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This exploratory study aims to develop and test a generalizable process for measuring the relationship between long-term work in a particular occupation and the onset of chronic disease later in life. A secondary aim is to test the utility o that process for assessing the relationship between long-term exposure to various job characteristics and activities (e.g., handling materials and objects) and subsequent chronic disease outcomes (e.g., arthritis). The proposed project is based on a conceptual model that transcends traditional notions of the distinction between work-related and nonwork-related illness. We hypothesize that aggregated long-term work experiences over several decades in particular occupations with characteristic job requirements can potentially affect the propensity for workers to develop specific chronic conditions later in life. This will be perhaps the first sudy in the U.S using long-term longitudinal data to estimate the relative risks of chronic disease by occupational category. The study will help answer questions about whether the development of common chronic health conditions among workers 45 years and older, such as arthritis, asthma, and diabetes, may be related to long-term exposure to work in particular occupations and job activities. The proposed analytical process has broad relevancy and can potentially be applied to numerous occupations, job activities/characteristics, and chronic conditions. The study has practical relevance for the establishment of workplace-based chronic disease prevention programs and chronic disease surveillance. The proposed study uses publicly available de-identified data from the National Longitudinal Survey of Youth-1979 (NLSY79) and the Occupational Information Network (O*NET), and applies occupational codes from the U.S. Census and the Standard Occupational Classifications (SOC) coding systems. O*NET contains ratings (on a scale of 0 to 100) of 277 descriptors characterizing each of 974 occupations. We will conduct two demonstration studies to test the efficacy and utility of using these O*NET codes and ratings to estimate the relative risk of subsequent chronic disease. Study #1 has two components: a) an analysis of the association between aggregated exposure to handling and moving materials (one of the 277 O*NET job descriptors) and diagnosis of arthritis later in life (one of nine chronic conditions outcomes assessed by NLSY79) and b) a subanalysis of the data limited to arthritis outcomes initially reported in surveys years 2000 to 2010. In Study #2a, we will analyze the association between aggregated years in an occupational classification and the risk for arthritis (and potentially any of the other eight chronic conditions assessed by NLSY79: diabetes, asthma, hypertension, heart disease, non-skin cancer, chronic lung disease, emotional and psychiatric disorders, and general health limitations). Additionally, in Study #2b, we will assess arthritis outcomes in five broad occupational groupings compared to a referent group of white-collar occupations (e.g., clerical and sales). By extension, this same process could then potentially be extended to any of the other chronic disease outcomes. PUBLIC HEALTH RELEVANCE: The rising prevalence of chronic disease among people over 45 years old is perhaps the greatest health challenge facing America. Insufficient attention has been paid to investigating possible connections between long-term job history in specific occupations and the risk of eventual chronic disease. This study will greatly advance knowledge in this field, and help target appropriate interventions (e.g., workplace surveillance for chronic conditions) in high-risk occupations.

The innate immune system is a first line defense mechanism that relies on pattern recognition receptors (PRRs) to detect foreign pathogens by recognizing pathogen associated molecular patterns (PAMPs). Toll-like receptors (TLRs) comprise one family of membrane-targeted PRRs that respond to a variety of PAMPs. One of these receptors, TLR4, recognizes lipopolysaccharide (LPS) present on cell walls of gram-negative bacteria. TLR4 engagement initiates immediate, but regulated, signaling cascades leading to activation of Nuclear factor KB (NF-KB) and Interferon Regulatory Factor (IRF) proteins and induction of inflammatory cytokines, as well as interferons. Attenuated immune functions, including deregulated or chronic inflammatory states, are often coincident with increasing age. Intriguingly, aged peritoneal macrophages are sensitized to inflammatory stimuli, including LPS. However, the molecular mediators that may contribute to age-related immune disorders remain ambiguous and largely undefined. Here, we propose to employ a systems-based approach, which will integrate a series of functional genomics analyses, to identify novel molecular factors that contribute to increased LPS sensitivity in aged peritoneal macrophages. Specifically, we will optimize and execute genome-wide RNAi screen using a macrophage cell line stably transfected with an NF-KB-luciferase reporter. The cell line will be treated LPS to induce TLR4-dependent pro-inflammatory cascades, and activation of the pathway will be monitored by assessing luciferase activity. Putative hits generated from high-throughput screening will be subsequently validated, and will represent a subset of the genome affecting pro- or anti- inflammatory states in macrophages. In order to determine if genes modulating TLR4 signaling are also alternatively regulated in aging, microarray analysis will be used to generate transcriptional profiles of young and aged peritoneal macrophages, both in the absence and presence of LPS stimulation. A subset of genes which are identified to both regulate TLR4 signaling and are also differentially expressed in aged macrophages will be further characterized at a mechanistic level. These studies will enable the translation of systems-level analyses towards mechanistic and physiological understanding of macrophage response to lipopolysaccharides and age-related chronic inflammation. PUBLIC HEALTH RELEVANCE: The studies proposed here will promote global insights into the molecular bases of innate immune response, and provide novel therapeutic strategies to address age-related immune and inflammatory diseases.

This program is for predoctoral training of biological science PhD students for research careers in Cellular and Molecular Biology. This interdisciplinary program involves students and 40 faculty members from the Divisions of Biology, Chemistry, and Engineering. It is a continuation of a training program supported at Caltech for the past 31 years by NIH. Subjects of special emphasis within Cellular and Molecular Biology include genetics and genomics, regulation of gene expression, signal transduction, eukaryotic cell biology, synthetic biological circuits, biopolymers, and protein and cell structure. Interaction between the Divisions is evidenced by students who, although earning their PhD in one Division, carry out their thesis research mentored by a faculty member of another Division;by joint courses;by a less-formal interaction including research collaborations, and by interdisciplinary graduate programs in BioEngineering and in Biochemistry &Molecular Biophysics. The major components of the training activities are: 1) each student's individual research program, guided by faculty members and carried out within a group of other students and postdoctoral fellows having related interests;2) core graduate courses including courses in bioinformatics and writing;3) preparation for candidacy examinations;4) formal and informal an seminars and group meetings;5) a course in responsible conduct of research, and 6) a research seminar during which CMB students present their own research. Predoctoral trainees are admitted to graduate study in each option based on highly selective admissions criteria, especially high quantitative aptitude and strong motivation for research. Trainees will be selected from admitted students, and will be those who have a primary interest in research in Cellular and Molecular biology. Trainees are expected to pursue research careers that require training in Cellular and Molecular Biology;the superb record of our past trainees supports this expectation. Facilities are located in a complex of adjacent buildings. Multi-user facilities include cell sorting, biological imaging including cryoelectron microscopy, NMR and mass spectrometry, monoclonal antibody production, high throughput DMA sequencing, animal care and production of transgenic mice. RELEVANCE: Cellular and Molecular Biology will continue to underlie the major advances in understanding of human health and disease that can be expected in the next decades. Young researchers trained in this area will make substantial contributions to human welfare. We will help train the next generation of cell and molecular biologists, those who make fundamental, mechanistic insights using both classic and cutting edge methodology and technology, borrowing appropriately from a variety of scientific and engineering disciplines.

Sarcopenia is a major public health problem among the rapidly elderly population expanding elderly population in our society. Disabilities directly related to muscle weakness, and indirectly related to changes in body composition and metabolic dysfunctions, are causing a staggering toll in disability and health care costs. Osteopenia occurs almost simultaneously with sarcopenia in the elderly population and muscle weakness increases the risk for falls and, therefore, fractures. Although these issues have been separately addressed in several studies, an integrated investigational approach to better understand the pathogenesis of sarcopenia and other age-related metabolic abnormalities and to investigate the potential role of androgens have not been undertaken in a comprehensive manner. The program contains four independent research programs, each representing different research disciplines, and four separate cores supporting the four projects. The main focus on the project is to determine the effect of the replacement of testosterone in elderly men and DHEA in elderly men and women and to compare these effects with placebo treatment over a two-year period. Project 1, "Effect of Androgen Replacement on Muscle Metabolism" will specifically determine whether these interventions have a different effect on size and quality of muscle in terms of strength and metabolic functions. Project 2, "Effect of Androgen Replacement on Bone Metabolism," will determine the effects of this intervention on bone mineral density and markers of bone turnover. Project 3, "The Effect of Androgen Replacement on Carbohydrate Metabolism," will determine whether the age-associated decrease in circulating androgens contributes to the alterations in carbohydrate metabolism that are commonly observed in the elderly and on insulin action, insulin secretion, and glucose effectiveness. Project 4, "Effect of Androgen Replacement on Fat Metabolism" will determine whether changes in fat distribution that occur with aging could result from differences in regional fatty acid uptake and systemic fatty acid kinetics, and whether these determinants of fat distribution are altered by the interventions. The data emerging from these studies will be integrated to determine the intervention of sarcopenia with other metabolic change and hopefully will contribute to a better understanding of the relationship between sarcopenia, hormonal changes, and many associated metabolic dysfunctions of muscle, bone, carbohydrate and fat metabolism. This study will hopefully form the scientific basis for future trials of androgen replacement in the elderly.

Core C will provide all animals required in the 4 Projects. Lovelace Biomedical and Environmental Research Institute (LBERI) has outstanding animal facilities and unique experience in veterinary care. This is a critical premise for studies of physiological myocardial aging in small and large animal models. Additionally, this Core will be responsible for delivering 14C-labeled thymidine to wild-type and transgenic mice, and dogs. Importantly, animals will be maintained at LBERI until shipment to the investigators' laboratories.

For evolution to occur, there must be modifications in developmental process. However, the relationship between development and evolution is only beginning to receive experimental investigation. The studies proposed here experimentally address the nature of changes in gene expression that underly dramatic shifts in timing and cell fate determination that accompany replacement of typical larval development by direct development in a sea urchin. The use of this system allows us to test mechanisms of change in development at the level of cell lineage determination and behavior at the level of gene activity. We are able to directly examine the mechanistic underpinnings of one of the key concepts in the evolution of development, that is heterochrony, or changes in relative timing of developmental events. The experimental system is a molecular and developmental comparison of a direct developing sea urchin versus its dosest typical developing species. We have already demonstrated that homologous cell lineages can be identified, and we have observed heterochronies at the cellular and molecular levels in these species. We also have begun tracing cell lineages and cloning cell lineage-specific expressed genes. The major questions to be studied include the following: Are cell fates modified by autonomous determination or by modified patterns of induction? What are the mechanisms for the observed heterochronies in the evolution of direct development? How are gene expression programs changed in modified cell lineages? How distant are typical and direct developing species, and what are their precise phylogenetic relationships? The study of the evolution of developmental processes adds a new and potentially powerful means of understanding the underlying processes of developing systems, because closely related organisms that differ in specific developmental processes provide us with natural variants in these processes. The details of process modification by which these variants have come to differ provides a way of probing basic developmental processes and their controls.

The overall goal of this proposal is to develop appropriate rank based tests for clustered data when the cluster size is potentially informative and apply the resulting methods for various marginal comparisons (e.g., average condition of teeth before and after treatment) using existing dental database resources, specifically as obtained from the Piedmont 65 + Dental Study and Iowa Fluoride Study. Informative cluster size arises when the number of units in a cluster is non-constant/random and in correlation with the outcome of interest. In the context of dental data, all teeth belonging to an individual will form a cluster. Since tooth loss (in adult) is correlated with two of the diseases we are planning to study, namely, periodontal disease and dental caries, we have potentially informative cluster sizes in the Piedmont data sets. It is a methodological challenge to adapt a classical rank test to such situations. As for example, the two sample Wilcoxon rank sum test has difficulty maintaining the correct size/significance level under informative clustering even if it is adjusted for cluster dependence through appropriate variance estimate. This proposal has a goal of developing proper classes of rank based tests (and related R estimators) and studying their statistical properties for three classical problems adapted to marginal inference under cluster dependence with informative cluster size. These are the so called one sample location problem (Aim 1), the regression problem (Aim 2) and the association problem (Aim 3). In each of these problems, we will obtain a class of test statistics using general score functions that maintain proper asymptotic size under the informative cluster size scenario. We will also study the properties of the related R estimates of marginal parameters. Multivariate extensions of the first two problems will also be considered (Aim 4). Another signification component of the proposed research will be to extend these procedures to handle missing data where the missingness mechanism can be modeled using observable covariates (Aim 5). Finally, when the cluster size is not informative, as in the case of Iowa Study which comprises of children only, we will be able to increase the power of our tests by incorporating cluster specific weights in the construction of our test statistics (Aim 6).

This proposal is a continuation of our ongoing studies of lipid metabolism, focusing on the action of a lipid mediator: 1-0-alkyl-2- acetyl-glycerophosphocholine (platelet-activating factor, PAF). We have found, in corneal epithelium, that PAF activates the expression of the early genes c-fos and c-jun and then the expression of collagenase type 1, the metalloproteinase that degrades interstitial collagen, a major protein of the corneal stroma. A PAF antagonist blocks this effect. We will test two hypotheses: a) that PAF is involved in the mechanism of corneal remodeling and ulcer formation by acting as a lipid second messenger in the transcriptional activation of c-fos, c-jun and the collagenase l gene, and b) that lipoxygenase metabolites formed by the activation of phospholipase A2 have a modulatory effect on PAF. We propose to investigate the PAF signaling pathway and the role of lipoxygenase metabolites in the transcription of collagenase type 1. PAF antagonists and lipoxygenase inhibitors will be evaluated to determine their sites of action and to correlate their biochemical effects with the clinical evolution of corneal ulcers. Another goal of this proposal is to investigate the PAF receptors in the cornea in order to define the sites of action of PAF antagonists in the gene cascade. Powerful analytical procedures such as high performance liquid chromatography (HPLC) and gas chromatography-mass spectrometry will be used. Quantification of specific mRNAs will be accomplished by using Northern blots with storage phosphor-imaging. The results obtained will define the involvement of PAF and lipoxygenase metabolites in corneal remodeling and ulcer formation. The new mechanism of action of PAF also can be important in a number of tissue disorders in which normal control of the degradative activity of collagenase appears to be lost, e.a. rheumatoid arthritis, leading to pathological tissue destruction. The action of antagonists can be useful to define new therapeutic tools for control of collagenase type 1 activity.

Familial Mediterranean Fever (FMF) is a rheumatic disease caused by a single autosomal recessive gene. Patients with this disorder experience acute attacks of fever, arthritis, abdominal pain, and/or pleurisy; some develop amyloidosis as a long-term complication. The biochemical lesion in FMF, as well as the chromosomal location of the FMF gene, is presently unknown. This project is designed to find the chromosomal location of the FMF gene, and ultimately the gene itself. We have identified 55 Israeli families in which more than one individual suffers from FMF. Blood samples have been obtained from 347 individuals from these families, and Epstein-Barr virus-transformed lymphoblastoid cell lives have been established for each individual. Using cell line DNA from a particularly informative subset of these families, initial mapping studies have been undertaken by probing Southern blots with highly polymorphic DNA markers. To date we have excluded approximately 10% of the human genome as a potential site for the FMF gene. Large areas of exclusion have been established on chromosomes 1 and 9; smaller exclusionary area have been established on chromosomes 4 and 22. More importantly, we have found a DNA marker which shows modest evidence for linkage to the FMF gene (LOD=1.3 for Theta=0.1). From the data obtained thus far, the odds in favor of linkage to this marker are 20:1; a 1000:1 ratio is required for proof of linkage. Additional studies are in progress to determine whether this marker is in fact linked to the FMF gene.

Project 4 will focus on understanding the role that social determinants play in the link between obesity and cancer at the population level across the lifespan by developing a multi-cohort simulation model of otiosity and non-Hodgkin's lymphoma (NHL). Aim 1: Develop a multi-cohort system dynamics computer simulation model of obesity and NHL population level incidence, treatment toxicity and survival trends. This will extend the obesity modeling from childhood to adult populations, develop a population level model of NHL, and based on emerging individual level analysis from the VHA cancer registry database, integrate these two models over the life course. Aim 2: Analyze the resulting model to identify how social determinants influence obesity and NHL population level incidence and outcome trends. This analysis will identify dominant social determinants, delayed effects, and temporal relationships across the life course. Aim 3: Design guidelines along with their implementation strategies to identify the most effective way to reduce the impact of social determinants of NHL population level outcomes. This will generate different guidelines (e.g., policy, prevention, screening, treatment, and survivorship care) along with potential implementation strategies to determine the best combination of guideline-implementation strategy for reducing the burden of NHL. This project is transdisciplinary. It will complement existing and separately funded work to develop an innovative system dynamics model of childhood obesity by extending the model into adulthood across the lifespan and develop a NHL model that will be combined with the extended obesity model. Once established, additional cancers can be added to the model from this or other TRECs. This project is significant. It will not only provide rigorous conceptual models of how social determinants for obesity and NHL might interact over time, but also help identify key areas for future transdisciplinary research that have high potential for population level impact. Involvement of the expert panel in the modeling process will facilitate the development of transdisciplinary knowledge. The model will also be one of the first to link efforts from the NIH funded Cancer Intervention and Surveillance Modeling Network (CISNET) and the NIH/RWJF funded Comparative Modeling (CompMod) Network for obesity prevention.

Toxicity and carcinogenicity of laboratory animals (mice) for Hexachloro-1, 3-butadiene.

The recent Agricultural Health Study among 25,814 farm women conducted by the researchers from NIEHS, NIOSH, and NCI reported a highly significant association between any use of pesticides in farms and atopic asthma in 282 subjects. This association was strongest among women who grew up on a farm. Growing up on farms and application of pesticides were jointly associated with higher overall risk for atopic asthma in contrary to the protective effect of farm environment (as expected based on the Hygiene Hypothesis);which was in the same study demonstrated for women who grew up on farms and never applied pesticides. Another recent study showed that the production of several cytokines induced by endotoxin was suppressed in pesticide treated mice. Exposures to toll like receptor (TLR) ligands in farm environment including MAMPs (microbe-associated molecular patterns) from microorganisms and microbial products have significant influence on the suppression of allergic response. This suppressive response was implicated to the induction of regulatory T cells (Treg) by recent revisions of the Hygiene Hypothesis. Therefore we propose to explore and determine how combined airway exposures to TLR ligands and pesticides influence immune responses associated with atopic asthma in farm environments. In the first specific aim we will characterize exposures of environmental TLR ligands (endotoxin, peptidoglycan, and bacterial DNA) and two conventional pesticides relevant to atopic asthma in 10 farms of Ohio before and after pesticide application. A state-of-the-art bioaerosol sampling methodology will be used. It includes an inhalable sampler and a NIOSH-developed size- selective sampling system with two-stage cyclone (three particle size fractions: <1.0 5m, 1 - 1.8 5m, and >1.8 5m), for the collection of airborne TLR ligands and pesticides and a novel microbial source strength tester for the collection of aerosolizable TLR ligands and pesticides from soil surface. We will determine the respiratory deposition potential of particulate TLR ligands and pesticides for male, female, and children subjects using the LUDEP ICRP respiratory deposition model. Results from the first specific aim will thus allow us to understand the real world simultaneous exposure levels to inhalable TLR ligands and pesticides in farms. In second specific aim we will determine how unpurified but clinically relevant air and surface sample extracts from above-mentioned farms analogous to real world exposures influence host susceptibility towards ovalbumin induced hypersensitivity and induction of Tregs. The TLR4 dependence of these influences will be determined by using ovalbumin-sensitized wild type and TLR4 -/- C57BL/6 mice. The findings will provide vital preliminary information on the relationship between adverse effects of pesticide exposure versus protective effects of TLR ligand exposure in the development of atopic asthma among agricultural workers. PUBLIC HEALTH RELEVANCE: Pesticide usage can be significantly associated with atopic asthma among the farmers and therefore farming environment may not provide the protective effect predicted based on the traditional Hygiene Hypothesis. Wide range of immunostimulatory materials and pesticides are present in farming environments and traditional investigations with purified materials and pesticides may not provide precise information on relationship between adverse effect of pesticide exposures versus protective effect of TLR ligands in developing atopic asthma among farmers. In this R21 project we will explore immunological activities of unpurified but clinically relevant environmental samples collected in farms (before and after pesticide application) in ovalbumin allergen sensitized mice.

The long-term objective of this research is to develop integrated approaches to understanding drug use and abuse. Conceptually, this objective relies on (a) development of a biocultural anthropology approach to drug abuse, and (b) integration of the biocultural approach with more traditional theories of drug abuse, such as the biopsychosocial approach. Methodologically, this objective relies on (a) ethnographic research on ambivalence and drug use and abuse among adolescents in Bogota, Colombia, and (b) psychophysiological research testing a preliminary theoretical approach to drug abuse among these adolescents. This preliminary theory contains three aspects-wanting, frames, and self. Wanting refers to the implication of dopamine systems in drug abuse, frames to the role cognitive processes play, and self as an encompassing construct around wanting and frames. The psychophysiological research is the main emphasis of the present proposal, and relies on integrating physiological responses of the sympathetic nervous systems to verbal statements emphasizing "wanting" and "self" respects of drugs. Greater sympathetic response by drug abusers is predicted to wanting and self stimuli that emphasize drugs, as well as quicker response to a change (in frames) between these stimuli.

