression (SSGE); 4) compute the so-called "pathologs" of gene expression for parental and transfected cells; and 5) define gene clusters within the SSGE. We expect that these studies will not only improve our use of the microarray analysis, but will develop new concepts by which such expression patterns are generally used.

The Connective Tissue and Diseases Section began studying inflammatory myopathies (polymyositis, dermatomyositis, and related diseases) some years ago in an attempt to understand the relationship of autoantibodies to autoimmune disease. These diseases seemed to offer the best example of autoimmune diseases associated with highly specific disease-related autoantibodies and evidence of a viral etiology. They are very uncommon and hence relatively less studied than other autoimmune diseases, and they are very debilitating and hence in need of improved therapy. In order to attract patients here to allow more detailed clinical, immunological, genetic, and viral studies, we began doing trials of therapy and have completed a number of such studies. They are among the very few published controlled trials in this difficult to treat family of illnesses. These trials have encompassed a variety of approaches to immunosuppression. The most recent trial - a pilot trial of the anti-thyroid drug methimazole was undertaken to make use of an unexpected property of the drug which was discovered by Dr. Leonard Kohn of NIDDK and Dr. Dinah Singer of NCI: that it can down-regulate MHC. This trial was completed in the past year. Patients with active myositis received a 6-month trial, with muscle biopsies being performed for research purposes at the beginning and three months into therapy. The level of mRNA of MHC Class I & II in muscle tissue and simultaneously-obtained peripheral blood monocytes was measured by Northern analysis. Patients were assessed carefully clinically, and the presence of inflammation in the quadriceps was measured by MRI. The MRI analysis employed a computer-based measure of tissue edema on the STIR image which was recently published by our group along with the Department of Radiology in the Journal of Magnetic Resonance Imaging. An attempt is being made, in collaboration with the laboratory of Eric Hoffman at Children's National Medical Center, to determine the effect of the drug on gene expression in affected muscle tissue.In collaboration with the Alexion Corporation, we have begun a trial of a recombinant humanized antibody (H5g) directed at the late part of the pro-inflammatory complement pathway, anti-C5, in dermatomyositis. In that illness, activation of the late components of complement has been shown. The trial is double-blind, with a weekly infusion for two months. Dr. Lisa Rider has continued to examine patients with juvenile myositis at the Clinical Center and to lead a collaborative group of pediatric rheumatologists throughout the country and in Canada in a thorough clinical and laboratory description of the disease. Dr. Rider is a former member of ARB who now works at CBER, FDA and volunteers in the ARB clinic.

Duchenne muscular dystrophy (DMD) is a progressive, lethal, muscle wasting disease that affects 1 in 3500 newborn males. Although tremendous progress has been made in advancing cures for the disease through gene therapeutic approaches, clinical application of gene therapies remains a distant goal and strategies for ameliorating the pathology are needed to reduce disease severity until a cure is established. DMD muscle and mdx mouse muscle experience extensive inflammation that is believed to accelerate DMD pathology. However, recent discoveries have shown that the inflammatory infiltrate is complex and that some immune cells promote muscle regeneration. The ability of macrophages to modulate injury and repair of dystrophic muscles suggests that experimental manipulations of macrophage function could provide a strategy to influence DMD pathology. Furthermore, macrophages could provide a means for the targeted selective delivery of therapeutic molecules rapidly to sites where the disease is most active. The overall objective of this investigation is to use the mdx mouse model of DMD to examine whether immune cells can function as vectors to deliver therapeutic molecules that reduce the pathology of DMD. Our experimental strategy is to suppress the immune compartment of mdx mice by myeloablative irradiation, then transplant bone marrow cells obtained from mice expressing therapeutic transgenes or wild-type and then assay for effects on the mdx muscle pathology and function. The findings of this investigation will provide new insights into a novel mechanism for the delivery of gene therapy to dystrophic muscle and provide the foundation for new therapeutic strategies for addressing DMD, a significant health problem.

Childhood asthma affects roughly 10 million children under age 16 in the United States. Reduced early life exposure to microbial products, particularly endotoxin, has been proposed as an important factor which may account for increased risk of asthma through increased risk of allergy or of allergic lung inflammation. Low asthma rates observed in farm children has lent credence to this hygiene hypothesis. However, endotoxin is also associated with occupational airways inflammation, increased asthma morbidity, and wheeze in infants. The different influences of endotoxin on asthma development and exacerbation can only be clarified by prospective studies. Our current project, ES07036-05, is examining whether endotoxin and related microbial exposures reduce risk of developing asthma in a birth cohort of 505 Boston area children. With this supplement, we propose to increase our power to test the hygiene hypothesis by measuring endotoxin and related microbially derived exposures (lipopolysacchande (LPS), peptidoglycan (PG), total fungal biomass, and (1 -3)-Beta-D-glucan) for 1,002 children in the Yale Childhood Asthma Study birth cohort with 41 percent minority (Black and Hispanic) members. The Yale study also includes 1,002 older siblings with doctor-diagnosed asthma for whom we will prospectively determine whether microbial exposures increase asthma morbidity. The proposed expanded study population has significantly different hereditary and environmental influences than the Boston cohort. This supplement will provide sufficient numbers of subjects and statistical power to achieve the following objectives: (1) test the hygiene hypothesis in Black and Hispanic (mainly Puerto Rican) children; (2) determine the effect of endotoxin and other key microbial exposures on asthma morbidity in minority children; and (3) set the stage for future studies of gene-environment interactions between polymorphisms in the pattern receptors for innate immunity and endotoxin and other microbially derived exposures recognized by these receptors. Analyses will be controlled for both allergen exposure and a wide range of other demographic and environmental factors using data collected by the Yale study. This application requests funds for endotoxin, LPS, PG. fungal biomass (ergosterol) and (1 -3)-Beta-D-glucan assay of settled dust and for data analysis and interpretation. This supplement will be one of the first opportunities to test the hygiene hypothesis in a large minority population

Project Summary: This project aims to develop a novel non-invasive technique, coherent hemodynamics spectroscopy (CHS), for the local measurement of brain perfusion and autoregulation at the microvascular level. This new technique consists of three components: (1) An experimental method based on diffuse near- infrared spectroscopy (NIRS) to non-invasively collect brain perfusion data at the microcirculation level; (2) Mild perturbations in the systemic mean arterial pressure to evoke oscillations or transients in the cerebral hemodynamics; (3) The application of a new hemodynamic model to translate NIRS measurements of cerebral hemodynamics into measurements of physiological and functional quantities. Specifically, this proposal focuses on CHS measurements of dynamic cerebral autoregulation (dCA) and capillary transit time (CTT), which yields absolute cerebral blood flow (CBF). The capability of CHS to measure local CTT, CBF, and dCA will be capitalized by developing spatially-resolved CHS (for brain mapping) and depth-resolved CHS (for discrimination of extracerebral and brain tissues). We plan clinical tests at two units at the Tufts Medical Center, namely the dialysis unit and the neurological critical care unit, to demonstrate the clinical potential of CHS. The first clinical test, on diabetic hemodialysis patients, aims to validate CHS measurements of CBF and dCA in a comparative study with age-matched healthy controls, and to demonstrate the potential of CHS to detect a reduction in cerebral perfusion during hemodialysis. The second clinical test, on patients who suffered from aneurysmal Subarachnoid Hemorrhage (aSAH) and Traumatic Brain Injury (TBI), will directly test the clinical potential of CHS in monitoring cerebral perfusion and autoregulation deficits. The broad objective of this application is the development of a new tool for the quantitative, non-invasive assessment of local cerebrovascular hemodynamics at the microcirculation level. CHS offers a significant clinical potential as a result of its ability to non-invasively assess and monitor brain tissue perfusion, which is impaired in a variety of clinical conditions, including subarachnoid hemorrhage, traumatic brain injury, hemodialysis treatment, ischemic stroke, and cardiopulmonary bypass.

