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We propose a summer clinical immersion program at the Weill Medical College of Cornell University that brings Biomedical Engineering (BME) PhD students from Ithaca Engineering College Campus to New York City Medical College Campus for dedicated intensive clinical exposure in the summer between their first and second year of graduate training. Each student will take bioethics lectures, shadow clinical practitioners and participate in clinical related research under the direction of an assigned clinician mentor. This clinical summer immersion is designed to connect BME to healthcare and society and to steer and enhance students'research interests towards medicine. It will immediately impact their PhD thesis research by adding a medical conscience and influence their long term career towards improving healthcare. PUBLIC HEALTH RELEVANCE: We propose to connect Biomedical Engineering Students'laboratory work with healthcare and help them give their discoveries the best chance to benefit clinical medicine by immersing them in a major medical center to shadow medical practitioners and participate in clinical research. When BME students see patients'suffering, they appreciate the urgency and importance of developing medical technology solutions to healthcare problems;when students watch in person their clinician mentor diagnosing and treating patients, students appreciate the utility of medical technology in medicine;when students see the limitations of the patients and the clinical environment, they develop a perspective on what kinds of technology solutions can be clinically workable;when students participate in clinical research, they appreciate the impact of medical technology

Despite the use of highly sensitive serologic tests for screening blood donations for antibodies to HIV-1, HTLV-I/II, HCV and HBV in US blood centers, the delay between the onset of infection and the antibody response creates a "window" period during which the blood may be infectious but seronegative. As a result, a low risk of virus transmission due to contaminated blood products still exists. During PHASEI of this grant, we have developed a Multiplex RT-PCR based capture assay which permits the simultaneous direct detection of HTLV-I/II, HIV-1, HCV and HBV in a single assay. In the course of PHASE II, we propose to further refine this Multiplex RT-PCR assay, and to develop a kit format useful for routine screening of blood products in a clinical lab setting. The assay will be validated on a number of well characterized performance and seroconversion panels for all five viruses and on large numbers (>1,000) of blood samples from normal blood donors. This test will provide a highly sensitive, cost-effective direct measure of infectious virus in blood and help further reduce the antibody-negative "window" period between infectivity and detection.

DESCRIPTION The purpose of this CDA-1 proposal is to provide Dr. Naomi Bramhall with the support and mentorship necessary to make the transition from basic science research to clinical research of noise-induced hearing loss. Dr. Bramhall's previous research has focused on hereditary hearing loss and hair cell regeneration, but as a certified audiologist, she is eager to shift to clinical hearing research. This proposal will provide Dr. Bramhall with the tools and experience necessary to engage in clinical research and generate data which will be used in applying for future funding such as the CDA-2, will guide her path towards independence as a clinical researcher. Her primary mentor for this proposal is Dr. Dawn Konrad-Martin and Dr. M. Charles Liberman is a co-mentor. Loud noise exposure is routinely experienced during military service and hearing loss is the second most common service-related disability among Veterans. Individuals with hearing loss and poor speech discrimination ability often have little success with hearing aids because amplifying sound improves the audibility, but not the clarity, of the speech signal. Loud noise exposure may result not only in damage to outer hair cells, but also lead to auditory neuronal degeneration. Neural loss can result in decreased word recognition (Schuknecht 1994) and may explain why many Veterans have poorer speech discrimination than would be expected based on their ability to detect tones, particularly in noisy environments. Mice show rapid degeneration of auditory nerve peripheral terminals followed by a slow degeneration of spiral ganglion neurons over a period of several months following loud noise exposure, even when their pure tone auditory thresholds recover completely (Kujawa and Liberman 2009). The subpopulation of neurons most vulnerable to noise exposure consists of the low spontaneous rate fibers (Furman et al. 2013), which have a high sensitivity threshold and respond to higher intensity sounds. This neuronal loss is correlated with a decrease in the amplitude of the first wave of the auditory brainstem response (ABR). Age-related reduction in the amplitude of wave I has been demonstrated in humans (Konrad-Martin et al. 2012) and is consistent with temporal bone studies showing auditory neuronal loss with age (Makary et al. 2011), suggesting this metric of auditory neuronal survival is also applicable to humans. Previously, quantification of the degree to which auditory neuronal survival influences speech understanding has been difficult due to limitations in measuring auditory neuronal loss in humans, which could only be accomplished through post-mortem temporal bone studies. The main objectives of this proposal are to use electrophysiological methods to 1) determine if decreased ABR wave I amplitude is associated with noise exposure in humans and 2) examine the impact of noise exposure and decreased wave I amplitude on auditory perception. Our hypothesis is that noise exposure will be associated with decreased wave I amplitude, consistent with animal studies, and that this change will be correlated with decreased speech recognition at high sound levels, difficulty understanding speech in the presence of background noise, and decreased tolerance to loud sounds. Developing the ability to assess auditory neuronal survival with existing clinical tools and advancing our understanding of the specific auditory perceptual deficits resulting from neuronal loss will help guide future treatment options for Veterans suffering from hearing loss.

This research is a study of the design and synthesis of agents useful primarily in the treatment of cancer. The approach used is to study the chemical and physical properties of the metabolic sequence that may be one of the controlling or limiting processes in the synthesis of deoxyribonucleic acid and ultimately cell division. The sequence is the reductive methylation of deoxyuridine-5'-phosphate catalyzed by the enzyme thymidylate synthetase. The research objective of this problem is a continuing study of the synthesis and in vitro testing of potential thymidylate synthetase inhibitors. Specifically the effective binding characteristics of the cofactor N5N10-Methylene-tetrahydrofolic acid, for thymidylate synthetase and dihydrofolate reductase enzyme will be examined. In this manner and by application of the chemical mechanism of the reaction and the physical properties directing enzyme-substrate or inhibitor affinity the nature of the groups important to binding can be defined and by design, attempts can thereby be made for specificity of action. Samples will also be submitted to CCNSC for testing.

Project Summary/Abstract The overarching goal of the Cancer Epidemiology Program is to investigate the causes of cancer through epidemiological studies incorporating genetic and molecular epidemiology approaches as well as risk factors/environmental and biomarker research that are strongly grounded in the relevant biology. A hallmark of this Program has been the integration of epidemiology and biology, along with the development and application of state-of-the-art biostatistical approaches and a defined focus on ethnic diversity that is consistent with the populations within the USC Norris catchment area. The Program is led by Graham Casey, PhD, a molecular geneticist who is interested in the integration of cancer epidemiology and molecular biology to develop novel insight into the complex etiology of cancers, and Duncan Thomas, PhD, a biostatistician with extensive contributions to design and analysis methods for genetic and environmental epidemiology studies and has had numerous cancer epidemiology collaborations. Drs. Casey (cancer genetics), Thomas (biostatistics), and Haiman (cancer epidemiology) comprise the Program's Executive Committee. The scientific aims of the Program are to: 1) elucidate the role of environmental and lifestyle factors (e.g., obesity, diabetes, radiation) in the etiology of cancer and study population cancer trends; 2) elucidate the role of genetic factors in the etiology of cancer with an emphasis on different racial/ethnic populations using existing and new cohorts; 3) determine the mechanistic and biological basis for genetic risk variants using large-scale fine-mapping and comprehensive cellular and biochemical approaches, and integrate biomarker and tumor biology studies into epidemiologic research; and 4) develop and apply novel study design and statistical analysis methodologies for environmental and genetic epidemiology research in cancer. Accomplishments during the project period include seminal contributions to the genetic etiology of the cancer field with over 40 GWAS-related manuscripts since 2010, leadership roles in several international genetics consortia, expansion of the genetics emphasis to study the biological implications of genetic inheritance through strategic recruitment, development of an integrative genomics theme to study the relationship between inherited and somatically acquired mutations in tumors, submission of several P01s, and increased collaboration with Cancer Control Research and other programs, that includes collaborative grant submissions. The Program is composed of 27 members from four departments within the Keck School of Medicine. Current grant funding totals are $12M in peer-reviewed funding (direct costs), of which 58% is from NCI, 25% from other NIH sources, and 8% in other peer-review funding sources. The Program is highly productive with 730 publications of which 26% are inter-programmatic, 47% intra-programmatic and 62% inter-institutional.

Project #1. Impact of HIV-1 on the Rho GTPase-mediated neuronal cell development. It is now well accepted that the HIV-1 regulatory protein, Tat, has a neurotoxic effect and that treatment of both human and rodent neurons with Tat induces apoptosis by mechanisms that are not fully understood. The cellular protein, Pur-alpha, is a key target of Tat for directing the regulation of HIV-1 transcription, translation, and viral RNA transport. Indeed, both Pur-alpha and Tat have been detected in the same cells within HIVE infected lesions. Results in the Pur-alpha knockout mouse model have revealed a critical role for Pur-alpha during development, particularly in the coordinated development and differentiation of neuronal cells throughout the brain. Over the past several years, it has become clear that the RhoGTPases and related molecules play an important role in neuronal cell development, including neurite outgrowth, differentiation, axon pathfinding, dendritic spine formation, as well as neuronal cell maintenance. Results from our studies indicate that Tat has the ability to disturb this pathway by stimulating RhoA activation, an event that is accompanied by conversion of RhoAGDP to RhoAGTP by interacting with guanine nucleotide exchange factor (GEF) family members including PDZ-RhoGEF. Indeed, our preliminary results suggest that Tat may mediate RhoA activity via interaction with PDZ-RhoGEF. Upon its activation, RhoA is usually positioned in the plasma membrane, and via a series of signaling pathways that involve Rock, MAPK, and JNK1, NFkB induces neurite retraction. Moreover, activation of RhoA via heteromeric G-proteins including Ga12 and G<x13 can stimulate GSK3(3 which is also known to be involved in HIV-1 neurotoxicity. On the other hand, Tat can block activation of another key RhoGTPase, i.e. Rac1 and its partner p21-activated kinase 1 (PAK1), which appears to regulate actin cytoskeletal dynamics, maintain neuronal cell integrity, and promote neurite outgrowth. In addition, Tat has recently been shown to inhibit this pathway by acting through PAK1 and JNK1. Finally, Rac1 and RhoA were found to display aberrant expression and subcellular localization in the Pur-alpha knockout mouse model, suggesting that Pur-alpha is required for their proper function. Based on these preliminary observations, we hypothesize that Pur-alpha has a central role in the well-balanced activities of RhoA and Rac, and that its genetic ablation or its functional alteration by associating with Tat can derail several key parameters in RhoA and Rac signaling pathways including p115/PDZ and PAK, respectively, and results in pathological features that are commonly seen in neuronal cells of AIDS patients with CNS disorders. In this research project, we will perform a series of cell biological and molecular studies to unravel the molecular basis of Tat cross-talk with Rho GTPase and its impact on neuronal cell integrity.

The University of New Mexico Health Sciences Center (UNM HSC) has developed an innovative and widely applicable model to provide treatment for patients with chronic, common and complex diseases who do not have direct access to specialty healthcare providers. This knowledge-on-demand model is called Project ECHO - Extension for Community Healthcare Outcomes. Conceived as a means to treat HCV-infected patients in New Mexico's rural communities and prison system, Project ECHO can assist in diagnosing and successfully treat many other chronic illnesses and conditions. Project ECHO connects urban medical center disease experts with rural general practitioners and community health representatives over a telehealth network. This enables them to effectively treat patients on site who would otherwise have to travel to urban healthcare facilities for specialty treatment. Project ECHO has enormous potential as an affordable healthcare intervention in rural communities where certain chronic diseases have reached epidemic levels and healthcare resources and personnel are scarce. The purpose of Project ECHO is to educate, train, and support rural general practitioners or other available healthcare representatives on the best practice treatment protocols for complex diseases they encounter in their communities. This model focuses on the principles of case-based learning and disease management using the telemedicine infrastructure and internet-based technologies to co-manage patients in community-based practices. The ultimate goal of Project ECHO is to provide the same level of healthcare to rural patients with chronic diseases as can be obtained in an urban setting. A secondary goal is to provide rural healthcare practitioners with a level of interaction and support commensurate with their urban counterparts to enhance their technical competence and decrease their feelings of professional isolation. Project ECHO is being field-tested in weekly, regularly scheduled telemedicine clinics hosted by UNM HSC specialists in the areas of hepatitis C. Through use of the UNM HSC Telehealth pathways and internet-based access, community healthcare providers around New Mexico are afforded the opportunity to present cases which are discussed among the network participants to jointly reach treatment decisions.

Participants in the Baltimore Longitudinal Study of Aging aged 60 and older were examined to detect changes in psychological, neurological, and neuropsychological tests related to early signs of Alzheimer's disease. Six-year changes in immediate visual memory performance assessed by the Benton Visual Retention (BVR) test were used to predict Alzheimer's disease (AD). The subjects of this study were 279 community-dwelling adult participants in the Baltimore Longitudinal Study of Aging, eight of whom received AD diagnoses from a neurologist based on DSM III-R and NINCDS- ADRDA criteria. Subjects with diagnoses of AD had disproportionately larger changes in numbers of errors over the six-year retest interval prior to estimated onsets than subjects without AD diagnoses. In addition, significant risk for AD was associated with changes in BVR performance from a proportional hazards analysis. The relative risk for AD associated with six-year BVR changes of six errors was 5.9 (95% CI=5.0-7.1). These results suggest that symptoms of AD may be manifested by changes in immediate visual memory performance earlier than is detectable by clinical evaluation.

Lower levels of physical activity, an "unhealthy" diet and the presence of overweight/obesity are established risk factors for several types of cancer. There is a growing interest in exploring the potential links between environmental characteristics and chronic diseases, such as breast cancer in women. Previous research done by our group has demonstrated a link between neighborhood characteristics, such as walkability, and risk factors for chronic diseases. Therefore, using the San Diego cohort of the Women's Health Initiative (WHI), we propose to test the hypothesis that neighborhood characteristics would potentially moderate the association between common behavioral risk factors and incident breast, colorectal and total cancers. To do so, we propose to conduct a study employing the following specific aims: [unreadable] [unreadable] 1. To assess the baseline associations among the neighborhood environment factors (built, [unreadable] sociodemographic, and food) and common, behavioral cancer related risk factors (physical activity, diet, body mass index). [unreadable] [unreadable] 2. After adjustment for pertinent cancer risk factors, to determine the potential direct effects of the neighborhood environmental factors on incident cancer outcomes (breast, colorectal, total). [unreadable] [unreadable] 3. Using multivariable models, to ascertain the direct, mediated or moderated effects of both the neighborhood environment and cancer risk factors on incident cancer outcomes. Participants' neighborhood characteristics within a 1 kilometer buffer of their residence will be geocoded using geographic information systems (GIS) and linked to census, land use and business license data. This data will then be merged to the existing data from the San Diego WHI cohort, which includes extensive baseline characteristics (i.e. physical activity, dietary and body morphology measurements) as well as incident cancer events that were adjudicated using a standard protocol and central physician adjudicators. Once linked, appropriate analyses will be conducted to test the specific aims listed above. We believe the proposed study is innovative because of the prospective nature of the follow-up and it will test links between multiple neighborhood characteristics and not only cancer risk factors but multiple types of cancer. This research is important because it would extend the existing literature on neighborhood characteristics beyond common behavioral risk factors to incident cancers and it would inform and potentially influence theoretical models on the associations between neighborhood structure and cancer events. As such, this study could have implications for policy formulation on neighborhood design and development. PUBLIC HEALTH RELEVANCE: Lower levels of physical activity, an "unhealthy" diet and the presence of overweight/obesity are established risk factors for several types of cancer. There is a growing interest in determining if there are links between the environment in which one lives and chronic diseases, such as breast cancer in women. Therefore we propose to determine if the neighborhood environment influences the association between common behavioral risk factors and new breast, colorectal and total cancers. [unreadable] [unreadable] [unreadable]

Retinal pigmentation is one of several factors that profoundly influence visual system development. This is indicated by the fact that albino mammals (including humans), in addition to their external pigmentation deficits, have abnormalities of visual system development. Introduction of a functional tyrosinase gene into albino animals (mice and rabbits) corrects all of these abnormalities, proving that all aspects of this phenotype are due to the single gene defect in tyrosinase. Although it is known that the contribution of tyrosinase is crucial to visual system development, it is not known when or how tyrosinase influences development. I propose to determine the timepoints at which tyrosinase expression is necessary for the prevention of two albino abnormalities: decreased rod cell density, and a disruption of the retinogeniculate pathway through the regulated expression of tyrosinase during critical periods in the development of these two components of the visual system. Insight into the developmental function of tyrosinase will shed light on some fundamental issues of neuronal development, such as cell fate determination, axonal tracking, the timing of neurogenesis, and programmed cell death during development. I will control tyrosinase expression through the creation and use of a novel method of regulating transgenes in mice which involves modification of regulatory elements from the lac operon of E. coli for use in the mammalian genome. Tight regulation over the expression of transgenes will allow the investigation of a variety of previously unanswerable questions, including those addressed in the current proposal. This project will set the groundwork for a substantial broadening of the range of issues that can be addressed with transgenic mouse models.

DESCRIPTION: The research program completes a sample of infant twins for a multi-method longitudinal appraisal of temperament from the neonatal period through 30 months of age. Infant twins offer a powerful resource for detecting similarities and differences among genetically related pairs and examining the stability and organization of temperament characteristics over age. From the perspective of developmental behavior genetics, features of temperament may be examined for genetic influences on dimensions of temperament and their reorganization when transitions occur. Twins provide an opportunity to detect specific contributions of parents, family/home when two infants' temperament may differ markedly at a given time or over time. The principal aims of the program are to: (a) complete a sample of neonatal assessments of all twins in newborn nurseries for an initial measure of irritability, reactivity, and developmental status; (b) complete assessments of a longitudinal sample of twins so that 280 pairs have a multi-method appraisal of temperament at 3, 6, 9, 12, 18, 24, and 30 months, as based on direct observations of twins' behavior during standardized procedures in a structured laboratory setting, temperament questionnaires completed by parents, IBR ratings obtained during Bayley mental testing, and parental interviews; and (c) complete appraisals of the family/home environment and its contribution to temperament development. Laboratory sessions are videotaped. The results are ultimately addressed to the questions of origins, patterning among variables, sources of stability/instability over ages, and the joint collaboration of genetic and environmental factors for the determinants of temperament. This project, linked with other data sets, also demonstrates the potential relevance of newborn and infant temperament, examined longitudinally, for its contribution to preschoolers' liability for unintentional injuries.

Despite the prevalence of hyperactivity in school-age children, few attempts have been made to identify or treat this disorder prior to school entrance. Parent reports, however, suggest that in most cases hyperactivity and associated impulsivity and disciplinary problems are present in some form from infancy or toddlerhood onward. Follow-up studies of preschool and school-age hyperactive children further suggest that this disorder persists and is relatively resistant to treatment. Thus, the present research is aimed at the early identification and treatment of childhood hyperactivity. Hyperactive toddlers (2 1/2 - 3 1/2) are being compared with matched controls on a variety of parent report, family history, behavioral, and cognitive measures to determine the correlates of hyperactivity in toddlers and the usefulness of certain assessment devices with this age group. The efficiency of parent training groups as a treatment strategy is then being evaluated. Therapeutic effect is being assessed at the conclusion of eight weeks of structured group intervention and in a six-month post-treatment follow-up to evaluate short-term gains and maintenance of change in both child behavior and parenting skills.

The objectives of this research are to investigate (1) effects of ionizing radiation in the immature spinal cord, (2) the reparative and regenerative capacities of the immature nervous system, and (3) the manner in which ionizing radiation alters these capacities. All experiments are carried out in rats because the developmental state of these animals at birth makes it possible to study the immature nervous system ex utero. Neurologic, histopathologic and autoradiographic methods are used in this research. The responses of the immature nervous system to injury are compared and contrasted with those reported by others in the adult nervous system; this, perhaps, will provide some insight into the reactions and limitations occurring in the mature state. In addition to the experimental data, the information derived from intact, control animals will contribute to the understanding of normal maturation of the nervous system.

The design of a component to elicit broadly neutralizing antibodies is thought to be crucial for a successful HIV vaccine but is proving very difficult. In particular, immunogens designed from study of broadly neutralizing human monoclonal antibodies have generally failed to elicit antibodies with the appropriate specificities. A number of hypotheses have been advanced for these failures including restricted access to broadly neutralizing epitopes in recessed sites on the virus surface, proximity of the epitopes to viral membranes and epitope mimicry of self-antigens. In this new R01 proposal, we propose to generate transgenic mice carrying genes encoding two of the broadly neutralizing HIV antibodies 4E10 and b12 to investigate these hypotheses and to provide tools to study and rank potential HIV vaccine candidates. In the first instance, we propose to use mice carrying HIV antibody genes targeted to the physiological Ig loci to test if 4E10 and b12 have biologically relevant self-reactivity. If self-reactive, the B cells generated in 4E10 and b12 transgenic mice will be regulated by the immune tolerance mechanisms of receptor editing, anergy induction, or deletion and these mechanisms will be dissected. This may allow the design of immunogens in the future that do not evoke self-responses. If non-tolerant, transgenic mice carrying the 4E10 or b12 specificities should carry a greatly increased precursor frequency of B cells that are broadly neutralizing for HIV. We will then use an array of immunogens to explore the origins of any restrictions on eliciting 4E10 and b12 antibody specificities, to evaluate the most promising immunogens and ultimately to guide the design of modified and new immunogens to elicit broadly neutralizing antibodies. Relevance: To be successful, it is likely that an HIV vaccine must stimulate neutralizing antibodies. This proposal provides a mouse model to understand some of the problems in stimulating such antibodies by various vaccine candidates and to thereby suggest how better candidates can be designed.

