Datasets:

haritzpuerto

/

the_pile_00_NIH_ExPorter

like 0

My laboratory investigates the properties of natural and chemically modified nucleic acids. Working at the interface of cell biology and nucleic acid chemistry we have modified cellular activities including transcription, translation, allele-selectie inhibition of protein expression, splicing, and telomerase-mediated elongation of telomeres. For 2016-2020 our central goal is to examine the recognition of cellular RNA with a primary focus on nuclear RNA. We will apply that understanding to methods for controlling gene expression and new insights into natural pathways of gene regulation. Much of the genome is transcribed into RNAs that do not encode proteins. The function of many noncoding RNAs is unclear, as is the mechanism for how they might affect gene expression. Published reports offer little insight into the molecular details for how a specific noncoding RNA affects expression of a given gene. In the absence of this information, validation of proposed effects becomes problematic and the predictive power for studying novel genes is limited. This lack of guiding mechanistic principles is a major obstacle to progress. RNAi provides a potential mechanism for recognizing specific nuclear RNA species. While RNAi is well-known as a driving force for recognition of mRNA in the cytoplasm of mammalian cells, its potential to drive recognition in the somatic cell nuclei has been unclear. We propose to define the scope and mechanism of mammalian nuclear RNAi and to apply it to novel disease targets. Objective 1. Understand nuclear RNAi. We will characterize the protein and RNA interactions involved in nuclear RNAi. RNAseq will be used to identify RNA targets for functional validation. Mass spectrometry will be used to determine protein partners. We will explore the mechanism of nuclear RNAi and obtain insights into the how nuclear RNAi regulates gene expression in mammalian cells. These data will reveal how argonaute proteins and other RNAi factors function in the nucleus to control gene expression. Objective 2. Novel targets for nucleic acids inside cells. We will also expand recognition to novel nucleic acids targets including the expanded intronic repeats within the mutant frataxin and C9orf72 genes. These data will further develop our understanding of the mechanism of nuclear RNAi and show how nuclear RNAi can be applied to the control of disease gene expression. Compounds that control frataxin protein expression or disrupt structures formed by the expanded repeat within intronic C9orf72 would be lead compounds for drug development.

Project Summary/Abstract The purpose of this study is to advance the science of recovery from alcohol problems through development of a standardized survey measure of recovery capital. Recovery capital is a theoretical construct that encompasses the ways in which physical, social, human, and cultural resources shape a person's likelihood of sustaining recovery from addiction. Recent studies have highlighted the importance of recovery indicators beyond alcohol consumption/abstinence, such as gains in health and overall quality of life. Although research indicates that recovery capital plays an important role in holistic recovery outcomes and that deficits in recovery capital may partially drive socioeconomic disparities in such outcomes, the field is limited by the lack of a psychometrically sound quantitative measure. Currently, the predominant measure of recovery capital is the 50-item Assessment of Recovery Capital (ARC), recently made available as a brief 10-item version (BARC- 10). The ARC appears misaligned with the theoretical literature; for example, its sobriety subscale implies an abstinence-based definition of recovery, which is inconsistent with recovery capital theory. Building from a preliminary study indicating psychometric weaknesses with the ARC when used with a racially and ethnically diverse low-income sample (N=273), in the proposed study we will develop a new measure of recovery capital, the Multidimensional Inventory of Recovery Capital. We will employ best practices in measurement development to generate an item pool that is informed by theoretical conceptualizations of recovery capital, as well as the perspectives of diverse individuals in recovery. Initial items will be refined through focus groups and cognitive interviews with people in recovery from alcohol problems, stratified by income and race/ethnicity, and will include individuals recovering without assistance from formal treatment systems. The emergent measure will be rigorously pilot-tested in a national sample using Amazon MTurk (n=500) and will use graded response models as an extension of item response theory to assess and calibrate item performance. The study will also establish the psychometric properties of the final measure in a nationally representative sample (n=500). Using a sequential mixed methods approach, our study will address the following specific aims: (1) Develop a new theory-aligned measure of recovery capital, the Multidimensional Inventory of Recovery Capital, using qualitative analysis of focus group and cognitive interviewing data with an economically and racially inclusive sample of people in recovery from alcohol problems; (2) Pilot test the Multidimensional Inventory of Recovery Capital in an economically and racially diverse sample of people in recovery from alcohol problems and assess its feasibility, reliability, and item performance; and (3) Establish a final version of the Multidimensional Inventory of Recovery Capital through assessment of its reliability, validity, and factor structure in a nationally representative sample of people in recovery from alcohol problems.

Bone mineral is basically calcium phosphate, and both elements (Ca and P) are required for bone acquisition. Typical Ca intakes in the U.S. are lower than current recommendations, and typical P intakes, higher. Thus attention has been focused mainly on increasing Ca intake, by supplementation and food fortification. The salt most commonly used for these purposes is calcium carbonate. But while the average P intake tends to be adequate or generous, nevertheless substantial proportions of older women ingest less than 70% of the RDA for phosphorus on any given day, and hence may be said to be at risk for P deficiency. When these women are given a combination of anabolic therapy and supplemental Ca (as the carbonate), the added Ca may uncover and aggravate the latent P deficiency. This is both because Ca binds phosphate in the gut and reduces its absorption (thereby effectively lowering the P intake still further) and because the induced bone anabolism will itself consume phosphorus, as a result of which absorbed phosphorus may not be sufficient to support the increase in bone mineral mass made possible by this therapy. To test the possible importance and value of supplementing both of the components of bone mineral in support of anabolic therapy of osteoporosis, we propose a 1-year randomized trial, comparing, in two groups of teriparatide-treated postmenopausal osteoporotic women, calcium supplements with and without extra phosphorus (i.e., Ca phosphate vs. Ca carbonate). The principal outcome measure will be change in bone mineral content over the one year of the trial. A secondary outcome is measurement of bone resorption biomarkers so as to assess whether the phosphate salt elevates remodeling relative to the carbonate salt. A finding of superiority of the phosphate-containing cost-neutral change in Ca sources and a corresponding osteoporosis prophylaxis as well). PERFORMANCESITE(S) _an_a_n, ci_,s_ Creighton University Medical Center Osteoporosis Research Center 601N. 30th Street Omaha, NE 68131 KEY PERSONNEL. See instructions. Use continuation pages as needed Start with Pdncipal Investigator. List all other key personnel in alphabetical Name O_anization to provide the required order, last name first. Robert P. Heaney, M.D. Creighton University Robert R. Recker, M.D. Creighton University Creighton University Ca supplement would provide evidence leading to a improvement in osteoporosis co-therapy (and possibly information in the format shown below. RoleonP_e_ Principal Investigator Co-investigator Co-investigator Joan M. Lappe, Ph.D. Disclosure Permission Statement. Applicable to SBIPJSTTR Only, See instructions. [] Yes [] No PHS 398 (Rev. 05/01 ) Page _2 Form Page 2 Principal Investigator/Program Director (Last, first, middle): Heaney, Robert P. The name of the principal investigator/program director must be provided at the top of each printed page and each continuation page, RESEARCH GRANT TABLE OF CONTENTS Page Numbers Face Page .................................................................................................................................................. 1 Description,

The overall objective of this project is the identification and characterization of tumor-associated antigens of human colonic tumors. This will be initially investigated by using human colonic cancer xenografts established in unconditioned golden hamsters and in continuous propagation in these hosts. Of immediate interest is the carcinoembryonic antigen (CEA) originally described by Gold and Freedman and synthesized by our human colonic cancer transplants. Immunological and chemical characterization of CEA in these tumors will be undertaken and the findings compared to similar preparations from human tumor specimens. In addition to this work with CEA, the presence of other antigenic substances in these tumors will be explored with a view toward identifying colon organ-specific, normal or neoplastic, markers. The relationship of these other antigenic markers to CEA will be investigated by immunochemical techniques.

Lung endothelium has historically been considered a homogeneous and metabolically inactive cell layer. However, we now know that lung endothelium is richly diverse in structure and function, and actively participates in normal vascular homeostasis, the response to lung injury, and vascular repair following injury. This Program Project Grant is founded on the hypothesis that endothelium lining the lung's extra-alveolar and alveolar blood vessels is phenotypically distinct, and that the unique behaviors of cells from these different vascular locations is necessary for them to fulfill their site-specific functions. Indeed, work from multiple investigators in our program project grant has resolved a demarcation at approximately 25 pm in vascular diameter, where extra-alveolar vessels transition into the capillary plexus to become alveolar vessels, that is associated with a prominent change in endothelial cell phenotype. The pulmonary microvascular endothelial cells that reside within the alveolar vessels possess a tighter barrier function than do their macrovascular counterparts, have higher metabolic activity, and migrate and proliferate at a high rate, due at least in part to the enriched number of progenitor cells that reside within this vascular niche. In total, we know very little about the cells that make-up the alveolar capillary bed. Therefore, in this funding cycle (2006-2011), four inter-related projects have been supported, which collectively seek to address fundamentally important signal transduction networks that are responsible for pulmonary microvascular endothelial cell barrier function, susceptibility to apoptosis/necrosis, and regulation of hemostasis and coagulation. Project 3 was initially supported for three years, to examine whether calcium entry through T-type calcium channels is an important amplification step in release of von Willebrand factor and upregulation of P-selectin, specifically within capillary endothelial cells. Great strides have been made toward completing the specific aims in project 3, and critical new directions that greatly complement the program project grant have been developed. Thus, this competitive supplement seeks two years of additional support for project 3, extending through this application's funding cycle in 2011.

This is a competing renewal application for the Pacific Northwest (PNW) Node, which joined the NIDA National Drug Abuse Treatment Clinical Trials Network (CTN) in January, 2001. Our primary aim is to continue our active support of the CTN's mission, to "improve the quality of drug abuse treatmentthroughout the country using science as the vehicle" by conducting studies of behavioral, pharmacological, and ntegrated behavioral-pharmacological treatment interventions in rigorous, multi-site clinical trials to determine their effectiveness across a broad range of CTP settings and diversified patient populations and ensuring the transfer of research results to treatment providers. In order to effectively meet these objectives, the (PNW) Node proposes to: Maintain an effective, bidirectional partnership and working relationship among researchers, treatment providers, and policy makers Maintain and expand a highly successful and well-established treatment research infrastructure within the Node Expand the Node's geographical reach by incorporating a community-based treatment program (CTP) from Alaska, also broadening the ethnic diversity within the Node Enhance the Node's capacity to conduct pharmacotherapy trials and respond more rapidly to emerging public health priorities by adding a larger, highly research-experiencedCTP [unreadable] Broaden and deepen the clinical research expertise of the Node by adding a number of affiliated investigators who bring a wealth of experience from prior intervention trials on topic areas directly relevant to the CTN We have successfully contributed to the CTN's mission over the past 6 years and anticipate continuing to do so in the future. From a public health perspective, our continued success in conducting such research and dissemination as part of the CTN will facilitate empirically supported, evidence-based interventions being implemented in community-based treatment programs, with a resultant improvement in the quality and effectiveness of substance abuse treatment.

This 5-year multi-center randomized trial will assess the effectiveness of Early Revascularization (ERV) using approved mechanical and surgical procedures (primarily PTCA and CABG) in reducing the current high in- hospital mortality rate from cardiogenic shock (CS) complicating acute myocardial infarction (MI). Approximately 7.5% of all acute MI's which are diagnosed in an ER or in- hospital lead to CS and an in-hospital death rate of 70%-80% (usually within 1-2 days of diagnosis of CS). This high death rate has not changed in the last two decades. Non-random clinical series and animal studies suggest that rapid revascularization following CS complicating acute MI may substantially improve survival. However the apparent benefit reported in the non-random clinic studies could have resulted (partly) from a selection bias towards patients with a better prognosis. The primary goal of this trial is to assess the effectiveness of ERV (within 16 hours of CS diagnosis and within 40 hours post MI) in reducing in-hospital mortality by a minimum of 20% absolute reduction or more with 90% power comparing 130 patients randomized to ERV with 130 patients randomized to conventional therapy (CT) consisting of thrombolytics and a possible late attempt at revascularization (greater than or equal to 88 hours post MI). Secondary aims include: 1. comparing survival at 6 months post-MI; and 2. assessing the quality of life among survivors using three measures (a subjective Quality of Life assessment designed for this type of post-MI population, a physical functioning questionnaire from which NYHA functional classes I-IV can be constructed). All patients with a clinically suspected diagnosis of CS complicating MI will form a Registry, with limited information collected on in-hospital procedures, medications, length of stay and vital status at discharge. The subset of eligible, consenting patients randomized in the trial will have more detailed in-hospital information abstracted and will be followed for at least six months post MI with telephone interviews at 2 weeks post discharge, 6 months post-MI and (if recruited early) 12 months post-MI. The modified Naughton test will be completed at 6 months post-MI. A final telephone interview will be completed with a proxy if the patient expires before the next scheduled contact. An early stopping rule is proposed with interim analyses to monitor protocol adherence. The final analysis will be according to intention to treat. Some subgroup analyses within treatment groups are proposed to identify important subgroups most or least likely to benefit from ERV and other therapeutic combinations.

Elderly patients often face high-risk health care decisions about vascular disease, and informed decisions are needed to obtain the best results. Carotid revascularization, via surgical carotid endarterectomy or carotid artery stenting, is among the most frequently performed vascular procedures, and is widely recommended to prevent future stroke. Such treatment is commonly performed for asymptomatic stenosis, where the risk of future stroke is low without intervention, so the potential benefit of intervention is much less than in symptomatic patients. When considering revascularization for asymptomatic carotid stenosis, patients and providers must weigh the up-front risk of surgery against the long-term risk of stroke, taken in the context of the patient's life expectancy. The best decision in ths clinical scenario is revascularization for low-risk patients who will live long enough to benefit from surgery, and medical treatment for higher risk patients with shorter life expectancy. However, providers and policymakers have found it difficult to identify the key variables to inform these decisions, both in terms of the short-term risks of endarterectomy, as well as the long- term risks of stroke or death. Health IT can be leveraged to support two key facets of this health care decision: (1) short-term surgical risk stratification by using detailed patient and procedural variables, such as those present in a clinical registry, and (2) longitudinal follow-up o assess the effectiveness of the revascularization in preventing stroke during the patient's remaining life. Our group has demonstrated that the first component can be established using real world data from a regional registry, and the second can be achieved in a broad, cost-effective manner by using administrative claims data. In the present application, we will develop and implement a merged clinical registry and claims health IT tool that will support clinical decision-making, in the ambulatory setting, for patients with asymptomatic carotid artery disease. This approach leverages the clinical detail present in registries with the complete follow-up available from administrative claims. Specifically, we will: (1) identify which asymptomatic patients are likely to receive unnecessary carotid revascularization, using a merged registry-claims dataset, and design a Health IT tool to convey these findings to providers, and (2) determine the potential cost savings associated with avoiding unnecessary carotid endarterectomy in asymptomatic patients. This health IT tool will identify patients who are least likely to benefit from carotid revascularization and allow patients and providers to make more informed choices in the ambulatory setting regarding medical management versus revascularization. This tool will also serve as a model for broader implementation, through the Society for Vascular Surgery Vascular Quality Initiative, and will inform policy makers about opportunities for reducing health care expenditures by reducing unnecessary care.

Multiple expression forms of the hS0S1 and GRF genes were detected in polyA+ mRNA Northerns from human adult and fetal tissues, including brain. placenta. pancreas. lung. kidney and skeletal muscle. Consistent with this variety of mRNA transcripts, in soluble fraction (5100) and in membrane (P100) of different human tissues, several protein forms that were recognized specifically in Western immunoblots were detected. In both cases transcripts and protein bands that are common to all the tissues tested were found, while others are expressed in a tissue- specific manner. In this same line, a 45 nucleotide insertion in Sos mRNA from skeletal muscle, which is absent in Sos mRNA from fetal brain, was detected. This insertion is also present in other tissues, both in fetal and adult. All of these findings suggest that both SOS and GRF gene products suffer alternative splicing and that the multiple forms originated may contribute to the fine regulation of guanine exchange factors (GEF) activity in different tissues or at different stages of development.

Fungi, especially those which live in symbiotic association with other organisms, are rich sources of small-molecule natural products representing novel structures and eliciting diverse biological responses, but to date they remain underexplored. The goal of this project is to generate molecular libraries derived from a unique collection of carefully identified and curated endophytic and endolichenic fungal strains from four diverse U.S. eco-zones. In Specific Aim 1, selected strains of fungal species which have never been investigated previously for their secondary metabolites will be cultured in small-scale and subjected to HPLC analysis, LC-MS and 13C-NMR dereplication to select high-yielding strains producing new, rare, and/or carbonyl-containing compounds. In Aim 2, promising fungal strains will be cultured on large-scale and processed to isolate natural products new to the NIH Molecular Libraries Small-Molecule Repository (MLSMR). Aim 3 will focus on an innovative approach of chemically diversifying CO-containing natural products prevalent among fungal secondary metabolites in a high-yielding reaction with the diamine reagent, hydrazine. This will result in analogs containing N-N moieties very rare in nature but common among drug-like combinatorial and synthetic molecules. During the course of this project compounds possessing unique structural features and occupying biologically-relevant chemical space hitherto not represented in the MLSMR will be generated and submitted to the MLSMR, which constitutes Aim 4 of this proposal. The major strengths of this application are: (i) availability of a collection of a unique, geno-typed endosymbiotic fungal strains not previously investigated for their secondary metabolites; (ii) biological relevance of structures to be generated that are poorly represented in the MLSMR; (iii) the track record of the group in microbiology and natural products chemistry, as well as (iv) the success the group has had in converting carbonyl-containing fungal metabolites into their N-N bond-containing analogs, many with unprecedented structures and potential bioactivities. Thus, the likelihood of discovering activities relevant to human diseases and/or affecting the functions of genes, signaling pathways, and disease-related biological processes will be greater within these libraries due to their natural product origin and/or drug-like structures. PUBLIC HEALTH RELEVANCE: Microbial natural products have a rich history for their utility as tools for identification of novel therapeutic targets for human diseases and as lead structures for drug development. This project proposes to generate diverse molecular libraries from endosymbiotic fungi with potential to interact with cellular targets and serve as lead molecules for drug development and therefore will have a great impact on public health.

The prion hypothesis poses a new paradigm for both infectivity and inheritance in which self- replicating alternate conformers of a normal, cellularly encoded protein specify new traits. In order for a protein to act in these roles, which have been historically limited to nucleic acids, a prion protein must traverse a multi-step pathway of changes in physical state and localization to ensure continued production of the alternate conformer and, thus, stability of the associated phenotype. A major challenge in prion biology is to understand the complex interplay between different conformers of the same protein in the same cell and the cellular regulation of this process. Given the dynamic nature of these interactions and the interdependency of the events in the multi-step prion cycle, development of a quantitative model that can be modified at any step would serve as a predictive tool in which experimental manipulations to the system could be designed and interpreted. Toward this end, I will develop a stochastic model of prion propagation in vivo and adapt this model to study two aspects of prion biology with direct implications for our understanding of human disease: the competition between prion conformers or strains, which has been linked to the interspecies transmission of prion diseases, and the mechanism by which mutations in the prion domain dominantly interfere with prion propagation by the wildtype protein, a process that will inform the rationale design of therapeutic targets. The combination of experimental tractability in the yeast system, which allows direct observations of protein conformation and activity in live cells, and the development of an accurate mathematical model provide a unique opportunity to meet these challenges.

The Research Core will generate new knowledge on minority health and chronic disease, specifically cancer and cardiovascular disease, with emphasis on developing and testing interventions to reduce these disparities. The Core will combine oversight of two P60-supported full research projects with an innovative pilot project program that leverages NIH funding with institutional and philanthropic funding. The Research Core will capitalize on the successful research conducted in the previous two phases of our P60 Center of Excellence, and will make use of the infrastructure of the UAB-supported Minority Health & Health Disparities Research Center (MHRC). Thus it will advance our understanding of health disparities and enhance our efforts to eliminate them. Specific aims are: Aim 1: Oversee the implementation of two full research projects: 1.