While magnetic resonance imaging (MRI) is clinically very valuable, current imaging methods are subject to blurring and artifacts in the presence of physiologic (e.g., respiratory and cardiac) motion, as well as of arrhythmias, thus limiting the practical application of MRI in many patients. The currently used MRI methods are also limited in their ability to study the effects of free breathing and arrhythmias on the heart. The proposed research will further develop and evaluate a new approach to imaging in the presence of physiologic motion, which parameterizes such motions with a variable that is treated as an additional dimension to be reconstructed. This would not be practically feasible with conventional methods, due to the additional associated data acquisition that would be required. However, with the use of sparsity-based image reconstruction methods, the high degree of correlation of the images along these additional dimensions permits good quality image reconstructions, even with heavily undersampled imaging data. We already have made successful initial implementations of this new method for 2D cine imaging and 3D MR angiography. In the proposed research, we will further improve these initial implementations, and we will extend them to include implementations of our methods for other MRI sequences, particularly fully 3D cine data acquisitions. We will evaluate the image quality achievable with these new methods in the presence of free breathing and arrhythmias, as compared with conventional clinical imaging methods, using both numerical phantom simulations and clinical cardiac function analysis in pediatric patients to test the performance. We will also evaluate the potential for extracting new kinds of functional information from these multidimensional image sets, including the effects of free breathing and arrhythmias on the heart, using analysis tools that we will be developing. If this research is successful, these new methods will provide significantly improved MR image quality in the presence of free breathing and arrhythmias, as well as providing potentially valuable new kinds of information on the function of the heart. They may also be able to be used for performing MRI in the presence of exercise, which could be useful for both cardiovascular and musculoskeletal applications, as well as in combination with other kinds of imaging, such as with integrated PET/MRI systems. This research should thus further increase the clinical utility of MR imaging for many patients.

Abstract Iron is an essential element for normal physiological functions. However, excess it can cause extensive tissue damage and participates in numerous ocular pathologies including cataractogenesis and retinal degenerations such as age-related macular degeneration. The study of ocular iron metabolism has been a focus of this laboratory for many years. We have made recent novel observations about iron's physiological role. We found that iron regulates synthesis and secretion of the neurotransmitter glutamate by ocular tissues and neurons. This is of fundamental clinical relevance since iron and glutamate are both dysregulated in neurodegeneration. In high quantities, glutamate can be excitotoxic in the central nervous system as well as the retina. Additionally, in retinal pigmented epithelial cells (RPE) and lens epithelial cells (LEC) iron regulates the activity of the transcription factor, hypoxia-inducible factor, which in turn regulates the synthesis of dozens of proteins. Our preliminary data indicate that hypoxic conditions stimulate glutamate release, another critically important observation since hypoxic conditions occur in stroke and retinal ischemia. Furthermore, there are profound changes in the structure of the iron storage protein ferritin in lenses that occur with age, cataractogenesis and differentiation. We will continue to explore how these changes affect iron storage in ferritin and the protection against iron damage such storage provides. Unfortunately, little is known about how iron levels are regulated in the eye which is isolated from the systemic circulation by blood ocular barriers (BOB). The proposal's hypothesis is that intraocular tissues have unique and independent systems for regulating iron uptake into and efflux from the eye across the BOBs. Their polarized location and iron-regulated quantity within ocular tissues allows for proper control of intraocular iron levels. Hypoxia, hemorrhage and inflammation significantly impact iron uptake storage, utilization and efflux. The resulting dysregulation of iron metabolism plays a critical role in ocular pathology. We will use an innovative integrated approach to determine how the BOB's regulate iron levels in intraocular tissues. The two specific aims utilize normal and pathological human eyes as well as normal canine eyes and tissue cultures of cells which form the BOBs, e.g., RPE and CE. Additionally, the lens will be used to assess how iron handling strategies adapt for survival in a normally hypoxic environment. We will utilize a state-of-the-art live-cell imaging quantitative fluorescence microscope with total internal reflection fluorescence for quantifying events at the plasma membrane and allow for measurement of dynamic processes underlying these complex interactions in four dimensions (4D) in living cells. It is the goal of this proposal to determine how intraocular iron levels are controlled and the specific role(s) iron has in ocular pathology in order to provide a basis for development of therapeutic modalities needed for prevention and treatment of ocular disease.

Factors affecting hard-of-hearing (HOH) listeners' use of formant transitions in speech will be investigated in three experiments. Listeners will be selected on the basis of: 1) hearing loss:(a) moderate-to-severe or (b) severe-to-profound, 2) audiometric configuration: (a) flat or (b) sloping, and 3) amount of auditory experience: (a) extensive or (b) limited, as defined by age of hearing loss onset, years of amplification use, and home/school mode of communication. The three experiments will use different approaches for the study of formant transition use by HOH listeners. These are: 1) investigation of listeners' use of formant transitions for glide and voiced stop consonant identification as a function of hearing loss and auditory experience. Spoken stimuli will be modified so that formant transitions are the only available acoustic cues for consonant identification. Transition audibility will be determined based on threshold measurements for each of the transition onset and offset frequencies. An audibility metric will be developed and applied to describe the effects of transition audibility on transition use. The relationships between use of formant transitions and the listeners' auditory experience will also be examined. 2) comparison of listeners' frequency/temporal discrimination for transition-like stimuli with transition use for consonant identification. The transition-like stimuli will simulate the transition characteristics found in the glide/stop identification stimuli. Relationships between transition discrimination and transition use will be described. 3) investigation of HOH listeners' perceptual weighting of formant transitions compared to other acoustic cues to the stop/glide contrast. Both synthesized and natural speech stimuli will be developed that vary along two acoustic continua (formant transition and amplitude onset). Analysis of the perceptual importance of the two acoustic cues, auditory experience, and degree of hearing loss will be completed. In all experiments, listeners with normal hearing thresholds will be used as controls.

DESCRIPTION (adapted from the Abstract): The Undersea and Hyperbaric Medical Society (UHMS) is a professional, non-profit scientific and medical association with a mandate to expand and disseminate the body of knowledge of diving and hyperbaric medicine. It was founded in 1967 and incorporated in 1972. It is a professional membership association of approximately 2800 physicians and researchers. Its purpose is to provide scientific and medical information to protect the health of sport, military and commercial divers and to improve hyperbaric oxygen research and treatment protocols. The Society also provides medical guidelines for physicians and scientists in the fields of diving and hyperbaric medicine. One of its most valuable resources is the Charles W. Shilling Library, which is the repository of the most complete collection of literature relating to these subjects. The long-term objective of this project is to provide wider access to the unique holdings of this special library. The specific aims are: 1) To organize and catalogue the collection for the on-line use of researchers and doctors working, worldwide, in the fields of Diving and Hyperbaric Medicine. 2) To update and systematize the bibliographic database in preparation for on-line accessibility. 3) And, ultimately, to make the entire library collection, including the database, available through the UHMS Internet Website. Reference work is done by the librarian who responds to phone, e-mail and fax requests. Researchers rarely come into the library in person. The librarian will catalogue the collection, including detailed subject indexing and assignment of classification numbers. She will work with a part/time data entry clerk who will assist her in preparing the database for on-line access. In order that the members of the UHMS are familiar with and understand what will be available on line, a training class conducted by the librarian will be offered at the next UHMS Annual Scientific Meeting. Additionally, full instructions for the use of the database will be available on line.

The primary thrust of the Specialized Population Research Center is to investigate mechanisms concerned with regulation of gonadal function. Most of the fundamental studies are directed towards questions of relevance to work dealing with the development of contraceptives. Applied studies deal directly with investigations of the feasibility of using hormonal preparations for male contraception and with problems related to diagnosis and treatment of gonadal disorders. To approach the question of contraception in a scientifically valid and clinically safe way a body of fundamental knowledge concerned specifically with mechanisms involved in regulation by the gonadal steroids of gonadotropin synthesis and release, with the role of gonadotropins and steroids in gonadal function and with the biochemical mechanisms of gonadotropins, androgen, and estrogen action needs to be accumulated. The studies proposed in this application will be directed towards acquisition of this knowledge and elucidation of these mechanisms. In addition, studies dealing with the kinetics of population and the role of androgens in it from physiologic and behavioral viewpoint will be conducted. These studies should place our entire research effort in a broader perspective through an inquiry into the models of natural mechanisms of population control operating in lower species.

Project Summary P-glycoprotein (P-gp) is an ATP-dependent efflux transporter that plays a critical role in drug and xenobiotic distribution, drug-drug interactions, and drug-nutrient interactions. Efforts to modulate P-gp activity to control cellular drug resistance or to modulate the action of existing drugs have been only modestly successful. A barrier to progress in the design of P-gp inhibitors has been the uncertainty about its catalytic mechanism. Different types of drugs or ligands elicit different behaviors, wherein some stimulate ATP hydrolysis and are transported, while others stimulate ATP hydrolysis but are not transported. Other drugs inhibit P-gp without stimulating ATP hydrolysis. The mechanism by which different drugs elicit different behaviors is unclear. Specifically, the conformational changes that mediate communication between the nucleotide binding domains (NBDs) that hydrolyze ATP and the transmembrane helices (TMHs) that bind and release xenobiotics remain unknown. One aim of this proposal is to map by H/D exchange mass spectrometry the ligand-dependent conformational changes in the NBDs and the TMHs. This will be performed with Pgp incorporated into lipid bilayer nanodiscs of defined lipid composition. By monitoring the nucleotide-dependent and drug-dependent changes in solvent exposure and dynamics of specific peptides in the sequence of each protein, with inhibitors, substrates, uncouplers and allosteric modulators, the conformational changes that correlate with each behavior will be identified. A second aim of these studies is to measure the on rates and off rates of drug binding to and dissociating from P-gp in varying conformational states. There are currently no data concerning these rates, which are likely to define ligand behavior, as a substrate vs. inhibitor vs. uncoupler. These measurements will be made via surface plasmon resonance and fluorescence correlation spectroscopy with P-gp nanodiscs. In order to correlate the conformational mapping and off rate information with physiologic behavior, cell based transport activity will be measured for representative drugs with different behaviors. Finally, this proposal aims to explore the methodological advancements offered by nanodiscs with an related drug transporter, BCRP. These studies will add methodological infrastructure to the larger transporter field, increase our fundamental understanding of P-gp, further inform pharmacokinetic models, and facilitate drug design aimed to modulate P-gp.

Project Summary Learned associations between environmental contexts and experience are the basis of decision-making and allow organisms to guide behavior towards advantageous outcomes. Dysfunction in the neuronal processes that regulate these associations, especially in the nucleus accumbens (NAc), is a critical factor in the pathology of addiction. The NAc is a heterogeneous region primarily composed of two opposing cell types: D1 and D2 medium spiny projection neurons (MSNs). Optogenetic stimulation of these cells results in divergent behavioral outputs; thus, it is important to study these populations in isolation to understand the cell-type specific signals that underlie NAc-mediated learning processes. Under the primary mentorship of Dr. Eric Nestler and Dr. Paul Kenny at Icahn School of Medicine at Mount Sinai in New York, the Pathway to Independence Award will provide the opportunity to build on my expertise in cocaine self-administration and synaptic function while simultaneously developing my training and expertise in in vivo calcium imaging and optogenetics. In the mentored K-phase of this grant fiber photometry calcium imaging will be paired with cocaine self-administration in transgenic mouse lines that express Cre-recombinase in D1 or D2 MSN populations. These mice allow for cell-type specific expression of molecular targets, such as calcium indicators (GCaMP6f) and opsins (ChR2; NpHR). By expressing GCaMP6f in D1 or D2 MSNs, the temporally specific signals that mediate cue-induced cocaine seeking will be determined. Further, optogenetic stimulation and inhibition will allow for direct manipulation of these cells and the associated seeking behavior. The innovative combination of these tools will enable the mapping of how D1 and D2 MSNs encode cue information and concurrently establish causality. In the independent phase (R00), these cutting-edge techniques will be combined with the inducible ArcCreERT2 mice which express constructs (GCaMP6f/Opsins) selectively in cells that are activated by environmental stimuli during a temporally specific window. This will allow for the recording and manipulation of neuronal ensembles that are activated by cocaine or cocaine-paired cues to determine their role in drug seeking. Together, these data will elucidate the underlying neural processes that control associative learning and how cocaine exposure dysregulates MSN signaling to drive relapse following abstinence, which will expand our basic understanding of addiction and may lead to the development of novel therapeutic avenues. In summary, the research proposed in this Pathway to Independence Award will elucidate the neural mechanisms involved in addiction while simultaneously preparing me to develop a fully independent research program capable of integrating a wide range of circuit based and behavioral approaches to dissect the neurobiology of addiction. .

Avian-human influenza A reassortant viruses that contain the human influenza hemagglutinin and neuraminidase genes and the six "internal" genes of the A/Mallard/78 (H2N2) avian virus were attenuated for monkeys and man. A similar reassortant derived from the avian A/Pintail/119/79 and the human A/Washington/80 (H3N2) viruses was also attenuated in monkeys and man. The M gene of the A/Mallard/78 virus, which is capable of effecting attenuation by itself, was sequenced and significant divergence from the corresponding gene of a human influenza A virus was observed in the M2 cistron. the RNA 1 and NS genes of the A/Mallard/78 virus, in combination, also contributed to attenuation of the A/Mallard/78 reassortants viruses for monkeys. Vaccinia-influenza HA recombinants were immunogenic and protective in hamsters.

Proposed is a continuation of a 20 year cohort study of the epidemiology of injection drug use and HIV risk among HIV-uninfected injection drug users (IDUs) in Baltimore, MD known as the ALIVE-2 study. The ALIVE cohort is unique in that it comprises a community-based IDU population of both genders who are largely out of drug treatment, with significant representation of African-Americans and those with limited access to appropriate medical care;these populations have been underrepresented in research on persons at risk for HIV infection. ALIVE-2 has provided critical insight into the dynamics of infection and risk behavior while serving as a comparison group to a parallel cohort of HIV positive IDUs (ALIVE-1, DA04334). Continuation of the ALIVE cohort will allow us to characterize current trends in the incidence of blood-borne infections and changing patterns of morbidity and mortality among our unique cohort of aging IDUs. Further, we will consider the broader contextual determinants of risk behavior, disease incidence and mortality. Building upon 20 years of follow-up and experience in studying the health effects of injection drug use, our specific aims are to: 1) Examine temporal trends in risk of HIV and other blood-borne infections (e.g., HCV) among injection drug users in Baltimore, MD;2) Assess mortality and morbidity indices among IDUs according to the burden of blood-borne infections and the spectrum of drug use;3) Characterize the effects of macro-level determinants (e.g., urban redevelopment and drug treatment policy) on HIV risk behaviors, blood-borne infection incidence and morbidity and 4) Continue to serve as a platform for independently funded collaborative investigations of HIV and drug use, including interventions to prevent morbidity and mortality associated with drug use, HIV and HCV infection. To achieve these aims, we will continue follow-up of a cohort of HIV negative IDUs (1000 in active follow-up) with semiannual visits involving interviews and collection of biological specimens for HIV antibody testing and repository. We will implement standardized assessments of chronic disease morbidity that include annual testing of early biological markers of disease as well as detailed medical record abstraction. Further, we will supplement the rich history of individual-level risk data with external data on changes in Baltimore's population, economic situation, and health and drug treatment policies so that we can characterize the effects of macro-level processes on risk behavior and the pathways through which they operate by mapping individuals to their neighborhoods of residence. Multiple external sources including the US Census and the Baltimore Neighborhood Indicators Alliance will be used. To enrich our analyses, we continue a parallel protocol of HIV-infected IDUs (ALIVE-1) to facilitate distinction of the effects of HIV and drug use on our outcomes of interest. The proposed aims will inform HIV prevention and structural interventions and will help to project future health care needs for aging IDUs with a changing spectrum of morbidity. PUBLIC HEALTH RELEVANCE: Injection drug users (IDUs) are at high risk for HIV and hepatitis infections and the long-term complications of these infections as well as drug use itself. The findings from this study will inform HIV prevention and structural interventions and will help to project the future health care needs for aging IDUs with a changing spectrum of morbidity.

In 1992, The University of California San Diego initiated this annual international research symposium in San Diego. Since 1998, the program [now jointly sponsored by the University of Heidelberg], alternated locations so that it is held every other year in Germany. The 2003 program is scheduled in San Diego, CA, where participants receive CME credit. This meeting flourishes because it is based on the exchange of innovative ideas shared among scientists and clinicians from academia and the biomedical/industrial community. Stem cell technology has evolved, with hematopoietic cell transplantation becoming standard therapy for various malignant and hereditary diseases. In the past, most successful transplants were conducted in patients under the age of 55 years. Current advances have significantly broadened the safety of these procedures, so that non-myeloablative transplants can be performed with relative safety in patients up to 75 years of age. Recognizing the importance of the immune system in controlling malignant disease, major advances continue to be reported in immunotherapy. Hematopoietic stem cell therapy provides a platform on which to demonstrate its efficacy. Non myeloablative transplants may permit innovative approaches to stem cell transplantation including tandem autologous transplantation to achieve cytoreduction followed by non-myeloablative allogeneic transplants to achieve the immunotherapeutic effect of the allogeneic cells. An exciting development in the field of stem cell biology has been the observation that hematopoietic stem cells may be capable of trans-differentiation into other tissue types. Reports of hematopoietic stem cells differentiating into hepatocytes, myocytes, neurons, and epithelial and endothelial cells have stimulated intense interest internationally. These findings suggest that hematopoietic stem cells may be important tools for the treatment of a variety of disease conditions by aiding in the repopulating of damaged tissues. Given the current national debate on stem cell research and clinical applications, the potential role of hematopoietic stem cells in regenerative medicine has profound importance. Advances in supportive care, including the diagnosis and treatment of fungal disease and graft vs. host disease, contribute greatly to the increasingly successful outcomes of stem cell transplantation. These issues and others form the major focus on the 11th International Stem Cell Transplantation symposium to be held in May 2003. [unreadable] [unreadable] [unreadable]

Regionally-advanced and metastatic carcinoma (Ca) of the breast is amenable to a variety of therapeutic modalities. Combination chemotherapy can produce response with improvement in survival. Radiotherapy can provide local control but patients (pts) still die of metastasis. This approach utilizes combination chemotherapy to shrink primary masses and treat overt or micrometastases. Seventeen pts (8 stage III, 9 stage IV) were treated with combination chemotherapy including 5 with inflammatory Ca. After tumor regression, 15 of the 17 underwent surgery, achieving local control. All 5 with inflammatory Ca had elimination of the inflammatory component. A variety of combinations are available for recurrent or metastatic disease, but there are few prospective trials which compare the various treatment regimens and stratify for the prognostic variables. This program randomly compares 3 active regimens for response rates and duration of survival, and evaluates the effect of MER immunotherapy. A group effort is necessary to stratify for the variables. Interim evaluations of the 335 pts entered show that prior postoperative adjuvant radiotherapy is a detrimental factor for determining response. MER immunotherapy also had a detrimental effect upon response rate (p equals .003) and survival was shorter. Further randomization to MER has been discontinued. Interim evaluations of the treatment arms revealed slight but not significant differences among the chemotherapy regimens.

The neutral crest is a population of migratory cells that arise from the ectoderm of vertebrate embryos and give rise to a diverse range of cell types, including most of the peripheral nervous system, melanocytes and the craniofacial skeleton. It has been classically assumed that the neutral crest is a segregated population in the early ectoderm, lying between the neutral plate and presumptive epidermis. However, our recent studies on avian embryos show that individual precursor cells within the "neutral folds" can form neutral tube (central nervous system), neutral crest (peripheral nervous system) and epidermal derivatives. This led us to explore the interactions that impart the potential form the neural crest. Interestingly, we found that neural crest cells are generated when epidermis and neural plate are juxtaposed-a classic type of embryonic induction. The proposed experiments aim to characterize this inductive interaction that leads to neural crest formation and to examine the plasticity of early ectodermal derivatives. To continue our studies on the mechanisms responsible for genesis of the neural crest, we will further characterize the molecular nature of the inductive interaction and will test the function of some candidate inducers in vivo and in vitro. We will examine the ability of ventralizing signals to compete with induction of the neural crest, using both grafting and ectopic expression paradigms. Finally, we will examine the lineage relationships and plasticity between neural crest cells and other ectodermal derivatives by challenging their prospective fates via transplantation. Much of the experimental design will involve in vivo experimental manipulations coupled with cell marking techniques, molecular biological approaches, as well as in situ hybridization to examine patterns of gene expression. Specific experiments will: 1. Characterize the molecular nature of the inductive interaction underlying neural crest formation. 2. Determine the ability of ventralizing signals to repress neural crest formation. 3. Determine the competence of ectoderm and neural plate to assume a neural crest fate. 4. Determine the competence of neural crest cells to assume a neural tube fate after transplantation into the ventral neural tube.

This is a 48-week, open-label, AUC-controlled, multicenter study. Sixty children will be enrolled. All subjects will receive DMP 266 and nelfinavir. Concomitant use of nucleoside reverse transcriptase inhibitors (NRTI) will be permitted, but they will not be supplied through this protocol. The initial target AUC for DMP 266 will be between 190 and 380 (M(h. The initial starting dose for children will be 600 mg adjusted for body size. The starting dose for subjects will be adjusted on the basis of the tolerability and DMP 266 plasma concentrations of the first six subjects receiving that dose for two weeks. The target AUC is considered to have been achieved if the dose was tolerated and at least 4 of 6 subjects reached the target AUC. Enrollment of subjects will be ongoing and those begun on a given starting dose will continue on that dose until individual subject dose adjustments are needed according to their individual pharmacokinetics evaluations. Baseline and study efficacy evaluations will include plasma HIV RNA levels, CD4 counts, and viral genotypic and phenotypic resistance analyses. Baseline and study safety evaluations will include the monitoring of adverse experiences, clinical laboratory tests, physical examinations and vital signs. At the end of 48 weeks, subjects will be given the option to continue DMP 266 off-study through a DuPont Merck protocol.