The proposed research is to continue fundamental studies on the physical-chemical functions and chemical reactivity of hydrophilic colloids (food stabilizers) in biological systems, for the purpose of obtaining information by which these additives may be judicially selected for use in food stabilization. The investigation will continue to deal with the significance of the chemical structure and configuration of selected polysaccharides, including carrageenan, carboxymethylcellulose, alginates, and locust bean gum for their interactions with proteins, lipids, lipoproteins, and salts in model systems. Improved methods will be used to delineate the fate of specific stabilizers in processed foods in order to determine if fragmentation, hydrolysis, or chemical modifications may occur and which may cause adverse nutritional effects. Particular emphasis will be given to the determination of molecular weights by sedimentation equilibrium approaches to screen the hydrocolloids for low molecular polysaccharides which have been demonstrated to be potentially dangerous to health. A search will be initiated for alternative stabilizing agents which may replace carrageenans for use in evaporated milk and manufactured infant formula. Studies will continue to examine possible unrecognized health problems related to the complexing of proteins with food stabilizers in the digestive tract. The complex formation between hydrocolloids and enzymes will be examined for reversibility and functional properties.

Project Summary The prevalence of obesity has rapidly increased in the US since the late 1970s, which has serious population health and economic ramifications. Over this same period, the US population has also exhibited shorter average sleep duration and increased prevalence of sleep-disruptive disorders. The concomitant timing of these trends suggests that they may be related?a theory supported by several observational and short-term experimental studies indicating that short sleep promotes weight gain and obesity. However, there is no consensus regarding the impact of sleep on body habitus among adults because extant research has produced a range of findings (e.g., linear, U-shaped, or null associations). There are also unexplored questions about the relative importance of sleep duration versus sleep quality with respect to weight gain and obesity. Furthermore, there is scant longitudinal evidence that links short or disrupted sleep to long-term alterations in body habitus through specific physiological mechanisms (e.g., appetite-regulating hormones). To address these limitations, our investigation will address the following study aims: Aim 1: Comprehensively characterize the longitudinal associations of sleep duration and sleep quality with body habitus trajectories. Aim 2: Determine how the longitudinal associations between sleep parameters and body habitus trajectories characterized in the investigations of Aim 1 are mediated by several neuroendocrine hormones. To achieve these aims, we will conduct sophisticated longitudinal analyses of data from the population-based Wisconsin Sleep Cohort Study (WSCS). The WSCS has collected self- reported and objective measures of sleep duration, sleep quality, and body habitus on >1,100 adult subjects for nearly three decades. In addition, we will assay stored blood from WSCS participants?collected at the time participants had sleep and body habitus assessments. These assays will produce longitudinal neuroendocrine hormone data, providing us with an unparalleled data resource to examine the long-term associations among sleep, neuroendocrine hormones and body habitus.

Harold C. Simmons Cancer Center Clinical Protocol and Data Management Project Summary/Abstract The Simmons Cancer Center's Clinical Protocol and Data Management, or Clinical Research Office (CRO) serves as the clinical coordinating center for all activities related to clinical cancer research at UT Southwestern. The office has 55 full-time-equivalent employees and is overseen by Dr. Joan Schiller, Deputy Director of the Simmons Cancer Center and Medical Director of the Clinical Research Office (CRO) who has direct oversight and responsibility for all clinical oncology research at UTSW and the Cancer Center. Day-to- day management of this office is headed by Ms. Erin Williams, Associate Director of Clinical Research Administration. The CRO provides infrastructure for oversight, management, and monitoring of oncology clinical trials, thus facilitating high-quality clinical research across our campus. Centralization of these activities ensures standardization of all aspects of clinical research, ensuring patient safety and coordination of clinical research activities. The resource also provides a central location for cancer protocols, an updated list of currently active studies, status reports of protocols, and quality control and training for clinical research personnel The goals of Clinical Protocol and Data Management Core include: Oversight. Provide central management and oversight functions for the coordination, facilitation, and reporting of cancer clinical trials at the Simmons Cancer Center (SCC); Diversity. Contribute to the research mission of the SCC by enrolling patients with diverse ethnic and racial backgrounds to therapeutic and non-therapeutic cancer clinical trials; Scientific knowledge. Contribute to the scientific direction of the SCC by bringing the basic, translational, and imaging research conducted at UT Southwestern Medical Center (UTSW) into hypothesis- driven clinical trials; Patient care. Provide high-quality health care by offering state-of-the-art clinical trials to our cancer patients. In 2009, at the time of our initial CCSG award, 633 patients were accrued to clinical cancer trials at UTSW and the SCC. In 2013, that figure had risen to 1242 patients enrolled on non-population science studies?close to a twofold increase. Accrual associated with the Population Science and Cancer Control Scientific Program increased from 726 in 2009 to over 37,000 in 2013. In addition, as suggested in the prior CCSG review, we increased our accrual to therapeutic studies from 290 in 2009 to 461 in 2013.

To determine whether dietary intake of oxalate influences urinary excretion of oxalate and calcium and the urinary saturation of calcium oxalate in patients with primary hyperoxaluria.

Abstract Liver disease is a cause of substantial morbidity and mortality in the U.S., and cholestasis (impairment of bile flow) is a common and devastating manifestation of liver disease. The focus of the studies proposed in this application is upon increasing our understanding of biological mechanisms that influence the nature and severity of the manifestations of cholestatic liver disease. This work will be accomplished through continued study of a mouse model of FIC1 deficiency. Mutations in the gene encoding FIC1, a protein that plays a role in aminophospholipid transport, underlie some cases of progressive familial intrahepatic cholestasis (PFIC) and benign recurrent intrahepatic cholestasis (BRIC). The severity and manifestations of FIC1 deficiency can vary notably between patients who share the same FIC1 mutation, and we have demonstrated that genetic background influences manifestations of cholestasis in the Fic1-deficient mouse. We have genetically mapped 7 quantitative trait loci (QTL) which influence phenotypes in the Fic1-deficient mouse, and thus, represent modifier genes of the Fic1-deficient phenotype. In Specific Aim 1, we will perform high-resolution genetic mapping studies and comparative haplotype analysis, using existing samples, to refine the localization of these QTL. In Specific Aim 2, we will use a novel, hybrid genetic approach to efficiently and simultaneously further refine the locations of these modifier genes to regions of modest genomic size, thereby decreasing the number of candidate genes that need be considered for each locus;specific genetic variants that are strong candidates for the causative variation underlying some QTLs will also be identified. In Specific Aim 3, gene expression studies and mining of available functional data will contribute to identification of strong QTL candidate genes. Studies proposed in Specific Aim 4 will yield increased understanding of specific phenotypes detected in the Fic1-deficient mouse, and related QTLs. Identification of the genetic variants underlying such QTL will a) shed light on mechanisms of disease in FIC1 deficiency;b) identify candidate modifier genes for FIC1 deficiency, and possibly other liver disorders, in people;and c) provide information of use to the broad community of researchers studying mouse models of liver disease, regarding susceptibility of the commonly used mouse strains to liver disease-related phenotypes.

We have been studying the molecular genetic mechanisms that control the development switch from embryonic to fetal adult hemoglobins with respect to their basic biology and their relevance to developing new therapies for diseases of hemoglobin. To study these processes we have been using two model systems: the K562 erythroleukemic cells and transgenic mice. K562 is an erythroleukemic cell line used for the last decade as a model for the study of the control of the human globin gene expression. These cells do not support transcription of beta-globin gene but do express transcripts of epsilon and gamma-globin genes at a very high levels when exposed to a number of inducing agents. Results from this and other laboratories suggest that the control of this pattern of expression is mediated by the presence and/or absence trans-acting factors which exert their action on sequences corresponding to the promoters of these genes. In the last few years transgenic mice have become an excellent model system for studying globin gene expression, and are supplementing the use of human cell lines. We have previously reported the presence of a transcriptional control element with properties of a silencer extending from -392 to -177 bp relative to the cap site of the human epsilon-globin gene. Using deletion mutants and synthetic oligonucleotides in transient expression assays, DNA sequences responsible for this effect have been further delimited to 44 nucleotides located between -294 and -251bp. Gel electrophoresis mobility shift assays and DNase footprinting assays demonstrate that these negative regulatory sequences are recognized differently by proteins present in nuclear extracts obtained from HeLa and K562 cells. The protein present in K562 cells, but not in HeLa cells, that interacts specifically with this silencer binds to the same sequence to study the epsilon-globin silencer; constructs with the silencer sequence intact, as well as constructs with the silencer mutated, have been injected into fertilized mouse ova to study the function of the silencer in this assay system. The results should help clarify the molecular mechanisms of silencing, an important aspect of hemoglobin study.