The Chicago Center for Reproductive Research Molecular Technology Core is established to function as a common facility where specific technical services and expertise are available to all project investigators. The Core facility will ensure increased efficiency and functionality of each of the individual projects. The molecular biology section of the core will provide a centralized facility that will provide a number of services to each participating investigator in the U54. This list includes, but is not limited to, the following services. Design and production of DNA constructs and targeting constructs for the production of transgenic and knock out mice, DNA sequencing, western blotting, genomic DNA preparation and plasmid DNA preparation. This section will also identify positive founder and chimeric mice produced by the transgenic section. The transgenic section of the core will be available to the participating investigators of the U54 to produce transgenic and chimeric/gene knockout mice. This will be done on a pay per use basis with the Transgenic/ES Cell Facility of the University of Chicago Cancer Research Center and the Diabetes Research Training Center. The histology section of the core will be available to all participating investigators to streamline the anatomical analysis of samples provided by each project. The services provided by this section would include sectioning and mounting of tissues, immunocytochemistry, in situ hybridization and double labeling histology. This section will have the resources and skills necessary to pertbrm radioactive and nonradioactive labeling, including enzymatic based detection systems and fluorescence.

In recent years, we have discovered that certain drugs (methylxanthines, phosphodiesterase inhibitors and selective A1 adenosine receptor antagonists) will induce plasticity in the respiratory pathways which results in recovery of the diaphragm after cervical spinal cord injury. Furthermore, we discovered that after rats are exposed to 3 days of multiple systemic drug administrations (3 injections/day);the drug-induced recovery persists long after the animals are weaned from the drug. In an effort to test our basic science work at the clinical level, we conducted 3 clinical studies using the methylxanthine, theophylline. The results of these theophylline studies were mixed. Some patients showed promising results whereas others did not following the required systemic administration of the drug (either by mouth or intravenous administration). The lack of complete success at the clinical level could not be attributed to any single factor. However, it was very clear that the majority of the patients could not tolerate the theophylline doses necessary to induce recovery without experiencing significant side effects (nausea, nervousness, vomiting, etc.) which forced them to discontinue drug therapy. To directly address the problem of side effects following systemic theophylline therapy in cervical SCI patients, we recently developed a novel approach which combines nanotechnology and proven neurobiological principals to "selectively target" only the respiratory motor (phrenic) and pre- motor (rVRG) neurons responsible for diaphragmatic function to induce recovery. We have shown that by using this method we can induce recovery of the diaphragm in spinal cord injured rats using 1/160th the dose of theophylline necessary to induce recovery following systemic drug administration. This approach to inducing motor recovery after SCI has never been taken by any other laboratory in the world. The purpose of this application is to optimize this technique so that it may be developed for clinical use. There will be three specific aims to address the following hypotheses: 1) that following a non-systemic administration (injection into the paralyzed hemidiaphragm) of one of three different drug-delivering conjugate nanodevices, optimal recovery will be induced. 2) that the "site(s) of action" of the conjugate nanodevice in inducing recovery will be determined by using different retrograde transporters to selectively target different respiratory neurons. 3) that following the single administration of either a slow-drug release, a fast-drug release, or a combination (cocktail) of both conjugate nanodevices, long-lasting functional recovery will be achieved in the hemidiaphragm paralyzed by spinal hemisection. PUBLIC HEALTH RELEVANCE: The ability to induce diaphragmatic recovery in SCI rats with conjugate nanodevices using only a small fraction of the theophylline previously necessary to induce recovery, has major translational implications for the use of this drug (and others like it) in treating SCI patients with respiratory muscle weakness. The obvious advantage is the low dose of the drug should reduce (or completely eliminate) the adverse side effects normally associated with administering theophylline systemically (i.e., orally or intravenously). The purpose of this application is to optimize our newly developed technique so that it may be further developed for clinical use as soon as possible.

Preliminary experiments on effects of microwave irradiation (1 mWatts/cm2) on barbiturate induction time and sleeping time in mice indicate the following interactions. Barbital and phenobarbital induction times were shortened by irradiating for 10 minutes immediately before intravenous administration of the barbiturate. The second distinct interaction seen was that the sleeping time to these two barbiturates were prolonged. Sleeping times to hexobarbital and pentobarbital were less affected. Since it is the long acting barbiturates (where metabolism is not as important a factor in terminating drug action) which were affected more by irradiation and the shorter acting barbiturates which penetrate into the central nervous system quickly (and are the ones where metabolism is important in terminating action) were not affected as much, the working hypothesis is that microwave irradiation increased blood brain permeability. Another separate effect was manifested when mice which were already asleep from phenobarbital were continuously irradiated. These mice regained their righting reflex. This separate effect appears to be an analeptic effect. Another indication of microwave irradiation increasing blood brain barrier permeability was indicated by the increased mortality seen in Japanese Encephalitis Virus (JEV). Two days after innoculation with JEV, mice irradiated with 1 mWatt/cm2 for 5 min showed increase neurotropic mortality to JEV. This irradiation dosage was well below that which caused any lethality by itself. In the coming year, the experiments will be aimed toward substantiatig the postulated increase in blood brain permeability. This will be examined by determining time dependent brain concentrations of the barbiturates and the increased presence of JEV in the brain.

The several time-bound effects of prestimulation on the human startle reflex offer the possibility that different levels of neural processing may be distinguished and may elucidate the nature of changes occurring in early development. The first approach will be to determine the age at which effects ascribable to a short-time processing system can be demonstrated. This will involve manipulating duration, rise time, and spectral composition of startle and of lead stimuli and measuring the effects on primary and secondary components of startle. The second approach will be to determine whether facilitatory effects, presumed to depend on the classical activation system mediated via brain core mechanisms, are present at birth and are modulated with increasing age and cortical maturation. The third approach will investigate the development of effects presumed to depend on orienting-attentional processes.

Cardiac imaging has emerged as an important tool for assessing the diagnosis and prognosis of patients with coronary artery disease. The extent and severity of coronary arterial obstruction (provided by coronary arteriography), along with the degree of myocardial hypoperfusion (provided by perfusion tomography) and the extent of myocardial dysfunction (provided for instance with the measurement of myocardial thickening by magnetic resonance imaging (MRI) together, have long been recognized to be directly related to morbidity and mortality in patients with coronary artery disease. Yet, in clinical practice these imaging studies are most often viewed independently of each other and conventionally displayed in only 2 dimensions, severely limiting the clinicians ability to synthesize the true extent of the abnormalities. Accurate assessment of the extent and severity of coronary artery disease requires the multidimensional (equal to or greater than 3) integration of anatomic and physiologic information obtained independently from these cardiac imaging modalities. However, this integration has traditionally been subjective, time consuming, lacking standardization and difficult to conceptualize multidimensionally. The long term objectives of the proposed research in this competing continuation application are to overcome these limitations by continuing to develop and validate computer-based methods to multidimensionally quantify, unify and visualize the coronary arterial tree and the distributions of myocardial perfusion and thickening. During this next funding period, collaborators from Emory University and the Georgia Institute of Technology propose to continue to focus their efforts in fully completing the development and validation of each of the following methodologies: 1) complete automation of multidimensional reconstruction of the coronary vasculature from arbitrary, non-orthogonal biplane angiographic projections using non-parallel geometry, 2) multidimensional sampling and rendering of myocardial perfusion distributions, 3) multidimensional count based quantification of the amplitude and phase of the onset of myocardial thickening from multigated perfusion tomographic studies, and 4) complete automation of the unification of the multidimensional coronary vasculature with myocardial distributions. In addition, the unification concept will be extended to include myocardial thickening distributions from MRI. The extensive progress thus far achieved in these aims during the initial funding period is strong evidence to support further research, development, and clinical implementation of this multidimensional unified approach.

Introduction: This is an interdisciplinary bioengineering project to develop and evaluate dynamic magnetic resonance imaging (MRI) techniques for the clinical management and surgical planning of pediatric patients suffering from deteriorating renal function. The long term goal is to replace the existing renal tests, which involve significant costs and risks to patients, with a comprehensive, cost-effective, noninvasive examination. Methods: 1) A model of the kidney developed to predict the velocity of the contrast media in the late proximal tubule and thin descending loop of Henle. Assuming that the initial velocity of the filtrate is directly related to the pressure (both osmotic and hydrostatic) gradients between the glomerulus and the initial proximal convoluted tubule, velocities in different parts of the nephron can be extrapolated, in principle, if the relevant spatially dependent pressures are known. Using lumped parameter element design theory, the goal would be to predict these relevant pressure gradients given a priori knowledge of glomerulus and collecting system pressures, fluid viscosity, diffusion constants, and tubule geometry (area and length). 2) A model of the kidney may be developed so that absolute Gd-DTPA concentrations can be ascertained. This would give a direct measure of regional concentrating ability similar to the nuclear medicine studies that are currently used. Since, for a given sequence (e.g., GRASS), the signal intensity is a complicated function T1, T2, T2* and Gd-DTPA concentration, many factors must be considered. Conclusions: During the next year we will compare interleaved spiral, short-TR GRE, single-shot EPI pulse sequences in terms of spatial resolution, temporal resolution, and sensitivity to inhomogeneities and motion. The ultimate choice of dynamic MRI pulse sequence will also be constrained by the minimum temporal and spatial resolution requirements needed to visualize renal pathology. The data analysis and image processing algorithms will focus on techniques such as correlation imaging that emphasize the detection and visualization of regional renal defects rather than absolute quantitation of kidney function.

Efficient muco-ciliary clearance is maintained by a multi-regulatory system complex. The prime objective of the proposed work is to elucidate the fundamental organization and behaviour characteristics of the regulatory system complex by a comprehensive approach. Further refinement of the model of muco-ciliary clearance and development of the basis for a rational approach to prevention and treatment of chronic lung diseases related to environmental noxious agents is our ultimate goal. Relying on a chemical and rheological joint approach, studies will be carried out to fully explore the molecular structure of respiratory mucus glycoproteins with a special focus on the nature of intermolecular crosslinks, and to elucidate the rheological and structural relationship. Based on the structural information, efficient and meaningful micromethods for chemical profile characterizations of major components of mucus secretions will then be developed. As such methods become available, the study will further be extended to elucidate various physological and environmental factors influencing the physicochemical states of mucus secretions and to evaluate the functional significance, in terms of efficiency of muco-ciliary clearance, of such influence.

With the development of rapid, accurate, high-throughput autoantibody and potentially HLA assay systems, we are rapidly achieving the ability to screen and detect pre-diabetic subjects in the general population. Intervention studies in the NOD mouse suggest that antigen specific therapy can be effective in preventing diabetes. However, our current knowledge of the cellular response in human type 1 diabetes has not yet progressed enough to define relevant antigens to use in a similar fashion. The overall of this program project is to identify and assess peptide- specific immunomodulation strategies suitable for intervention therapy in patients with new-onset type 1 diabetes and pre-clinical islet autoimmunity. It rests on the hypothesis that by utilizing a novel approach of determining peptide immunogenicity in human HLA-transgenic mice bearing alleles associated with type 1 diabetes, we can establish the dominant epitopes of pre pro-insulin, GAD65 and ICA512 for a particular HLA allele. This analysis has been used by several groups including Project 1 to identify novel peptides that appear to be reactive in human type 1 subjects. This particular project will attempt to validate the analysis of peptide immunogenicity in HLA-transgenic mice (Project 1) by testing recognition of these peptides in HLA-defined new onset diabetic subjects and other unique pre-diabetic populations. We will test the hypothesis that identification of peptide specific responses is dependent on the HLA peptide interaction (identified in Project 1 and quantitated in Project 4). We will attempt to define this reactivity in proliferation and cytokine assays performed on selected antigen and peptide-specific T cell lines. We will extend these initial observations to include development of stable T cell lines and clones which can then be used to define the TCR alpha and beta chain usage of these antigen specific cells. Efforts will also be directed at obtaining a greater understanding of the immunologic phenotype associated with disease development by analyzing the differences in cellular and humoral immunity between individuals with high risk and protective HLA phenotypes identified by the genetics core (Project 6). This information will be important in defining hard immunological endpoints for potential future intervention trials. Additionally, we will determine the optimum dose, timing and frequency of administration of peptide in vaccination protocols in a animal model of type 1 diabetes. This type of information will be critically important to design to implement future clinical trials of peptide immunotherapy in human subjects. Together with our collaborators we hope to identify peptides of islet autoantigens which are immunodominant and recognized in human type 1 diabetes populations which in native or modified form might eventually form the basis of a trial of immunotherapy for this disease.

The general objective of this project is to provide support and care for the use of transgenic, severely compromised immunodeficient (SClD), knockout, and specific pathogen free (SPF) mice at Stony Brook University (SBU). The Institution has both short-term and long-term goals to improve animal care and services for the faculty. This project will purchase equipment, and perform minor renovations for equipment installation, to upgrade maximum isolation overflow housing of SPF mice. Twenty-two basic and clinically applied research laboratories, that conduct research in gene identification, mapping, analysis and regulation; bacterial and viral receptor regulation; targeted gene therapy; infectious disease pathogenesis; oncology; cerebral amyloid angiopathy; vaccine development; and membrane transport, currently house animals in this overflow space. The project will purchase and renovate two existing animal rooms to install twenty-five ventilated racks. Four mobile, Class 100 animal transfer stations will be purchased and used within the animal rooms for cage servicing and experimental procedures. This equipment will improve the care of animals and address expanding research program needs by creating high density housing space for SPF mice, improving environmental conditions at the cage level to safeguard and preserve animal health, reducing occupational health risks for animal care and research personnel and decreasing labor costs.

It has been generally assumed that secretin is a physiological stimulant for pancreatic water and bicarbonate secretion. There is, however, no proof for this assumption. In fact, there is some recent evidence that it may be invalid. In order to qualify as physiologic stimulant, secretin has to be released after intake of a meal. Using a radioimmunoassay which increased sensitivity (6 pg/ml) and determining IRS concentrations in peripheral and portal venous serum we plan to investigate whether or not secretin is released after a meal in dogs and in human subjects. Existence of molecular heterogeneity has recently been demonstrated for secretin. In this study, we plan to separate different forms of secretin immunoreactivity by Sephadex Gel Filtration and by polyacrylamide gel electrophoresis and to study their physicochemical and immunochemical characteristics, their metabolic half-life and their biological activity. There is some evidence to suggest that ectopic secretin production by tumors exist in humans. We hope to obtain information on the incidence of ectopic secretin secretion, on the kind of tumor it is most frequently associated with and whether or not it is associated with a clinical syndrome by determining the secretin content in extracts obtained from solid tumors. Somatostatin may have therapeutic potential in insulin requiring diabetics, treatment of diabetic ketoacidosis, acromegaly and the Zollinger-Ellison syndrome. However, the tetradecapeptide also has serious unwanted side effects. We have recently demonstrated that somatostatin inhibits HCl stimulated release of secretin and pancreatic secretion of bicarbonate and enzymes. In this study we plan to investigate whether or not meal stimulated pancreatic exocrine secretion is similarly affected by somatostatin.

The objective of this proposal is to understand how people perceive tactile patterns. The project will focus on the processing of tactile spatial patterns. Studies will be conducted comparing interactions between spatial patterns presented to the same location and to separate locations. Results from the psychophysical and perceptual measures will be related to neurophysiological studies of peripheral and central responses to tactile stimuli. Tactile spatial patterns will be generated by means of several types of displays. One is the newly-developed dense array, which consists of 400 independently-controlled tactors in a 20 x 20 matrix. The center-to-center spacing of the contactor points can be varied from 0.5 mm to 1.0 mm. The dense array will be used to generate spatial patterns that selectively activate different populations of mechanoreceptors and to study spatial sensitivity on the fingerpad and palm. The dense array will also be used to examine the conditions for producing perceptual changes related to sensory plasticity. Studies will be conducted with the dense array as well as arrays from the Optacon, a reading aid for the blind, to investigate such perceptual phenomena as temporal integration, temporal masking, response competition, and selective attention. Measurements will also be made of subjects' abilities to resolve spatial gratings. One of the aims of the project is to compare the results obtained with tactile stimuli to studies conducted with visual and auditory stimuli. Results from the project will be relevant to the development of cutaneous communication systems for the deaf, blind, and deaf-blind and to the measurement and understanding of neurological problems.

Molecular dynamics simulations of oligopeptides in solution will be used to study the ways in which hydrogen bonds characteristic of protein secondary structure are made and broken. Structures considered will include 13-membered rings closed by a hydrogen bond (found in alpha-helices) and 10-membered rings (found in beta-turns and 3-10 helices). The simulations will consider both sequences known (by NMR analysis) to contain significant populations of secondary structure as well as sequences of more general interest (e.g. X-Pro-Gly-X for the case of tight turns.) Two general computational approaches will be used: in the first, "umbrella" sampling techniques will generate free energy profiles (potentials of mean force) for reaction coordinates corresponding to making or breaking particular hydrogen bonds. The second approach will use thermodynamic perturbation theory to estimate the free energy changes due to sequence changes for both folded and "random" conformers. The polypeptides studied will be from four to twelve amino acids in length. This work should have important application in understanding the earliest events in protein folding, when initial pieces of secondary structure are formed. In addition, a deeper understanding of the solution structure of polypeptides should aid in the design of novel peptide hormones and neurotransmitters. Antibodies to peptides appear to recognize nascent structures similar to those seen in a corresponding sequence in intact proteins; hence, an understanding of the forces and sequence specificity involved in forming internal hydrogen bonds in polypeptides may aid in understanding antibody-antigen recognition and in the design of synthetic vaccines. The study will be carried out in parallel with NMR analyses of polypeptides from the laboratory of Dr. Peter Wright. Sequences studied by both experimental and theoretical techniques will include those derived from influenza virus hemagglutinin, from a malaria parasite circumsporozite protein, and from myohemerythrin.

Depression is among the most prevalent of all psychiatric disorders. Consistent with the NIH objectives of promoting discovery in the brain and behavioral sciences to fuel research on the causes of mental disorders and charting mental illness trajectories to determine when, where, and how to intervene, a critical public health priority is the development of methods for identifying and altering factors and mechanisms that increase individuals' vulnerability to depression. Offspring of depressed parents are known to be at elevated risk for developing depression; consequently, assessing these children has been an important strategy for elucidating factors associated with the increased risk for psychiatric disorder. To date, however, few studies have gone beyond documenting the magnitude of this risk to examine specific mechanisms that might underlie the intergenerational transmission of risk for depression. Over the last four years we have worked hard to recruit and test a large, carefully diagnosed, sample of 10- to 14-year-old never-disordered girls at either high or low familial risk for depression. Each girl has a biological mother who either has experienced recurrent episodes of Major Depressive Disorder (MDD) during her daughter's lifetime (high risk) or has never experienced an episode of any Axis-I disorder (low risk). Although the high-risk girls are asymptomatic, we have found that they nevertheless already exhibit characteristics of MDD, including selective attention to sad faces, higher and more prolonged cortisol secretion in response to a laboratory stressor, higher levels of awakening cortisol, smaller hippocampi, and anomalous neural functioning both in response to reward and punishment and while experiencing and regulating a sad mood. Because we began recruiting the girls in this study at this young age only four years ago, we have not yet been able to examine fully whether these difficulties predict the subsequent onset of a first episode of MDD. Therefore, we propose to conduct a 54-month follow-up diagnostic session with this sample and to add a second fMRI scan to assess differences in the stability of neural structure and function between high-risk girls who do, and who do not, develop MDD. We also propose to recruit a new sample of high-risk daughters, to teach them either through attentional bias training or through real-time neurofeedback training to alter mechanisms that we posit underlie the development of MDD, and to assess both short- and long-term effects of modulating these mechanisms. We propose to examine immediate changes in stress reactivity, cognitive biases, and reward processing as a function of training. We also propose to conduct an 18-month follow-up assessment to examine longer-term effects of training on specific clinical constructs, such as levels of depressive symptoms, coping strategies, and the onset of MDD. This project will yield new insights concerning psychological and biological risk factors for MDD, and will provide important information that can be used to develop innovative and effective approaches to the prevention of depression.

