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Timaeus 12

Influenza, yearly, causes approximately 36,000 deaths in the United States. Many of the deaths are secondary to pneumonia or cardiac complications and are much more common in the older than in the younger populations. In the past several years there has been increasing concern over rising rates of P&l deaths in the elderly despite higher vaccination rates than ever before. This concern became a public debate in 2005 with the article by Simonsen that asserted that observational studies of vaccination overestimated the benefits of vaccination. Due to the disproportionate morbidity and mortality of influenza for people > 65 years of age, public health officials in the United States have recommended routine yearly influenza vaccination to individuals = 65 years of age and more recently added all adults over 50 years of age to the recommendation. These decisions were intended to reduce the morbidity and mortality of influenza but were not based on randomized clinical studies of vaccine efficacy in older individuals. Only three randomized clinical trials (RCTs) testing the efficacy of trivalent inactivated influenza vaccines have been conducted in elderly adults and only one used laboratory-confirmed influenza as an endpoint. Even though vaccine efficacy (VE) was 56%, this study was not adequately powered to examine VE in adults > 70 years of age and only looked at healthy elderly. Hence many questions still exist about the effectiveness of influenza vaccination in older adults. This application is a sub-study of a larger surveillance program conducted by Dr. Talbot's mentor, Dr. Kathryn Edwards. This surveillance study will prospectively test hospitalized patients for influenza with culture, antigen and PCR. With this information the following aims will be addressed: [unreadable] 1. To estimate the effectiveness of trivalent inactivated influenza vaccine (TIV) against laboratory-confirmed influenza-associated hospitalizations in individuals aged > 50 years of age. [unreadable] 2. To determine the sensitivity and specificity of both the CDC-defined influenza-like illness (I LI) and rapid influenza antigen detection using the gold standard diagnosis of either a positive influenza culture or a positive reverse transcriptase - polymerase chain reaction (RT-PCR) for influenza in individuals aged > 50 years of age hospitalized for acute respiratory illness. [unreadable] 3. To describe the morbidity associated with hospitalized episodes of influenza in individuals a 50 years of age, using prospectively collected population-based data. [unreadable] [unreadable] [unreadable]

This grant application seeks financial support for participation at the University of Vermont in the breast cancer adjuvant therapy protocols of the National Surgical Adjuvant Project for Breast and Bowel Cancers (NSABP). We currently follow 109 patients who have been treated on NSABP protocols 4, 5, 6, 7, 8, 9 and 10, since 1972. We anticipate continued entry of approximately 25 patients per year. The NSABP project headquarters' proposal should be sought for details of protocol rationale and overall scientific achievements of the NSABP. The availability of this patient population at our Clinical Research Center has made and will continue to make possible longitudinal studies on the mutagenic effects of adjuvant chemotherapy and chemoimmunotherapy using the recently developed Albertini/Strauss technique of assaying 6-thioguanine resistant peripheral blood lymphocytes. Additional collaborative studies planned at this institution utilizing NSABP patients will include psychological assessments to monitor the effect of group counseling programs for these patients.

This is an application to identify and intensively study panic attacks and panic disorder in children and adolescents who present to pediatric emergency rooms, pediatric clinical services and psychiatric services. Isolated panic attacks are prevalent in adults (10%), and although the lifetime prevalence of panic disorder is less (1.6%), it is a disabling condition with high morbidity. Until recently, panic disorder was considered a disorder of adulthood that did not occur in children or adolescents. Our preliminary work, recent isolated clinical reports, epidemiological studies and retrospective reports of adult patients reporting age of first panic episode strongly indicate that panic disorder does occur in children. Children and their parents, like some adults, may interpret their symptoms as physical in nature and many will visit pediatricians and emergency rooms to rule out medical illness. Pilot data will be collected within the confines of the limited time period and budget of a small grant to lay the ground work for a comprehensive validation study on panic in children. This pilot study is designed to collect preliminary data in four areas and to use the data and experience gained for future grant applications. The four areas where work is necessary and where we will begin to collect pilot data are: 1) thorough assessment of symptoms, medical history, risk factors, psychiatric history, social functioning; 2) detailed family psychiatric history of the children's first degree relatives; 3) assessment of associated biological factors of panic disorder studied in adults such as cardiac auscultation, echocardiogram, lactate infusion and C02 inhalation; 4) a longitudinal follow-up to document clinical course. 'ne feasibility of our methods will be assessed and approaches to promising new leads will be incorporated into later projects. Children with symptoms suggestive of panic attacks will be fully assessed using a structured diagnostic interview and diagnostic instruments as well as echocardiography where possible to obtain data on phenomenology, course, suicidal behavior, comorbidity, family history, medical history, biological markers and social functioning. Information will be obtained from child and from parent about child. Children with disabling panic symptoms meeting DSM-IIIR criteria for panic disorder will be treated with standard treatment interventions including behavioral, cognitive and pharmacological therapies. This sample of child and adolescent onset panic attacks or disorder will become a cohort for future follow-up studies so that we can plot the course, recovery, recurrence biological markers and familial aggregation of the illness. Such data beginning with a cohort of children in non-psychiatric settings is not currently available. Specific aims of this study are to: identify and intensively study a sample of children and adolescents (n=60) ages 6-18 years with panic attacks or panic disorder meeting DSM-IIIR criteria, specifically to describe symptoms, risk factors, psychiatric history and family history; identify and collect family history data on the child's first degree relatives from mother or father; initiate biological marker studies, specifically auscultation and echocardiogram; sample maintenance.

Acute adjustments in ventilation represent a powerful homeostatic mechanism for the maintenance of arterial and/or brain P02, PC02 and pH. Sensory feedback to the ventilatory control centers occurs via peripheral and central chemoreceptors, but there are several unresolved questions regarding the mechanisms and relative importance of specific subpopulations of neurons thought to act as central C02/pH chemoreceptors. What confers upon select neurons the intrinsic ability to sense pH? What are the signal transduction pathways for pH? Which neurons perform this task? Under which circumstances are they important (sleep wakefulness)? The overall goals ofthis proposal are to address some of these questions regarding central chemoreception by taking advantage of unique inbred rat strains with inherent, and large differences in C02 sensitivity; the Brown Nonway (BN: low responder) and Dahl Salt-sensitive (SS: high responder) rats. First, we will determine if the deficit in C02 sensitivity in BN rats is due to decreased cellular C02/pH sensitivity of individual chemosensitive neurons in vitro, which will be aided by the development of transgenic rats expressing fluorescent proteins in select neuron pools for direct patch clamp recordings. We will also determine the relative effects of creating focal acidosis in brainstem regions harboring these neuronal subpopulations in BN and SS rats in vivo. Finally, our preliminary data suggests that deficits in multiple neuromodulators in the brainstem may contribute to the blunted C02 sensitivity in the BN rats, and thus we will determine whether if augmenting these neuromodulators individually or collectively can restore C02 sensitivity in the BN rat. The data obtained from these unique rat strains will provide significant insights into the mechanisms and relative importance of specific chemoreceptor subpopulafions in regulafing eupneic ventilation and ventilatory C02 sensitivity. They will also provide insights into respiratory-related diseases, and provide a framework for future genomic investigations aimed at idenfifying the genefic determinants and the unique cellular mechanisms by which central chemoreception occurs.

Antibiotic resistance is growing at a rapid pace and is a major threat to human health. The need for new antibiotics is eminent, though there is a lack of research programs in the industry devoted to this cause. At the Institute for Genomic Biology at the University of Illinois, we aim to identify and study potential antibiotics by mining microbial genomes. Phosphonates and phosphinates, compounds containing one or two direct carbon-phosphate (C-P) bonds, respectively, are a growing class of natural products with antibacterial and antifungal activity. The biosynthetic pathways that produce these compounds have remained largely unexplored until recently. The study of antibiotic biosynthetic pathways is necessary for our goal to eventually produce analogs through engineering of the biosynthetic machinery. Elucidation of the activity of specific enzymes in these pathways will help better predict the structure of new, undescribed C-P containing natural products and the function of the biosynthetic machinery that produces them. Phosphoenol pyruvate mutase (Ppm) is responsible for constructing the first C-P bond in the biosynthesis of the antifungal agent phosphinothricin tripeptide (PTT). In close proximity to the gene for this enzyme is a gene encoding for hydroxyethyl phosphonate dioxygenase (HEPD), which converts 2-hydroxyethylphosphonate (2-HEP) to hydroxy-methylphosphonate (HMP), a later step in PTT biosynthesis. Two enzymes have been identified in Nitrosopumilus maritimus, an archaeal marine species, one of which is homologous to Ppm and the other is a potential downstream dioxygenase with homology to HEPD. These enzymes represent the first examples of archaeal involvement in phosphonate biosynthesis and provide a link to methane production in the oceans, a major contributor to the greenhouse effect. We have already shown the new dioxygenase to convert 2-HEP to methylphosphonate (MPn). We believe that this HEPD homolog, or methylphosphonate synthase (MpnS), catalyzes this transformation via a similar hydroperoxylation mechanism predicted for HEPD. This proposal involves comparison of native activity of both dioxygenases by analysis of kinetic parameters and identification of reaction intermediates. Next, the substrate flexibility of MpnS will be studied to aid in elucidating its mechanism. Finally, the active sites of both enzymes will be probed through mutagenesis. The work proposed will expand on current research of the mechanism of HEPD, will aid in further elucidating the function of MpnS in N. maritimus, and will provide a better understanding of both mechanisms so as to afford better predictions a priori of in vivo function of new HEPD homologs for use in antibiotic bioengineering. PUBLIC HEALTH RELEVANCE: Antibiotic resistance is a major threat to human health and it is imperative that new classes of antibiotics are developed. This work will investigate how certain types of compounds with antibiotic properties are made by microbial organisms and will enable the identification of additional antibiotic candidates produced by other organisms based on their genomes.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Microcrystallography at MacCHESS greatly extends the capability of the stations and significantly increases the success of MacCHESS users with difficult samples, as has been illustrated in the accomplishments,Section C. In the coming project period, we will work closely with key collaborators to further develop microcrystal methodology to facilitate the structural analysis of challenging biological systems such as: 1) complex aggregates such as those that make up the amyloid fibrils associated with Alzheimer's disease (Eisenberg, UCLA) [102], 2) membrane proteins grown in lipidic mesophases, particularly those associated with Pseudomonas aeruginosa, an opportunistic pathogen responsible for many hospital-acquired infections (Caffrey, Univ. of Limerick, Ireland and Ohio State Univ.), 3) the gating properties and conformational transitions necessary for ion channel function (MacKinnon, Rockefeller Univ.) and biomedically important G protein-coupled receptors (Navarro, U. Texas Medical Branch). Below is a brief summary of the challenges confronted by these collaborators that motivate the microcrystal technical program. More information about the collaborators'work is given in section D.2.2. A number of important human diseases involve the harmful aggregation of proteins. Best known are Alzheimer`s disease, transmissible spongiform encephalopathies, and Type II diabetes mellitus. The Eisenberg group has managed to produce microcrystals of key amyloid-forming peptides, in spite of their tendency to form fibers rather than regular crystal lattices. These ultra-small needles, typically 1 micron in the narrow diameter, require special harvesting and mounting techniques. To date, usable diffraction data have only been obtainable using the microcrystallography beamline at the ESRF in Grenoble, France, a facility that is not often available to US researchers. These fibril crystals strain the limits of optical light microscopy used for positioning at beamlines. They are a unique example of sample dry mounting and their smallness serves as an important benchmark for mechanical precision of sample positioning. The smallness of X-ray illuminated volume combines with the relative durability of the crystals and their small unit cell to produce an excellent test case for the proposed micro CCD detectors (described below). Fibrils also exhibit highly variable quality, making it necessary to screen multiple samples to obtain optimal data. The challenging membrane protein crystals grown by the MacKinnon group are also often small (<20 microns) and tend to be variable in their diffraction quality. The variability can sometimes mean that a few percent of the crystals are suitable for data collection. For this reason, Dr. MacKinnon had encouraged us to develop methods to optimize data collection on small crystals and to implement robotics to rapidly screen large numbers to identify useful crystals. Membrane associated protein crystals grown by the Caffrey and Navarro groups pose additional challenges. Beyond the fact that they are small, fragile, and of significant unit cell size, the unusual matrices in which the crystals are grown (such as cubic lipidic mesophases), present unique visualization and harvesting challenges. The use of more sophisticated visualization methods, such as confocal microscopy, should prove valuable in this case. We propose to explore how a combination of microbeams, sample manipulation and advanced visualization methods can be used to identify good quality regions on otherwise defective crystals. In this regard, Cornell is home to one of the world centers for multiphoton confocal microscopy. The Developmental Resource for Biophysical Imaging Opto-Electronics (DRBIO) is currently developing a laparoscopic version of their confocal microscopy technology which has similar form factor and optical requirements to what would be needed for beamline use. We propose to leverage DRBIO expertise (Prof. Warren Zipfel) to investigate the feasibility of either adapting our current optics or using an inexpensive aspherical lens system to achieve submicron 3D imaging crystal samples based on natural (tryptophan) or dye-induced flourescence. All four collaborating groups encounter many cases of sample inhomogeneity and crystal imperfection. We propose to also work with a wide range of our users in using microbeams, as part of the MacCHESS service, training, and dissemination missions, to examine crystal quality, to help develop strategies for locating good portions of crystal, and to help users obtain useful data.

The broad, long-term objective of this application is to define in molecular terms the linkage between the accumulation of soft drusen below the retinal pigment epithelium (RPE) in the macula and the increased risk of developing age-related macular degeneration (AMD). The presence of soft drusen in the macula is the hallmark risk factor for developing AMD. Surprisingly little is known of the composition or origin of drusen. To this end a novel method for drusen isolation has been developed that allows the collection of microgram quantities of drusen from donor eye tissue. At the time of isolation, different drusen sub-types can be identified and separated for use in studies that will characterize their molecular composition. The diagnostic utility of drusen in AMD can be likened to that of blood levels of cholesterol in atherosclerosis. The presence and abundance of drusen, like the level of cholesterol in the blood, indicates the degree to which a patient is at risk for developing the disease. Because of the relationship of drusen and AMD, understanding the composition of different drusen sub-types will provide important information on possible pathways that are causally involved in drusen development. Novel proteins or common modifications of proteins present in drusen, should provide insight as to potential drug targets of therapeutic agents to treat AMD. The current application is focused on exploiting this drusen isolation procedure to define the molecular composition, distribution and cellular origin of drusen sub-types in normal and AMD tissues. The three specific aims are: (1) To test the hypothesis that different sub-populations of drusen can be isolated from donor eye tissue. (hard vs. soft, foveal vs. peripheral, old vs. older, spherical vs. flat, amber vs. clear, opaque vs. granular, etc.). (2) To test the hypothesis that different structural features of drusen sub-populations reflect different molecular composition (light microscopy, histochemistry, electron microscopy, SDS/PAGE, Western blotting, mass spectrometry). (3) To test the hypothesis that some of the molecules present in drusen are novel and are not found in Bruch's membrane.