Abstract (Administrative Core) The Administrative Core will oversee coordination and integration of all U19 Program functions; guide and facilitate interactions between the Project and Core leaders, Principal Investigators, and research staff; provide rigorous and regular fiscal oversight of the research projects and cores; ensure that the U19 TeamBCP maximizes the utilization of existing and established resources; and facilitate data, technology and information sharing and collaboration with key BRAIN Initiative stakeholders and the greater neuroscience research community. The core will be lead by our Team Directors, Rui Costa and Tom Jessell, and have an Internal Advisory Committee, a Data Science management sub-committee, an External Advisory Committee and a dedicated staff Program Manager. The core will a) provide overall coordination and support for the TeamBCP U19 research activities, b) foster growth of the Motor Control research within and beyond our research team, c) oversee and ensure resource dissemination and outreach, and d) fiscal and administrative management of cores and research projects. The Internal Advisory Committee will meet monthly to discuss all administrative issues and to provide scientific direction for the program. Drs. Costa and Jessell will co- chair these meetings. The needs, usage and effectiveness of the Cores will be assessed at these meetings and any obstacles to progress will be identified. Each Core, including the Administrative Core, will present a status update on any outstanding scientific, administrative or budgetary issues that require immediate attention. These regular meetings will ensure that the program can address problems in a timely manner. They will also serve to make decisions about the resources, priorities, efforts and data access for the whole program, and to resolve potential conflicts. The Internal Data Science Subcommittee will meet at least once a year, with more frequent ad hoc meetings with individual advisory members to review Data Science Core specific issues and report back to the IAC.

Primary immunodeficiency can be caused by reduced immune cell number or function. Lymphocytes are essential components of the adaptive immune system. Mutations in DNA repair factors compromise lymphocyte development and contribute to three of the eight classes of human primary immunodeficiencies recognized by the International Union of Immunological Societies. XLF (also called Cernuous or NHEJ1) is a member of the non-homologous end joining (NHEJ) family of DSB repair genes. Human patients with loss of function mutations in the XLF gene develop a primary immunodeficiency characterized by age-progressive lymphocytopenia and occasional myeloproliferative diseases. The NHEJ pathway as a whole is required during lymphocyte development at the step of V(D)J recombination that assembles the antigen receptor gene products in developing B and T cells. While the role of XLF in V(D)J recombination, was thought to explain the lymphocytopenia in XLF-deficient patients, we and others reported that XLF is NOT required for chromosomal V(D)J recombination in developing murine lymphocytes, underscoring the need for an alternative mechanism. In this proposal, we investigate an alternative and novel mechanism that might contribute to the XLF related immunodeficiency. Specifically, we hypothesize that XLF-mediated NHEJ is essential for the maintenance of genomic stability in hematopoietic stem cells (HSC). XLF-/- HSCs recapitulate the phenotype of aged HSCs - i.e. loss of self-renewal function, impaired lymphocyte (versus myeloid) differentiation and a relative expansion of the myeloid compartment. The lymphocytopenia observed in XLF-/- mice and in human patients is therefore secondary to HSC dysfunction and is accelerated with age. The deficiencies in HSC function and differentiation in the XLF-deficient patients or mice could be due to defects in the HSCs (cell autonomous) or the microenvironment (cell non-autonomous). HSCs are known to reside in bone marrow niches defined by osteoblasts, mesenchymal cells and endothelial cells, all of which regulate HSC function. Notably, prior studies by the co-investigator- Dr. Siddhartha Mukherjee, revealed that genetic alterations in osteoblasts can impair HSC differentiation. To test of hypothesis, we will determine (Aim 1) the cell autonomous function of XLF and DNA repair in HSC maintenance, function and B cell differentiation and (Aim 2) how XLF deficiency in the bone marrow niche environments affects HSC renewal, function and B cell differentiation. In particular, the later study will provide information on the long term therapeutic effects of bone marrow transplantation, the only curative therapy that is currently available for patients with primary immunodeficiency associated with DNA repair defects. The completion of this proposal will establish a link between HSC and primary immunodeficiency that will have general implication in other genomic instability syndromes.

The research will develop improved measures of social connection through coordinated analyses of intensive ethnographic data and a longitudinal demographic data about the same population in a rural area of South Africa. A great volume of research conducted in various contexts demonstrates that social connection is an important determinant of well-being measured by outcomes such as education, nutritional status, employment prospects, access to health care, and support of the aged and infirm. The most common measure of social connection in population and public health research is co-residence, which has critical limitations. Failure to attend to the full range of social relationships limits our ability to understand the social context of health and well-being. To redress this failure, we will use existing ethnographic data and longitudinal demographic data from rural South Africa to develop new measures of social connection that can be used in social surveys. The ethnographic data is from an intensive fieldwork investigation of children's networks of support in the Children's Well Being and Social Connection project (CWSC) which we conducted between 2002 and 2004. The quantitative data is the result of fourteen rounds of a population-wide census in a sub-district population of 70,000 people conducted by the Agincourt Health and Demographic Surveillance System (AHDSS). The project has three specific and coordinated aims. First, we identify meaningful and efficient indicators of social connection by using all the available data from the CWSC. This is done by identifying instances of resource transfer and behavior that directly impact children's well-being, analyzing these instances to determine the relevant social relationships, individual, household and community characteristics, and spatial distribution of the actors and resources involved, and classifying the coded instances and reduce the list by selecting the most frequent and important patterns. Second, we determine which indicators of social connection identified in Aim 1 can, and which cannot, be measured by manipulating the data from the AHDSS by linking new data files to the AHDSS database, adding links between existing files, and deriving new variables. Third, we assess the value of the new indicators of social connection added to the AHDSS database in Aim 2 by comparing the explanatory power of models that do and do not include these variables in predicting measures of child well-being such as education and mortality. This research will increase the utility of the AHDSS, and potentially, of other censuses and surveys, in explaining child well-being outcomes. By integrating ethnographic, spatial, and longitudinal population data it will contribute to the development of interdisciplinary methods for studying population and public health. PUBLIC HEALTH RELEVANCE: Resources, such as money, food, protection, opportunities, care, and emotional support that flow directly from person to person are vital for the well-being of children in poor countries that do not have strong market or government institutions. To understand why some people thrive and others do not, and to direct policy interventions effectively, we must understand the web of social connections that surrounds and supports them. This project will produce improved measures of social connection by integrating ethnographic data and longitudinal demographic data about the same population in a poor, rural area of South Africa.

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 Administrative Core of the Program will be responsible for theoverall oversight, organization, and management of the scientific aspects of the COBRE grant. In addition to the scientific oversight, the Core will also provide a number of support services to Program-associated investigators, including fiscal/budgetary management, preparation of progress reports, scheduling of research meetings for project-associated investigators, coordinating travel arrangements and meetings of the external science advisory board, and organizing a regular scientific seminar program, liver club, "school of hard knocks," amid various work sessions as described in the mentoring section.

Cancer has been the leading cause of disease-related deaths in human beings, yet, its major non-surgery treatments have been chemotherapy and radiotherapy, both of which are quite toxic and cause severe side effects. Also, the survival rates of cancer patients have had little improvement. Thus, it still remains remarkably important, though challenging, to develop more potent and specific molecule-targeted therapies. The inactivation of the most important tumor suppressor p53 is one highly cancer-related molecular alteration, as its gene is mutated in ~50% of all types of human cancers while its activity or leve is often markedly reduced in the remaining 50% of cancers that harbor wild type TP53. The p53 deactivation is primarily due to the negative feedback regulation by its two chief suppressors, MDM2 and MDMX, which are over expressed in cancers and form a complex that mediates p53 ubiquitination and degradation as well as inhibits p53 activity directly. This negative regulation s further facilitated by SIRT1, which is highly expressed in some cancers, as the deacetylation of p53 by this deacetylase favors MDM2/MDMX-mediated ubiquitination of this protein. Thus, re-activation of p53 in cancers by targeting SIRT1 can be utilized to screen small molecules for the development of an anti-cancer therapy. Indeed, our recent work has identified a small molecule named Inauhzin (INZ) that inhibits the activity of SIRT1 and induces p53 acetylation, level and activity, leading to p53-dependent apoptosis and senescence in p53-containing human lung and colon cancer cells and suppressing the growth of xenograft lung and colon tumors. Our further studying INZ surprisingly reveals another target, IMPDH2, which is also highly expressed in human cancers. Our previous study shows that inhibition of this enzyme causes ribosomal stress by reducing the level of nucleostemin (NS), which is essential for rRNA processing. Consistent with this, INZ also binds to IMPDH2 and reduces NS levels, leading to the activation of the ribosomal stress-p53 pathway. In light of these interesting findings, I hypothesize that INZ can activate p53 by simultaneously targeting SIRT1 and IMPDH2 and thus eliminate cancer cells via a p53-dependent mechanism. We will test this hypothesis by addressing two specific aims. 1. To determine if INZ induces ribosomal stress and activates p53 by inhibiting IMPDH2 and downregulating NS. Since we have recently reported that INZ inhibits SIRT1 activity, here in this aim we will determine if INZ targets IMPDH2 by verifying INZ as a specific inhibitor of IMPDH2, consolidating if inhibition of IMPDH2 by INZ reduces NS levels and induces consequent ribosomal stress, and determining if RPL11 and RPL5 are critical for INZ-induced p53 activation. 2. To determine the role of INZ-14, a more potent INZ analog, in p53 activation and tumor suppression. In this aim, we will test our newly synthesized INZ derivatives, particularly potent INZ-14, by further modifying it, characterizing it in our established biochemical, cellular and animal tumor model systems, and testing the cooperative effect of INZ-14 with doxorubicin or -irradiation in xenograft and orthotopic tumor model systems.

Oral administration of low doses of antibiotics (chlortetracycline and penicillin) to rats results in substantial changes in bile acid patterns, especially in the lower bowel. Hyodeoxycholic acid formation is suppressed, and omega-muricholic acid becomes the dominant bile acid. These changes persist after discontinuation of treatment, and may lead to reduced fecal bile acid output. In time certain animals revert to a "normal" fecal bile acid pattern, but in others a predominance of omega- muricholic acid has been found to last up to three months. To extend these preliminary observations, we propose to study the effect of oral administration (via drinking water) of chlortetracycline, penicillin and neomycin to adult conventional rats. Two dose levels will be used: low (5.00, 5.00 and 15.00 micron M resp.), and high (250.00, 250.00 and 750.00 micron M resp.), the latter within range of therapeutic application. Treatment will last 10 days. Intestinal and fecal bile acids will be determined at various time intervals after discontinuation of treatment. Those treatments which result in the most pronounced and persistent changes will then be used to investigate possible alterations in cholesterol absorption and catabolic turnover of cholesterol, presumably starting 30 days after discontinuation of treatment. Germfree rats will be used to detect any systemic influence of the antibiotics on cholesterol-bile acid metabolism. Conventional and germfree gerbils will be included later in the program. The projected study will probe the influence of routine antibiotics administration on bile acid and related cholesterol metabolism. The investigation aims especially at the long term effects of such treatment as depicted by persistent shifts in bile acid metabolism as they may relate to cardiovascular disease, and possibly to cancer of the lower bowel.

The vascular smooth muscle cell (VSMC) provides dynamic regulation of contractile tone during homeostasis, and is often dysfunctional in diseases involving vascular obstruction and hyper-proliferation of the vessel wall. VSMC modify their gene expression and contractile proteins in response to a variety of external stimuli, particularly during disease progression. The Notch signaling pathway is critical for proper cardiovascular development and is implicated in the pathogenesis of vascular disease. Human mutations in the JAGGED1, NOTCH2, or NOTCH3 genes lead to syndromes with cardiovascular structural defects and susceptibility to stroke. In addition, dysfunction of the Notch pathway is associated with vascular obstructive disease, cerebral vascular disorders, angiogenesis, arteriovenous malformations, and vasculitis. Despite the high significance of this pathway to human vascular disease, there is limited understanding of signaling mechanisms mediated by distinct Notch ligands and their corresponding receptors. Because components of the Notch pathway are therapeutic targets in cardiovascular disease and cancer, it is critical to clarify these signaling pathways. Our studies previously defined an important role for Jagged1/Notch signaling in promoting VSMC differentiation and suppressing proliferation. Part of this mechanism is via the transcriptional activation of miR143/145, which are important for VSMC maturation and function. In addition to Jagged1, a second Notch ligand, Dll1 is also produced during vasculogenesis and remodeling in response to vascular injury. Studies with human primary cells showed that Jagged1 and Dll1 activate distinct pathways consistent with Jagged1 regulating early commitment and differentiation of VSMC, and Dll1 promoting late maturation. These activities correlate with inverse regulation of miR143/145. We propose that the selective ligand effects are mediated through differential transcriptional complex assembly and gene regulation, leading to ligand-specific protein signatures. In addition, we discovered a novel function of Jagged1 signaling via Notch2 to regulate p27kip1 and suppress VSMC proliferation. Thus, the coordinated activities of Jagged1 and Dll1 regulate all phases of the VSMC life cycle from embryonic development to re-establishment of homeostasis following vascular injury. The aims of this project are to: 1) Elucidate Jagged1- and Dll1-induced differential pathways and functional outcomes on VSMC recruitment and differentiation, 2) Discover novel components of differential Notch-mediated Jagged1- and Dll1-induced transcriptional complexes and their impact on gene expression and protein signatures, and 3) Identify mechanisms by which Jagged1 suppresses VSMC proliferation to maintain the contractile phenotype. These studies will provide novel insight into molecular signaling of the Notch pathway, which is a potential therapeutic target for the treatment of cardiovascular diseases.

The Laboratory of Epidemiology and Biometry National Institute on Alcohol Abuse and Alcoholism and the National Institute on Drug Abuse is in the planning stage of a large (N=46500) national survey, the National Epidemiologic Survey on Alcohol and Related Conditions III, that will collect both environmental and biological data (i.e., saliva samples). The assessment battery includes extensive questions on sociodemographic characteristics and risk factors for alcohol and drug use and alcohol and drug use disorders (i.e., abuse and dependence) and their associated physical and mental disabilities. These include detailed measures of alcohol consumption and drug use for ten substances, along with their abuse and dependence measures. Major physical disorders (e.g., liver cirrhosis) and mental disorders (e.g., major depressive disorder) highly comorbid with alcohol and drug use disorders are also measured. Risk factors include sociodemographic characteristics, physical and mental impairment, discrimination, acculturation, stress, childhood adverse experiences (e.g., sexual abuse), needle use and HIV, stigma due to substance use disorders, objective measures of weight and height, sexual preference and behaviors, traumatic experiences, tobacco use and dependence, treatment utilization for alcohol and drug use disorders, permanent and temporary disability, academic functioning and achievement, and cognitive functioning.

A basic requirement of all cells is the ability to sense and respond to changes in the environment. Osmolarity is one of the most basic conditions to which cells must respond. Despite the ubiquitous nature of osmosensing systems, the molecular mechanism by which osmotic pressure is sensed is largely unknown. Studies involving microbial model systems have played an important role in identifying the sensors and signal transduction pathways that respond to changes in osmolarity. We plan to use the yeast osmotic stress sensor, Slnlp, as a paradigm for eukaryotic osmosensors. Changes in osmotic conditions regulate Slnlp causing changes in phosphate flux between the individual proteins comprising the two-component signaling system. The fundamental question addressed by this proposal is how Slnlp activity is regulated in response to changes in osmolarity. Using computational, genetic, and biochemical techniques, we have identified a coiled-coil dimerization domain that plays a key role in mediating the stimulus-activation step. It is located in the linker region between the membrane and the kinase domain. The specific objective of this application is to perform a detailed structure-function analysis of the coiled coil (CC) region of the yeast Slnlp osmosensor to test the hypothesis that the unusual composition of the HK CC contributes to the regulation of the HK family of sensor kinases. The specific aims of the proposal are to (1) Genetically dissect the Sin1 CC domain, (2) Determine the structure of the CC domain and CC mutants, and (3) Develop membrane-based assays for Sin1 function. The elucidation of the unique structural and mechanistic features of the two-component type coiled-coil domain in Slnlp will serve as a model for this class of signaling molecules and may lead to the development of histidine kinase inhibitors for antifungal therapy.

Recent advances in human genetics have enabled the identification of various mutations responsible for disease. Such advances include collections of families and populations presenting clinical phenotypes, genome sequence and polymorphism databases, mathematical algorithms and computer programs for computational purposes, and novel applications of pedigree and linkage disequilibrum (LD) analyses. LD is the non-random association between alleles at different loci. By tracking genetic markers in LD with a disease phenotype, a genomic region harboring a causal variant can be localized. With association studies, it is necessary to determine the haplotype structure of any given region. Recent studies have demonstrated that many regions of the human genome are characterized by segments or blocks of limited haplotype diversity due to high levels of LD between genetic markers. Chemotactic cytokines are known to direct the migration of specific subsets of leukocytes to sites of infection and inflammation. Further, the natural chemokine receptors CCR5 and CXCR4 are HIV-1 coreceptors. About 40 human chemokine genes are found at eight different chromosomal locations, with major clusters on chromosomes 4 and 17. We are focusing on 16 Chemotactic cytokines (CC) genes located at 17q12-21. Nine of these genes (CCL2, CCL7, CCL11, CCL5, CCL14, CCL3, CCL4, CCL3L1, and CCL4L1) have been implicated in HIV-1/AIDS pathogenesis based upon tissue culture, cellular immunological and virus infection assays. In fact CCL3L1 has the strongest binding affinity of all ligands for CCR5, and is thus an excellent candidate for an HIV-1 entry inhibitor. A comprehensive haplotype analysis is being carried out using 80 single nucleotide polymorphisms (SNPs) covering 400 kb in 200 individuals from each of three racial groups: European Americans, African Americans and Chinese. Approximately 40 'tagged' SNPs will be selected and subsequently genotyped in 4000 subjects enrolled in HIV-1/AIDS cohorts. We have already identified and published genetic influences on HIV-1/AIDS for two gene regions, one containing CCR5 (RANTES) and one containing CCL2-CCL7-CCL11 (MCP-1, MCP-3, and EOTAXIN). In addition, SNP CCL3 459 C/T is associated with an increased rate to AIDS-87 in European Americans (p=0.003), and SNP CCL4 662 C/G is associated with apparent resistance to infection in African Americans (p=0.01). Subjects are also available for lung cancer, skin cancer and Hodgkin's disease studies, and are being developed for hepatitis and nasopharyngeal carcinoma. Once the studies 'tagged' SNPs have been identified they will be genotyped in all disease populations.

Nevi (moles) are important precursors and risk markers for melanoma. Current knowledge, derived largely from cross-sectional studies, indicates that adolescence is a critical period for the appearance and evolution f nevi. There is further compelling evidence that nevus phenotype is largely genetically determined with a significant modifying effect of sun exposure. The primary objective of this study is to evaluate specific genetic and environmental factors as risk factors for nevus development and growth in early adolescence. A secondary aim is to document the clinical and dermoscopic evolution of individual nevi in this age group. We will apply a combined cross-sectional and longitudinal study design to the cohort of all consenting 5th graders in the Framingham, Massachusetts school system (estimated n=735) to address three aims. Aim #1 utilizes a cross-sectional study of the 5th grade students to test the hypothesis that germline melanocortin receptor (MC1 R) variants, intense childhood sun exposure, and lack of sun protection in childhood are associated with increased numbers of nevi and large nevi in early adolescence. Aim #2 applies a longitudinal study design in the same student population followed through 8th grade to test the hypothesis that MC1R variants, ongoing intense sun exposure, and ongoing lack of sun protection are associated with increased numbers and increased size of nevi during adolescence. Aim #3 utilizes digital photography and dermoscopy, a recently developed imaging technique, to document the clinical and subsurface appearance and evolution of common nevi in the cohort under study. We will use parent and child surveys conducted at baseline and repeated annually to ascertain sun sensitivity, childhood sun exposure and current sun exposure. We will conduct examinations of the skin of the back including high resolution overview photography and close up digital photography and digital dermoscopy of four index nevi at baseline (5th grade) and repeated in 8th grade to document pigmentary phenotype, the prevalence of nevi by size, the incidence of new and changed nevi, and the clinical and dermoscopic features of individual nevi. Mouthwash derived DNA collected at the baseline examination will be used for MC1R genotyping. The insights into nevus risk factors and evolution gleaned from this study will have significant implications for reduction of melanoma mortality through improved risk stratification and more informed prevention.

The current work is concerned with investigating the role of opiate peptides in the brain. Since there are multiple systems containing naturally occurring opiates, we have chosen to focus on the so-called 31K system - a neuronal pathway within brain containing beta-Endorphin, ACTH, alpha-MSH and related peptides. Our overall goal is to characterize these peptides as neurotransmitters or neuromodulators by studying their anatomy, storage, release, receptor binding and behavioral effects. The techniques being employed include immunocytochemistry, autoradiography, receptor binding, extraction of peptides from brain, blood and CSF, radioimmunoassays and HPLC. Furthermore, we employ behavioral techniques and microinjection procedures in an effort to study the in vivo function of these peptides. It is expected that these studies will shed light on the role of endogenous opiates in pain control, drug addiction and motivated and affective behavior.