The neuropharmacological basis for organochlorine-induced tremor and hyperexcitability was studied in rats. Both chlordecone and p,p'-DDT increased the release of brain norepinephrine and serotonin, while having marginal effects on dopamine; p,p'-DDT, but not chlordecone, was found to increase tissue levels of excitatory amino acids such as aspartate and glutamate in the brain stem and spinal cord. Pharmacological experiments to determine the functional significance of these neurochemical changes showed that cholinergic and serotonergic receptor antagonists attenuated the tremor produced by chlordecone, but enhanced that produced by p,p'-DDT. Blockade of alpha-noradrenergic receptors attenuated tremor produced by both organochlorines. Previous studies showed that pretreatment with the anticonvulsant phenytoin attenuated the tremor produced by p,p'-DDT, but enhanced that produced by chlordecone. Recent work extended this observation to augmentation of acoustic startle response produced by p,p'-DDT and chlordecone. Permethrin, a Type I pyrethrin believed to have the same mechanism of action as p,p'-DDT, produced the same neurochemical effects as p,p'-DDT; pretreatment with phenytoin also attenuated the neurological effects of permethrin. Intraventricular administration of calcium prior to the administration of p,p'-DDT or chlordecone attenuated or enhanced the tremorigenic effects produced by these agents, respectively. These experiments demonstrate the neurological manifestations produced by many of the organochlorines, such as tremor and augmented startle responsiveness, are similar, suggesting that they may activate a final common pathway; however, the neuropharmacological basis for the effect may be different.

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 use of functional MRI has been applied to a number of brain systems such as language, sensory (motor, visual), pain, and higher level cognitive functions such as memory or spatial attention. Typically, there is a known model of the experiment (i.e. the subject moves their fingers at the appropriate time);however, in the case of acupuncture this may not hold true. There is known model of the presence of the needling but the brains response may differ. This response may be variable from subject to subject. We are proposing to use a combination of principal components analysis followed by independent component analysis to identify the primary brain responses to acupuncture of the visual system. These experiments have a large number of voxels (64x64x32) for a large number of time points (500) making this analysis difficult on standard computational hardware. Furthermore, we plan to investigate how the independent components interact across a population of subjects to identify the population-based time course of acupuncture. We seek the use of the super-computing resources (storage, processing and memory) to conduct our large scale analysis of within and across subject responses to acupuncture as measured by functional MRI collected using NIH funds from the National Center for Complementary and Alternative Medicine.

The product that will result from this proposal is a nucleoside-modified mRNA encoding erythropoietin for treatment of red blood cell deficiency (anemia). In vitro transcribed mRNAs encoding physiologically important proteins have considerable potential for therapeutic applications. However, mRNA is naturally labile, inefficiently translated and immunogenic and has therefore been traditionally unsuited for therapy. RNARx is developing a technology that modifies mRNA by incorporating non-classical nucleosides, such as pseudouridine. Our preliminary data suggest that this improves the translational efficiency and overall stability of mRNA, as well as diminishing its immunogenicity in vivo. These favorable new properties provide an opportunity to develop deliverable pseudouridine-modified mRNAs as vectors for expressing clinically beneficial proteins safely and effectively in vivo. RNARx will collaborate with the University of Pennsylvania in this Fast-track proposal to develop the first of these therapeutic vectors for delivery of human erythropoietin (EPO). In Phase 1, nucleoside-modified mRNA encoding EPO will be developed, characterized and delivered to mice for verification of biological EPO activity in the absence of immune activation. In Phase 2, a suitable (preferably non-injection) delivery system will be developed and modified mRNA will be tested in small and large animal systems. In addition, based on new models of autoimmunity immunopathogenesis, we will investigate the potential of mRNA to exacerbate a model of autoimmunity (SLE) and whether non-immunogenic nucleoside-modified mRNA avoids this potential. At the completion of these studies, we plan to file an IND to initiate clinical trials of EPO-encoding modified mRNA. Future objectives will include the use of the mRNA platform for other biologics and for intracellular protein delivery (gene therapy), an important therapeutic need for which there are currently no products beyond human clinical trials. Deliverable proteins such as erythropoietin, insulin, and clotting factors are an enormously important arsenal of medical therapies that nevertheless carry a risk of dangerous allergic reactions. In addition, many proteins in the human body, including cell structural proteins, cannot be replaced by conventional protein administration and alternative therapies, such as gene therapy, have not performed to expectation. The product we are developing, a structurally-modified messenger RNA (the intermediary between DNA and protein), is an alternative to protein delivery and gene therapy that will enable the safe (non-allergic) and efficient replacement or enhancement of proteins (in this proposal erythropoietin) including cell structural proteins. [unreadable] [unreadable] [unreadable]

The selection and refinement of appropriate psychophysical methods for the separate measurement of the various aspects of taste perception is a primary and continuing concern of this project. Normal variation in taste perception with chronological age is assessed by procedures which quantify not only the taste detection threshold but also the intensity and pleasantness of the subject's taste experience. Both naturally occurring anomalies and therapeutically induced changes in taste are investigated in parallel studies which emphasize the role of saliva in distortions of taste perception. Finally, the possibility that taste perception differences exist between individuals with and without caries experience is being investigated.

Eye-movement analysis software employing new algorithms has been successfully tested in Phase I for its ability to automatically quantify caloric, rotational, smooth pursuit and optokinetic tests in routine clinical use. In the Phase II project, some refinements in the basic algorithms suggested by results of the Phase I work will be implemented, the capabilities of the system will be expanded to include the saccade velocity and rotation tests, clinical trials comparing the sensitivity achieved by a computer and hand measurements in quantifying the caloric test to detect peripheral vestibular pathology will be completed, and development of software and hardware necessary to commercialize the system will be undertaken. The ultimate objective of the project is to create a low-cost, easy-to-operate, general-purpose clinical vestibular and oculomotor test system based on the new eye-movement analysis software.

The administrative shell has an organizational rather than a scientific focus. While the goals of the individual Cores are to serve the user base, the goal of the administrative shell is to serve the individual cores, overarching needs and the overall Research Core Center mission. The charge to the administrative shell is therefore to: Specific Aim 1: Integrate and supervise the Research Core Center activities Specific Aim 2: Facilitate the operation of the individual cores by resolving issues of space and facilities Specific Aim 3: Assume administrative tasks for all cores such as personnel management and financial operations Specific Aim 4: Carry out or facilitate contacts with University of Michigan administration and granting agencies Specific Aim 5: Coordinate and facilitate the submission of progress reports and renewal grant applications Specific Aim 6: Track user group research and publications associated with their use of the Core Specific Aim 7: Coordinate training across Core components Specific Aim 8: Coordinate and maintain the Research Core Center web site

This application is in response to a Request for Application (RFA-HD-10-018) to conduct community-linked studies to investigate the role of prenatal alcohol exposure in the risk for SIDS, stillbirth and FASD, and to determine how these different outcomes are inter-related, The proposed research will be conducted by the investigators the Prenatal Alcohol, SIDS and Stillbirth (PASS) Research Network in a cooperative agreement with NICHD and NIAAA. This research involves the collaboration of: 1) two comprehensive clinical sites serving populations that are high risk for prenatal alcohol exposure, SIDS, and stillbirth, i.e. the American Indians in the Northern Plains and the Cape Coloured in Cape Town, South Africa; 2) a central Developmental Biology and Pathology Centre (DBPC); 3) a central Data Coordinating and Analysis Center (DCAC); 4) a central Physiology Assessment Center (PAC); and 5) program scientists and officers at the NICHD and NIAAA. The mission of our site is to accomplish the 9 Specific Aims of the Safe Passage Study through the recruitment and enrollment of 7,000 women and the performance of all of the hypothesis-driven study protocols involving the maternal/fetal dyads. The SCCS is overseeing the performance and analysis of Specific Aim 3. This aim is to determine the role of prenatal alcohol exposure, as potentially modified by other environmental, genetic, and placental factors, upon the facial features, somatic growth, and neurological/brain development in the fetus and infant utilizing fetal ultrasonography and infant facial imaging and standardized feature assessment. We will test the hypotheses that: 1) prenatal alcohol exposure adversely impacts facial, somatic, and/or brain growth in the human fetus as early as 20-24 gestational weeks, i.e., the earliest time-point examined by us; 2) prenatal alcohol exposure is associated with facial dysmorphology that is identified by imaging of the face as early as 20-24 weeks and remains present at 1 postnatal month and 12 postnatal months; and 3) this alcohol toxicity is modified by other environmental and placental factors, such as maternal nutrition, maternal smoking, and placental perfusion failure.

The current proposal recognizes that individuals do not operate in a vacuum but rather our lives are inextrica- bly intertwined with family, friends, and community. Taking a multilevel and ecologically valid approach, we plan to identify barriers and conduits to cervical cancer prevention, detection, and treatment among Hispanic women at the individual, interpersonal, and community levels and identify interactions between levels. To achieve this goal, we will conduct a quantitative survey using touchscreen technology with audio narration on a random sample of 2,000 of the 18,000 Hispanic women between ages 21-45 who will receive cervical cancer screening in Years 1 and 2 at LA County-USC Medical Center in East Los Angeles. This large random sample is necessary to enroll approximately 450 women who will later be identified as having an abnormal pap result-- 300 of who will return for follow up treatment and 150 of who will not return--and 1,550 women who will subse- quently receive normal pap results. Because we are interested in predicting differences in screening status, screening results, and follow-up, we will also survey 300 who have never been screened for a total of 2,300 respondents. Individual, interpersonal, and community level factors will be analyzed in isolation and, more im- portantly, working together within a system. As a theoretical framework we employ and elaborate Fishbein and Cappella's Integrative Model of Behavioral Prediction (IMBP). IMBP acknowledges both proximal barriers (i.e., language barriers, lack of insurance) and more distal or background barriers (i.e., education). An innovative aspect of the proposed research is that it extends IMBP by incorporating interpersonal networks and commu- nity-level factors as predictors of cervical cancer-related beliefs, knowledge, and behavior. Replicating a pro- cedure used successfully by The Metamorphosis Project, we will identify the strength of the community's health storytelling network as played out through conversations generated by local or geoethnic media, com- munity organizations, and interpersonal networks. To add further context and depth to the survey findings, we will conduct two sets of focus groups with women who vary in terms of their screening status and results, com- pliance with further treatment, and level of acculturation--12 in Year 1 to aid in survey construction and 12 in Year 3 for interpretation. By taking this multilevel and ecologically valid approach we anticipate that our find- ings will inform practitioners and health researchers not only about how to reach Hispanic women regarding cervical cancer but how to communicate with women about cancer more generally. This work is timely and sig- nificant because Hispanic women are simultaneously the highest risk major racial/ethnic group for cervical cancer and the fastest growing major demographic group in the U.S. By the year 2050, the Hispanic popula- tion in the U.S. will triple from 46.7 million to 132.8 million, growing to 30% of the population. It is critical that we understand the multilevel determinants of compliance with cervical cancer prevention, detection, and treatment guidelines in this high-risk segment of our population.

The increased use of amphetamine-type stimulants (ATS), which refer to the group of stimulants including methamphetamine (MA) and amphetamine, has become a major global health problem in recent years. There is also an increasing concern about the high rate of the non-medical use of prescription stimulants among adolescents. In this competing renewal of DA024070, an international collaborative study aimed to determine the effects of MA on the developing brain, we propose to extend our studies in ATS-using adolescents to evaluate a novel treatment strategy for this population. On the basis of preliminary findings identifying adolescent-specific brain and cognitive vulnerability to stimulant exposure and the neuroprotective properties of cytidine-5'-diphosphate choline (CDP-choline), we propose to conduct a 12-week, randomized, double-blind, placebo-controlled trial of CDP-choline in ATS-using adolescents. As outcome measures, multi-level assessments will be performed at molecular, neural circuitry, cognitive, and clinical levels. It will be determined whether CDP-choline administration, compared to placebo administration, will 1) repair ATS-induced neural cell damage within the target brain regions, which will be assessed using multinuclear magnetic resonance spectroscopic imaging, 2) improve cognitive deficits and normalize the relevant target neural circuits, which will be assessed using neuropsychological tests and multimodal imaging analyses including cortical thickness analysis, diffusion tensor imaging analysis, and network analysis, and 3) reduce ATS-taking behaviors, which will be assessed using a combination of urine monitoring and self-report of ATS use. This study will demonstrate that CDP-choline engages the treatment target and thereby provides clinical benefits to ATS-using adolescents. In the context of the unique opportunity to recruit a special population, such as ATS-using adolescents, in Seoul, South Korea, this project will be performed based on the international collaboration between Perry F. Renshaw at the Brain Institute, the University of Utah, UT, USA and In Kyoon Lyoo at the Ewha Brain Institute, Ewha W. University, Seoul, South Korea. We believe that this research proposal is highly responsive to the International Research Collaboration on Drug Abuse and Addiction Research Program Announcement (PA-12- 040) that encourages the utilization of unique resources or subject populations which would otherwise be difficult to access domestically. As is also acknowledged in the '2007 Distinguished International Scientist Collaboration Award' and '2008 International Program Award for Excellence [Collaborative Research]', our well established collaboration will contribute to the success of this proposal.

This grant is the major support of pre- and postdoctoral trainees in the Graduate Group of Nutrition. The program at UCD is unusual in many respects and a model for training in an applied science, such as Nutr. There are 30 faculty with basic and clinical emphases who are committed to mentorship and didactic training. The training program is establish-ed and successful. There is a history of strong scientific collaborations among basic and clinical nutrition faculty scientists through joint academic appointments and an NIH-funded Clin. Nutr. Res. Unit (CNRU). There is also strong support from the Univ. (e.g. the Div Graduate Studies matches the number of predoc fellows supported on this grant and there are financial commitments from Med., Vet Med, and Agr.). The program provides training for pre(n=3 NIH funded + 3 univ match) and post-doc fellows (n+4). Fellows are immersed in academic/research programs using modern model systems and are trained in integrative approaches from the whole organism to cellular processes. Fellows chose from one of 5 research tracks in nutrition and metabolic regulation: 1. obesity and energy metabolism ; 2. growth, development, and aging; 3. lipid metabolism and disorders; 4. alcohol metabolism and disorders; 5. nutrition, inflammation and immunity. The program is supervised by the Director and Co-Director with input from the Exec. Advis. Board (EAB) and External Advis. Committees (EAC) and participating faculty. Criteria for selection of predoc trainees include evidence of research ability, MS or BS in nutrition or other biological science, excellent academic record, high GRE scores, and strong recommendations. Pre-doc training leads to a PhD in Nutrition or closely related discipline. Postdocs will hold MD, DVM, or PHD in nutrition, biochem, physiol, or molec. biol. Selection criteria include prior educational/residency record and commitment to a career in nutr. research and/or teaching. One or more of the post-doc fellows will be MDs aspiring to career in academic medicine. Trainees participate in graduate courses in nutr. and related subjects. Clinical training of MD fellows is supervised in the Div. Clin. Nutr. and Metab. (DCNM) prior to initiation of the traineeship. Trainees meet 2X/ wk with training faculty via 1) Journal club to discuss published work and to get critical feedback on their own research, and 2) in weekly seminars held by the Graduate Group of Nutr. Fellows meet 2X/yr with their indiv. academic advisory committee. Fellows prepare an annual formal research proposal which is evaluated by the EAC and EAB. Upon completion of training, fellows are expected to have gained a fundamental understanding of the interactions of nutrients and genetic background in metabolic regulation in addition to skills in experimental design, execution, data analysis, interpretation, and presentation of completed research. Fellows are prepared primarily for careers in research/teaching. We also spend a considerable amount of training efforts in insuring that our graduates have the vision and adaptability to work in both government and business environments where, in many cases, the skills and views of health professions trained in nutr. are in urgent demand.

Cortical thickness cocaine dependence is one of the most difficult substance-use disorders to treat and currently has no FDA- approved interventions. While current pharmacological strategies modulate neurochemistry in a spatially- unbiased manner, the development of non-invasive brain stimulation strategies would enable us to directly modulate neural activity in a circuit-specific manner. We recently demonstrated that direct attenuation of the medial prefrontal cortex (a cortical hub of frontal-striatal circuitry tht governs craving) through theta burst stimulation (MPFC cTBS) decreases baseline frontal-striatal activity, neural responses to cocaine uses, and craving in non-treatment seeking cocaine users. The effects of a single session however, erode over the first few hours after treatment. Sustainable effects require multiple days of treatment. GOAL: The primary goal of this proposal is to collect critical feasibility and effect size data which is necessary before moving forward wih multisite clinical trials. The questions that need to be addressed are: 1) Does MPFC cTBS improve retention and abstinence in treatment-engaged patients? and 2) Does it produce an acute and sustainable change in frontal-striatal connectivity among these patients? DESIGN: In this double-blind active sham controlled cohort study, cocaine users enrolled in a 4 week intensive outpatient treatment program will be randomized to receive 3 weeks of real or sham MPFC cTBS. Clinical assessments and neuroimaging data will be acquired four times: before the first cTBS session, after the last cTBS session, at the patient's 1 month follow-up visit, and at the 2 month follow-up visits. Aim 1 - Clinical effects. We will test the hypotheses that real cTBS treatment will enhance retention and decrease cocaine use during the treatment program (part A) and at the follow up visits (part B). Aim 2- Neurobiological effects. We will also test th hypotheses that real cTBS treatment will produce a selective decrease in MPFC-striatal functional connectivity (part A), and that this will be sustained in the individuals that return an have remained abstinent at the follow-up visits (part B). The scientific rationale of this study is an extension of optogenetic studies in animal models of addiction which have demonstrated a causal link between activity in this circuit and drug taking. The experimental design of this study is an extension of the initial clinical trials that led to FDA-approval of repetitive brain stimulaion as a treatment for major depression disorder. Taken together the results of this study may have a high impact and direct relevance to development of brain stimulation as a novel, innovative treatment option for cocaine dependent individuals.

The debate regarding the reality of repressed and recovered memories of childhood sexual abuse (CSA) has occurred almost entirely uninformed by data on memory functioning in individuals reporting recovered memories of CSA. The purpose of the proposed research, therefore, is to test hypotheses about memory in women who either 1) report recovering memories of CSA that have been corroborated, 2) report recovering memories of CSA that have not been corroborated, 3) report never having forgotten their abuse, 4) report believing that they harbor repressed memories of abuse, or 5) report never having been exposed to CSA. Proponents of both the recovered memory and false memory perspectives agree that people who report recalling long- forgotten memories of CSA differ cognitively from those who report never having forgotten their abuse. Proponents of the first perspective suggest that people reporting (corroborated) recovered (or repressed) memories of CSA are characterized by impairments in autobiographical memory and by heightened ability to forget disturbing material relative to people reporting continuous abuse memories or no history of abuse. Proponent of the second perspective hold that people reporting (uncorroborated) recovered memories of CSA are characterized by deficits in reality monitoring (ability to distinguish perceived events from imagined events) and by proneness to "recalling" events that never happened to them. Four laboratory experiments are proposed that test each of these hypotheses, and a fifth study designed to identify individual difference variables (e.g., fantasy proneness, imagery ability) that predict performance in these memory tasks is proposed.

Our established gonadotroph cell lines will be used to better define the regulatory mechanisms for the secretion of gonadotrophins (FSH and LH). We plan to examine direct effects of a number of agents such as neurohumoral substances and steroids which are known to influence gonadotrophin-secretion using our established gonadotroph-rich cell line, or cell lines derived from neonatal or immature rat pituitary glands. Morphological studies of gonadotroph cell lines will be performed by immunocytochemistry in order to define the localization of FSH and LH in the cells. We will also continue our electron microscopic examinations on gonadotroph cell lines.