The St. Louis-Cape Girardeau CCOP is a consortium of five hospitals serving parts of eastern Missouri and western Illinois. The mission of this organization is to provide access to clinical trials for community oncologists and their patients. St. John's Mercy Medical Center, the primary institution in the CCOP, is a tertiary care teaching hospital in St. Louis County. The other component hospitals are primarily located is smaller, rural communities with underserved patient populations. Providing access to state-of-the-art cancer treatment to these patients in their own community, and thus increasing involvement of these populations in clinical research, are important goals of the CCOP. One-thrid of all patients accrued to NCI treatment and preventiontrials are enrolled at CCOP sites. The program includes cancer treatment, prevention and control studies that benefit not only people with cancer but also people at high risk for cancer. It offers a variety of Phase II and III protocols including radiation therapy through RTOG, adjuvant treatment of breast and colorectal cancer through NSABP, and chemotherapy-based treatment of all major cancer types through SWOG. The repertoire of clinical trials is supplemented by using the CTSU menu as needed. Over the next five years approximately 80 patients per year will be enrolled in cancer treatment trials through the St. Louis-Cape Girardeau CCOP. SWOG, NSABP, and RTOG also offer cancer prevention and control studies which are a major focus of the CCOP. The St. Louis-Cape Girardeau CCOP was one of the top accruing sites for the STAR breast cancer prevention study and anticipates similar success with the upcoming STELLAR study. Approximately 60 cancer prevention and control credits are expected during each year of this grant period. By providing access to clinical trials in cancer treatment and prevention, the CCOP hopes to improve cancer care in the communities of western Illinois and eastern Missouri. When clinical trial participants are drawn from community hospitals and not just from universities, the study results are more applicable and relevant to the general population. The St. Louis-Cape Girardeau Community Clinical Oncology Program will do its part to achieve the NCI's goal to eliminate suffering and death due to cancer. [unreadable] [unreadable] [unreadable]

Complex interactions between nerves and hormones regulate pancreatic exocrine secretion. Peptidergic neurones are an important component of the vagal, sympathetic, sensory and intrinsic innervation of the pancreas. The peptides are synthesized and transported by these nerves and serve as important neurotransmitters. Examination of the role of neuropeptides in the regulation of pancreatic exocrine secretion has been hampered because specific antagonists were not available. Although neuropeptides almost certainly participate in the enteropancreatic reflex, by which food in the intestine induces pancreatic secretion, the relative importance of neural and humoral pathways in this reflex is unknown because satisfactory models for complete extrinsic denervation were lacking. We now have specific antagonists for neuropeptides and can successfully transplant the jejunum to achieve total extrinsic denervation. We propose, therefore, three specific aims which will elucidate the role of neuropeptides in the neural control of pancreatic exocrine secretion. Specific aim I examines the contribution of peptidergic neurones to the vagal control of pancreatic secretion by using specific antagonists to cholecystokinin, vasoactive intestinal polypeptide, gastrin releasing peptide and somatostatin; these peptides are contained in vagal as well as in intrinsic pancreatic neurones. Specific aim II examines the role of the sensory innervation of the pancreas in two ways. Firstly, low doses of the sensory neurotoxin, capsaicin, will be administered to the isolated, vascularly-perfused pancreas to release neuropeptides (calcitonin gene-related peptide and substance P) from sensory nerve fibers. Secondly, the sensory innervation of the pancreas will be ablated by the chronic administration of high doses of capsaicin. The consequences of these treatments to pancreatic exocrine secretion will be studies. Specific aim III examines the importance of enteropancreatic reflexes by small intestinal transplantation, a well- established technique in our laboratory. By this technique and by using specific antagonists of cholecystokinin, secretin, vasoactive intestinal polypeptide an gastrin releasing peptide we will be able to determine the mechanism by which intestinal nutrients stimulate pancreatic secretion. The proposed work has important application to our understanding of human pancreatic diseases and to their treatment by surgical intervention and transplantation.

In the U.S., metastatic colon carcinoma is second only to lung cancer as the cause of death from malignancy in the United States, accounting for 60,000 fatalities a year. 80 percent of the patients who died of colon cancer have metastases in the liver, and half of them have only liver metastases at the time of death. Current treatments include surgery and chemotherapy, with a mean survival time of only 37 months. Using an orthotopic murine model for pre-established hepatic colon cancer, we have shown that intratumoral injection of an adenoviral vector expressing murine interleukin-12 (Adv.mIL-12) led to tumor regression and prolonged survival of treated mice. However, the anti-tumor immunity induced by this type of gene therapy was mediated primarily by natural killer (NK) cell. In order to also induce a tumor specific T cell response as well, we co-administered an agonistic monoclonal antibody against murine 4-1BB, a co-stimulatory molecule on activated T cells, to induce CD8+ T cell activation and proliferation. The combination treatment resulted in the induction of a strong anti-tumoral immunity and 80 percent long-term survival in the tumor-bearing animals (5x5 m2), even with an eighteen-fold reduction of the Adv.RSV-mIL-12 dose. However, the combination treatment can only induce an NK and CD8+ T cells mediated anti-tumor responses and is less effective for large tumors (lOx10 mm2). In order to achieve persistent anti-tumor immunity and improve therapeutic effect when treating large tumors, the activation of CD4+ T helper cells will be incorporated into the combination therapy via agonistic antibodies against the co-stimulatory molecule OX40, an activation molecule on activated CD4 T helper cells, or CD40, an activation molecule on activated antigen presenting cells (APC). Three specific aims will be pursued: 1) To determine whether intra-tumoral IL-12 gene delivery in conjunction with agonistic antibodies against 4-1BB plus OX40 and/or CD40 can achieve better therapeutic effect for large tumors and generate persistent anti-tumor immune responses to prevent residual and metastatic tumors. 2) To examine the role of CD4 T cells in the accentuated development of tumor specific CD8 memory T cells in combination treated animals. 3) To investigate whether the CD4O and OX 40 activated immune cells can overcome the immune tolerance in mice with a large tumor burden. Successful completion of these studies will result in a better understanding of the interaction of APC, CD4, and CD8 cells in the establishment of anti-tumor memory CD8 T cells and immune suppression/tolerance mediated by a large tumor burden. These therapeutic strategies will be utilized as the scientific foundation for active immune modulation of patients with metastatic colorectal cancer.