Tobacco use causes about one of every five deaths in the United States and is the single most preventable cause of death and disease in our nation. At least 70 percent of smokers visit a physician each year, but most are not advised or assisted in any attempt to quit. While more than 90 percent of children visit a pediatrician annually, little is known about the prevalence of smoking prevention counseling. Few practicing physicians are prepared to prevent smoking or help patients stop smoking and a majority of medical school graduates are not adequately trained to counsel families, treat nicotine dependence, or minimize smoking initiation among youth. Shortcomings in tobacco control curriculum in medical schools is a well-recognized deficit. Since 1996, Boston University has developed a curriculum with 10-15 new hours of tobacco control curriculum for students graduating in the year 2000, interwoven into major preclinical and clinical courses, Boston University?s effort follows successful integration of tobacco related curricula at the University of Massachusetts (since 1990). Other Universities have begun tobacco control education with many opportunities to test and evaluate new curriculum in multiple contexts. In this project, II medical schools from throughout the United States, with a wide range of depth and breadth of tobacco curricula, will collaborate with Boston University to develop, refine, and integrate new modules, train medical school faculty, evaluate teaching content, assess opportunities for diffusion to and adoption by other schools, and disseminate teaching guides, We anticipate that exemplary universities of tobacco teaching can be developed and serve as regional and national role models. We are aided in this effort by national representatives of major primary care prance organizations, preventive health specialists, medical student organizations, and cancer control advocates. With expertise in medical student education, curriculum development, faculty training, and evaluation for tobacco prevention and cessation, we have the following specific aims: Aim1 - Assess current curriculum and convene a national conference Aim2 - Develop new modules, plans for integration, and faculty training Aim3 - Conduct trial implementation Aim4 - Conduct a comprehensive, formative, process and impact evaluation Aim5 - Disseminate teaching guides to other medical schools

Caloric restriction (CR: reducing caloric intake 30-40 percent below ad libitum levels) has been repeatedly shown to extend lifespan, reduce the incidence and delay the onset of age-related disease, enhance stress protection, and attenuate functional decline in mammals. The NIA longitudinal study of CR and aging in rhesus monkeys is in its 29th year and 25% of the animals remain on study. In 2012, we reported findings of improved health; however, unlike findings from a similar study at the University of Wisconsin, the NIA CR monkeys did not have improved survival compared to the controls. Both studies are ongoing and we will continue to monitor the monkeys for age-related changes in many health parameters. Specifically we have ongoing studies assessing behavior including locomotor and motor performance. We have identified traits traits consistent with macular degeneration. We are analyzing samples to identify markers consistent with intestinal cancers that might be similar between monkeys and humans. Monkeys are currently being trained for learning and memory tasks.

Reactive oxygen species (ROS) and related oxidative stress are linked with various diseases including cardiovascular disease, cancer, chronic lung diseases, and diabetes mellitus. Interventions favoring the scavenging of ROS to attenuate the oxidative stress may prevent oxidant stress associated diseases. Recent studies suggest that various fruits and vegetables contain high concentrations of antioxidants. Quercitrin is a glycosylated form of flavonoid compounds, widely distributed in nature, and is ubiquitous in plants, fruits, seeds, and vegetables. Our preliminary studies show that this compound displayed a stronger antioxidant activity than that of ascorbic acid over the same concentration range. It blocked TPA-induced neoplastic transformation in JB6 P+ cells. Pretreatment of JB6 cells with quercitrin down-regulated activation of AP-1, NF- :B induced by UVB or TPA. In the skin of AP-1-luciferase transgenic mice, topical treatment of mouse with quercitrin markedly blocked the TPA-induced AP-1 activation. Further studies indicated that these inhibitory actions appear to be mediated through the inhibition of MAPKs phosphorylation, including ERKs, p38 kinase, and JNKs. In addition, quercitrin stimulated the activation of NF-E2-related factor (Nrf2) and GST ARE- luciferase activity. Comet assays showed that quercitrin could block DNA damage induced by UVB. These preliminary studies indicate that quercitrin may function as a potential chemopreventive and chemotherapeutic agent. The overall hypothesis of this application is that quercitrin functions as an antioxidant and protects UV- induced carcinogenesis. The goal of this proposal is to test this hypothesis. Aim 1 will investigate the antioxidant properties of quercitrin in both non-cellular and cellular. We will study whether quercitrin scavenges free radicals or inhibits their generation and determine reaction rate constants of the reactions between quercitrin and oxygen radicals. Moreover, we will detect the UV-induced free radical generation from UV- irradiated skin of living animal using in vivo electron spin resonance (ESR) spin trapping and identify the radicals generated. Aim 2 will investigate the inhibitory effects of quercitrin against the UV-induced oxidative damage to lipid, protein, and DNA, and UV-induced tumorigenesis and cell proliferation in SKH-1 hairless mice. This study represents first detection of free radicals generated by UV-irradiated skin of living animals and may potentially open a new avenue to evaluate the properties of antioxidant against free radicals generated in living animals. Another significance of the study is the identification of qucertrin as a preventive agent against UV-induced skin cancers. PUBLIC HEALTH RELEVANCE: Reactive oxygen species (ROS) and related oxidative stress are linked with various diseases including cardiovascular disease, cancer, chronic lung diseases, and diabetes mellitus. Interventions favoring the scavenging of ROS to attenuate the oxidative stress may prevent oxidative stress associated diseases. This proposal will test the hypothesis that quercitrin, a compound found in blackberries and other foods, functions as an antioxidant and protects UV-induced carcinogenesis.

CENTER-DRIVEN RESEARCH PROJECT 1: 3D HIGH-CONTENT SCREENING A. INTRODUCTION &SPECIFIC AIMS High-throughput cellular screens interrogate more biologically relevant processes than do cell-free screens, but they grow in complexity proportionally to their ability to mimic the in vivo state. While most high-content (cellimage- based) screening (HCS) is performed on standard cell culture models, it is well known that many normal and malignant cells lose key phenotypic and functional characteristics when grown in monolayer culture. Introduction of organotypic 3-dimensional (3D) culture and screening systems into mainstream small-molecule and drug discovery processes is increasingly discussed but severely limited by complex methodological requirements and a lack of sophisticated biological model systems, miniaturized screening methods, and 3D image analysis and instrumentation (21, 22). Burnham's Center-driven component will apply our multidisciplinary expertise in cell biology, cellular imaging, and high throughput microscopy (HTM) algorithm and instrumentation development to address this fundamental unmet need in chemical genomics and drug discovery. The broad objectives are to: (1) develop validated 3D model cellular systems for screening;(2) develop software analysis and instrumentation tools for 3D visualization and screening of the model systems;and (3) utilize the 3D culture protocols and 3D image analysis tools to perform screens and disseminating the tools and screens to the scientific community. Realization of these 3D culture models and HTM screening tools will fill the gap between traditional monolayer cultures and in vivo animal models, allowing all MLPCN users to generate their own 3D-HCS primary and secondary assays to enhance and expand the development of biologically meaningful Probes. We will pursue these goals through the following Specific Aims: Aim 1. Develop a toolbox of model organotypic 3D systems for screening. We propose to develop a wide variety of 3D culture systems, including spheroids, multi-layered cultures, and co-cultures of interacting cell types, which are of high interest to the scientific community, in 384- and 1536-well formats amenable to medium and high throughput screening. Aim 2. Create image acquisition and analysis tools for screening of 3D models. A range of image acquisition and analysis tools will be developed to enable primary and secondary 3D-HCS ~ from fixed endpoint 3D imaging for fast primary screens to high-resolution 3D time-lapse analyses for in-depth secondary screens. The following approaches will enable completion of this Aim: 2.1. Development and validation of 3D image acquisition using existing confocal and non-confocal HCS instruments, including deconvolution protocols. 2.2. Development and implementation of 2D assay read-outs from optical sections of 3D images. 2.3. Development and implementation of 3D assay read-outs directly from the 3D images. 2.4. Development and implementation of parallel confocal microscopy instrumentation to speed 3D image acquisition. 2.5. Implementation of 3D time-lapse image acquisition and analysis tools for living cells. Aim 3. Integrate and validate tools from Aims 1 and 2 for primary and/or secondary 3D-HCS screens. Here we will integrate the tools developed in Aims 1 and 2 into assays that will be screened within the MLPCN, thus validating the technology for wider use in the scientific community.

Increased expression of the Fibroblast growth factor 8 (Fgf8) gene plays an important role in the progression of both breast and prostate cancer. To understand how abnormal Fgf8 expression affects cell function, we are studying its normal role during vertebrate embryogenesis, using the mouse as a model system. Fgf8 is expressed in a variety of regions of the embryo that may be termed "organizers": regions that are a source of signals that pattern and thus "organize" the surrounding tissue. An allelic series generated at the Fgf8 locus (Meyers et al. 1998 Nature Genetics 18:136), as well as Cre-mediated tissue-specific knockouts (Lewandoski et al. 2000 Nature Genetics, 26:460; Lewandoski 2001 Nature Reviews Genet. 2:743) has revealed a role for Fgf8 in organizers that control gastrulation, limb, and brain development. Recently we have produced a valuable mouse line (TCre) that expresses Cre throughout all embryonic mesodermal lineages but not ectoderm/neuroedctoderm, thus allowing us to control gene expression in these lineages. This line is useful to bypass the embryonic lethal phenotypes of genes that affect early development, yet allows the study of the role of such genes throughout much of the embryo. Inactivation of Fgf8 with TCre has revealed that Fgf8 plays a central role in cell survival and gene expression during kidney development. Another surprising insight emerging from these studies is that Fgf8 is not require for several mesodermal signaling centers where it was thought to play a role. To investigate this, we are studying mutants in which Fgf8 and each of the other five Fgfs expressed in these regions are simultaneously inactivated. One of the intriguing insights that has emerged from these studies is that at different stages of embryogenesis FGF signaling plays different roles in cell migration, proliferation, patterning, and survival. How is this diversity of response achieved? To answer this question, we are studing downstream targets of FGF signaling. One set of such target genes is the homeobox genes Gbx1 and 2. The role of the mouse Gbx2 gene during neurulation and particularly in defining the mid/hindbrain organizer has been well documented (Wasserman et al. 1997 Development 124:2923). We are currently extending this analysis by studying a hypomorphic (partial-loss-of-function) Gbx2 allele, which has revealed that Gbx2 is required at certain threshold levels for different parts of the brain. Compared to Gbx2 relatively little has been reported about Gbx1. We recently described the cloning and embryonic expression pattern of Gbx1 and defined regions of potential molecular redundancy with Gbx2. (Waters et al 2003 Gene Exp. Patterns. 3:313). We are currently generating mice with an allelic series at the Gbx1 locus to study it's function during development, including its role in FGF signaling.

This is proposal to study how the frequency and complexity of pretend play among 30, 3- and 5-yr-old black girls vary as a function of age and toy structure (high vs. low). High structure toys are defined as those which have highly specific, readily identifiable, and conventional functions (e.g., tea set). Low structure toys are defined as those which have less specific, relatively ambiguous functions (e.g., piece of cloth, cardboard box). Data for this study have already been gathered. This proposal requests support for processing and analyzing them. Same-age girls were randomly grouped to contsitute 10 triads, each of which was observed in a playroom for four 30-min. sessions. High structure toys were present in two of the sessions and low structure toys in the remaining two. Several dependent indices of frequency and complexity of pretend play will be examained. The significance of this study derives from the fact that the interactive effects of age and toy structure on spontaneous pretend play will be examined in one design, dissimilarity between high and low structure toys is increased, providing a more powerful test of their effects, threats to validity present in previous psychological studies are eliminatated, the range of dependent variables is increased, and a previously neglected population is the focus. This study will lay groundwork for more comprehensive research in which comparative data for children who vary on specific population parameters (e.g., social class) will be generated to test hypotheses about the interactive effects of toy structure and individual differences. The theoretical and practical implications of such research are many. Empirical research and theoretical formulations suggest that pretend play facilitates or is associated with basic social and cognitive skills and mental health. Hence, research is warranted to examine situational and ecological factors with a view toward clarification of conditions conducive to pretend play. Results of this study could identify at least one way in which socializers may structure the play environment, with minimal intervention, to ehhance children's pretend play.

This project will apply the technique of hyperspectral imaging, via the development of an acousto-optic tunable fiber, to enhance endoscopic imaging for the discrimination of tumors, malignant, and normal tissue. With this system, tissue identification will be based not only on a spectral ratio but on the entire spectrum, thus enhancing the system's sensitivity to very small spectral variations. This system will aquire spectral images of drug or laser induced tissue fluorescence, and will perform near real-time mapping for tumor localization.

DESCRIPTION: Demyelinating diseases resulting from different etiologies may share common secondary pathogenic mechanisms. The overall objective of the proposed research is to advance the understanding of the role of cytokines in the pathogenesis of demyelinating diseases. This project will focus on one demyelinating disease, globoid cell leukodystrophy, which has several advantages for experimental investigations: 1) it has an authentic genetic animal model called the twitcher mouse, 2) it has an inflammatory response that is simple by comparison to other demyelinating diseases such as multiple sclerosis and adrenoleukodystrophy, and 3) it can be treated with bone marrow transplantation (BMT). The rationale for the proposed research is based on the findings that TNFa and IL-6 expressions are elevated in twitcher mice, and twitcher/IL-6 deficient mice have a different pathological course than twitcher mice. If twitcher mice are given BMT, donor macrophages enter the brain and slow the development of pathological changes. However, donor macrophages and endogenous glial cells are still capable of producing cytokines, which may affect the therapeutic course of BMT. Based on these facts, two central hypotheses are advance: 1) IL-6 modulates the pathogenic course in twitcher mice and it promotes the therapeutic effectiveness of BMT, and 2) activated TNF receptors negatively affect twitcher mice by promoting pathogenesis, and limiting the therapeutic effectiveness of BMT. These hypotheses will be tested by the following specific aims: 1_ produce double mutant mice that carry both the twitcher and IL-6 mutations, and use them to analyze the effects of the IL-6 mutation on the clinical and pathological course in twitcher mice with and without BMT, and 2) produce double mutant mice that carry both the twitcher and TNF-receptor(R) 1 mutations or the twitcher and TNF-R2 mutations, and use them to analyze the effects of the TNF-R1 or TNF-R2 mutation on the clinical and pathological course in twitcher mice with and without BMT. The proposed studies should provide insights about the role of TNF receptors and IL-6 in the pathogenesis of globoid cell leukodystrophy, which should be relevant to other demyelinating diseases in the CNS. The long range objectives are to utilize this information to design treatment strategies that modulate cytokine expression in demyelinating diseases in general and to enhance the therapeutic effectiveness of BMT in Leukodystrophies. The project will utilize the following techniques: production of double mutant mice, immunohistochemistry, pathological and clinical evaluations, electron microscopy, PCR, ELISA and BMT.

The proposed research is planned to study the functional development of sympathetc innervation of the heart directly in puppies and indirectly in newborn infants. Experiments are carried out in open chest newborn puppies up to 6 weeks of age. After bilateral dissection of the sympathetic cardiac nerves, refractory period changes are determined during maximal stimulation of the individual nerves. The findings from different age groups including adult dogs are compared and functional maturational changes as well as differences between newborn and adult dogs are found. Serial electrocardiograms are obtained in full term premature infants during the first three months of life, starting 24 hours after birth. The data will be processed through a computer and any evidence of sympathetic nerve imbalance such as ST-T waveform abnormalities and tachyarrhythmias will be identified, followed and correlated with maturation. Finally, the results of the electrocardiographic studies in newborns will be correlated with results of the animal studies.

As a part of a continuing study of the neurophysin-peptide hormone system, a cell-free translation system is used to obtain the radiolabelled precursors of neurophysin I and II. The principal technique to be used involves passage of the translation product through two affinity ligand columns, methionyl-tyrosyl-phenylalanyl-aminohexyl agarose and lysine vasopressin agarose, shown by previous studies to specifically bind neurophysin I and II. The interaction of the precursors with the affinity ligands should yield information on the expression of the neurophysin hormone binding site. In the event that the translated precursors do not recognize the ligands, two enzymatic preparations are known that may produce modified forms of the precursors. Microsomes from dog pancreas are reported to convert the pre-pro-neurophysin to the pro-neurophysin form. Treatment of the precursor with trypsin reportedly produces a neurophysin-like species. Differences in the interaction of these precursors with the affinity ligands may suggest a processing scheme wherein the initially translated precursor is converted to a functional neurophysin through a series of proteolytic cleavages.

The proposed research concerns the attempt to discover the brain substrates, both anatomical and pharmacological, that are responsible for intracranial self-stimulation (ICSS). ICSS is the phenomenon seen when an animal can be trained to perform tasks for no reward other than the electrical stimulation of its own brain. Because of the hypothesized link between this behavior and certain psychopathological states, a better understanding of the central mechanisms responsible for it is needed. Proposed experiments include: 1) the identification of all neural systems in the area of ICSS electrodes, making use of silver impregnation, fluoresence, and orthograde as well as retrograde axonal tracing techniques; 2) the experimental elimination of these systems with lesion or neurotoxins to assess their contribution to the behavior; and 3) the administration of psychoactive drugs such as the stimulants and neuroleptics before and after the lesions to assess the contribution of various systems to the facilitatory or inhibitory effects of those drugs.

Ricin, a natural product of the castor bean (Ricinus communis) and a Category B toxin, is significant as a biological weapon because of its heat stability, worldwide availability, and ease in production. It can be disseminated as an aerosol, a likely route that terrorists may use. The ricin toxin is a lectin consisting of two polypeptide chains linked by a disulfide bridge and cellular entry is required for toxicity. The ricin B-chain (RCB) facilitates entry of the toxin into the cell and the ricin A-chain (RCA) possesses RNA N-glycosidase activity that attacks a specific site on the 28S rRNA, preventing polypeptide chain elongation, thereby inactivating ribosomes (ribotoxic) and leading to cell death. Since the ricin toxin is free in the circulation for only a brief period of time (hours to days) before cellular internalization, the greatest clinical benefits will be derived from therapeutics capable of blocking RCA enzymatic activity at intracellular sites. We have recent evidence that a high dose of a novel primatized anti-ricin A-chain antibody (43RCA IgG) administered directly to the lung can offer 83% and 75% survival when administered at 44 hr and 54 hr post-ricin challenge, respectively. The primatized 43RCA IgG was generated by fusing the V domains from macaque-derived 43RCA scFv to the constant regions of the human IgG1. The 43RCA scFv sequence is very similar to that for human IgG germline genes, with 90% sequence identity for the VH and VL regions, thereby increasing the potential for this human-like antibody to be used as ricin antidote in humans. In the case of human exposure, whether it is accidental or deliberate, it is anticipated that there will be delays in post-exposure treatment. It is, therefore, imperative to develop antidotes with a therapeutic window beyond 2 days to allow sufficient time for treatment of exposed individuals. We therefore hypothesize that the therapeutic index and/or window for post- exposure treatment can be improved and extended by cytosolic delivery of neutralizing anti-RCA antibodies to block intracellular ricin activity. To achieve our goal, we will develop cell-permeable monoclonal antibodies (or TransMabs) by conjugating antibodies to transport peptides or cell-penetrating peptides (CPPs) and the therapeutic effects will be evaluated as described in the following specific aims. Specific Aim 1: To Ascertain the Protective Effects of Cell-Permeable antibodies against Ricin Cytotoxicity in Cell Culture Models. Specific Aim 2: To Ascertain the Efficacy of Cell-Permeable Antibodies for Post-Exposure Treatment of Ricin-Induced Lung Injury and Lethality using the Lung Aspiration Model. Specific Aim 3: To Validate the Efficacy of Cell-Permeable Antibodies for Post-Exposure Treatment in an Aerosolized Ricin Mouse Model. PUBLIC HEALTH RELEVANCE: Since there is currently no treatment for the ricin toxin, development of a specific antidote for the treatment of ricin after exposure will contribute significantly to the protection of our civilian and military populations. Additionally, the availability of an effective antidote may also significantly reduce the threat of the use of ricin as a biological weapon.

More than half of all human proteins are glycosylated, and the physiological significance of glycosylation is exemplified by the numerous instances in which variable glycosylation compromises protein function and causes developmental defects and disease. Despite this, the factors that control which glycans are assembled on proteins are not well understood. Polysialylation is a striking example of a protein specific modification that can dramatically change protein function. Polysialic acid (polySia) is best known for its ability to block neural cell adhesion molecule (NCAM)-dependent cell adhesion and signaling, and for its roles in cell migration, axon guidance, synaptic plasticity, an nervous system development. PolySia is also upregulated on damaged peripheral neurons and facilitates their regeneration, and on the surface of several different types of cancers where it promotes their growth and invasiveness. Remarkably, polySia is found on only five proteins in addition to the polysialyltransferases (polySTs) that modify their own N-glycans. The recent identification of two of these polyST substrates, SynCAM 1, a synaptic adhesion molecule, and neuropilin-2 (NRP-2), a semaphorin and VEGF co-receptor, suggests that the roles of polySia may be more extensive than previously thought, and raises the question of how the polySTs recognize and modify these distinct substrates. Our long-term objectives are to determine the mechanism of protein specific polysialylation, what factors regulate the polymerization of polySia chains on specific substrates, and how polySia modulates the functions of the proteins it modifies. In this proposal we will test the hypothesis that the polySTs recognize common amino acid and structural features of their substrates and that this interaction allows an initial polymerization of the polySia chain on a substrate's glycans, and that this is followed by a polyST-polySia chain interaction that promotes further chain elongation. To do this we will evaluate the domain and sequence requirements for polyST recognition and polysialylation of NCAM, SynCAM 1 and NRP-2, and determine whether residues in a conserved polyST polybasic region mediate substrate protein and/or polySia chain interaction to promote protein specific polySia chain polymerization. We will also test the hypothesis that changes in the length of the stalk regions of SynCAM 1 and NRP-2, generated by alternative splicing, alter their alignment with membrane- associated polySTs and control the polysialylation of these proteins in a cell- and tissue-specific manner. We anticipate that these studies will allow us to identify points in the polysialylation process that are subject to physiological regulation, and that will b amenable to experimental and therapeutic manipulations to control substrate polysialylation and function during development, repair, and disease. PUBLIC HEALTH RELEVANCE: Polysialic acid is a developmentally regulated sugar polymer found on a small group of adhesive and signaling proteins. It is critical for nervous system development, and plays roles in cell migration, synaptic plasticity, neuronal regeneration, and the growth and invasiveness of a wide variety of cancers. The proposed work will elucidate the mechanism of polysialylation, and identify points of physiological regulation that can be experimentally and therapeutically manipulated to control the functions of polysialylated proteins during development and disease.