The overall objectives of the proposed project is to investigate and clarify the pharmacological and physiological interrelationships between calcium and the secretion and effects of calcemic and gastrointestinal (GI) hormones. The goal is to relate the findings to calcium homeostasis and gastrointestinal function in health and disease. Primary emphasis will be placed on secretion, synthesis and, actions of calcitonin (CT), gastrin, and parathyroid hormone (PTH), but studies will also involve several other gastrointestinal hormonal peptides (e.g., cholecystokinin, somatostatin and bombesin). Problems to be investigated include: 1. In vivo in the pig: a) Effects of various GI hormones, esp. gastrin, bombesin and somatostatin, on secretion of CT and the mechanisms involved. b) Possible physiological significance of effects of PTH, CT, somatostatin, and bombesin, on antral gastrin secretion and the mechanisms involved. In vivo in the rat: a) Application of an immunoassay for rat PTH and its use in conjunction with that for rat CT to explore potential GI factors affecting release of calcemic hormones during feeding - esp. in suckling baby rats. b) Examination of possible detrimental skeletal consequences of CT removal in early life in the rat. 3. Evaluation of concurrent secretion of rat CT and PTH in vitro to elucidate mechanisms involved in biosynthesis and release of PTH and CT and to study putative secretagogues and endogenous factors which may influence secretion of CT and PTH.

In recent years, diminished governmental support, dwindling faculty numbers and the need to educate greater numbers of students in the face of declining public health funding have made it increasingly difficult for the University of Zimbabwe College of Health Sciences (UZCHS) to meet the healthcare training needs of Zimbabwe. In this application, we propose to address this dilemma directly by proposing a program whose overall objective is to transform the UZCHS academic environment by - compensating for diminished support with improved efficiency by leveraging information technology introduced as part of the programmatic award to which this application is linked. - reversing the decline in faculty numbers by improving the retention of current UZCHS faculty and the recruitment of UZCHS faculty from the ranks of recent graduates through creation of new educational opportunities, clinical partnerships, and research opportunities. - expanding the scope of medical education in Zimbabwe into cardiovascular conditions of critical national importance through its partnership with University of Colorado School of Medicine (UCSOM) cardiology faculty. The proposal focuses on three conditions that are of critical public health importance in Zimbabwe and provide opportunities to enhance students' educational program broadly: heart failure, rheumatic heart disease, and hypertension/stroke. Using these three focus conditions as a foundation, we will enhance the medical school curriculum for all students and create a track for developing future faculty we call the Cardiovascular Clinical Scholars Program. Scholars will work on projects of public health relevance that also will be a substrate for clinical research, mentored by teams of UZCHS and UCSOM faculty. The proposal includes a formal evaluation component that will provide ongoing feedback on the progress and will guide redirection of the program if necessary. PUBLIC HEALTH RELEVANCE: Because of economic and political instability, and necessary attention on communicable disease, the capacity of the healthcare system in Zimbabwe to deal with cardiovascular disease has declined tremendously in the past decade. This proposal seeks to reverse this decline by training Zimbabwe's future generation of medical faculty in cardiovascular care.

Onychomycoses are fungal infections of both the fingernails and the toenails accounting for up to 50% of all the nail disorders. They are currently treated by oral and topical administration of the antifungal drugs. Oral antifungal therapy is generally associated with serious adverse effects independent of or associated with a number of significant drug interactions. Topical monotherapy is "less successful" in treating onychomycosis due to poor trans-nail permeation of antifungal drugs. The poor drug absorption across the nail plate could be attributed to reasons such as unfavorable physicochemical properties of the drugs, lack of formulations that can overcome the barrier properties of the nail plate, short residence time of topical formulations and extensive binding of drug to keratin. Topical monotherapy would be successful only when consistent free drug level >MIC is maintained in the nail stratums during the course of treatment. This requires a drug delivery method that can rapidly drive an effective quantity of drug across the nail plate. We propose to develop a novel "Electropulsation" method for the treatment of onychomycosis. We believe that Electropulsation will significantly improve the success rate of topical monotherapy and shorten the duration of treatment of onychomycosis. Electropulsation of the nail plate, in vitro (at 10V/cm2, 50ms pulse duration at 1Hz) delivered an order of magnitude higher amount of drugs (within a 20 minutes treatment period) as opposed to that achieved by conventional topical application (in 24 hours duration). We hypothesize that application of an appropriate Electropulsation protocol can rapidly deliver therapeutically effective quantity of drugs across the nail plate by kinesis of the drug molecules and also by permeabilizing the nail plate as well. In our Aim 1, we propose to study the effect of different Electropulsation protocols on the trans-nail delivery of drugs and to investigate the mechanisms of trans-nail drug transport enhancement. We further hypothesize that the Electropulsation protocols do not cause any significant discomfort in human subjects. In our Aim 2, we propose to assess the tolerability of Electropulsation protocols applied on the nail plate in the healthy human subjects. The completion of these Specific Aims will result in Electropulsation protocols that are effective and safer for clinical applications. [unreadable] [unreadable] PUBLIC HEALTH RELEVANCE: Currently the fungal infection of nails (onychomycosis) is treated with oral and topical antifungal drugs. Oral antifungal therapy is associated with severe side effects and topical monotherapy is less successful due to poor drug penetration into the nail plate. We propose to develop a novel technique; the "Electropulsation" for rapid delivery of drugs across the nail plate, which in turn is likely to significantly improve the success rate of topical monotherapy and shorten the duration of treatment of onychomycosis as well. [unreadable] [unreadable] [unreadable]

The overall objective of the proposed activity is to acquire, by purchase or otherwise, death index records from the 56 U.S. registration areas for compilation into a consolidated death index covering some period of time prior to 1979. The consolidated file will be for the direct use and benefit of the government and will be maintained and operated by the government, either directly or through the services of a contractor. The file will be used by epidemiologists, both intramural and extramural, for the purpose of determining the vital status of study subjects. Such subjects are followed to determine the health effects of exposures or events which have occurred at some time in the past.

Anorexia Nervosa (AN) is a serious disorder associated with significant psychiatric and medical morbidity and mortality and high treatment costs due to the use of intensive treatment. Evidence suggests that FBT for adolescent AN is an effective outpatient treatment both at the end of treatment and 4-5 year follow-up. Using FBT, about 50% of participants remit (%IBW>95 + EDE >1SD of the mean) and 90% of those fully remitted at post-treatment, remain so at 12-month follow-up. In contrast, 75% of those not fully remitted at post-treatment are still not remitted at follow-up. Because there are no known effective treatments for adults with AN, and adolescents with AN appear to be more responsive to treatment, efforts to improve outcomes in this age group are critical to prevent the development of a chronic and unremitting course. However, when response to FBT is inadequate, more intensive programmed treatment (e.g. day treatment, residential care, or psychiatric hospitalization) is often recommended. Despite the common use of such programs, available data do not suggest that these interventions are more effective than outpatient interventions in adolescents. An alternative strategy to improve outcomes for those that do not respond to FBT would be to provide additional and targeted outpatient help directly to families themselves. Data suggest that families that are not likely to be successful in FBT can be identified as early as one month into treatment. Consequently, providing an alternative therapy early in the treatment course may enhance overall outcome. To develop a new treatment - Intensive Family-Focused Treatment (IFT) to improve outcomes in those who do not show an early response to FBT we propose a 2-phase treatment development study which aims to (1) identify modifiable family factors/behaviors that interfere with accomplishing weight restoration, and (2) develop a new family treatment (IFT) targeting unhelpful behaviors and promoting helpful ones (Phase 1 - Iterative case series (n=40)), and (3) pilot IFT (for those not responding early to FBT) vs. FBT alone in a small RCT, and (4) explore familial and individual factors as predictors, moderators, and mediators of treatment (Phase 2 - Small RCT (n=50)). To achieve these aims, this study will recruit 90 adolescents with AN (DSM-IV, exclusive of amenorrhea) and their families at two sites (45 participants at The University of Chicago and 45 at Stanford University). Primary outcome will be full remission from AN (%IBW>95 + EDE >1SD of the mean). Assessment of primary outcome as well as potential predictors, moderators and mediators will occur at baseline and at the end of treatment and will include eating related psychopathology in the parent and child (Eating Disorder Examination), Yale- Brown-Cornell-Eating Disorder, height/weight, general psychopathology in parent and child, family status, and family functioning. PUBLIC HEALTH RELEVANCE: Anorexia Nervosa (AN) has the highest mortality rate compared to any other psychiatric disorder. The most promising treatment for adolescents with AN is family-based treatment (FBT). However, only 50% of patients receiving FBT fully remit at 12-moth follow-up. Consequently, providing an alternative therapy early in the treatment course for those not responding to FBT may enhance overall outcome. This study aims to develop a new treatment - Intensive Family-Focused Treatment (IFT) - to improve outcomes in those adolescents, aged 12-18 years, who do not show an early response to FBT.

The long term objectives of this proposal are to elucidate the exact sequence of biochemical events that are involved in the priming and stimulation of superoxide (02-) production by neutrophils. Superoxide is a key component of the oxygen-dependent antimicrobial mechanisms of these cells which provide a major defense against infectious organisms. Certain cytokines prime neutrophils to release increased amounts of 02-. Therapeutic value of these factors is now being tested clinically in a variety of cancer and AIDS patients with promising results. Thus, experiments outlined herein are relevant to an understanding of host- defense mechanisms and immune deficiency diseases. Specifically, this project addresses two largely unknown areas. These are: (1) the mechanism(s) of action of the hydroxylated eicosatetraenoic acids (HETE) in modulating 02- production, and (2) the link between protein phosphorylation and 02- generation. Neutrophils produce 5- and 15-HETE under various circumstances. 5-HETE dramatically potentiates 02- release, whereas 15-HETE inhibits it. We propose that 5- HETE primes neutrophils by increasing the flux of Ca2+ across the plasmalemma, perhaps by opening a channel for this cation. This will be measuring the rates of 45Ca2+ uptake by these cells in the presence of different concentrations of 5-HETE. The effects of a variety of calcium channel antagonists on this process will be evaluated. The relationships between the rates of 45Ca2+ uptake and 02- generation will be defined, and possible antagonistic effects of 15-HETE will be sought. With regard to protein phosphorylation, we have observed that stimulation of 02- release by neutrophils is always accompanied by an intense phosphorylation of two proteins with molecular weights of ca. 47 and 49 kDa. Quantitatively, these are the two major proteins which are phosphorylated upon stimulation of these cells. While the 47 kDa protein has been extensively characterized, virtually nothing is known of the 49 kDa species. Experiments are detailed herein to identify, characterize and purify this protein. Techniques of biochemistry and cell biology will be employed. We will determine whether the 49 kDa protein is a component of the 02- generating system, or is involved in some other fashion in the mechanism of stimulation of these cells (e.g., phospholipose D). Changes in the activity of the 49 kDa protein after phosphorylation will be sought to forge a link between this modification reaction and cell stimulation.

Many cases of sensorineural hearing loss (SNHL) likely are caused by interruption of cochlear blood flow (CBF). Disruption of the internal auditory artery during surgical procedures to remove acoustic neuromas (ANs) probab'y account for many cases of postoperative deafness. Also, many cases of clinical SNHL, such as sudden SNHL, are thought to involve reduction of CBF. Previous experiments have demonstrated the utility of measuring cochlear blood flow using laser-Doppler flowmetry techniques. This proposal involves the refinement and clinical human testing of a novel otic probe and instrumentation capable of measuring CBF and electrocochleographic potentials. The probe will also have treatment capability in the form of irrigation and suction to present medications to the round window and inner ear. Phase I results demonstrated the feasibility of these functions in animal experiments using a prototype probe. In Phase II, a refined probe will be used to test safety and efficacy during human surgery. Ultimately, it is anticipated that this instrumentation will be used (1) intraoperatively to assist surgeons in preserving hearing during AN removal; (2) in research applications to elucidate the role of CBF in SNHL; (3) in the clinic for diagnosis and treatment of SNHL caused by vascular insufficiency.

A series of experiments have been proposed to elucidate the mechanisms of two enzymes, dopamine Beta-monooxygenase and peptidylglycine alpha-hydroxylating monooxygenase. These enzymes play an important role in the synthesis and regulation of hormones. Many aspects in the mechanisms of these enzymes are still unresolved. We propose to trap and characterize several of the intermediates generated by the enzymes throughout the catalytic cycle, using Co(ll) complexes. These complexes will also allow us to probe the redox properties of the copper cofactors involved in these processes. Studies will be carried out on both enzymes in order to draw comparisons between the two systems.

The long-range goal of this project is to study bacteriophages and genetic exchange (mainly lysogenic conversion, transduction and transformation) in streptococci, and to investigate how such mechanisms may affect the pathogencity and immunology of these organisms. Currently we are searching for the nature and site of the genetic coding for a rare, Group A, non-lysogenic, erythrogenic toxin (ET) producing strain. We have attempted: 1) to isolate a defective prophage by phage-genetic recombination, 2) to induce ETB production in ETB strains by lysogenization with phages from ETB ion strains, and 3) to find toxin production in a selected number of Group C and G streptococci. Other studies concern extracellular product of Pseudomonas fluorescens which causes morphological changes and lysis in strains of Trypansoma cruzi. We are trying to identify the factor and assess its significance.

During the coming year (1974) work on this project will include the following: 1. Completion of a detailed study of the locations, fine structural details and sources of the synaptic vesicles containing dense cores that are in or near the substantia gelatinosa of the spinal cord. The study will use cats, dogs and monkeys and include normal animal animals, animals subjected to dorsal root resections and to double hemisection of the spinal cord. 2. Attempts will be made to determine the active ingredients of the dense cores and especially to establish whether or not they contain biologically active amines. 3. Tests will be made to determine the feasibility of obtaining samples of astrocyte mitochondria without cristae from the dorsolateral region of cat spinal cord. 4. A study of specialized cell junctions between astrocytes and neural elements will be initiated.

This proposal describes the continuation of a project that involves the synthesis of biologically active molecules containing, or derived from molecules containing the cyclopentenone ring system. Target molecules currently under investigation, and proposed target molecules for future research include quadrone, laurenene, the pentalenolactones, carbacyclins, and hydrazulenes. These molecules display a wide range of biological activity and include antibiotics, cytotoxics and anti-cancer agents, and therapeutic agents for circulatory disorders. The methodology to be used is derived from the organometallic cycloaddition processes which have been highly successful in the past at permitting synthetic schemes to bypass much conventional synthetic methodology. The current state of the art permits prediction of success with considerable confidence. This proven methodology is proposed to be supplemented with several new concepts that, taken together, are aimed at addressing those situations in which the methodology does not succeed satisfactorily, and at modifying the existing procedures to permit ready access in a practical way to the desired target molecules in optically active form. The overriding goal of the proposed research will be to generate methodology derived from unique new combinations of several technologies, to permit access to molecules of biomedical importance in their most biologically active forms, as efficiently as possible from readily available materials.

The identification of tumor suppressor genes has led to new insights into the mechanisms of human cancer development. The normal functions of these genes often lie in the control of gene expression, especially in the realm of cell cycle control and cellular differentiation. Several recent studies have implicated aberrant activity of chromatin remodeling complexes in the development of human cancer. Mutations in the INI1/SNF5 gene, a component of the SWI/SNF chromatin remodeling complex, occur in the majority of malignant rhabdoid tumors. The SWI/SNF complex acts as a global transcriptional activator that alters nucleosome positioning on DNA via an energy-dependent mechanism. Others and we have also demonstrated the loss and/or mutations of both human SWI2 homologs, BRG1 and BRM, in human tumor cell lines and primary tumors. Loss of expression of both proteins abrogates Rb-mediated cell cycle arrest. However either protein appears to regulate the expression of key cancer progression genes including Ecadherin and CD44. Furthermore, while BRG1v/- mice develop adenocarcinomas, BRM-/- mice do not show an increased tumor incidence. Therefore, the mechanism by which loss of expression of either or both proteins contributes to the etiology of human cancers remains unresolved. We have observed an association between BRG1-induced gene expression and loss of promoter methylation in human tumor cells, especially Non-Small Cell Lung Carcinomas (NSCLC). Based on these studies, we hypothesize that loss of SWI/SNF complex activity through inactivation of BRG1 and/or BRM fuels genomic instability during human tumor progression by facilitating gene silencing through DNA methylation. To test this hypothesis, we require a better understanding how loss of expression of BRG1 and BRM proteins alters the biological properties of tumor cells and the chromatin structure of cancer related genes. In this application, we will identify overlapping and independent biological and biochemical functions of these proteins in the first specific aim. In the second specific aim, we will determine how the loss of these proteins contributes to lung tumor development using a human tracheobronchial cell culture model. In the last specific aim, we will characterize the effects of BRG1 and BRM loss on the chromatin structure and DNA methylation status of important target genes. The dissection of the role of these genes in human cancer development will broaden our understanding of their normal biological and biochemical functions, provide new insights into the control of DNA methylation and impact upon treatment and detection of this clinically important tumor.

This research utilizes the Operations Research approach to develop a method for effectively allocating high cost health services throughout a planning region. A set of mathematical models are formulated for forecasting service demands, optimally specifing service locations and quantity level, and simulating political restrictions. In order to consider the possible political and social factors which hinder the implementation of optimal solutions, the model will be developed with the collaboration of the local planning agencies in Allegheny County. The extensive data souuces which are available in Western Pennsylvania will be utilized in an effort to develop models whose solutions can be implemented. It is recognized that previous Operations Research approaches to health planning have emphasized abstract models which have not been useful to planning agencies. This research seeks to move towards developing models that can, in fact, be implemented by involving local planners in the formulation process in order to avoid proposing models which do not have implementation potential. This research is concentrating on those planning problems involving the allocation of emergency medical services.

The proposed work is based on our observations that human blood group MN precursors, T (Thomsen-Friedenreich) and Tn (precursor of T) antigens are human carcinoma associated and that T antigen present on human breast carcinomata elicits both humoral and cell-mediated immune responses in patients with these malignancies. We will perform the leukocyte migration inhibition assay in agarose with T antigen on a large population of breast carcinoma patients and include a comparable population of similar size consisting of patients with benign breast disease as well as of apparently healthy people. We will also test patients with carcinomata of the gastrointestinal tract and lung for their cellular immune responses to T antigen since we found such carcinomata also to contain T-specific antigen. We intend to evaluate the specificity and sensitivity of leukocyte adherence inhibition assay in comparison with other methods for measuring cell-mediated immunity. We plan to investigate more patients with carcinomata of the gastrointestinal tract and lung, for their serum anti-T antibody levels. We hope to complete establishment of a method for rapid quantitation of T-specific structures in human body fluids. We will isolate and characterize T- and Tn- specific structures from human breast carcinoma tissues.

This proposal requests partial support for a meeting on Iron Sulfur Enzymes as part of a the Gordon Research Conference series to be held in Stonehill College (Easton, MA) on June 15-20, 2014 with a broad and long-term goal of bringing together the world's leading experts in iron-sulfur enzymes, focusing on enzymatic mechanisms, model complexes, biogenesis, and roles in regulation and human disease in a uniquely cross-disciplinary manner. First organized in 1994 to focus on nitrogenase and its unique iron-sulfur chemistry, knowledge about iron-sulfur clusters has grown exponentially in the last two decades and, in 2006, the name of the conference was changed accordingly to Gordon Conference on Iron-Sulfur Enzymes. The conference has tremendous breadth, featuring sessions on a wide range of iron-sulfur enzymes in unicellular organisms, animals and plants, which provide important insights into how these versatile proteins are synthesized and utilized. Proteins containing iron-sulfur cofactors perform a variety of biological functions, ranging across electron transfer, acid-base catalysis, and sensing where they are agents for cell regulation through transcription (DNA) or translation (RNA). They are redox catalysts for radical-based reactions and the activation of H2, N2 and CO2, processes that offer scientific and economic challenges for industry. Iron-sulfur centers provide the focus for fundamental investigations of chemical bonding, spectroscopy, structure and molecular mechanism, and their functions have numerous implications for health, medicine and applications for technology, including renewable energy. The specific aim of this conference is to include young and distinguished speakers and discussion leaders from many fields, and topics will range from fundamental iron-sulfur chemistry, mechanistic investigation of iron-sulfur enzymes and iron-sulfur biogenesis to eukaryotic iron/sulfur metabolism and an emerging class of diseases related to abnormality of iron-sulfur biogenesis. The health relatedness of the meeting is clear, and mixing chemists, biochemists, structural biologists, molecular biologists and medical researchers together for the first time has the potential to catalyze progress in the field.