Pulmonary infection is a common occurrence in immunocompromised patients and is associated with high mortality. Immunosuppressing drugs, such as glucocorticosteroids and cyclophosphamide have been associated with specific defects in systemic immune function but little is known regarding their effects on local pulmonary host defense mechanisms. This project will evaluate drug-induced pulmonary immunopathology in an animal model of immunosuppression and based upon this information, attempts will be made to enhance pulmonary host defense during periods of peak susceptibility. Guinea pigs will receive one week courses of parenteral cortisone acetate, 100 micrograms/kg/day plus cyclophosphamide, 15 micrograms/kg/day or 30 micrograms/kg/day, Bronchoalveolar lavage fluids will then be obtained for cell enumeration differentiation and studies of the functional capabilities of cells (alveolar macrophages and lymphocytes) during immunosuppressed states. Normal animals will serve as controls. Besides cell number, an evaluation of antibacterial functions, phagocyte chemotaxis, complement synthesis by macrophages, and lymphocyte-lymphokine production will be made. Attempts to augment acute pulmonary inflammation in immuno- and myelosuppressed animals using granulocyte and monocyte transfusion therapy will be made. Likewise, efforts to augment cell-mediated immune responses in the lung with immunoadjuvants will be made. Finally, human bronchoalveolar cells, obtained from immunosuppressed patients by lung lavage, will be studied with similar techniques, in an attempt to

Obesity and gestational diabetes (GDM) increase the risk of fetal overgrowth, which is associated with perinatal complications and development of metabolic syndrome in childhood or later in life. Maternal adiponectin (ADN) levels are reduced and placental nutrient transporters are up regulated in these pregnancies, however the effects of ADN on placental function are largely unknown. Our preliminary studies show that ADN regulates placental amino acid (AA) transporters. The objective of this proposal is to determine the mechanisms by which ADN regulates placental nutrient transporters and influences fetal growth. The central hypothesis is that ADN binds to the AdipoR2 in the placenta and, mediated by APPL1, activates p38MAPK and PPAR-1, which inhibit the insulin/IGF-I signaling pathway resulting in a down-regulation of nutrient transport and reduced fetal growth. Our hypothesis has been formulated on the basis of strong preliminary data demonstrating that ADN (i) inhibits insulin-stimulated AA transport, (ii) activates PPAR-1, (iii) abolishes insulin-stimulated Akt and IRS-1 phosphorylation in cultured trophoblast cells, and (iv) chronic infusions of ADN in pregnant mice inhibits the placental insulin signaling pathway and reduces fetal size. We propose three Specific Aims: (1) Determine the effects of ADN on placental nutrient transport and identify the receptors and APPL isoforms mediating these effects. We will incubate cultured human primary trophoblast cells (with and without siRNA knock-down of AdipoR1 & 2 and APPL 1 & 2) in physiological concentrations of ADN and/or insulin and study the activity, gene and protein expression of transporters for glucose and amino acids. (2) Identify the intracellular signaling pathways involved in mediating the effects of ADN on placental nutrient transporters. We will incubate cultured human primary trophoblast cells in ADN and/or insulin and study the phosphorylation and expression of p38MAPK, PPAR-1, IRS-1, and Akt. In cause-and-effect experiments we will transfect cultured primary human trophoblast cells with siRNA targeting key components of these signaling pathways and re-examine the effects of ADN on insulin signaling and nutrient uptake, and (3) Establish the effects of maternal ADN on placental insulin signaling, nutrient transport and fetal growth in vivo. We will infuse ADN during the last week of gestation in mice by mini- osmotic pumps. At embryonic day 18.5 we will study maternal metabolism, placental nutrient transport in vivo, placental nutrient transporter expression and placental p38MAPK, PPAR-1, IRS-1 and Akt signaling. This research is innovative because we propose a novel model where ADN, in contrast to its action in maternal peripheral tissues, causes decreased insulin sensitivity in the placenta. Furthermore, this proposal will lead to the discovery of a novel mechanism by which the endocrine functions of maternal adipose tissue influence fetal growth. This is significant because this model predicts that low maternal ADN in obesity and GDM increase placental insulin sensitivity, which contributes to enhanced placental nutrient transport and fetal growth. PUBLIC HEALTH RELEVANCE: Statement Fetal overgrowth is common in mothers with obesity and/or gestational diabetes, and predisposes for perinatal complications and increases the risk for her child to develop obesity, diabetes and cardiovascular disease later in life. The mechanisms underlying fetal overgrowth in these pregnancies are not fully understood and no specific treatment is available. We propose that the low maternal levels of the hormone adiponectin cause fetal overgrowth by stimulating transport of nutrients to the fetus. This new knowledge will facilitate the development of novel intervention strategies in pregnancies with fetal overgrowth with the exciting potential for adiponectin 'replacement therapy' in pregnancies complicated by gestational diabetes or obesity.

Project 1: Rac1 induces PCK-dependent myosinIIA phosphorylation to regulate association with focal adhesions and cell migration. Pasapera AM1, Fischer RS1,Plotnikov SV1,Egelhoff T2, Waterman CM1. Cell Biology and Physiology Center, NHLBI, NIH1;Department of Cell Biology, Lerner Research Institute NC-10, Cleveland Clinic.2 Cell migration requires coordinated assembly of focal adhesions and contraction in the actomyosin cytoskeleton. The small GTPase Rac1 is critical to cell migration through its known functions in regulation of focal adhesion and actin cytoskeletal assembly dynamics, but its role in regulation of myosin II is not known. Myosin II dynamically assembles into minifilaments at the leading edge of migrating cells, and PKC-mediated phosphorylation in Ser 1916 in the non-helical tail is one of the main regulators. We hypothesized that Rac1 may regulate myosin II minifilament assembly dynamics during cell migration via downstream regulation of PKC and Ser 1916 phosphorylation. To test this, we analyzed the effects of Rac1 activation on the phosphorylation and dynamics of myosin IIA in U2OS cells. We found that transfection of active Rac1 (Rac1V12) induced PKC- and integrin-dependent myosin IIA phosphoryation on Ser 1916. Live cell imaging of GFP-myosin IIA revealed that Rac1 activation promotes rapid assembly, motion, and turnover of myosin IIA minifilaments, as well as perpendicular orientation to the leading edge, resulting in its accumulation specifically in focal adhesions. To determine the role of Ser 1916 phosphorylation on myosin IIA dynamics and localization, we expressed phospho-mimetic (S1916D) and non-phosphorylatable mutants (S1916A) of myosin IIA. This showed that phosphorylation is critical to the Rac1-induced rapid assembly and turnover of myosin IIA minifilaments as well as to the focal adhesion association of myosin IIA. Thus, Rac1 acts as a master regulator of cell migration by coordinating actin assembly-mediated protrusion, adhesion, and actomyosin contraction dynamics. This work is being prepared for publication Project 2: PROTEOMIC ANALYSIS OF FOCAL ADHESION MATURATION Proteomic Analysis of Myosin II-mediated Focal Adhesion Maturation Reveals a Role for -Pix in Relaxation-mediated Rac1 Activation J. Kuo, X. Han, C.T Hsiao, J. Yates, C. M. Waterman Focal adhesions (FAs) undergo contraction-mediated maturation wherein they grow and change composition to differentially transduce signals from the extracellular matrix to modulate cell migration, growth and differentiation. To determine how FA protein composition is globally modulated by myosinII contraction, we developed a proteomics approach to isolate native FAs, identify their protein composition, and compare specific protein abundance in FAs from cells with and without myosinII inhibition. We reproducibly identified 905 FA-associated proteins, half (402) of which changed in FA abundance in response to perturbation of myosinII activity, thus defining the myosinII-responsive FA proteome. FA abundance of 75% of proteins in the myosinII-responsive FA proteome were enhanced by contractility, including those involved in Rho-mediated FA maturation, stress fiber formation, and endocytosis- and calpain-dependent FA disassembly. Surprisingly, 25% of the myosinII responsive FA proteome, including proteins involved in Rac-mediated lamellipodial protrusion, were enriched in FA by myosinII inhibition, establishing for the first time negative regulation of FA protein recruitment by contractility. We focused on the role of the Rac guanine nucleotide exchange factor, -PIX, documenting its depletion from FA during myosin-mediated FA maturation and its role in negative regulation of FA maturation to promote rapid FA turnover, lamellipodial protrusion and fast cell migration. A mthods paper describing our method was published this year. Project 3: Analysis of traction stress variation across single focal adhesions. Sergey V. Plotnikov, Benedikt Sabass, Clare M. Waterman The ability of eukaryotic cells to sense mechanical properties of the extracellular matrix (ECM) and to exhibit durotaxis (directed migration toward stiffer environments) is thought to underly many biological processes including angiogenesis, neurogenesis and cancer metastasis. ECM stiffness sensing is achieved by integrin-mediated focal adhesions (FA), protein assemblies that couple contractile actomyosin bundles to the plasma membrane and transmit force generated by the cytoskeleton to the ECM. Although it has been shown that both structural and signaling components of FA are crucial to translate mechanical cues into cell behavior, the molecular mechanism of mechanosensing remains unknown. Here we demonstrate that a molecular clutch, a mechanical link between the actin cytoskeleton and ECM-engaged integrins, acts as a mechanosensor in FAs, and the strength of the clutch determines range of ECM stiffness cells are able to sense to mediate durotaxis. Using high-resolution traction force microscopy on polyacrylamide ECMs of varying stiffnesses, we found that the mechanical behavior of the integrin-actin interface at FA exhibited ECM stiffness-dependent switching between a load-and-fail compliance sensing regime and a frictional slippage regime as described in the clutch oscillation model (Chan and Odde, 2008). In the load and fail regime, the position of peak traction within the FA resided on average at the distal FA tip, but oscillated over time towards the FA center and back to the tip. As ECM rigidity was increased, the traction peak did not oscillate and remained in the FA center, signifying the frictional slippage regime. We found that perturbing the gradient of paxillin phosphorylation across FA by expressing Y31/118E- or Y31/118F-paxillin mutants or by inhibiting FAK weakened the molecular clutch and switched FAs from load-and-fail compliance sensing to frictional slippage regime on compliant ECMs. In agreement with the clutch oscillation model, the load-and-fail regime could be rescued by further decreasing either substrate stiffness or myosin II contractility. Since paxillin phosphorylation on tyrosine residues 31 and 118 mediates vinculin recruitment into FAs, we demonstrated that vinculin and the paxillin-vinculin interaction are essential to strengthen the molecular clutch and to enable mechanosensing over a wide range of ECM compliances. We demonstrated the physiological importance of the load-and-fail compliance sensing regime by showing a requirement for this FA behavior in durotaxis, but not in chemotaxis in a boyden chamber assay or in random cell migration. This work resulted in 2 publications