Overall: Summary/Abstract Over the past four decades, MIT has had a focused effort in cancer research, first in the form of the MIT Center for Cancer Research (CCR) and, since 2007, as the Koch Institute for Integrative Cancer Research at MIT. This effort has been continuously supported by a Cancer Center Support Grant (CCSG) from the National Cancer Institute (NCI), providing the designation as an NCI-designated Cancer Center at MIT. By supplying infrastructural support for Core Facilities and other organizational components of the Koch Institute as well as funds for faculty recruitment and pilot projects, this CCSG is a critical resource for cancer research at MIT. From the establishment of the CCR in 1974 to the transition to the Koch Institute and continuing to the present, the NCI Cancer Center designation has had a strong influence on the MIT administration, leading to significant institutional support over this entire period. The investment in construction of the Koch Institute building (opened in late 2010) is a recent indication of this support. The building brings together 27 cancer scientists and cancer-oriented engineers to form a highly inter-disciplinary and collaborative research environment. The building is also the hub of cancer research on the MIT campus, with a nearly equal number of Members of the Center having their laboratories in other research buildings nearby. The 56 Center Members are drawn from eight academic departments at the School of Science or School of Engineering at MIT. Beyond the discovery research and technology development being pursued by the Members of this Center, significant emphasis is placed on translational research in the form of collaborations with clinical centers and industry partners. Research in the Koch Institute is organized into three Programs. Each of these Programs has made significant advances over the current grant period. Program 1: Genetic & Cellular Programs in Cancer is co-led by Drs. Phillip Sharp, J Christopher Love, and Eliezer Calo. Program 2: Cancer Biology & Immunology is co-led by Drs. Richard Hynes, Dane Wittrup, and Stefani Spranger. Program 3: Systems and Engineering Approaches to Cancer is co-led by Drs. Michael Yaffe, Scott Manalis, and Angela Koehler. These Programs function to stimulate new research initiatives by their Members as well as to foster intra- and inter-programmatic collaborations. In aggregate the 56 Members of this Center have published 1006 cancer-related articles over the past grant period. Of those, nearly 18% have involved multiple Members. The Center has a cancer-related funding base of $58,596,507 TDC (see Data Tables 2A/2B).

The identification of biomarkers that enable the early detection and prognosis of disease, or that facilitate measurement of the efficacy of response to a specific therapeutic intervention, holds great promise in advancing the capabilities of individualized medicine. Recent technological advances, particularly in the development and application of sensitive and robust mass spectrometry approaches, have enabled large scale biomarker discovery efforts to be initiated, including within the emerging field of lipidomics. Lipids are a diverse group of compounds, including fatty acyls, sterols, glycerolipids, glycerophospholipids and sphingolipids that play key biological roles as the main structural component of cell membranes, in energy storage and metabolism, and in cell signaling. A large number of studies have demonstrated that the disruption of lipid metabolism or signaling pathways can play a key role in the onset and progression of human disease. Thus, a comprehensive comparative analysis of changes in lipid profiles that occur between normal and diseased cells, tissues or organs, may enable the identification and characterization of specific lipids that can serve as effective biomarker signatures of disease. Here, we propose to (i) critically evaluate and optimize extraction and sample handling procedures associated with the isolation of lipids from specific tissue types and (ii) develop a comprehensive strategy, based on the use of electrospray ionization and complementary tandem mass spectrometry approaches, coupled with automated data analysis and principle component analysis methods for lipid identification and quantification. Then, we will apply this lipid biomarker identification approach to examining changes in lipid profiles that are observed between normal tissue and the end organs of two common human disease models in which lipids play a major role, namely diabetes (retina) and hepatocellular carcinoma (liver), and to correlate these changes with those occurring in the blood fractions (plasma, erythrocytes and leukocytes) of these subjects for use as potential clinically relevant biomarkers for early disease diagnosis. PUBLIC HEALTH RELEVANCE: The successful completion of these studies will lead to the development of robust and sensitive bioanalytical mass spectrometry methods for comprehensive lipid analysis, and for the identification of biomarkers of diabetes and diabetic complications, and hepatocellular carcinoma. The results from this work will therefore provide information leading to the development of effective clinical methods for the early detection and monitoring of these diseases.

The goal of this protocol is to determine whether staphylococcal toxins (superantigens) contribute to the T cell activation found in atopic dermatitis (AD) and study the role of S. Aureus infection in the pathogenesis of this common skin disease. Specific Aim One will determine whether AD is associated with a selective expansion of T cells in their skin lesion. Specific Aim Two determines whether the selective stimulation of T cells in AD is clonotypic or diverse by cloning and sequencing the individual T cell receptor (TCR) transcripts amplified from AD skin lesions. The final specific aim investigates whether S. Aureus growing on the skin of patients with an exacerbation of AD due to infection, produce toxins known to act as bacterial superantigens E.G. Staphylococcal enterotoxins (SES) and correlate these findings with the TCR VB gene usage of the t cell infiltrate from skin biopsies of the culture site. In addition, nonlesional skin of patients with AD and normal controls will be patch tested with staphylococcal exotoxins to determine they can induce eczematoid skin lesions.

Classic antipsychotics treat schizophrenics's hallucinations, but not their social withdrawal and flattened affect. These drugs also produce extremely unpleasant motor side effects, because these compounds were originally selected based on their ability to produce motor disturbances in rats. Clozapine is an atypical antipsychotic which treats both types of symptoms with very few motor side effects, but it can also produce a potentially lethal side effect, agranulocytosis. Thus, a tremendous demand exists to develop safer and more effective atypical antipsychotics. In order for a compound to show promise as an antipsychotic in preclinical trials, it must have appropriate behavioral and biological actions. The goal of this research is to develop a better method for detecting atypical compounds by testing the hypotheses that atypical antipsychotics will differ from classic antipsychotics in terms of their effects on PCP-induced social withdrawal with repeated administration. In particular, atypical antipsychotics are predicted to be more efficacious at alleviating PCP- induced social withdrawal with repeated administration. In particular, atypical antipsychotics are predicted to be more efficacious at alleviating PCP-induced social withdrawal. Atypical antipsychotics are also predicted to produce greater tolerance to the initial increases in extracellular dopamine in mesocorticolimbic structures, while having a limited effect on striatal dopamine, compared to typical antipsychotics. This hypothesis will be examined using simultaneous sampling from pairs of structures using in vivo dialysis coupled to HPLC. The results will provide scientists with: 1) a profile of social behaviors in a rodent model for candidate atypical compounds to restore in order to be effective at treating both types of symptoms in schizophrenia; and 2) a neurochemical model with limited effects on striatal dopamine and only transient elevations in mesocorticolimbic structures. These studies would improve the manner in which compounds are selected for human clinical trials and enhance our understanding of biological mechanism involved in the atypical antipsychotic effect.

Several diverse metabolic events become compromised when mammalian cells are made deficient in essential amino acids or when charging of their tRNA is blocked by amino acid analogs. This rapid general demise of cell function can be due to inhibition of phosphofructokinase (PFK) by uncharged tRNA. It has now been demonstrated that when tRNA is added to PFK in any assay dependent upon the reassociation of inactive, dissociated enzyme subunits, nanomolar concentrations cause complete inhibition. The model for control suggests that charged tRNA becomes associated with EF-1, which is specific for aminoacyl-tRNAs and is present in sufficiently high concentrations in cells to sequester the charged forms from an inhibitory role. Support for this model include: (1) the rapid onset of inhibition of glycolysis and glucose uptake upon amino acid deficiency; (2) the unique role of the product of PFK activity, fructose-1,6-diphosphate, in reactions of peptide chain initiation, particularly its role as a co-factor for purified eIF-2B, the GDP/GTP exchange factor; (3) the correlations of this interaction with the cellular and molecular lesions of insulin insufficiency; (4) the recognition that the anomalous role of high concentrations of cAMP as a stimulant of peptide chain initiation in energy depleted or gel-filtered cell lysates correlates with its stimulatory action on PFK as an analog for the positive effector, adenosine-5'-monophosphate; and (5) the role of fructose-1,6-diphosphate in the formation of glyceraldehyde-3- phosphate, a substrate for synthesis of ribose-5-phosphate via the non- oxidative portion of the pentose phosphate pathway, which, as precursor of phosphoribosylpyrophosphate, is essential for nucleic acid synthesis.

PROJECT SUMMARY/ABSTRACT The project ?Expansion of Training and Program Components to Maintain Manufactured Food Regulatory Program Standards? is designed to further advance the manufactured food program in the State of Wyoming. Wyoming Department of Agriculture, Consumer Health Services (WDA/CHS) has made great strides in improving the manufactured food program statewide with the assistance of the Manufactured Food Regulatory Program Standards (MFRPS). In October 2017 WDA/CHS was elated to learn that after the 60 month Program Assessment Validation Audit (PAVA) the program was in conformance with the MFRPS. WDA/CHS takes great pride in providing a quality program for our inspection personnel and industry to be part of. This project will address four (4) goals. The first goal is to adequately train our inspection personnel and continue to meet MFPRS requirements. This training will equip inspection personnel with the knowledge and ability to conduct quality inspections and further advance WDA/CHS to be part of an Integrated Food Safety System. The training received will also be used to provide education to industry. The second goal of this project is for WDA/CHS to continue to show incredible growth in outreach to our clientele. Through this project WDA/CHS will develop and produce a guide for Manufactured Food processors addressing 21 CFR 117 and Food Safety Modernization Act (FSMA) regulations and publish the Wyoming Food Safety Rule revision. This project includes outreach to establishments required to have a Preventative Controls Qualified Individual by providing FSPCA Preventative Controls for Human Food Training. The third goal of this project is to adequately equip our inspection personnel to complete the inspections. Additionally, WDA/CHS plans to expand our routine sampling program. All of the goals listed above are incorporated into the final goal of this project, which is to advance the manufactured food program and continue to conform to the MFRPS.

OBJECTIVE To determine whether mRNAs are expressed in the rhesus placenta which could encode a soluble MHC class I molecule. RESULTS An unusual feature of both the human placental MHC class I molecule HLA-G and the rhesus molecule Mamu-AG is expression of multiple protein isoforms and splice variants. Splice variants also exist for HLA-G which encode a soluble protein but the presence of a mRNA encoding a soluble rhesus MHC class I molecule has not previously been detected. We cloned a fragment of the Mamu-AG gene from rhesus monkey genomic DNA to define the sequences of intron 4 and 5 and noted that the sequence of intron 4 was highly homologous to that of HLA-G (88% identical), which is similar to exon 4 homology with HLA-A sequences (~91%), but higher than that for intron 5, suggesting that evolutionary drift of intron 4 has been restrained and implying selection for functionally important sequences. We then conducted RT-PCR analyses with placental mRNA using an upstream primer specific for Mamu-AG exon 4, and a downstream primer which would amplify through the 3U-untranslated region and allow diagnostic identification of Mamu-AG transcripts via the premature stop codon homologous to HLA-G. RT-PCR products were amplified from placental RNA, cloned and sequenced. A stop codon (TAG) is present in the exact codon location of the TAA stop codon in the soluble HLA-G reading frame of intron 4. The predicted amino acid sequences of the carboxy-terminal end of the 4th exon and the intron 4 peptides indicate identity with the human sequence at 16/21 residues, and conservative substitutions (e.g., G to S) at the other 5 residues. Further studies cloned and defined the sequences of the other introns of Mamu-AG. FUTURE DIRECTIONS These results suggest that the rhesus monkey will be a model for addressing the physiology of soluble Mamu-AG. We will evaluate 1) the expression of soluble mRNA in the fetal membranes, 2) whether antibodies against soluble HLA-G recognize the rhesus molecule, and 3) rhesus amniotic fluid and fetal serum for the presence of soluble MHC class I molecules. KEY WORDS maternal-fetal immune tolerance, placenta, amniotic fluid, splice variant FUNDING NIH HD 26458, HD34216

Hematopoietic stem cell transplantation (HSCT) is now the second most frequent major organ transplant in the US and is used as the treatment of a variety of malignancies and bone marrow failure disorders. An estimated 45,000 transplants are performed each year, 2000 in patients under 20 years of age. There are more than 90,000 survivors of this procedure in the US. HSCT recipients and their families are extremely vulnerable as a result of the physical and psychological demands of the treatment, the geographic dislocation, and physical and social isolation. During the transplant recovery process, care shifts back from the transplant center to the child's home and local treatment center with the parents assuming the primary responsibility for the child's care. Traditional hospital-based interventions have focused on the peritransplant period, but given the prolonged and demanding period of recovery (6-12 months), alternative interventions are needed. To address these issues, we are collaborating to develop a highly transportable transplant-specific module, adapted from the well-established Comprehensive Health Enhancement Support System (CHESS), HSCT-CHESS. This module will be an interactive, web-based, health information and support system for pediatric HSCT parents and families. Through a randomized controlled trial of 190 parents at four pediatric HSCT centers across the US, we will evaluate the impact on health-related quality of life (HRQL) of HSCT-CHESS, comparing it to standard care over a nine-month intervention period. Information about the impact of pediatric HSCT on the family will also be collected over the same time period. This proposal sheds new light on the application of interactive health communication systems to a new population, by formally exploring the mechanism of action and identifying who may benefit most by this approach. The study also directly investigates the link between interactive health communications and HRQL. The results of the proposed family-centered evaluation will have important implications for family adaptation in other intensive cancer treatments. This intervention has the potential to serve as a model in other clinical situations in which complex care is shared by health care providers with different expertise in geographically distant sites, particularly those in which the patient and family must be actively engaged in care coordination and disease management. [unreadable] [unreadable] [unreadable]

Leveraging Family Data to Identify Genetic Variants for Sleep Apnea PROJECT SUMMARY (ABSTRACT) Obstructive Sleep Apnea (OSA) affects more than 10% of the population, especially Hispanic- and African- Americans, and is associated with profound cardio-metabolic morbidity. Through the Cleveland Family Study (CFS), a genetic epidemiological study of rigorously phenotyped families ascertained through probands with OSA, we have established that OSA has a strong genetic basis and have identified promising areas of linkage to inform genetic association analysis and sequencing efforts. To meet the objectives of this RFA, we intend to efficiently utilize existing data from th CFS as well as newly available genotype and sleep phenotype data from major NHLBI cohorts (Sleep Heart Health Study cohorts of ARIC, CHS and Framingham Heart; MESA; MrOS- Sleep, Starr County Health Study, and the Hispanic Community Health Study). Our primary phenotype is the continuous trait, the apnea hypopnea index (AHI), derived from sleep studies rigorously analyzed and archived at our central Sleep Reading Center. In toto, the sample includes 1200 CFS family members and ~11,000 members from NHLBI cohorts, including admixed populations at high risk for OSA and likely to harbor rare variants. Capitalizing on the power of family designs combined with focused sequencing efforts and modern statistical tools, including methodological advances by our research team of leading genetic statisticians, geneticists and sleep epidemiologists, we propose to use complementary strategies designed to identify common as well as low frequency and rare variants associated with OSA. We will: 1) leverage information from areas of linkage to the AHI to prioritize genes for further testing and for targeted exon sequencing; and 2) perform whole exome sequencing in individuals selected from our most informative families and extreme OSA phenotypes, a sample likely enriched with rare variants. Our hypotheses and aims will be addressed using advanced gene-based analysis (SNP-set kernel association tests) and meta-analysis in a multi-stage design consisting of discovery and validation phases. Linkage information will be incorporated into association analysis to improve the chance of true discovery. Weighted methods and bioinformatics approaches (using gene networks) will be used to increase analysis power for detecting rare variants. Analyses will control for covariates including population stratification using principal components, local ancestry, and family relatedness. Multiple comparison adjustments will be carried out to control for overall type I error. Additionally, we will develop novel statistical approaches to meet the challenges of this study as well as other studies supported by this RFA, including methods for analyzing family and unrelated samples when multiple rare and common variants contribute to phenotypic variation. RFA HL-07-012 provides a critical opportunity to leverage the family data from the CFS, newly available data from large cohorts, statistical advances and advanced sequencing technology to together fill a major need to discover and replicate functionally important variants for OSA that may serve as targets for novel therapies for this serious health condition.

Vitamin D deficiency affects between 31% and 82% of the U.S. population, depending on race/ethnicity, and is associated with hypertension, as well as other cardiovascular outcomes. Several randomized clinical trials of the efficacy of vitamin D supplementation in reducing blood pressure have been conducted with mixed results. These inconsistent clinical trial findings may be explained by the substantial inter-individual variabiliy in the ability of a given vitamin D dose to raise circulating 25-hydroxyvitamin D [25(OH)D] concentrations and, thus, to influence vitamin D-related outcomes. This variability is influenced by genetic variants. However, these variants have not been used to guide the dose of vitamin D supplementation. Our long-term goal is to use Genotype-gUIded vitamin D supplEmentation (GUIDE) to improve vitamin D-related health outcomes. The objective of this application is to conduct a randomized, double-blind, placebo-controlled clinical trial of daily oral vitamin D3 for three months in 558 healthy participants between the ages of 30 and 70 from an existing population-based study. The vitamin D3 dose will be determined by the number of risk alleles in two vitamin D- related genes. The specific aims are to determine the efficacy of GUIDE in 1) achieving 25(OH)D concentrations of 20-50 ng/mL and 2) lowering blood pressure.

We described 3 patients with hypertrophic cardiomyopathy who after septal myotomy-myectomy had severe mitral regurgitation. All 3 patients had complete relief of the left ventricular outflow obstruction by the myotomy-myectomy operation but nevertheless, severe symptoms of cardiac dysfunction persisted post-operatively. At reoperation, ruptured mitral chordae tendineae were found in 2 of the 3 patients and severe posterior leaflet systolic anterior motion in the third patient. It is likely that the mitral valve damage was done at the time of the myotomy-myectomy operation. Thus, this potential danger must be kept in mind at the time of myotomy-myectomy in these patients.

Immunoperoxidase studies are designed to determine the presence and intracellular location of measles virus polypeptides in CNS cells of chronically infected hamsters. Virus isolated from acutely and chronically infected hamsters will be compared by polypeptide composition. To maintain the characteristics of neural cell growth, all virus will be maintained in reaggregated brain spheroid cultures prior to biochemical characterization. Immunoprecipitation methods will be used to measure the serum antibody response to acute and chronic SSPE virus infection in the hamster. Defined measles polypeptides will be used to "vaccinate" hamsters of various ages prior to exposure to intracerebrally inoculated measles virus. Following this, animals will be analyzed to determine the protective value of individual antibodies to specific polypeptides or the ability of such antibodies to induce "antigenic modulation" of virus leading to persistent infection.

Role of cytoplasmic and nuclear skeletal structures in the function and metabolism of RNA will be further investigated. The apparent association of poly(A) with the cell skeleton will be pursued and the apparent solubility of Friend cell polyribosomes will be investigated and compared to HeLa. A new set of small RNA molecules located in the nucleoplasm and apparently synthesized by a polymerase of one-type activity will be further studied. These RNAs show a profound change in different animal species and are possibly related to the state of cellular differentiation. Macromolecular metabolism will be further studied in anchorage dependent cells suspended in methocell and reattaching on plastic surfaces. Profound changes in protein synthesis and messenger processing are noted and will be characterized. Changes in message composition have been detected and will be measured. Changes in plasma membrane composition of suspended anchorage independent cells will be measured. Evolution of phenotype between anchorage dependence and independence can be seen at the biochemical level and will be studied with cell lines having intermediate phenotypes.

OBJECTIVE: To conduct a research program on the biology and treatment of murine colon carcinoma. APPROACH: The general goal of our colon tumor project is to obtain improved treatment methods (mainly through the use of chemotherapy or surgery in combination with chemotherapy) for colon tumors in mice. These methods will then function as leads for clinical trials against the human disease. The general method of research involves the testing of various chemotherapy agents and combinations of agents against transplantable colon tumors in mice. We are currently using four transplantable colon tumors that were developed in these laboratories. One of these colon tumors is highly metastatic and surgical removal of 1/2 g tumor growths will not cure the animals because cells are shed prior to surgery through the blood and lymph to distant organ sites. We are testing active single agents and combinations of agents following surgical removal of the primary tumors in an effort to determine the curative potential of the various drug treatments.

PROJECT SUMMARY It has long been known that methylation of genomic DNA correlates with gene expression. However, the structural mechanisms that underlie these observations remain obscure. In this project, we will pursue several innovative strategies for studying how methylation affects transcription factor (TF) binding. First, we will use Methyl-SELEX-seq ? a novel experimental method developed in the previous cycle of this grant that uses barcoded mixtures of methylated and unmethylated DNA ligands ? to create detailed maps of the effect of methylation on binding affinity for a broad panel of human transcription factors from various structural families. Second, we will perform detailed computational analyses and follow-up experiments to test the hypothesis that methylation causes local changes in DNA shape, which in turn modify TF binding affinity. We have shown that adding a methyl group in the major groove changes the geometry of the minor groove and enhances the electrostatic interaction between negative charges in the DNA minor groove and positively charged amino- acids in the TF. We will extend these analyses to other DNA modifications, as well as a wider range of DNA shape parameters and associated flexibility parameters. By building interpretable TF-DNA recognition models that integrate base, shape, and flexibility features using a powerful new machine learning framework developed in the previous funding cycle, we will make specific predictions regarding sequence and methylation readout mechanisms, and validate these using SELEX experiments with mutated TFs. To assess to what extent our quantitative models for binding to naked DNA built from SELEX data are predictive of binding to genomic DNA in the context of the living cell, we will perform detailed parallel analyses of SELEX and ChIP- seq data for Hox proteins and other TFs. Finally, to study the relationship between DNA binding and gene expression control in human cell lines, we will exploit Survey of Regulatory Elements (SuRE-seq), a novel massively parallel reporter assay that provides unique information about the autonomous transcriptional activity for each of >108 overlapping genomic fragments.