Increasing problems with rigidity, bradykinesia, tremor and gait disturbances are seen with advancing age. These movement dysfunctions are associated with a significant increase in morbidity and mortality in the elderly. Our studies in rhesus monkeys will initially focus on delineating the relationship between age-associated changes in the nigrostriatal system and the decline in motor functions during normal aging processes. Our research program project has been designed to critically test four hypotheses. Hypothesis 1: that rhesus monkeys undergo declines in motor functions which closely model those seen in humans. Hypothesis 2: that there is a gradual, continuous change in nigrostriatal dopaminergic function in rhesus monkeys with age. Hypothesis 3: that there is a gradual continuous loss of midbrain dopamine neurons in rhesus monkeys in aging. Hypothesis 4: that upregulation of the nigrostriatal dopaminergic system in older rhesus monkeys by intracerebral administrations of the potent dopaminergic trophic factor GDNF improve motor function. The program project is organized into three research projects which are supported by two core units. Project 1-3 are carefully integrated and coordinated to analyze motor functions and the nigrostriatal dopaminergic system in rhesus monkeys. An Administrative Core supports all three project, coordinating collection, pooling and analysis of data. A Primate Core Facility supervises and coordinates the use of all rhesus monkeys in these studies.

Gradual initiation of TMP/SMX as primary PCP prophylaxis.

This K23 award will allow Dr. Misty Hawkins, a clinical psychologist with expertise in behavioral medicine and obesity, to further develop into an independent investigator proficient in physiological, neuropsychological, and self-regulatory research methods and interventions that will allow her to study novel mechanisms of and treatments for obesity maintenance. She has a career interest in intervening on the neuropsychological factors related to obesity. Her research goal is to identify novel cognitive and self-regulatory mechanisms of obesity in this pilot trial which can then be targeted in larger RCTs to reduce obesity development or to maintain weight loss. Obesity continues to be a global epidemic, yet successful interventions for obesity are rare, with 80% of individuals in treatment programs being unsuccessful at achieving long-term weight loss. The training and research activities in this K23 application will allow her to examine the complexities of physiological dysregulation, cognitive deficits, and self-regulatory failure in an obesity treatmen study. The application proposes an intensive, 5-year program of mentored research and formal training activities to enhance Dr. Hawkins' skills and experience in: 1) basic research on and assessment of cognitive function; (2) assessment of obesity-related physiological changes; (3) research with patient populations; (4) advanced assessment of self-regulation (SR); and (5) the conduct of randomized clinical trials and advanced statistics. In the long term, Dr. Hawkins will apply these translational research skills to study targeted cognitive and self-regulation interventions as potentially effective treatments for persons with obesity who may exhibit cognitive deficits or chronic self-regulatory failure. The research component of this career development award is a clinical trial examining the impact of two different weight loss treatments on physiological markers, cognition, self- regulation, and health behaviors in 64 obese persons compared to 32 no-treatment controls. The specific aims are to: 1) Confirm that baseline obesity-related physiological dysregulation is linked to cognitive deficits, poorer self-regulation and obesogenic behaviors, 2) Demonstrate that the two treatment groups have greater improvements in biomarkers, cognition, SR, and obesogenic behaviors, less weight gain, and greater weight loss, and 3) Evaluate whether the acceptance-based treatment (ABT) group has greater improvements in biomarkers, cognition, SR, and obesogenic behaviors, less weight gain, and greater weight loss than the standard behavioral treatment group (SBT) from pre- to post-treatment and 1-year follow-up. Kent State University and its research partner Case Western Reserve University provide exceptional environments for Dr. Hawkins to gain the skills needed to achieve her goals. The training component uses academic resources including the College of Arts and Sciences, the Department of Psychology, and the Clinical Research Scholar Program. Dr. Hawkins' mentors are highly regarded scientists in the areas of obesity, neuropsychology, psychophysiology, and patient-oriented research.

Project Summary/Abstract The National Cancer Institute (NCI) established the Cancer Genetics Network (CGN) in 1998 as a multi-centered national project designed to support collaborative investigations on the genetic basis of cancer susceptibility and to explore mechanisms to integrate this new knowledge into medical practice. A fundamental issue in this high-risk population is how to optimally screen for early detection of disease. Recent discovery of new biomarkers that change with early stages of disease offer promising non-invasive strategies for screening. However, two aspects that are fundamental for determining an optimal strategy are: for what cancers is the individual at elevated risk and how can longitudinal measurements on a biomarker be used to predict risk of disease. To focus on these issues in the context of the Cancer Genetics Network project, there are two aims in this proposal. The first aim will develop and apply methods for analyzing data on family history to discover what cancers tend to aggregate in individuals and families. We will develop methodology that can be applied to the CGN Registry (family history) data and appropriately accounts for age and mode of ascertainment of participants. We will apply these methods to identify clusters of cancer sites that occur together in families. The second aim of this proposal focuses on the development and application of a method to assess the relationship between longitudinally collected biomarkers and early evidence of disease onset. The method will allow us to analyze data from the CGN Ovarian Cancer (CA125) Biomarker Screening study, as it will appropriately handle this longitudinal and event time data even though the screening schedule for these women varied due to missed or delayed visits. The PI of this proposal is the PI on the Statistical Coordinating Center of the Cancer Genetics Network, and is responsible for overseeing data acquisition and analysis of CGN studies, but is not funded for statistical methods research. This grant would provide the support needed to complete the research for and apply the required statistical methods. This research has the potential of a major impact on the vulnerable population of individuals with family history of cancer, as it will allow the screening strategy to be more focused on cancers to which the individual is at elevated risk, and potentially improve the chances that the disease will be detected in a treatable stage.

Amyotrophic lateral sclerosis (ALS) and spinal muscular atrophy (SMA) are fatal neurological disorders that involve the selective degeneration of spinal motor neurons. SMA ? the most common genetic cause of infant mortality ? is a monogenic disorder caused by widespread deficiency in the survival motor neuron (SMN) protein due to deletion of the SMN1 gene. In contrast, ALS is predominantly a sporadic disorder, but in a minority of familial cases, mutations in over 20 different genes cause motor neuron degeneration. Genetic and molecular studies increasingly suggest that ALS and SMA may share common underlying mechanisms of disease. This project focuses on one form of familial ALS caused by mutations in the RNA binding protein fused in sarcoma (FUS) - which are associated with a broad range of clinical phenotypes including some of the most aggressive, juvenile-onset forms of the disease - and the possible role of SMN biology in the pathogenesis of FUS-dependent motor neuron degeneration. SMN has a well-established function in the assembly of small nuclear ribonucleoproteins (snRNPs) involved in diverse mRNA processing pathways and increasing evidence links SMN-dependent RNA dysregulation with the etiology of SMA. Remarkably, recent studies in cultured mammalian cells and ALS patients' fibroblasts have shown that FUS depletion or expression of ALS-linked FUS mutations disrupt the normal localization of SMN to nuclear bodies known as Gems. Furthermore, FUS has been shown to associate with SMN as well as specific snRNPs whose biogenesis is SMN-dependent and might be disrupted by ALS-linked FUS mutations. Together, these findings suggest that FUS and SMN are functionally linked through a shared molecular pathway(s) and support the view that SMA and ALS are related motor neuron diseases. However, the normal requirement of FUS for snRNP biogenesis and the pathogenic impact of FUS mutations on SMN biology have not yet been defined mechanistically, and the contribution of SMN dysfunction to FUS-ALS pathology remains unknown. To address these outstanding questions directly, our project takes a systematic, multi-disciplinary approach involving novel mouse models of FUS-dependent ALS to explore potential SMN-dependent mechanisms of FUS-mediated motor neuron degeneration. In Aim 1, we will investigate the phenotypic effects of both reduced and increased SMN expression on FUS-dependent motor neuron pathology in mouse models of ALS. In Aim2, we will employ a comprehensive set of molecular approaches to establish the functional relevance of normal and pathogenic FUS-SMN interactions in the pathway(s) of snRNP biogenesis in motor neurons using a combination of cellular and animal model systems. Collectively, these studies aim to establish convergent mechanisms in ALS and SMA and will yield a more complete understanding of the biology of FUS and SMN that is relevant to motor neuron survival. Identification of shared molecular pathways contributing to death and dysfunction of motor neurons in SMA and ALS may also expand the range of therapeutic targets for these diseases.

DESCRIPTION This is an application from the Association of American Medical Colleges (AAMC) that requests funding for four years to conduct a series of seminars and workshops involving 2 cohorts of 25 minority junior medical school faculty members. Each cohort would participate in a program lasting two years ( a total of 50 participants over 4 years). The program is designed to develop the participants' health services research (HSR) skills. It is focused on helping the participants develop a concept paper that would eventually grow into an R01-type grant application, over an 18-month period. This goal will be accomplished by demanding a commitment from the participant's department to free 20% of his/her time; by pairing each participant with a mentor; and by all participants attending four especially convened seminars (one being a mock study section to critique the applications), as well as two AHSR annual meetings. The investigators also state that the program will be evaluated using both process and outcome measures. The application models the project closely on predecessor projects which were funded by AHCPR in 1991 and 1993 which have taken 2 previous cohorts of 25 and 27 participants respectively through a similar process. The first cohort completed the program in 1994, and the second cohort will complete the program in 1996.

Loss of life and non-fatal serious injuries are among the public health costs associated with alcohol related rashes. In order to develop successful public policies for reducing alcohol related crashes, it is necessary that we understand the causes of these crashes and how effective alternative public policies are in reducing the incidence and severity of these crashes among various at-risk groups. Because of their increasing proportion on the nation's highways, older drivers are at greater risk. This raises two questions. What are the underlying determinants of alcohol related crashes among older drivers? And what public policies are most effective in reducing alcohol related crashes among this group of drivers? The purpose of this project is to analyze each of these questions using California as a case study and based upon data over a 16 year period, 1981 through 1996. The focus of the analysis will be upon four specific areas of public policy interest: the per se BAC level for driving under the influence of alcohol; alcohol availability; traffic enforcement; and speed limits. The analysis will comparative analyze the extent to which each of these public policies affects the incidence and severity of alcohol related crashes among older drivers relative to the other-age population. Further, in order to better our understanding of older driver crashes and regulatory policy, three different methodological approaches will be used: aggregate time series analysis using monthly data over a 16 year period; time series-cross section analysis based upon county-wide observations over the 16 year period, and ordered probit analysis using a sample of individual crash data from 1996.

We have been examining polymorphisms in genes involved in the leptin signaling pathway to identify gene variants impacting on body composition. We are currently intensively studying a variant MC3R that is associated with adiposity in children and which appears to have functional significance for MC3R signal transduction (1). Children who are homozygous for two rare polymorphisms (Thr6Lys and Val81Ile) have significantly greater fat mass and leptin compared with wild type or heterozygous children. Recent studies have confirmed greater adiposity in homozygous adults. Ongoing studies attempt to understand the mechanisms by which these sequence alterations impact body weight. We have initiated a study comparing energy balance during adaptation to a high-fat diet in humans with double-mutant and wild type MC3R. We have also successfully bred and studied novel knock-in mice expressing the human wild type MC3R(hWT/hWT) and human double-mutant MC3R(hDM/hDM). MC3R(hDM/hDM) have greater weight and fat mass, increased energy intake and feeding efficiency, but reduced fat-free mass compared with MC3R(hWT/hWT). MC3R(hDM/hDM) mice did not have increased adipose tissue inflammatory cell infiltration or greater expression of inflammatory markers despite their greater fat mass. Serum adiponectin was increased in MC3R(hDM/hDM) mice and MC3R(hDM/hDM) human subjects. MC3R(hDM/hDM) bone- and adipose tissue-derived mesenchymal stem cells (MSCs) differentiated into adipocytes that accumulated more triglyceride than MC3R(hWT/hWT) MSCs. MC3R(hDM/hDM) impacted nutrient partitioning to generate increased adipose tissue that appeared metabolically healthy. These data confirmed the importance of MC3R signaling in human metabolism and suggested a previously-unrecognized role for the MC3R in adipose tissue development. Current studies are seeking to understand better the roles of MC3R in peripheral metabolism. We have previously found that leptin is an important predictor of weight gain in children and identified children with hyperleptinemia and leptin receptor mutations. We have also found hyperleptinemia out of proportion with body fat mass in children with psychological loss of control (LOC) over eating. Such data suggest the importance of leptin resistance as a factor stimulating weight gain and have led to recent explorations of other syndromes associated with obesity that may cause dysregulation of leptin signaling, including Bardet Biedl syndrome. Current studies are directed at understanding additional genetic, physiological, psychological, and metabolic factors that place children at-risk for undue weight gain (2-14). We have recently examined how obesity and thyroid hormone (4) are interrelated as well as how leptin impacts bone in children (2) Two recent initiatives target insulin resistance. One trial studied the role of depressive symptoms in childrens insulin resistance. A randomized clinical trial to study the effects on insulin resistance of preventing depression in obese adolescents with a family history of type 2 diabetes (14,15) found at one-year follow-up, among girls with moderate baseline depressive symptoms (N = 78), those given a cognitive behavioral therapy (CBT) program for prevention of depression developed lower 2-hr insulin than those in a health education control group (Delta-16 vs. 16 muIU/mL, P < .05). Further studies are required to determine if adolescents with moderate depression show metabolic benefits after CBT. Another pilot study initiated in 2013 tested if short bouts of activity may improve glucose tolerance in children. In non-overweight children, we found that Interrupting sitting resulted in a 32% lower insulin Area Under the Curve (AUC; P < .001), 17% lower C-peptide AUC (P < .001), and 7% lower glucose AUC (P = .018) vs continuous sitting. We have recently found similar decreases in insulin and c-peptide response to glucose challenges in children with overweight and obesity. Interrupting sedentary time with brief moderate-intensity walking thus improved short-term metabolic function. A new trial beginning in 2017 will test if these acute improvements are sustained over 1 week. These studies may help lead to community studies examining if interrupting sedentary behavior is a promising prevention strategy for reducing cardiometabolic risk in children. Investigations concentrating on binge eating behaviors in children suggest that such behaviors are also associated with adiposity in children and predict future weight gain in children at-risk for overweight. The ability to consume large quantities of palatable foods, especially when coupled with decreased subsequent satiety, may play a role in the greater weight gain found in binge eating children. Two protocols have examined efficacy of interpersonal therapy (IPT) as a weight gain preventive strategy among children and adolescents who report binge eating behaviors (11-13). IPT was not superior to health education (HE) to prevent excess weight gain at 1- or 3-year follow-up the entire cohort (11,12). However, among girls with high self-reported baseline social-adjustment problems or anxiety, IPT, compared to HE, was associated with the steepest declines in BMIz (p < .001) and fat mass (p <= .03). Thus, in obesity-prone adolescent girls, IPT was associated with improvements in BMIz over 3 years among youth with high social-adjustment problems or trait anxiety. Future studies are planned to test the efficacy of IPT for obesity prevention among at-risk girls with social-adjustment problems and/or anxiety. We also have evaluated feasibility and acceptability of a preventive family-based interpersonal psychotherapy (FB-IPT) program (13). FB-IPT was compared to family-based health education (FB-HE) in 29 children, 8 to 13 years who had overweight/obesity and LOC-eating. At post-treatment, children in FB-IPT reported greater decreases in depression (95% CI -7.23, -2.01, Cohen's d = 1.23) and anxiety (95% CI -6.08, -0.70, Cohen's d = .79) and less odds of LOC-eating (95% CI -3.93, -0.03, Cohen's d = .38) than FB-HE. Family-based approaches that address interpersonal and emotional underpinnings of LOC-eating in preadolescents with overweight/obesity show preliminary promise, particularly for reducing internalizing symptoms. Whether observed psychological benefits translate into sustained prevention of disordered-eating or excess weight gain requires further study. A new study is underway to examine if retraining attentional biases away from palatable foods can help children avoid weight gain. Given the rapid increase in the prevalence of obesity, the development of treatments for obesity is urgently needed (16-18). In clinical protocols, we have studied pharmacotherapeutic approaches to the control of body weight (19-21). Metformin 1000 mg BID was studied in 100 severely overweight children (6-12y) who manifested hyperinsulinemia and insulin resistance. We concluded that metformin, added to a monthly behavioral program, significantly improved weight loss, insulin resistance, and cholesterol over a 6-month interval in severely overweight, insulin-resistant children. We recently reported how the pharmacokinetics of metformin is affected by polymorphisms in genes controlling metformin metabolism (21). We also participated in a multi-site randomized-controlled trial of beloranib, a methionyl aminopeptidase 2 inhibitor, to treat the hyperphagia of patients with the Prader Willi Syndrome (19). We have recently initiated additional translational trials related to modulation of the leptin signaling pathway using a melanocortin agonist called setmelanotide. Finally, we have initiated studies to examine the hypothesis that administration of colchicine can decrease NLRP3-activated inflammation and improve obesity-related metabolic dysregulation (20).

Camptothecin displays unprecedented antitumor activities against human colon cancer. To date its full therapeutic utility has been limited by poor water-solubility and the aqueous instability of the lactone ring moiety. Ring opening is rapid, resulting in a complete loss of biological activity. Int his communication, we demonstrate that liposome-bound camptothecin is stable, thus suggesting that liposomes may serve as useful drug delivery systems for solubilizing camptothecin and conserving both its lactone ring and antitumor activity. In order to study the equilibrium associations of camptothecin with lipid bilayers, we exploited the drug's intense intrinsic fluorescence. Fluorescence is associated with the extended conjugation of the quinoline ring system. Upon association with small unilamellar vesicles (SUVs) composed of L-`-dimyristoyl phosphatidylglycerol (DMPG), the lambda_max value of camptothecin's emission spectrum shifts to lower wavelength, or blue shifts, some 16 nm.

MITOCHONDRIAL DYSFUNCTION is an important component of many of the pathologies associated with aging, such as type 2 diabetes mellitus, Alzheimer's disease, Parkinson's disease, and some cancers. Indeed, mitochondria have been implicated overall in the aging process, although the mechanisms are not fully understood. One of the most widely accepted theories of aging, the oxidative stress theory, suggests that the aging process involves the accumulation of oxidative damage to mitochondria and other cellular components. Oxidative damage is induced by reactive oxygen species (ROS), produced primarily as a by-product of mitochondrial oxidative phosphorylation. As mitochondrial ROS can cause damage to mitochondrial DNA, proteins, and membrane lipids, a self-perpetuating and destructive cycle can ensue in which increased ROS production leads to incremental damage and further ROS production. Calorie restriction (CR), without malnutrition, is a well-known dietary intervention that consistently increases life span by delaying the aging process in a wide variety of animal species. The mechanisms underlying aging retardation by CR are poorly understood. However, it has been suggested that they may involve a decrease in cellular oxygen consumption and ROS production. Based on solid preliminary data in both pre-clinical rodent models and in humans participating in a 6 month study of CR (phase 1 of this U01), we propose the hypotheses that 2 years of 25% CR will increase mitochondrial biogenesis and increase coupling in vivo;that these changes will be associated with a reduction in ex vivo mitochondrial ROS production and accumulated mitochondrial ROS damage. To test this hypothesis, we will conduct an ancillary study to the multi-center CALERIE phase 2 U01 at the Pennington Biomedical Research Center. This multi-disciplinary project will use newly developed state-of-the- art in vivo measurement of skeletal mitochondrial capacity / resting ATP synthesis [31P MR spectroscopy] combined with in vivo measurement of O2 uptake [deconvolution of high resolution optical spectrometry] to measure mitochondrial coupling during a two year 25% CR. These measures will be paired with measures of mitochondrial enzyme content and mitochondrial ROS production assays performed on fresh skeletal muscle biopsies. Importantly, we will measure mitochondrial ROS production and ROS damage to fully test the hypotheses at hand;namely that CR will increase coupling, reduce ROS production and decrease ROS damage. These totally novel studies of mitochondrial adaptations to caloric restriction will directly test the first and critical aspects of the oxidative stress theory of aging in the longest controlled study of CR in humans planned to date. PUBLIC HEALTH RELEVANCE: The energy producing cellular organelles called mitochondria produce free radicals producing oxidative stress. Over time, it is thought that this oxidative stress damages mitochondria as a major part of aging. This protocol aims to test the hypothesis that reducing energy intake in non-overweight people will make mitochondria more efficient at producing ATP, thereby reducing oxidative stress and accumulated oxidative damage.