The Behavioral & Social Sciences Core (BSSC) is structured to provide services that facilitate interdisciplinary AIDS research among established, new, and transitional investigators. Although the BSSC Core does not direct the science of the behavioral AIDS program at Penn, our Core services are guided by a set of scientific priorities and principals that program members have identified as crucial to scientific discovery, translation, and public health impact. These priorities include investigations that seek to define, measure^ahd^xparrdthe~understahdingof the role of contextual factors (e.g. social, sexual, and drug using networks; community/geography; Mental health and substance abuse) within which HIV transmission occurs and infection exists. These factors are viewed as critical to understanding risk of infection, access and adherence to treatment. The BSS Core at Penn is also well positioned to expand the role of behavioral sciences in the design and evaluation of clinical trials of biomedical interventions (microbicides, vaccines, and therapeutics) and to facilitate scientific collaborations with other investigators (behavioral and interdisciplinary), both domestically and internationally is critical to the development of sustainable programs of AIDS research. The Core plans to accomplish its objectives through the delivery of services which include: 1) Research application and implementation support.through the provision of consultation on AIDS research application as investigators conceptualize and prepare applications for funding; 2) Behavioral and biological assessment services to facilitate the expanded use of biological assays in behavioral research; 3) Target population services to identify and recruit populations and individuals of scientific relevance to AIDS research; 4) Support for the Penn CFAR Community Advisory Board (CAB) to develop a highly visible and active agenda of community education and research; and 5) Educational activities to stimulate integrated biological and behavioral research and advance our scientific priorities.

The synthesis of ribosomes is a complex but tightly regulated process in which the cell synthesizes equimolar amounts of four ribosomal RNAs in the nucleus, and of seventy-five ribosomal proteins in the cytoplasm, and assembles them into a ribosome in the nucleolus. We propose to study in Saccharomyces cerevisiae the mechanisms controlling both the transcription of ribosomal RNA and the synthesis of ribosomal proteins. Ribosomal RNA: We have identified a sequence in the rDNA repeat with many of the properties of an enhancer element. We plan to identify the precise sequences involved in this effect. We will ask whether this enhancer element: a) binds a specific transcription factor? b) is involved in the regulation of transcription of 35S prerRNA? c) is responsible for the 1:1 ratio of the transcription of 35S prerRNA and 5S RNA, whose gene lies between the enhancer and the 35S initiation site? Ribosomal Protein: The rates of transcription of most, if not all, ribosomal protein genes are tightly coupled. To understand the mechanism behind this coupling we will identify the sequences essential for the transcription of one ribosomal protein gene and then determine if similar sequences are used for other ribosomal protein genes. We will ask if the same sequences are involved in regulation of their transcription and/or whether there is a transcription factor specific for these genes. Finally, we will analyze the mechanism for translational control of ribosomal protein synthesis, with emphasis on testing the hypothesis that a regulatory molecule interacts with the m7G cap and its adjacent sequences.

Fetal alcohol syndrome (FAS) is an important cause of mental retardation. Exposure of the developing brain to alcohol can induce the death of neurons, which contributes strongly to learning deficits and neurological problems associated with FAS. Understanding the factors that influence neuronal vulnerability to alcohol induced loss in the developing brain is of considerable significance. We hypothesize that as neurons mature, they acquire a neuroprotective, signaling pathway that protects them against alcohol toxicity. We have discovered a neuroprotective pathway, which is mediated by nitric oxide (NO). This proposal will elucidate the function and molecular mechanisms of the NO-signaling pathway in protecting the developing brain against alcohol-induced neuronal loss. Our experiments will utilize a mouse strain carrying a null mutation for neuronal nitric oxide synthase (nNOS-/-), thus these animals lack NO-mediated neuroprotection. Specific Aim 1 includes morphological experiments examining the importance of NO-mediated neuroprotection on neuronal survival. Stereological methods will measure alcohol-induced neuronal losses in the hippocampus and cerebellum of mice that express or lack the protective pathway. Alcohol exposure will occur at a variety of ages in order to determine the impact of NO-mediated neuroprotection at different stages of brain development. Specific Aim 2 utilizes behavioral testing (eyeblink conditioning, Morris Water Maze) to determine whether the enhanced alcohol-induced neuronal losses in nNOS -/- mice are linked to greater functional deficits. Alcohol-induced deficits on these tests will be compared in nNOS / and nNOS+/+ mice and linked with neuronal losses. Specific Aim 3 includes molecular studies to determine the mechanism by which NO-signaling provides neuroprotection against alcohol toxicity. Experiments will determine whether nNOS is developmentally regulated in vivo and link alcohol vulnerability to expression levels and activity of nNOS. "Rescue" experiments in which the nNOS gene will be transfected into pathway-deficient neurons in culture derived from nNOS -/- mice will determine whether neuroprotection against alcohol toxicity can be restored. Immunohistochemistry and quantitative RT-PCR will explore the role of cAMP-responsive element binding protein (CREB) as a downstream effector molecule for the NO-signaling and elucidate CREB's role in regulating apoptotic proteins, which may underlie this neuroprotection.

Picture Archiving and Communication Systems (PACS) are expected to replace the currently existing film systems within the decade. However, presently there are no formal measures to evaluate the cost-effectiveness of image management systems. The objective of this Program Project is to develop a methodology to compare the cost-effectiveness of a PACS and the existing film management system. We will measure cost-effectiveness in the Medical Intensive Care Unit, the Emergency Department, and in the outpatient Neurology suite. The effects of diagnostic outcomes on health outcomes will also be estimated. The first project develops and evaluates a model that describes the diagnostic value of an image management system. The model will be validated and tested to determine its predictive capability. The second project evaluates workstations for clinical effectiveness, analyzes the radiologist-referring physician interaction, and studies physician acceptance. Teleconsulting between the radiologist and the referring physician including the transmission of outside images from the referring physician to the radiologist will also be studied. The third project utilizes clinical-history derived rules to optimize the display with diagnostically-relevant information. The fourth project uses the results of the other projects to measure the cost-effectiveness of PACS. Both direct and indirect costs before and after implementing PACS will be combined with data from the diagnostic value model to derive the incremental cost-effectiveness and to compare the usefulness of PACS to other medical interventions. The Operations core is responsible for software development, data collection, and quality control. This Program Project will provide the methodology for other institutions to determine the value of implementing PACS in their environment. Additionally, it may be applicable for the assessment of other medical technologies and to aid in the comparison of investments in such technologies.

Intercellular RNA transport is a potentially important means of cell-cell communication. In the nematode C. elegans the RNA channel SID-1 is essential for the cell-to-cell transport of silencing RNAs, SidTI and SidT2 (SidT1/2) are the highly conserved vertebrate homologs and published reports indicate that fish, mouse, and human versions of SidT1/2 have RNA transport activity. Analyses of RNA transport in cultured cells derived from SidT2 knockout mice confirm these reports and establish SidTI and SidT2 knockout mice and derived cultured cells as valuable tools for the identification and characterization of RNA signaling pathways in mammals. Extracellular RNAs are present in many body fluids and are being explored as disease indicators. How these RNAs are selected, transported, and whether they have a biological function is unknown. To address these issues the RNA transport activity of SidTI/2 will be exploited to selectively identify RNAs that are purposefully extracellular. These SidT1/2-dependent extracellular RNAs will be validated and then, to unambiguously establish the fact of intercellular RNA transport, their expression and transport will be rigorously examined. This analysis will be collaborative with consortium partners, whom have established powerful experimental systems to detect intercellular RNA transport. Importantly, the dependence of RNA mobility on SidTI/2 provides the means to disrupt their transport between cells, which will be important for deciphering the physiological and developmental of these mobile RNAs. Parallel to these discovery efforts, the RNA transport mechanism and the RNA features that permit or restrict RNA transport will be elucidated. These efforts will also be collaborative among the consortium partners. These findings will have bearings on how RNA transport is regulated and how the RNA transport pathway can be exploited to develop RNA-based therapies, including the safe and effective delivery of siRNAs.

In this project we will define a role for copper-zinc superoxide dismutase (SOD1) in the regulation of cell survival and death. While the wild-type (WT) SOD1 is a pro-survival protein, amyotrophic lateral sclerosis (ALS)-linked SOD1 mutants are toxic both in vitro and in vivo. We recently found that both WT and mutant SOD1 interact with the anti-apoptotic protein Bcl-2. However, the nature of the mutant SOD1 binding with Bcl-2 differs from WT SOD1. Contrary to WT SOD1, mutant SOD1 specifically localizes to spinal cord mitochondria where it forms SDS-resistant high molecular weight aggregates that bind and entrap Bcl-2. (Pasinelli et al, 2004, Neuron 43: 19-30). These studies suggest a potentially novel function for WT SOD1 in regulating cell survival and death, and a novel, toxic gain-of-function for mutant SOD1. Thus, while WT SOD1 may protect against cell death through its interaction with Bcl-2, mutant SOD1 may become toxic by aberrantly binding to Bcl-2 and converting Bcl-2 into a toxic or non-functional protein. In support of this hypothesis, we now have preliminary data indicating that both WT and mutant SOD1 might require Bcl-2 to exert their anti-and-pro apoptotic function respectively. With the present proposal we intend to characterize the anti and pro-death function of WT and mutant SOD1 and their respective interactions with Bcl-2. The ultimate goal is to understand the mechanism(s) of mutant SOD1-mediated toxicity and to define a potential role for the mitochondrial mutant SOD1/Bcl-2 complex in ALS pathogenesis. The specific aims are: 1) (A) To determine whether WT SOD1 pro-survival activity depends on its binding to Bcl-2, and (B) to determine whether mutant SOD 1-mediated toxicity depends on the aberrant interaction with Bcl-2. 2) (A) To identify the region(s) in SOD1 essential for the binding with Bcl-2, and (B) to study the difference in binding strength between WT SOD1 and Bcl-2 and mutant SOD1 and Bcl-2. 3) (A) To determine whether Bcl-2 undergoes conformational modifications upon binding with mutant SOD1 and.(B) to test the potential benefit of Bcl-2 and SOD1 like-peptides that abolish binding between Bcl-2 and mutant SOD1 on our cell culture model of mutant SOD1-linked ALS. 4) To determine whether Bcl-2 mediates mutant SOD1 mitochondrial translocation. 5) To study the correlation between mutant SOD1/Bcl-2-containing aggregates and ALS using transgenic ALS mice and patients.

In this project, the investigators propose to study the pathophysiology of mitochondrial long-chain fatty acid ?-oxidation (FAO) disorders. Patients with a FAO disorder can present with fasting-induced hypoglycemia, cardiomyopathy, heart beat disorders, sudden infant death, myopathy and (exercise-induced) rhabdomyolysis. Current therapeutic interventions need to be improved, but development of novel therapies is hampered by limited insight into pathophysiological mechanisms as well as small patient groups and insufficiently developed animal models. Therefore the overall objective of this proposal is to increase our knowledge of the pathophysiology of FAO disorders by studying newly identified molecular mechanisms and metabolic pathways that contribute to the different disease presentations using well-established mouse and cell models. The investigators hypothesize that perturbations in metabolic signaling pathways and the resulting impaired protein homeostasis play crucial roles in the pathophysiology of FAO disorders and that peroxisomal metabolism is an important alternative pathway for fatty acid degradation when mitochondrial FAO is defective. They will test this hypothesis by addressing two specific aims. In AIM 1, the investigators will study the molecular mechanisms underlying perturbed protein homeostasis in a well-established mouse model for mitochondrial FAO disorders. For this they will determine the dynamics of metabolic signaling during fasting in liver, heart and skeletal muscle and will quantify metabolic fluxes in protein synthesis and degradation. They will use a specific therapeutic diet to assess whether the depletion of tricarboxylic acid cycle intermediates that results from impaired protein mobilization can be delayed. In AIM 2, the investigators will study peroxisomal metabolic pathways that are alternatives for fatty acid degradation when mitochondrial FAO is defective. Using genome editing technique in a cell line, they will elucidate the molecular players participating in one of these alternative pathways. They will also assess the physiological importance of these peroxisomal pathways by studying the biochemical and clinical features of a unique mouse model with a combined defect in mitochondrial FAO and peroxisomal ?-oxidation. Combined these two aims will yield not only the much needed refined mechanisms, but also novel pathophysiological insights in FAO disorders and thus novel potential targets for treatment of these diseases.

The proposed research will investigate some of the biochemical and biophysical properties of structural proteins isolated from normal and cataractous human lenses. Gamma-crystallin, and other low molecular weight crystallins will be fractionated and characterized. The study will include: amino acid composition, molecular weight determination by ultracentrifugation and gel electrophoresis. Secondary and tertiary conformational parameters will be investigated by sensitive circular dichroism measurements. Effects of concentration, ionic strength, pH, and temperature on the conformation will be monitored by absorption, fluorescence and circular dichoism measurements. The properties of proteins isolated from normal and cataractous lenses will be compared. The properties of fluorescent pigments found in normal and cataractous lenses will be studied by using absorption and fluorescence measurements on whole lenses, lens thin-sections and lens homogenate. Low temperature (77 degrees K) absorption spectra will be utilized to increase the resolution. Plasma membrane proteins will be isolated from normal and cataractous human lenses. The study will include amino acid compositon, molecular weight determination, and carbohydrate analyses. Structural studies using circular dichroism and laser Raman spectroscopy will be performed. The effects of various detergents on the conformation of the membrane proteins will be investigated.

Biliary atresia (BA) is a progressive, inflammatory, sclerosing cholangiopathy that presents in infancy and leads to bile duct obstruction, biliary cirrhosis and the need for liver transplantation in the majority of patients. The etiology of BA is not known; a proposed theory is that the bile duct injury is initiated by a viral infection, followd by a progressive, autoimmune-mediated response targeting bile duct epithelia. Our laboratory and others have established the contribution of T cell-mediated inflammation and autoimmunity to bile duct injury in the rotavirus-induced mouse model of BA and in limited human studies. Recent data from our laboratory reveals that B cell-deficient mice are protected from BA, suggesting that B cells are essential to the development of bile duct injury. Research in murine models and humans have demonstrated the significant contribution of B cells to the onset and progression of many different autoimmune diseases, despite the fact that the organ-specific injury in these diseases was traditionally thought to be solely due to T cell-mediated inflammation. The specific hypotheses to be tested in this proposal are two-fold: 1. B cells play a critical role in the development and progression of bile duct injury and obstruction in murine BA; and 2. BA patients have circulating serum autoantibodies that may provide clues to disease pathogenesis and serve as prognostic biomarkers of disease severity. Specific Aim 1: Establish the contribution of B cells to development of bile duct injury in murine BA through use of B cell knockout and transgenic mice. Investigations of knockout and transgenic mice will establish the B cell mechanism involved in bile duct injury, specifically B cell antigen presentation versus immunoglobulin production. Specific Aim 2: Determine the contribution of B cells to progression of bile duct injury in murine BA through administration of B cell-depleting agents. This aim has direct translational implications to potential new therapies for human BA. Specific Aim 3: Define serum autoantibodies in BA patients and determine correlation with disease severity. Serum autoantibodies will be identified from a protein autoantigen microarray. The utility of autoantibodies in BA as serum biomarkers of disease severity will also be assessed. Significance: These investigations will add a unique perspective and increase our understanding of how B cells function in the setting of virus-induced autoimmunity. Discovery of autoantibodies in BA would provide clues to autoimmune mechanisms of pathogenesis and function as useful biomarkers to gauge severity of disease or response to novel therapies. The potential benefit of B cell depleting agents in alleviating progression of disease could change the paradigm of how physicians care for BA patients.

The effects of lithium in emotionally disturbed children having at least one manic-depressive lithium responsive parent will be examined. Thirty children, ages 6-12, of both sexes, having psychiatric symptoms that are in some way incapacitating and are of major concern to the child, parent, or teacher will be selected. Children will be given lithium and placebo, in a double-blind, crossover design for 16-18 weeks. Behavior ratings will include: parent's and teacher's assessment, psychophysiological measures, child interview, and cognitive testing. The lithium administration will be monitored by an "unblind" coinvestigator. This project has been discontinued.

The grant proposes a program of basic and clinical research in stem cell transplantation. Preclinical studies in dogs will develop nonmyeloablative conditioning programs for patients with aplastic anemia, who only require immunosuppression for hematopoietic engraftment, and for patients with genetic diseases, e.g. sickle cell disease, where 20-30% donor-type hematopoiesis may suffice to relieve symptoms. The dog model, which allows for in vivo analysis of stem cell function, will be used to define the limits of cord blood transplantation, with emphasis on in vitro expansion that is needed to increase cell yield for transplantation into adults and for gene therapy. Additional studies will focus on improved retroviral delivery systems for gene therapy, to improve upon clinical gene therapy studies already begun in patients with Gaucher's disease. Clinical studies will address ways of increasing the application and efficacy of stem cell transplantation for treating aplastic anemia (AA), myelodysplastic syndromes (MDS), and autoimmune disease, as well as genetic diseases, in conjunction with these projects, in vitro studies of cellular interactions will define how diseased cells in MDS or AA may be capable of suppressing normal hematopoiesis, possibly leading to alternative forms of therapy for these diseases as well as other marrow failure syndromes. Marrow grafts from HLA-identical siblings will be continued with emphasis on developing new regimens for preventing graft rejection and acute GVHD. Efforts to increase the number of patients eligible for transplantation will focus on the use of less well matched family members and of unrelated individuals as donors. As more patients become long-term survivors, long- term follow-up studies have become imperative to understand, treat, and prevent secondary malignancies and chronic GVHD. All of the clinical and laboratory studies are supported by core components which provide sample acquisition and analysis, as well as study design, data processing, and statistical analyses. Other cores deal with the outpatient department and grant administration. The principles derived from the proposed studies will make it possible to provide more effective treatment to a greater number of patients.

Experiencing the empty nest during marriage is highly normative in middle adulthood in the United States, yet scant longitudinal marital research has been conducted during this period. Establishing how spouses and marriages fare during the empty nest is important because marital functioning predicts an extensive set of older adults' outcomes in the later years of life, including physical, psychological, and financial health, and mortality. As well, midlife health and well-being reliably predict the developmental transition to old age. In this proposal, I set forth two specific aims to address gaps in our understanding of the role of the empty nest in middle adulthood marital functioning and health. First, I propose to examine empty nest status as a predictor of both marital functioning (quality and course) and health (mental and physical) for men and women over time, and to then test moderators of the longitudinal associations. Second, I propose to use dyadic data obtained from participants and their spouses to test within-couple associations between concurrent empty nest status and husbands' and wives' marital quality and health, respectively, and to again test moderators of these linkages. This proposal seeks to conduct these aims with value-added secondary analysis of the Wisconsin Longitudinal Study (WLS). The WLS is uniquely suited to accomplish the proposed aims for several reasons. First, detailed interviews that spanned middle adulthood tracked participants' empty nest status, relationship functioning, and health over time. Here, sophisticated quantitative methods will appropriately handle longitudinal data. Additionally, the WLS incorporates marital functioning indicators of quality and course in a single study, offering the potential to reconcile disparate findings in the existing empty nest literature. Second, the WLS includes a spousal sub-sample, which will facilitate direct statistical tests comparing effects of the empty nest on marital quality and health for husbands versus wives. Again, appropriate dyadic quantitative modeling will be employed. Third, a broad array of theoretically-informed spouse and marriage characteristics will be tested as covariates and potential moderators, thereby elucidating protective and risk factors. Thus, the proposed research holds important implications for translational efforts designed to prevent and alleviate distress in partners and relationships. Identifying protective factors that encourage some spouses and marriages to thrive and endure in middle adulthood is critical to understanding the determinants of healthy aging and promoting public health.

Psychophysical methods will be used to study perception of temporally ordered visual stimulation. Two novel techniques have been developed in the course of this project: one that employs temporal delays to mimic color differences resulting ordinarily from changes in the luminance ratio of complementary colors, and another which provides a measure of monocular sensitivity during binocular viewing. The program has two main objectives; one is an extensive investigation of spatiotemporal relationship existing in the visual field (specific questions include: the relationship between temporal asynchrony and spatial disparity of binocular stimulation, in the absence of stimulus velocity; the relationship between perceived vertical and the vertical horopter; an evaluation of sensory correlates of perceptual suppression during binocular rivalry); the other is development of the method of temporal visual resolution as an aid in neuro-ophthalmological diagnosis (the specific questions, here, are all addressed to anisotropies which may appear across the visual field; thus, hemispheric differences in visual-temporal resolution are expected to precede explicit field defects discovered, at a later stage, by traditional techniques such as campimetry). Data obtained so far support the conclusion that the central nervous system does not compensate for differences in conduction times.

Although the human immune system has evolved to repel many forms of biological invasion, its ability to identify which cells are detrimental to the body is not perfect. The research proposed herein has two main objectives: to explore a new fundamental strategy for directing the human immune system to kill target cells and to apply that strategy to treating several forms of metastasis-prone cancer beginning with HT-29 human colon cancer cells. The strategy is partially inspired by the concept of prodrugs. A molecule will be made such that when it interacts with urokinase-type plasminogen activator (uPA, an enzyme overexpressed on the surface of many types of cancer cells, especially those that are prone to metastasize), it will be chemically transformed into an electrophile that will covalently attach to the cancer cell's surface. The molecule will be bifunctional, and the other end will bind naturally occurring human antibodies. Once antibodies have bound the cancer cell, the human immune system will be activated to kill the target. This general strategy should also be applicable to treating a variety of other medical conditions. The research is divided into the following specific aims. (1) To design and synthesize a substrate analogue that functions as a latent electrophile to be activated by uPA- mediated hydrolysis. (2) To use this enzyme-activated electrophile to covalently attach antigens to the surface of cancer cells. And, (3) to study the immunological response against human cancer cells labeled by this target-activated antigen. This research will employ various methods. Bifunctional molecules will be chemically synthesized using standard organic chemistry reactions. Analysis of uPA-mediated activation of these molecules will be assayed by high-performance liquid chromatography (HPLC). Analysis of covalent attachment of the activated electrophiles to the activating protein will be assayed by mass spectrometry. Noncovalent binding between the antigen-labeled protein and human antibodies will be assayed by enzyme- linked immunosorbent assay (ELISA). Binding of antibodies to cells will be assayed by immunofluorescence microscopy. Antibody-mediated cytotoxicity will be assayed in various cancer cell lines beginning with commercially available HT-29 colon cancer cells. PUBLIC HEALTH RELEVANCE: The research proposed herein offers a strategy to use small organic molecules to direct the natural human immune system to fight cancer. Specifically, this approach will use organic chemistry to make molecules that can selectively attach to cancer cells and direct the immune system to destroy those cells. Although the first application will be against the HT-29 human colon cancer cell line, the method proposed is broad enough in scope to target several types of cancer, especially those that are most likely to metastasize throughout the body.