The number of underrepresented minority students entering into medicine, nursing, and related health care disciplines is declining. To illustrate, in the past 5-6 years, the number of underrepresented minority students entering into our medical college has dropped by about 82% (from 50 in 1998 to 9 in 2002). This gradual decline in enrollment is not unique to our medical college, for it is observed in many institutions of higher education across the nation. A similar trend is also witnessed in graduate education programs. One key approach that could be adopted to reverse this trend is to involve underrepresented minority students in biomedical and behavioral research during the course of their undergraduate education. Drexel University College of Medicine (Drexel Med) has been educating students in biomedical sciences for over 150 years. Its recent merger with Drexel University, a respected leader in engineering and computer sciences, has unveiled yet additional opportunities. At Drexel Med, we have the required experience and the expertise to successfully initiate and implement summer research programs. We currently have three programs which offer short-term summer research training opportunities to local high school, undergraduate, and medical students. These successful programs serve as an archetype for initiating, in partnership with NHLBI, similar research opportunities for underrepresented minority students in undergraduate and graduate programs in various schools and colleges within Drexel University and at Cheyney University - the oldest black historical university in the country. Since the primary goal of NHLBI is to support research in the areas of cardiovascular, pulmonary, blood, and sleep disorders, minority students enrolled under this training grant will be assigned to one of the many (> 19) investigators in basic and clinical departments who are working in areas of similar interests. We believe that by providing such an opportunity to underrepresented minority students in undergraduate and graduate programs, we will not only enrich their academic experience but would also facilitate their entry and retention into health care related professional pathways. This outcome would meet the growing needs of our racially divergent communities and help alleviate the existing discord between the representation of minorities in the general populace as compared to that in the health care profession.

Homeodomains are a class of DNA-binding protein domains which have important roles in control of transcription and development in eukaryotes, and in some cases are involved in human oncogenesis. We are using the MAT a2 homeodomain of yeast (Li et al., 1995, Science 270, 262-269) as a model system to characterize the thermodynamics of protein folding and sequence-specific DNA binding by this protein motif. Using differential scanning and isothermal titration calorimetry, we measured the enthalpy, entropy, heat capacity, and Gibbs free energy changes of these processes for the wild-type sequence of a2 homeodomain (Carra & Privalov, 1997,Biochemistry 36, 526-535). The protein-DNA interaction is enthalpically driven at physiological temperatures. Protein folding and DNA binding are linked, as for several other DNA binding proteins. A comparison of the circular dichroism spectra of the free and DNA-bound protein species revealed that formation of protein structure is induced by DNA binding. The energies measured for association therefore include a component due to folding. We are currently extending these studies to mutant versions of the a2 homeodomain. Site-directed mutagenesis was used to create versions of the homeodomain that contain alanine substitutions in the DNA recognition helix, removing important contacts to DNA. Changes in the core packing of the protein were also made to assay the importance of certain conserved residues to folding, and the ability of the protein core to accept compensating substitutions. Deletion of the N-terminal arm of the protein, which wraps around the back of the DNA in the complex, results in a drastic loss of DNA binding affinity. The MATa2 homeodomain can also bind DNA cooperatively with the MATa1 homeodomain, as a heterodimer. This protein-protein interaction, and DNA binding by the heterodimer, will be studied using the isolated homeodomains and a genetically produced fusion of the two.

This project responds to Notice NOT-OD-09-058, Enabling RPGs to Leverage NCRR Center and Center-like Programs. Plasmacytoid dendritic cells (PDC) represent a unique secretory cell type that plays a critical role in innate immune responses. During viral infections, PDC secrete massive amounts of type I interferon (IFN), prime anti-viral T cell responses, and activate multiple immune cell types. In addition to infectious immunity, PDC are implicated in many immunological phenomena including anti-tumor immunity, allergy/asthma and autoimmunity. The PDC possess unique features and gene expression patterns that facilitate their direct recognition of viruses and high level IFN secretions. However the molecular mechanisms that regulate lineage commitment, maturation, and function of PDC remain poorly understood. The overall goal of our research is to dissect the regulation of PDC development and function at the molecular level. Our work on the parent grant has indentified E protein E2-2 as a key transcriptional regulator of PDC development in mice and humans. We also found the E2-2 directly activated transcriptional factors and receptors critical for pDC function and identified novel E2-2 targets in PDC development and function. In collaboration with the NCRR Program, the Knockout Mouse Project (KOMP), we will generate and analyze loss-of-function mouse mutant strains for several PDC-enriched genes regulate by E2-2. These studies would help elucidate the molecular basis of PDC development and function and thus pave the way for immunotherapeutic approaches directed at the PDC. PUBLIC HEALTH REVELANCE: The study is aimed at molecular and genetic analysis of plasmacytoid dendritic cells (PDC), a unique immune cell type that plays a major role in anti-viral immune responses and in autoimmunity. The elucidation of molecular mechanisms that govern PDC development and function would pave the way for novel therapeutic approaches targeting PDC function in infections and autoimmune diseases.

The recognition of soluble protein antigens by T cells during the induction of immune responses appears to require that the antigen be "processed" and "presented" by accessory or antigen presenting cells (APC), the nature and origin of which have not been clearly established. These studies will use a model protein antigen, hemoglobin (Hb), which has a well-characterized structure, to examine structural effects exerted on this antigen by APC required for its recognition by T cells. Recent advances in the ability to clone antigen-reactive T-cells, the identification of APC that can be isolated as relatively homogenous populations (such as antigen-presenting tumor, cell lines), and in the development of extremely sensitive analytical and separation techniques for complex mixtures of proteins and polypeptides (such as high-performance liquid chromatography (HPLC)) make these studies feasible. Various cell populations including macrophages, tumor cells, and B cells will be studied for their ability to present Hb to murine Hb-reactive T-cell clones. The specificities of the T cells for various epitopes on Hb will be localized through the use of polypeptide fragments obtained by cleavage of Hb with cyanogen bromide. The effects that APC exert on the structure of Hb, particularly with regard to its proteolytic degradation, will be examined by attempts to detect (by HPLC) and identify peptide fragments of Hb generated after uptake of radiolabeled Hb by APC. If these studies are successful, a correlation may be made between the molecular structures of epitopes recognized by T cells and structural effects exerted on antigen by APC. Such findings will shed considerable light on the events that lead to the immune recognition of antigens by T cells and will be significant with regard to developing approaches for manipulating immune responses in both health and disease. (LB)

HIV in sub-Saharan Africa is almost exclusively transmitted by heterosexual sex. Prospective randomized clinical trials (RCTs) in Africa, including 2 conducted by Hopkins researchers in Uganda, demonstrate that adult male circumcision reduces HIV acquisition by 60% and herpes simplex virus-2 (HSV-2) acquisition. Little is known about the immunology and pathophysiology of the foreskin. We propose to describe the immunology of foreskin inflammation and assess its role in HIV and HSV-2 acquisition and transmission using data and foreskin specimens obtained from the circumcision RCTs. The Hopkins Uganda circumcision trials enrolled both HIV+ and HIV--index male subjects and their female partners and archived the surgical foreskin specimens. With this repository, we have a unique opportunity to evaluate this hypothesis and to understand inflammation in the foreskin. The K23 candidate, Dr. Kristine Johnson, has specific interests in HIV immunology and sexually transmitted infections. The proposal provides for structured training and mentorship support to develop a translational research program to explore these relationships. Using methods already developed by Dr. Johnson, she will define local lymphocyte populations within the foreskin tissues, by confocal microscopy and immunohistochemistry. Using tissue PCR, she will quantify and characterize associated inflammation patterns and tissue HIV and HSV-2 viral content. After this work is completed, using the rich data collected on index subjects and partners, she will assess the impact of the foreskin cellular components on HIV acquisition events among initially HIV-uninfected men and the impact of foreskin cellular and viral components on HIV transmission events to serodiscordant female partners. Kristine Johnson, MD, joined the faculty of Johns Hopkins University Division of Infectious Diseases in July 2008. This application integrates epidemiology and immunological approaches using a unique sample set to answer critical questions in HIV/HSV-2 transmission. There is an especially strong mentorship team of experts in HIV/STI transmission and prevention, including Drs. Jonathan Zenilman, Ronald Gray and Thomas Quinn. RELEVANCE: As a beginning, we propose here to evaluate relationships between foreskin inflammation, epithelial lymphocyte populations, cytokines and HIV and HSV-2 infection and transmission. The results of this work will lead to a better understanding of the male foreskin immune milieu and may inform the development of new modalities for targeted HIV and STI prevention.

Normal cellular function is dependent on the ability of environmental signals to elicit distinct responses through activation of specific cell surface receptors, and yet these signals often converge on the same members of the MAPK family. How can cells maintain specificity of signal transduction when so many different receptors ultimately activate the same enzyme? My previous work helped establish that components of the endocytotic machinery, (e.g. b-arrestin), can assemble scaffolding complexes that direct the subcellular localization, duration and outcome of MAPK activity. These studies investigate the mechanism by which these endosomal scaffolds can direct distinct cellular outcomes, by characterizing a molecular scaffold formed in response to protease-activated receptor-2 (PAR-2) that promotes chemotaxis by sequestering and prolonging MAPK activity at the leading edge and comparing it to one formed in response to a receptor that promotes nuclear translocation of MAPK and proliferation. We pose the following questions: 1) Does the PAR-2 endosomal scaffold sequester MAPK to a subcellular domain where it directs localized reorganization of the cytoskeleton? 2) Do receptor/b-arrestin interactions and molecular contacts within each endosomal scaffold and ultimately determine the outcome of receptor activation? 3) How does the endosomal scaffold control kinase activity in a specialized region of the cell? Understanding the mechanism by which endosomal scaffolds determine signaling specificity is important from a fundamental cell biological perspective as recent studies suggest this mechanism is utilized by a wide variety of receptors. Furthermore, the receptor on which this study focuses, PAR-2, is involved in a plethora of normal physiological and pathological processes; two areas studied in our laboratory are tumor metastasis and maintenance of colonic epithelial integrity. Therefore, understanding the molecular basis of PAR-2 signaling may ultimately lead to new targets for the treatment of metastasis and inflammatory bowel diseases.

Fluorescent Molecular Oscillator for DNA Damage Detection In this proposal, we will design and test a new and useful nanodevice [unreadable] a fluorescent molecular oscillator. We will develop an application for this device as an ultra-fast nanosensor for detection of DNA damage and apoptosis in cell suspensions. The sensor is the first representative of a class of semi-artificial molecular machines, a category of molecular devices which we recently introduced. In our nanodevice, the molecular motor protein continuously separates and religates two different fluorescently labeled DNA parts, which participate in fluorescence resonance energy transfer (FRET). When specific DNA breaks are present in cells, energy transfer between parts of the sensor is interrupted, leading to a fluorescence color change signaling detection of DNA damage and apoptosis. The detection reaction is very fast and takes less than a minute. Specific Aims of the project are: 1. To design and test this new type of molecular device [unreadable] a light-emitting molecular oscillator, which will self-assemble from fluorescently labeled DNA and a molecular motor protein [unreadable] vaccinia topoisomerase I. 2. To develop the first practical biomedical application of the newly created fluorescent molecular oscillator as a FRET-based nanosensor for the ultra-fast detection of specific DNA damage and apoptosis in live and fixed cells. In experiments we will determine sensitivity, specificity of the sensors and the time of reaction sufficient for the accurate detection of DNA breaks. The time of survival of molecular oscillators in reaction conditions will also be measured. Based on our results, we will modify the prototypical design and apply it for ultra-fast detection of apoptotic DNA breaks in suspensions of fixed and live cells. The nanosensor will be useful in theoretical and clinical research in apoptosis and drug development, and in evaluation of the effects of therapy in diseases in which the extent of apoptosis and DNA damage have prognostic value.

A major aspect of the pathogenesis of BPD in newborns is the inflammatory response to injury caused by mechanical ventilation at high FiO2. Ventilated fetal baboons develop the pathophysiological, histologic and biochemical features of the infant with BPD, and provide an opportunity to test hypothesis for the pathogenesis of BPD and to evaluate preventative and therapeutic measures. We study the cell surface heparan sulfate proteoglycans (HSPGs) known as the syndecans. These single membrane spanning protein contain extracellular domains (ectodomains) that bear HS chains near the N- termini, distant from the plasma membrane. Syndecans are on the surface of every adherent cell where they act as co-receptors for a variety of growth factors, cytokines, chemokines and cell adhesion molecules, as well as coordinate, in part, the highly complex reparative response to tissue injury. Specifically, syndecan-1 and -4 are induced in response to tissue injury, agents produced during tissue injury cause syndecan ectodomains to be released intact from cell surfaces, yielding soluble effectors that modify the activities of wide variety of proteins and act as dominant negative inhibitors. Mice containing a targeted deletion of the syndecan-1 gene and mice that transgenically over-express syndecan-1 under the broadly acting CMV promoter show major defects in wound repair: the null mice show slow epithelial response to injury and the over-expressing mice shed excessive amounts of the soluble syndecan-1 ectodomains, causing a major disturbance in repair of injury. We hypothesize that the persistent injury of the lung by mechanical ventilation at high FiO2 leads to excessive shedding of the soluble syndecan ectodomains, that the biological activities of these PGs delay the reparative response and contribute to the changes recognized pathologically and clinically as BPD, and reducing the level of these ectodomains will ameliorate, in part, the progression to BPD. Specifically, we aim to (i) to correlate syndecan expression and shedding with histopathological changes in ventilated pre-term baboon lungs; (ii) determine the biological activities of the soluble ectodomains from ventilated lungs; (iii) establish baboon lung cultures to investigate mechanisms of syndecan induction and shedding and to produce syndecan ectodomains; (iv) assess whether the soluble ectodomains are casually related to development of BPD in the baboon models.

The aerodigestive system is comprised of a complex of integrated anatomic structures that support both ingestive and respiratory physiologies throughout the human lifespan. The developmental origins of this system---in both its form and function, begin in utero where prenatal morphologies and associated processes form the foundations for extrauterine survival at birth. When this prenatal development is disrupted, alterations to normal physiologic functioning may serve as antecedents of later neonatal respiratory distress, dysphagia, or upper gastrointestinal dysfunction. Further, premature birth before upper airway processes are fully established may exacerbate many of these conditions. In turn, this may prompt a cascade of respiratory-related dysfunctions throughout postnatal life. It is therefore important to understand how the respiratory system develops, how mechanisms regulate normal emerging physiologic processes, and the events that predicate fetal and neonatal compromise. To do this work in the living human fetus, we apply a novel noninvasive ultrasound imaging and analysis method to measure the role of altered amniotic fluid exchange in developing prenatal respiratory and ingestive mechanisms. The key components of this protocol are to: 1. Extend the use of prenatal ultrasound sonography to detect and measure the developing upper aerodigestive region. 2. Prospectively characterize differences in ingestive versus respiratory fluid flow-related dynamics across maturation in normal fetuses and those with conditions that may influence airway development. Using spectral Doppler-derived fluid flow analyses, we will map across the spectrum of gestation the fluid dynamics of upper airway development and the factors that influence functional integrity of upper airway amniotic fluid exchange. 3. Identify how deviations in amniotic fluid regulation within the upper aerodigestive system may be associated with fetal and neonatal morbidity and mortality and, the predictive utility of these indices in conditions such as oligohydramnios or polyhydramnios. This study (approved by NIH IRB on August 5, 2003; MOU approved May 24, 2004) is a collaborative effort with National Naval Medical Center Bethesda, Childrens and Womens Health. Data colllection began in June, 2004. The project uses a novel standardized 4-axis sonographic examination to quantify growth and respiratory-related fluid flow mechanics in the upper airway of the living human fetus. The use of this noninvasive ultrasound technique as part of the clinical prenatal examination will not only discriminate function at four upper airway sites (perinasal, oral, pharyngeal, and tracheal), but provide estimates of amniotic fluid flow volumes, inspiratory-expiratory fluid flow velocities and durations, and Doppler waveform patterns associated with fetal breathing and ingestive processes. This provides a method to explore how deviations in amniotic fluid regulation may be associated with morbidity and mortality and, the predictive utility of these indices in understanding conditions such as oligohydramnios or polyhydramnios. The germinal database will include healthy fetuses 16.0 to 39.6 weeks gestational age and test cases with polyhydramnios/oligohydramnios. By elucidating how developing structures integrate with emerging upper respiratory behaviors, this work will document the maturational events underlying normal function at birth that in turn may facilitate future clinical strategies for successful postnatal care.