This project will examine the beneficial effects of growth hormone and IGF-I in AIDS patients who are wasting. Ninety-five percent of this project is funded by Genetech, Inc. However, they have refused to pay for some ancillary studies which they feel are not necessary for FDA drug approval.

The San Antonio/Dallas Asthma and Allergic Diseases Cooperative Research Center represents an integrative, collaborative and innovative multidisciplinary effort to investigate the role of a unique Mycoplasma pneumonias toxin in asthma and related airway diseases. This toxin, designated Community Acquired Respiratory Distress Syndrome Toxin (CARDS TX) remarkably replicates the proinflammatory cytokine/chemokine profiles and histopathology that accompany M. pneumoniae infection. This consortium between The University of Texas Health Science Center at San Antonio and The University of Texas Southwestern Medical School in Dallas combines 4 projects, which focus on basic, clinical and animal modeling strategies, with 2 support cores (administrative and pathology) to bring a totally new approach to defining the relationship between M. pneumoniae and the pathogenesis of asthma. A substantial literature, which has accumulated over thirty-five years, connects M. pneumoniae to onset, exacerbation, and chronicity of asthma, yet no single mycoplasma virulence determinant, or mycoplasma molecule for that matter, has been shown to be a mediator of symptoms and associated pathologies. This lack of definable M. pneumoniae pathogenic factors has greatly hampered an understanding of how M. pneumoniae influences the development and progression of airway diseases. This is especially challenging in complex diseases like asthma, where genetic, immunologic, infectious and environmental variables appear to affect disease development and progression. A major focus of the AADCRC is to directly link the biochemical, molecular and immunological properties of the ADP-ribosylating, vacuolating M. pneumoniae CARDS TX (Project 4), to diagnosis and treatment of asthmatic patients (Projects 3 and 4). By so doing, we hope to demonstrate that CARDS TX is a key mediator of asthma-associated pathobiology in humans (Project 3) and in experimentally infected or intoxicated mice (Projects 1 and 2). Therefore, we intend to (a) directly connect CARDS TX to asthma pathogenesis through novel and effective CARDS TX-targeted diagnostic assessments (ELISA, immunohistochemistry, antigen capture and PCR methodologies) using patient's nasal lavage, sputum and serum samples;(b) use mouse models of M. pneumoniae infection and CARDS TX intoxication to examine both acute and chronic stages of asthma and therapeutic interventions as well as the impact of CARDS TX on airway hyper-reactivity;and (c) further characterize ADP-ribosylating activities of CARDS TX and develop effective and rapid diagnostics to assist in the treatment and control of asthma and related pathologies. The key investigators of each project and core have strong track records and expertise in asthma, airway-related pathologies, immunopathogenesis and M. pneumoniae biology and virulence as well as a history of collaboration and co-publication.

Multiple Myeloma (MM) is a disease of plasma cells with specific localization in the bone marrow. Recent studies in several malignancies including MM have shown intraclonal architectural heterogeneity at diagnosis and at different stages of disease progression over time. The presence of clonal tides in MM represent a novel paradigm in myeloma evolutionary biology which will revolutionize the current modeling of MM tumorigenesis and progression and are likely to have profound therapeutic implications. However, the role of the supporting bone marrow niche, specifically mesenchymal stromal cells (MSCs) in the clonal evolution of MM and other malignancies has not been previously elucidated. Although many factors regulating tumor progression are tumor cell autonomous, they are insufficient to induce progression and metastasis, and a permissive microenvironment is required for frank malignancy to emerge. In this grant, we focus on MSCs as critical regulators of clonal evolution in MM that allows for more rapid dissemination and drug resistance during disease progression. Our overarching hypothesis is that MSCs are integral regulators of clonal evolution in MM inducing both tumor dissemination and drug resistance during progression. We will examine this in 3 Specific Aims. Specific Aim 1 will elucidate sequential molecular events that occur in MSCs during MM progression and explore mechanisms of cooperativity of these events with tumor clonal evolution. MM patient samples at different stages of disease progression (MGUS to MM) will be used to determine molecular changes that occur in MSCs that correlate with, or drive tumor clonal diversification. Specific Aim 2 will determine the role of MSCs in clonal evolution in MM that leads to disease progression. The hypothesis of this aim is that MSCs confer selective advantage of specific clones for tumor progression in MM. We will use in vivo tracking of clones distinguished by fluorophores, where clonal subsets can be molecularly interrogated sequentially to track the biography of cells that emerge as winner or loser MM clones in response to loss-of or gain-of-function studies of specific genes deregulated in MSCs. Specific Aim 3 will investigate the role of the MSCs in the regulation of drug resistance in MM. Our hypothesis is that specific molecular changes that occur in MSCs after high-dose chemotherapy allow outgrowth of aggressive drug-resistant subclones of MM. In this aim, we will use in vitro and in vivo model systems to determine molecular changes that occur in MSCs after high dose chemotherapy used in stem cell transplant in MM and investigate how this in turn plays a role in clonal evolution and drug resistance in MM. This grant is focused on using innovative and diverse methods to understand the role of MSCs in clonal evolution in MM. We combine patient samples with mouse models to examine, in high-throughput unbiased methods, the role of MSCs in inducing tumor growth, clonal heterogeneity and drug resistance.

PROJECT SUMMARY Diarrheal disease causes close to one million deaths in children under five each year. Although its incidence is much lower in the more affluent nations, diarrhea remains one of the two most common visits to pediatric emergency rooms and is also common among the institutionalized elderly. NHE3 is a major sodium transporter in the brush border membrane of the small intestine and proximal colon. Abnormal NHE3 expression and function are associated with diarrheal diseases resulting from acute pathogenic infection and inflammation in the gut. This application aims at understanding post-translational modification of human NHE3. The impetus of the proposed study comes from our recent finding that NHE3s of human and primates differ from NHE3s of lower mammals, including rodents and rabbits. Nedd4-2 is an E3 ubiquitin ligase that interacts with substrate proteins via PY (PPxY) motif. We found that human NHE3 (hNHE3) interacts with Nedd4-2, which ubiquitinates hNHE3 and mediates endocytosis, and the response of hNHE3 is much greater to forskolin/PKA than rabbit NHE3. Although mice have widely been used to understand the physiological role of NHE3, mice are relatively resistant to development diarrhea. We will examine the idea that ubiquitination of hNHE3 contributes to the increased severity of acute diarrhea in man. To begin, we will compare the regulation of hNHE3 and mouse NHE3 in vivo using transgenic animals. We will study how hNHE3 is endocytosed, recycled and degraded by Nedd4-2 mediated ubiquitination. Ubiquitination is counteracted by deubiqutination. We have identified several putative deubiquinating enzymes (DUBs) that interact with hNHE3. We plan to investigate how Nedd4-2 and DUBs dynamically regulated hNHE3. Our work is expected to reveal new mechanism for the control of intestinal brush border NHE3, and identify Nedd4-2 as a novel target for the therapeutic of diarrheal diseases caused by abnormal sodium and water balance in intestinal epithelium.

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. Sphingolipids are essential components of eukaryotic membranes, and many unicellular eukaryotes, including kinetoplastid protozoa, are thought to synthesize exclusively inositol phosphorylceramide (IPC). Here we characterize sphingolipids from Trypanosoma brucei, and a trypanosome sphingolipid synthase gene family (TbSLS1-4) that is orthologous to Leishmania IPC synthase.

Keratoepithelin (KE) is an extracellular matrix protein encoded by the Big-h3 gene on human chromosome 5q. It is expressed in human cornea, skin and other tissues and its expression is up-regulated with TGFb. KE promotes the adhesion and spreading of dermal fibroblasts in vitro and cell-extracellular matrix interaction in corneal development and wound healing. Recently, DNA point mutations have been identified in KE in patients with a hereditary form of comeal stromal opacities-corneal stromal dystrophies. We postulate that KE plays a universally important role in maintaining corneal optical clarity and in the wound healing of epithelial tissues in cornea and skin. In this study, we plan to create a transgenic mouse model for the human lattice and Avellino corneal dystrophies with KE mutant constructs. We will make DNA constructs, each of which contains the KE mutant cDNA that corresponds to one of the two human corneal dystrophies, lattice (R124C) and Avellino (R124H) respectively. By using cornea-preferred promoters, we will test the KE constructs first in vitro to examine the expression of the KE mutant protein. These KE mutant constructs will then be injected into mouse embryos and the genotypes of these founder pups examined to confirm the presence of the transgenes. Phenotypically, these transgenic mice will be characterized using sit-lamp biomicroscopy to detect the presence of corneal dystrophies, histologic studies of the amyloid studies for the amyloid studies for the amyloid precipitation, and immunohistochemical characterization of the KE deposits. This transgenic mouse model will be valuable in our future wound-healing experiments designed to characterize the biological function of KE, its role in maintaining corneal optical clarity and in wound healing.

Core A. Abstract. The objective of Core A is to maintain a large collection of venoms and tissues from different Conus species and a collection of purified venom peptides, synthetic peptides and their analogs. Biological material will be obtained mainly from various locations in the Philippines. While the venom will be used as a direct source of purified peptides, tissues will be used as a source of mRNA and genomic DNA for the molecular biological identification of toxins. Peptides will be identified by calcium imaging assay (Project I), testing on heterologously expressed channels and receptors (Projects II and III), and by molecular biological identification of peptide-encoding nucleic acid sequences. Venom peptides will be purified by biochemical methods. From knowledge of the evolutionary relationship among the Conus species we will systematically analyze the venoms for specific ligands. Genetic markers for the cladistic analysis will be obtained by PCR amplification of genomic DNA using specific primers. Complete toxin (pre-pro-mature toxin) sequences will be determined by identification of corresponding cDNAs from cDNA libraries constructed from venom-duct mRNA. PCR amplification of cDNA libraries with family-specific primers will be used to determine the repertoire of expressed toxins. We will use Next Generation sequencing methods to obtain the complete list of toxin sequences and other proteins, expressed in the venom duct. This will enable us to design primers to identify related toxins in other cone snails. We will also implement a conotoxin discovery pipeline combining proteomics and next-gen sequencing by direct analysis of venoms and sequencing the venom-duct transcriptome. Molecular biological methods offer a means of identification of potential ligands (peptides) from very limited amount of biological tissue. The core with extensive molecular biological experience will assist in the detection and quantification of receptors and ion channels expressed in dissociated neurons initially using real-time PCR methods. The peptides isolated by the core are used by all the projects. The equipment requested, is for the production and isolation of the peptides. The real time PCR instrument will be used by the different projects to quantify receptor/channel expression (see equipment budget justification for details). The core will continue to provide peptide and venom samples to the scientific community.

This randomized, controlled, open-label multi-center trial is designed to compare the intracellular delay in onset of suppression of HIV-1 viral replication within the first 72 hours of initiation of antiretroviral therapy using indinavir, or nevirapine, or combination zidovudine(ZDV)/3TC. All subjects receive four-drug combination therapy from 72 hours forward. Additionally, the trial evaluates whether (GM-CSF and IL-12) cytokines which stimulate the proliferation and activation of monocytes and macrophages, when added to antiretroviral therapy, can hasten the clearance of HIV from these cells. The study also measures several aspects of T cell recovery. Twenty-four HIV-1 infected subjects will be enrolled in total; 8 will be enrolled at the Rockefeller GCRC. All will be nanve to prior antiretroviral therapy, and will have been HIV-1 infected for at least 6 months. The study duration is 72 weeks. If preliminary results suggest a significant increase in the clearance rate of HIV-1 and the sponsor (ACTG), the FDA and IRB approve, the study may be amended to extend immune modulation.

This proposal is designed to provide information concerning the development of bacterial resistance to antibiotics that are frequently used as adjuncts in the treatment of destructive periodontal disease and to elicit the molecular mechanisms involved. Patients have been encountered whose disease condition does not respond to conventional therapy combined with the adjunctive use of tetracycline antibiotics. The microbiotas associated with sites undergoing active destruction were initially susceptible to tetracyclines prior to therapy but demonstrated significant resistance following therapy. The post-therapy microflora closely resembles that associated with refractory periodontitis. The predominant cultivable bacteria present before and after therapy have been characterized and identified. Minimal inhibitory concentrations (MICs) will be determined by agar dilution technique for these bacteria to determine if significant differences exist within species present both before and after therapy. Mechanisms of bacterial resistance which may account for the failure of a patient to favorably respond to antimicrobial therapy will be investigated both functionally and at the genetic level. B-lactamase enzymes produced by periodontal strains of Bacteroides intermedius and Eikenella corrodens will be characterized to determine if differences exist in the enzymes produced within the species and if such enzymes are chromosomal or plasmid mediated. The in vivo effect of bacterial B-lactamases on B-lactam antibiotics in gingival crevicular fluid will be determined. Gingival fluid concentrations of amoxicillin, a B-lactamase susceptible penicillin, and Augmentin, which contains amoxicillin and a B-lactamase blocker, will be individually monitored over time in sites with and without B-lactamase activity. Comparisons will be made of the antibiotic levels maintained to determine the in vivo effect of B-lactamase on these antibiotics. Plasmid carriage, an important mechanism which often accounts for acquired antibiotic resistance in bacteria, will be investigated in strains of B. intermedius and E. corrodens to determine if plasmids encode for resistance in these organisms. Experiments will be conducted t determine if these plasmids encode for single or multiple antibiotic resistance by curing the strain of its plasmid(s) and determining the effect of the loss of plasmid carriage on susceptibilities to specific antibiotics. Confirmation of plasmid-encoded resistance will be performed by transferring the plasmid into a susceptible donor either by conjugation or electroporation. Plasmid transmission will be investigated to determine if antibiotic resistance may be transferred between strains of a species as well as between species. Plasmid transfer will be tested by conjugation using a membrane filter mating technique. DNA probes will be constructed of the portion of the plasmid or chromosomal sequence which encodes for resistance to an antibiotic. These probes will be used to determine if similar DNA sequences are present in strains within the same species and in different species.

This proposal is a renewal of our EDRN Biomarker Reference Laboratory (BRL) which is located within the Clinical Chemistry Division (CCD) of the Johns Hopkins Medical Laboratories (JHML) at the Johns Hopkins Hospital. The BRL will continue to serve as a network resource for clinical and laboratory validation of biomarkers, which includes technological development and assay refinement. The JHML are certified by the College of American Pathologist (CAP) and regulated by CLIA (Clinical Laboratory Improvement Amendments of 1988). As such, the JHML follow stringent, good laboratory practice (GLP) guidelines for quality control and quality assurance. The proposed product development will be conducted at the Center for Biomarker Discovery and Translation (CBDT) at The Johns Hopkins University. The project goal is to validate serum proteomic biomarkers, including two biomarkers discovered in our current EDRN BRL developmental study, for the prediction of aggressive prostate cancer in men prior to prostate biopsy. The product AGPC is an in vitro diagnostic multivariate index assay (IVDMIA) that combines a panel of biomarkers into a single-valued numerical index score. We have assembled a strong team of research and clinical scientists with many years of experience with cancer biomarkers and in technology development, study design, bioinformatics, validation, and translation. Dr. Chan, the PI, is the Director of both CCD and CBDT. He has over 30 years of experience in clinical chemistry and has conducted many (> 75) research studies funded by industry on cancer diagnostics leading to the approval by the FDA. Since the inception of the EDRN in 2000, five EDRN developed assays have been approved by the FDA for clinical use. Of these five, our BRL led the development of serum OVA1 for ovarian cancer, serum proPSA (phi) for prostate cancer, and served as the reference lab for the urine PCA3 study for prostate cancer. In fact, OVA1 which is based on our original multiplex proteomic study published in Cancer Research, was the first proteomic IVDMIA ever cleared by the FDA for clinical use. In addition, three leading diagnostics/biotechnology companies have agreed to collaborate with us in our product development proposal. In this renewal, we plan to continue our strong commitment to the EDRN mission through network collaborations, and provide leadership in biomarker validation, technology/assay improvement, and product development. With this multi-disciplinary team of scientists, the BRL at Johns Hopkins offers the best opportunity for the success of cancer biomarker validation and translation into the clinic for the early detection of cancer.

An integrated series of structural studies on the frog, xenopus and bovine retinal rod outer segment (ROS) is proposed. Studies include 1) determination of the z-projection of frog rhodopsin from 2-dimensional sheets (space group P22121; unit cell 47 A x 151 A), using glucose and auro-thio-glucose; 2) determination of factors controlling hexagonal and lamellar phases of ROS lipids; and 3) determination of potential form components contributing to the birefringence banding in Xenopus photoreceptors.

Ataxin-1 (Atx1) is a neurodegenerative disorder protein whose expanded glutamine-repeat form causes spinocerebellar ataxia type 1 (SCA1) in humans and which exerts cytotoxicity in mice and in Drosophila. Although the linkage between Atx1 and SCA1 has been established for more than a decade, the exact properties of Atx1 and their connection to the pathogenesis of SCA1 remain unclear. It was long thought that the expanded glutamine-repeat in Atx1 is the main culprit that brings about toxic effects, yet growing evidence indicates that the protein's inherent properties also play an important role in SCA1 pathology. In our research, we recently established that Atx1 binds directly to a class of conserved transcriptional co- repressors, including vertebrate SMRT (Silencing Mediator of Retinoid and Thyroid hormone receptors) and its Drosophila cognate SMRTER, both in vitro and in vivo. Consequently, we hypothesize that one key aspect of AtxVs properties involves transcriptional regulation, and that perturbation of conserved SMRT/SMRTER-dependent regulatory pathways is partly responsible for its cytotoxicity in both vertebrates and invertebrates. Because both SMRT and SMRTER are nuclear receptor co-repressors, this proposal probes whether Atx1-mediated cytotoxicity involves SMRT/SMRTER and their associating nuclear receptors. In Specific Aim 1, we propose various molecular biology approaches, including the chromatin immunoprecipitation method, to investigate whether Atx1, by means of its interaction with SMRT, is involved in thyroid hormone receptor (TR) signaling in vertebrates. In Specific Aim 2, we will use genetic approaches to elucidate the relationships among Atx1, SMRTER, and ecdysone receptor (the functional equivalent of TR) in Drosophila. Finally, in Specific Aim 3, building on our recent discovery of two functional domains of Atx1, the SMRT/SMRTER interacting AXH (Atx1 and HMG-box protein) domain and a self-association NBA (N-terminal region of Boat and Atx1) domain, we will investigate the specific consequences that these two domains may have for Atxl's cytotoxicity. Connecting the functions of Atx1 to nuclear hormone receptor signaling will have major implications, since it suggests that intervention with hormones or with chemical compounds that affect the operation of nuclear hormone receptors can be exploited to develop therapeutic agents for treating SCA1. [unreadable] [unreadable] [unreadable]

Introduction. The mechanism of fluid transport across epithelia is not yet known with certainty. It is most frequently explained in terms of local osmosis. However, we have generated recent evidence suggesting that corneal endothelial fluid transport is instead due to electro-osmosis through the paracellular junctions. Hence, our specific aims are: 1) To investigate whether endothelial fluid transport behaves as a local osmotic or electro-osmotic process. Distinctive criteria for electro-osmosis include 1-1) presence of current-induced fluid movements; 1-2) much faster response of flow to imposed electrical currents, 1-3) dependence of the fluid movement/electrical current coupling ratio on junctional electrical charges, 1-4) dependence of fluid movement on ambient osmolarity, 1-5) dependence of fluid movement on intercellular junctional integrity, 1-6) presence of paracellular (rather than transcellular) fluid movements associated with fluid transport. Techniques include determinations of fluid transport by specular microscopy and volume clamp at high time resolution (~1 s), theoretical estimates of transendothelial osmotic flows driven by solute buildups, determinations of fluxes of paracellular markers, and determinations of the coupling ratio after junctional modification by agents including cadmium chloride, protamine, and polylysine. 2) To confirm and characterize novel routes for trans-endothelial membrane movements of ions which would contribute to local osmotic or electro-osmotic fluid transport. We propose to use fluorescent dyes to monitor intracellular electrolytes and intracellular potential of cultured cells, and radiolabeled Na+ to determine the sidedness of Na+ entry via phenamil-inhibitable pathways. Using immunocytochemistry and molecular biology, we will seek to pinpoint which cell membranes express electrogenic transport components consistent with electro-osmotic fluid movement. Through the use of genetically modified mice and cultured cells with transiently modified expression, we propose to determine to which extent fluid transport and osmotic permeability across cultured bovine corneal endothelial cells depend on functional expression of aquaporin 1, sodium bicarbonate cotransporter(s), and epithelial sodium channels. 3) To develop a model for fluid and electrolyte transport across the endothelial layer, and a theory for electro-osmotic coupling across the endothelial junctions. Clarification of local osmotic flows may require a time-dependent extension of the model. We propose to utilize Brinkman's ideas to describe electro-osmosis in narrow channels filled with a charged gel matrix, rather that in the conduits covered by the classical Helmholtz-Smoluchowski treatment. The understanding to be gained may provide insights on how to modulate fluid transport so as to offset endothelial dysfunction leading to the loss of normal vision.