Despite the fact that HIV infection has soared amongst heterosexual women of color, Latinas are consistently underrepresented in HIV research (Carmona, Romero & Loeb, 1999). Latinas are at increased risk for HIV due cultural inhibitions of condom use, idealized gender roles, and idealization of romantic relationships (East, 1998). Yet these relationships, when chronically stressful, can exacerbate mental health problems such as depression (Hammen, 2005), and long-term effects of poor immune response (Robles & Kiecolt-Glaser, 2003); it is thus imperative to examine the effects of couple conflict and depression on HIV+ Latinas' morbidity and mortality. This study aims to investigate the links between relationship functioning and immune functioning, as measured by CD4 cell count and prevalence of gynecological problems, over time and how depression may mediate these links, in both HIV+ and HIV- heterosexual Latinas. Its results will thus be useful in understanding and creating new public health prevention and intervention methods aimed at ethnic minority women. I am using a sub-sample of HIV-positive (N=97) and HIV-negative Latinas (N=52) from the University of California-Los Angeles Charles Drew Medical Center Women and Family Project, a longitudinal study examining the impact of HIV on women's lives (see Wyatt & Chin, 1992). The data has been collected and I will specifically analyze data spanning across two years, using hierarchical linear modeling (HLM) and path analysis and focusing on measures of relationship quality, depression, CD4 cell levels, and gynecological problems. In accordance with the stress/social support hypothesis, a theory that accounts for the varied effects of stress and social support in couple relationships on physiological health (Burman & Margolin, 1992), I predict that poor relationship functioning over time will be associated with decreased CD4 levels and a higher prevalence of gynecological problems, and that depression will mediate these associations. My proposed study is highly relevant to public health research in innovative and important ways. First, my project is unique in that I am looking at HIV-positive Latinas, a population that has been overlooked in HIV research despite the fact that HIV infection is the fourth leading cause of death amongst Hispanic women (CDC, 2004). Moreover, as there is a dearth of longitudinal studies in HIV research, my project will contribute a more comprehensive picture of the intricate interplay between relationships, immune regulation, and mental health. [unreadable] [unreadable] [unreadable]

A major theme of this SCCOR is discovery and elucidation of genetic defects that result in congenital cardiovascular malformations, particularly those affecting valvulogenesis. The objectives of Core B are twofold: 1. To ensure the availability of cohorts of patients with specific cardiovascular anomalies in whom phenotype has been well characterized by echocardiography. To catalogue and store echocardiographic and additional relevant clinical material along with DNA from both probands and parents so that the information is easily accessible, both by the SCCOR participants and other interested parties who satisfy the privacy governance issues that will be put in place. 2. To provide echocardiographic characterization of extended family members for probands with bicuspid or bicommisural aortic valve (BAV) (Project 1) or Noonan syndrome (NS) (Project 2). The resources obtained by these critical Core functions will facilitate identification of novel candidate genes and their genotype-phenotype correlations. As candidate genes become apparent, they can immediately be moved into mechanistically based studies by all of the SCCOR's participants.

The Program in Cancer Biology is focused on investigations into mechanisms and signaling pathways involved in the malignant transformation of cells. The goals are to advance basic understanding of cancer pathogenesis and to translate basic science discoveries into the clinical arena to improve the outcome of patients with cancer. The Program encompasses three major thematic groups: 1) Mechanisms and Pathways of Oncogenesis;2) Cell Cycle, Cytoskeleton and Genomic Stability;and 3) Epithelial Neoplasia (including skin, prostate and lung cancer subgroups). Investigations are focused on mechanistic studies of oncogenes and tumor suppressor genes, the control of cell division, genomic stability and DNA damage checkpoints, as well as disease-specific interests in hematologic malignancies, skin, prostate and lung cancers. The 40 investigators represent two different schools (the School of Medicine and the School of Humanities &Sciences), 16 Departments, and five Divisions within the Department of Medicine. The research activities of the 40 investigators are supported by 73 peer-reviewed, investigator-initiated grants (R01s and others), and five T32 training awards. Total NCI support is $4.3 million, other NIH support is $7.2 million, and non-NIH support is $3.2 million. Since 2005, program investigators have published over 400 manuscripts relevant to cancer biology in peer-reviewed journals. The Cancer Center will continue to be invaluable in assisting with the translation of the basic science findings from this Program into new clinical approaches for the diagnosis and treatment of patients with cancer.

The National Institutes of Health (NIH) has defined health literacy as the degree to which individuals have the capacity to obtain, process and understand basic health information and services needed to make appropriate health decisions. Unfortunately, fully 22% of US adults have only basic and an additional 14% have below basic health literacy. The relationship between limited literacy and adverse health outcomes has been well documented, and seminal reports about health literacy have been issued by the Institute of Medicine, Agency for Healthcare Research and Quality, and the American Medical Association among others. Each of these reports advocates further research to develop an effective response and research in health literacy has begun to flourish. An important next step to cultivate this emerging research field is to create a national professional interdisciplinary home for investigators dedicated to health literacy research. In this grant application we propose to deliver a sixth annual conference along a multi-year trajectory dedicated to the promotion of the field of health literacy research. This meeting will be designed to provide a venue to focus attention on health literacy research, emphasizing the role of health literacy research in the elimination of racial and ethnic health disparities and on patient empowerment. This venue serves crucial functions for the field of health literacy by providing an incubator for young investigators and encouraging a cross-disciplinary exchange of knowledge, collaboration, and creativity. We aim to create an environment in which controversial topics within the research community can be openly discussed. Dr. Paasche-Orlow has served as a guest editor for special journal issues emerging from each of the past meetings; we plan to continue our successful dissemination efforts with another special issue on Health Literacy to be published by the Journal of Health Communication. We also plan to improve the meeting's Web site so that it can serve as a public resource for sharing information about health literacy measurement instruments. This year the meeting will take place on November 3-4, 2014 in Bethesda, MD.

A major goal of this proposal is to develop a novel experimental paradigm to engineer preventative HIV-1 vaccines. The V1/V2 domain of HIV-1 envelope is an important target for vaccine development for several reasons. V2 interaction with the 47 integrin of mucosal T cells may play a key role in the efficient capture of HIV-1 virus at the site of initial exposure during sexual transmission. The V1/V2 domain is also a key player in regulating neutralization sensitivity of HIV-1, and consists of epitopes recognized by some of the most potent broadly neutralizing antibodies reported to date. Here, we hypothesize that induction of antibodies by vaccination with V1/V2 immunogens that interferes, specifically with the V2 47 interactions, confers protection against HIV-1 acquisition. Since the conformation of V2 is highly variable, the V2 domain will be constrained by transplanting it into the compactly folded 9 kDa small outer capsid protein (Soc) from bacteriophage T4. To select for the functionally important V2 conformations, libraries of V2 domain variants consisting of founder/acute virus sequences, different glycosylation patterns, and random mutations will be constructed and expressed in CHO cells as secretory proteins. High throughput assays will be developed, one to select 47 binding conformations and another to select V2 conformational antibody binding variants. The biologically active Soc-V2 conformational variants will be arrayed on phage T4 nanoparticles by incubating the purified Soc-V2 proteins with Soc-minus Hoc-minus phage, up to 870 copies per particle. A Hoc- fused targeting ligand such as Dec205 mAb will also be assembled on the same capsid, up to 155 copies per capsid, to target the T4-V2 nanoparticles to the antigen-presenting dendritic cells. The immunogenicity of T4-V2 nanoparticles will be evaluated in mice by intramuscular route as well as transcutaneous (skin) route using heat labile enterotoxin as an adjuvant. The immune responses will be quantified for V2- 47 interfering antibodies, virus neutralizing antibodies and transmission-blocking antibodies. The best V2 variants down-selected from these assays will then be tested in the rabbit model. A novel HIV-1 transmission assay will be developed, which will precisely quantify the number of virus genomes that cross the host membrane after a few minutes of exposure to CD4+, 47+ and CCR5+ T cells. Based on real-time PCR, this assay would be extremely sensitive, rapid, and high throughput. Using this assay, V2 variants that can induce antibodies which block virus entry by interfering with the initial virus-host interactions will be selected. These might potentially serve as preventative HIV-1 vaccine candidates for further studies in nonhuman primates and clinical trials.

Surgical resection for cancer is becoming more minimally invasive with smaller incisions and less patient morbidity. However, this process switches the burden to the surgeon to remove all the disease with suboptimal visualization. This project proposes to develop a technique to employ pre- operative injection of ultra-sound contrast enhancement stationary microbubble and microshells to enable intraoperative detection of tumors even in cases with small skin incisions to allow surgeons to better identify and completely resect tumors and decease patient morbidty. Aims: To determine the optimal dose and lifetime of stationary microbubbles in a rabbit model as an ultrasound contrast agent in order to localize small breast tumors for surgical resection. To study the localization ability of a novel gas filled silica microshells compared to microbubbles as an ultrasound contrast agent and localization method for occult breast cancers using a rabbit model. PUBLIC HEALTH RELEVANCE: Because surgical resection of small early cancers remains so important in local control and prevention of metastases, techniques to improve its efficacy in visualizing and removing tumors completely can have a major impact on breast and other cancer treatment. We believe that improving targeting of nonpalpable cancers is crucial. This fact is particularly important in this era in which tiny cancers are detected by imaging and more advanced techniques are needed for localizing tumors. This project may be able to decrease positive margins, local recurrence and possible cure of early stage cancers.

Taste controls an animals food intake. For the past fifteen years, our focus has been the isolation and characterization of genes encoding taste receptors and using these to mark the cells, define the corresponding signaling pathways, dissect receptor specificity, generate topographic maps, and trace the respective neuronal connectivity circuits. This research continues to involve a long standing and wide ranging collaboration with Charles Zuker and his groups at Columbia University. Together, we have now established that at the periphery there are 5 distinct classes of taste receptor cells that are selectively tuned to respond to sweet, bitter, sour, sodium salt and savory (umami) tastants and have demonstrated that activation of these cells elicit either appetitive or aversive responses accordingly. Taste information is transmitted to the nucleus of the solitary tract (NST) in the hind-brain through sensory neurons with cell bodies in the geniculate and petrosal ganglia. Projections from the NST diverge: conscious perception of taste quality is thought to involve a pathway that innervates the primary gustatory cortex in the insula; in addition, less well characterized circuits are believed to mediate immediate responses to tastants. Our current work focuses on understanding how taste information is transmitted to the brain and represented there to generate defined percepts and to guide behavior. We are using a battery of modern molecular genetic approaches to define and trace circuits involved and ultimately to relate these circuits to taste perception. These include: Ca-imaging techniques to assess the activity of neural ensembles; screening and sequencing approaches to identify markers for select subsets of taste responsive neuron; and optogenetic, pharmacogenetic, activation and silencing as well as other silencing and ablation approaches to modulate activity of specific parts of the taste circuitry. This reporting period has seen major progress in uncovering the logic for the transmission of taste information from the tongue to the brain (manuscript in press). Specifically, the tuning of taste primary afferents appears to closely match the tuning of the taste receptor cells pointing at a highly specific transfer of information. In mice, the odorant receptors are encoded by a family of more than 1000 genes. A fundamental feature of the mammalian olfactory system is that each olfactory sensory neuron expresses just a single member of this vast family of genes. However, the details of the control of odorant receptor gene expression remain unexplained. In a collaborative project with Leonardo Belluscio, we have demonstrated new aspects of regulation that contribute to the control of odorant gene expression and have devised a system that can reliably generate mice expressing a single odorant receptor in the vast majority of olfactory sensory neurons. In this reporting period we have continued to characterize the behavioral responses of a transgenic mouse that we generated that expresses an octanal receptor in almost all olfactory sensory neurons. Although this transgenic mouse line has the potential to express a second olfactory receptor from the same synthetic promoter as the octanal receptor, surprisingly it rarely does. Indeed, whereas the octanal receptor is found in 90% of all olfactory receptor neurons, the other receptor (that responds to acetophenone) is present in just 5% of cells and these do not overlap with the cells expressing the octanal receptor. We are therefore examining the mechanisms that account for this completely unexpected gene expression pattern and believe that they will provide insight into the unusual expression pattern of this remarkable gene family of sensory receptors.

This protocol evalutes the growth, physical development and pituitary function in children surviving oncology diseases. Possible alterations of physiologic mechanisms resulting in hormonal failure, including treatment modalities will be studied.

The overarching goal of the proposed research is to identify the underlying causes of speech understanding problems in older adults. Older listeners, with or without significant hearing loss, often have much greater difficulty understanding speech in the presence of competing sounds than do younger listeners (CHABA, 1988; Dubno et al., 1984; Duquesnoy, 1983). For hearing-impaired listeners, differences in degree of hearing loss (or audibility) account for a large portion of the variance in listeners' ability to understand speech in the presence of competing sounds, but other factors (e.g., cognitive or central auditory factors) play an important role for both hearing-impaired and normal-hearing listeners (see Houtgast & Festin, 2008; Akeroyd, 2008; Humes, 2007; Humes & Dubno, 2010; Humes, Kidd, & Lentz, 2013). One promising hypothesis, which has received relatively little attention to date, is that effective speech understanding in the presence of competing sounds depends strongly on the ability to follow the temporal structure of an utterance and to accurately anticipate the timing of speech events through a process of attentional entrainment. According to the entrainment hypothesis (see Large & Jones, 1999; McAuley et al., 2006) the ability to attentionally track the temporal patterns of speech helps listeners to perceptually isolate and resolve the details of a single talker's speech by enhancing attention to speech events that correspond to a talker's temporal pattern. Moreover, accurate extrapolation of temporal patterns through inaudible or partially audible portions of an utterance is proposed to facilitate the integration of intermittent glimpses of speech by incorporating them in a common temporal framework. This general hypothesis will be investigated through a series of experiments that test the predictions of entrainment theory in a speech context, in combination with a new test battery that assesses individual differences in entrainment ability. A sample of 1000 young and older listeners with and without hearing loss will be tested, using shaped amplification to ensure audibility for hearing- impaired listeners. The project will employ a combination of behavioral and neurophysiological measures to determine how entrainment ability is related to speech understanding, while controlling for individual differences in basic auditory capabilities and cognitive abilities previously shown to be important for speech perception.

DESCRIPTION (Applicant's Description): The investigators hypothesize that the angiostatic factor IP-10 is an endogenous inhibitor of tumor-derived angiogenesis. In this proposal, they will test this hypothesis by characterizing the role of IP-10 in regulating overall tumor-derived angiogenic activity in an animal model of human non-small cell lung cancer (NSCLC). They will demonstrate that underexpression of IP-10 imparts increased angiogenic activity to NSCLC cell lines. Next, they will reconstitute IP-10 production in NSCLC cell lines, thus restoring the angiostatic balance, using two methods. (1) In vitro treatment of NSCLC cells with interferon gamma (IFNgamma) to induce IP-10 expression, and (2) Gene transfer technology to indirectly (IFNgamma) or directly (IP-10) augment IP-10 expression. They will then show that IFNgamma and IP-10 gene transfected cells have reduced in vitro angiogenic activity and reduced tumorigenicity in SCID mice. Finally, they will treat established NSCLC tumors in SCID mice with locally administered IFNgamma or IP-10 and show that IFNgamma inhibits tumor growth via an IP-10 dependent increase in angiostatic activity. These studies will lead to important new information about the pathogenesis of NSCLC, and enable the Principal Investigator to acquire the necessary skills to develop into an independent investigator.

The study of MHC restriction of T cell specificity and more particularly the relationship of this phenomenon to Ir gene function, has been a major concern of our laboratory. This project addresses three aspects of the specificity of T lymphocytes restricted to react with antigen and self Ia molecules. 1. The nature of Ia-antigen interactions, and the specificity of T cell receptors for self Ia. 2. The control of IL-1 production at the level of the antigen presenting cells and the role of Ia molecules in this process. 3. The selection of a differentiating T cell population in the thymus restricted to react specifically with self Ia molecules.

The paternal age effect (PAE) refers to an increased risk for a particular phenotype or mutation with increasing age of the father. This is of major relevance in psychiatry where three of the most severe disorders (autism, schizophrenia, and bipolar disorder) all show a PAE. These disorders are roughly twice as likely with fathers >40 years of age and three times more likely with fathers >50, compared with 20-25 year old fathers. The birth rate for males >40 has increased by 50% in the past 25 years (now 10% of all US births), so this is becoming a significant public health issue. Recent discoveries show an increased burden of de novo copy number variants (CNVs) in autism and schizophrenia, supporting the involvement of spontaneous mutations in severe mental illness and potentially helping to explain the PAE for these disorders. However, paternal age has not been explicitly shown to increase the mutational burden of offspring in any organism. Since a highly-powered search for a PAE on CNV and point mutation burden in humans is difficult to assess because of low numbers of offspring, we propose to use the laboratory mouse to answer this question. We already have DNA samples in hand to perform this experiment via next-generation sequencing and request funds to calculate the PAE on mutational burden. Furthermore, we have collected a range of fertility-related phenotypes in old and young males and psychiatrically-related behavioral phenotypes in the offspring of young and old fathers, allowing a direct comparison of genotype to phenotypes. We have collected all of the DNA samples and relevant phenotypes, and are ready to proceed with high-throughput sequencing in order to calculate mutation rates of great relevance to biomedicine. In Aim 1 we focus on CNVs, which are readily detected at low coverage sequencing due to our selection of mice for this purpose, and in Aim 2 we focus on point mutations, which require higher coverage. Clearly, the potential reward of this project is substantial. We know that PAE is a risk factor for multiple neuropsychiatric disorders but the mechanism underlying risk is unknown. In conclusion, if this project is successful, it would lay the foundation for extension to human psychiatric samples and detailed mechanistic studies within a next-generation mouse systems biology platform, the Collaborative Cross.

Three peptidic transition-state analogs have been proposed from highly-conserved polypeptide segments of the HIV-1 envelope glycoprotein and two of these analogs have been synthesized and used as immunogens for the production of murine monoclonal antibodies. Several hundred hybridomas have been shown to bind to the transition-state analog used for immunization and several of these show differential binding to the transition-state analog vs the original-sequence peptide.

Research within the Laboratory for Integrative Neuroscience, Section on Synaptic Pharmacology, continues to focus on mechanisms underlying neuromodulation and plasticity and the effects of alcohol and other drugs of abuse on these neuronal functions. Our main interest is the function of the dorsal striatum (DS), a brain region involved in action control and selection, as well as action learning. Striatal Synaptic Plasticity and Effects of Drugs of Abuse We have examined many forms of synaptic plasticity at synapses within the DS. One of the most prominent is long-term depression (LTD) at glutamatergic corticostriatal synapses produced via activation of the Gi/o class of G-protein-coupled receptors (GPCRs). In addition to LTD induced by activation of cannabinoid type 1 (CB1) and serotonin type 1B receptors, we have also observed that activation of the mu, delta and kappa receptors for opioid peptides can induce LTD. Likewise, inhibition of opioid peptide catabolism induces LTD, and this synaptic depression appears to involve actions of endogenous opioid peptides at the three receptor subtypes. The expression of the LTD induced by mu and delta opioid receptors involves decreased probability of glutamate release, resembling the mechanism of CB1-dependent LTD. Indeed, mu opioid receptor and CB1-dependent LTD can occlude on another, indicating that they share mechanisms at an overlapping population of synapses. In contrast, delta opioid receptor-dependent LTD cannot be occluded by mu-dependent LTD, indicating separate sites of action for these two opioid-mediated forms of synaptic plasticity. We have examined effects on striatal LTD of in vivo exposure to the widely-prescribed and heavily abused opioid receptor agonist oxycodone as well as ethanol. A single injection of oxycodone in vivo leads to loss of mu- and CB1-dependent LTD. The effects persist for up to 4 days following a single drug injection. The in vivo oxycodone injection has no effect on delta receptor-mediated LTD. Similar loss of mu- and CB1-dependent forms of LTD is observed following 2-4 weeks of in vivo exposure to intoxicating concentrations of ethanol. Thus, impairment of certain forms of presynaptic LTD appears to be a common impairment produced by different drugs of abuse. Loss of LTD likely alters cortical control of striatal medium spiny neurons, perhaps contributing to alterations in striatal-dependent learning produced by drugs such as ethanol, and perhaps contributing to habitual drug seeking. It will be important to figure out how mechanisms involved in LTD are impaired by in vivo drug exposure. The metabotropic glutamate receptor 2 (mGluR2) is another presynaptic receptor implicated in modulation and long-term depression of corticostriatal glutamatergic synpases. In collaboration with the laboratories of Drs. David Goldman and Markus Heilig here at NIAAA we have confirmed that a polymorphism that produces a premature stop codon in this gene (the grm2 gene) leads to loss of function of this receptor in alcohol-preferring (P) rats. Presynaptic receptor function is completely lost at corticostriatal synapses. Studies performed by the Goldman and Heilig laboratories indicate that loss of this receptor contributes to increased ethanol drinking and preference in both rat and mouse. It will be interesting to determine the contribution to these phenotypes of mGluR2 at corticostriatal synapses.