Creatine kinase is an enzyme found in rapidly respiring tissues such as heart, muscle and brain. It is a well-studied enzyme, but to date, there is no X-ray crystal solution for the enzyme. In collaboration with Professor Robert Stroud here at UCSF, we are trying to accomplish this goal. In lieu of a three-dimensional structure, we are utilizing mutagenesis and homology studies to try to identify amino acid residues that may be important in substrate binding and catalysis. It has come to our attention that the crystal structure of a structural homolog will soon become available. The Computer Graphic Laboratory will be an indispensable asset in this regard, as it will provide us the means and resources through which we can carry on even more meaningful investigations in this research. The Computer Graphics Laboratory also provides another important asset, allowing us to access numerous databases and search for proteins that show similarity to creatine kinase. By aligning these protein sequences using programs provided by CGL, we may be able to find amino acid residues that may be conserved throughout evolution. This information allows us to assess the importance of the various amino acid residues with respect to substrate binding and catalysis. We then use site-specific mutagenesis to test our hypotheses concerning specific amino acid residues. We have already done so in one instance, and may have determined the locale of an important residue in the active site of creatine kinase.

To help understand the nature of physical/chemical interactions in biomolecules and biomolecular assemblies, we have developed an experimental approach to study their structure (morphology) and thermodynamic properties simultaneously as a function of the length scale (spatial resolution). The method combines macroscopic osmotic swelling pressure measurements and small angle neutron scattering (SANS). Macroscopic swelling pressure measurements probe the system in the large length scale range, thus providing information on the overall thermodynamic response. The SANS measurement simultaneously provides information about the size of different structural elements and their respective contribution to the osmotic properties. Combining these measurements allows us both to separate the scattering intensity arising from thermodynamic concentration fluctuations from the intensity scattered by large static superstructures (e.g., aggregates), and to determine the length scales relevant to the macroscopic thermodynamic properties. This thermodynamic and structural information cannot be obtained by other techniques. Specifically, we have applied this approach to study interactions between multivalent cations (e.g., calcium) on various biomolecules. Such ions are ubiquitous in the biological milieu, yet existing theories do not adequately explain their effect on and interactions with charged macromolecules in general, and biomolecules in particular. Moreover, experiments to study these interactions are difficult to perform, particularly in solution, because above a low concentration threshold multivalent cations cause charged molecules to phase separate or precipitate. We have overcome this limitation by cross-linking our biopolymers. Since macroscopic phase separation does not occur in cross-linked gels, we are able to extend the range of ion concentrations in which the system remains stable and can be studied. In pilot studies, this new method has been applied to investigate cross-linked gels of a model synthetic polymer, polyacrylic acid, and of DNA. Concentrated DNA solutions and DNA gels have never before been investigated using SANS in conjunction with osmotic measurements. The proposed method also provides a unique framework for analyzing the osmotic and scattering behavior of other biopolymer systems. Recently, we have begun to apply this methodology to study interactions between macromolecular constituents of extracellular matrix (ECM).

During Phase I, Innovation associates demonstrated the feasibility of developing a fiber optic sensor for measuring lead at levels of interest to blood screening clinics. The Lead Ion Selective Fiber Optrode (LISFO) operates in the linear portion of the response curve between 2-200 microg/dL (the new CDC level of concern). Use of fiber optic technology will provide a rugged, compact portable instrument and allow low cost screenings of blood lead concentrations to be performed. During Phase II, conditions will be optimized to insure inter-fiber reproducibility, sample preparation methods will be optimized and automated, and a pre- prototype will be constructed and validated by parallel measurements on clinical samples comparing LISFO with AAS. The instrument will interface with a PC which will regulate automatic operations and process and report data so that minimally trained operators can achieve reliable results with little opportunity for sample contamination. A user interface will permit entry of patient information. PROPOSED COMMERCIAL APPLICATION: Since the CDC lowered the level of concern for lead in blood to 10 microg/dL, the instruments which have previously been used in lead screening clinics no longer give accurate reading at the level of concern. Consequently, there is a need for a new instrument which is usable by personnel with limited training and which can provide large scale, low cost testing of blood lead levels. The proposed instrument will meet the requirements of lead screening clinics and will find acceptance in that market.

DESCRIPTION (Applicant's Description) Environmental agents cause the majority of human cancer with greater than 60 percent of human cancer attributed to tobacco products and diet. Specific chemicals play a significant role in this human disease. While it is generally understood that chemical carcinogens produce DNA damage and DNA damage initiates the carcinogenic process, the precise mechanisms by which this occurs are only beginning to be understood. Most carcinogens generate an array of DNA adducts but only a fraction of this damage induces mutations that initiate cancer. A better understanding of the chemical and biochemical properties of carcinogens and their DNA adducts will aid in understanding the mechanisms of this complicated disease. In the last part of the 20th century, great strides were made in the identification of potential human carcinogens, the detection of their presence in the environment and characterization of DNA damage caused by these agents. Future studies are required to understand the precise mechanisms by which these compounds exert their biological effects. Recent advances in genetic engineering, DNA chip technology and chemical synthetic methods have allowed for exciting new studies in the field of chemical carcinogenesis. These studies will lead to a better understanding of how compounds initiate the carcinogenic process and will allow for the development of new prevention strategies to control human cancer. The Division of Chemical Toxicology, American Chemical Society, will hold a symposium entitled "Chemical Perspectives on Human Cancer" at Pacifichem 2000 in Honolulu, Hawaii December 14-19, 2000. Our goal is to mount a stimulating symposium that highlights the contribution that chemistry has made to the understanding of how chemicals cause cancer. The purpose of this symposium is to assemble the leading Pacific Basin experts in this field to assess the current status of the field and to define future directions for this exciting area of research. We are requesting partial support for the symposium through this grant application.