This proposal seeks to support and enhance Down Syndrome research capabilities via the DS-CONNECT registry by leveraging PCORnet, the National Patient-Centered Clinical Research Network. Since 2014, the Patient-Centered Research Institute?s investment in creating PCORnet has resulted in a diverse national network covering over 100 millon lives: providing curated, electronic health record data and claims as real world evidence along with heighted patient/clinician/health system engagement across participating organizations. We will link PCORnet to DS-CONNECT and test capability in three dimensions: 1) increasing DS-CONNECT registry enrollment, 2) extract clinical observations, treatments, and outcomes from PCORnet for DS-CONNECT patients, and 3) conducting cognitive assessment via DS-CONNECT in the PCORnet population. People with Down syndrome and intellectual and developmental disabilities experience poorer health-related quality of life than people without a disability. For people with Down syndrome, person factors, such as communication ability and social skills can also impact health in terms of access to quality health care, opportunities for social participation, and challenging behavior. Self-determination which is defined as people with disabilities engaging in goal-directed actions that enhance quality of life has been shown to be a critical factor in improving employment and health-related outcomes. Specifically, the relationship between sleep quality and cognition may play a significant role in community living and employment for adults with Down Syndrome. We will invite PCORnet DS-CONNECT participants to complete the Self-Determination Inventory (SDI) and link SDI scoring of problem solving and goal setting cognitive deficits for Down Syndrome patients with Obstructive Sleep Apnea (OSA) diagnoses recorded in PCORnet. This will be one of the first studies to robustly integrate data regarding the relationships between health and community participation for people with DS, which is made possible linking health data from registry participants in PCORnet using DS-CONNECT and the Self-Determination Inventory (SDI) System Data Dashboard. As such, our proposed research will have far reaching implications for supports and services for people with DS and the influence of health on participation. The addition of self-determination will help researchers better tailor interventions to support specific aspects of cognition that interfere with health and participation outcomes and create a framework for exploring other associations by integrating data to inform intervention development and future clinical trials.

Recent evidence suggests that H2O2, produced in response to various extracelluar stimuli, functions as an intracellular messenger. H2O2 is also a precursor of hydroxyl radicals that cause irreversible damage on cellular components. This dichotomous function of H2O2 predicts that the production timing and local concentration of H2O2 be stringently regulated. The thioredoxin peroxidases, TPxI and TPx II, are members of the peroxiredoxin (Prx) family that participates in growth factor and cytokine signaling by modulating intracellular concentration of H2O2. In an effort to understand how TPx enzymes cooperate with different agonist signalings, the distribution of TPx proteins has been studied in subcellular fractions obtained by differential centrifugation. TPx I and II were detected in organelle and membrane fractions, as well as cytosol. When the membrane pool was further analyzed on Nycodenz-density gradient, TPxs I and II showed wide distribution throughout light and heavy membrane structures. Furthermore, we detected a part of TPx proteins in Triton X-100-insoluble membrane fraction. Immunofluorescence staining showed an obvious punctuate pattern of TPx II protein in HeLa cell, similar to endosomal staining. These results indicate that TPxs I and II may localize in various membrane structures, including endosomes and lipid raft. Interestingly, treatment of HeLa cells with EGF or H2O2 induced the accumulation of TPx II in perinuclear region, suggesting a stimulation-dependent translocation of TPxII. We also observed that TPxs I and II, but not other Prx isoforms, are present in nuclei fraction purified from HeLa and NIH3T3 cells. The amount of TPxs I and II in nuclei fraction increased in response to serum stimulation and decreased in response to treatment with H2O2 and UVB. TpxI and TpxII contain a site [TP(K/R)K] of phosphorylation by cyclin-dependent kinase (CDK). CDK2, CDK4, and CDK6 immunoprecipitated from cell extracts phosphorylated wild type TpxI and TpxII, but not mutant Tpx proteins with the Ala for Thr substitution at the putative phosphorylation site. The threonine phosphorylation was also confirmed with CDKs co-immunoprecipitated by antibodies against cyclin A, D, and E. Taken together, Tpx enzymes appear to be mobile protein that respond to the local need by translocation. Either translocation to nucleus or their catalytic activity in nucleus is likely regulated by CDK-dependent phosphorylation.

Large inhalable particles exist in the workplace and can constitute a substantial amount of inhaled exposure (50% or more). Particles >20 m primarily deposit in the oro-nasal cavities. Symptomatic health effects (e.g. acute or chronic rhinitis, chronic pharyngitis, chronic sinusitis, nasal cancer, chronic laryngitis and gastro- intestinal diseases) occur in many industries; such effects may be due to the presence of large inhalable particles in these workplaces. However, measurement capabilities for counting and sizing particles between 20 and 100 m are limited. Although large particles settle through air quickly (terminal settling velocity of 100 m particle = 0.3 m/s), these particles are also capabl of delivering very high doses to affected tissues (a 100 m particle weighs approximately 1 g). Thus, a real-time instrument is needed to capture the timing and magnitude of such exposures. There are two specific aims for this project. The first aim optimizes the design of a portable inhalable particle spectrometer to segregate inhalable aerosols as a function of particle size and incorporates optical detection for real-time measurement of particle size and concentration. The second aim evaluates the sampling efficiency, cutoff curves, and other performance measures in calm air and low-wind speed environments. The career development plan outlines the activities that will be taken during the K-award period. This plan describes training in three specific skillset areas: engineering design, aerosol optics, and experimental techniques. The career development plan also describes the specific training the mentors will provide, as well as important milestones, steps, and a timeline for achieving the short and long term career goals. One-on-one mentoring (for both research and career development) is described.

A multi-center study of oxacillin resistant S. aureus is in place and should be completed within a year. Phage types have been, and will continue to be used to fingerprint strains of the oxacillin resistant species. We have evidence that strains with oxacillin resistance not reversible by clavulanic acid are few in number and that oxacillin resistant S. aureus is represented in many U.S. hospitals by a single predominant strain. S. aureus in which oxacillin resistance is reduced in the presence of clavulanic acid, the hyperbetalactamase producers, have another, distinct phage pattern. We also are studying loss rates of oxacillin resistance after the strains are cultured from the treated patient. This phase will include initial sensitivity tests done directly from clinical samples, followed by tests done during serial subcultures. This has important bearing on sensitivity testing of these S. aureus strains, since they begin to lose resistance immediately, on initial cultures.

This investigator-initiated new R01 application, in response to PA-10-067, proposes to reduce depression and cancer-related emotional distress, while also improving health behaviors in adult hematopoietic stem cell transplantation (HSCT) survivors using an internet-based intervention in a multi-center randomized controlled clinical trial (RCT). Among adult long-term cancer survivors, those who received HSCT live with some of the highest risks for life-threatening chronic health conditions including cardiovascular disease, diabetes, osteoporosis, and recurrence or second cancers along with other risks. Even more prevalent in HSCT survivors are psychological symptoms that inhibit quality of life, specifically depression and cancer-related distress. Our previous internet intervention has demonstrated efficacy in reducing depression and distress with survivors at a single site, and has pilot tested the efficacy of providing a survivorship preventive care plan. With the proposed improvements and the extension of the intervention to multiple transplant centers we expect to strengthen both the reach and efficacy of the intervention while we advance the science of internet-based providing of health behavior change models to cancer survivors. Specific aims of the project are to: 1) determine whether 2-10 year HSCT survivors with elevated depression or cancer-related distress who are randomized to receive access to a tailored internet-based program report reduced depression and distress when compared with control group survivors who receive a survivorship care plan by mail and delayed access to the internet site (enhanced usual care), and 2) determine whether survivors with low survivorship preventive care adherence (PCA) who are randomized to receive access to the tailored internet-based program, including a survivorship preventive care plan, report increased PCA when compared with control group survivors. The study will examine main effects of the intervention as well as moderating (age <40, chronic graft versus host disease, rural residence, lower income) and mediating effects (health self-efficacy, knowledge of survivorship needs) per Protection Motivation Theory. Further analyses will identify risk factors for disparities in survivorship preventive care adherence in HSCT survivors. The five-site multi-center RCT will enroll N=1200 adult HSCT survivors with internet access. Assessments will occur at baseline, 3 and 12 months. Participants without internet access will be invited to complete the baseline assessment for participation in the risk factor analyses. To our knowledge, this study will be the first to explore the efficacy of a tailored internet-based intervention, enhanced with options for alerts and reminders through individualized SMS messaging, mobile applications and social networking media, to target health behaviors in a cancer survivor population. The program is cost-effective and readily tailored. If effective, the program could be disseminated nationwide for long-term HSCT survivors in all transplant centers and would provide a framework for similar survivorship care models in non-HSCT cancer survivor populations. PUBLIC HEALTH RELEVANCE: Among adult long-term cancer survivors, those who receive hematopoietic stem cell transplantation (HSCT: also called blood, marrow or umbilical cord blood transplantation) live with some of the highest risks for life-threatening chronic health conditions including cardiovascular disease, diabetes, osteoporosis, and recurrence or second cancers. Also prevalent in HSCT survivors are symptoms that inhibit quality of life such as depression and cancer-related emotional distress. In a study with five sites across the nation, this research is designed to test whether a tailored internet-based survivorship program with methods for managing health behaviors improves depression, emotional distress and adherence to health care guidelines in adult HSCT survivors.

Normal tissue complication probability (NTCP) models can be used to individualize radiation therapy treatment planning, potentially by guiding the design of the dose distribution. Our goal is to produce improved NTCP models, based on dose-volume, patient, and disease characteristics, for head and neck and lung treatment complications. Under the previous grant, we developed a software system, which enables the construction and convenient analysis of databases of 3-D treatment plans, including datasets from multiple institutions. Using data thus obtained, we will construct predictive models using multi-metric logistic regression methods, which include dose-volume terms as well as other patient and disease-related factors. The robustness of variable selection will be tested with bootstrap methods. For lung treatment plans, we will recompute lung and esophagus dose-volume histograms using a novel Monte Carlo-based technique, to improve the consistency and accuracy of the database dose distributions. Under Specific Aim (SA) #1, Improvements in post-RT late pneumonitis/fibrosis NTCP models, we will: (a) expand the currently available Wash. Univ. dataset (from 166 pts. to an estimated 450 in 4 years), (b) study the inclusion of new factors such as spatially-varying sensitivity and pretreatment pulmonary function tests, (c) test and refine our model using the RTOG 93-11 dataset (113 pts.), and (d) test and refine our model against data contributed by Duke University and the Netherlands Cancer Institute (an estimated 550 pts.). Under SA #2, Improvements in acute esophagitis NTCP models, we will: (a) accrue more patients (from 166 to an estimated 450 in 4 years), (b) incorporate new factors such as partial-circumferential irradiation and other metrics based on the shape of the high dose region, and (c) test and refine our model using new data contributed by the Netherlands Cancer Institute (an estimated 300 pts.). Under SA #3, Improvements in post-RT parotid salivary function/xerostomia models, we will: (a) test the effect of spatial placement of high-dose regions, (b) use the model to analyze the radio-protective effect of Amifostine on salivary function in an ongoing intensity modulated radiation therapy trial, and (c) test/refine our model against the University of Michigan xerostomia dataset. In addition, we will establish publicly archived databases with convenient and freely available software tools. We hypothesize that this research will result in a significantly improved ability to predict, on an individualized basis, the risk of xerostomia, pneumonitis, or esophagitis, and could thereby lead to improved radiation therapy treatments.

Atherogenesis in the adult-onset diabetic is difficult to explain satisfactorily on the basis of existing knowledge concerning insulin, glucagon and growth hormone. Additional controlling hormone(s), particularly those involved with the mobilization of depot fat, would seem to be required for a more nearly complete description of the mechanisms involved in diabetogenesis and in the concomitant or subsequent atherogenesis. The existence of such hormones, which are probably produced by the pituitary or hypothalamus, has been clearly indicated by previous investigations, but they have not been isolated and characterized and their relation to various pathophysiological states established. We propose that the over-production of such hormones leads to increased levels of plasma fatty acids which can then lead to diabetes because of insulin resistance and to accelerated atherogenesis because of hyperglyceridemia. These hormones occur in the urines of fasted, normal adults and of diabetics. The hypothesis will be tested by: (a) isolating the active materials from these sources using chromatographic procedures, (b) determining their amino acid sequences, (c) developing radioimmunoassays for their quantitation in plasma, and (d) using such procedures to ascertain the circulating levels of the active materials in pre-diabetics, diabetics, in persons at risk for atherosclerosis. Such measurements will also be carried in persons in other pathophysiological states with the objective of ascertaining the factors which control the release of these materials from their gland of origin.

The overall objective of this research is to understand the mechanism for the enzymatic assembly of cholesterol, the control of this process, and the relationship of this process to lipoprotein assembly in the liver. Specific objectives include the isolation and purification of Sterol Carrier Proteins and delineation of the relationship of these substances to lipoprotein assembly. Other objectives include the determination of chemical structures of intermediates in cholesterol biosynthesis, as well as the mechanism of inhibition for hypocholesterolemic drugs.

Project Summary Alzheimers disease (AD) is the most common late-onset neurodegenerative disease. It is known to have strong genetic influences. Over the last decade using advanced high-throughput genotyping/sequencing technology, researchers have identified nearly 30 AD susceptibility genes/loci. However, these loci account for only a portion of AD heritability. Furthermore, almost all of them have been identified for the risk of developing AD, where disease status is the primary outcome of interest. In our paper (Li et al. AJHG 2002) we highlighted the hypothesis of genetic predisposition for age-at-onset (AAO) of AD, estimating the heritability for AAO of AD to be ~42% and reporting linkage to new genetic quantitative trait loci for AD AAO. Recently, two genome wide association studies for AAO of AD confirmed findings in the APOE gene, but otherwise, there was little overlap. Overall, much remains to be discovered about genetic factors for AAO of AD. If AAO genes can be identified, they will contribute new knowledge about the genetic modifiers of AD, provide excellent intervention targets for delaying the onset of AD, and improve our ability to predict AAO in an individual. The increase in large genome- wide genetic datasets for AD, provides an excellent resource for the comprehensive investigation of the genetic basis of AAO of AD through a well-conceived statistical analysis plan and methods development. Our central hypothesis is that genetic variants exist that regulate the variation of AAO and/or one's age-associated risk. To test this hypothesis, we will use datasets from the Alzheimer's Disease Genetics Consortium (ADGC) and Alzheimer's Disease Sequencing Project (ADSP). These two large datasets will allow us to search for common and rare variants for AAO of AD. Our analysis strategy is unique from previous studies, because we will not only analyze AAO as a quantitative trait in a case-only design as used in the past, but we will also treat AAO as a censored trait by applying novel survival-analysis methodology. The survival-analysis approach will include unaffected subjects whose AAO was censored at the age of enrollment, and allow us to identify genetic variants that are associated with AD risk in an age-dependent manner. To uncover novel genes tied to AAO and develop predictive models of AAO of AD, we propose the following aims: (1) Conduct discovery analysis in the ADGC dataset to identify common and/or rare variants associated with AAO of AD; (2) Replicate AAO findings in the independent ADSP dataset and additional cohorts; (3) Develop a suite of analysis programs to support the proposed analysis in Aims 1 and 2 and accommodate the different data structures included in the Discovery and Replication datasets; and (4) Develop a polygenic risk score model to predict AAO of AD. This proposal promises to provide targets with the potential to delay onset of AD and to advance the future development of personalized medicine utilizing individual genetic burden to predict AD AAO. Furthermore, it will provide innovative tools for data analysis and risk modeling to the genetic research community.

We plan to study two groups of 5-8 adolescent chimpanzees, each group living in a 1 1/2-acre enclosure partially simulating a natural habitat. The initial focus will be on the adaptation of each group to its new environment. Thereafter the principal focus will be on changes in behavior that occur during adolescence and beyond, noting sex differences and individual differences. We will especially focus on changes in behavior in the sphere of aggression, sex, and attachment, and hormonal correlates of these behavior patterns. We expect to delineate these patterns, to measure changes in their frequency and duration over the years of adolescence, and to search for endocrine correlates. We will differentiate such behavior patterns by age; sex; phase of menstrual cycle; hormonal condition as occurring spontaneously and as manipulated experimentally; and environmental conditions. Our semi-natural chimpanzee facility will permit repeated endocrine measures over several years; the administration of hormones for experimental purposes; continuous, close-range observation of behavior; a simple social system focusing on peer relations; systematic comparison of behavior in this semi-natural habitat with behavior of chimpanzees in their fully natural habitat and with human adolescents in roughly similar situations. Hormone assays will be conducted on a regular basis over the next two years including measurement of several androgens, estrogen, progesterone, the gonadotropin LH, and biogenic amines. The research will be conducted by an inter-disciplinary group: ethology, psychiatry, biochemistry, statistics, and sociology. There will be a long-term, collaborative arrangement between the Stanford research and a longitudinal study of chimpanzee behavior in the natural habitat in Tanzania.

Mutations in parkin are largely associated with autosomal recessive juvenile parkinsonism (AR-JP). The underlying mechanism of pathogenesis in parkin-associated Parkinson's disease (PD) is thought to be due to the loss of parkin's E3 ubiquitin ligase activity leading to accumulation of parkin substrates due to failure of the ubiquitin proteasome system. A large number of possible parkin substrates have been identified, yet their role in the pathogenesis of PD due to parkin mutations have yet to be clarified. Moreover, the post-translational modifications that potentially regulate parkin's function are not known. We propose to generate and characterize parkin knockout mice to test the role of proteasome dysfunction in PD. Furthermore, we propose to characterize and identify parkin substrates, and identify potential post-translational modifications of parkin that regulate its function, thus providing important new information about the role of UPS dysfunction in PD. To accomplish these goals we propose the following specific aims. In Specific Aim #1 we will generate and characterize parkin knockout mice. In Specific Aim #2 we will evaluate the sensitivity of parkin knockouts to proteasome inhibitors. In Specific Aim #3 we will characterize the Role of Nitrosative Stress on Parkin Function. In Specific Aim #4 we will identify additional and potentially authentic parkin substrates in parkin knockout mice in response to proteasome inhibition. Identification and characterization of parkin substrates and regulatory mechanisms of parkin function may provide novel therapeutics and targets to prevent the toxic effects of this familial associated gene in the degenerative process of PD.

Adolescents are at particular risk for acquiring HIV, and increasing numbers of young African American women are affected by the epidemic. We have developed a new theory-based model from the literature to investigate the impact of provider-patient interaction, mutual exchange of information and psychosocial development on the return for recommended visits to the Fulton STD clinic in Atlanta, Georgia. Previous studies indicated that health care providers could influence adherence to treatment and management of chronic diseases in adults. Most of these constructs have not been operationalized or tested with adolescents. Some questions concerning consumer opinions of providers have been adapted to the adolescent population especially with respect to patient satisfaction and access to care. In addition, a model of adolescent psychosocial maturity suggests that interpersonal skills may increase the ability of adolescents to communicate with providers. However, there is little information about the impact of providers on adolescents who are being treated for STDs to increase their likelihood of returning for future STD visits. For our last submission, we piloted the questionnaire with 60 African American adolescent girls and young women from the Fulton Clinic, obtained feedback about the draft questionnaire, and found that 33% of participants indicated high mutuality scores. After the second review, we have adjusted the plan in response to reviewers to refine our outcome variables, address potential confounders and developmental issues, and incorporate focus groups in the initial and final phases of the project. This project "develops and tests new methods" under the NIMH RO3 grants program, using a unique window of opportunity to complement the currently NIMH funded grant (R01 MH61210) designed to test an HIV prevention intervention (the HORIZONS Project) at a state supported STD clinic that serves a large population of African American adolescent girls aged 15-19 years old. This new initiative will develop and test culturally and developmentally sensitive measurement tools specifically for adolescent girls being treated for STDs concerning the association of provider-patient relationships and mutuality of exchange of information with return for subsequent health care visits. If these measures are useful, then an intervention can be designed to enhance provider-patient mutual exchange of information and reduce the future risk of STDs and HIV infection.

Natural products are extremely important sources of bioactive compounds for agricultural and pharmaceutical applications. Enzymes involved in secondary metabolism hold great potential as biocatalysts that may be used in the efficient synthesis of fine chemicals and high value pharmaceuticals. In this collaborative work between a metabolic engineering group and a structural biology group, we will harvest this potential towards the one-step synthesis of the blockbuster drug simvastatin (Zocor.). Simvastatin is currently synthesized from the natural product lovastatin via inefficient, multistep processes. Our proposed biosynthesis of simvastatin will result in a completely novel process that can be an attractive alternative over the current chemical routes. The central enzyme in this study is LovD, an acyltransferase from the lovastatin biosynthetic pathway. We have performed extensive, preliminary biochemical characterization of this enzyme to show that LovD is a simvastatin synthase, and can be potentially engineered into a powerful biocatalyst for simvastatin biosynthesis. This proposal will examine the following specific aims: AIM 1: Directed Evolution of LovD. We will use directed evolution methods to improve the catalytic efficiencies of LovD towards simvastatin synthesis. We have developed a high throughput screening assay based on the formation of simvastatin. AIM 2: Structure-Based Engineering of LovD. The X-ray crystal structure of LovD will be pursued in the Yeates Lab. Rational mutagenesis of key residues identified from structural analysis will be performed to probe LovD function and improve LovD catalytic properties towards simvastatin synthesis. AIM 3: Metabolic Engineering of E. coli as a whole cell biocatalyst. We will engineer the multidrug transporter system of E. coli to improve its efficiency in exporting simvastatin to the extracellular space. This will improve the conversion of the whole cell reactor at high product concentrations. AIM 4: Direct Biosynthesis of Simvastatin from A. terreus. We will metabolically engineer A. terreus to be blocked in lovastatin biosynthesis, but robust in simvastatin biosynthesis. Project Narrative We have proposed biochemical and structural studies to investigate a simvastatin synthase recently identified from our laboratories. We will use protein and metabolic engineering methods to develop a whole cell biocatalyst that can biosynthesize simvastatin. This work will represent an important milestone in biocatalysis, application of enzymes towards the synthesis of a compound as commercially important as simvastatin has not been reported. The successful outcome of engineering a natural product biosynthetic enzyme into a useful biocatalyst may lead to additional efforts to examine this class of fascinating enzymes from a biocatalysis prospective. At the same time, the outcome of the proposed work will provide important scientific insight into protein engineering, enzyme structure and function, E. coli membrane transport, and Aspergillus metabolism.