Since graduating from medical school, my goal has been to become an academic surgeon with great emphasis on a career exploring basic science issues related to clinical problems. In vascular surgery, the need for improved small diameter vascular grafts has been recognized. Through the excellent research training I've received, I have developed an interest in the heterotypic endothelial (EC) and smooth muscle cell (SMC) interactions occurring within the vascular wall. I have used a SMC-EC co-culture system to model these interactions, and have developed a novel flow device to study the effect of flow on SMC-EC interactions. My objective is to further characterize the biologic mechanisms involved in this model, but I have found I lack the specific expertise in molecular biology to proceed. At Robert Wood Johnson Medical School, I have a unique opportunity to purse my career development by working with outstanding scientists in fields that will enhance my abilities and intellect. I plan to achieve my objective through course work at Rutgers University, attendance of basic science conferences, and regular meetings with my mentor and advisors designed to monitor and enhance my progress. This program will improve my fundamental knowledge base in basic science (molecular biology, biochemistry, and biomedical engineering), and allow me to expand the breadth of techniques used in my laboratory. This will allow me to continue to define the biologic mechanisms responsible for the complex cellular and cell-material interactions. Using this co-culture flow model to study heterotypic cellular and cell-matrix/biomaterial interactions in the presence of shear stress, we have observed that SMCs promote EC adhesion and adaptation to flow. We hypothesize the failure of biomaterials to support the essential processes of FC adhesion and migration is due to a lack of key matrix components, and the development of a matrix optimal for EC attachment and migration is dependent on SMC-EC communication. I now propose to study the mechanisms by which SMCs enhance EC adaptation to flow. Using BC detachment rates and migration in the co-culture flow model as bioassays, we will evaluate if SMCs enhance BC adhesion and migration by altering cell-matrix and cell-cell interactions. We will also determine if BC apoptosis is in involved in the detachment process, and whether BC apoptosis is modified by SMCs. We will partially characterize the factor(s) present in SMC conditioned media and determine by bioassays and immunodepletion if it is likely to correspond to a previously identified compound. If the factor(s) is not identified in this manner, we will continue the purification process to isolate the factor(s) and determine its identity. Conceivably, the responsible mediator(s) could be used as an adjunct in the development of new prosthetic or tissue- engineered biosynthetic grafts.

Coxiella burnetii is the causative agent of both acute Q fever and chronic endocarditis in humans. Both plasmid and chromosomal differences correlate with the type of disease (acute or chronic) caused by an isolate suggesting that the different isolates have different virulence determinants. We have designed experiments to extend our knowledge of C. burnetii genomic and plasmid DNA and their relationship to disease. Unique plasmid regions will be analyzed by cloning, restriction enzyme, Southern hybridization and sequencing analyses. We will also identify the plasmid origin of replication for studies on transformation and mutagenesis of C. burnetii. Plasmid encoded surface proteins (possible virulence factors) will be identified by looking for protease-sensitive surface proteins produced by maxicells containing pUC/C. burnetii plasmid constructs and also by TnphoA mutagenesis. We will identify chromosomal encoded surface antigens employing both cosmid and 1 ZapII gene libraries in E. coli, by screening for colonies/plaques reacting with C. burnetii-specific antisera and monoclonal antibodies to isolates with differing disease potential. The distribution of antigens in C. burnetii isolates will be determined using surface iodination, PAGE, and western analyses. To understand the role of gene in virulence and intracellular growth of C. burnetii, we have designed experiments to evaluate gene function directly within the organism. We will use previously cloned C. burnetii chromosomal and plasmid genes as specific probes to assay their transcriptional activation at different times and under different conditions, in vitro and in vivo. Concurrently, we will examine qualitative differences in C. burnetii RNA production. Genes specifying unique or differentially expressed transcripts produced at different time points in vitro or post-infection will be cloned and sequenced. Proteins being synthesized under these conditions will also be evaluated by 1 and 2 -D PAGE. In this fashion we will develop an understanding of gene expression in C. burnetii during the host-parasite interaction. We also will examine plasmids and the chromosome for alternative virulence functions; factors that enhance intracellular survival and growth and thus pathogenesis. We will clone genes encoding key metabolic functions as well as those shown to be important for intracellular survival of other organisms. These genes will be cloned from C. burnetii employing the methods of PCR, mutant complementation and using homologous genes as probes. And because the development of genetic exchange systems is vital to an understanding of rickettsial genetic mechanisms, we will continue to develop transformation and mutagenesis systems for C. burnetii using electroporation and antibiotic selection in cell cultures.

Stroke is the leading cause of long-term disability in the United States. Although acute revascularization therapies can be used to abort or reduce stroke burden, there are currently no drugs that improve recovery after a stroke has happened. The inflammatory response is a promising target for such therapies as it occurs in the days and weeks after a stroke and can be both detrimental and beneficial. A major unanswered question is how the injured brain modulates immune responses, and if there are molecular pathways that can be utilized to exert beneficial or limit detrimental effects on functional recovery via modulating the overall immune response. Astrocytes are a key component of the brain&#700;s injury response so-called reactive astrocytes are ubiquitous after brain injury. They are also increasingly recognized as key components of the brain&#700;s innate immune system. We propose to ask if Transforming Growth Factor Beta (TGFbeta) signaling in astrocytes modulates inflammation after stroke because it is a master regulator of immune responses. TGFbeta can resolve immune responses after injury and drive immune cell phenotypes towards less inflammatory states. Our preliminary experiments show that TGFbeta signaling is increased in the brain after stroke, persists for weeks, and occurs in reactive astrocytes. To test if TGFbeta&#700;s function in reactive astrocytes mirror its role in other types of immune cells we constructed mice in which TGFbeta signaling is decreased only in astrocytes. We have found that primary astrocytes from these mice exhibit a more pro-inflammatory phenotype after oxygenglucose deprivation, and the mice themselves demonstrate increased inflammatory responses after stroke. Based on this data we hypothesize that after stroke, TGFbeta signaling (1) occurs in reactive astrocytes, (2) limits the inflammatory response, and (3) improves functional recovery. We plan to test our hypothesis in three Specific Aims. In Aim 1 we will use reporter mice and immunohistochemistry to determine patterns of TGFbeta signaling after stroke. We hypothesize that there are increased responses to TGF-beta for weeks after stroke, and that reactive astrocytes are responding to TGFbeta after stroke. In Aim 2 we will test the function of astrocytic TGFbeta signaling in the neuroinflammatory response to ischemia, using genetic and pharmacological approaches and in vivo and in vitro experiments to target TGFbeta signaling in astrocytes. We hypothesize that astrocytic TGFbeta signaling drives resolution of the immune response to stroke. In Aim 3 we will use a genetic mouse model to ask if stroke-induced astrocytic TGFbeta signaling is beneficial or detrimental for functional recovery. We predict that astrocytic TGFbeta signaling improves recovery from stroke. With the completion of the proposed experiments we will have defined the length and cell specificity of TGFbeta responses after stroke. We will gain insight into how astrocytes influence the immune response to stroke, and into the functional diversity of reactive astrocytes. Our findings may lead to therapies that will target the brain&#700;s immune responses and benefit patients who present for medical care in the days after stroke.

Project Summary/Abstract The evidence for the degree to which indirect effects (i.e. the protection conferred to unvaccinated individuals) of influenza vaccination reduce the risk of infection is lacking. Much of our current knowledge of indirect effects of influenza vaccination comes from ecologic studies comparing communities in which school children were vaccinated to those in which they were not. Subsequently, several randomized clinical trials vaccinated children and then compared the risk in their contacts to the risk in contacts of unvaccinated children. This previous work is generally limited to one year of follow-up and has not used laboratory-confirmed infection, limiting the quality of the evidence and the ability to draw conclusions about different influenza types and subtypes. The objective of this project is to determine the indirect effects of influenza vaccination in households over multiple years. We hypothesize that indirect vaccine effects can manifest in two ways at the household level. First, vaccinated individuals may be protected against community acquired infection thus preventing them from exposing unvaccinated household contacts. Second, unvaccinated household members can be protected through reduction in infectiousness of a vaccinated household index case, and a subsequent reduction in the secondary infection risk among household contacts. Using longitudinally collected data from the Household Influenza Vaccine Effectiveness (HIVE) cohort study we plan to conduct analyses to address the following specific aims: Aim 1) Quantify the indirect and total effects of influenza vaccination in reducing the risk of influenza infection; Aim 2) Estimate the indirect effect of influenza vaccination in reducing the risk of exposure to influenza at the household level; and Aim 3) Estimate the indirect effect of influenza vaccination in reducing the risk of influenza infection in unvaccinated members following introduction of influenza to the household. The expected outcome of the work proposed in these aims is to quantify the protection of unvaccinated individuals by influenza vaccination of household contacts by influenza type/subtype. We further expect to clarify how that protection is conferred, provide clearer evidence for targeting vaccination by age in the event of vaccine shortage, and to estimate the total effect of vaccination in preventing infection. Indirect effects of influenza vaccination are assumed, despite insufficient evidence. Thus, the impact of these findings will be to improve public health messaging by informing vaccine advisory bodies and other decision makers of the total benefits influenza vaccination.

This protocol will evaluate the antitumor activity of melphalan (L-PAM) and L-buthionine sulfoximine (BSO) in patients with metastatic melanoma. The effect of BSO pretreatment on glutathione metabolism in peripheral blood lymphocytes and tumor tissue, as well as BSO pharmacodynamics, will be studied.

The objective of the proposed research project is to study the processes of ventilation of the middle ear and sinuses. The purpose is to obtain knowledge that will have direct application to clinical diagnosis. An approved dosage of Xenon-133, a radioactive gas, is administered to normal subjects by means of a bilateral negative-positive pressure air displacement technique that yields an initial concentration of the gas in the sinus and middle ear cavities. The rate of egress of the gas from the individual cavities is measured using a scintillation camera that is interfaced to a PDR-15 computer. Simultaneously, two small probes (6 mm diameter) are placed at counters. The computer data and the portable equipment data will be directly comparable, so the portable equipment can be assessed for its value in providing reliable rates of egress. Normal rates of ventilation of the mentioned cavities are being determined from the data. Subjects with confirmed pathology of the middle ear or sinuses will then be tested to determine if a desired correlation is obtained in relationship to normal standards. It is hoped that this research will help solve some of the most crucial problems of otology.

DESCRIPTION (Applicant's abstract): The elimination of neurotropic viruses such as herpes simplex virus (HSV) from the nervous system is likely to be complex, and to involve other mechanisms besides direct lysis of infected cells by cytotoxic T cells (CTL) because MHC antigens are not normally expressed in the nervous system. Preliminary studies have revealed an extensive and prolonged inflammatory response in the mouse trigeminal ganglion (TG) and brain stem during latency. T cells associated with secreted IFN-g were focused around neurons that appeared morphologically normal, some of which were latently infected with HSV. TNF-a and IL-2 transcripts were also detected during latency. Based on the investigators preliminary studies and published studies of others, it is hypothesized that IFN-g and TNF play an important role in suppressing HSV replication at the acute stage contributing to the survival of infected neurons, and it is speculated further that they might also function to modulate the frequency of detectable reactivation. In this application an immune-PCR assay will be used to determine whether HSV antigens are expressed in the ganglion and brain during latency and the cytokine profile in these tissues will be determined by RNAse protection assay. The investigators will determine the extent to which IFN-g and TNF-a synergize to control HSV replication in the nervous system by comparing the course of infection in control, IFN-g knockout (gko) and TNF receptor knockout (TNFR1,2) mice inoculated with wild type HSV, or a HSV recombinant (HSV-g) that expresses an IFN-g transgene. An HSV mutant (HSV-gR) engineered to express the IFN-g receptor (IFN-gR) will be used in studies with IFN-g receptor knockout (Rgko) mice to determine whether the effects of IFN-g are mediated through interaction with the receptor on infected neurons and other cells. The potential of IFN-g and TNF to modulate the frequency of detectable HSV reactivation once initiated will be examined in latently infected Rgko mice, gko mice, and control mice inoculated with type HSV-gR, and subjected to transient hypothermia as a reactivation stimulus. Studies proposed here aim to enhance our understanding of the role of IFN-g and TNFa/b in the control of acute HSV infections in the nervous system and clarify their role in latency. Since episodes of chronic IFN-g secretion have been demonstrated during HSV infection in the nervous system, HSV may be involved in a variety of neurological disorders associated with increased cytokine expression in the brain.

The myeloproliferative neoplasms (MPN), originate at the level of the pluripotent hematopoietic stem cell. Each MPN can undergo clonal evolution and hematologic progression that terminates in bone marrow failure, marked by progressive man'ow fibrosis, extramedullary hematopoiesis, and/or transfonnation to acute leultemia (AL). The leading causes of morbidity and mortality in PV and ET are thrombotic complications, followed by myelofibrosis and AL. PMF has a much graver prognosis due to marrow failure, the development of massive hepato-splenomegaly and a higher rate of transformation to AL. The somatic mutation JAK2 V617F, present in between 30-95% of patients with MPN, confers constitutive tyrosine kinase activity and plays a role in the pathophysiology of MPN. MPN have not been the subject of an organized group of investigators focused on studying their pathophysiology in a manner which would lead to the development of translational clinical trials since the demise of the P Vera Study Group two decades ago. Although individual investigators have conducted clinical trials and two multi-institutional phase III studies have been completed establishing the efficacy of aspirin in reducing risk of thrombosis, and the utility of hydroxyurea in the long term in patients with ET none of these treatments has changed the natural history of the MPN. We have established the Myeloproliferative Disorders Research Consortium (MPD-RC) an intemationai group of 24 academic centers in the US and Europe to conduct translational clinical trials in MPN. The goals of this project are to conduct a series of hypothesis driven MPD-RC investigator initiated translational clinical trials, based on interactions with the laboratory based projects in the MPD-RC, in order to identify active new agents and approaches that alone or in combination, can change both the treatment paradigm and natural history of the MPN leading to improved outcomes for such patients. The specific aims include plans to conduct phase l/ll trials in patients with PMF testing such novel approaches as: CEP 701, a JAK2 inhibitor;azacitidine and vorinostat an epigenetic approach to disease modification based on observations made in Project 5: plitidepsin an inhibitor of protein tyrosine phosphatases known to change the natural history of MF in a Gata(low)'mouse model developed in project 4, and allogeneic stem cell transplant as a potential curative approach. In PV, the effects of pegylated interferon alpha-2a as well as anti-thrombotic agents will be studied in high risk PV and ET in phase 2/3 trials. These investigator initiated trials are completely dependent on the clinical research infrastructure of the MPD-RC

The primary purpose of this project is to evaluate the effects of a culturally relevant genetic cancer risk assessment (GCRA) intervention on a population of underserved women of Hispanic descent, and examine a social-cognitive-cultural model of post-GCRA cancer screening and prevention behaviors that can guide future GCRA and psychosocial interventions for this population. The following aims will be addressed: 1) Assess cognitive, social and cultural factors influencing cancer screening and prevention behaviors following culturally relevant GCRA; 2) Conduct a focus group with Latina women who recently underwent GCRA to identify important cultural components in order to individualize GCRA to this population; and 3) Evaluate changes in breast and ovarian cancer screening and prevention behaviors after culturally relevant GCRA. Two focus groups will be conducted in separate single sessions consisting of Latina women that have recently undergone GCRA. The focus group will entail completion of researcher-developed instruments and participation in facilitated discussions of the instruments, key study variables, and other culturally relevant issues. Based on focus group qualitative data specific modifications will be made to assure that the GCRA protocol is culturally relevant. All participants will undergo culturally relevant GCRA following referral by their primary physicians. Genetic testing for a BRCA mutation will be offered when there is medical necessity and a reasonable calculated probability of a detectable mutation. Cancer screening and prevention recommendations will be given based on genetic test results or empiric risk estimates. Participants will complete a baseline medical history questionnaire and survey instruments measuring acculturation, fatalism, self-efficacy, expected outcomes, and social support pre-GCRA and 3-months post GCRA. A hereditary cancer knowledge survey will be completed by participants pre GCRA, immediately post GCRA and 6- months post GCRA. A follow-up questionnaire will be administered to participants 6-months post GCRA to assess adherence to risk management recommendations. The proposed pilot study is designed to develop and implement culturally relevant GCRA services for low income Latinas and to place the intervention within a theoretical context. The long-term objectives of this study are to utilize these pilot data to develop future interventional and longitudinal studies with experimental designs which can help reduce the disparity in the prevalence, recurrence, and mortality of cancer between the majority and minority population. [unreadable] [unreadable] [unreadable] [unreadable]

The potency and safety of TRAIL has prompted clinical trials with the recombinant protein as a novel treatment for human cancer. While TRAIL is very active in killing tumor cells, recombinant TRAIL possesses drug properties that limit its efficacy such as short serum half-life, instability, and the inability to cross the intact blood-brin barrier. To overcome these limitations we identified a small molecule inducer of the TRAIL gene, TIC10 that is superior to recombinant TRAIL in terms of stability, bioavailability, ability to cros the blood-brain barrier, cost of production, and spectrum of activity. Importantly, our data shows that TIC10 is highly active in several aggressive and therapy-resistant cancers. On a mechanistic level, TIC10 causes potent antitumor effects and TRAIL-induction that is mediated by the transcription factor Foxo3a, which directly regulates the TRAIL gene promoter. Furthermore, we found that TIC10 results in the dual inactivation of Akt and ERK, thereby inhibiting their constitutive phosphorylation of Fox3a and potentiating its translocation to the nucleus and binding to the TRAIL gene promoter. We hypothesize that TIC10 induces potent antitumor effects that require Akt- and ERK-mediated Foxo3a nuclear translocation and transcriptional activation of the TRAIL gene. To address the hypothesis we propose the following specific aims: Specific Aim #1: Identify TIC10-induced effects on Foxo3a expression, phosphorylation, and binding to 14-3-3; Specific Aim #2: Elucidate the role of Akt and ERK kinases in the mechanism of action of TIC10; Specific Aim #3: Determine the differential regulation of TRAIL gene transcription by FOXO family members. These studies will create a comprehensive molecular understanding of how TIC10 harnesses Foxo3a to achieve its potent antitumor activity and also has the potential to undercover novel regulatory mechanisms of Foxo3a activity that are biologically significant. Preclinical cancer models including orthotopic xenografts and transgenic mice along with primary human tumor specimens will substantiate the efficacy and validate the mechanistic findings regarding TIC10 and Foxo3a. Together, these studies will elucidate key and novel regulatory mechanisms involving Foxo3a with the first-in- class molecule TIC10 to yield insight regarding its mechanism of action as well as in clinical samples in the context of colon cancer disease progression. Our studies will facilitate the clinica translation of a novel anti- cancer therapeutic agent through further development in preclinical laboratory studies.

Age-related hearing loss (ARHL) is an epidemic influencing the lives of 48 million Americans6. Current clinical protocols to detect ARHL leave room for much improvement so that early diagnosis and intervention can be achieved. One way to improve current clinical protocols is through the implementation of fast, objective, and sensitive tests of cochlear function using tools such as stimulus frequency otoacoustic emissions (SFOAEs). SFOAEs are currently not utilized clinically, despite their potential for assessing the cochlea in a frequency- specific manner and sensitivity to minor insults to the cochlea. Before clinical implementation of SFOAEs can be pursued, three major gaps in knowledge must be addressed. First, the relationship between SFOAEs and psychophysical measures of hearing must be thoroughly examined across the entire range of human hearing. Second, the sensitivity of SFOAEs must be evaluated for detecting cochlear changes due to developmental and aging processes. Third, using SFOAEs, cochlear contributions in functionally relevant auditory deficits must be determined. Recent findings suggest that SFOAEs may be more sensitive than behavioral thresholds to age-related cochlear changes. However, a deeper examination is needed to determine how SFOAEs are influenced during a critical period of human life, which shows early aging effects in distortion product OAEs. In evaluating the potential of SFOAEs in the accurate and early detection of cochlear dysfunction due to ARHL, the specific aims of this proposal are to investigate the relationship between SFOAEs and psychophysical measures of hearing up to the highest measurable frequency (Aim 1), to examine the developmental and aging effects of OHC function using SFOAE amplitudes and phase slopes (Aim 2) and to apply this understanding to validate the clinical utility of SFOAEs in detecting OHC contributions to speech perception in noise (SPIN) deficits (Aim 3). In Aim 1, the relationship between thresholds derived from SFOAE input-output functions with those obtained behaviorally will be evaluated. Additionally, tuning estimates derived from SFOAE phase slopes will be compared to psychophysical tuning curves. In Aim 2, a cross-sectional investigation of cochlear function across a significant portion of human lifespan (7 - 45 years) will be performed using both behavioral thresholds and SFOAE amplitudes and phase spanning a significant range of human hearing (0.5 - 20 kHz) using state- of-the-art calibration techniques. In Aim 3, SFOAEs as well as neural measures will be examined in individuals with SPIN deficits. By addressing the aims of this proposal, the long-term objective to advance the clinical applicability of SFOAEs for detecting age-related cochlear dysfunction can be pursued.

Our long term goal is to elucidate the principles by which cAMP controls cell development, proliferation and diffrentiation. cAMP regulates a striking number of physiologic processes, including intermediary metabolism, cellular proliferation, and neuronal signaling by altering basic pattrens of gene expression via activation of cAMP response element (CRE)-directed transcription. The mechanism of the CRE-directed transcription in cell proliferation, however, is largely unexplored. To elucidate the role of the cAMP enhancer (CRE) in the control of cell proliferation, we used transcription factor-decoy oligonucleotide approach. Our studies revealed that a synthetic single-stranded oligonucleotide composed of the CRE sequence, which self-hybridizes to form a duplex/hairpin, can penetrate into cells, compete with CRE enhancers for binding transcription factors and specifically interfere with CRE- and Ap-1-directed transcription in vivo . This oligonucleotide restrained tumor cell proliferation, without affecting the growth of non-cancerous cells. These results suggest that the role of PKA in cancergenesis may involve its transcription of array of genes. Recent development of high throughput DNA microarray enables parallel analysis of expression profiles of thousands of genes in a single hybridization for complex biological systems. We examined by the microarray analysis the expression of global cellular genes that are involved in PKA-cAMP-pathways by overexpression/suppression of the wild type and mutant PKA R and C subunit genes that cause changes in the growth patterns and phenotype of cancer cells. We found that approximately 240 cDNAs, representing 10% of the total DNA elements on the array, were found to have significantly altered levels of expression after treatment with RIa antisense oligonucleotide (ODN)(48hr, 0.2 mM) which produced 50% growth inhibition. The cDNA expression for these candidate genes all exhibited greater than 2.5 fold alterations (either up- or downregulated). Importantly our results showed that in comparison to cells treated with antisense ODN, the antisense gene overexpressing cells exhibited striking overlaps in the expression profiles. The same DNA elements up- or downregulated in the antisense ODN treated cells were similarly up- or downregulated in the antisense gene overexpressing cells. On further analysis by Scatter plots, we found that less than 2% of the DNA elements which exhibited altered expression were selectively up- or down-regulated in either antisense ODN treated cells or antisense gene overexpressing cells. The microarray analysis of gene profiling is also underway in cells treated with CRE-decoy oligonucleotides. This approach will lead us to potentially survey all the genetic pathways and also to discover hitherto unrecognized novel genes that may be involved in tumor growth and tumorigenesis. Furthermore, the discovery of novel genes by this approach coupled with the genetic and biochemical analyses may unravel the mechanism of cAMP-deregulation underlying cancergenesis and offer new targets for drug development and novel treatment strategies for cancer.