SUMMARY Atherosclerotic plaque regression as a result of lipid-lowering treatment has been limited at best, with coronary artery disease (CAD) related event rates remaining unacceptably high. In this application, we propose that targeted immunomodulation of macrophages will resolve plaque inflammation and beneficially impacts myocardial infarction-induced monocytosis? detrimental effects. To that aim, we have developed novel ?nanobiologic?, a bioengineered version of our body?s own high density lipoprotein (HDL) nanoparticle that specifically targets (plaque) myeloid cells6,7. The HDL nanobiologic contains a small molecule inhibitor (TRAF6i), directed against the binding domain of CD40 on TRAF6, to specifically block CD40-TRAF6 interactions. Based on these preliminary data in Apoe?/? mice and nonhuman primates, we propose ? in two independent Specific Aims ? TRAF6i-HDL?s application to (i) prevent atherosclerotic plaque aggravation due to myocardial infarction in Apoe?/? mice and (ii) induce plaque regression in a nonhuman primate atherosclerosis model. In Aim 1, we will execute a longitudinal and imaging-guided therapeutic study, involving a one-week high-dose and a four-week low-dose TRAF6i-HDL regimen in atherosclerotic Apoe?/? mice. In the same mouse model, we will apply TRAF6i-HDL nano-immunotherapy to prevent plaque aggravation after myocardial infarction, by preventing the detrimental accumulation of inflammatory monocytes in the vessel wall. In Aim 2, based on our extensive mouse efficacy data and imaging data in nonhuman primates, we will employ TRAF6i-HDL nano- immunotherapy to regress established plaques in nonhuman primates. Positron emission tomography with magnetic resonance imaging (PET/MRI) methods will serve as readouts for TRAF6i-HDL?s in vivo behavior and therapeutic efficacy. In light of the promising CANTOS trial data, demonstrating a reduction in recurrent rates in cardiovascular patients that were treated with a targeted anti-inflammatory therapy, successful completion of this application?s aims will pave the way for potential clinical translation.

DESCRIPTION: (applicant's abstract) The study of virus structure is both scientifically interesting and medically relevant. We seek to gain information on viruses by taking advantage of the inherent speed and accuracy of mass spectrometry and by using the techniques we have developed to study viral structure. While viruses can be analyzed by other spectroscopic techniques, those methods are by no means routine or complete. Since viruses are composed of multiple protein-protein subunits, viral proteolysis followed by MALDI-MS will be used to map the amino acids that are exposed on the viral surface and to provide information about the protein dynamics through comparisons with crystallographic data. In order to perform these analyses we have initiated studies on a model system, the flock house virus (FHV), which is a small RNA icosahedral animal virus that is well-characterized crystallographically. Our MALDI-MS/proteolysis approach has already been shown to be useful on less than 10 ug of FHV, whereas crystallographic data typically requires 100 mg of material. Once the approach is established we will move to viruses that have not been crystallized or fully characterized such as cucumber mosaic virus. Viral autocatalytic proteolysis events will also be examined and, through identification of protein fragments, we will investigate their role in initiating viral infectivity. The FHV also exhibits a structural feature termed the "pocket factor" that is analogous to a feature observed in human rhinoviruses. Interestingly, crystallographic studies do not reveal the structure of the organic molecule, most likely due to structural disorder or low occupancy. The existence of the "pocket factor", however, is closely associated with viral infectivity in FHV and rhinoviruses and has not yet been identified. Our goal is to investigate/characterize this factor using methods that we previously developed. In addition to characterizing viral substructure, we are also designing a mass spectrometer to perform mass analysis on whole viruses, preliminary research has allowed us to demonstrate the retention of virus ultrastructure and infectivity throughout the ESI mass analysis process. The ability to mass measure whole viruses and gain structural information on viruses could provide virologists with an accurate means of viral identification and characterization. The aim of our work is to develop new methods for understanding local and global viral structure.

Successful orthopaedic management of displaced intra-articular fractures, to forestall post-traumatic osteoarthritis (OA), depends on avoidance of a mechanical environment that is deleterious to articular cartilage. Clinically, there are many reports of cases or series where patients have done surprisingly well in the presence of substantial incongruency, provided that joint stability is maintained, whereas minimally displaced or congruously repaired intra-articular fractures often fare poorly in the presence of joint instability. To date, almost no attention has been directed to the causative mechanisms by which instability induces post-traumatic OA. Many confounding factors, especially heterogeneity of injury, preclude systematic human clinical study of the relative importance of instability versus incongruency as causes of osteoarthritis secondary to intra-articular fractures. We propose laboratory studies, to investigate mechanisms of how global joint instability manifests itself in terms of mechanical anomaly at the tissue and cellular level, where osteoarthritis metabolically originates. And, we propose to study how local incongruity and global instability interact in that regard. Three specific aims will be pursued. First, in an established cadaver model of stepoff tibial plafond fracture, we will measure transient intra-articular contact stresses under quasi-physiologic loading throughout plantar-dorsiflexion cycles, for meta(stable) versus unstable articulations. Second, using a contact finite element model of a tibial plafond fracture stepoff incongruity that incorporates a (rate-dependent) poroelastic constitutive formulation for cartilage, we will compute internal cartilage stresses for loading and motion inputs spanning the instant of transition of transition from (meta)stable to unstable articulation. Third, in an established rabbit knee defect model, we modulate instability by means of partial sectionings of the anterior cruciate ligament, and document the speed/severity of the resulting secondary degenerative changes. If dynamic instability can be shown to be a more potent determinant of post-traumatic OA following intra-articular fractures than is chronically elevated contract, this would strongly argue that orthopaedic management of these difficult injuries ought to prioritize attaining suitable thresholds of joint stability, rather than the presently dominant strategy of aggressive interventions to attain precise congruency in order to minimize contact stress elevations.

Cervical cancer is second to breast cancer as the most common form of malignancy in both incidence and mortality for women worldwide. The population-wide utilization of screening cervical cytology (Pap tests) has been associated with a dramatic decrease in morbidity and mortality from cervical cancer in the United States and in other industrialized nations. Despite this success, the cytologic diagnosis of cervical lesions is plagued by a persistent problem of low specificity for clinically significant high-grade lesions in patients with low-grade cytologic abnormalities. As a result, over four million women each year receive a cytologic diagnosis that requires further evaluation to rule out the possibility of high-grade dysplasia or cancer. In most cases, further evaluation does not identify underlying high-grade lesions in patients with low-grade cytologic abnormalities. Although HPV testing can play an important role for the triage of some patients, it is not useful for several cytologic diagnoses. Complicating the situation is that simple detection of high risk HPVs does not predict an underlying high grade lesion, since infections do not indicate clinically significant cervical lesions. The long-term goal of this project is to apply emerging technology to develop a high-throughput cell-based analysis with suitable specificity to identify high grade premalignant and malignant lesions of the cervical mucosa. The methods to be used in this project will employ, test and validate the approach of cytometry-based molecular diagnostics to detect false negative cervical specimens. Under the guise of the previous grant phase, an application of protein expression of p16INK4a and Mcm5 (cervical cancer biomarkers) with high- throughput flow cytometry and cell sorting has been used to identify and capture the rare cancerous cells in cervical specimens. Furthermore, a multiplexed HPV genotyping assay has been implemented to analyze the rare cells isolated in this approach. Importantly, the work flow has been implemented using common sample preparation with current pathology testing protocols. The technology and methodology being applied in this application will be implemented using an integrated workflow, with substantial automation, to assess feasibility of further translation to accommodate clinical need and to improve the standard of care worldwide. The ultimate goal is to establish a primary assay with potential to supplant slide based cervical cytology with greater sensitivity, less subjectivity, and less labor intensiveness. PUBLIC HEALTH RELEVANCE: This proposed project will apply new technology, for detection of abnormal cervical cells, to clinical samples from previous Pap tests. We will use automated analysis of protein content of cells to isolate abnormal cells, after which automated DNA analysis will determine whether cancer-causing human papillomavirus types are present. These results will be compared to the Pap smear and biopsy results of the same samples in order to determine how well our test can detect early stages of cervical cancer.

Ten thousand cord serum samples obtained from infants born from 1967 through 1971 are to be tested for cytomegalovirus IgM antibody by an indirect fluorescent method described by the principal investigator and his associates in 1968. Infants found to be positive will be followed for four to seven years after birth for evidence of neurological sequelae. Control infants will be selected and similarly evaluated.

T. gondii is a major cause of neurologic disease in AIDS patients. Currently very few drug regimens have been shown to be effective in treating T. gondii infection, and thus alternative agents are needed. Potential therapeutic agents are screened in standard in vitro culture assays, as well as by an assay specific for folate metabolism in T. gondii that utilizes incorporation of tritiated para-aminobenzoic acid into reduce folates. By these techniques, we are in the process of screening agents that interfere with folate metabolism, including those that will inhibit specific enzymes in the folate metabolism pathways. In collaboration with investigators at UCSF, we have identified one DHFR inhibitor with potent antitoxoplasma activity that is also selective for protozoan compared to mammalian DHFR. In animal studies, we have been able to show that this inhibitor is able to prolong survival of mice acutely infected with T. gondii, and in combination with sulfadiazine will prolong survival of acutely infected animals for the entire six week observation period. To facilitate our studies, we have also developed a recombinant DNA library using T. gondii RNA. We are in the process of screening this library with a degenerate oligonucleotide probe that is based on the dihydropteroate synthase (DHPS) of cryptococcus, pneumocystis and a variety of bacteria, to try to identify the gene coding for the DHPS of T. gondii. The goal of such studies would be to identify the genes coding of target enzymes, and to produce such enzymes in large quantities so that specific chemotherapeutic agents can be assayed in vitro prior to proceeding to some of the more cumbersome techniques outlined above. The ultimate goal of these studies is to identify agents that would be alternatives to the currently available regimens for treating T. gondii infection in immunosuppressed patients, including patients with AIDS.

NCI Workforce Analysis Communication Products for the National Cancer Institute Division of Cancer Control and Population Sciences

Dengue hemorrhagic fever (DHF) is a severe manifestation of mosquito-borne dengue virus (DV) infection that is characterized by plasma leakage. Several factors are believed to be important in the pathogenesis of DHF, including host factors (e.g. HLA, pre-existing memory B and T cell responses), viral factors and entomologic factors. The goal of this project is to better define the mechanisms of disease pathogenesis and control utilizing three distinct clinical studies in Thailand to address the following specific aims: * Aim 1 conducts a prospective study of symptomatic children in the early phase of suspected dengue infection to quantitate the DV-specific T cell responses, plasma and cellular viremia levels and markers of immune activation, as well as shifts in body fluid compartments, to better define predictors and outcome measures of disease severity. * Aim 2 focuses on the characterization of virus infection and localized cellular immune responses in skin compared to that seen in blood in a cohort of DV-infected adults with skin rash. * Aim 3 conducts a prospective, school-based study of children to determine correlations of disease severity with pre-existing memory T and B cell responses, immunogenetic factors, and serotype of the infecting virus. Case-based cluster investigations, in conjunction with Project 3, will characterize DV transmission in the home to define relationships between disease burden, illness severity, vector density and activity, and quantitative measures of virus in both host and vector. The long-term objectives of this Project are to better understand the pathophysiology of DHF for improved early diagnosis and novel therapeutics. An improved understanding of the correlations between host immune response and subsequent risk of DHF will help guide future vaccine trials in areas where dengue is endemic. The knowledge gained from these unique clinical studies will also improve our understanding of the roles human immune response genes and cross-reactive T and B cells may play in other emerging flavivirus infections such as West Nile Virus and in developing safe and effective vaccines against dengue and WNV.

In recent years monoclonal antibodies (mAbs) recognizing tumor-associated antigens have increasingly been integrated into the clinical management of neoplasia and this trend is expected to continue. Prominent targets for antibody-based therapies are ErbB family members, including the epidermal growth factor receptor (EGFR/ErbB1) itself and ErbB2. EGFR-specific antibodies currently in clinical use typically inhibit EGFR-dependent signal transduction and induce antibody-dependent cell-mediated cytotoxicity. Administration of these agents to tumor patients is, however, associated with dose-limiting adverse side effects due to inhibition of EGFR signaling in normal tissues, specifically skin and the gastrointestinal tract. It is, therefore, highly desirable to develop antibody-based therapeutics that specifically recognize their target antigen in the tumor environment but not on normal tissues. Here we propose a novel approach to achieve this goal. It is based on the construction of antibody derivatives in which antigen recognition sites are reversibly masked by antigen fragments. Association of the antigen fragment with the mAb CDRs is expected to occlude the antigen recognition site of the fusion protein and, thus, reduce normal tissue reactivity. To restore high affinity antigen- antibody interaction at tumor sites, a proteolytic cleavage site is engineered into the linker between the blocking moiety and the antibody fragment. This site is designed to be susceptible to proteases with high activity in tumor tissues (i.e. matrix metalloproteases (MMPs)) but little or no activity in normal tissues including peripheral blood under homeostatic conditions. Upon cleavage at tumor sites, the 'unmasked'antibody is predicted to partition to the tumor-associated antigen due to mass action principles. The proposed work will test biological properties of antibody derivatives based on the EGFR antagonistic mAbs C225 and 425 that recognize distinct epitopes on the extracellular domain of the human EGFR (EGFRdIII). In preliminary work dual (Ab-Ag)2 complexes were made whereby C225 and 425 scFvs were covalently linked to EGFRdIII sequences engineered to encourage dimerization and reciprocal occlusion (crossmasking) of the antigen recognition sites of each other. Upon MMP9 treatment these complexes were shown to dissociate followed by markedly enhanced binding to purified EGFR and to tumor cells expressing EGFR. These promising results form the basis for the reformatting and in vitro testing of crossmasked Abs in an IgG-like format (Specific Aim 1) and the determination of tumor homing and growth inhibition human tumor xenotransplants in mice by such constructs in vivo (Specific Aim 2). In future work, the utility of these reagents as diagnostic agents (imaging) will be further tested in appropriate animal models, i.e. human tumor xenotransplants in mice. PUBLIC HEALTH RELEVANCE: In recent years monoclonal antibodies (mAbs) have been used increasingly in the clinical management of certain forms of cancer and select other diseases. Most mAbs currently used in cancer therapy recognize tumor-associated antigens, which are also present on normal tissues. This circumstance leads to adverse side effects that limit the application and efficacy of these agents in patients. This proposal describes a novel concept to engineer antibody derivatives that overcome these limitations. To this end, we will focus on molecular modification of two EGFR antagonistic monoclonal antibodies currently in clinical use or clinical trials, i.e. C225 and 425, respectively. If successful, the concept to be tested here may be applicable to a wide range of mAbs currently in clinical use or development.

There is concern about the long-term use and abuse of benzodiazepine anxiolytic/hypnotic drugs by patients and polydrug abusers. One of the most insidious adverse effects of benzodiazepines is memory impairment. Five double-blind, placebo-controlled outpatient experiments are proposed in healthy volunteers to test specific hypotheses regarding the acute and chronic effects of benzodiazepines on memory processes, guided by recent conceptual and methodological developments in human memory research. Experiment 1 will test the hypothesis that benzodiazepines selectively impair specific processes involved in working memory (a type of short-term memory that enables the temporary maintenance and on-line manipulation of information in the service of behavioral goals, and is critical for the performance of many higher-order cognitive functions), and that the benzodiazepine-induced working memory impairment differs qualitatively from that induced by the anticholinergic drug scopolamine. Experiments 2 and 3 will test the hypothesis that benzodiazepines selectively impair specific processes involved in metamemory (people's knowledge and awareness of their own memory; a fundamental prerequisite to acquiring new information and modifying one's behavior), and that the benzodiazepine-induced metamemory impairment differs qualitatively from that induced by both scopolamine and alcohol. Experiment 4 will test the hypothesis that the memory-impairing effects of benzodiazepines can be dissociated from the drugs' sedative effects by examining changes in the benzodiazepine hypnotic triazolam's amnestic versus sedative effects as a function of administration of varying doses of the stimulant d-amphetamine, conjointly with varying doses of triazolam. Experiment 5 will test the hypothesis regarding dissociation of amnestic versus sedative effects by examining the degree to which tolerance, over the course of repeated benzodiazepine administration, develops differentially to effects on sedation vs. memory; results will also provide information of direct clinical relevance to long-term benzodiazepine users. In addition to enhancing the understanding of benzodiazepine-induced amnesia and its neurochemical specificity, data from this novel project, which uniquely combines pharmacological and cognitive manipulations, will contribute to a better understanding of the specificity of human memory processes and of the relationship between memory and arousal. Information from this project can also be used to develop cognitive rehabilitation programs and to tailor drug abuse and anxiety treatment interventions to the specific capabilities of long-term users.

The objectives of this study are to uncover the mechanisms by which aerogenic infection with facultative intracellular parasites is overcome. Syngeneic rats will be infected with either L. monocytogenes or M. tuberculosis which produce acute and chronic lung disease, respectively. Rats will be infected either aerogenically, intravenously or intratesticularly in order to compare the fate of the parasite in various organs following different routes of inoculation. The progress of infection will be monitored by means of viable counts. The mechanisms underlying the responses to infection will then be analyzed in three parts. 1. The initiation of the immune response. The transport of bacilli from the portal of entry to lymphoid organs will be traced by means of viable counts. 2. The lymphoproliferative response will be masured by incorporation of 3H-thymidine into lymphocyte DNA. Adoptive immunity will be used to quantitate the output of specifically sensitized lymphocytes. 3. The effector mechanism. The assembling of lymphocytes and macrophages into the lungs will be studied by in vivo radioactive labelling techniques. By judicious timing of the pulse of 3HT it will be possible to separate the migration of lymphocytes and monocytes into the pulmonary DTH reactions elicited by inhalation of tuberculin as an aerosol. Cell migration will also be studied in rats adoptively immunized with lymphocytes radioactively labelled in vitro. Finally, the influence of local conditions in the lung, such as oxygen tension, will be examined.