Application of the human tumor stem cell cloning assay to drug screening to determine the efficacy of antitumor agents.

Request is made for support of Pediatric Nephrology Seminar 32, to be held in Miami Beach, FL, February 25 to March 1, 2005, and the four following seminars. The seminars are organized to include, on an international basis, faculty and other participating scientists and clinicians, ranging from young trainees to established clinicians and investigators. The seminars take place over a 4-day period and deal with a diverse array of current nephrologic and urologic issues, ranging from basic science to clinical practice. Topics are chosen to relate research advances to the understanding of mechanisms of renal disease and clinical diagnosis and management. Presentations are carefully timed to permit maximum opportunity for discussion by participants and faculty. The atmosphere is deliberately maintained at an informal level, encouraging participation by even the most junior attendees. A number of major themes are selected for each seminar, each usually involving three or more presenters, allowing comprehensive coverage of each area. The aims of the seminar are two-fold. First, and foremost, is to stimulate interest in pediatric nephrology as a career choice. This is accomplished by actively encouraging the attendance of young trainees who have evidenced an interest in nephrology. The informal setting facilitates interaction between the young trainees and the established faculty. The faculty provide successful role models and offer direct evidence of satisfaction with careers in research as well as in the clinical practice of nephrology. The second aim is to create opportunities for extensive exchange of ideas and knowledge among the faculty and the participants as well as to foster international collaboration. This is accomplished by including outstanding faculty and limiting the size of the meeting, permitting extensive discussion and exchange of ideas. The faculty and participants have many opportunities to interact at the formal sessions and during scheduled and nonscheduled social encounters. The success of the seminars in achieving these goals is attested to by the fact that this format, creatively adjusted annually to accommodate changing ideas and needs, has proven viable for over 30 years as an effective, participatory teaching and learning tool.

Asthma is a polyetiologic disease with a complex pathogenesis. Atopy predisposes patients to developing asthma, and a Th2-type immune response plays a dominant role in the pathogenesis of asthma. However, natural asthma exacerbations are frequently triggered by upper respiratory infections, and physical, environmental and emotional stress. Some of the foregoing asthma triggers are likely to stimulate a non-Th2 type immune response and thereby, cause asthma exacerbation. The exact mechanism of asthma exacerbation induced with these diverse triggers is unknown. The main objective of this proposal is to identify a common signaling mechanism that links allergens (Th2 trigger), viral (Th1 trigger) and bacterial (innate trigger) infections-induced asthma exacerbations. Our central hypothesis is that the p38-MK2 signaling pathway links the innate and Th1 immunity to allergic inflammation and thereby, plays an important role in asthma exacerbation. In order to test this hypothesis we will study the role of the p38 MAP kinase and its downstream effector MK2 in induced asthma in a mouse model of multiple allergic sensitivity and chronic exposure. The activation and the role of p38 and MK2 will be examined following challenge of sensitized animals with allergens, virus and bacterial peptidoglycans (TLR2 ligand). The importance of p38 will be examined by pharmacological inhibition as well as by overexpression of a dominant negative mutant. We will examine MK2 knockout mice to establish its biological relevance in asthma. Finally, we will examine the activation of p38 and MK2 in biopsy samples from patients with increasing asthma severity and from control subjects. Our preliminary results suggest that both p38 and MK2 play an essential role in airway inflammation, mucus production, airway hyperreactivity and airway remodeling. Therapeutic interventions targeting the p38-MK2 pathway are likely to prevent asthma exacerbations induced with diverse triggers.

This project aims to study the molecular structure of the oligomeric enzyme RNA polymerase from E. coli and the structural basis for some of its interactions with templates, substrates, products and effectors. This is achieved by a combination of high resolution electron microscopy (CTEM and STEM) and analog and digital image processing techniques applied to ordered polymers or two-dimensional crystalline arrays of native and specifically labeled core and holoenzyme. Specific labels include subunit-, template-, substrate-, Product-and effector-specific antibody fragments and heavy metal labeled derivatives of these interacting molecules.

This protocol is designed to provide guidelines for the compassionate use of Daunoxome in the treatment of patients with advanced Kaposi's sarcoma, who are not eligible for inclusion in the Phase III comparative trial. It will provide information concerning the efficacy (as measured by response rate) and safety of Daunoxome.

APPLICANT'S ABSTRACT: Although certain gender differences in drinking patterns and practices are acknowledged to exist, a closer examination of the rate, or pace, of alcohol intake on drinking occasions is needed in order to advance knowledge of possible important gender differences or similarities in alcohol exposure that typically occur during discrete drinking episodes. This approach is motivated by the understanding that the toxic insult suffered from drinking is related to the blood and tissue concentrations of ethanol achieved during drinking episodes. In refining the approach to alcohol exposure, established gender differences in the volume of distribution for ethanol (total body water) and its effect upon the blood alcohol levels (BAC) achieved during drinking also need to be recognized and factored into estimations of exposure. Thus, the widespread belief that women are more susceptible than men to the chronic health consequences of alcohol abuse needs to be interpreted in the light of current information on gender differences in the bioavailability of ingested ethanol. Suspected gender differences in susceptibility to alcohol toxicities may not be as great as previously believed when the biological impact of discrete drinking episodes (for example, estimated peak BAC achieved during drinking), rather than simply the number of drinks consumed, is taken into consideration and used as a measure of exposure. The proposed research will address the above issues by assessing, for the first time, the rate of alcohol consumption during typical drinking occasions (i.e., drinks per hour on drinking days) in a national general population sample of subjects. Furthermore, by combining information on the pace of alcohol intake with estimates of the total body water in subjects, estimates of the peak blood ethanol levels (BAC) typically achieved during drinking episodes in both male and female subjects will be obtained by age decade and level of drinking. We expect that estimated peak BAC achieved during drinking will decline with age in both genders in response to an age-related increase in central nervous system sensitivity to alcohol. Because the pace of drinking may vary with the level of involvement with alcohol, analyses will examine the relationship of dependent variables to the quantity and frequency of drinking and the presence of alcohol problems, and possible interactions with age and gender. This information will address the issue of basic gender similarities or differences in alcohol appetite and will provide an improved measure of ethanol exposure for epidemiological investigations into the health-related effects of alcohol consumption in men and women. These improvements, in turn, ultimately will allow for the more accurate formulation of guidelines and precautions regarding the safe levels of drinking in men and women.

As a participating institution in the Primary Breast Cancer Therapy Group (NSABP), we are contributing data from patients entered into the various group protocols. We have made a major contribution to Protocol, which is a protocol to evaluate the relative efficacy of radical mastectomy alone in patients with clinically negative axillary nodes. A comparison between radical mastectomy and total mastectomy with radiation is also made in those with positive axillary nodes. Patients have also been entered into Protocol 5, which compares L-PAM and Placebo and Protocol 6 which evaluates L-PAM with L-PAM plus 5FU. The present application seeks funding: (A) to permit continued patient accrual in on-going protocols, (B) to continue follow-up of patients entered into protocols which have terminated, and (C) to collect appropriate biological material and information relevant to such protocols. The new protocols are: (1) No. 6: A trial to compare the worth of segmental mastectomy with and without breast radiation with total mastectomy plus axillary dissection. All patients will undergo axillary dissection and those with histologically positive axillary nodes will receive adjuvant chemotherapy. (2) No. 8: A trial to compare the worth of 3 drug chemotherapy (L-PAM, 5-FU and Methotrexate) with 2 drug chemotherapy (L-PAM, 5FU). (3) No. 9: A trial to determine whether the anti-estrogen Tamoxifen when combined with chemotherapy the value of the immunopotentiator C. parvum combined with chemotherapy vs. chemotherapy alone. (5) No. 11: A trial to evaluate various treatment modalities in the management of patients with Clinical Stage III breast cancer. In addition, the NSABP membership has decided, because of its unique membership to implement adjuvant protocols for primary colo-rectal carcinoma. The group has the same capability to accomplish this as it has done for breast cancer. This institution will participate in such protocols. Finally, an aim of this proposal is to enhance the multidisciplinary approach of this institution in clinical trials.

Venous anastomotic intimal hyperplasia (VAIH) is the most prevalent cause of hemodialysis arteriovenous (AV) graft failure. The economic impact of AV access and its related morbidity approaches one billion dollars annually in the US. Previously, this laboratory has demonstrated that hemodynamic forces regulate key in vivo molecular and structural events in artery wall intimal hyperplasia. We will apply and extend this expertise to investigate the independent role(s) of turbulence-induced solid and fluid dynamic forces in inducing VAIH following AV graft implantation. The investigators have established a realistic experimental in vivo model of the human AV circuit and VAIH to test the following hypotheses: 1) VAIH is modulated by turbulence-induced vein wall vibration levels, with elevated vein wall vibration enhancing, and reduced vein wall vibration attenuating, VAIH; 2) This relationship is independent of regional variations in wall shear stress magnitude within venous anastomoses subjected to turbulent flow conditions; 3) Elevated vein wall vibration upregulates the level and activity of the extra-cellular regulatory kinase (ERK1/2) and the stress activated protein kinase (JNK and p38) required for transcriptional activation of the immediate early genes (IEGs) Egr-1, c-jun and c-fos involved in VSMC differentiation, proliferation, apoptosis and VAIH. To verify the proposed hypotheses we will correlate the degree and localization (transmural, circumferential, and axial) of the above-mentioned molecular and cellular events with the corresponding magnitude and spatial distribution of the venous anastomotic biomechanical variables under conditions of elevated and reduced levels of vein wall vibration. It is anticipated that the results of these novel investigations will provide seminal information regarding the pathogenesis and detection of AV grafts at risk for accelerated VAIH, and for the design of interventions to inhibit VAIH and extend patency of hemodialysis AV grafts.

The Epidemiology/Statistics: Research Operations Core is responsible for support and consultation in the following areas: 1) identification and characterization of populations for the proposed behavioral interventions, 2) measurement and data collection, 3) data base management, and 4) quantitative analyses. The core staff will use its collective expertise to anticipate the data collection requirements of the proposed projects to propose methods, evaluate their relative efficiencies, and facilitate implementation of the optimal methods. As a whole, these projects constitute a marked expansion in the number of individuals from whom data will be collected on a periodic basis in comparison to the present CCSP. The experience of the core staff will allow us to schedule, disseminate materials and reinforce recommended behaviors in an efficient and timely manner, incorporating findings of the screens where appropriate. In addition, appropriate verification of behaviors and periodic followups for data collection will require quality control/quality assurance. Needs assessments, which this core staff will perform regularly, will provide support for commitment of resources (e.g., computer systems) to address these components of any and all of the proposed projects. The sharing of the core staff expertise in data management will facilitate selection and implementation of optimal approaches for the following: file creation, modifications (edit/update), determining storage requirements (size, regularity of acquisition, medium) documentation and archiving. Throughout the execution of the proposed program projects, the core staff will provide for the timely acquisition, processing and quality control/quality assurance of all data collected to facilitate the management of each project. To realize the overall efficiencies of collaborative research, it is essential that the core staff has time to review and adapt materials and methods, and the opportunity to create standards and procedures applicable across projects. This pooling of professional talent will result in marked savings. A major commitment on the part of core staff to one or more projects provides the basis for developing and maintaining the lines of communication and authority required to implement the proposed standards and procedures.

PROJECT SUMMARY The Sts phosphatases negatively regulate signaling pathways within cells of the mammalian immune system. Mice lacking Sts expression (Sts-/-) are profoundly resistant to infection by clinically relevant fungal and bacterial pathogens, including Candida albicans, Francisella tularensis, and Staphylococcus aureus. Resistance is associated with rapid pathogen clearance and reduced inflammation. Our preliminary data demonstrates that phagocytes lacking Sts expression have enhanced microbicidal functions. We have shown that the Sts phosphatase domain is structurally and mechanistically distinct from other classes of protein phosphatases. We also demonstrate that the Sts active site sits in a distinct binding pocket, and enzyme activity can be competitively inhibited with drug-like compounds. We hypothesize that drug-mediated inhibition of Sts-1 will recapitulate the Sts-/- phenotype and lead to beneficial clinical outcomes. Based on this hypothesis, the objective of this proposal is to identify and characterize small molecule inhibitors of Sts-1 that can enhance leukocyte anti-microbial responses and demonstrate efficacy in whole animal infection models. To achieve this objective, we will conduct a high-throughput screen (HTS) of the 530K compound Scripps Drug Discovery Library. Active compounds will be extensively characterized and thoroughly validated, using our established assays and in vivo models. We will accomplish our objective by completing the following Specific Aims: Specific Aim 1: Conduct a 530K compound HTS to identify inhibitors of Sts-1 phosphatase activity. Specific Aim 2: Validate and characterize hit compounds. Specific Aim 3: Determine the effects of Sts inhibitory compounds on host responses to microbial infection. Successful completion of the proposed studies will yield a set of validated small molecule inhibitors of Sts phosphatase activity that will serve both as chemical probes of function and as leads for the development of novel therapeutic agents. In the long term, this work is expected to provide a foundation for the development of new clinical protocols that will significantly reduce the morbidity and mortality attributed to systemic pathogen infections, by enhancing host immune responses.

Partial support is requested to host the Third International, Academic Conference on Immunology Related to Otolaryngology to be held under the aegis of the University of California, San Diego, in November of 1990. The specific aims of this symposium are to critically assess current applications of immunology in Otolaryngology research, and to foster the interaction of basic immunologists with individuals who are in the field of Otolaryngology performing applied immunology research. Since the field of immunology is moving ahead at such a rapid pace, the interaction of immunologists with otolaryngologists will enhance collaboration and application of new ideas for advancement of this important field. Recent advances on basic immune mechanisms operating in autoimmunity, mucosal immunity, allergy, virology and tumor biology will be reviewed by immunologists selected by the Program Committee. Specific lectures on immunological aspects in otolaryngology, rhinology, head and neck oncology and the nervous system, will be provided by experts in these fields. Free papers will be solicited and abstracts reviewed by the Program Committee. The design and environment of the conference have been chosen to foster discussion and interaction between the basic scientists and clinician researchers. The proceedings of this symposium will be published and disseminated by Kugler Publishing Company.