This proposal is designed to elucidate some of the mechanisms by which differentiation occurs in animal cells. Animal cells with the same genetic information produce a variety of different proteins, and different amounts of the same proteins. I propose to investigate the mechanism by which the amount of ornithine transaminase (E.C.2.6.1. 13) is regulated in differentiating animal cells. By using cells in culture, it will be possible to use cells of a single, defined type, whose environment can be controlled. The Chinese hamster ovary cell line will be used.

The Ohio Quality of Work Project is a social experiment which has been undertaken with the direction of representatives of labor, management and State government to improve the quality of work and productivity. The major objective of the Project is to set up throughout Ohio well-documented models of new work structures and cooperative labor/management/worker relationships in both goods manufacturing and service (including public sector). Each demonstration will be a three and a half year process including (1) an initial research portion measuring both human and economic factors of the work, (2) a continuing education process, (3) a work change program instituted by the demonstration participants and (4) remeasurements of human and economic indicators after 18 and 36 months. The instrument measuring the quality of work and its human outcomes will be used across all demonstrations. It will allow analyses to be made over time of the relationship between the quality of the working environment and a variety of outcomes. This proposal seeks funding for a portion of the research and evaluation component of this Project, to include outside evaluations of process, management systems and industrial relations.

The goal is to develop a cell deposition system (CDS) prototype that can be used for slide preparation of diagnostic samples. The CDS has been constructed by interfacing the opto-electronics of a flow cytometer into a computer whose output can be data from analyzed cells or a word for each cell suitable for sorting that cell. The concept of CDS can be enlarged to include arbitrary sorting formats because the format is determined by a computer program. Hence, it is a matter of rewriting the computer program to alter the sorting format of the system and its associated data storage. The prototype will be used in several applications to determine its utility with fixed and living cells. Three useful classes of sorting have been identified and will be implemented. Applications in cancer detection include preparation of microscope slides by placement of cells in a highly ordered fashion. The cells are then identified morphologically and correlated with their two-dimensional histograms; other correlations include flow cytometry data with high-resolution image analysis data. In addition, there is the potential use of this device for obtaining high-frequency chromosome spreads on a microscope slide. Under computer control, the device performs the cell deposition function. With a recently purchased computer-controlled microscope, we are able to correlate each cell's visual characteristics with its flow cytometry parameters. Cells are deposited onto microscope slides covered with albumin; additionally, cells encased in a suitable growth medium can be grown on microscope slides. (3)

Project Summary/Abstract This proposal details a 5-year training program for career development and advancement in academic cardiovascular medicine for Dr. Michael A. Burke, M.D., the principle investigator. Dr. Burke is a physician- scientist at Emory University (EU) School of Medicine. He completed clinical and research training in Internal Medicine and Cardiovascular Diseases at Northwestern University through the ABIM research pathway. He then completed subspecialty fellowship training in Advanced Heart Failure (HF) and Transplant Cardiology at Brigham and Women's Hospital (BWH). Finally, he completed a post-doctoral research fellowship in the lab of Drs. Christine E. (study co-mentor) and Jonathan G. Seidman in 2015. Dr. Burke has recently established his own laboratory at EU where he is embarking on a research and career development program under the combined mentorship of Drs. Ahsan Husain (EU) and Christine Seidman (BWH). Dr. Husain is a professor of medicine and expert in cardiomyocyte biology and Dr. Seidman is a physician-scientist and cardiovascular geneticist; both have an extensive track record of training future leaders in academic cardiology. Dr. Burke's research interest focuses on characterizing the epigenetic mechanisms that regulate gene expression with progression of dilated cardiomyopathy (DCM) to HF. His long-term career goals are to translate this research into clinical advances for patients with HF. He has published important research demonstrating temporal changes in cardiac transcription using a genetic model of DCM that suggests a key role for early activation of pro-fibrotic signaling. He has recently generated new evidence suggesting that epigenetic reader proteins are a key nodal point for pathologic gene transcription in the progression of DCM. The objectives of this research proposal are (1) to characterize the roles of specific TGF? isoforms and the bromodomain and extraterminal (BET) family of epigenetic reader proteins in DCM, (2) to establish a possible mechanistic link between TGF? signaling and BETs, and (3) to define the mechanism of BET recruitment to target genes. Understanding these mechanisms will provide important fundamental insight into the biology of HF and could unlock potential therapeutic targets for this common and morbid disease. This research will teach Dr. Burke the use of advanced molecular techniques including viral vector delivery in animals, chromatin immunoprecipitation with sequencing (ChIP-seq) and single-cell RNA-seq. Dr. Burke's career development plan also includes educational resources to further his scientific knowledge. Drs. Husain, Seidman and Burke have formulated a clear timeline for career development, including publication of research, presentations at national meetings and development of a plan for his subsequent transition to independent investigator. The support provided by EU and this comprehensive career development program will optimally position Dr. Burke to compete for independent grant funding by the end of the program period.

DESCRIPTION: The major goals of this project are: 1) To investigate the mechanisms underlying the anabolic effects of thalidomide, 2) To determine the dose of hGH which achieves the optimal anabolic effect, 3) To determine whether a combination and thalidomide act synergistically to improve the functional lean body mass (LBM). The applicant proposes that administration of a lower doses of hGH may avoid the metabolic and structural side effects of hGH and combination with thalidomide may be additive or synergistic. He proposes to study the effects of these two agents under metabolic ward conditions, using metabolic balance studies, whole body kinetic studies, direct determination of muscle protein synthesis, and body fluid compartments. The specific aims are: 1) to find an optimal dose of hGH which will achieve the protein-anabolic effects without causing (or minimizing), the hypermetabolism and accumulation of non-functional extracellular tissue. 2) to determine whether thalidomide decreases the net protein degradation without increasing lipid oxidation and REE. 3) to test whether administration of thalidomide in combination with lower doses of hGH achieve a selective increase in the functional, as opposed to non-functional, lean tissue and causes a smaller increase in the REE. Total number of 42 HIV+ patients and HIV- control subjects will be included over a period of 5 years. All subjects will participate in a 2 week inpatient study, which will involve treatments with hGH or thalidomide or both. Then the HIV+ patients will continue their treatments for another 10 weeks. At the end of these period HIV+ patients will be readmitted for a 5 day period for the repeat studies. The investigations will include 1) the measurements of body weight and composition (by DEXA, bioelectrical impedance, D20 and Na bromide dilution, and midarm circumference measurement techniques), 2) metabolic balance studies of nitrogen, K, Na, SO4, 3) determination of resting energy expenditure and substrate oxidation rates by indirect calorimetry, 4) protein and lipid metabolism studies using stable isotopes of leucine and glycerol, 5) determination of muscle protein synthesis by measuring the incorporation of leucine to muscle protein and abundance of myosin heavy chain mRNA, 6) measurements of hormones, metabolites, TNFalpha and other cytokines, immunologic and virologic parameters. This study will address the mechanisms of HIV-induced wasting and investigate the role of two potential medical treatments.

Injection drug use is a serious public health problem, leading to blood-borne infections including HBC, HCV, and HIV. In spite of advances in the prevention of HIV/AIDS and hepatitis, injection drug use continues to contribute to new infections both directly through the sharing of injection equipment, and indirectly through sexual transmission from injection drug users (IDUs) to non-IDU sex partners. Behavioral interventions with IDUs are an important component of a comprehensive HIV prevention strategy. Enhanced interventions with IDUs have shown only modest effects over and above standard interventions. The key to improving interventions with IDUs may lie in understanding variations in responses to these interventions. Latent variable approaches (also known as mixture modeling) can be used to identify subgroups of individuals with similar response patterns. These approaches are often referred to as person-centered, in contrast to the traditional variable-centered approaches. To better understand how HIV prevention interventions influence injecting and sexual behavior of IDUs, we will conduct secondary analyses of data from the Third Collaborative Injection Drug Users Study / Drug Users Intervention Trial (CIDUS-3/DUIT). This study recruited young injection drug users in five U.S. cities to test a novel peer education intervention (PEI) against an attention control on changes in HIV/HCV risk behavior. We will use mixture modeling approaches, including latent class analysis, growth mixture modeling, and latent transition analysis, to analyze program outcomes. Our aim is to identify specific subgroups of IDUs for whom the intervention was most beneficial. Moreover, we seek to identify predictors of group membership which would enable us to assign individuals to the intervention that is most likely to have an impact. The proposed analyses will increase our understanding of the effects of the standard and enhanced interventions on injection and sexual risk behavior, and lead to recommendations for improving intervention efforts with injection drug users. The results may also have wider implications for strategizing prevention interventions with other populations.

Vitamin A and its metabolites (retinoids) play a critical role in cardiac morphogenesis. Retinoids are potent human teratogens linked to several types of birth defects, including cardiovascular anomalies, while vitamin A deficiency can produce ventricular chamber hypoplasia, ventricular septal defects, and aortic abnormalities. A mutation in a single members of the retinoid receptor family (RCRalpha) reproduces many of the cardiovascular defects seen in vitamin A deficient embryos. The specific aims of the present proposal will capitalize on recent advances in CRE- lox technology ti specifically mutate the RXRalpha gene within specific cardiovascular cell type compartments during in vivo cardiac development: 1) to determine the role of RXRalpha pathways within atrial and ventricular muscle cells in control of cardiovascular morphogenesis by creating mice which harbor an early, complete atrial and ventricular muscle cell lineage restricted deficiency in RXRalpha via CRE-lox technology; 2) to determine the role of RXRalpha pathways within cardiac neural crest cell lineages in the control of cardiovascular morphogenesis by creating embryos which harbor a neural crest cell lineage restricted deficiency in RXRalpha; 3) To determine the role of RXRalpha pathways within endothelial/endocardial cushion cell lineages in cardiovascular morphogenesis by creating mice which harbor an endothelial cell lineage restricted deficiency in RXRalpha; 4) To determine the role of all retinoid receptor dependent pathways in specific cardiovascular cell types (atrial muscle, ventricular muscle, endothelial, and neural crest) in cardiovascular morphogenesis by creating mice which conditionally express a dominant negative retinoid receptor in specific cardiovascular cell types via a Cre-lox switch strategy. These studies should lead to the definitive identification of the specific cardiovascular cell types that generate the RXRalpha dependent signals required for specific steps in cardiac morphogenesis. Since mutations in a number of transcription factors have been implicated in human congenital heart defects, these studies of RXRalpha could lead to establishment of a new paradigm by which an individual transcription factor may be required in distinct cardiovascular cell types at precise stages of cardiogenesis to provide the molecular cues that lead to complex steps of cardiac morphogenesis.

Exposure to isocyanate, the essential cross-linker for making polyurethane, is a leading cause of occupational asthma world-wide. Three major types of isocyanate are used commercially, methylene-diphenyl diisocyanate (MDI) and toluene diisocyanate (TDI), or aliphatic, hexamethylene diisocyanate (HDI). Despite being the newest of the three, MDI has overtaken the isocyanate market in production and consumption, for many reasons, including its unique applications and properties. Importantly, at room temperature, MDI has a much lower vapor pressure than HDI or TDI, and exists as a solid, rather than a (volatile) liquid. Thus, MDI is presumed to be safer, because the potential for respiratory tract exposure is thought to be restricted to times when the chemical is heated or aerosolized (sprayed), i.e. during application. MDI is widely used in making flexible and rigid foams, coatings, elastomers and numerous other polyurethane-based products. Exposure to MDI is the best-recognized risk factor for the development of MDI asthma and exposure reduction is the primary strategy of disease prevention. Contemporary methods of monitoring MDI exposure are severely limited, and new approaches are needed to help protect millions of people exposed at work. In this new phase II application for the project Biomonitoring Methylene Diphenyl Diisocyanate (MDI) exposure and body burden, we aim to develop innovative biomonitoring approaches to exposure surveillance for MDI. The investigative team consists of scientists and physicians from Yale University School of Medicine and the small business, L2 Diagnostics, who are applying their expertise with immunoassays, to develop blood tests that measure (2) different MDI exposure biomarkers. One biomarker is MDI-specific antibodies (IgG), produced by the immune system in response to exposure. The second biomarker is the chemical (MDI) itself conjugated to albumin, the major protein adduct in vivo. To date, we have successfully completed the (4) Specific Aims of the projects 1st phase, and published supportive findings (Wisnewski et al Analytical Biochem 2010; PMID: 20123080), including the generation of key reagents, and their extensive characterization. Completion of these milestones demonstrates our readiness to embark on the specific aims of the project's 2nd phase (listed below), which should lead to new commercial assays for biomonitoring MDI exposure, which will help prevent ongoing exposures and new cases of MDI asthma. (Aim 1) Define the analytical performance characteristics of two new laboratory assays to measure (biomarkers of) MDI exposure. (Aim 2) Determine the clinical sensitivity of new MDI exposure (laboratory-based) assays, among MDI exposed workers and different populations of control unexposed individuals. (Aim 3) Using the new MDI exposure assays, establish the kinetics of MDI immune sensitization, and indications for use in evaluating exposed workers.

Organic cation (OC) transporters play important roles in the disposition and clearance of many endogenous and foreign OCs in the body. In contrast to the kidney and liver, little is known about OC elimination mechanisms in the brain. This is rather unfortunate because many CNS active compounds, including monoamine neurotransmitters (e.g. dopamine, serotonin), CNS drugs (e.g. amantadine, nicotine) and neurotoxins (e.g. 1-methyl-4-phenylpyridinium (MPP+)) are small hydrophilic OCs that rely on transporters to regulate their brain levels. This proposal focuses on plasma membrane monoamine transporter (PMAT), a novel brain OC transporter first cloned in our laboratory. While structurally related to the equilibrative nucleoside transporter family (SLC29), PMAT possesses a unique and surprisingly diverse substrate specificity, transporting structurally heterogeneous OCs such as biogenic amines, clinically used drugs and neurotoxins. In humans and rodents, PMAT is most abundantly expressed in the brain and highly concentrated in the blood-cerebrospinal fluid (CSF) barrier (i.e. BCSFB or choroid plexus). We hypothesized that PMAT is the principal OC transporter at the BCSFB and is responsible for removing a variety of endogenous and xenobiotic OCs from the brain. Three Specific Aims (SAs) have been proposed. SA1 is focused on elucidating the molecular mechanisms governing transporter-substrate interactions to explain the unique and versatile substrate specificity of PMAT. SA2 is focused on elucidating the transport mechanism of PMAT and developing a cellular model for OC flux at the BCSFB. Lastly, in SA3, we will construct and validate a transgenic animal model, which will allow a variety of mechanistic studies to reveal the in vivo action of PMAT. We will apply several experimental methods, ranging from molecular biochemical techniques, computational biology, electrophysiology, immunohistochemistry to transgenic approach, to elucidate the structure, function and biological significance of PMAT in clearing neurotransmitters, drugs and toxins in the brain. The proposed studies have broad implications in our understanding of normal and pathophysiological functions of the brain. Detailed knowledge of OC transporters at the CNS barriers can also help to explain and predict the pharmacokinetics and pharmacodynamics of OC drugs and toxins in the CNS compartment and aid in the development of new strategies for drug targeting to the brain. [unreadable] [unreadable] [unreadable] [unreadable]

Stress echocardiography is a clinically established, cost-effective technique for detecting and characterizing coronary artery disease by imaging the left ventricle (LV) of the heart at rest and then after either exercise or pharmacologically-induce stress to reveal ischemia. However, acquisitions are heavily operator dependent, two-dimensional (2D), and interpretation is generally based on qualitative assessment. While a variety of quantitative 2D approaches have been proposed in the research literature, none have been shown to be superior to the still highly variable qualitative visual comparison of rest/stress echocardiographic image sequences for detecting ischemic disease. Here, we propose that the way forward must focus on a new computational image analysis paradigm for quantitative 4D (three spatial dimensions plus time) stress echocardiography. Our strategy integrates information derived from both radiofrequency (RF) and B-mode echocardiographic images acquired using a matrix array probe. The integrated analysis system will yield accurate and robust measures of strain and strain rate - at rest, stress and differentially between rest and stress - that will identify myocardial tissue at-risk after dobutamine-induced stress. This work wil involve the development of novel (1) phase-sensitive, correlation-based RF ultrasound speckle tracking to estimate mid-wall displacements, (2) ma- chine learning techniques to localize the LV bounding surfaces and their displacements from B-mode data, (3) a meshless integration approach based on radial basis functions (RBFs) and Bayesian reasoning/sparse coding to estimate dense spatiotemporal parameters of strain and strain rate and (4) non-rigid registration of rest and stress image sequences to develop unique, 3D differential deformation parameters. The quantitative approach will be validated with implanted sonomicrometers and microsphere-derived flows using an acute canine model of stenosis. The ability of deformation and differential deformation derived from 4D stress echocardiography to detect new myocardial tissue at-risk in the presence of existing infarction will then be determined in a hybrid acute/chronic canine model of infarction with superimposed ischemia. The technique will be translated to humans and evaluated by measuring the reproducibility of our deformation and differential deformation parameters in a small cohort of subjects. Three main collaborators will team on this work. A group led by Matthew O'Donnell from the University of Washington will develop the RF-based speckle tracking methods. An image analysis group led by the PI James Duncan at Yale University will develop methods for segmentation, shape tracking, dense displacement integration and strain computation. A cardiology/physiology group under Dr. Albert Sinusas at Yale will perform the acute and chronic canine studies and the human stress echo studies. A consultant from Philips Medical Systems will work with the entire team to bridge the ultrasound image acquisition technology.

Farm workers living in the United States (US) carry a burden of HIV infection that is 10 times that of the general population. Migrant farm workers (MFW) who come from Mexico to the U live in rural areas, are marginalized from mainstream society, are commonly undocumented, and often lack access to necessary health and prevention services. Females MFW in particular comprise a unique group that experience high risk of HIV infection. This sexual risk is related to the threat of intimate partner violence, low social support, low HIV knowledge, high-risk partners, and in particular, cultural norms that may influence relationship power and the ability to negotiate for protective sex in the face of infidelty. HIV risk is closely tied to interpersonal power dynamics and patriarchal ideologies that can influence relationships. Moreover, migration to a new country is a life-changing event that can alter family structure and bring about changes in cultural values and beliefs, which could be harmful or protective in relation to sexual risk. Although a number of studies have investigated HIV risk among male MFW, very little research has approached the topic of sexual health for female MFW. Therefore, understanding the perceived sexual risks and strategies used to maintain sexual health by female MFW who were born in Mexico will assist in understanding what personal factors (intrinsic strengths) and which resources (physical resources, material resources, financial resources, supportive resources, relational resources, etc.) help recent migrants to maintain sexual health. Moreover, this research will form a foundation for planning population-specific and culture-appropriate interventions for female MFW. The specific aims of this study are: 1) To describe the perceptions and experiences of female MFW of Mexican descent in relation to their health risks: 2) To Identify the beliefs/values about culture, migration, gender roles, and power that are related to sexual health risks for female MFW of Mexican Descent: 3) To describe what resources and self-protective factors female MFW of Mexican descent use to mitigate these perceived risks: and 4) To generate a theoretical framework that is grounded in the voices and narratives of female MFW of Mexican descent regarding their experiences and perspectives on sexual health, power relations, and self-protective strategies. PUBLIC HEALTH RELEVANCE: Migrant farm-workers in the United States carry a burden of HIV infection that is 10 times that of the general population, and farm-worker women are particularly at risk for HIV in relation to interpersonal power dynamics, gendered social norms, and cultural beliefs about sexual health and nursing knowledge regarding appropriate interventions to increase the health and well being of this group. This qualitative study will focs on examining perceptions of risk and self-protective strategies and resources used by farm-workers women to maintain sexual health.

DESCRIPTION (from Applicant's Abstract): The Ah receptor (AHR) is a ligand activated transcription factor that binds ligands such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and is thought to mediate many of the highly toxic and carcinogenic actions of TCDD. The AHR and its DNA binding partner ARNT (Ah receptor nuclear translocator) recognize specific DNA sequences, referred to as dioxin responsive element (DREs) resulting in the activation of gene transcription. Both the AHR and ARNT are basic/helix-loop-helix proteins. Basic/helix-loop-helix proteins are characterized by their ability to form homo- or hetero-dimers, contact DNA via their basic domains and form a variety of DNA binding species that recognize distinct DNA sequences and regulate specific sets of genes. Contrary to the generally accepted theory that TCDD exerts its adverse affects through the regulation of DRE responsive genes, we propose that TCDD actions include nuclear localization of the AHR, dimerization of ARNT and the subsequent removal of ARNT from the regulatory regions of other genes. This hypothesis suggests that ARNT, in the absence of the AHR regulates genes and that this regulation is adversely affected by the presence of TCDD and the AHR. Our preliminary data has shown that ARNT can form multiple partnerships. The ARNT homodimer recognizes the CACGTG sequence, the recognization site of the Myc/Max heterodimer involved in regulating cellular proliferation and differentiation. We will use Myc/Max gene regulation as a model of AHR-independent ARNT gene regulation. First, we will determine whether the ARNT homodimer will recognize the CACGTG site with high affinity required for biological significance and compare this binding affinity to that of Myc and Max. Then we will use transient transfection assays to determine whether theARNT homodimer formation may repress biological processes such as the induction of cytochrome P4501A1 and Myc induced cellular transformation. To determine why the AHR ARNT recognize different DNA half-sites, we will perform site-directed mutagenesis of their DNA binding regions followed by footprinting analysis. Finally, we will use cell-free transcription to identify the interacting transcription factors that facilitate AHR and ARNT gene regulation and determine whether different AHR and ARNT ratios found in vivo may affect the ability of the AHR and ARNT to heterodimerize and resulting differential gene regulation. The broad, long-term objectives of this proposal are to understand how the AHR and ARNT regulate and the mechanisms that dictate the tissue-specific effects of TCDD toxicity and carcinogenicity.