The American Cancer Society estimates that a total of 192,370 new invasive breast cancers and 62,280 new cases of in situ breast cancer are diagnosed annually. Women with operable breast cancer undergo Breast Conserving Surgery (BCS), also called lumpectomy or partial mastectomy. On average between 50 to 75% of patients with breast cancer undergo BCS followed by radiation therapy. Approximately 20-70% of women undergoing BCS have to return for a repeat surgery due to incomplete removal of the cancer at the first BCS. There is a significant unmet need for a rapid and practical tool for intra-operative assessment of breast tumor margins. The broad goal of this BRP is to bring multiple investigators from Duke University and the University of Wisconsin, Madison together to develop and validate a miniature optical spectral imaging strategy for intra- operative imaging of tumor margins in patients undergoing BCS. Achieving this goal requires significant innovation in optical physics and engineering, and will leverage advances in thin film detectors, waveguides, and on-board electronics. The specific aims of the proposed work are: (1) to design, construct and refine a miniature spectral imaging device and software for quantitative optical spectral imaging, (2) to verify the spectral imaging technology performance metrics through rigorous bench testing, (3) to develop and optimize an automated device-tissue interface platform and to minimize sources of systematic and random errors at the device-tissue interface, and (4) to test the miniature spectral imaging device for the detection of margin positivity in approximately 150 patients undergoing BCS. The proposed clinical studies will serve as an important basis for larger scale successor clinical studies. It is theorized that the miniature optical spectral imaging system developed and tested in this BRP grant will result in a practical clinical tool for rapid intra- operative assessment of breast tumor margins, i.e., it will help guide the surgeon to either complete the surgery, if margins are negative, or re-excise additional tissue, if margins are positive at the time of first surgery, thus avoiding an un-necessary re-excision. The implications are significant in that tens of thousands of unnecessary surgeries could be prevented each year. The technology proposed in this research could also be extended to the assessment of tumor margins in patients with other types of cancers, including the brain, prostate and head and neck. This novel technology also has the potential to serve as a low cost solution for global cancer diagnostics.

[unreadable] [unreadable] ACIP has now recommended influenza vaccination for all children 6 months to 18 years of age. While many school-aged children will be vaccinated in the medical home, the large number of children for whom the vaccine is now recommended exceeds the capacity of many primary care settings. Schools have been recommended as potential sites for influenza vaccination, yet little is known about the feasibility, implementation requirements, costs, or effectiveness of school- based influenza vaccination (SIV) programs. This project will implement and rigorously evaluate the feasibility, acceptability, billing and reimbursement components, costs, cost-effectiveness, and overall effectiveness of an SIV program that targets diverse populations and different intensities of a recruitment advertising intervention. The Monroe County SIV will integrate five sets of activities: 1) SIV program implementation in 24 elementary schools; 2) a randomized controlled trial (RCT) testing SIV vs. standard-of-care and an RCT of high- vs. low-intensity SIV; 3) economic analyses regarding income generation and costs of SIV with extrapolation to the U.S.; 4) associated quantitative studies that will shed light on implementation issues including vaccine refusal, parents' attitudes and beliefs regarding SIV, and teachers' perceptions of vaccination in participating and non-participating schools; and 5) qualitative studies examining attitudes and beliefs of various groups including parents, mass vaccinators, school personnel, and insurers. Key factors of strategic importance to this proposal include: the significance to the nation of results that will be generated by the proposed body of work, the approach that interweaves collaborative program implementation and carefully constructed evaluation; an innovative nesting of randomized controlled trials (RCT) testing both SIV vs. Standard-of-care and High-Intensity vs. Low-Intensity interventions; a team of investigators with one of the U.S.'s top records in immunization research including RCTs, economic studies and modeling, quantitative surveys, and qualitative research; and an environment that ensures success because of long-standing collaborations between the Monroe County Department of Public Health, a nursing organization experienced in mass vaccinations, the NYS Department of Health, academicians at the University of Rochester Medical Center, the local school districts, pediatric and family physician professional organizations, and health care insurers. [unreadable] [unreadable] PUBLIC HEALTH RELEVANCE: [unreadable] [unreadable] The relevance of this research to public health is that - just as ACIP has recommended influenza vaccination for all children 6 months to 18 years of age - the proposed Monroe County project would both implement a school-based influenza vaccination (SIV) program and study the program's costs and benefits (vaccine uptake) while examining billing and reimbursement from the perspective of a party capable of billing insurers for vaccination. The innovative nesting of randomized controlled trials testing both SIV vs. Standard-of-care and High-Intensity vs. Low- Intensity interventions is further strengthened by a carefully crafted set of economic studies and modeling, quantitative surveys, and qualitative research. [unreadable] [unreadable] [unreadable]

The major goal of this project is to give new directives for studies in tobacco carcinogenesis by identifying potential carcinogens in smokeless tobacco, mainstream and sidestream cigarette smoke, and environmental tobacco smoke (ETS). As in the past, newly identified suspected tobacco carcinogens (e.g. polynuclear aromatic hydrocarbons, aromatic amines, tobacco-specific N-nitrosamines) need to be quantitatively assessed, their formation has to be studied and they must be assayed in vitro and/or in vivo for tumorigenicity. We plan to investigate N-nitrosamino acids in chewing tobacco and to examine their carcinogenic activity. We will also study the fate of N-nitrosamino acids during cigarette smoking. The significant carcinogenic activities of some nitroalkanes, nitroalkenes, and protein-derived heterocyclic amines make it highly desirable to assay these pyrolysis products in tobacco smoke. We will develop new analytical methods on the basis of supercritical fluid extraction (SFE) and GC-MS. 3-Vinylpyridine, a pyrolysis product of the Nicotiana alkaloids, will be assayed as a marker that is well suited to determine the contribution of volatile tobacco smoke constituents to indoor air pollution. This far, only nicotine has been used as marker for ETS, but a reliable indicator for volatile smoke carcinogens in ETS is urgently needed. 3-Vinylpyridine appears to be suitable for this purpose, since unlike nicotine it remains static in the polluted air over a period of several hours. We have identified 4-(methylnitrosamino)-4-(3-pyridyl)butyric acid (iso- NNAC) in smokeless tobacco, but did not find it in tobacco smoke. Since iso-NNAC can be a nitrosation product of nicotine and/or its metabolites, and since it is not carcinogenic and is excreted in the urine without undergoing metabolism, it may serve as a marker for the endogenous formation of tobacco-specific N-nitrosamines (TSNA) in the urine of smokers. It is likely that Nicotiana alkaloids inhaled by the smoker are potential precursors to endogenously formed carcinogenic TSNA. Therefore, an in-depth analytical study is proposed to determine iso-NNAC in the urine of smokers. Epidemiological studies have demonstrated an increased risk for cigarette smokers for cancer of the uterine cervix. Nicotine and cotinine as well as nitrite have been identified in cervical mucus; it is likely therefore, that carcinogenic TSNA can be formed in the cervical vault of smokers. Preliminary studies utilizing highly sensitive methods, based on SFE and GC-TEA, have indicated the presence of TSNA in some samples of cervical mucus. We plan to explore this question by analyzing the cervical mucus of cigarette smokers and nonsmokers. The results of this project will answer important questions in tobacco carcinogenesis and will give impetus to new biochemical studies in chemical and environmental carcinogenesis.