The proposed MARC U*STAR Program at the Rio Piedras Campus seeks to improve an already exemplary program by increasing the number of trainees who enter prestigious graduate programs and with new strategies designed to ensure that more of its trainees will be in positions to enter into the mainstream of the nation's scientific community. To this end, the Program will continue to recruit highly talented students and provide them with a mentored intramural research experience that includes a well-defined project that leads to results which will be presented at scientific meetings or published and which will be described in a tesina. Emphasis will also be placed on an appropriate summer research at an external site, counseling and assistance in graduate school applications. In order to meet these objectives it will continue to seek positions for 34 trainees. It will establish direct communications with research supervisors who have recruited its ex-trainees in order to gain feedback on research training strategies. It will provide its trainees with workshops that improve their communications skills, particularly English writing, and will emphasize their participation in technical workshops. The Program also seeks funding to accelerate the development of the entry level Biology and Mathematics courses and to further improve those in Chemistry. These activities will not only increase the retention rate and reduce the time to graduation of all Biology and Chemistry majors, but in five years should increase the pool of MARC eligible rising Juniors by at least a 100 students. A series of science promotions will be included in order to direct more of these into careers into biomedical research. [unreadable] [unreadable]

This Facility provides a variety of molecular biology techniques, genotoxicity tests and general tissue and bacterial culture techniques, as well as training in these, to AHFCC members. The Facility is both responsive and proactive to the community it serves. Room 201 in Unit 2 is used for tissue culture and molecular biology and room 324 for bacterial work. The Facility has equipment needed for basic molecular biology: small centrifuges, speed vacs, electrophoresis equipment for general analysis sequencing, and blotting. A tissue culture hood and incubator are in the large of the two laboratories. Artek counters are used to score the Ames and UDS assays. To tube-based thermocyclers are available for PCR experiments. Other major equipment includes an oligonucleotide synthesis unit and Pharmacia SMART HPLC system for micro-purification of proteins. The senior investigators requested the following new services: expression of human and mouse genes using Clontech's 588 Atlas arrays; 32/P-post- labeling the DNA adducts and the single cell gel electrophoresis (COMET) assay for their repair, and Telomerase assay. The requested deep freezer will enable us to offer controlled long-term storage of plasmids, bacterial and mammalian cells. We shall work with the Histopathology Facility to develop analytical routines for image analysis using the requested fluorescence/transmission microscope upgrade and software system. Services currently available include the requested fluorescence/transmission microscope upgrade and software system. Services currently available include: The Ames Salmonella mutagenicity assays, with or without metabolic activation. The 7000 strains, containing defined mutations, will be made available. The hepatocyte DNA repair assays (Williams' test) uses human or rodent hepatocytes in vitro and rats in the in vivo/in vitro version. Oligonucleotide syntheses include carcinogen- modified nucleotides. DNA analysis via Souther blot hybridization and sequencing of enriched PCR analysis. Individual RNA analysis via Norther blot hybridization will be supplemented by the Clontech array. Protein analyses include Western blotting and protein-DNA interactions via gel retardation assays and micro-purification. We supply S9 and bacterial and mammalian cultures. The two excellent technicians provide an experienced and stable resource of experimental techniques and train other AHFCC personnel in tissue culture methods and molecular biology techniques.

The goal of this research is to define murine B cell subsets by surface markers, to determine the functional capacity of each subset with regard to stimulation requirements and effector molecules secreted, and to determine the interrelationships of the subsets. We will search for differences in mitogen receptors, and differentiation antigens and will relate these to known differences in membrane immunoglobulin and complement receptors. The functional capacity of each subset will be measured by stimulation with panels of mitogens and antigens known to elicit different types of responses in mixed cell systems. Neonatal suppression with anti-Lyb reagents should allow determinations of developmental relatedness of each subset. Finally, we will determine whether each subset has the ability to produce any immunoglobulin or whether limitations exist.

Glaucoma is the second most common cause of blindness worldwide. Our broad, long-term objectives are to investigate innovative technologies and methods that will accurately and reproducibly provide the earliest possible evidence of glaucoma and its progression so as to prevent blindness. We have improved on the program that we have pursued for the past ten years in a number of ways, but importantly we have assembled a unique collaborative group for this project. This team includes ophthalmologists, engineers, computer scientists and statisticians, as well as appropriate support personnel, from the University of Pittsburgh, Tufts University, Massachusetts Institute of Technology and Carnegie Mellon University. We have brought excellent investigators from disparate fields to bring new insights, knowledge and skills from outside of ophthalmology to bear on innovations in technology for glaucoma disease and progression detection. We will accomplish this via cross-sectional and longitudinal studies using cohorts of healthy, glaucoma suspect and glaucomatous subjects. Our Specific Aims are to (1) detect the earliest possible evidence of glaucomatous damage and progression. We will compare objective, quantitative ocular structural measurements obtained by ocular imaging and functional measurements, to test the prediction that changes structural functional change, and to characterize those changes, (2) advance optical coherence tomography (OCT) software innovations that assess the intra-retinal layers in the peripapillary and macular areas as well as the optic nerve head (ONH). This aim includes employing innovative image processing and image analysis techniques, as well as new techniques to improve scan quality post hoc, (3) identify the particular clusters of clinical characteristics distinct to specific glaucoma diagnostic technologies resulting in the earliest detection of glaucoma and its progression. We will use innovative automated machine classifiers and state-of-the-art statistical methods which use the best combination of parameters generated by the imaging devices in order to determine the optimal use of each device in assessing disease and progression, (4) advance micron-scale tomographic imaging using OCT for improved understanding of the anatomical and biomechanical properties of the ONH and intra-retinal substructure in the macular and peripapillary regions in health and in glaucoma. These are key areas involved in the glaucomatous process. This experiment is designed to improve our understanding of glaucoma and potentially create a new glaucoma diagnostic. Swept-source and ultra-high speed spectral- domain OCT will be used to obtain detailed information in the ONH, lamina cribrosa and retina. Rapid image acquisition by these devices will minimize OCT scanning artifact, and modulation of OCT light source wavelengths will allow optimization of imaging at various tissue depths. We expect that these studies will lead to our ability to detect glaucoma and its progression earlier than ever before with high sensitivity and specificity, enabling early intervention to prevent glaucoma blindness.

Hutchinson-Gilford Progeria Syndrome (HGPS) is a premature aging disorder that is lethal by age 16. It is caused by a defect in a nuclear envelope protein called Lamin A. Lamins are structural proteins that line the inner surface of the nuclear envelope. Humans have two types of lamins: A-types (Lamin A and C) and B-types (Lamin B1 and B2). All lamins, except Lamin C, are processed for membrane anchorage by the addition of a hydrophobic prenyl (lipid) group at the C-terminus. But for unknown reasons, Lamin A is further processed to remove the anchor. Thus, Lamin A is transiently rather than stably prenylated. Mutations that cause HGPS delete a recognition site in the Lamin A protein required for removal of the anchor. This defective Lamin A protein is called progerin, and is stably rather than transiently prenylated, resulting in organism-wide rapid aging by unknown mechanisms. The enzyme thought to be responsible for removing the prenyl anchor from Lamin A is called Zmpste24, which is a zinc-dependant prenyl protease. The experiments described in this proposal will establish the fruit fly Drosophila as a model to study human Lamin A processing. It is possible to incorporate human genes into the fly genome (so-called transgenic flies). When transgenically expressed in flies, normal human Lamin A is stably, rather than transiently, anchored in the fly nuclear envelope, with no apparent ill effects. This is in contrast to the human situation, in which stable membrane anchorage of Lamin A leads to disease, and suggests that no conserved prenyl proteases in flies are removing the anchor from human Lamin A. Thus the Drosophila nuclear envelope can serve as an ideal environment in which to confirm the interaction between human Lamin A and the protease Zmpste24, which is a critical step that goes awry in HGPS. In Specific Aim 1, normal or processing-defective human Lamin A proteins (including progerin) will be transgenically expressed in fruit flies together with human Zmpste24, and the localization patterns of Lamin A examined with immunofluorescence microscopy. Drosophila larvae possess huge, easily manipulated nuclei in their salivary glands, which permit detailed dissection of nuclear protein dynamics. Since human Lamin A is confined to the nuclear envelope in flies, co-expression with Zmpste24 should release membrane anchorage and cause re-localization into the nuclear interior, thus demonstrating a direct interaction between these two proteins. Specific Aim 2 comprises a genetic and molecular dissection of the Drosophila prenyl protease functions, firstly to confirm they do not interfere with the human processing assay from Aim 1, and secondly to establish the degree to which lamin processing is conserved in flies. Results from this work will support the use of Drosophila as a non-human in vivo environment in which to study human lamin biology, and will reveal underlying generalities about the nuclear envelope and its role in aging. PUBLIC HEALTH RELEVANCE: Hutchinson-Gilford Progeria Syndrome is a devastating and extremely rare premature aging disorder that kills children before the age of 16. The research described in this proposal will contribute to world-wide efforts to find a cure or at least a palliative. But it is also very probable that unraveling the biology of this unusual disease will go some way to explaining the root causes of aging in general, a genetic disease which affects us all.

Project Summary Objectives. The goal of this project is to train a DVM/PhD anatomic pathologist in the field of translational biomedical research to facilitate transition to an independent established investigator. The research questions addressed in this fellowship application focus on the paucity of information regarding the pathogenesis of HIV- and FIV-induced oral disease in juveniles, and the corresponding lack of effective therapies to treat these syndromes. The aims described in this proposal will investigate the virologic impact, contributing factors, and pathogenic mechanisms of this juvenile condition, thereby providing baseline data to establish a novel approach and more efficacious treatment strategy in HIV-infected children. Specific Aims. 1) Characterize oral kinetics of in vivo FIV infection and the relationship to oral dysbiosis, immune dyscrasia, and oral lesion development. 2) Evaluate treatment response of retroviral-induced oral lesions to HAART and microbial-targeted therapy. Relevance to Public Health. Common therapies used to treat HIV-induced oral disease in adults are often unsuccessful in children, resulting in frequent disease recurrence and resistant microbial infections. There is a lack of information regarding virologic effects and HAART on the oral microbiota. It is of exceptionally high impact to define the pathogenesis of juvenile HIV associated oral disease, and test practical, attainable therapies for outcomes that result in reversal of underlying pathologies. Functional understanding of the pathogenic effects of virus-induced immunosuppression on oral disease in HIV-infected children is the first objective of this proposal. Implications of HAART and symptomatic therapy for gingival-stomatitis will be tested in Aim 2 to guide appropriate therapies for this complex disease syndrome. Collectively, this proposal will provide a focused analysis of both cause and cure, aimed to substantially improve management of this condition for more than 2.5 million HIV-infected children. Experimental design. Specific pathogen free (SPF) cats will be orally inoculated with a strain of FIV that reproducibly produces oral lesions. FIV viral and proviral loads in blood and saliva will be monitored over time and compared with hematologic data. Oral lesion development will be evaluated grossly and histologically, and changes in the oral microbiota and cytokine expression will be evaluated by 16S rRNA NGS and MIA, respectively. Following collection of baseline data, changes in viral/proviral status, oral microbiota, oral cytokine expression, and oral lesions will be measured in the presence and absence of highly active antiretroviral therapy (HAART). The synergistic effect of HAART on the resolution of oral lesions will be subsequently measured in the presence and absence of a novel microbial-targeted therapy comprised of dental hygiene, antimicrobial, and probiotic treatments.

The proposed research takes a social psychological perspective to gender differences in group interaction and performance. Previous research has documented consistent sex differences in group members' style of interactions, such that females (vs. males) display a greater amount of positive social - emotional activity and males (vs. females) display more task-oriented activity. Yet we know little about the origin of these differences or their consequences for task performance. The first two studies evaluated the extent to which gender differences in interaction (a) are manifestations of males' (vs. females') greater instrumental traits and females' (vs. males') greater expressive traits, (b) arise from the perception that males are generally more competent than females, and (c) arise from the perception that males have greater legitimate authority than females. A number of researchers have argued that group task performance is highly dependent on aspects of group interaction (e.g., Steiner, 1972), and thus gender differences in interaction would be expected to have implications for performance. Consistent with this idea, preliminary work by the PI suggests that all-male (vs. all-female) groups' higher task-oriented activities and lower positive social activities facilitate performance at brainstorming tasks. The third study will extend this work to assess whether all-female (vs. all-male) groups' interaction style facilitates performance at discussion tasks. The fourth study will then explore the link between the interaction style of mixed-sex groups and task performance. Finally, the fifth study will use meta-analytic techniques to estimate the magnitude of gender differences in group performance in prior research and to examine the relation between the size of this difference and attributes of the group task and number of group members.

Anticipated impact on Veterans' Healthcare: This project will strongly impact VHA patient care because: (a) VHA devotes significant resources to long term care, (b) the prevalence of dementia in long term care facilities is high, (c) the prevalence of dementia in the veteran population is high and is increasing, and (d) the VHA has identified pain control as a priority clinical and research area. Project Background: The goal of this study is to develop a pain intensity measurement strategy for persons with dementia and limited ability to self-report pain. Though many available instruments capture information about the pain of those with limited ability to self-report, there remain four major barriers in developing a practical and clinically relevant pain intensity measurement strategy for this vulnerable population: 1) uncertainty regarding the specificity of some behaviors typically counted as indicative of pain; 2) lack of a measure that goes beyond identification of pain presence to estimate pain intensity; 3) lack of an inviolable criterion or gold standard; and, 4) uncertainty regarding whether clinicians' globa ratings of patients' pain add incremental validity as compared to using observational pain behavior checklists alone. The research proposed here addresses these barriers. Objectives: Phase I: Building a Pain Behavior Measure for Persons with Dementia. Specific Aim 1: Develop an efficient and specific measure comprised of behavior observations that best discriminate pain from other constructs (e.g., agitation, boredom). Specific Aim 2: Evaluate the validity of item response theory-calibrated PIM-D scores in the cross-sectional sample of NH residents. Phase II: Responsiveness and Incremental Validity of Pain Measures. We will measure pain and relevant additional variables pre- and post-analgesic therapy in X nursing home residents who have dementia and limited ability to self-report. Specific Aim 3: Evaluate the responsiveness of the PIM-D to analgesic therapy. In a sample of 50 NH residents who receive analgesic therapy, a) evaluate the responsiveness of PIM-D scores, and b) compare responsiveness of the PIM-D to that of existing behavioral pain measures. Specific Aim 4: To evaluate the incremental validity of PIM-D scores compared to existing pain behavior measures and compared to nurse and NA gestalt pain intensity ratings in predicting changes in Expert Clinician Pain Evaluations. Project Methods: Item reduction will be achieved using a modified Delphi method with an expert panel. The total patient sample will be comprised of residents from 4 large VA Community Living Centers (CLCs; Philadelphia, Coatesville, Atlanta, Tuscaloosa). We will enroll veterans with moderate to severe dementia. Study measures will include evidence-based observer-recorded indicators of pain (pain-related behaviors, clinical gestalt pain ratings), and known correlates of pain (sleep, agitation, and depression). All participants will also be clinically assessed by a nurse practitioner.

A recent focus of development biology has been to isolate and study genes through to make critical decisions of cell fate and patterning in embryogenesis. Genetic studies in Drosophila indicate that genes containing the homeobox DNA-binding motif participate in such decisions.Other genes such as the vertebrate Myo-D gene, also have the compelling properties of developmental regulators. We propose to study regional specification and pattern formation in Xenopus embryos using as a focus the Xhox-1 homeobox genes and the Myo-D gene. In previous work we devised an overexpression assay to address the function of the Xhox-1A gene and demonstrated that it is likely to be involved in somite formation. Somitogenesis is a primary segmentation event which shapes the vertebrate body play. This finding is significant because it is the very first indication of a function for a vertebrate homeobox gene and it fulfills the broad expectation that homeobox genes will participate in critical developmental events. We propose to study further the role of Xhox-1A and a linked gene. Xhox-1B, in somite formation. We will use antibodies to investigate at cellular resolution the normal expression pattern of these genes. Furthermore, we will design dominant-acting mutants of the Xhox-1 genes which will be used to antagonize the action of the endogenous genes and thus perturb somitogenesis in new ways. We will also approach homeobox gene function at the biochemical level by studying the binding of Xhox-1A protein to DNA. We believe that in the long term this biochemical approach will reveal how stable developmental changes are affected. In Xenopus, most of the somite tissue becomes muscle. Nothing is known about how the patterning of muscle tissue (somitogenesis) meshes with commitment to the muscle lineage. Evidence suggests that the Myo-D gene is instructive in this commitment step. We will characterize the expression of the Xenopus Myo-D during early development and attempt to devise functional assays to test whether Myo-D gene expression can commit multipotent, embryonic ectoderm directly into the muscle lineage. These experiments are designed to investigate specific examples of cell commitment and patterning in vertebrate development. In addition, we will take a more general approach to the fundamental problem of how regional specification occurs in embryos. We will screen a pre-gastrulation cDNA library with probes for developmentally interesting genes such as other homeobox genes, believing that many of them will show restricted spatial expression and will indicate zones of cell commitment at early stages. The strength of this proposal lies in the fact that we already have a functional assay for the homeobox genes that we study and in the fact that we can relate these studies to the wealth of cellular and developmental information available for Xenopus embryos.

The radiation sensitizer Misonidzaole (MISO) can selectively potentiate the effectiveness of certain conventional chemotherapeutic agents in vitro and in vivo. In preliminary experiments we have demonstrated that this chemopotentiating effect of MISO can be significantly augmented by mild hyperthermia (41 degrees C, 1 hr) under oxygen-deficient, but not aerobic conditions, suggesting that the addition of hyperthermia to drug-sensitizer combinations might result in a significant therapeutic advantage. The overall objectives of the proposed research are 1) to evaluate this hypothesis in vivo comparing enhancement of tumor response (KHT sarcoma) and normal tissues damage when MISO-drug combinations are administered in conjunction with whole-body hyperthermia, 2) define parameters influencing the interactions among the three variables in vitro in controlled experimental environments and 3) determine the mechanism(s) of the interactions and the influence of oxygen concentration on their expression. Experiments indicate that hyperthermia, independent of its effect on the chemotherapeutic agent, increases the chemopotentiating efficiency of a dose of MISO, possibly by increasing nitroreductase (NR) activity, an oxygen sensitive reaction. This enhanced chemopotentiating efficiency might prove clinically significant as the success of chemopotentiation in patients may be limited by an inability to deliver adequate MISO doses. The proposed research will make extensive use of statistically- motivated experimental designs to determine the significance of temperature, oxygen-concentration, sensitizer dose, and sequencing on expression of chemopotentiation using cell survival, tumor-growth delay and nitroreductase activity as endpoints. Other experiments will utilize the multi-cell tumor spheroid model and selective trypsinization techniques to determine the effect of treatment on aerobic and hypoxic populations exposed simultaneously and to determine whether thermal enhancements are possible at MISO concentrations and pharmaco-kinetics achievable in the clinic. These later experiments will make use of a spheroid treatment device designed to permit pharmacokinetic modeling in vitro. Finally, the effect of heat on the chemopotentiation of nitrosourea-resistant, chemopotentiation- resistant Mer+ tumor cells (about 80% of human tumors express the Mer+ phenotype) will also be evaluated as preliminary experiments indicate that the addition of heat can overcome the resistance of this phenotype.

OBJECTIVE: Obesity and chronic sleep deprivation have both become increasingly pervasive medical problems in recent years. Average sleep time has decreased over the last century by two hours. Chronically sleeping less has been associated with increased weight, various endocrine changes such as an increase in cortisol levels, a decrease in growth hormone levels and changes in the adypocite-derived hormone leptin and the gastric produced hormone ghrelin that, together may increase appetite and food intake. Immune alterations associated with obesity and sleep deprivation include subclinical inflammation, and a decreased resistance to infections, respectively. We have recently demonstrated that increased weight in subjects suffering from sleep disturbances and depression is associated with an increase in proinflammatory cytokines and with a decreased in anti-inflammatory cytokines. Such changes are detectable in the serum as well as in the sweat, as non invasively collected over 24h by the means of a sweat patch, specifically developed by us to study endocrine immune relationships in freely living subjects. The general goal of this proof-of-concept, controlled trial is to investigate the impact of increasing sleep time on endocrine and immune parameters in chronically sleep-deprived, obese subjects. STUDY POPULATION: 18 to 50 year old, obese (BMI 30-55) men and premenopausal women, who usually sleep less than 6 hours, mainly recruited from the Baltimore-Washington metropolitan area. Sleep duration will be assessed by the use of sleep logs and actigraphy. Secondary causes of sleep deprivation such as insomnia, psychological (depression), and medical conditions associated with poor sleep quality (including obstructive sleep apnea) will be exclusionary criteria. DESIGN: This is a randomized, 12-month duration, comparison-controlled clinical trial of a minimum additional 90 min of sleep per night (Intervention Group) or continuation of habitual short sleep schedule (Comparison Group). The proposed treatment is an educational and behavioral intervention aimed at increasing sleep in a non-pharmacological fashion. The main analysis of the study will be to determine if additional sleep will result in changes in the endocrine and immune profiles opposite to those associated with chronic sleep deprivation. Specifically, we hypothesize that 12 months of sleep extension will result in weight loss and subsequent corrections in the following hormonal levels: leptin, adiponectin, ghrelin, cortisol, and growth hormones, among others. The immune and inflammatory profile will be characterized by a decrease in C-reactive protein, TNF-alpha, IL-6 and other inflammatory cytokines, and a return of anti-inflammatory cytokines to a healthy range. At the end of the 12-month intervention study (Phase 1, Efficacy Study), all participants will be given information about the potential benefit of more sleep and encouraged to increase sleep time. Health teaching about proper nutrition and adequate exercise will also be provided at that time to the Comparison Group. All participants will be evaluated 6 months later to assess the effects of this intervention in a real-life situation (Phase 2, Effectiveness Study). As of August 31, 2008 70 subjects have been randomized. Two third of the participants are women and ore than 50% are minoritites. Subjects'retention in this prospective study has been excellent with less than 15% of subjects interrupting the study before completion. Most common reasons for interruption include move to a different geographical area and the clinical need for bariatric surgery or the need for drastic change sin life style (diet, physical exercise) above and beyond the standard of care. OUTCOME PARAMETERS: body weight, food intake, hormones, and cytokines.