Characterization of surface markers on B cells at successive stages along the differential pathway from early precursor to antibody forming cells (AFC); a) H chain class and allotypes of surface immunoglobulins (Ig): relationship to Ig produced by progeny AFC; ontogeny; functional role; b) VH markers: relationship to Ig produced by progeny AFC; genetic control; c) Non-Ig surface markers: correlations with H chain class; ontogeny; genetic control; functional role with respect to antibody production. Isolation and characterization of subsets of T or B cells and definition of their function(s) in humoral immunity. Development and study of Ig allotype (CH) congenic strains: a) New congenic strain development; maintenance and genetic testing of existing congenic strains; b) Study of Ig regulatory abnormalities in congenic strains. Production of (B cell myeloma) hybrid cell lines which survive indefinitely and produce a single species of Ig: a) Lines which produce antibody to Ig class, allotype or idiotype determinants; b) Lines producing antibody to non-Ig surface determinants on B or regulatory T cells; c) Lines which produce Ig of classes or allotypes where no myeloma tumors currently exist. BIBLIOGRAPHIC REFERENCES: Sato, Vicki L., S.D. Waksal, and Leonard A. Herzenberg. 1976. Identification and separation of pre T-cells from nu/nu mice: differentiation by preculture with thymic reticuloepithelial cells. Cell. Immunol. 24: 173-185. Stout, Robert D., Donal B. Murphy, Hugh O. McDevitt and Leonard A. Herzenberg. 1977. The Fc receptor on thymus-derived lymphocytes. IV. Inhibition of binding of antigen-antibody complexes to FcR-positive T cells by anti-Ia sera. J. Exp. Med. 145:187.

A recent appellate court ruling has vacated the FDA's Final Rule that implemented the 2009 Family Smoking Prevention and Tobacco Control Act (FTA) requirement of graphic warning labels (GWLs) on cigarette packs. According to the FTA, the GWLs are to include one of nine mandatory textual warnings and a picture illustrating the negative health effects of smoking. The court decided that the GWLs selected by the FDA infringed on the tobacco company plaintiffs' First Amendment rights. The court opined that FDA's evidence of effectiveness of GWLs in general and GWLs producing strong emotional arousal in particular was insufficient to justify the infringement. Specifically, the court noted te lack of data supporting the effectiveness of GWLs in reducing actual smoking. The ruling highlights the limitations of existing data on GWLs that have been obtained predominantly through large-scale surveys using self-report outcome measures. In addition, the ruling has touched on a more basic controversy on the mechanisms underlying the effectiveness of strongly salient format and content in persuasive health communications. This project will take a unique trans- disciplinary approach to address this issue from both a clinical and neurophysiological perspective. We shall use biochemical (urine cotinine levels) and neuroimaging (Functional Magnetic Resonance Imaging, fMRI) assays, as well as traditional measures of persuasion, to compare the brain and bio-behavioral (urine cotinine) impact of GWLs that were rated high or low on the emotional response (ER) scale, used by the FDA to select GWLs for its Final Rule. We shall study 150 non-treatment seeking smokers' brain response to GWLs and monitor their urine cotinine levels, before and after a 4-week long exposure to real-life cigarette packs carrying High or Low ER GWLs. Based on our preliminary data, we hypothesize that smokers exposed to high ER GWLs will exhibit larger reductions in UC than those exposed to low ER GWLs. Based on our preliminary data and prior literature pointing to the mPFC and the amygdala as key opponent brain regions mediating the brain response to audio-visual anti-smoking ads, we hypothesize that acute exposure to high ER GWLs will be associated with higher amygdala and mPFC response than low ER GWLs. In addition, we predict that the amygdala response to GWLs will decline after 4-week long naturalistic exposure to GWLs, while mPFC response will increase, with both effects more prominent in the High ER group. Secondary hypotheses will evaluate the predictive value of acute mPFC response on urine cotinine levels change, as well changes in cigarette craving, daily intake, and brain fMRI response to visual cigarette cues prior to and after the 4-week exposure period. By applying measures of brain (fMRI), behavior (cotinine levels), persuasion and motivation (attitudes and intentions towards smoking quitting) to a cohort of smokers exposed to different affective levels of GWLs in a real-life setting, the project will provide integrated results that would complement existing population-level data and inform science-driven regulation of cigarette packaging that balances constitutional law and public health interest.

This proposal is aimed at continuing our ongoing efforts to identify and characterize novel AD genes involved in presenilin-related pathways. AD candidate genes implicated in presenilin-related pathways will be derived from three different pools: Pool 1. Positional candidate genes mapping to established AD genetic linkage peaks, including UBQLN1, VPS26A, VPS35, VDAC1, VDAC2, NCSTN, PSEN1 and TFCP2; Pool 2. Candidate genes derived from systematic meta-analyses performed on ourAlzGene.org database, including CHRNB2, DAPK1, SORCS1, SORL1, TNK1, HMGCS2, CH25H, and SOAT1; and 3. Novel AD candidate genes from our ongoing whole-genome association (WGA) screens of the NIMH AD family sample in which >1400 subjects from 457 uniformly ascertained and evaluated AD families have been genotyped using three different Affymetrix genotyping arrays: 500K genomic single nucleotide polymorphisms (SNPs), 100K genomic SNPs, and 20K coding SNPS (cSNPs). Follow-up analyses of presenilin pathway-related AD candidate genes will include genetic confirmation/replication testing, linkage disequilibrium analyses, and mutation identification. In collaboration with the other P01 projects and cores, we will also carry out biological and functional validation studies of specific candidate genes based on our genetic results. In specific aim 1, genotyping of the NIMH sample will be completed for all genes in all three pools. In specific aim 2, SNPs exhibiting genome-wide significance for family-based association with AD in the NIMH sample will be subjected to replication testing in four independent AD family samples: CAG (224 families; 505 subjects; AD: 245), NIA (353 families; 1117 DNAs; AD: 815), NCRAD (369 families; 1266 DNAs: AD: 895), and NIMH African American (24 families, 58 subjects; AD: 49). For genes exhibiting the strongest association with AD, we will carry out extensive linkage disequilibrium mapping of additional SNPs and re-sequencing in probands and unaffected individuals of specific associated families for each locus. In specific aim 3., we will perform bioinformatic (in silico) analyses of AD candidate genes to identify which SNPs represent potentially pathogenic gene mutations/variants for AD. Finally, in specific aim 4., we will collaborate with the other P01 projects and cores to carry out biological validation and functional analyses of novel AD candidate genes, including effects of RNAi silencing and overexpression of wild-type and potentially pathogenic mutations/variants on presenilin function, e.g. APP trafficking/processing as well as A(3 and AICD generation, y-secretase activity, APP-PS1 interaction, and PS1 conformation. Lay Summary: The four known AD genes (APP, PSEN1, PSEN2, and APOE) are the subjects of the vast majority of current biological research on AD. Yet, these genes represent only ~30% of the genetic variance of AD. The goal of this project is identify the additional AD genes implicated in presenilin-related biological pathways to increase our knowledge of the causes of AD and the role of the presenilins in AD pathogenesis.

Malignant gliomas represent one of the most challenging tumors to treat. Gene-based therapeutics have been advocated as a novel treatment modality. Although initial results from clinical trials have revealed the safety of this approach, evidence for efficacy has been elusive. Analysis of tumor tissue has been shown that gene delivery mediated by replication-defective vectors occurred in the immediate vicinity of the needle infection delivery tract. A potential avenue that could increase the volumetric extension of gene delivery consists of using replication- conditional (tumor-selective, oncolytic viruses as gene delivery vehicles. Over the last four years, we have engineered herpes viruses (HSV) that are defective in the function of he viral ICP6 (encoding the viral ribonucleotide reductase function) and/or the ICP34.5 (encoding a virulence function) genes. Our studies have revealed that the viral oncolytic effect can produce tumor growth inhibition and regression and that and regression and that this effect can be augmented by delivery of pro-drug-activating genes. We thus propose to increase the oncolytic efficiency of these mutants by cloning into their genome the CYP2B1 and RED genes, responsible for the activation of pro-drugs of the oxazosphorine class, such as cyclophosphamide (CPA), and the CE gene, responsible for the activation of pro-drug, irinotecan (CPT11). This new viral mutant (MGH2) will be employed to test the following hypotheses: 1) Combined intratumoral transfect of two pro-drug-activating gene therapies enhances tumor cell oncolysis by MGH, 2) This strategy provides for effective treatment in animal models of herpes simplex encephalitis, and 3) Modifications in the function of viral genes responsible for host cell toxicity and production of viral progeny alter the extent of vector-mediated pro-drug activation. Taken in conjunction, these studies will provide not only justification for a treatment modality that we will be able to test in phase I clinical trials, but also a mechanistic insight into the variables affecting cytotoxicity by the viral and pro-drug activation.

The field of evolutionary biology has long been interested in the molecular basis of morphological evolution; however a clear understandingremains elusive. Abdominal pigmentation in the subgroup Drosophilinae is a well studiedtrait suitable for studies to identify the genetic changes culminating in the emergence of similar and diversepigmentation patterns. In addition to diverse pigmentation patternsit is apparent, based on phylogeny, that similar patternsof pigmentation haveappeared independently. In the melanogaster speciesgroup sexually dimorphic, segmentspecific pigmentation has recently evolved. The terminal abdominal segmentsof males are dark, whereas all segmentsare lightly pigmentedin females. Closely related groups of species are monomorphic, i.e. all segments are lightly pigmented in both sexes. In all species, pigmentation is prevented where the gene products of bric-a-brac (bab), a represserof pigmentation, are expressed. In dimorphic species, a newly evolved circuitry involving homeotic and sex-determination inputs repressesbab expression in the terminal segments of the male, but not the female. This proposal will analyze how Drosophila melanogaster bab gene regulatory elements has evolvedto integrate responsiveness to homeotic and sex-determination inputs and the extent that both similar and diverse abdominal pigmentation patterns haveevolved through the modification of orthologous bab sequences. Insight from the mechanisms of enhancer evolution and function in Drosophilinae will very likely apply to the understandingof cis-regulatoryelements in vertebrates as similar sequence alterationshave significant medical implications inhumans.

This project will investigate how Drosophila controls the expression of its Alpha-tubulin genes. The four genes of this small multigene family offer a good opportunity to investigate the control of tissue- and time-specific transcription and to investigate mRNA characteristics that determine mRNA stability and location. The issues to be dealt with in this investigation are: 1) the location and complexity of DNA sequences that cause one family member's transcription pattern to deiifer from that of another member; and 2) whether or not there are sequences in the mRNAs from different family members that cause them to differ in fate, that is to differ in intercellular transport, in cellular location and in stability. In pursing the first issue, regions and sub-regions will be exchanged between the Alpha-tubulin genes and the resulting genes used to transform Drosophila. The pattern of expression of these altered genes will indicate which sequences are necesssary for a particular tissue- or time- specific pattern of transcription. In pursuing the second issue, portions of the mRNA encoding regions of Alpha-tubulin genes will be excahanged with portions of other genes and then a transformation assay will be used to test the effect of 3 feet and 5 feet untranslated regions, introns and translated regions on the intercellular transport, the location and the stability of Alpha-tubulin mRNAs. The initial subjects for these exchanges will be an Alpha-tubulin gene which codes for a maternal mRNA, one which codes for an mRNA that first appears between egg laying and cellular blastoderm formation and one which appears to be transcribed at all times in all tissues.

The long term goal of the research in this proposal is to understand the neural mechanisms controlling the expression of arousal. The research will use combined behavioral, biochemical, and electrophysiological approaches to investigate the role of peptide transmitters and co-transmitters in the expression of arousal states. lt is hypothesized that intrinsic co-transmitter modulators in buccal motoneurons function to stabilize motor contractions under varying conditions. Extrinsic modulatory systems may override this autoregulation to produce plasticity in the system, which may be needed during different behavioral states or during different classes of behavior executed by the buccal mass. The proposed projects include: 1) Purification and sequencing of bioactive peptides In the ARC muscle. 2) Study of the characteristics of the release of peptides from motoneurons. 3) Localization and functional characterization of peptidergic neurons innervating the ARC muscle. 4) Analysis of a model of the functional organization of the motor control of the buccal mass.

Infections with Plasmodium parasites, the causative agents of malaria, constitute one of the world's largest disease burdens with up to 300 million infections and 1.2 million deaths each year. The parasite maintains an incredibly complex life cycle between mosquito vector and mammalian host. After transmission, it will pass through the skin and bloodstream as sporozoites, which infect liver hepatocytes, then develops as liver stages and emerges back into the blood as red blood cell-infective merozoites. The pre-erythrocytic (sporozoites and liver stages) phase is an ideal target for vaccine development as it is asymptomatic and has limited parasite numbers. However, as only one parasite can cause a fulminant blood stage infection, preventing blood stage disease with pre-erythrocytic immunity faces a challenge. Immunization with attenuated parasites which invade and infect the liver but fail to progress to blood stage have been highly effective in preclinical and clinical studies. The conventional understanding is that this protection relies predominantly by CD8+ T cells. However, I have shown that both monoclonal antibodies (mAb) against the sporozoite protein CSP and polyclonal antibodies (pAb) elicited by immunization with whole parasites are capable of providing robust protection against an infectious mosquito bite. The Abs elicited by whole parasite immunization (WPI) can also control a direct blood stage infection independent of T cell help. Little is known about the antibody effector mechanisms mediating this protection at either stage, but the cross-stage protection afforded by WPI provides an ideal platform on which to investigate these mechanisms. I propose to use this model to investigate the contributions of neutralization, complement-mediated lysis and opsonization underlying Ab-mediated protection in a rodent model of malaria. Aim 1 will focus on protection against sporozoite infection by passive transfer of mutant mAb which lack specific FC-mediated effector functions. These mutant mAb will be compared to WT mAb for their ability to reduce liver stage burden following infection by mosquito bite. Furthermore, passive transfer of WPI serum followed by mosquito bite challenge in mice deficient in complement, FC receptor binding or both will elucidate the role of each mechanism in the context of WPI. Aim 2 will expand this model to examine antibody-mediated protection in the blood stage of disease. Again, passive transfer of WPI serum to mice deficient in complement, FC receptor binding or both will be followed by direct blood stage challenge. By monitoring subsequent parasitemia, we will be able to determine the respective contributions (if any) of each effector mechanism to control of blood stage malaria. The studies proposed here represent the first comprehensive and exhaustive analysis of the mechanisms conferring antibody-mediated protection against both the sporozoite and blood stages of Plasmodium. Knowledge of the specific type of antibody response required for effective protection at these stages will guide the rational design of the next generation of malaria vaccines aimed at preventing infection.

The long term goal of this research is to understand the mechanisms by which cell adhesion to extracellular matrix regulate cell cycle progression. Dr. Guan and his colleagues have recently found that integrin signaling through FAK plays a major role in cell cycle regulation in cell adhesion. Their analyses using an inducible expression system suggested a critical role for focal adhesion localization of FAK and its associated signaling molecules, and identified Erks and cyclin D1 as important mediators of cell cycle regulation by the integrin-FAK signaling pathways. Studies from his laboratory and others have also suggested that FAK can interact with multiple intracellular signaling molecules to trigger several downstream signaling pathways. In addition, they have identified a novel cellular protein that can associate with both FAK and its related kinase Pyk2 and may function as an inhibitor of the FAK family kinases in intracellular signaling. In this proposal, the investigators plan to dissect the molecular mechanisms underlying cell cycle regulation by integrin-FAK signaling pathways. In Aim 1, they will first analyze the relative contributions of FAK/Src vs FAK/PI3K complexes in cell cycle regulation by FAK. They will then examine the roles of signaling molecules downstream from the FAK/Src and FAK/PI3K complexes including p130cas, Grb2 and Akt. Lastly, they will determine the role of focal adhesion localization of the FAK signaling complexes. In Aim 2, the group will examine FAK regulation of cyclin D1 at transcription level by analyzing transactivation of cyclin D1 promoter reporter by FAK signaling pathways as well as the effect of inducible expression of FAK and its mutants on endogenous cyclin D1. They will also investigate regulation of cyclin D1 protein synthesis and degradation and the potential role of eIF-4E, 4E-BPs and cyclin D1 phosphorylation as mediators of cyclin D1 regulation by FAK signaling pathways. Finally, they will determine whether overexpression of cyclin D1 can rescue cell cycle inhibition caused by disruption of FAK signaling. In the last Aim, they will identify the binding sites for both FAK and FAP200. They will then investigate the potential mechanism by which FAP200 may function as an inhibitor of FAK to regulate cell cycle progression, and the alternative possibility that FAP200 may serve as a novel FAK substrate which plays a role in cell cycle regulation by FAK. The investigators hope that these studies will provide new insights into the mechanisms by which cell adhesion to extracellular matrix regulate cell cycle progression.

Development of an effective HIV vaccine is the long-term solution to control HIV/AIDS world-wide. The failure of adenovirus type 5 based HIV-1 vaccines in humans that was designed to elicit primarily antiviral T cells strongly suggests the need to develop novel vaccine approaches that generate high levels of antiviral T cells with improved function as well as protective antibody. Development of novel adjuvants is necessary to enhance the immunogenicity and efficacy of HIV vaccines. In this HIVRAD, we will use molecules that target mTOR (mammalian target of rapamycin) and CD40 pathways as novel adjuvants to elicit high levels of polyfunctional virus-specific CD8 T cells, CD4 T cells that are resistant to HIV infection and high avidity protective antibody that will lead to enhanced control of HIV infection. This PPG has two projects and two cores. Our project 1 will test the potential of CD40L expressed on the surface of virus-like particles as an adjuvant to enhance the immunogenicity and efficacy of DNA/MVA SIV vaccine. Our project 2 will test the safety, immunogenicity and efficacy of rapamycin (inhibitor of mTOR) as an adjuvant for DNA/MVA SIV vaccine. In addition, we will test for the effects of rapamycin on other replication defective and replication competent viral vectors. Finally, we will test the synergy between inhibiting mTOR and activating CD40 pathways for enhancing the immunogenicity and efficacy of DNA/MVA SIV vaccines. The macaque studies that will be conducted in projects 1 and 2 will be supported by a non-human primate core. An administrative core will provide coordination between projects 1 and 2, and core B, and help with data management and data analyses. RELEVANCE: WHO estimates that there are currently 32 million humans living with HIV/AIDS. Drugs and improved treatment regimens have successfully prolonged the lives of infected individuals in first world countries. However, these are not affordable for the vast majority of HIV-infected individuals. Even in developed nations these are limited by toxicity, affordability, the need for rigorous adherence to therapy and the emergence of drug resistant viruses. Thus, there is a great need to develop a safe and effective HIV vaccine that provides a low-cost, low toxicity solution to long-term control of viral replication - the main goal of this PPG. PROJECT 1: Title: CD40L-Adjuvanted Vaccines for HIV/AIDS Project Leader: AMARA, R PROJECT 1 DESCRIPTION (provided by applicant): The failure of Ad5 based HIV-1 vaccine in humans that is designed to elicit primarily antiviral T cells strongly suggests the need to develop novel vaccine approaches that generate high levels of anti-viral T cells with improved function as well as protective Ab. The goal of this project is to adjuvant the cellular and humoral immunity elicited by our DNA/MVA vaccine that has just entered phase II testing in humans in US. Specifically, we propose to target the CD40 pathway on dendritic cells (DC) and B cells using CD40L. Stimulation of CD40 on DC results in production of IFNa and IL-12 that are critical for generation of highly functional antiviral CD8 response. Activation of B cells through CD40 is necessary for germinal center formation where affinity maturation of B cells occurs leading to generation of high avidity Ab. Here, we will express CD40L on the surface of a SIV virus-like particle (CD40L-VLP). These CD40L-VLPs are potent activators of DC and B cells in vitro. In addition, they can be targeted to DC (through interaction between gpl20 on VLP and CD4 on DC) and allow presentation of Env in its native form that is critical for eliciting broadly cross-reactive neutralizing Ab. In specific aim 1, we will test the potential of CD40L-adjuvanted DNA/MVA SIV vaccine to enhance control of a pathogenic SIV challenge. In addition, we will test whether delivering DNA by electroporation enhances the immunogenicity of the adjuvanted and non-adjuvanted DN/VMVA vaccines. Recent studies from Dr. Rafi Ahmed's lab (PI of project 2) demonstrated that mTOR regulates memory T cell development and inhibition of this pathway following infection or vaccination using rapamycin enhances the magnitude and functional quality of antigen-specific CD8 T cells. Rapamycin has also been shown to down regulate expression of CCR5 on CD4 T cells that results in marked reduction of HIV replication. This could be an added advantage for HIV vaccines, because the vaccine-elicited CCR5'virus-specific CD4 T cells may not be infected by the virus. Essentially we may be reducing the frequency of potential virus target cells while preserving the much-needed CD4 T cell help following infection. In specific aim 2, we will test the synergy between inhibiting mTOR and activating CD40 pathways for adjuvanting the immunogenicity and efficacy of DNA/MVA vaccines. RELEVANCE: WHO estimates that there are currently 32 million humans living with HIV/AIDS. Drugs and improved treatment regimens have successfully prolonged the lives of infected individuals in first world countries. However, these are not affordable for the vast majority of HIV-infected individuals. Even in developed nations these are limited by toxicity, affordability, the need for rigorous adherence to therapy and the emergence of drug resistant viruses. Thus, there is a great need to develop a safe and effective HIV vaccine that provides a low-cost, low-toxicity solution to long-term control of viral replication - the main goal of this PPG.

In collaboration with Dr. Michael Tarlov, we have further investigated the optimal sensing layer design for the characterization of the carbohydrate moiety of glycoproteins. This resulted in the recognition that multi-valency of lectins is an important factor in the choice of immobilization strategy. Previously we had recognized surface site heterogeneity is a key problem of surface binding analysis, and developed a method for extracting affinity and kinetic rate distributions from experimental data. For this purpose we had programmed a software tool that is now widely used. We have now further improved this program by facilitating the application of corrections for mass transport limited binding, broadening the applicability of this approach.