DESCRIPTION (Applicant's Abstract): The goal of the present research is to use Functional Magnetic Resonance Imaging (fMRI) of the brain to discover the functional components of visual recognition. This application takes as its starting point the recent discovery of an area in extrastriate cortex which is specifically involved in the analysis of visual object shape. The experiments described here will test subjects in an MRI scanner on a wide variety of visual stimuli and tasks in order to 1) zero in on the specific processes that take place in this cortical area and 2) discover new brain areas specialized for different functional components of visual recognition. Part I will test whether visual recognition of different classes of stimuli (objects, faces, words and biological-motion displays) engage the same or different cortical areas. Two aspects of the experimental design will permit a more precise answer to this question than has previously been possible. First, stimuli will be constructed which control for low-level feature components of complex images. Second, detailed within-subject analyses will allow the investigators to dissociate different functional areas even if the anatomical loci of these areas varies considerably across subjects. In Part II, a more fine-grained analysis of the different components of visual shape analysis will be carried out in an effort to distinguish between cortical areas involved in figure-ground segregation, part decomposition, depth interpretation, and other components of shape processing. The investigators will also look for cortical areas that respond differently to familiar and unfamiliar visual stimuli in order to determine whether visual memories and memory-matching processes are separable from bottom-up stages of visual processing. In order to explore the role of attention in visual object recognition, in Part III the investigators will manipulate the subjects' attention with a variety of tasks to ask whether the shape of an object is analyzed even when attention is focused on 1) the color of the same object, 2) another object in the visual field, or 3) a difficult perceptual task in another sensory modality. Using fMRI imaging in conjunction with carefully-controlled stimuli and tasks to characterize and localize specific visual computations is a new way to exploit the modular structure of the brain to discover the functional components of the mind. The present approach may provide the crucial missing link between the considerable knowledge of the neural basis of vision and the understanding that has been gained from computational and cognitive approaches.

Consumption of ethanol (Et) alters certain regulatory aspects of the hypothalamic-pituitary-adrenal axis (HPAA). Because the integrity of this system depends on the coordinated synthesis and secretion of specific regulatory substances at the hypothalamic (e.g., corticotropin-releasing hormone (CRH); vasopressin (AVP); biogenic amines), pituitary-gland (e.g., beta endorphin (BE); ACTH) and adrenal-gland (e.g., catecholamines; glucocorticoids) level, we have been evaluating the impact of Et at each level of the HPAA. Activation of the HPAA or hypercortisolism accompanies both short- and long-term consumption of Et and the Et withdrawal syndrome. Alcoholics often present with a pseudo-Cushing's syndrome in which some 17- 40% of alcoholics do not respond to the dexamethasone suppression test during the first week of abstinence. Since a relative state of elevated glucocorticoids (chronic continuous or chronic intermittent) can lead to neural changes and even cell death, particularly in the hippocampus, the progressive loss of cognitive capacity in many alcoholics may indeed be due in part to hypercortisolemia and subsequent irreversible neural damage in the hippocampus and other areas of the central nervous system. Furthermore, armed with the concept of the bidirectional communication between the HPAA and the immune system. We are exploring whether or not certain immune system-derived cytokines may be ameliorating or accelerating neural death through endocrine or paracrine actions. Certainly cytokines stimulate diverse cell types in an attempt to repair cellular damage through intracellular signal amplification which could in concert with Et and glucocorticoids overstimulate selected neural populations leading to their demise.

Project D: Hypocretin and Histamine studies in the Zebrafish Hypocretin and histamine are now known to be important arousal signals. Anatomical correlates c hypocretin, histamine, and other cell groups relevant to sleep regulation, as well as the cognate homologou genes, have been identified in zebrafish and appear to anatomically interconnect similarly as in mammals We have identified a sleep-like state in zebrafish and can objectively characterize alterations in this behavio with an automated digital video system. The goal of this revised proposal is to take advantage of th attributes of the zebrafish as a genetic model organism, to develop a better understanding of factors relevan to sleep regulation in humans. Specifically, we will validate the zebrafish as a model for the study of sleep disturbances in relationship to hypocretin and histamine through the use of transgenic and gene-targeted zebrafish by characterization of the behavioral effects of loss of, or overexpression of hypocretin (HCRT) o histidine decarboxylase (HOC) and loss of expression of their relevant receptors (hypocretin recepto (HCRTR), histamine H1 receptor (H1). We hypothesize that animals deficient in hypocretinergic o listaminergic transmission will have altered rest/activity patterns. These hypothesized altered rest/activity patterns will demonstrate functional conservation of these systems in the zebrafish, and validate the use o zebrafish as a model in the study of study of sleep disturbances in relationship to hypocretin and histamine, We will also exploit the forward genetics advantages of the zebrafish to identify novel developmental genes affecting the hypocretin and histamine systems by screening for ENU-induced mutations resulting in loss of expression of HCRT, HOC and their receptors, or abnormal placement of these cells. Such novel genes may be relevant to the development of narcolepsy and hypersomnia in humans. We will also characterize these dentified mutants in terms of cellular specificity, and gene expression profiles, and behavior as a preliminary step toward positional cloning. Finally, we will genetically map the most interesting mutations (those with cell specificity and/or robust behavioral phenotype). These studies will be valuable in our understanding of the etiology of narcolepsy and hypersomnia.

Project Summary Nearly half of mechanically ventilated (MV), critically ill adults develop ventilator-induced respiratory muscle weakness (particularly of the diaphragm), which impairs successful weaning from MV, often leads to re- intubation, and is associated with higher post-ICU mortality. We have recently shown that respiratory muscle weakness is associated with extubation failure in critically ill children, but we still lack crucial information on the mechanisms and timing of this weakness, its importance for ventilator weaning, and its potential prevention through promoting more physiologic levels of patient effort of breathing during MV. Diaphragm weakness is most common in patients exposed to high levels of controlled ventilation. In children, we have shown that usual care ventilator management is frequently associated with minimal patient effort of breathing. To enhance patient effort, we developed a novel computer-based approach (Real-time Effort Driven ventilator management (REDvent)), which offers systematic recommendations to reduce controlled ventilation during the acute phase of MV, and uses real-time measures to adjust supported ventilator pressures such that patient effort of breathing remains in a normal range during the ventilator weaning phase. Through a Phase I clinical trial, we demonstrated that patients managed with REDvent spent fewer days on MV than historical controls, and bedside providers could easily implement REDvent. Our central hypothesis is that REDvent use will reduce ventilator-induced respiratory muscle weakness, leading to shorter time on MV by enhancing the patient?s capacity for effective, unsupported ventilation and by facilitating MV weaning. This application proposes a Phase II controlled clinical trial that will obtain comprehensive, serial assessments of respiratory muscle strength and architecture to understand the evolution of ventilator-induced respiratory muscle weakness in critically ill children, and test whether REDvent can preserve respiratory muscle strength and reduce time on MV. Three integrated yet independent Specific Aims are proposed: SA 1 will determine the clinical impact of REDvent by examining whether REDvent use for either acute or weaning phase ventilator management results in shorter MV weaning times compared to usual care; SA 2 will use a sophisticated, multi-modal approach to obtain a comprehensive view of respiratory muscle capacity, effort, load, and architecture to quantify the importance of respiratory muscle weakness on weaning outcomes in children, and identify when this weakness develops; SA 3 seeks to determine the independent effect of patient effort of breathing on the development of respiratory muscle weakness, after controlling for known or suspected risk factors for respiratory muscle weakness. Upon completion, this study will provide important information on the pathogenesis and timing of respiratory muscle weakness during MV in children, whether this weakness can be mitigated by promoting more normal patient effort during MV via the use of REDvent; forming the basis for a larger, Phase III multi-center study, powered for key clinical outcomes such as 28-day Ventilator Free Days.