Objectives: 1. Research and work of the past three years has resulted in the refinement of a computer-controlled language training system. Through use of that system we have evidence that a young female chimpanzee (Pan; Lana, now 4 yrs old) has mastered a variety of linguistic-type skills--reading, sentence-completion, object naming, color naming, ability to engage in the exchange of novel sentences as used in conversation, ability to use sentences in response to the challenge of new problematic contexts, ability to request the names of things and then to use those names forthwith to request that they be given to her, etc. The mastery of these skills by this chimpanzee subject leads us to conclude that the system, with appropriate modifications, might be applied to the study of language processes of mentally retarded children with language deficits. 2. It is proposed that the present computer be moved to the Georgia Retardation Center for studies that will assess the feasibility of using the language-training system with mentally retarded children and, that contingent upon the success of those studies, that the system be expanded in Year 3. It is further proposed that work be undertaken at the Yerkes Primate Center in Year 1 to extend the capabilities of the present system for research with two groups of chimpanzees to assess the contributions of diverse experiences to the acquisition of language- relevant skills. 3. Studies of language-learning processes in mentally retarded children, the development of and studies with a portable language-training unit (the Conversation Board), continued studies with the present chimpanzee subject (Lana), and a systematic study with two groups of chimpanzees (N equals 8, total) to better understand the contributions of various experiences to language acquisition and to assess the potential of the ape as an animal model for linguistic research.

Quantitative measurement of tumor biomarkers has shown great promise in predicting patient outcome and response to treatment. Recently, we have developed an automated system for assessing biomarker expression on tissue sections (AQUA - Automated Quantitative Analysis). AQUA provides quantitative analysis and sub-cellular localization of biomarkers on immunohistochemically stained tissues. AQUA, however, is limited by 1) the resolution of light microscopy and 2) the non-linear effects of enzymatic amplification used to identify biomarkers. The progression toward bio-specific therapies and associated pharmaco-diagnostics will require linear, quantitative measures of protein expression within each patient's tumor. Individual tumor profiling can also be advanced by developing methods for assessing the functioning of tumor-related pathways, in particular by studying specific protein-protein interactions. To acomplish this, we propose the following specific aims: 1: To develop methods for identifying specific protein-protein interactions in tissue sections. And 2: To develop methods for increasing the dynamic range of assessing biomarker expression. To assess protein-protein interactions - which are far below the resolution of light microscopy - we will develop new techniques that combine heterobifunctional crosslinking reagents, fluorescence energy resonance transfer (FRET), and catalyzed tyramide-amplification, making them suitable for use in tissue sections. To improve the dynamic range of the AQUA technology, we will adapt several recently described methods for building large fluorescent molecules in situ (Christmas trees, rolling-circle and ImmunoAT-tailing). Once these techniques are developed, they will provide new linear, quantitative, and highly specific assessments of biomarkers and pathways in tissues that can ultimately be used to advance personalized pharmaco-diagnostics. [unreadable] [unreadable] [unreadable]

Lower extremity prosthesis fit results from a complex biomechanical interaction at the interface between the residuum and socket. The quality of fit determines subject comfort, acceptance, and potential for ambulation. The long term goal of this research is comprehensive assessment of lower limb prosthesis fit that is highly correlated with subjective metrics and has high predictive value for complications and functional outcome. In situ static 3D determination of prosthesis socket fit by a valid, repeatable, practical, and comprehensive method is sought to aid prosthesis design and evaluation, and to improve outcome. Volumetric imaging based on x-ray spiral CT will be used for static in situ lower limb prosthesis evaluation with and without axial loading. From these image volume data sets, tissue composition volume fractions (fat, muscle, bone, skin, other) will be extracted, mass properties estimated, and shape information mapped onto a 3D display. Shape and fit will be visualized by 3D. The measurement methods will be validated in vitro with phantom test objects and cadaver parts, and in vivo with adult amputees. Tissue composition estimates will be tested on extremity remnants phantoms, and on cadaver limbs. Prediction of the biomechanical characteristics for a given socket and limb remnant will be achieved by mathematical modeling. Using the CT volume data, we will synthesize polynomial version (p-version) finite element models that are static, fully 3--dimensional, incorporate separate tissue compartment (bone, muscle/fascia, fat, skin, and prosthesis), anisotropic, and nonlinear with large deformations. The models will be validated experimentally and used to predict the quality of socket fit. Measurements using x-rat spiral CT imaging and finite element tools will be used in a logistic regression model to determine the biomechanical characteristics associated with good and poor fitting prostheses. The feasibility of diagnostic fit evaluation using x-ray spiral CT and finite element mapping methods will be determined through ROC and repeatability analysis. Image-derived fit measures will be tested for correlation with subjective reports corrected for covariation due to age, gender, race, nutritional status, diabetes, smoking, amputation characteristics (stump length, reason for amputation), and other factors known to influence fit. On completion, this project will provide a valid and repeatable practical comprehensive volumetric image-based method to aid lower limb prosthesis design and evaluation. Prediction of the biomechanical characteristics of a given socket and limb remnant by p-version finite element modeling will be developed and tested.

Protein trafficking and epithelial adaptation to physiological stimuli are well-developed in the kidney collecting duct, where intercalated cells (IC) and principal cells (PC) show chronic and acute responses that include: a) regulation of gene and protein expression; b) alteration of the composition of the plasma membrane by vesicle trafficking. We will use novel transgenic mice that express EGFP or Beta-galactosidase in PC or IC, and new technology including laser capture microdissection and mass spectrometry to dissect epithehal remodeling (in PC and IC) and V-ATPase trafficking (in IC) at three levels. Our aims are: 1) To examine cell-specific gene expression, protein expression and cell "plasticity" in rodent collecting ducts during epithelial remodeling. Remodeling, induced by acetazolamide treatment (carbonic anhydrase inhibition) and acid/base manipulation, could occur by phenotypic switching among epithelial cells and/or by apoptotic cell loss and/or cell division. We will explore these mechanisms using immunocytochemistry, laser capture microdissection, real-time PCR, Western blotting and in situ hybridization to follow changes in the expression and localization of cell-specific proteins and mRNA (V-ATPase subunit, AE1, AE4, NHERF, AQP2, AQP4, V2R) during cell remodeling. 2) To examine vesicular pathways involved in the acute regulation of V-ATPase trafficking in IC. We propose that different pathways of protein targeting exist in A-IC and B-IC, that transcytosis is responsible for the heterogeneous distribution of V-ATPase in B-IC, and that VATPase endocytosis occurs via a novel molecular mechanism. We will: a) identify vesicle trafficking pathways in subtypes of IC using specific cell markers coupled with quantification of FITC-dextran and HRP endocytosis, b) determine the effect of acute acid-base manipulations on exocytosis, endocytosis and transcytosis, c) examine the proteome of purified V-ATPase transporting vesicles from rat kidney using 2D Gel electrophoresis and mass spectrometry; d) examine the function of identified "accessory" proteins on V-ATPase trafficking by transfection of cultured IMCD cells. 3) To elucidate the role of the PDZ protein NHERF in V-ATPase trafficking and function: Based on our finding that the 56 kD B 1 V-ATPase subunit binds to the PDZ protein NHERF, which is concentrated in B-IC, we will: a) characterize the site of interaction of NHERF-GST fusion proteins with the VATPase complex; b) examine the expression, localization and rearrangement of NHERF, ezrin, actin and the VATPase in IC under different acid/base conditions in vivo; c) express mutant constructs of NHERF in IMCD cells in culture by transfection and TAT-mediated protein transfer to determine their effect on polarized trafficking and function of the V-ATPase, using immunocytochemistry, membrane fractionation and self-referencing, proton-selective microelectrodes; d) use isolated cortical and medullary endosomes to determine the effect of PDZ proteins on V-ATPase function (by fluorimetry) and V-ATPase structure (by freeze-fracture electron microscopy). Together, these studies represent a multidisciplinary approach to examine epithelial acute and chronic adaptive responses of epithelia that will be relevant not only to renal physiology, but also to cell physiology in general and pathophysiology as more and more "diseases of protein trafficking" are uncovered.

Objective: To achieve the total synthesis of the aglycone of the antileukemic drug daunomycinone and of the alkaloid dendrobine. Approach: The regiospecific synthesis of daunomycinone is to be carried out using a photochemical o-Fries rearrangement of an o- cyanoester derivative of a bicyclic tetralin derivative. Subsequent ring closure would give 9-deoxydaunomycinone. Introduction of C-9 hydroxyl by direct or indirect routes would yield synthetic daunomycinone. Coupling of either aglycone with daunosamine by the Stanford Research Inst. procedure would give total synthetic 9- deoxydaunomycin and daunomycin, respectively.

The goal of these studies is to enable persons paralyzed by spinal cord injury (SCI) to drive powered wheelchairs and interact with computers by acting through an interface that utilizes and adapts to their residual upper-body motor capabilities. This is called a body-machine interface because it maps the motions of the upper body -detected by wearable sensors- (arms and shoulders) to the space of device control signals in an optimal way. In this way, paralyzed persons who cannot operate a joystick controller because of lack of hand mobility can effectively use their whole upper body as virtual joystick device. An important characteristic of the proposed approach is that it incorporates an interactive learning process, in which the interface adapts to the subject's mobility and the subject learns to act through the interface. This study aims at developing and testing the customization of this interface to a group of SCI participants with tetraplegia, resulting from hig-level cervical injury. The proposed research is organized in three specific aims: (Aim 1) To develop new functional capabilities in persons with spinal cord injury by customizing a body- machine interface to their individual upper body mobility. After fitting the interface to the residal movements of each subject, participants will practice computer games aimed at training two classes of control actions: operating a virtual joystick and operating a virtual keyboard. This study will test the ability of the subjects to perform skilled maneuvers with a simulated wheelchair. (Aim 2.) To test the hypothesis that practicing the upper-body control of personalized interfaces results in significant physical and psychological benefits after spinal-cord injury. Rehabilitation of secondary complications is important in SCI. A study will evaluate and quantify the impact of the practicing functional upper-body motions on the mobility of the shoulder and arms by conventional clinical methods and by measuring the subjects' ability to generate coordinated upper body movements and to apply isometric forces. Other studies under this aim will evaluate the effects of operating the body-machine interface on musculoskeletal pain and on the mood and mental state of the participants. (Aim 3) To train spinal-cord injury survivors to skillfully operate a powered wheelchair using their enhanced upper body motor skills and customized interface parameters. The goal of this study is to transfer the skills learne in the virtual environment to the control of an actual powered wheelchair. After reaching stable performance in the simulated wheelchair, subjects will practice the control of the physical wheelchair via the same body-machine interface within safe a testing environment. If successful, this study will lead to effective operation of powered wheelchairs using a customized interface that adapts to the residual motor capability of its users. Physical and psychological benefits are expected to derive from the sustained and coordinated activity associated with the use of this body-machine interface PUBLIC HEALTH RELEVANCE: People with tetraplegia often retain some level of mobility of the upper body. The proposed study will develop personalized interfaces, which utilize this residual mobility to enable paralyzed persons to control computers, wheelchairs and other assistive devices. If successful the project will result into the establishment of a new family of human-machine interfaces based on wearable sensors that adapt their functions to their users' abilities.

The overarching goals of this research are to screen older persons living in the community to identify those who are mobile but at increased risk of future disability, to evaluate limitations to participating in an exercise program, and to describe the kind of exercise program that would be appropriate. Specific questions to be addressed include: 1. What is the effectiveness of a three-level screening and recruitment program that progresses from a telephone screen, to a home evaluation, to a clinic exam? 1. What proportion of people who say that they can walk a mile and climb stairs can actually walk 400 meters? 2. What proportion of potentially eligible people (meeting screening criteria below) are already walking for exercise or doing other forms of exercise? 3. What are the diseases, physical impairments, or symptoms that will prevent potentially eligible people from participating in an exercise program or some aspect of the program? 4. Among those who can exercise, what specific exercises can they do and at what level of intensity can they begin these exercises? 5. What is the readiness of this group of persons to begin an exercise program and what disease, functional, and psychosocial characteristics are associated with readiness to exercise? We have screened and evaluated study subjects using a 3-stage approach. The subjects for this pilot study are a convenience sample of volunteers who met specific criteria. Subjects were entered into the screening process until 101 had qualified for and completed the full three stages of evaluation. Data from this study are currently being analyzed.

NIAID supports a comprehensive portfolio of contract resources to discover and develop novel therapeutic agents for the prevention and treatment of infections caused by HIV-1, AIDS-associated opportunistic pathogens, and other infectious agents. Animal model resources constitute one element of this drug discovery and development program. NIAID initiates the preclinical evaluations of the efficacy and tolerability of novel HIV-1 therapeutics and selects the agents to be tested. The purpose of this contract is to provide small animal models that can be used to evaluate potential therapeutics for HIV-1 infection.

Our groups have common interests in the development of improved techniques for generating pluripotent stem cells, directing their differentiation into relevant tissues, and in disease modeling in two major systems of central interest to the NHLBI-the cardiovascular system and the blood. While the causative genetic lesion has been identified for many conditions, certain inborn and acquired hematologic disorders continue to cause significant morbidity and mortality. The limitations of animal and in vitro models is particularly relevant to the hematopoietic system, where engineering gene defects into mouse strains has failed to phenocopy cardinal features of diseases like Fanconi anemia and Down Syndrome. Human models would offer a relevant system to study these diseases and to develop therapeutics. We have pioneered methods for somatic cell reprogramming to generate mouse and human induced pluripotent stem cells (IPS) and bring considerable experience to the directed differentiation of embryonic stem (ES)/ IPS cells into hematopoietic lineages. We wish to exploit these new humanized research tools to complement our traditional expertise in zebrafish and murine models to study hematopoietic development and disease pathophysiology. In this proposal we plan to create and study human IPS cells for genetic blood diseases that: disrupt genomic stability (Fanconi's anemia and Dyskeratosis congenita), specify aberrant nucleolar or ribosomal proteins (Shwachman-Bodian-Diamond Syndrome and Diamond-Blackfan Anemia), and represent a constitutional' trisomy with prominent hematologic and cardiac anomalies (Down Syndrome). With these IPS cells, we will explore disease phenotypes, pursue strategies for gene repair, and search for novel therapeutics that might ameliorate these conditions. This proposal is part of a collaborative R03 application with Drs Ken Chien and Kit Parker, cardiovascular researchers at the Massachusetts General Hospital, and Doug Melton, a stem cell researcher at Harvard University, and has three specific aims: Aim #1: Generate human induced pluripotent stem cells from patients with genetic and acquired disorders of the hematopoietic system. Aim #2: Explore the hematopoietic phenotypes of disease-specific IPS cells. Aim #3: Investigate methods for gene repair, and pursue chemical and genetic screening to identify novel small molecules and genetic pathways to ameliorate the disease phenotypes in vitro.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. There is great individual variation in the structure and function of the brain. Discovering the determinants of this variation will not only advance our knowledge of normal brain development, but further our understanding of developmental disorders and diseases that affect the brain. The long-term objective of this application is to characterize the differential roles of genes and environment in shaping brain structure and function, to map and identify the genes involved, and to characterize the impact of brain relevant genetic polymorphisms. We shall collect structural images on a genetically informative sample of 700 Australian twins and their non-twin singleton siblings (aged 20-26 yrs) from whom we have previously obtained measures of cognitive functioning, and for whom we have extensive genotyping already available. Using state-of-the-art brain mapping techniques we will establish the genetic architecture and show the trajectory of genetic effects on brain morphometry (Specific Aim 1). In addition to structural MRI, twins and their siblings will participate in diffusion tensor (DTI) (Specific Aim 2) and functional (fMRI) imaging (Specific Aim 3). Our brain mapping techniques will be extended, for the first time, to model genetic and non-genetic influences on white matter micro-structure, and to reveal the genetic topography. The functional imaging study will apply the widely used 'N-back'working memory task, since this is the process most directly linked to individual differences in cognitive ability. We shall assess the extent of genetic mediation and generate the first detailed heritability maps of neuronal activity during working memory. Multivariate genetic modeling of twin/sibling covariance will be used to estimate the relative contributions of genetic factors to co-variance between regional brain structures, areas of working memory-related brain activation, and cognitive ability (Specific Aim 4). Genome-wide linkage and association scans (funded elsewhere) will be used to identify genetic loci that contribute to heritability of brain morphometry and functioning (Specific Aim 5). We will also screen three brain relevant genetic polymorphisms for affects on selected neural phenotypes using sib-pair allelic association analysis (Specific Aim 6). No other imaging study in twins has been designed specifically to use such a QTL-mapping strategy, and this will be at marked cost savings due to the employment of an existing well-genotyped sample. A final aim is to provide a unique and valuable resource base for future investigation (Specific Aim 7). The results of this study will provide fundamental information on genetic mechanisms influencing variation in brain structure and function. This will provide new insights into the origin of individual differences in cognitive functioning and vulnerability to brain disorders.