This 3-site collaborative U01 aims to improve identification of individuals who will develop schizophrenic psychosis (including brief psychotic disorder, schizophreniform disorder, schizophrenia, or schizoaffective disorder) at the initial prodromal stage of illness, prior to the onset of the full schizophrenic syndrome. Accurate identification of schizophrenic psychosis risk offers what may be the field's best hope for developing more effective treatment strategies, including secondary prevention of this typically devastating disorder. Without sensitive and specific prodromal diagnosis strategies, intervention studies are controversial, and the results of any studies will have limited impact on clinical practice. Identification efforts to date have focused on attenuated positive symptoms, but these criteria do not consider negative symptoms that occur in the prodromal stages of psychosis and are fundamental to schizophrenia. To enhance the potential sensitivity of prodrome evaluation we have developed a modified version of the "Criteria of Prodromal Syndrome" (COPS) that retains attenuated positive symptoms, but also considers selected negative symptoms in the diagnosis of prodromal state. We propose to develop a schizophrenic psychosis risk prediction model, and our proposed risk factors are selected based on the hypothesis that schizophrenia results from a pathological neurodevelopmental process that occurs during a critical stage of forebrain development in gestation and affects the development of neurons primarily in the thalamic, prefrontal and frontal cortical, and limbic regions of the brain (thalamolimbic- cortical circuitry [TLCC]). These neurodevelopmental abnormalities are likely to be expressed premorbidly by subtle behavioral, cognitive, and structural "vulnerability markers". In most cases, these abnormalities require specific maturational processes (i.e., synaptic elimination, myelination), which occur around puberty, to unmask the vulnerability and trigger dysfunction, resulting in the development or worsening of attenuated positive and negative symptoms (clinically defining the "at risk" state), as well as diverse but specific impairments in social function, social cognition, neurocognitive function, olfaction, and motor function. We hypothesize that as connectivity of the TLCC becomes more dysfunctional, a consequence will be increased severity of measurable impairments with more domains being affected to a greater extent. Thus, the number and severity of symptomatic manifestations of TECC circuit impairment are indicators of a biologically high-risk state for schizophrenic psychosis. Furthermore, we hypothesize that these vulnerable neural circuits may be further perturbed by environmental events that typically occur during adolescence, such as stressful life events or drug abuse. Such stressors may exceed the adaptive capacity of relevant circuits producing the characteristic symptoms that signal the onset of the illness. To develop the schizophrenic psychosis risk assessment model we propose a 3-site prospective study of 180 individuals meeting modified "Criteria for Prodromal Syndrome", and 80 help-seeking control subjects who will be prospectively evaluated over 2-5 years for risk of developing schizophrenic psychosis. The collaborative team has developed leading instruments in this field and has substantial expertise in social cognition, neurocognition, developmental psychopathology, statistics and data management. Each site has provefi its ability to recruit prodromal patients in a previous collaboration.

Graded changes in light intensity at dawn and dusk are a hallmark of the natural illumination cycle, yet their contribution to circadian entrainment has received little experimental attention. The program described below will make use of recently developed twilight simulation technology, for systematic investigation of the role of dawn/dusk transitions in the entrainment of circadian rhythms of nocturnal fossorial rodents (rats and hamsters) and of diurnal primates (squirrel monkeys)--animals adapted to vastly different lighting environments. In keeping with our long-term goal of elucidating the process of photic entrainment under naturalistic, ecologically valid conditions, the rodents will be provided with a dark nest compartment and will thus have the opportunity to self-select their daily light exposure. Experiments are designed to test a number of specific hypotheses, formulated on the basis of preliminary evidence from our laboratories and those of others. We aim (1) to examine entrainment and light self-exposure patterns under twilight cycles with seasonally-varied photoperiods and with different cycle lengths, and under corresponding rectangular light-dark (LD) cycles; (2) to compare the zeitgeber strength of these two cycles, as well as any after-effects they might exert on circadian period measured in constant darkness (DD): (3) to identify distinctive features of the self- selected daily illumination pattern and determine their contributions to period and phase control; (4) to evluate potential light adaptation effects occurring during the early portion of the dawn twilight signal; and (5) to analyze the temporal relations between the circadian rhythm of visual sensitivity, as measured by psychophysical signal-detection methods, and dawn/dusk illumination cycles. Recent observations in winter-depressed patients suggest that low-intensity twilight signals can have marked therapeutic consequences, and elicit shifts in sleep phase and in melatonin secretion patterns previously thought to require bright-light interventions. This, in seeking a systematic account of twilight-induced circadian effects in both nocturnal and diurnal mammals, within the present proposal, there is already a strong basis for expecting that refined clinical treatments of sleep phase and seasonal affective disorders will ensure.

The polyamines, putrescine, spermidine, and spermine, are major polybasic compounds in all living cells. These amines are important for many systems related to growth and differentiation. For many years we have been studying how these polyamines are synthesized, how their biosynthesis and degradation are regulated, their physiologic functions, how they act in vivo, and the structure of the various biosynthetic enzymes. For this purpose we have constructed null mutants in each of the biosynthetic steps in both Escherichia coli and Saccharomyces cerevisiae, and have prepared overexpression systems for the biosynthetic enzymes. Our overall studies have aimed at the use of these mutants plus those of S. pombe to elucidate the physiological functions of the polyamines, and, in particular, to ascertain the physiological effects of polyamine deprivation. In our last report we reported our studies on the effects of polyamine-deprivation on cell-cycle progression in S. pombe; we showed that in the absence of polyamines the cell cycle stops in the G1-S phase, and that these cells showed profound morphological changes. During the current year we have completed our studies showing that polyamines are important in protecting E. coli cells from the toxic effects of oxygen and hydrogen peroxide. In these studies we showed that, whereas wild type cells can be grown in an 100% oxygen atmosphere, polyamine-deficient cells are killed under these conditions. Addition of polyamines to the medium prevents this toxicity. The involvement of superoxide in this toxicity was shown by the ability of the Mn-SOD plasmid to protect the polyamine-deficient cells from the oxygen toxicity. Partial protection was also achieved by the addition of certain amino acid mixtures or by addition of sucrose or sorbitol. In our most recent work we have studied the relative importance of spermidine and spermine for the growth of S. cerevisiae. In these studies we have shown that spermidine is specifically required for the growth of S. cerevisiae, and that spermine will not substitute for this requirement unless it is first converted to spermidine by the FMS1 encoded amine oxidase. In these in vivo studies we have shown that in yeast, as in other cells, spermidine, and not spermine, is required for the hypusine modification of the protein initiation factor eIF5a, and that presumably this is one reason for the specific requirement of spermidine for growth. In addition to the in vivo studies we have purified the FMS1 encoded amine oxidase from an overexpressed yeast strain, and studied the amine oxidase activity of the purified protein in vitro. Current work is directed at extending the use of the spe3 ( delta) fms1 (delta) mutants to elucidate the relative importance of spermidine and spermine in a variety of physiological functions.

The objective of the proposed research is to study the steps in the enzymatic formation of conjugated bile acids in order to enhance understanding of the control of bile acid conjugation. Using methods developed in this laboratory, it is now possible to independently assay each of the enzymatic reactions necessary for conjugation. These assays will be used to study the subcellular distribution of the enzymes involved. The enzyme proteins involved in each reaction will be purified and the substrate affinities and substrate range will be studied for each step. Physiologic and pharmacologic controls of conjugation will be studied by assaying the individual reactions. Initial work will be performed in animal models as a prerequisite for future study in human tissue.

It is well known that Ca2 ion is required for normal cell growth and is involved in the regulation of a number of cell functions (i.e., cell division, motility, secretion, and maintenance of cell shape). In order to achieve a better understanding of the precise role of Ca2 ion, it is first necessary to determine the specific interaction of Ca2 ion with intracellular components. The specific tasks of this proposal are to study the biochemical mechanisms of Ca2 ion in cells by studying the intracellular Ca2 ion-binding proteins. This will be accomplished by using, in a complementary manner, biochemical and cell morphological techniques. The expected common denominator for mediating Ca2 ion stimulated processes in a ubiquitous low molecular weight protein which binds Ca2 ion with high affinity. Current data suggest that this protein may prove to be the intracellular Ca2 ion-receptor protein. The general approach of this proposal is to study this intracellular Ca2 ion-receptor with regard to its biological synthesis, intracellular localization, and involvement in cell division and motility. In particular, the Ca2 ion regulation of microtubules and microfilaments will be investigated. It this manner it is hoped that further insight will be gained regarding the specific mediation of calcium regulated events in the cell.

Abstract This is a revised renewal application for years 11-16 of the University of Florida ?Training Program in Regenerative Medicine? T32 Institutional Research Training Grant DK074367. The current renewal application seeks an additional five years of funding for a multi-disciplinary training program in Stem Cell Biology & Regenerative Medicine to support fuor pre-doctoral trainees and two post-doctoral fellows. This represents the addition of one additional pre-doctoral training slot over our current grant award, as recommended by the previous reviewers. The goal of this training program is to produce independent investigators capable of sustaining productive research programs studying the molecular and cellular aspects of stem cell biology and the use of stem cell products in patient care. Stem cell biology, outside of hematopoietic stem cells, is a relatively new area with great potential for development of novel treatments for both human and animal diseases ? Regenerative Medicine. With the emerging interest in stem cell biology and Regenerative Medicine, the need for trained researchers cannot be overstated.

DESCRIPTION: Epidemiological studies of US nuclear weapons workers allow evaluation of the effects of low dose, low dose rate radiation exposures accrued in an environment of mixed radiological and non-radiological exposures associated with the USDOE weapons complex. In the proposed study, we will examine a cohort of nearly 22,000 badge-monitored workers at the Savannah River Site (SRS) who will be followed over a fifty-year period. Past research on this epidemiologically important population has focused on analyses of standardized mortality ratios. The proposed study, in contrast, will focus on radiation-mortality associations in this cohort while investigating potential sources of bias and effect modification. We will examine radiation-mortality associations under varying lag assumptions, and investigate potential changes with age-at-exposure in susceptibility to the carcinogenic effects of radiation. Next, we will investigate differences between workers in the carcinogenic effects of radiation exposure due to non-radiological exposures accrued at SRS. Mechanistic models of carcinogenesis suggest that initiating exposure to some non-radiological carcinogens may modify the effects of subsequent ionizing radiation exposures. We will use a job-exposure matrix to identify workers with routine potential for exposure to several non-radiological agents, and apply innovative methods to examine the joint effects of radiological and non-radiological exposures. Finally, we will examine the role of tritium and neutron exposures in these analyses of radiation-mortality associations. We will investigate whether variation in radiation risk estimates between subgroups of workers reflects heterogeneity in radiological exposures. Study results for this large USDOE cohort will be evaluated in relation to observations from studies of other DOE facilities; and, study data will be compiled in a manner that will facilitate future pooled analyses. In this way, the proposed work will substantially strengthen the available epidemiological information about low level radiation effects in USDOE cohorts.

The novel neoclerodane diterpene Salvinorin A is the most potent naturally occurring hallucinogen. It has been reported to be the first subtype selective opioid ligand. In a screening against an array of receptors, Salvinorin A inhibited only kappa-opioid receptors (KORs). The activation of KORs has been documented to induce a wide range of behavioral effects, including sedation and perceptual distortions. Given its high selectivity, Salvinorin A could be used to investigate the precise role of KORs in the modulation of human perception. This has promoted interest in the development of a concise chemical synthesis of Salvinorin A. The synthetic scheme proposed for Salvinorin A should construct the molecule in an efficient manner. The key step is an intramolecular double Michael reaction, which assembles the tricyclic core and five of the seven stereocenters. A chiral bis(oxazolinyl) copper(II)-catalyzed vinylogous aldol condensation and a chirl auxiliary-mediated aldol reaction will be used to install the remaining two stereogenic centers. A late stage intermediate is readily amenable toward the preparation of analogues for use in a structure-activity relationship study.

HIV is unable to establish infections in macaque monkeys. We previously reported that an HIV-1 derivative, designated HIV 1NL-DT5R, containing a 7 amino acid segment from SIV CA and the entire SIV vif gene was able to initiate spreading infections in cultured macaque PBMC whereas wild type HIV-1 could not. Those experiments indicated that the presence of 666 SIV nucleotide sequences (6.7%) at these two specific locations within the full-length 9894 nucleotide HIV-1 genome was sufficient to counteract innate restriction factors residing in simian cells such as APOBEC3 and TRIM5alpha family members, which otherwise block HIV-1 replication. These results have now been extended to the organismal level by showing that HIV 1NL-DT5R, which is more than 93% HIV, was able to establish infections in all 5 pig-tailed macaques inoculated and elicited humoral responses against all of the HIV-1 structural proteins using commercially available Western blotting strips.[unreadable] [unreadable] In other studies, we have continued investigations of CCR5-utilizing SHIVs that are able to consistently replicate to high titers in inoculated macaques and cause immunodeficiency in a timely manner. Our approach has been to serially passage such R5 SHIV candidates animal-to-animal and introduce genetic changes associated with augmented replicative properties into the starting molecularly cloned virus. Presently we have generated R5 SHIVs exhibiting peak plasma viremia levels reaching 7 to 8 logs of viral RNA copies/ml and set points of 4 logs of viral RNA copies/ml. In most cases, memory CD4+ T lymphocytes are exclusively targeted for infection and depletion. These R5 SHIVs can be used in vaccine experiments in which efficacy is measured from virus load determinations.

The mechanisms by which the complement system regulates the metabolism of connective tissue continue to be investigated. In organ cultures of fetal rat bones, complement activation initiated by antibodies reactive with cell surface antigens results in the enhanced synthesis of prostaglandins which, in turn, causes resorption of the bone. A cell similar to a monocyte may be responsible for the elevated levels of prostaglandins found in these cultures since complement activation by immunoglobulins stimulates prostaglandin production by macrophages in culture. Immunoglobulin fragments which are incapable of directly activating these cells but which activate the alternative complement pathway are effective in this regard. Following interaction of the components of the complement system with macrophage membranes, enhanced incorporation of prostaglandin precursors into prostaglandins occurs. These studies demonstrate that both steroid as well as non-steroid inhibitors of prostaglandin biosynthesis abrogate this response. Complement activation also inhibits the growth of bone in organ cultures of calvaria. Although prostaglandin production is stimulated in these cultures, this event is unrelated to bone growth since indomethacin inhibits prostaglandin production but does not restore bone growth.

The project on Behavioral Pharmacology is primarily aimed at elucidating interactions between functions of (a) the glutamate-mediated corticostriatal system and related anatomical systems and (b) dopaminergic systems, including the nigrostriatal system, and functional effects of stimulant and neuroleptic drugs. Related to this primary aim are several related goals: 1. Elucidation of the functional effects of the "AA2" or quisqualic acid excitatory amino acid receptor, in relationship to stimulants, compounds with possible anticonvulsant effects, and alcohol withdrawal. 2. A search for more effective antagonists of the AA2 receptor. 3. Studies on the hypothetical non-specificity of postsynaptic denervation supersensitivity in the central nervous system. 4. Studies of CNS effects of sulfur-containing amino acids. The ultimate purpose of these studies is to enhance our understanding of the mode of action of neuroleptics and possibly brain dysfunction in schizophrenia as well. Secondary purposes are to enhance our understanding of mechanisms of stimulant drug effects, alcohol addiction, to develop compounds with potential utility for the acute treatment of stimulant overdose, schizophrenia, and seizure disorders, and to understand the possible forms of plasticity in the corticostriatal system.

The Animal Models Core provides critical support for the animal research proposed in all 4 projects. Support is provided for the maintenance of the transgenic mouse colonies used in the Program Project Grant. These colonies provide necessary models for the study of erythropoiesis, hemoglobin switching, and for vector-induced insertional mutagenesis. This Core provides funds for cage costs, and a research technician for genotype and breeding services. This Core also provides the expertise and resources necessary for the rhesus macaque autologous transplantation model used in projects 3 and 4. This model is essential for the further development of safe and effective gene therapy approaches to sickle cell disease. For example, the rhesus macaque model provides an accurate assessment of the efficiency by which lentiviral vectors can transduce human hematopoietic stem cells (HSCs), and whether expression of the transferred globin gene will occur at potentially therapeutic levels. Primates are also necessary to develop in vivo HSC selection strategies, given our prior experience that mouse models alone are insufficient for these studies. The Core provides expert technical services for the entire transplantation procedure, the supplies necessary to accomplish the transplant and the support the animals will require through the period of immunosuppression. We have developed a state of the art primate facility, with particular emphasis on the humane care and psychological well being of the animals, and have the necessary expertise to perform these experiments at a level commensurate with human stem cell transplantation.

Integrative molecular biology requires understanding interactions of large numbers of pathways. Similarly molecular medicine increasingly relies on complex macromolecular diagnostics to guide therapeutic choices. A fundamental argument for laser capture microdissection (LCM) of tissues is that without separation of specific cell populations from complex tissues, we will miss critical control functions of thousands of regulated transcription factors, cell regulators, and receptors that are expressed at low copy number. Without detecting changes in many of these critical effectors, the integrative understanding of tissue function and pathology will not proceed effectively. In complex tissues - particularly among pathological variations - it is exceptionally difficult to measure the majority of molecules that are at low copy number per cell without first isolating specific cell populations. For example, among Cancer Genome Anatomy Project partially sequenced cDNA libraries, only LCM-dissected ovarian cancer cDNA libraries are exceptionally informative about ovarian cancer biology. After LCM isolation of pure target cells, the library construction protocol used had selectively amplified a small number of rarer transcripts to the level that allowed statistical comparison of their expression between highly purified low and high malignant potential cancer cells. Many of these ?overamplified? genes overexpressed in the high malignant potential compared to low malignant potential ovarian cancer libraries are known to be oncogenes, and genes associated with invasion and metastatic processes in other tissues. The LCM techniques that we started developing ten years ago are now widely used in molecular analysis of genetics and gene expression changes within target cells within complex tissues. However, in global proteomic and lipid studies without molecular amplification methods, the quantity of isolated cells sufficient to perform accurate characterization of less abundant species is problematic as the microscopic visualization, targeting, and isolation in laser microdissection has a maximal rate of about 1 to 20 cells per second depending on the cells? microscopic distribution within the tissues. Recently, in collaboration with NCI and CIT, we invented and are now refining an automatic ?target-directed microtransfer? technique based on macromolecule-specific labeling of cells not requiring user visualization or microscopic targeting and capable of much higher throughput rates. This technique (patent pending) is built on our solid physical understanding of thermoplastic microtransfer and uses a much simpler device and transfer films than commercial laser microdissection instruments. Our current prototype is capable of isolating all specifically immunolabeled cells or organelles within 1 cm2 region of a standard immunostained tissue section in about five seconds, which corresponds to specific separation from approximately 50,000 cells per second. With this technique we can exceed the cell separation rates of standard technologies such as fluorescence-activated cell sorting while preserving our ability to harvest directly from standard sections of complex tissues. This rapid, automated microtransfer method has improved spatial resolution (~1 micron) and is consequently particularly well-suited to isolate highly dispersed, specific cell populations (e.g., stem cells or only those neurons in the supra-optic nucleus that express vasopressin) or specific organelles (e.g., neuronal nuclei in the brain). The spatial relationships (morphology) among the specific cells in the tissue are preserved on the transfer film. As this technology becomes more robust, we will seek to integrate the microtransfer with molecular profiling of specific cells within tissues, including routine proteomic and lipidomic analyses, particularly for the large number of less abundant molecular species. If microdissection and molecular analysis can be made clinically practical, the expression levels of sets of approximately 20 to 100 critical, stage-specific disease markers within a selected cell population might provide reliable diagnosis and intermediate endpoints of response to molecular therapies in individual patients. Our analysis of large gene expression and protein databases suggests that a significant fraction of all genes is expressed in any specific cell type and that the levels of gene products universally exhibit a highly skewed power-law distribution similar to those characterizing many other complex systems. We have developed mathematical models for the evolution of such distributions that predict the observed distributions of genes, protein domains, and gene expression observed in species of increasing biological complexity. We foresee an evolution of molecular diagnosis from one based on the qualitative or quantitative analysis of a few key biomarker macromolecules to one in which special clustering algorithms analyze complex multivariate databases. Such analyses should permit a more complete identification of highly correlated clinical cases and allow us to characterize their response to molecular therapies specifically designed to prevent progression. We are attempting to develop new approaches for better integration of our thermoplastic microtransfer methods of microdissection with downstream macromolecular analysis to permit more routine and simpler multiplex molecular diagnostics. A key feature is using the polymer matrix in which target cells are embedded for affinity purification and then for direct optical detection within the transparent polymer. We are using a variety of microscopy techniques in our lab to quantitatively characterize protocols for incorporating affinity nanoparticles in the tissue and polymer matrix. In the longer term, we foresee using in situ optical labels to quantify the spatial distributions of specific molecules captured within the microtransfer and retained following simple purification steps. Coupling the robust and simple automatic microdissection with rapid purification and detection of species might provide unique abilities to follow macromolecular changes in normal tissue development and in pathologies such as cancer progression within prostate, colon, breast, lung, and ovary tissues. In continuing collaborations with NCI, we have developed standard procedures for the isolation of normal and pathological cells from clinical specimens. We have used our models of the statistics of expression levels in cell populations to identify genes differentially expressed in cancer progression. With future integration of microdissection and macromolecular analysis, we believe the critical role for many less abundantly expressed genes in determining normal function and pathological changes will be more easily studied and integrated into molecular diagnostics and selection of clinical therapies.