Alcohol use and alcoholism are presently viewed as governed by antecedent factors that include childhood environment and genetically determined biological characteristics. Less attention has been paid, however, to how these antecedents influence later drinking. In addressing this issue, the psychosocial and biological literature both refer to acquired (learned) behavior, which is possibly influenced by differing reinforcement values of alcohol for different subsets of drinkers. It is, therefore, important to understand the precise nature of this learning and how it influences ongoing decisions about whether, and how much, to drink. Recent research on alcohol-related expectancy, considered in the context of cognitive (information processing) psychology, has opened a new research "window" on this learning. Exploration of the antecedent factors which influence expectancy development is just beginning, however, and there has been no exploration of the operation of expectancies immediately proximal to actual drinking opportunities. The present project will comprehensively investigate whether alcohol expectancies may be one (mediational) mechanism by which psychosocial and biological antecedents these influence later drinking behavior. Three research strategies for investigating mediational relationships will be used. A prospective design, using covariance structure modeling (LISREL) techniques, will assess the capacity of alcohol expectancy to serve as a psychological mediator. Since expectancies are theorized to influence drinking at a point proximal to a drinking opportunity, a study of the feasibility of monitoring expectancies in the natural environment, proximal to actual drinking occasions, will be also undertaken. Finally, since the most powerful test of mediation is effective experimental manipulation, an extensive experimental challenge of alcohol expectancies will also be conducted. Specifically, this research program will: assess whether antecedent variables which have been shown to influence later drinking can be usefully understood as carrying over their influence by psychological/information processing mechanisms; increase our understanding of which antecedent variables most influence the psychological mechanisms; assess expectancy operation proximal to drinking decisions; test the possibility of actually manipulating drinking via challenging expectancies; and refine expectancy challenge procedures for application to primary and secondary prevention.

The overall goal of this project is to develop oligonucleotide analogs that can effectively interact with specific RNA or DNA target sequences within the HIV genome and as a consequence of these interactions repress HIV replication. We will continue to investigate novel, nuclease resistant antisense and triplex forming 2'-0- methylribo-oligonucleoside methylphosphonates (mr-OMPs) that are targeted to critical sequences in the HIV-1 genome. The project has three Specific Aims. In Specific Aim 1, linear and hairpin antisense mr-OMPs complementary to the apical stem loop of the transactivation response element, TAR, RNA or the stem IIB of Rev response element, RRE, RNA will be prepared and their interactions with TAR and RRE RNA targets will be studied by gel electrophoretic mobility shift assays (EMSA). Chimeric anti-TAR and anti-RRE mr- OMPs that contain a tract of six deoxyribonucleotides flanked by four or five 2'-O-methylribonucleosides will be studied for their ability to form RNase H cleavable hybrids with their RNA targets. Imidazole-conjugated mr-OMPs will be prepared and their ability to catalyze hydrolysis of specific phosphodiester linkages in their RNA targets will be investigated. In Specific Aim 2, triplex forming oligonucleotides (TFOs) in which 5- methylcytosine is replaced by pseudoisocytosine will be prepared and their interactions with purine tracts found in the env- and gag-genes of HIV-1 proviral DNA will be investigated. The ability of TFOs to recognize purine tracts that contain one or more pyrimidine interruptions will be explored by synthesizing derivatives of cytosine and uracil that are designed to interact with C-G base pairs in the target DNA. To enhance efficacy, TFOs conjugated with an alkylating group will be prepared and their ability to form covalent adducts with their target DNAs will be evaluated. Specific Aim 3 will explore the biochemical and biological activities of the oligonucleotides. The ability of anti-TAR or anti-RRE mr-OMPs to inhibit Tat protein-TAR or Rev peptide- RRE interactions and to inhibit Tat- or Rev-mediated gene expression in cells will be evaluated in collaboration with Dr. Tariq Rana, University of Massachusetts Medical School. The effects of TFOs on in vitro DNA transcription will be investigated. The uptake of antisense and TFO mr-OMPs by U-937 cells (human promonocytes) in culture will be characterized and the stabilities and cellular toxicities of the oligonucleotides will be investigated. The antiviral activities of the antisense and TFO mr-OMPs will be investigated in collaboration with Dr. Jeffrey Laurence, Cornell Medical Center.

The current treatment options for metastatic colon carcinoma are limited. Our preclinical research indicates that immune-modulatory gene therapy may be effective for the treatment of advanced metastatic colorectal cancer. Using an orthotopic murine model for pre-established hepatic colon carcinoma, we have shown that intratumoral injection of an adenoviral vector expressing murine interleukin-12 (Adv/mIL-12) and 4-1BB, a co-stimulatory molecule normally found on activated dendritic and T cells, can induce dendritic cell and CD8+ T cell activation and proliferation. This leads to tumor regression and prolonged survival of treated mice. However, this therapeutic effect is significantly compromised by large tumor burdens (>8x8-10x10mm2). Significant accumulation of myeloid suppressor cells (MSC) and T regulatory cells (Treg) were found in animals bearing large tumors. The lack of proper antigen presentation and Treg mediated immune suppression are two of the main means by which tumor cells evade the immune system and present the major obstacles for cancer immune therapy. We found that CD 40 engagement greatly improved the long-term survival rate of mice bearing large tumors when used in conjunction with IL-12 + anti-4-lBB combination therapy. Furthermore, agonistic anti-CD40 antibody prevented MSC-mediated Treg induction. We hypothesize that CD40 activation by agonistic antibody or Ig-CD40 ligand fusion protein can enhance anti-tumor responses through multiple mechanisms: 1) CD40 ligation can prevent Treg induction through CD40 signaling on MSC;2) CD40 activation can induce the differentiation of MSC into dendritic cells and downregulate the inhibitory receptors, PIR-B (paired Ig-like receptor B) and gp49b, which are highly expressed on MSC and immature DCs. Three specific aims will be pursued in order to understand the mechanisms underlying the CD40 mediated regulation of MSC function and reversion of immune suppression in the large tumor setting: 1) Study the mechanisms underlying the prevention of MSC-induced Treg development by anti-CD40 ligation;2) Investigate the negative receptor signals as the potential mechanism behind the ineffective anti-tumor response to synergistic IL-12 and 4-1BB activation in mice with large tumor burdens;3) Determine the therapeutic effect of blocking negative inhibitory receptors and reversing immune suppression by co-stimulatory molecules on the persistence of an effective anti-tumor response following immune activation in the large tumor bearing host. The completion of these studies will not only provide a new insight into the mechanisms of tumor mediated suppression and tolerance, but also facilitate effective clinical translation of our immune enhancing therapy.

DESCRIPTION: The goal of this renewal proposal is to characterize inhibition of antitumor immune responses by tumor gangliosides in vivo. The general hypothesis underlying this work is that gangliosides are shed by tumor cells, act as intercellular signaling molecules, block the cellular immune response, and protect tumor cells from host immune destruction. Findings of significant shedding and of potent immunosuppressive activity of human neuroblastoma gangliosides, and new preliminary data showing inhibition of murine cellular immune response to syngeneic tumor cells by these molecules, lay the foundation to directly test the hypothesis that tumor gangliosides downregulate host cellular immune responses to syngeneic tumor cell both in vitro and in vivo. Two approaches will be used to obtain neuroblastoma tumor gangliosides, homogeneous in both carbohydrate and ceramide structure, in large quantities: isolation from human neuroblastoma tumors (natural) and chemical synthesis (synthetic). All gangliosides will be further purified by HPLC to remove potential traces of contaminants prior to biological study. Molecular structures of the gangliosides will be confirmed by mass spectrometry and chemical methods. The delineation of inhibitory effects of tumor gangliosides upon both the afferent (priming) and efferent (effector function) phases of the cellular immune response in murine tumor models in vivo will be complemented by determination of the effects of these same gangliosides on the human cellular immune response in vitro. The in vivo studies will be performed in two murine syngeneic tumor models, FBL erythroleukemia and B78H1 melanoma. The proposed studies will conclusively demonstrate downregulation of syngeneic antitumor immune response by neuroblastoma tumor gangliosides in vivo. They will provide the foundation for future investigations (longer term aims) to elucidate the molecular mechanism(s) of inhibition by tumor gangliosides and to develop strategies to eliminate these immunosuppressive molecules (such as removal of gangliosides from the circulation or modulation of ganglioside biosynthesis and shedding by pharmacologic agents), with the ultimate goal of achieving improved therapeutic approaches to neuroblastoma and other neuroectodermal tumors such as melanoma.