The present R13 grant requests support for a joint Gordon Research Conference and Gordon Research Seminars (GRC/GRS) on Cannabinoid Function in the CNS to be held in 2013. We will exploit the innovative GRC/GRS format to accomplish three Specific Aims: 1. To promote and sustain a scientific forum that allows for in depth presentation and adequate discussion of cutting edge unpublished data in the area of Cannabinoid Function in the CNS. 2. To foster opportunities for cutting edge collaborative science that propels the next generation of scientific advances and unites basic researchers studying cannabinoid-related neuroplasticity with clinical researchers studying long term impact of altered neuoroplasticity in disease states. 3. To promote and mentor the next generation of cannabinoid scientists while encouraging diversity and inclusiveness in the cannabinoid field through the Gordon Research Seminar. The joint meeting format is warranted based upon the critical role of the endocannabinoid system in controlling neuronal excitability, the contribution f endocannabinoid dysregulation to disease states, the therapeutic potential of endocannabinoid modulators and the increasingly widespread use of both recreational (e.g. spice) and medicinal (e.g. Sativex, medical marijuana) cannabinoids in humans. The recent advent of cannabinoid- based medicines requires that continued research emphasis be placed on understanding cannabinoid effects in humans, especially long-term consequences (i.e. abuse liability, adverse side-effects, safety considerations). Clinical studies of cannabinoids have not been represented at any prior Gordon Research Conference on cannabinoids, mandating that program organizers specifically reach out to clinical researchers that would not typically attend a Gordon Research Conference focused on basic as opposed to clinical science. The format of the GRC thus provides an important opportunity to build bridges in interdisciplinary science between preclinical researchers studying neuroplasticity and clinical researchers studying human disease states marked by altered neuroplasticity (e.g. pathological pain, addiction, neurodegenerative diseases). The Gordon Research Seminar (GRS 2013: Endocannabinoids in Neurophysiology and Neuropathology) is organized by and for graduate students and post-doctoral fellows and has been highly ranked by trainees as a venue for providing mentoring and networking opportunities at a critical stage of trainee development. The GRS directly precedes the GRC (GRC 2013: Synapses, Circuits and the Human Brain). R13 support of the 2013 joint GRC-GRS is expected to promote mutually beneficial increases in diversity in the cannabinoid field. The present R13 grant is expected to foster opportunities for cutting edge collaborative science while ensuring the continued success and sustainability of a highly rated GRC devoted to cannabinoid research.

This application requests a further five years of support for an Institutional National Research Service Award to support multidisciplinary post-doctoral training in Genetic Epidemiology and Molecular Genetics and Neurobiology. We request support for six postdoctoral fellows (2 M.D. and 4 Ph.D.) for training in Genetic Methodology, Family Epidemiology and Behavior Genetics, Gene Mapping and Bioinformatics, Molecular Genetics, and Molecular Neurobiology. Recruitment will be staggered over two years to assure a high caliber of fellows. In addition to training in a primary area, fellows will be encouraged to obtain a broad understanding of the diverse skills in Psychiatric Genetics to facilitate their collaboration in (and leadership of) cross-disciplinary research teams. The fellowship will usually last three years, but one or two years may suffice for those with much pertinent experience. Fellows with a wide variety of backgrounds will be recruited including; Psychology, Psychiatry, Genetics, Statistics, Mathematics, Anthropology, Sociology, Biology, and Neuroscience. The training program uses an apprenticeship model, combining research under the mentorship of one or more experienced mentors with more formal training through seminars, didactic courses and individual reading. Major strengths of the program are; (i) the participation of a large multidisciplinary group of well-funded preceptors (n=l8) with expertise in statistical and computational genetics, molecular genetics and neuroscience; (ii) the study of quantitative and qualitative traits and the development of methods for the analysis of multivariate phenotypes and (iii) the availability of major epidemiological and genetic data sets (phenotypes and genotypes). Fellows may participate in ongoing linkage and candidate gene studies of Schizophrenia, Bipolar Disorder, Alzheimer's Disease, ADHD, Alcohol Dependence, Electrophysiological measures of CNS activity, Personality traits and Nicotine Dependence. Preceptors in this program have approximately 62 federally and non-federally funded grants providing many opportunities for training in all aspects of Psychiatric Genetics. The program is located in one of the nations leading Medical Schools with a rich array of basic and applied genetic research studies and educational opportunities. Thus, we expect the long tradition of successful mentoring and research training of scientists and physician-scientists from diverse intellectual backgrounds to continue.

Project Abstract - IICA With this current proposal, the Inter-American Institute for Cooperation on Agriculture (IICA) seeks to build on previous successes and implement high-impact interventions that directly reduce risks associated with importing food into the United States. Specifically, we propose to develop and test alternative technologies to greatly augment the availability of high-quality training opportunities using the existing Produce Safety Alliance (PSA) curriculum and other training materials, such as the On-Farm Readiness Review (OFRR), in support of the FSMA Produce Safety Rule (PSR). We will leverage the wide availability of mobile telephones throughout the Americas and develop web- and application-based training materials that not only incorporate requisite training per the curriculum but also provide easy links to relevant portions of the rule and other important documents so that the user can refresh their knowledge as needed. While a variety of training materials are available in English, we propose to strengthen the reach and impact of existing and new materials by making them available in Spanish as well. This approach will take advantage of the prevalence of access to mobile technology throughout the Americas to create a group of tools that address the needs of adult learners and non-traditional students. We will use well-tested approaches and learning techniques to ensure sustainable knowledge building and understanding around FSMA implementation. Working with partners in academia, we will design controlled experiments to test the efficacy of alternative training materials against in-person training techniques to better understand what is and what may be possible to achieve through alternative approaches. Using an iterative process, we will develop alternative training materials, package them into new delivery methods, and test and validated both the materials and delivery approaches through pilot projects. Based on the results of these pilot projects, we will modify and further test our approaches, with the goal of releasing final, validated products to a large, hemispheric audience. These ?final? products will then be evaluated over the life of the project to ensure their continued efficacy and provide inputs for corrective actions. IICA is the specialized agency for agriculture within the Inter-American System. Counting on over seventy-five years of experience and a network of over thirty offices across the Americas, IICA is an ideal partner to support FDA to address technical capacity needs around FSMA implementation by foreign governments and their industries in Latin America. IICA is and has been a strong U.S. government partner in Latin America and the Caribbean for many years and has the demonstrated capacity to implement FSMA training in the region.

Many cancer patients who are candidates for high dose chemotherapy do not have available a suitable related or unrelated donor of bone marrow or peripheral blood progenitor cells. Human umbilical cord blood (CB) is a unique source of transplantable hematopoietic cells that offers an alternative source of hematopoietic cells for these patients. The use of CB cells to date, has primarily been limited to small pediatric patients due to the low cell numbers in CB products. The focus of this proposal is to optimize ex vivo culture of CB to provide increased numbers of cells to enable the use of CB in adult patients. In addition, optimization of the expansion cultures will enable more rapid hematopoietic and immune recovery post transplant and potentially minimize other complications associated with transplantation of CB. The first aim of these studies is to evaluate improved expansion culture conditions in clinical trials. Preclinical studies will also be performed to evaluate other culture conditions that may provide enhanced platelet recovery. In addition, analysis will be performed of primitive cells in the expanded products as an indication of the effect of expansion on long term engrafting cells (stem cells). The second aim will optimize transduction cultures of expanded cells and subsequent clinical studies will incorporate these conditions to use marked expanded cells in patients. This will allow the evaluation of the contribution of expanded cells to long to engraftment. Correlative analysis will be performed comparing the contribution of the marked cells in patients with in vitro assays of primitive progenitor cells to determine the predictive value of the in vitro assays. The third aim will evaluate the immune recovery in patients receiving ex vivo expanded cells. Both phenotypical and functional analysis of T cells subsets will be performed The final goal of these studies is to determine culture conditions for the expansion of T cells in vitro for use a source of donor lympohocytes to induce an anti tumor effect in patients who relapse. In the absence of cultured T cells the patients who relapse have no alternate source of donor lymphocytes and would die from disease progression. The outcome of these studies will provide us with a better understanding of the potential clinical utility of CB. Optimal expansion cultures will enable the use of CB in adults and provide more rapid engraftment. Overall these studies will lead to improved outcome in these patients.

Leptin (L) and its receptor (L-R) are key players in the regulation of energy homeostasis and body weight. Complex formation between leptin and the extracellular portion of L-R results in the activation of Janus kinase 2 (JAK2) that is constitutively bound on the intracellular regions of the receptor. Leptin-instigated JAK2 signaling in hypothalamic nuclei reduces food intake and stimulates energy expenditure, while functional defects in either leptin or L-R result in morbid obesity, hyperglycemia, decreased insulin sensitivity, and hyperlipidemia. Despite the crucial impact of the leptin system on body weight and other physiological responses, little is known about the structure of the L/L-R complex and its association with JAK2. One of the reasons for this lack of insight is that both L-R and JAK2 have a relatively long and flexible multi-domain arrangement that has proved to be very challenging for both large-scale purification and implementation of X-ray crystallography. The present proposal aims to overcome these limitations in addressing the architectural prerequisites of L-R signaling by applying single- particle cryo-electron microscopy (cryo-EM) to characterize the holo-complex of full-length L/L-R and JAK2. Single-particle EM has emerged as a very powerful tool for the characterization of dynamic protein assemblies in relatively small concentrations and without the need for crystallization. We anticipate that the application of single-particle EM techniques on this system will reveal the architecture of the L/L-R assembly and JAK2 independently, and in complex. Given the underlying importance of this membrane-localized signaling complex in obesity, energy metabolism, and heart disease, the structural results obtained will be of very broad biomedical interest. Considering the current lack of structural information on any receptor/JAK complex, our studies will provide the general architectural framework for understanding how extracellular ligand binding on cytokine receptors results in intracellular JAK activation.

Research Summary (For the funded R01 GM118553 proposal). Malaria, a disease caused by parasites of Plasmodium species, re- mains one of the most relevant infectious diseases; in 2015 over 200 millions of individuals had clinical malaria and over 500,000 individuals, mainly children, died from it. The infection starts when a Plasmodium-infected mosquito injects into the skin a small dose of sporozoites, a speci?c form of the parasite, which travel via the blood to the liver, infect hepatocytes, and form liver stages. Several vaccine candidates, including the most re- cent RTS,S vaccine, are aimed at eliminating sporozoites from the skin, blood, or hepatocytes. However, the low ef?cacy of such vaccines highlights the problem with lack of basic understanding of how Plasmodium sporozoites are eliminated by host immunity. CD8 T cells, a subset of lymphocytes, have been shown to play an important role in preventing clinical malaria by eliminating Plasmodium liver stages, speci?cally in radiation attenuated sporo- zoites (RAS)-based vaccines. Using intravital imaging, we have recently discovered that activated CD8 T cells form clusters around Plasmodium-infected hepatocytes in mice and that these clusters are important in parasite elimination. Mechanisms driving the formation of such clusters remain poorly de?ned and how activated CD8 T cells eliminate liver stages from the whole liver is not well understood. Another layer of complexity arises as the level of immunity needed for protection depends on a speci?c host-parasite combination. By combining math- ematical modeling and experiments we will provide quantitative insights into potential mechanisms that explain contribution of CD8 T cells to elimination of Plasmodium liver stages in mice. We will provide such insights via three complementary speci?c aims. In speci?c Aim 1, we will discriminate between alternative mechanisms of formation of CD8 T cell clusters around sporozoite-infected hepatocytes (T-cell intrinsic vs. T-cell extrinsic), de?ne the role of T cells, speci?c to irrelevant antigens, in the formation of clusters, and quantify the impact of T cell cluster size on the ef?ciency at which liver stages are eliminated. In speci?c Aim 2, we will determine the impact of structure of liver sinusoids on the ef?ciency of CD8 T search for rare sporozoite-infected hepatocytes and de- termine the speed at which moving CD8 T cells can localize the site of infection. Finally, in speci?c Aim 3 we will discriminate between alternative mechanisms for a larger number of memory CD8 T cells required for sterilizing protection against exposure to Plasmodium yoelii sporozoites as compared to Plasmodium berghei sporozoites. Completion of these aims will lead to a better understanding how CD8 T cells localize and eliminate Plasmodium liver stages from one of the major peripheral tissues, the liver. This understanding may help in designing more ef?cient immunization protocols of RAS-based malaria vaccines. In addition, deeper understanding of the mech- anisms by which CD8 T cells eliminate infections at peripheral sites may be also useful for the improvement of several others CD8 T cell-based vaccines such as those against HIV, HCV, and HSV.

Arterioles undergo major morphological changes during vasoconstriction. These changes consist of formation of an orderly pattern of periluminal ridges whose size and number vary systematically with the level of constriction. The cellular interactions which produce these changes have not been studied in detail, hence little is known about how the arteriolar wall coordinates rearrangement of its components as the vessel changes its diameter. The purpose of the present study is to explore the relationships between the degree of microvessel constriction, mural configuration, and the frequency and distribution of myoendothelial junctions (MEJ's). The working hypothesis is that points of attachment between the arteriolar endothelium and smooth muscle (i.e. MEJ's) provide focal areas of force transduction between these two cell types, and thereby determine deformations in the wall which occur by selectively exerting force along the luminal perimeter. The present study seeks to address this issue by undertaking an ultrastructural analysis of MEJ's in both dilated and constricted arterioles. Knowledge of the mechanical interactions between the cell of the arteriolar wall is important to our understanding of the pathology of hypertension and other cardiovascular lesions (e.g. cerebral vasospasm). To obtain arteriolar vessels for ultrastructural analysis, male golden hamsters are anesthetized with sodium pentobarbital (IP) and tracheostomized. Supplemental anesthetic is administered through a femoral vein cannula. The left cheek pouch is gently exposed, prepared for intravital observation as previously described (Duling, 1973) and suffused with a physiological salt solution at pH 7.4. Arterioles displaying appropriate responses to vasoactive substances are selected as experimental vessels. Vessels dilated with adenosine or constricted with norepinephrine are fixed for ultrastructural analysis in situ by immersing the entire pouch in fixative. Thin cross sections of arterioles are cut, stained and examined with a TEM. The frequency and distribution of MEJ's present in both dilated and constricted microvessels will be compared. Morphometric analysis of vessel cross sections will determine: 1) If MEJ's are labile entities, 2) any organized array of MEJ's associated with the periluminal ridges of constricted vessels, 3) the type of MEJ's extant between the endothelium and smooth muscle (e.g. tight junction, gap junction, etc.) and 4) the cellular origin of the process which ultimately produces the MEJ.

The approximately 1 ,000 G protein-coupled receptors (GPCRs) of humans mediate key signals - triggered by photons, odorants, hormones, and neurotransmitters - in brain, heart, blood vessels, white blood cells, and virtually every organ and endocrine gland. The fact that most of these signals represent potential targets for drug therapy justifies the broad-based strategy of this proposal, which aims to understand the conserved molecular mechanisms responsible for transmitting G protein-mediated signals between signaling molecules, in vitro and in the context of the cell. The first two aims test relations between structure and function of the GPCR and the G protein trimer at the level of individual molecules. Experiments with GPCRs aim to: a. use molecular probes to determine how the extracellular surface of a GPCR actually binds the activating ligand; b. engineer metal binding sites that activate a GPCR by inducing coordinated movement of its transmembrane helices, allowing us to infer how the natural ligand induces similar movements; c. identify sites on the GPCR's intracellular surface that interact specifically with peptides representing different parts of the trimeric target. To understand the conformational changes in a G protein trimer that mediate its activation by the GPCR, a second set of experiments will: a. test the hypothesis that the GPCR uses the beta-gamma subunit of the G protein trimer as a lever to open a route for bound GDP to exit from its binding pocket in the alpha subunit and thereby activate the trimer; b. determine how key structural elements of the G protein alpha subunit cooperate during the activation process, by constructing metal binding sites that restrict movements of these elements, relative to one another. The third set of experiments uses fluorescent probes and fluorescence energy transfer (FRET) to determine the locations of G protein alpha and beta-gamma subunits in intact cells and ask how hormonal activation affects their interaction. Biochemical experiments with pure G protein subunits indicate that activation in the test tube causes the alpha subunit to dissociate from the beta-gamma heterodimer; it is not known, however, whether such a dissociation accompanies activation in an intact cell responding to a hormone. Investigation of this question begins by constructing functioning G protein alpha and beta-gamma subunits attached to fluorescent tags; the fluorescent subunits are used to assess their subcellular distributions in intact cultured cells, and FRET between co-expressed tagged beta-gamma and alpha subunits will reveal whether hormones cause them to dissociate.

DESCRIPTION (adapted from the abstract): Caring for critically ill patients suffering from multiple organ dysfunction syndrome (MODS) represents one of the most difficult challenges in critical care medicine. The high mortality of adults and pediatric patients with MODS warrants a greater understanding of pathophysiology at the molecular level. Elucidating the molecular mechanisms that cause MODS is a daunting task because critically ill patients are typically subjected to multiple, simultaneous, and/or sequential injurious stimuli. The "multiple hits" hypothesis of MODS states that the interactions of multiple stimuli, and the unexpected cellular responses that they may generate, can ultimately lead to organ failure. The investigators propose to undertake a reductionist approach whereby they will study the in vitro interactions of two fundamental, yet distinct, cellular responses that are ubiquitous during various forms of critical illness: the heat shock response, and activation of the transcription factor NF-kappaB. They have demonstrated that the heat shock response, a primitive cellular defense mechanisms, inhibits activation of NF-kB, which is involved in the regulation of many proinflammatory genes. Preliminary data indicate that inhibition occurs by two mechanisms. The proximal mechanism involves inhibiting phosphorylation and subsequent degradation of the NF-kappaB inhibitory protein, I-kappaBalpha. The distal mechanism involves heat shock response-mediated de novo expression of the I-kBa gene. Specific Aim I is designed to determine the mechanism by which the heat shock response inhibits phosphorylation of I-kBa. Specific Aim II is designed to determine the mechanism by which the the heat shock response induces expression of I-kBa, and proposes a fundamental reclassification of I-kB as not only an inhibitor of NF-kB, but also as a novel heat shock protein. Specific Aim III is designed to determine if heat shock response-mediated expression of I-kBa independently inhibits activation of NF-kB. By elucidating the mechanisms proposed in this application, they will provide novel insight regarding the interactions of these two fundamental cellular responses. Ultimately, it is hoped that this mechanistic insight, at the molecular level, will lead to a greater understanding of mechanism at the physiologic level, which in turn can form the basis of more rational and specific therapeutic strategies.

PROJECT SUMMARY/ABSTRACT Incident epilepsy is more common in the elderly than at any other time of life. While some cases are due to stroke or known Alzheimer?s disease (AD), many cases have no known etiology. This proposal hypothesizes that late-onset epilepsy (LOE; epilepsy starting at age 60 or later, in the absence of stroke or other identified cause) is associated with cognitive decline and preexisting neuropathology such as amyloid deposition; i.e., LOE may be a marker for future cognitive impairment and dementia. The research will use data from the ongoing longitudinal Atherosclerosis Risk in Communities (ARIC) cohort study (Aims 1 and 2), as well as from a new cohort recruited as part of this grant (Aims 3 and 4). The specific aims are: 1) To test the hypothesis that late-onset epilepsy (LOE) is associated with cognitive decline, and the subsequent development of MCI or dementia. 2) To test whether A? 42/40 ratio is associated with LOE. 3) To determine whether individuals with clinically-identified LOE have lower cognitive scores than individuals without LOE. 4) (Exploratory) To compare AD biomarkers in patients with and without LOE, and correlate subclinical epileptiform activity with cognitive performance in patients with LOE. Defining whether there is an association of LOE with an elevated risk of future cognitive impairment could allow patients to have earlier interventions, which may help slow future cognitive decline. This is an application for a Mentored Patient-Oriented Research Career Development Award (K23). The overarching training goal of the proposed project is to provide the candidate with the skills necessary to become an independent researcher in epilepsy related to aging. Specific training goals are to obtain advanced training in epidemiology and biostatistics; to obtain training in recruiting and leading cohort studies; to acquire and apply knowledge of AD pathophysiology and the use of biomarkers; to gain didactic and experiential training in cognitive assessments in neurodegenerative diseases; and to advance professional development skills in preparing manuscripts and presenting at conferences. The career development plan includes classes in biostatistics and epidemiology at the Johns Hopkins School of Public Health; regular meetings with mentors; seminars in biostatistics and the epidemiology of aging; seminars in AD pathophysiology and research; and attending/presenting at national conferences. The candidate?s primary appointment is in the Johns Hopkins Department of Neurology, an extremely supportive environment with a strong history of junior investigators successfully transitioning to independent investigators.