Human papillomaviruses (primarily HPV 16 and 18) play a central role in the development of in situ and invasive cervical cancer. Based on this observation and the well-recognized shortcomings of Pap smears, several groups have examined the use of HPV DNA testing as an adjunct to cytologic screening and found it to be cost effective. Consequently, a number of laboratories throughout the U.S. have begun offering HPV DNA tests, and in doing so, have left many clinicians and their clients with questions that currently do not have answers. Although the potential consequences of genital HPV infection are well documented, we know relatively little about the long-term implication; of a single or repeatedly positive type-specific HPV DNA test result. Furthermore, it is likely that HPV vaccines will introduced to prevent cervical cancer and, perhaps, genital warts in the future. However, knowledge of the infectivity and natural history of specific HPV types is essential for evaluating the impact and feasibility of vaccines. Much of this required knowledge is currently lacking. Since 1991, we have been studying the short term natural history of HPV in a cohort study of 600 freshman women and are now in a position to build on and extend these studies to gain an understanding of the longer-term natural history of genital HPV infection and of male to female transmission rates. Our specific aims are to l) define the natural history of genital HPV infection over ten years with respect to persistent detection of HPV DNA, SIL, genital warts, and HPV type-specific antibodies, 2) determine the prevalence, seroprevalence, and behavioral predictors of genital HPV infection among a random sample of male undergraduate students, and 3) estimate per partner and per act transmission rates of specific HPV types and define characteristics of partnerships (e.g., courtship behavior, condom use, and frequency of intercourse) and of partners (e.g., age, race, occupation, number of partners, circumcision status, and report of concurrent relationships) that affect transmission. The proposed study is likely to provide important information relevant to the development of effective HPV prevention strategies, including the synthesis of accurate and informative public health messages concerning the transmissibility of HPV and the meaning of a single or repeatedly positive HPV DNA test. Additionally, these data will provide investigators working on HPV vaccine development with information that is needed to guide the selection of appropriate target populations, outcome measures, and immunization strategies.

Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) is epidemic in the United States and other countries and poses a significant health and economic burden. As CA-MRSA is now the predominant MRSA clone in the community and in many healthcare settings, clinicians and infection control practitioners face new challenges. The community is now an important and expanding reservoir for the spread of virulent CA-MRSA strains into hospitals, likely increasing the severity of nosocomial MRSA infections. Existing infection prevention strategies are based on risk factors associated with the transmission of traditional healthcare-associated MRSA strains. To effectively mitigate the spread of contemporary CA- MRSA strains in both healthcare and community settings, we must first understand the transmission dynamics of this epidemic clone, identify factors associated with acquisition and infection, and determine the significance of environmental surface contamination in the spread of the organism. The clustering of CA-MRSA infections in households makes this a logical setting in which to study CA- MRSA transmission dynamics. Our prior studies revealed a high prevalence of MRSA colonization in household contacts of children with MRSA disease and demonstrated that decolonization of all household members resulted in a decreased incidence of skin and soft tissue infection (SSTI) in index patients and household contacts compared to decolonization of the index patient alone. Pediatric patients with CA-MRSA infection and their household members will be recruited to participate in a one-year prospective natural history study of CA-MRSA household transmission dynamics. Commonly handled household objects and surfaces, and pet dogs and cats, will be cultured. Serial colonization status and infection incidence in participants will be ascertained over 12 months. Molecular typing will be performed on all recovered MRSA isolates to illuminate the directionality of transmission and to determine whether infecting strains resemble endogenous colonizing strains, strains recovered from household contacts, or strains recovered from environmental surfaces. Using mixed effects logistic regression modeling, the relationships among risk factors at multiple levels (including household colonization pressure, host behavioral practices, and contamination of household surfaces and pets) will be elucidated to identify factors conferring the greatest risks for CA-MRSA colonization, infection, and transmission. Subsequently, study participants will be randomized into an intervention study to compare the effectiveness of decolonization measures (application of nasal mupirocin and dilute bleach water baths) in preventing recurrent SSTI when these measures are performed by all household members compared to decolonizing only household members with a history of SSTI in the prior year (as determined during the natural history study). Colonization status and infection incidence will be followed for an additional 12 months. These studies will inform optimized interventions to reduce the burden of CA-MRSA colonization and disease.

Neural reflexes rapidly adjust the cardiovascular system yet little is established about mechanisms governing their central nervous system pathways. The initial neurons are located within nucleus tractus solitarius (NTS). NTS integrates primary afferent information with CNS inputs. Our Research Plan will provide a cellular understanding of NTS integration and autonomic control. Heterogeneity in neuronal phenotype and pathway may reflect inherent patterns of specificity and, ultimately, represent therapeutic opportunities. Our Research Plan will address questions regarding heterogeneity of cellular properties as well as pathways of network organization within and beyond NTS. Our approach capitalizes on knowledge of cranial sensory neurons to probe the identity of NTS neurons. Our major long-term goal tests the hypothesis that sensory synapses within NTS are sites of major transformation of information. Our work features a cellular electrophysiological approach to NTS utilizing unique in vitro preparations of brain stem slices and dissociated cells. This renewal proposal focuses on synaptic processing (pre- and postsynaptic) as well as defining particular pathways and subsets of NTS neurons. The combination of molecular/cellular phenotype together with pathway information will allow us to define the roles of particular neurons within pathways. In brain stem slices and dissociated neurons with intact native boutons, we use live cell imaging, electrophysiology including paired recordings, plus retro- and anterograde labeling. We will target key areas in NTS sensory processing: presynaptic mechanisms regulating transmitter release, transmitter interactions, and potassium channels. Our Aims focus on GABAb receptors at 2nd order NTS neurons, regulation of GABAergic NTS neurons, cell-cell coupling, mechanisms of vasopressin action, and PVN and CVLM-projecting neurons. These Aims will provide new and direct information about NTS that leads to a better understanding of the normal basis of the neural control of the circulation as pathophysiology.

The Minority Biomedical Research Support Program at SUNY/Old Westbury is designed to provide undergraduate minority students with experience in conducting meaningful contemporary biomedical research. The ultimate aim of the program is the encouragement of minority students to pursue advanced study and research careers in the biomedical sciences. The vehicle which provides this encouragement is involvement in well-conceived and well-designed biomedical experimentation in research areas of wide current interest. The program at Old Westbury currently includes nine subprojects in chemistry, biology, and psychology. These projects provide students with a broad range of disciplines to choose from and thus provide opportunities for students with widely varying interests.

This project is a joint collaborative endeavor on the part of NIDR and of the Division of Computer Research and Technology, NIH. It is a laboratory automation project which is involved in the concurrent on-line acquisition and real time processing of data from a number of analytical instruments and experiments. The project also involves the control of bacteria growth experiments. The computer complex (Honeywell H-316 and H-516) operates under the OLERT multi- programming operating system. The system is interfaced with an amino acid analyzer, liquid scintillation counters, a spectrophotometer, a gas chromatograph, X-ray diffractometer, a fermenter-pumping system and neurophysiology microelectrode experiments. Telecommunication with the NIH center IBM 370 and PDP-10 computers has been established. Extensions this past year have included a neurophysiology laboratory for behavioral studies and a second spectrophotometer. Projected additions include another spectrophotometer, a third neurophysiology laboratory and additional scintillation counters.

The Na-K-Cl cotransporter is a plasma membrane protein that plays a vital role in cellular and systemic electrolyte homeostasis. In non-polarized cells it is involved in regulation of cell volume and possibly of local extracellular potassium concentration; in transporting epithelia, it is a key element in balancing the transcellular flow C1. In secretory epithelia, the cotransporter functions in concert with C1 channels (CFTR), the Na pump, and K channels to bring about regulated salt movement; in the mammalian kidney another isoform of the transporter mediates salt absorption and is the site of action of the loop diuretic drugs furosemide and bumetanide. Previous work from this laboratory has demonstrated that the Na-K-C1 cotransporter is a glycosylated membrane protein 150 to 195 kDa in size, depending on tissue and species. The goal of this project is to understand the molecular mechanism of the cotransporter including the structural and functional features underlying ion translocation and its regulation, as well as the role of the transporter in various tissues. The proposed studies will be carried out using mammalian cell lines with protein expressed from cDNA's encoding the secretory form of the Na-K-C1 cotransporter, as well as with membranes isolated from a salt-secreting gland of the shark, and with rabbit tissues. The experiments will be greatly aided by probes and technique that are available from the recent work of this laboratory. The Specific Aims of the project are: 1) The membrane topology of the Na- K-C1 cotransport protein will be determined by identifying the sequence location and membrane sidedness of proteolytic cleavage sites, antibody epitopes, glycosylated residue(s), and specific regions labeled by the insertion of epitope tags. 2) Structure/function relationships in ion translocation and bumetanide binding will be determined by a combined approach utilizing photoaffinity labeling and mutagenesis strategies; the hypothesis that transport is mediated by a single polypeptide will also be tested. 3) The mechanism by which the Na-K-C1 cotransporter is activated by intracellular stimuli will be addressed, with particular attention to a role of intracellular [C1] in modulation of the level of protein phosphorylation. Phosphorylated residues of the cotransporter will be located, and the kinase involved will be identified; these studies will also address the question as to whether intracellular ions affect phosphorylation status by binding to the kinase, to a phosphatase, to a modulatory site on the transporter, or to the transport sites. 4) The role of the Na-K-C1 cotransporter in gastrointestinal secretory epithelia, vascular endothelium, nerve, and muscle, will be examined using cDNA and antibody probes to determine cellular distribution of cotransporter isoforms.

PAR Leadership Training Foundation proposes to prepare for broadcast on public television an innovative series of thirteen videotaped workshop programs, for parents and others who work with young children, which will demonstrate the communication and learning values inherent in play, making it possible for adults of all levels of income, education and resourcefulness to experience constructive confidence in handling their key role in the child's early development process. Workshop concepts and format will be based on the experimental methods and materials and the practical experience of Parents As Resource (PAR), a training organization founded in 1968 by three teachers and a social worker concerned about communicating an understanding of the use of creative play to strengthen and enrich adult-child relationships. PAR materials and procedures have developed in the course of some fifty parent training workshops with Headstart parents, daycare mothers and other community groups. PAR Leadership Training is based on the premise that the parent is the most important person, the prime resource in the child's emotional, intellectual and social development, especially during the crucial first six years of life; and the conviction that parents need to be helped to recognize this key role and to be encouraged and supported in its enhancement. To these ends, PAR proposes to offer many more people throughout the country insights, information, concrete ideas, and assistance concerning positive shared experiences between the child and adult through the effective mass communications medium of television, working with child care agencies to assure viewer group participation and maximum utilization of the workshop program series at the individual and community level.

Circadian rhythms are present in species throughout the animal kingdom. In humans, disorders of circadian timing contribute to circadian-based sleep disorders, maladjustment of shift workers and during jet lag, and may contribute to neuropsychiatric disorders including depression and seasonal affective disorder. A transcriptional-translational feedback loop is at the center of the circadian clock mechanism. The known positive elements driving circadian transcription are CLOCK and BMAL1, two basic helix loop helix proteins that dimerize to activate expression of responsive genes. We have recently generated mice with a null mutation of the Clock gene. Unexpectedly, these CLOCK-deficient mice retain circadian rhythmicity in behavior in constant conditions. Our studies will characterize physiological and molecular rhythms in CLOCK-deficient mice, and assess mechanisms of rhythmicity in the absence of CLOCK. In mice with the previously described dominant negative mutation of Clock, the CLOCK-delta19 protein likely disrupts circadian rhythmicity by interfering with the activity of other key bHLH-PAS proteins, indicating that a major circadian transcriptional activator remains to be identified. A major objective of this project will be to identify this apparent second mechanism for transcriptional activation. We will test the hypothesis that NPAS2, a bHLH-PAS transcription factor closely related to CLOCK, can substitute for CLOCK and thus maintain rhythmicity in CLOCK-deficient mice. We will also determine whether BMAL1 is necessary for rhythmicity in the absence of CLOCK, expecting a finding that will enable studies based on assessment of the functional importance of candidate BMAL1-interacting proteins. The proposed studies are necessary to understand the function of CLOCK, a central component of the circadian clock mechanism, and thus are relevant to understanding and possibly developing novel treatments for circadian-based sleep and psychiatric disorders. In addition, the circadian clock plays diverse roles in regulating reproduction, metabolism, cell growth and tumor progression, so the importance of understanding basic mechanisms of circadian rhythm generation has many implications. [unreadable] [unreadable] [unreadable]

The purpose of this revised application is to expand and continue the research begun in the previous funding period, Treatment of Nicotine Dependence in an HMO Setting, in which 1,524 individuals enrolled in a health care system received treatment with bupropion SR and behavioral counseling and were then followed for 12 months after their target quit date to determine smoking status. The results of this study demonstrate that: (1) an effectiveness trial can be conducted in a large health care system; (2) 150 and 300 mg doses of bupropion SR are equally effective for smoking cessation at 12 month follow-up; (3) proactive telephonic based (PTB) treatment is more effective than a tailored mailing (TM) program; (4) female gender, lower education, and higher levels of depression are independent risk factors for post-treatment smoking; and (5) there is substantial heterogeneity in treatment outcome among women and men at one year follow-up. In addition, cost-effectiveness analyses showed that 150 mg bupropion SR is more cost effective than the 300 mg dose from both the payer (i.e., employer) and societal perspectives. The next generation of this research program will evaluate the effectiveness of three different intervention approaches in combination with 150 mg bupropion SR: a proactive telephone-based (PTB) tobacco cessation program, a web-based (WB) tobacco cessation program, and a combined PTB+WB program. Participants (n=1,200) will be randomized to one of the three behavioral treatment conditions, receive an eight week course of 150 mg bupropion SR, and then followed at various points throughout a 12-month period to determine indices of medication adherence, treatment utilization, point-prevalent smoking outcomes and continuous nonsmoking. The specific aims of this project are: (1) To determine the relative effectiveness of each version of the behavioral treatment in combination with 150 mg bupropion SR; (2) To determine group differences in adherence to both the bupropion SR and behavioral conditions; (3) To examine heterogeneity in responsiveness to the three treatment conditions with Classification and Regression Tree (CART) Analysis; (4) To characterize process variables related to recruitment, implementation, barriers to treatment, exposure to intervention, satisfaction with treatment, treatment contamination, and program maintenance; (5) To determine the cost-effectiveness of the different delivery modes offered in the proposed trial; and (6) To disseminate the results of the proposed trial to the adult GHC consumers (approximately 500,000), providers, and other key stakeholders in GHC's integrated care model (n=1,400).

PROJECT SUMMARY Background: Effective scalable water, sanitation, and hygiene (WASH) interventions are urgently needed to reduce pediatric diarrheal disease globally. Phone based reminders is an emerging low cost approach shown to lead to improved disease prevention practices. Objective: The purpose of this study is to design, implement, and evaluate a WASH mobile health (mHealth) intervention that can be delivered nationally as a mass phone messaging campaign in Bangladesh to increase handwashing with soap and water treatment practices. Previous studies: Our research group recently developed the Cholera-Hospital-Based-Intervention-for-7-Days (CHoBI7, picture in Bangla), a WASH intervention which promotes handwashing with soap and water treatment to diarrhea patients and their household members in a health facility setting. Our recent randomized controlled trial (RCT) in Bangladesh of CHoBI7 demonstrated this intervention was effective in significantly reducing cholera, and led to sustained handwashing with soap and improved drinking water quality. Building on this work, we are currently conducting a RCT evaluating the impact of delivering mHealth messages to diarrhea patient households as a low cost approach to reinforce the CHoBI7 intervention delivered in a health facility setting. Design and Setting: The Director of Disease Control at the Bangladesh Ministry of Health and Family Welfare is interested in taking our WASH mHealth intervention to scale through incorporating this intervention into the National Operational Plan for Communicable Disease Control. To build evidence for scalable approaches to deliver this intervention our study will be divided into three phases: (1) Formative research and planning; (2) Intervention implementation and evaluation; and (3) Dissemination and policy planning. During the formative research and planning phase will develop an evidence and theory based WASH mHealth intervention through in-depth interviews, focus group discussions, workshops, and a pilot. During the intervention phase we will conduct a two-arm RCT to prospectively follow 300 households and 1200 participants (4 per household) to evaluate the effectiveness of the developed intervention in terms of increasing handwashing with soap, stored drinking water quality, and reducing caregiver hand contamination. The first arm will receive the WASH mHealth intervention developed for the mass phone messaging campaign and the second arm will serve as a control arm. During our dissemination phase, we will work closely with the Director of Disease Control to disseminate the study findings and inform policies for WASH. Significance: This will be the first study to develop a theory and evidence based WASH mHealth intervention that can be delivered nationally as a mass phone messaging campaign.

The purpose of this application is to request partial support of the travel expenses for invited speakers to the 42nd Gordon Research Conference on Cancer. The conference is entitled "Cancer-Genetics" and will take place August 12-17 at Salve-Regina College in Newport, Rhode Island. The Cancer Conference is the oldest of the Gordon Conferences and has played a unique role in providing a combination of topical research themes and clinical applications. The aim of the 1990 Conference is to consider the development of cancer as a genetically based disease. To this end, it has been organized around an opening lecture followed by none oral sessions and three poster sessions. The oral sessions have been arranged to provide consideration of the genetic aspects of tumor development the following order: 1) Advances in cytogenetic technology and tumor specific alterations; 2) genetics of predisposition to cancer; 3) genetics of tumor progression; 4) genetic mechanisms involved in tumor development; 5) cellular genetics of tumor suppression; 6) epigenetics and genome imprinting in tumorigenesis; 7) oncogenetics and development; 8) genetics of metastasis; and, 9) the clinical significance of genetics alterations in cancer diagnosis. We anticipate selection of about 130 scientists from universities, research institutes, industry and government for participation and each will be asked to present their work orally or in poster session format. We expect that the 1990 Gordon Research Conference on Cancer will provide a lively and pivotal forum for consideration of the genetic basis of cancer, one of the fastest moving and contemporary areas of cancer research.

Our objective is to provide a centralized, publicly available resource with comprehensive, well-annotated data and analysis tools that informs design and interpretation of environmental health studies and promotes novel insights into the etiologies of environmentally influenced diseases. Most human diseases involve interactions between genetic and environmental factors; however, the basis of these complex interactions are not well understood and limit improvements in toxicity prediction, risk assessment, research prioritization and therapeutic interventions. We developed the Comparative Toxicogenomics Database (CTD; http://ctdbase.org) to enhance understanding about environment-disease connections by providing manually curated data describing chemical-gene interactions and chemical- and gene-disease relationships from the peer-reviewed literature and integrating these data with select external data sets (e.g., pathways and biological process data) and novel data analysis tools. We propose to develop and implement a new module of manually curated data describing cross-species chemical-phenotype information and analytical capabilities that will incorporate these data into the broader biological context of CTD. These additions will significantly increase the impact of CTD and specifically aim to advance: a) understanding of environmental disease progression via pre-disease phenotypes, b) identification of potential biomarkers of exposure, c) the capacity to conduct and interpret studies across species and experimental systems, and d) development of biological networks that associate chemicals, genes, phenotypes, and diseases. This project will leverage our interdisciplinary team's expertise in toxicology, software development, curation, bioinformatics and statistics, as well as the flexible infrastructure and demonstrated value of CTD to facilitate understanding of critical environmental health issues in direct alignment with emerging research priorities.

Evidence is presented which suggests a) that the pathogenetic basis of the syndrome of absorptive hypercalciuria is a disordered control of 1,25-dihydroxyvitamin D (1,25-OH)2D production, b) that "dietary" hypercalciuria, apparently largely sodium-induced, is a common clinical syndrome, contributing to the hypercalciuria observed in as many as one-half of patients found to be hypercalciuric on their habitual diets, and c) that innate parathyroid suppressibility may be an important pathophysiological feature in patients with primary hyperparathyroidism. The specific aims of the proposal are five: 1) to dissect out the disordered control of 1,25-(OH)2D production in patients with absorptive hypercalciuria by protocols involving phosphate deprivation, parathyroid hormone (PTH) infusin, and the challenge with supraphysiological dose of 25-hydroxyvitamin D, 2) to evaluate the biochemical responses to other-than-conventional therapeutic agents in these patients (Timolol and Indomethacin), 3) to investigate the biochemical natural history of the syndrome by reevaluating patients five years after initial diagnosis, 4) to examine the basis and/or mechanism of apparent sodium-induced hypercalciuria in four carefully defined study subpopulations, and 5) to evaluate the unanticiapted suppressibility of the PTH-1,25-(OH)2D axis in patients with primary hyperparathyroidism to an increase in calcium intake in longer-term study as well as to examine and directly compare parathyroid suppressibility in vivo and in vitro in surgical candidates with this disease.