DESCRIPTION: Current uveitis therapies are inadequate and have significant side effects. Recent research provides evidence that oxygen free radicals and oxidative damage are involved in experimental uveitis and other ocular inflammations. Experiments completed under the Phase I SBIR Grant indicate that certain NRTs are active in an endotoxin uveitis model. The PI proposes to extend this research to determine if NRTs can control ocular inflammation using two additional models of uveitis. Both are models of autoimmune uveitis, with one affecting primarily the anterior chamber of the eye (bovine melanin-protein) and one affecting the posterior segment of the eye (S-antigen). In human patients, the pathological damage to the retina and choroid in posterior uveitis can result in blindness. Both animal models are clinically relevant models of uveitis and closely mimic the human uveitis condition. Three candidate NRTs will be tested by the topical route in the bovine melanin-protein model of anterior uveitis. These candidate NRTs will also be evaluated by the systemic route in the S-antigen-induced model of posterior uveitis. If NRTs demonstrate efficacy in one or both of these models, we will proceed with the selection of an NRT for clinical development. PROPOSED COMMERCIAL APPLICATION: NOT AVAILABLE

Complicated Grief in Older Adults: Physiological Substrates of Emotion Regulation This application for a Career Development Award in Aging describes an integrated training plan and research project to allow the candidate to become an expert in the physiological underpinnings of emotion regulation in older adults. Conjugal bereavement is highly prevalent in older adults, with more than 900,000 people widowed each year in the U.S. Although most adjust gradually to widow(er)hood, others experience a complicated course. Complicated grief, which persists despite anti-depressant treatment, has been found to be an independent predictor of cognitive decline, poor health, depression and suicidality in older adults, making it a major public health concern. Cognitive factors, such as the inability to shift attention away from intrusive thoughts of the deceased are hypothesized to impair adjusting to a life as a widow(er). Furthermore, physiological mechanisms, such as low-grade inflammatory processes, may contribute to emotional dysregulation found in this population. The proposed research will assess neural activation, immune markers, and neuropsychological tests of attention and emotion regulation in older adults. We will recruit 135 older adults (45 with complicated grief, 45 with uncomplicated grief and 45 non-bereaved, determined by a structured interview with new consensus criteria). The specific aims are: Aim 1) To examine attentional deficits in older adults with complicated grief as measured by the e-Stroop as compared to controls (uncomplicated grief and non-bereaved);Aim 2) To examine the neural substrates of attentional deficits in a subsample of 36 older adults with fMRI in these three groups;Aim 3) To evaluate circulating markers of inflammation (e.g., sTNF-RII, IL-6, slL-6R and IL-1Ra) in older adults with complicated grief as compared to controls (uncomplicated grief and non-bereaved). The long-term goal of the project is to explore whether neural activity mediates the relationship between circulating markers of inflammation and attentional deficits. The Cousins Center for Psychoneuroimmunology at UCLA has both excellent resources (e.g. 3T imaging scanner) and excellent mentors (including Dr. Michael Irwin, MD) necessary for advanced training in bereavement, immunology, and neuroimaging research that will facilitate development of the candidate, Dr. Mary-Frances O'Connor, into an independent investigator and allow her to develop an R01 proposal to evaluate treatment for complicated grief, based on both physiological and psychological mechanisms.

Obesity, diabetes and heart disease are characterized by resistance to insulin as a glucoregulatory hormone. African Americans, particularly African American women suffer disproportionately from these three conditions. We believe that sex differences in sensitivity to insulin?s ability to suppress lipolysis contribute to the accelerated development of obesity, diabetes and heart disease in African American women. The hypothesis of this research is that African American men are resistant to insulin as a glucoregulatory hormone and resistant to insulin as a fat regulatory hormone. In contrast, African American women are resistant to insulin as a glucoregulatory hormone but sensitive to insulin as a fat regulatory hormone. The frequently sampled intravenous glucose tolerance test is used to measure insulin?s action as a glucoregulatory hormone. Unfortunately there is no index of insulin?s action as a fat regulatory hormone. Hence we are working on the development of this index by obtaining free fatty acids during the frequently sampled glucose tolerance test. Our goal is be able to do one test to obtain information about both actions of insulin. However, the development of an index of free fatty acid sensitivity to insulin during the frequently sampled intravenous glucose tolerance test involves sophisticated mathematical modeling and will need to be validated. As we work towards developing this index, we have studied sex differences in body fat content and distribution in African Americans. The distribution of body fat has a major impact on the interaction between insulin and body fat. We determined using serial slice computerized tomographic scans and dual X-ray absorptiometry (DXA) that there are sex differences in central body fat distribution in African Americans. These findings were published in the American Journal of Clinical Nutrition in November 2002. In addition, we have found that beta cell secretion of insulin is impaired in obese glucose intolerant African American women and this work was published in Diabetes Care in 2001. Now we are concentrating on sex differences in the free fatty acid profile during the frequently sampled intravenous glucose tolerance test. It is the intent that this free fatty acid work through the complicated process of modeling will lead to the development of an index of free fatty acid sensitivity to insulin. The development of this index is required so the research hypotheses can be fully tested. Further we are investigating the ability of insulin and lipid related variables to predict heart disease risk in African Americans.

Hematopoietic cell transplantation (HCT) has curative potential for individuals with sickle cell disease. While the results of conventional HCT have been good, this treatment carries risks of significant short- term and longterm toxicities. For this reason, HCT has been reserved for children who have experienced severe symptoms that predict a poor outcome. Of interest, some patients developed stable donor-host hematopoietic chimerism after conventional HCT. Due to a natural enrichment of donor erythrocytes in the blood, those who developed stable chimerism had a significant clinical benefit, even when there was a minority of donor cells. These observations have paralleled efforts to develop less-toxic, non-myeloablative preparative regiments for transplantation, proved first in a canine model of transplantation, and subsequently translated successfully in a clinical trial for older adults with hematological malignancies. Thus, this proposal, based on these supporting pre-clinical and clinical investigations, aims to investigate a modified transplant procedure for sickle cell disease that significantly reduces the toxicity of HCT, yet retains its therapeutic benefit. This is a novel approach, conducted in the outpatient setting, which will rely upon the ability to establish and maintain donorhost chimerism. It will be achieved by combining less toxic, non-myeloablative pre-transplant therapy with modulated post-grafting immuno-suppression aimed at controlling host-versus-graft and graft-versus-host reactions. This investigation will employ an existing network of collaborative sickle cell and transplant centers to identify and enroll eligible patients. The primary endpoint of stable donor cell engraftment will be determined and secondary endpoints to measure the impact on sickle cell-related symptoms and end-organ damage will be followed. If successful, this novel approach will expand the availability of HCT for patients with clinically significant hemoglobinopathies.

The purpose of this study is to assess the effects of pre- and postnatal exposure to the organophosphorus (OP) pesticide chlorpyrifos (CPF) on neurobehavioral functioning in a cohort of inner-city children who have reached 9 years of age. This work builds on, and shares a cohort with, a prospective cohort study of ambient air pollutant effects on child development and respiratory health, being conducted by the Columbia Center for Children's Environmental Health (NIEHS R01 ES08977, P.I. Perera:11/01/03-10/31/08;5 P01 ES09600/EPA RD-83214101, P.I. Perera, Co-I Rauh:11/01/03-10/31/08). Previous findings from the Children's Center study reveal adverse effects of prenatal exposure to CPF (validated with biomarkers in cord blood) on birth weight (Whyatt et al., 2004), cognitive and motor development, and neuro-behavioral problems at 3 years (Rauh et al., 2006). Affected neurobehavioral areas include attention and ADHD-- conditions with serious long-term clinical implications. Based on these findings, and evidence from the experimental literature showing long-term, possibly irreversible effects of CPF exposure on brain development and sensorimotor activity (Dam et al, 2000;Levin et al, 2002;Slotkin et al, 2005), we now propose to evaluate the longer-term neurodevelopmental effects of CPF in 300 9-year-old children. We will use Magnetic Resonance Imaging (MRI) to define the intermediary neurobiological effects of CPF exposure on the structure, metabolism, and anatomical connectivity of the brain in all 300 children. CPF was selected as a model for translational research because it is representative of the class of OP pesticides, well characterized, and well-studied in animals. Furthermore, agricultural and commercial uses continue, despite the 2001 residential ban by EPA. Specifically, we propose to: (1) assess the 9- year effects of prenatal and early childhood exposures to CPF on neurobehavioral (inattention, hyperactivity and depression) and neuropsychological functioning (attentional capacity, memory, impulse control and sensory motor functioning);and (2) assess the 9-year effects of prenatal and early childhood exposures to CPF on measures of brain structure (using anatomical MRI), brain chemistry/cellular metabolism (using magnetic resonance spectroscopy), and anatomical connectivity (using diffusion tensor imaging) in the same children. PUBLIC HEALTH RELEVANCE: This application is for a 5-year study to assess the effects of exposure to the organophosphorus pesticides, chlorpyrifos and diazinon, on neurobehavioral functioning in a cohort of inner-city children (n=400) who have reached 8-9 years of age. This work shares a cohort with a longitudinal study of ambient air pollutant effects on child development, from the prenatal period to 9 years of age, being conducted by the Columbia Center for Children's Environmental Health. The project has access to biomarkers of exposure, includes neuroimaging assessments on a subset of 60 children, and has potential for further informing regulatory safety standards for OP use in the United States.

This project's long-range goal is to define the biological role of fibronectin (Fn), a plasma and tissue matrix protein, in inflammatory processes. This project addresses two subjecta: epithelial wound healing and macrophage activation. Fn mediates cell attachment to collagen and fibrin (Fg), binds and activates macrophages, and is chemotactic for several cell types. Epithelial cells migrate over a substrate matrix of Fn/Fg in healing corneal and skin wounds. We hypothesize that the melecular form of Fn critically influences these diverse biological effects. We are determining whether this provisional matrix promotes epithelial migration by studying the effects of local application of polyclonal and monoclonal Fab antibody fragments to Fn, Fg, and laminin in healing guinea pig corneal wounds in vivo and in vitro (organ culture). We are characterizing the molecular form of Fn and Fg in the matrix by SDS-PAGE autoradiography and by antibody blot techniques and using this information to construct substrates for testing epithelial cell attachment in vitro. We have obtained one monoclonal antibody that distinguishes solid-phase and liquid-phase fibronectin. Our preliminary trials have suggested that exogenous fibronectin can promote corneal epithelial healing for deep, but not superficial, cornea wounds. Monocytes or macrophages recognize, bind, and synthesize Fn, yet the biological control of these processes is unknown. Macrophage interaction with Fn is being characterized in three ways by analyzing the form of Fn recognized by the macrophage by in vivo and in vitro binding studies; the modulation of antigen presentation, collagenase, and PGE2 production and tumor killing by different forms of Fn; and the control of Fn synthesis by lymphokines. These studies employ analytic gel techniques, immunoelectron microscopy, in vivo animal experimentation, and in vitro studies of human cells. We have shown that gamma-interferon increases the number and effectiveness of macrophage fibronectin receptors. We expect to learn which forms of Fn are most active in promotion of epithelial wound healing and macrophage activation, which may lead to more effective control of these vital processes in humans. (MB)

Investigations into regulation of cell growth of several growth factors and by certain oncogenic viruses have recently converged, focusing attention on tyrosine-specific protein kinases. The binding of several polypeptide growth factors, including platelet-derived growth factor (PDGF), epidermal growth factor (EGF), insulin, somatomedin C, and tumor growth factors to their respective cellular receptors, results in very rapid (less than 1 min) activation of a receptor-associated tyrosine kinase. The activated kinases cause rapid tyrosine phosphorylation of the respective growth-factor receptors as well as several cellular proteins. Further implicating tyrosine-specific protein kinases in the control of cell growth has been the finding that oncogenic transformation of cells by certain viruses is dependent on the presence of virally encoded tyrosine kinases. However, progress in these studies has been impeded by the extreme rarity of phosphotyrosine (PTYR)-proteins and the difficulty in distinguishing them from the 1000-fold more prevalent phosphoserine-\and phosphothreonine-proteins. To facilitate these studies, a monoclonal anti-PTYR antibody that has effectively purified PTYR-proteins both from cells stimulated by growth factors and from cells transformed by certain retroviruses was prepared. Making extensive use of this unique antibody, these studies seek to determine the significance of tyrosine phosphorylation of growth-factor receptors (e.g., to determine the stoichiometry of receptor phosphorylation in vivo, and to determine whether tyrosine phosphorylation of receptors modulates receptor function), to elucidate the physiology and metabolism of phosphorylated receptors, and to begin to identify and characterize physiologically relevant PTYR-proteins. Parallel studies will probe the significance of tyrosine phosphorylation of viral transforming proteins and will seek to characterize their physiologically relevant substrates. (J)

: Aim 1 will determine the optimal combination of supplemental AOE required to protect cells from hyperoxia. This will be done using a pulmonary epithelial cell culture system exposed to hyperoxia. Aim 2 is to improve the distribution and uptake of AOE in the lung. Stabilized suspensions will be tested in cell culture and in mice exposed to hyperoxia for their effects on cell viability, virus infectivity, multiple gene delivery and expression, and degree of injury. Aim 3 will use viral constructs containing AOE genes under the control of the human SP-B promoter (to target Type II or Clara cells) or the CMV promoter. Aim 4 is to compare the effects of PFC-protein or adenoviral-associated AOE suspensions on normal or injured, preterm or term lamb lungs, using assays for levels and duration of transgene expression.