Human apolipoprotein A-IV (apo-IV) is a plasma protein whose most distinctive property is its labile affinity for the surface of plasma lipoprotein. Although the specific function of apo A-IV in lipid metabolism has not yet been determined, studies, conducted by the Principal Investigator have contributed to a growing body of evidence which implicates apo A-IV as an important factor in the intravascular metabolism of high density lipoprotein (HDL). Recent studies now suggest that the labile binding of apo A-IV to lipid surfaces may be central to its physiological function. Specifically, we have proposed that the labile binding of apo A-IV to HDL constitutes a barostatic mechanism which maintains the surface pressure of HDL in a critical range required for optimal activity of lecithin-cholesterol acyltransferase (LCAT) and cholesterol ester transfer protein (CETP). The purpose of this proposal is to investigate the impact of molecular structure on the interaction of apo A-IV and phospholipid, with particular regard tot he molecular mechanism of the CETP reaction. We therefore propose four specific aims: 1) Recombinant apo A-IV deletion mutants will be studied by spectroscopic and surface balance techniques to examine the effect of specific amino acid domains on its ordered structure, thermodynamic stability, and lipid affinity; 2) The interaction of recombinant apo A-IV deletion mutants and phospholipid will be studied by physico-chemical techniques to examine the impact of protein structure on the properties and LCAT reactivity of apo A-IV/lipid complexes; 3) The interaction of CETP and HDL will be investigated using fluorescent labelled recombinant and native HDL to determine the mechanisms by which apo A-IV facilitates CETP-catalyzed lipid exchanges and the interconversion of HDL subspecies; 4) CETP will be studied using surface balance techniques to elucidate the mechanisms of the reaction and the role of apo A-IV facilitates CETP-catalyzed lipid exchanges and the interconversion of HDL subspecies; 4) CETP will be studied using surface balance techniques to elucidate the mechanisms of the reaction and the role of apo A-IV in lipid exchange phenomena. The investigation of the structure and function of the plasma apolipoproteins has led to major advances in the diagnosis, treatment, and prevention to atherosclerotic cardiovascular disease, the leading cause of death in the USA. The long term goal of this proposal is to delineate the physiological functions of human apo A-IV.

There is an urgent need to develop novel approaches to accelerate osteogenesis for the treatment of skeletal injury and disease. Combat troops and aging Veterans alike suffer from devastating orthopaedic injuries. While the young soldier suffering from a blast injury requires difficult limb reconstruction due to massive bone and soft tissue loss, the senior Veteran suffering from age-related bone loss is incapacitated by osteoporotic fractures, which are often associated with diminished osteogenic capacity, delayed healing, and non-union. Challenges in both acute trauma- and age-related fracture repairs converge on one critical deficiency: diminished ability to make bone due to a depleted stem cell pool. Autologous bone grafting, the gold standard for replenishing the stem cell pool in young healthy patients, can be unpredictable in cases of extreme tissue loss and in older patients. This is where our motivation lies: to mobilize and recruit a patient's own stem cells to the site of injury, and then activate these cells to produce bone in a timely manner. Our studies focus on a soluble stem cell recruitment factor, SDF-1, and its role as a paracrine signal regulating bone homeostasis and regeneration in response to mechanical loading and injury. The overall objective of this work is to determine the role of SDF-1 signaling during osteogenesis and regeneration using both loss-of-function models and exogenous SDF-1 treatment. Our central hypothesis is that SDF-1 acts as a paracrine mediator of progenitor recruitment and osteogenic differentiation in load- and injury-induced osteogenesis. We further hypothesize that SDF-1 treatment can accelerate bone repair in aged mice, a model of diminished regenerative capacity, possibly by enhancing cellular recruitment. To our hypotheses, we propose the following Specific Aims: (1) Determine the influence of osteoblast- and osteocyte-expressed SDF-1 on skeletal homeostasis and load-induced osteogenesis; (2) Determine the influence of osteoblast- and osteocyte-expressed SDF-1 on bone regeneration; and (3) Demonstrate that locally delivered SDF-1 can augment bone regeneration in aged mice, a model of suboptimal healing. If our hypotheses are correct, our studies will provide convincing evidence that SDF-1 works in a paracrine fashion in bone, a finding that would significantly advance our understanding of skeletal homeostasis and serve as a basis for developing therapeutic protocols to treat devastating orthopaedic injuries and disease in our Veterans.

The program aims to examine the significance of changes that phagocytic leukocytes undergo when they ingest bacteria, with respect to the role those changes play in protecting the host. The leukocytes may be stimulated by solid particles, or by some more soluble substances, to produce and release compounds of both leukocytic and bacterial origin. These phenomena are relevant to maintenance of health, because they provide a bulwark against infection. On the other hand, some biochemical sequelae of this stimulation may injure host cells. Generation and release of activated oxygen species is a key phenomenon in these contexts. Studies of the nature of the enzymes involved, of the "trigger" mechanism for stimulation of phagocytic cells, and of morphological changes the leukocytes undergo, have been made, and will be extended. The focus will be on the effects of some to the very molecules that phagocytes release from bacteria upon the phagocytic leukocytes themselves. Some other cells may also be affected. For example, monomeric breakdown-products of bacterial cells walls are well known as immuno-adjuvants and pyrogens, and have now been found to be somnogens. Binding sites (receptors?) for these substances have been found on macrophages, and, in preliminary work, on glial but not neuronal cells. These binding sites will be characterized, their variation followed as a function of the condition of the animal, and physiological effects ascribed to the ligand related to occupancy of the cellular binding sites. Radioactive (125I) muramyl ligands have been made and the conditions for binding studies are established. Attempts to isolate the receptor (binding) proteins will employ methods that include affinity columns with immobilized peptidoglycans. Muramyl peptides (exogenous substances) and an endogenous substance (serotonin) mimic each other "pharmacologically." Thus, muramyl peptides induce slow- wave sleep in which serotonin has been implicated. Indications are that the latter substance "activates" macrophages, e.g., by conditioning them to release activated oxygen species upon later stimulation - a function of muramyl peptides. Work will examine aspects of the synergism between stimuli and ionophores (for Ca++) and possible physiologically significant synergistic situations. Further, environmental (bodily) components may affect the lipid matrix of the phagocytic cell - membrane, triggering release of active oxygen species as shown in vitro. Both these matters could be of importance in vivo in bacterial infections, as may be the implications of the somnogenic activity of muramyl peptides.

This consortium proposal represents a joint effort to study the Omega- and (Omega-1)-hydroxylation of prostaglandins E, A, and F2Alpha by the microsomal fractions of liver, lung, kidney and male sex accessory organs of humans, rabbits, and pigs. The multi-faceted approach outlined in this proposal is intended to determine the sites of formation of hydroxylated prostaglandins, the physiological states regulating their levels, and their mode of metabolism by cytochrome(s) P-450. Human male accessory sex organs and seminal fluid will be examined for metabolic capacity or hydroxy prostaglandin content. These studies will utilize autopsy material, organs obtained surgically, and seminal fluid derived from patients seen for infertility for a variety of reasons. Rabbit lung and kidney will be studied from pregnant or progesterone-treated animals which are induced in their hydroxylation capacities toward prostaglandins E and F2Alpha. The transcriptional aspects of these induction processes will be examined by isolating mRNA from lung tissue induced 100-fold by pregnancy or progesterone treatment and translating in a cell-free system to determine the amount of mRNA synthesized with time after treatment or pregnancy. Pig kidney and lung tissue will be utilized as sources for purification of the cytochromes P-450 involved in prostaglandin Omega- or (Omega-1)-hydroxylation in untreated and progesterone-treated animals in order to study the physicochemical properties of these cytochromes P-450. By correlating the results of these studies in the various species, it is hoped that some insight will be gained with regard to the metabolic sources, fate, and significance of these hydroxylated prostaglandins.

Sepsis is the leading cause of deaths in US hospitals contributing to or causing 52% of all US hospital deaths and is the most expensive condition treated in US hospitals, costing more than $17 billion annually. More than 300 million blood culture bottles are produced annually, with a market size greater than $1.2B in the US and EU alone. The two-year phase II period spanned February 2013 to February 2015 (5 R44 AI094781-03). The developments, funded in part by this grant, have created a new paradigm to diagnose blood stream infection causing sepsis. The work of this grant has assisted in creating the SpecID blood culture system, which the company is now commercializing. The SpecID solution replaces the present 3-step process for characterization of blood samples, currently divided into 1) detection of infection; 2) an obligatory Gram stain for all positive cultures performed on the bench by trained technician; and then 3) species ID by MALDI-TOF, (again requiring skilled sample preparation), The SpecID solution combines faster detection, more accurate Gram status determination than the benchtop stain, and ID more accurate than MALDI, in a single fully automated step which reduces the time to ID, streamlines lab workflow, and saves significant cost. The result is improved patient outcomes and improved lab operating performance. Phase IIB aims address application to clinical human specimens along with development of instruments and media appropriate for commercial use. The objectives of this proposal reflect the major goals required to achieve commercial entry of the SpecID smart culture system into commercial, high volume laboratories including development of the SpecID blood culture instrument, blood culture bottle sensor attachment, and the clinical and analytical studies to prove performance, gain regulatory approval and make first sales.

This application is submitted is in response to PAR-09-173: Pilot Intervention and Services Research Grants (R34). We propose to use this grant mechanism to address three priority areas of the NIMH including decreasing the morbidity associated with depression, improving the mental health of females, and reducing the racial disparity in mental health. We address these issues by developing a novel approach to preventing depression in preadolescent African American girls growing up in urban, low-income environments. Aims: The specific aims for the proposed application are to: 1) Develop methods and infrastructure for carrying out tests of proof-of-principle, and 2) Establish proof-of principle through evidence of feasibility and acceptability and preliminary studies of efficacy and determination of settings for deployment. Approach: Our approach will be to target individual and interpersonal vulnerabilities that have been shown to confer risk for depression in our existing research on African American girls living in low- income, urban settings. Specifically, we aim to increase assertiveness and active coping in order to reduce early emerging depressive symptoms during the preadolescent period. In addition, we propose that increasing interpersonal connectedness with the mother via maternal engagement in the prevention program is necessary to yield effects that generalize and are maintained. Investigators: The Principal Investigators, Drs. Kathryn Keenan and Kathryn Grant, are among the leading researchers in the field of developmental psychopathology who focus on children living in urban poverty. They are currently testing developmental models of depression in girls growing up in poverty. The present application is a joint venture in which their collective expertise in depression research and recent interest in translating their empirical findings to the prevention of depression will be applied. Innovation: The three novel aspects of our approach to preventing depression in females are targeting the preadolescent period, deriving individual vulnerabilities from existing datasets to target in the prevention program, using the mother as the method of delivery of the preventive intervention, and addressing barriers that have limited participation of low-income urban African American families in treatment.

It has repeatedly been reported that dietary Na + induces an increase in blood pressure (BP) i.e. elicits "salt-sensitivity" (SS) only when loaded as NaCI. However, in both normotensive and mildly hypertensive, middle-aged African-Americans (blacks), we find that: 1) In the great majority of those with SS, dietary NaHCQloading induces a robust increase in blood pressure that is fully two-thirds that induced by dietary NaCI loading. 2) In those with such "selective Na + sensitivity" (sNaS), NaHCO-Ioading and NaCl-loading induce similarly robust increases in renal vascular resistance (RVR). We will test this hypothesis: In most blacks, the Na + component of NaCI selectively elicits SS mainly by inducing an increase in peripheral vasoconstriction that increases RVR and is mediated by, and varies in extent directly with, the plasma concentration of Na +, but does not require, as the hypothesis of Guyton does, a NaCI-mediated increase in plasma volume that entrains an increase in cardiac output. We will study healthy, salt-sensitive and salt-resistant blacks with normal to mildly increased blood pressures. Under controlled metabolic conditions, dietary NaCI and K + will be set at 30 and 50 mmol/70kg/d, respectively. Immediately after a 7-day control period, a dietary Na-salt will be loaded, 250 mmol/d, over a 7-day period. In each subject, we will measure the effects both of NaCI and of NaHCO3-1oading on: 1) BP and Na + balance (NAB), throughout; 2) extracellular volume (ECV), plasma volume (PV) and RVR, on the 7th day of the control period and on the 2nd and 7th days of Na+-Ioading; 3) cardiac output (CO) and total peripheral resistance (TPR), daily. The hypothesis predicts that in those with sNaS: 1) Oral Na+-Ioading with either salt will induce robust increases in BP, RVR and TPR that precede any increases in CO. 2) The preceding increases will persist, and they, and the absolute values each attains, will vary directly with the concomitant plasma concentrations of Na +induced, but not with the concomitant values of CO. 3) NaHCO3-1oading will not induce greater, and may induce lesser, increases in PV, CO, ECV and NaB than those induced by NaCl-loading in salt-resistant subjects. 4) Either hypertonic NaHCQ or NaCI intravenously administered over a 2 hr-period, will induce acute increases in BP, RVR and TPR greater than those induced by an equimolar but larger volumes of hypotonic NaCI that are more PV-expanding. 5) The acute increases in plasma Na + concentration will predict the extent of acute increases in BP, RVR and TPR.

Non-uniformity of myosin ATPase activity has been observed within single cells of rat ventricles during reperfusion following a brief period of ischemia and in old rats. In the reperfused hearts the non-uniformity is transitory. If the period of ischemia is sufficiently brief the contractility of the heart returns to pre-ischemic levels and the non-uniformity in myosin ATPase activity disappears as the contractility recovers. The specific aim of this project is to determine the cause of this non-uniformity and the conditions that either predispose to it or make it less likely. The abnormality of the myofibrils may have important clinical implications because non-uniformity of myofibrillar function within a single cell may impair the ability of these cells to shorten uniformly and consequently may hamper mechanical performance of the heart. There are two possible explanations for the non-uniformity in myofibrils of old hearts: 1) non-uniform distribution of myosin isozymes; and 2) non-uniform specific activity of a single myosin isozyme. In the reperfused heart, the more likely cause is non-uniform specific activity because the rapidity of appearance is probably too great for change in myosin isozyme content of myofibrils. Ca-activated and actin-activated myosin ATPase will be determined by a microphotometrical technique that can localize and measure ATPase activity in a single A-band. The concentration of a specific isozyme of myosin will be measured in the adjacent serial sections by quantitative immunocytology. Since sections can be as thin as 3u, isozyme distribution in a cell and specific activity of myosin ATPase in a cell can be resolved within 2 sarcomere lengths. The relation of non-uniformity to activity of a cAMP dependent system that regulates myosin will be examined. The spatial relationship of the myofibrils with different myosin ATPase activity to other organelles and of cells with non-uniformity to tissue vasculature will be examined to determine if some causal relationship might exist. The effect of the duration of ischemia, the conditions of reperfusion and the degree of catecholamine stimulation on the appearance and disappearance of non-uniformity will be studied.

Marval Biosciences Inc. has developed a novel liposomal X-ray contrast agent (NCTX)1. With prior SBIR funding (1R43EB04700), we demonstrated facile blood pool imaging, and enabled the simultaneous imaging of cardiac and pulmonary features, something that is not possible to do with conventional contrast agents. Marval is currently developing large scale production facilities for NCTX, and has demonstrated 4 liter production of the agent. We anticipate producing NCTX at the 50L scale under GLP conditions within the next 12 months, and pursuing an IND application shortly afterwards. In a recent, highly significant discovery it was shown that NCTX can predict the outcome of chemotherapy using Stealth Liposomal Doxorubicin (SLD). NCTX consists of a clinically used Iodinated contrast agent encapsulated in a Pegylated liposome. One expects therefore, that NCTX will extravasate in tumors in the same way that SLD (e.g. Doxil(r)) does, by the so-called Enhanced Permeation and Retention (EPR) effect. In addition to the molecular effect of doxorubicin in inhibiting topoisomerase-II, the efficacy of SLD in treating a tumor is fundamentally dependent on (1) the extent of its localization in the tumor and (2) the release of the active drug from the liposomes after localization. It is well-known that the degree of tumor vasculature leakiness differs not only among same type tumors but even spatially within the same tumor. It was therefore hypothesized that the extent of extravasation of NCTX in individual tumors would be a predictor of the extravasation of SLD, and therefore of the efficacy of SLD in treating a specific tumor. To test these hypotheses, NCTX was used to image rat mammary adenocarcinomas using a clinical mammography instrument. It was demonstrated that in individual rats bearing tumors of identical size and morphology, those specific tumors that exhibited high NCTX uptake as visualized by X-ray imaging were the most susceptible to treatment by SLD. There are currently 128 ongoing clinical trials in the US (www.ClinicalTrials.gov), of Stealth Liposomal Doxorubicin (Doxil(r) and various generic equivalence candidates). Even if a small fraction of these trials are successful, one anticipates that the number of patients treated with SLD will dramatically increase in coming years. While liposomal doxorubicin does reduce the side effects of chemotherapy, it still has significant side effects of its own, including cardiotoxicity and hand-foot syndrome12. The ability to predict the efficacy of liposomal doxorubicin in treating a specific tumor, in a patient-specific manner, would therefore be of enormous clinical utility. In Phase 1 of this SBIR project therefore, we seek to develop NCTX as a prognosticator of SLD efficacy. If successful, this project will lead to a new indication for NCTX. To date, we have demonstrated the prognostication ability in a syngeneic rat mammary tumor, the MAT B-III adenocarcinoma. Yet, liposomal doxorubicin (specifically Doxil) is primarily used for Ovarian cancer. In this project, we therefore propose to test the prognostic ability of NCTX in relevant ovarian cancer mouse models. PUBLIC HEALTH RELEVANCE: Marval Biosciences Inc. has developed a novel liposomal X-ray contrast agent (NCTX)1. With prior SBIR funding (1R43EB04700), we demonstrated facile blood pool imaging, and enabled the simultaneous imaging of cardiac and pulmonary features, something that is not possible to do with conventional contrast agents. Marval is currently developing large scale production facilities for NCTX, and has demonstrated 4 liter production of the agent. We anticipate producing NCTX at the 50L scale under GLP conditions within the next 12 months, and pursuing an IND application shortly afterwards. In a recent, highly significant discovery it was shown that NCTX can predict the outcome of chemotherapy using Stealth Liposomal Doxorubicin (SLD). NCTX consists of a clinically used Iodinated contrast agent encapsulated in a Pegylated liposome. One expects therefore, that NCTX will extravasate in tumors in the same way that SLD (e.g. Doxil(r)) does, by the so-called Enhanced Permeation and Retention (EPR) effect. In addition to the molecular effect of doxorubicin in inhibiting topoisomerase-II, the efficacy of SLD in treating a tumor is fundamentally dependent on (1) the extent of its localization in the tumor and (2) the release of the active drug from the liposomes after localization. It is well-known that the degree of tumor vasculature leakiness differs not only among same type tumors but even spatially within the same tumor. It was therefore hypothesized that the extent of extravasation of NCTX in individual tumors would be a predictor of the extravasation of SLD, and therefore of the efficacy of SLD in treating a specific tumor. To test these hypotheses, NCTX was used to image rat mammary adenocarcinomas using a clinical mammography instrument. It was demonstrated that in individual rats bearing tumors of identical size and morphology, those specific tumors that exhibited high NCTX uptake as visualized by X-ray imaging were the most susceptible to treatment by SLD. There are currently 128 ongoing clinical trials in the US (www.ClinicalTrials.gov), of Stealth Liposomal Doxorubicin (Doxil(r) and various generic equivalence candidates). Even if a small fraction of these trials are successful, one anticipates that the number of patients treated with SLD will dramatically increase in coming years. While liposomal doxorubicin does reduce the side effects of chemotherapy, it still has significant side effects of its own, including cardiotoxicity and hand-foot syndrome12. The ability to predict the efficacy of liposomal doxorubicin in treating a specific tumor, in a patient-specific manner, would therefore be of enormous clinical utility. In Phase 1 of this SBIR project therefore, we seek to develop NCTX as a prognosticator of SLD efficacy. If successful, this project will lead to a new indication for NCTX. To date, we have demonstrated the prognostication ability in a syngeneic rat mammary tumor, the MAT B-III adenocarcinoma. Yet, liposomal doxorubicin (specifically Doxil) is primarily used for Ovarian cancer. In this project, we therefore propose to test the prognostic ability of NCTX in relevant ovarian cancer mouse models.

Project Summary/ Abstract Bioassay-guided fractionation of cells often uncovers small molecules that bind macromolecular targets in new and unexpected ways. Exploration of the chemical reactivity and target selectivity of these metabolites has laid the chemical foundation for the development of new biological tools and therapeutics. The molecular architecture of secondary metabolites is challenging and different than the chemical space explored by most medicinal chemistry campaigns: there is recognized `natural product-like' space and we lack the tools to explore it with the same depth as `drug-like' space. Our lab has developed new tools to more easily access natural products, and has focused our efforts on secondary metabolite families that appear to covalently modify their targets as the basis for their phenotypic effects. Our chemical syntheses are deliberately concise and easily-scaled to enable subsequent investigation into reactivity and biological activity. The current application significantly advances these efforts, provides compelling preliminary data as a foundation for the proposed work, and delves into new areas of chemistry. In this proposal, two areas of research are described: 1. the identification and investigation of covalently-reactive pharmacophores associated with the asmarine alkaloids, Nuphar dimers and isocyanoterpenes; and 2. the development of cross-coupling technology to access `natural product-space' more generally. In the first area, we disclose a preliminary cellular target of the unusual N-hydroxydiazepine purine (HAP) pharmacophore of the asmarine alkaloids and address unsolved problems posed by this motif. As part of a theme that runs throughout our work, we show how the aims of chemistry and biology intersect in the study of covalently reactive secondary metabolites. We also investigate the sulfur-electrophilicity of the Nuphar dimers, a property recently demonstrated by our lab to operate in organic solvent and cellular environments. We propose solutions to the challenging stereochemical problems posed by the monohydroxy dimers and show how chemistry developed in our lab can generate a combinatorial library of sulfur electrophiles. In a third area, we investigate the challenging architecture and reactivity of the isocyanoterpenes, which we recently demonstrated can kill Plasmodia by an alternative mechanism to the heme detoxification pathway often suggested. We also propose a novel caging strategy to render the isonitriles systemically-viable. In part two, we investigate a bimetallic catalytic cycle capable of solving long-standing problems in chemical synthesis. The proposed methodology is supported by proof-of-principle examples and provides chemists the reaction vehicles necessary to `escape from flatland.'