abstract

Summary: Chronic infection with hepatitis B virus (HBV) is a major cause of chronic liver disease, cirrhosis and hepatocellular carcinoma (HCC) worldwide. Globally there are an estimated 400 million persons infected with HBV. In the United States, there are 1.25 million affected individuals and the epidemiology of the infection is changing due to immigration of persons from endemic regions. The natural history of chronic hepatitis B (CHB) also appears to be changing with an increasing prevalence of HBeAg negative chronic hepatitis B. Knowledge of the rate of progression between individuals with HBeAg positive and negative CHB is unknown. An equally important and related issue is the clinical assessment of disease severity in patients with CHB. Unfortunately, there are no good laboratory markers of disease severity. Liver biopsy is the accepted gold standard for assessing disease severity and cirrhosis but is costly, invasive, and associated with complications, which often limits patient acceptability as well as being subject to sampling error ranging from 15% to 25%. Non-invasive methods to assess disease severity are highly desirable for physicians caring for patients with CHB. Despite the availability of safe and effective oral nucleoside analogues for treatment of CHB, therapy remains problematic due to the need for prolonged therapy and limited effectiveness of alternate treatment -interferon. Identifying the optimal regimen, defining when to treat, for how long and when to stop therapy are major unresolved issues. In addition, defining the best parameters to monitor patients both on and off therapy are not clear. Hypotheses/problems addressed: 1) Define the host, viral and environmental factors that determine the natural history and outcome of HBV infection. To study this problem, we have analyzed a large database of untreated and treated patients with CHB (n750), focusing on the outcome of infection after hepatitis B e antigen (HBeAg) seroconversion. We analyzed factors that predict progression to HBeAg negative chronic hepatitis B as well as factors that predict HBsAg loss. These analyses identified that 10% or greater of patients do not fit one of the traditional phenotypes used to classify the phase of HBV infection. The natural history of these subjects are being evaluated. The database is being used to develop a non-invasive model to predict fibrosis progression in patients with CHB. We also plan to evaluate the role of transient ultrasound elastography (Fibroscan) to assess fibrosis stage in persons with CHB. These results will be compared to liver biopsy, MRI elastography and plasma will be stored for future proteomic analysis. The goal is to develop a series of blood and imaging tests that will obviate the need for liver biopsy in most patients with CHB. The Liver Diseases Branch is participating in a large multicenter study, the Hepatitis B Research Network, to define the natural history of HBeAg positive and negative chronic hepatitis B. This study has enrolled >1900 patients at 13 North American sites. Primary questions that will be spearheaded by our group will be to identify the predictors of spontaneous HBeAg loss in immunetolerant patients and to evaluate the natural history of patients with elevated HBV DNA levels but normal ALT who are also HBeAg negative-so called indeterminate phenotype. 2) Develop and evaluate novel, safer and more effective therapies for chronic viral hepatitis. Current therapy of CHB remains less than optimal. Relapse is common if treatment is discontinued after one year in the absence of HBsAg loss. Consequently, nucleos(t)ides must often be administered long-term or indefinitely. However, long-term use is associated with the development of antiviral resistance with resulting loss of clinical benefit and increased risk of side effects. Therefore, the focus of current studies is to develop strategies to induce HBsAg loss (functional cure) to permit discontinuation of therapy We are taking multiple approaches to this problem. The first is to compare the combination of tenofovir and emtricitabine compared to tenofovir for patients with CHB. Tenofovir is a nucleotide analogue that is more effective than adefovir at suppressing HBV DNA and has an excellent resistance profile. The first approach is to discontinue therapy in patients receiving long-term nucleos(t)ide analogues. To address this question we have initiated a prospective trial to withdraw patients from medication and observe for benefits (HBsAg loss) or adverse events (ALT flares, HBeAg seroreversion and fulminant hepatitis). To date 15 patients have been withdrawn from therapy. The second approach is to combine peginterferon alfa with tenofovir compared to tenofovir alone. This is being conducted under the auspices of the hepatitis B research network and is a multicenter trial that will enroll 200 subjects. The third approach is to add peginterferon to ongoing long-term nucleos(t)ide analogue therapy for a period of 6 month. This trial recently started enrolling and 9 subjects have been enrolled. We will examine the long-term efficacy and safety of tenofovir and emtricitabine versus tenofovir alone in patients with HBeAg positive and negative CHB with the goals of maintaining long-term viral suppression to preventing the emergence of viral resistance and promote HBsAg loss. To date 33 patients have been enrolled and started on therapy. Long-term results are pending. 3) Elucidate the viral pathogenesis of HBV infection and mechanisms of anti-viral resistance The course of CHB is variable and affected by host, viral and environmental factors. We are investigating the role of pre-core and basic core promoter mutations on the natural fluctuations in HBV DNA levels observed during the natural course of HBeAg negative chronic hepatitis B. We hypothesize that elevation in HBV DNA levels are due to an increase in variant virus over wild type virus. To achieve this we are quantifying the relative amounts of pre-core and basic core promoter mutations relative to wild type virus at different time points when virus levels are fluctuating -both high and low. The level of hepatitis B virus among the infected population is broad and ranges 9 log10 IU/ml. The cause for this is unknown. We are performing whole genome sequencing from patients that span the range of viral loads and patients with atypical HBV phenotype to identify mutations that may affect viral replication. Novel mutations identified in the sequence analysis will be engineered into a laboratory replicative construct (wildtype adwR9), using site directed mutagenesis. Replication competence of these mutant constructs will then be assessed after transient transfection of HuH-7 cells. Comparison of replication levels (measured by HBV DNA and HBsAg quantitation) will be made with wildtype adwR9 to determine whether these mutations upregulate or downregulate HBV replication. Finally, we are exploring potential mechanisms for clearance of HBsAg-the viral protein associated with clearance of HBV-by measuring the kinetics of HBsAg loss using a quantitative HBsAg assay.

The specific aim of this study is to determine whether lifibrol lowers LDL cholesterol levels in patients who are deficient in LDL-receptors. These patients are resistant to drug therapies that induce synthesis of LDL-receptors such as competitive inhibitors of 3-hydroxy-3methylglutaryl coenzyme A (HMGCoA) reductase. Lifibrol has been shown to inhibit cholesterol biosynthesis buy not as a competitive inhibitor of HMGCoA reductase. Although the specific mechanism of action of lifibrol is not known, the drug has been shown to lower cholesterol in a dose-dependent manner. Optimum doses lower LDL by about 40%. The magnitude of cholesterol-lowering is similar to the HMGCoA reductase inhibitors.

It is proposed to test the levels of phospholipase B activity in the small intestines, and the numbers of eosinophils in the bone marrow, of nonsensitized and sensitized rats after challenge with Nippostrongylus brasiliensis. Our tests in mice and rats challenged with Trichinella spiralis showed a close correlation between intestinal inflammation, greatly elevated enzyme levels and increased numbers of eosinophils, all of which were related temporally to worm expulsion. With the view that expulsion of worms from tissues might have a common mechanism, tests in other models (N. brasiliensis-rat, etc.) are proposed.

As the population continues to age, the incidence of Alzheimer?s disease (AD) and AD-related dementias (ADRD) is dramatically increasing, resulting in an urgent need to identify at-risk older adults. Effective early identification will occur during the preclinical stages of disease, before the onset of clinically overt symptoms. Subjective cognitive decline (SCD) can represent a preclinical and early disease state that is easily captured in clinical and research settings. SCD is driven by multiple pathological pathways, including AD, neurodegeneration, and cerebral small vessel disease, all of which underlie clinical dementia. Neuropsychiatric symptoms also contribute to SCD and represent early symptoms and a risk for dementia. Current SCD assessment methods lack the specificity to tease apart the underlying etiology of SCD. Tool development has focused on the content of the questions, rather than identifying questions that relate to specific underlying contributors. Additionally, minimal investigation exists understanding the interplay of these mechanisms on the presence of SCD. The majority of research has thus far focused on SCD in relation to a singular pathological process. These approaches to assess definition and tool development limit the specificity of SCD. This study will leverage legacy data from the Vanderbilt Memory & Aging Project, a longitudinal study with a subset of individuals who are cognitively unimpaired and have minimal SCD. To supplement this cohort with an expanded range of SCD and neuropsychiatric symptoms, this proposal will enroll a prospective longitudinal cohort of cognitively unimpaired older adults with a range of SCD. Participants will undergo detailed assessments of cognition, neuroimaging, and lumbar puncture to capture multiple clinical and pathological markers. Leveraging this rich information, the study will shift how SCD items are selected and using feature selection methods will identify questions that relate to each SCD contributor to create profiles that SCD. Modifiers of these profiles will be examined, including age, sex, and concomitant pathologies. The delivery of these novel SCD profiles will enhance the utility of this cost effective and easily measurable early disease marker that can be easily implemented by clinicians and researchers.

Using somatic cell hybridization techniques, monoclonal antibodies are being prepared against antigens associated with human acute lymphoblastic leukemia. Studies are proposed to examine the pharmacokinetics and toxicity of these reagents after intravenous infusion. Methods are being sought to utilize or to prevent modulation of antigens from the surface of ALL cells. Monoclonal antibodies are being used to eliminate leukemic cells from bone marrow before antigenic modulation occcurs, facilitating autologous marrow transplantation.

Although inflammation is an essential host defense mechanism during lung infection, its timely resolution is critical for the host to prevent lung injuries resulting from uncontrolled inflammatory cells. Recently we discovered that MUC1, one of the membrane-tethered mucins expressed on the apical surface of airway epithelial cells, has an ability to control inflammation at the end of airway Pseudomonas aeruginosa (Pa) infection by suppressing toll-like receptor (TLR)5 signaling. In this renewal application, we propose to elucidate the mechanism by which MUC1 suppresses TLR5 during Pa infection. We hypothesize that MUC1 suppresses TLR5 signaling during Pa infection by a direct interaction with TLR5 which is mediated through activation of EGFR and propose the following aims to test the hypothesis. In Aim 1, we will determine whether anti-inflammatory effect of MUC1 during airway Pa infection is due to its direct interaction with TLR5 by examining the details of molecular interactions between MUC1 and TLR5 in cultured epithelial cells using both genetic and immunological methods. In Aim 2, we will determine whether the interaction of MUC1 with TLR5 is regulated by tyrosine phosphorylation of MUC1 by EGFR in cultured cells using genetic as well as immunological methods and such role of EGFR will be verified using EGFR deficient mice. In the final Aim 3, the complete sequence of the events that take place from Pa infection to the resolution of inflammation by MUC1 will be monitored in real time using both cultured cells and in vivo animals. Successful completion of these experiments will provide the detailed molecular interactions associated with the anti-inflammatory role of MUC1 during airway Pa infection and should provide insights into possible therapeutic strategies to control excessive and prolonged lung inflammation characteristic of chronic inflammatory lung diseases such as COPD and cystic fibrosis.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Cyclic AMP (cAMP) is an extremely important and universal small molecule second messenger involved in the regulation of a myriad of cellular events. A conserved cAMP binding domain is present in a large number of proteins, including protein kinase A (PKA), EPAC, the cyclic-nucleotide gated channels, and cAMP-regulated bacterial transcription factors that mediate the allosteric effects of cAMP on protein functions. As cAMP analogs are increasingly being used as therapeutics, especially for cancer, an understanding of the molecular features of activation may aid in the rational development of specific inhibitors and agonists that selectively target certain effector pathways.

The rates of HIV/AIDS among adolescents are alarming in Sub-Saharan Africa, where sexual transmission is the leading cause of infection with HIV. In Sub-Saharan Africa, the Republic of Botswana has the second highest rate of HIV/AIDS in the world. A limited capacity and infrastructure for rigorous HIV/STD prevention research has hampered efforts to curb the spread of sexually transmitted HIV infection among adolescents in Botswana. Accordingly, the broad long- term objective of the proposed research is to build capacity and infrastructure to develop, implement, and evaluate culturally competent, developmentally appropriate, sustainable interventions suitable for implementation in a variety of settings to dissuade Botswana adolescents from engaging in behaviors that increase their risk for sexually transmitted diseases (STDs), including HIV. This application is a collaborative effort of a multidisciplinary team of researchers at the University of Botswana and the University of Pennsylvania to build such capacity and infrastructure at the University of Botswana. The project will be directed by Professor Bagele Chilsa at the University of Botswana and Professor John Jemmott at the University of Pennsylvania. The capacity building will be organized around three cores. Qualitative and Quantitative Methodology Core, Social and Behavioral Intervention Core, and the Administrative Core. In addition, three research projects that draw upon the cores to address adolescents in different settings are proposed: School-Based HIV/STD Prevention, Church-Based HIV/STD Prevention, and HIV/STD Prevention for Adolescents Living with HIV. The Principal Investigator of each core and research project will be a University of Botswana faculty member and the Co-Principal Investigator will be a University of Pennsylvania faculty member. This partnership is committed to developing a creative comprehensive and interdisciplinary HIV/STD prevention research program on adolescents that is fully integrated within the research and education mission of the University Botswana and dedicated to addressing the urgent need to stem the devastating impact of HIV on one of the highest risk adolescent populations in the world.

Cerebral aneurysms (CAs) are tricky to manage, since their rupture is deadly, rupture rate is low and treatment complication risk is significant. The new guidelines for CA management by the American Heart/Stroke Association suggest the clinicians: consider morphological and hemodynamic characteristics of the aneurysm when discussing the risk of aneurysm rupture. However, morphometrics and hemodynamics require extensive engineering expertise to calculate. Currently there is no tool for clinicians to obtain such metrics. Through seamless collaboration between biomedical engineers from University's Clinical and Translational Research Center and neurosurgeons from the private healthcare provider Gates Vascular Institute in one building, we have developed the prototype of a clinical software platform, AView, for rapid assessment of patient-specific CA hemodynamics and morphometrics at the bedside. We have further deployed this prototype to 8 clinical centers to test its technical feasibility. All participating clinicians enthusiastically embraced this ground-breaking tool. All of them underscored the urgent need of such a tool to be integrated into the day-to-day clinical workflow in CA management. They further indicated that routine clinical use of AView requires to have minimal manual operation and to provide maximal clinical benefits. Therefore, in this grant we would like to take a leap to transform AView from the current prototype into a bedside tool to improve healthcare. In Aim 1, we will automate image segmentation procedure. Specifically, a small number of seed spheres will be positioned on image slides, which will be automatically centered within the vessel as well as automatically resized. Furthermore, in this aim, we will implement a treatment planning module including coil density calculation and stent positioning measurement. Coil planning tool will guide users in choosing type and number of coils based on specific packing density, aneurysm volume and a built-in coil library. Stent planning tool will guide users in stent placement based on aneurysms morphology, vessel geometry and inflow jet. In Aim 2, we will validate the morphometric and hemodynamic calculation of AView using experiments from 5 patient-specific CAs. For morphometric validation, we will compare key morphometrics from AView against the results from standard software. For hemodynamic validation, we will validate flow calculation by in vitro and clinical data from the same 5 CAs. We will compare flow solutions against Particle Image Velocimetry data from CA phantoms, and flow-derived virtual angiography against patient angiography. Successful completion of this project will transform AView into an easy-to-use, high-impact, and well-validated bedside tool. We believe AView will revolutionize the way how neurosurgeons approach CA at the bedside. Our long-term vision of AView is to extend this tool to every participating healthcare provider, in order to facilitate on-site treatment decision- making and planning.

Recent evidence suggests that a platelet-derived growth factor-like molecule (PDGFc) is produced by several normal cell types under circumstances which are associated with cell proliferation in vivo. This project will investigate the nature, regulation, and possible roles of PDGFc. Specifically, the project will address the following questions: (1) Does PDGFc differ from PDGF from platelets? PDGFc will be compared with PDGF by SDS-polyacrylaminde gel electrophoresis and isoelectric focusing followed by detection of receptor-binding activity after gel elution and of antigenic reactivity by immunoblotting with anti-PDGF antisera. (2) What cells produce PDGFc and under what circumstances? Using cultured cells, initial observations of developmentally regulated production of PDGFc production by rat aortic smooth muscle cells (SMC), and of thrombin-stimulated production of PDGFc by vascular endothelial cells, will be further investigated and extended to other species and cell types. Biochemical and histochemical techniques will be used to investigate PDGFc production in vivo. (3) Is production of PDGFc regulated at the level of transcription, translation, post-translational processing, or secretion? This will be investigated in culture using thrombin stimulation of endothelial cells and developmentally regulated differences between pup and adult rat SMC as model systems. mRNA levels will be determined by hybridization with sis cDNA probes; potential protein precursors will be identified by immunoprecipitation of [35]cysteine-labeled intracellular proteins and by immunoblotting with peptide-specific antisera; active PDGFc will be quantitated by radioreceptor (RRA). (4) Is the reduction in the number of PDGF receptors on cells which produce PDGFc a consequence of decreased receptor synthesis or of downregulation by endogenously synthesized PDGFc? If the latter, where does the PDGFc-receptor interaction occur? Techniques will be developed to study solubilized receptors and PDGF-receptor complexes, and to determine the rate of accumulation of complexes, the locus of formation, and the physiological consequences of formation of cell surface vs intracellular complexes. (5) Does PDGFc production play an important role in determining the phenotype of a PDGF-responsive cell, or is it just an epiphenomenon? The importance of the extracellular phase of autocrine exposure to PDGFc will be evaluated by incubating the test cells with antibodies to PDGF or with inhibitors of PDGF binding. The effects of specifically terminating PDGFc synthesis will be evaluated by transfecting PDGFc-producing cells with a construct expressing anti-sense mRNA under the control of an inducible promotor.

Steroid hormones are a large family of molecules that play pivotal roles during childhood development. During puberty in humans, elevated secretion of gonadal steroid hormones produces secondary sex characteristics such as breast development and the appearance of facial hair. Because of such important roles of steroid hormones in maturation processes, disruption of steroid hormone signaling during childhood can cause developmental defects that last into adulthood. Understanding the machinery and regulatory mechanisms of steroid hormone signaling in normal as well as pathological conditions, therefore, contributes greatly to the promotion of healthy childhood development. The ultimate goal of this project is to elucidate as-yet-unknown machinery and regulatory mechanisms of steroid hormone release and trafficking, by using the fruitfly Drosophila as a model organism. In order to accomplish this purpose, the Principal Investigator will test the hypothesis that the insect steroid hormone ecdysone is secreted from the steroidogenic tissue in a vesicle-mediated manner, challenging the conventional idea that all steroid hormones are secreted by free diffusion. During the first mentored phase of the project, the Principal Investigator will work closely with his mentor, Michael O'Connor, at the University of Minnesota to develop some key in vitro methods necessary to elucidate his hypothesis. Those methods include the immunohistochemical detection of ecdysone, in vitro transporter assay and in vitro steroidogenic tissue culture. This initial step of the proposed project will help the Principal Investigator master various biochemistry and cell biology techniques required to conduct the next step of the project. During the mentored phase, the Principal Investigator will also undergo extensive training on teaching and scientific communication, which will be helpful in the next independent phase of his career. In the subsequent independent investigator phase, the Principal Investigator will work on the regulatory mechanisms of the putative vesicle-mediated ecdysone release, by screening G protein-coupled receptors working in the steroidogenic tissue. He will also screen for a putative ecdysone importer required for its uptake by peripheral tissues. These approaches should tell us how well this novel machinery of steroid hormone secretion and trafficking is conserved among different organisms. In the long run, the Principal Investigator's work has the potential to shift the paradigm of steroid hormone action and will impact a vast range of research on developmental and disease processes. PUBLIC HEALTH RELEVANCE: Steroid hormones regulate multiple physiological processes and are involved in many types of developmental diseases and cancers. This project is designed to answer the question of how steroid hormone release and uptake are regulated at the molecular level. The expected outcome of this project will shift the paradigm of steroid hormone biology and thus will facilitate development of new strategies for disease treatment and human health improvement.

Traditionally sensory signaling in the urinary bladder has been attributed to direct activation of bladder afferents. New findings have highlighted urinary bladder urothelial cells as key players in the transduction of sensory events. We have shown that urothelial cells exhibit a number of "neuron-like" properties (including expression of sensor molecules that allow them to respond to chemical/thermal/mechanical stimuli and to release chemical mediators) making it likely that urothelial cells communicate "directly" with bladder nerves or indirectly via urothelial cell-cell interactions. Urothelial cells are constantly exposed to various forms of mechanical stimuli, yet the mechanisms by which these cells respond are not well defined. Mechanical forces are thought to "initiate" a series of signaling events (calcium waves) throughout the urothelium. In turn, the accompanying release of chemical factors induced by mechanical stimuli could activate urothelial cell surface receptors and amplify the signal within and adjacent to the urothelial cell. Although these data suggest that urothelial cells play an important role in cell-cell signaling, this is a relatively unexplored area with little information known about the mechanism for sensory transduction or the mode of communication between cells. Using a multidisciplinary approach involving pharmacology, siRNA, transmitter release, patch clamp and novel imaging techniques, our goals are to evaluate how urothelial cells receive and integrate multiple stimuli. Specific Aim 1 will characterize urothelial cell mechanosensors and their mechano-transduction pathways. Evidence has shown that TRPV1 is essential for mechanically-evoked purinergic signaling by the urothelium, yet the mechanisms by which urothelial cells respond to mechanical forces are not well defined. This aim in part will examine the involvement of TRP channels in urothelial mechanotransduction. Specific Aim 2 will evaluate the mechanism by which various transmitters are released from urothelial cells. The aim will utilize imaging with fluorescent dyes to study how chemical and physical stimuli stimulate movement and release of transmitters from vesicles in urothelial cells. Specific Aim 3 will elucidate the responses evoked by mechano-stimulation in coupled and adjacent cellular targets. This aim will utilize conditions of increasing complexity (signaling within a single cell; urothelial-neuron co-cultures and intact tissue) to examine mechanisms for cell-cell interactions. These imaging methods will enable us to evaluate urothelial-cell signaling between different regions of the bladder as well as within the urothelial layers. Results from these studies will help us to understand how urothelial cells receive and integrate multiple stimuli thus providing an important "link" in the transfer of information from the urinary bladder to the nervous system. Understanding these mechanisms may provide important insight for the identification of novel targets for the future clinical management of bladder dysfunctions. [unreadable] [unreadable] [unreadable]

The aging process in the human species leads to the deterioration of higher CNS functions. Age-related changes experienced by cerebral cortex neurons and their synapses are at the heart of these cognitive deficiencies observed in the old age. There is, however, scanty experimental data defining which neocortical neurons and which synapses are most affected structurally or functionally and in which temporal sequence. This project will test the hypothesis that 1) large pyramidal neurons are more sensitive than non-pyramidal cortical neurons to the aging process and that 2) an imbalance between excitatory and inhibitory influences upon pyramidal neurons might exist in the neocortex of cognitively impaired aged individuals. To test the above hypotheses a multidisciplinary team will apply a combination of behavioral, physiological (single cell electrophysiological, patch clamp) and morphological (light and electron microscopy immunocytochemistry of intracellularly labeled neurons) studies to define which particular cerebral cortex neurons are most affected with aging, what is their synaptic pattern and whether an imbalance between excitatory and inhibitory synaptic discharges occur in memory impaired aged animals when compared with aged non-impaired animals. This research should reveal which neuronal and transmitter-specific synaptic systems are more vulnerable to the aging process, thus providing valuable clues for their protection and/or corrective pharmacological treatments.

Women with polycystic ovary syndrome (PCOS) are characteristically hyperinsulinemia and insulin resistant. The hyperinsulinemia appears to play a pathogenetic role in PCOS both in terms of its effects upon ovarian androgen production an the increased prevalence of non-insulin dependent diabetes mellitus (NIDDM). An increased frequency of NIDDM in insulin resistant subjects is predictable whether insulin resistance induces NIDDM or is a necessary but insufficient predisposing factor. Yet, additional genetic and/or environmental factors are likely to be necessary to account for the increased prevalence of NIDDM in PCOS since only a subset of these insulin resistant women goes on to develop frank NIDDM. Our preliminary studies suggest that 1) beta cell dysfunction is more prevalent among women with PCOS than among similarly insulin resistant controls; 2) parents of women with PCOS have NIDDM or impaired glucose tolerance (IGT) at higher than population level frequencies. Thus, the apparent increased frequency of NIDDM in women with PCOS and their first- degree relatives may be attributable to high frequencies of both insulin resistance and beta cell dysfunction. We hypothesize a genetic basis for the beta cell dysfunction in PCOS. To test this hypothesis, we propose to quantitate insulin sensitivity and beta cell function in a large and readily accessible population of women with PCOS, their first degree relatives, and controls. This will then allow us to perform segregation analyses to characterize the transmission of insulin resistance and beta cell dysfunction in PCOS families and to determine the extent to which beta cell dysfunction contributes tot he risk imparted by insulin resistance to the development of NIDDM in PCOS. The following specific aims are addressed in this revised application; Specific Aim 1. To determine if beta cell secretory dysfunction is more prevalent among insulin resistant women with PCOS compared to similarly insulin resistant controls Specific Aim 2. To determine if first-degree relative sof women with PCOS are at increased risk of a) impaired glucose tolerance of NIDDM; b) insulin resistance; c) beta cell secretory dysfunction Specific Aim 3 To a) characterize the transmission of beta cell function and insulin sensitivity in families of women with PCOS; b) determine whether the transmission of insulin sensitivity is independent of the transmission of beta cell function: c) characterize the extent to which beta cell dysfunction contributes to the risk impaired by insulin resistance tot he development of NIDDM.

The global and local flexibilites of the L7/L12 domain are being investigated to identify the solution characterization of this protein. The results will be evaluated in term of the position and orientation of the two L7/L12 domains in the complete ribosome. In addition, the postulant binding affinity of L10, a second ribosome protein, and elongation factor Tu with L7/L21 will be examined. This data should help further our understanding on how these proteins cooperate within the ribosome during a protein elongation.

ABSTRACT The Colon Cancer Family Registry Cohort (CCFRC) has served as an international resource for studies of the genetics and epidemiology of colorectal cancer since 1997. It comprises approximately 37,000 subjects from 10,800 families recruited between 1998 and 2013 from the USA, Canada, Australia and New Zealand. In an effort to enhance access to CCFRC data to the scientific community, we will submit CCFRC data to NIH?s Database of Genotypes and Phenotypes (dbGaP). Datasets will be linkable via an individual dbGaP_ID number. The funds requested in this administrative supplement will support needed additional database staff required to develop this pipeline, and to submit the individual-level incident cancer and observation time data to dbGaP, thereby allowing us to expand the accessibility of the CCFRC resource to an even greater extent. In this administrative supplement application, we seek funding to support the submission of individual- level data to dbGaP. Funds are requested for CFR site staff to work with the Informatics Center for the CCFRC to perform the following aims: Creating a high-quality pipeline for future submissions of CCFRC data to dbGaP: Writing a series of database programs to create data dictionaries and related dataset documentation for the large number of variables to be submitted from questionnaire data including: conversion of fields to meet dbGaP requirements, including privacy; performing error checks to identify missing and erroneous data; and annotating variables as required by CCFRC IRB/Ethics boards to ensure subject privacy is protected including assigning new dbGaP ids; [Note: This aim would not have been done for the depositing of previously contracted submissions (the baseline data and the most recent follow-up data). It is required here to streamline the process for the incident cancers and observation time (Aim 3 of this application) and for future data submissions to ensure the highest possible quality and annotation thereby maximising the usefulness of the submitted data]. Supplement Aim 2: Registration of incident cancer data and observation time with dbGaP: Register the data submission; obtaining IRB/Ethics approval for the submission and Institutional Certificates; developing CCFRC-site-specific (if necessary) or CCFRC-wide dbGaP application materials for incident cancer data and years of observation; Supplement Aim 3: Submission of incident cancer data and years of observation to dbGaP: Uploading incident cancer data and observation time to dbGaP (note: the submission of baseline questionnaire data and new follow-up questionnaire data is not part of this application); reconciliation of submitted incident cancer data and observation time data.

In the last 20 years, studies have revealed that the right hemisphere (RH) processes language in a qualitatively different not just weaker -- manner than the "dominant" left hemisphere (LH). Most evidence comes from the subtle deficits RH- damaged (RHD) patients have when comprehending discourse, and from visual hemifield methods that show distinct patterns of processing for words directed to the right visual field-LH (rvf-LH) or to the left visual field-RH (Ivf-RH). Few studies have explicitly examined R H language processing with neuroimaging. We propose a 3-year program of 25 experiments to investigate semantic processing in both hemispheres of normal individuals using functional Magnetic Resonance Imaging (fMRI) and visual hemifield methods. Some proposed experiments will directly investigate fMRI and visual hemifield methods. Similar or dissimilar results across methods could support or challenge each method, with similar results providing converging evidence about phenomena related to hemispheric processing of language. The project is guided by a coherent theory of how both hemispheres process language, which specifies optimal conditions for revealing R H language processing. The proposed project tests the following hypotheses: 1) When subjects respond to lvf-RH words, greater fMRI signal will be observed in the R H than when subjects respond to rvf-LH words, 2) When subjects respond to foveally presented words, they will show greater fMRI signal in the RH for tasks that yield a lvf-RH advantage and greater LH fMRI signal for tasks that yield a rvf-LH advantage. 3) Both men and women will show these effects; some left-handers will show opposite effects. Also, subjects will show R H advantages in both methods when they: 4) Decide that target words are related to ambiguous words in sentences that bias against the target meaning; 5) Focus on multiple distant semantic relations rather than a single close relation; 6) Judge the relative typicality rather than category membership of exemplar words; 7) Focus on predictive rather than on coherence inferences or on explicit information. And 8) fMRI analysis and acquisition parameters can bias toward detection of either focused or diffuse fMRI signal; our theory implies that R H areas of fMRI signal will be more diffuse than LH areas. The proposed project will enhance knowledge of how both hemispheres process language, illuminating the critical features that make LH adept at many language processes and the R H adept at a few.

The purpose of this renewal application is to request continued NIH support for the q-bio Conference on Cellular Information Processing (http://q-bio.org), an annual systems biology conference attended by about 200 researchers. The renewed conference grant will support three specific aims, encompassing several new directions for the conference during the next funding period: Specific Aim 1. Advance predictive modeling of cellular regulatory systems. This is the overall goal of the conference. Specific topics of interest to the conference include: (1) Modeling of genetic regulatory and signal-transduction systems; (2) Theory of cellular information processing and general design principles of cellular regulatory systems; (3) Quantitative experimental studies at the systems level that are directly relevant for physics- and chemistry-based modeling and theoretical studies; (4) Emerging areas in systems biology, such as the role of ecology and evolution in determining the quantitative behavior of cellular information processing systems. The conference has become an important forum for work addressing the first three topics and we will continue to track these important areas. The fourth topic represents a new emphasis of the conference that will be pursued during the next funding period. Specific Aim 2. Promote new directions in quantitative biology. In the next funding period, we will actively support the inclusion of more talks outside of topics (1)-(3) above in the conference program. Our goal is to advance systems biology as an approach in all biological fields that can benefit from such an approach. Specific Aim 3. Increase participation of women and minorities in quantitative biology and advance the careers of young investigators. We will continue to aggressively support the participation of women and minorities in the conference. Many researchers in quantitative biology have backgrounds in physics and mathematics, fields in which women and minorities are underrepresented even in comparison to the representation of women and minorities in mainstream biological fields, such as cellular and molecular biology. We will place special emphasis on supporting young investigators financially, in partnerships with regional minority-serving institutions (MSIs), and on maintaining diversity within our program and advisory committees.

It is the purpose of this research project to continue to develop experimental designs and methods of statistical analysis applicable to dental and oral research. The kinds of studies we have considered and will continue to consider range from large scale field surveys to small scale clinical trials. The kinds of data we have considered and will continue to consider range from bona fide quantitative measurements to categorical ratings. We expect to apply in the future the kind of paradigm we have successfully applied in the past. Specifically, challenging statistical analysis problems are identified in an actual dental study, theoretical statistical research is conducted when necessary and the results of that research are applied to the original problem. Experimental designs developed to increase study efficiency in other disciplines will be adapted for use in basic oral biological research as well as in applied clinical and field investigations. The theoretical results would be (and indeed have been) published in a technical statistical journal, and their applications would be published in a dental journal. We plan to devote considerable attention to the analysis of multivariate data, both quantitative and discrete. We will give special emphasis to log linear and logistic regression methods for the latter kind of data.

In order to evaluate right ventricular function in septic shock, radinuclide-determined right ventricular ejection fractions with simultaneous hemodynamics are determined. The initial step in this project has been to evaluate a variety of techniques for determining right ventricular ejection fraction for inter- and intra-observer reproducibility. While first pass techniques have been recommended because there is no overlap activity with the left ventricule, an equilibrium technique using the E1 Scint collection and analysis programs has been shown to give better inter- and intra-observer reproducibility because the tricuspid and pulmonic valve planes are more readily identified. Using this technique to evaluate right ventricular function in patients with septic shock will provide valuable insight into the effects of septic shock on the myocardium.

Program Director/Principal Investigator (Last, First, Middle): Gantz, Bruce J. PROJECT SUMMARY ? PROJECT 3 The most important deficit faced by people with hearing impairment is understanding acoustic information? particularly speech?in noisy real-world environments. The central goal of this project is to understand the cognitive and neural bases of hearing impaired listeners' abilities to detect complex auditory objects in simulations of noisy real-life environments. Work on normal hearing listeners has identified fundamental cognitive and cortical mechanisms for perceiving auditory objects in noisy backgrounds, and there are established cortical mechanisms for perceiving of speech in noise. In both cases mechanisms in auditory cortex are active during the abstraction of complex objects from noisy backgrounds, and provide input into networks that allow further perceptual, attentional and semantic analysis. These brain networks have not yet been characterized in hearing impaired listeners, and it is not clear if auditory object detection contributes to speech perception in a way that cannot be predicted by peripheral hearing alone. Aim 1 applies a new measure of the ability to detect complex (non-speech) auditory objects in noise, and relates this to more standard speech in noise perception in a range of hearing impaired listeners. This is done to determine whether of speech in noise in hearing impaired listeners depends on mechanisms for cross-frequency grouping and examine how that dependence differs in patients with impaired acoustic hearing or a CI. Aim 2 relates brain activation (measured with high density EEG) to performance on these two tasks and measures of peripheral auditory function to identify differences in cortical activation in impaired listeners that are not solely a function of their poor peripheral input. Aim 3 conducts a longitudinal study to examine changes in the cortical systems for detecting generic auditory objects and speech in noise. We examine the same patients before implantation and 1, 3, 6 and 24 months post implantation using a combination of EEG, Positon Emission Tomography, (to achieve spatially precise measure of cortical activation)and behavioral measures. This will determine how the abilities to detect complex objects and speech in background noise?and the neural substrates that support these abilities?changes with experience with a hearing device.

The high 5-year relative survival rates for many cancer types mean that cancer survivors should receive the recommended clinical preventive services, as well as quality health care for other co-existing chronic diseases. It is important, as well as consistent with the Healthy People 2010 objectives, that older Americans, including those of each race and ethnic group, receive these services irregardless of whether they have survived cancer, or not. This study will test if recommended clinical preventive services, including cancer screening, as well as diabetes quality of care guidelines, are met as frequently among elderly Americans who have survived breast, uterine, colon and rectal, bladder, or prostate cancer as among elderly persons without cancer. The services we will study are (a) influenza immunization; (b) the American Diabetes Association's recommended diabetes quality of care measures: the determination of serum hemoglobin Alc (HbA1 c) and low-density lipids (LDL-C), eye examination, and testing for diabetic nephropathy in persons who have diabetes, and (c) screening for breast, colorectal and prostate cancer. We will study elderly Medicare fee-for-service beneficiaries identified in the Surveillance, Epidemiology and End Results (SEER)-Medicare linked database with incident breast, uterine, colorectal, bladder or prostate cancer who survived 5 to 25 years We will determine the use of appropriate clinical preventive and diabetes care services in the four-year period from January 1, 1998 through December 31, 2001. Specifically, we will (1) compare the rates of utilization of the recommended care in the cancer survivors to the rates in a 5 percent sample of elderly Americans living in SEER areas who have not had cancer; and (2) compare the use of services in the majority population (non-Hispanic whites) with the minority population groups: non-Hispanic blacks, Hispanics and Asians. We will do a sub-set analysis of use of these services among persons who are diagnosed with localized cancer compared with those with regional or distant disease. Important covariates in all analyses include the type(s) of initial treatment received (surgery or radiation), patient sociodemographic variables, comorbidities, and the specialties of the physicians providing care.

Recent policy initiatives emphasize that encouraging marriage may help an effective strategy for improving the lives of economically disadvantaged single mothers and their children. Although decades of research establish that, on average, married individuals are healthier, happier, and less likely to live in poverty than their unmarried counterparts, there is a paucity of research examining the consequences of entering marriage for the health and well-being of single mothers, per se. The result is that we simply do not know if marriage is beneficial, neutral, or even harmful to single mothers' mental and physical health or that of their children. Although evidence that marriage improves the economic well-being of single mothers when it endures suggests optimism, we lack research that follows single mothers through a range of union transitions to assess their long term impact on health and well-being across the life course and across generations. Determining whether marriage is associated with better long-term outcomes for single mothers and their children is a crucial first step in assessing the likely success of marriage promotion initiatives. McLanahan summarizes the importance of this issue: "If parents marry, but children are worse off, such initiatives will have failed." Because more than 1/3 of all births in the U.S. occur to single mothers, identifying factors that improve or undermine the health and well-being of this vulnerable population has substantial relevance to public health. The central goal of the proposed project is to determine the consequences of single mother's union transitions (including marriage, cohabitation, and union dissolution) for their own health and well-being in mid-life and for the health, well-being and union formation patterns of their young adult offspring. Data for our study will come from the 1979 National Longitudinal Study of Young, an ongoing panel survey of a nationally representative sample of 12,686 young men and women who were aged 14-22 in 1979 and their offspring. Data currently span a 25-year period and the majority of children are now between the ages of 14 and 29. The aging of the NLSY79 cohort provides an unprecedented opportunity to investigate the intergenerational consequences of union transitions for the health and well-being of single mothers and their offspring. [unreadable] [unreadable] [unreadable] [unreadable]

Project Summary/Abstract Over 3 million people in the United States carry a diagnosis of inflammatory bowel disease (IBD), a chronic and incurable condition of unknown cause that carries with it significant morbidity and healthcare costs. Intestinal dysbiosis is a hallmark of IBD that contributes to its pathogenesis, though specific mechanisms underlying this observation are lacking. As a gastroenterologist and physician scientist, the candidate seeks to elucidate specific molecular mechanisms that mediate host-microbe interactions that drive IBD with the ultimate goal of translating these findings into clinical applications. The proposed research builds on the candidate's published observations that adenosine (Ado) regulates colonization and virulence of the Salmonella enterica serovar Tyhpimurium and will provide the candidate with the additional training necessary to move forward as an independent investigator. Whereas it has been shown that Ado signaling in the GI mucosa promotes resolution of inflammation, the actions of Ado on the intestinal microbiota have not been examined. The proposed research tests the hypothesis that extracellular Ado produced in the gastrointestinal mucosa influences the composition and metabolism of the intestinal microbiota through inhibition of the bacterial stringent response (SR) to inhibit dysbiosis and promote intestinal homeostasis. This hypothesis is explored through three aims: In Aim 1, the mechanism by which Ado inhibits growth of enteric bacteria will be defined. Preliminary data show that Ado inhibits bacterial growth by inducing amino acid auxotrophies through suppression of the SR, a global bacterial adaptation to nutritional stress. The mechanisms underlying this observation will be determined through metabolomic and transcriptomic analyses of bacterial mutants deficient in purine metabolism. Aim 2 will elucidate the mechanisms by which Ado promotes intestinal homeostasis by modulating the intestinal microbiota. A novel mouse model wherein intestinal epithelial cells lack the ability to generate extracellular Ado (conditional knockout of ecto-5'-nucleotidase) will be used to assess how Ado shapes the intestinal microbiota. Murine fecal microbial transfer (FMT)-IBD will be used to examine the role of Ado in promoting intestinal homeostasis by shaping the intestinal microbiota. Finally, Aim 3 presents a translational application of the mechanisms under study by demonstrating that exogenously administered purines can alter intestinal homeostasis in an analogous manner to naturally-occurring purines in the intestine. The proposed work will be performed under the mentorship of Drs. Sean Colgan and Andrs Vzquez-Torres at the University of Colorado, who have extensive expertise in the role of adenosine in gastrointestinal inflammation and the role of nucleotide metabolism in the pathogenesis of enteric infectious diseases, respectively. This mentoring team is ideally suited to the proposed research, which is situated at the intersection of each of their areas of expertise and will provide the candidate with necessary additional training in the areas of murine models of IBD, metabolomic analysis, microbiology, and microbiome analysis.

Our recent studies have concerned the localization and function of calcium ions at the cell surface, topics that lie at the basis of many biological phenomena and that constitute one of the central problems in membrane biology. An understanding of the role of calcium will both require and enhance comprehension of various aspects of cellular calcium movement and regulation, and should have profound medical implications. Specific questions that interest us are the interaction of calcium with components of the cell membrane (lipids, carbohydrates, proteins, ATPases), the mechanism of calcium transport across epithelia, cellular aspects of calcification, and intracellular calcium homeostasis. These are complex and over-lapping problems requiring multi-diciplinary approaches for their solution. Although this is a rather vast topic for a research proposal, we have singled out several specific projects for further study. Our approach takes advantage of recent advances in the cellular localization of calcium ions. It has been found that under appropriate conditions of fixation and processing calcium ions can be retained within tissues and are sufficiently electron-opaque to be visualized in the electron microscope. New techniques of microprobe analysis, including use of frozen unfixed material, when correlated with physiological and morphological studies, should make possible significant advances in diverse aspects of the biology of calcium ions. While we may ultimately wish to obtain the apparatus for biological microprobe analysis, the microprobe aspects of these studies can be done in collaboration with other laboratories.

The Biobehavioral Core has as it overall goal the festering of new clinical research in dentistry utilizing concepts and methods from the disciplines of behavioral medicine, health psychology, and health services research. Research drawing upon these disciplines to address critical problems in delivering and evaluating dental treatment will be accomplished through the availability of a team of behavioral and health services by clinical faculty at the School of Dentistry and their peers and colleagues in other branches of the University and elsewhere. In addition, the Biobehavioral Core will make available a staffed, state-of-the-art behavioral data gathered in dental operatories dedicated to the Core. The combination of facilities and expert biobehavioral research consultants will also allow educational training programs to be provided for potential biobehavioral researchers.

PROJECT SUMMARY/ABSTRACT The goal of this proposal is to understand human vision in the context of natural behavior. This is of fundamental importance because relatively little is known about how vision functions in the natural world, and many important issues arise in this setting that are absent, or difficult to address, in standard paradigms. It is our contention that understanding many aspects of vision, such as selective attention and control of gaze will be impossible without investigation of vision in its natural context. Previous attempts to explain gaze patterns have almost exclusively concerned only static, restricted stimulus conditions, and focused on the properties of the stimulus. Such models cannot extend to natural behavior where the visual input is dynamic, and where the observer's behavioral goals play a dominant role. The pervasive effect of reward in the neural circuitry underlying saccadic eye movements, the development of the mathematics of Reinforcement Learning, and the application of statistical decision theory to understanding sensory-motor behavior all allow a novel framework for understanding the sequential acquisition of visual information in the context of normal behavior. We explore evidence for this framework in the proposal. Specifically, we examine the role of reward, uncertainty, and prior knowledge in the control of gaze in the natural world, with the goal of providing a formal structure for understanding complex behavioral sequences. The research focuses on dynamic environments where a central open question is how the visual system balances the need to attend to existing goals against the need to maintaining sensitivity to new information that may pose opportunities or threats. We investigate the role of reward, uncertainty, and prior knowledge in control of gaze in experiments in an immersive virtual walking environment. We then test the predictions of a model based on reinforcement learning in a simple divided attention task. We then use recently developed methods of Inverse Reinforcement Learning to estimate the intrinsic rewards of different behavioral goals in the walking environment. This will allow us, for the first time, to infer intrinsic human rewards directly, and to predict gaze sequences in novel situations. We also explore the nature and complexity of prior knowledge and the role of prediction in intercepting moving objects in a virtual environment. Although investigation of natural behavior is challenging, the potential benefit is that it provides the empirical basis and theoretical tools for understanding how complex behavioral sequences are generated. The experiments will lay the groundwork for investigation of clinical populations in contexts such as walking, and will also have direct relevance to safety issues in driving and any situation involving multi-tasking.

Parkinson disease (PD) is one of the most common neurodegenerative diseases. The histopathological hallmark of PD is neuronal degeneration in the substantia nigra, with Lewy body inclusions in many of the remaining neurons. Idiopathic PD is traditionally considered a disease of the extrapyramidal motor system, but there is growing realization that cognitive impairments are common in PD and often culminate in dementia, one of its most feared consequences: estimates of the cumulative incidence of dementia range from 38% to 65%, and prevalence as high as 78%. There are two main clinical diagnoses of dementia in PD: PD-dementia (PDD) and dementia with Lewy bodies (DLB). In PDD, motor signs and symptoms are the dominant features and dementia develops only after many years. In DLB, dementia occurs early, within one year of motor signs, and is often the initial and dominant feature. Although these clinical phenotypes are markedly different, the neuropathological findings in PDD and DLB are usually identical. By examining PDD and DLB patients early in their course of dementia, research proposed in this biomarker grant application will address this central question: Are these two forms of dementia variants of the same disease, or do they have different underlying processes? To resolve this question, we will examine three groups of patients - PD, PDD & DLB - with comprehensive neurological and neuropsychological assessments, and will perform innovative PET imaging using the dopamine transporter (DAT) ligand Altropane and the amyloid ligand PiB. The central hypothesis is that the extent and regional pattern of amyloid burden and mesolimbic dopamine impairment will distinguish DLB from PDD, and both from PD. This project draws upon the patient populations and rich research environment of the MGH/MIT Udall PD Center and the MGH Alzheimer's Disease Research Center. Successful completion of the project will establish DAT and PiB PET scans as potential biomarkers for dementia in the spectrum of Lewy body diseases. These advances will be important steps in devising improved treatments for dementia in Lewy body diseases. . Impaired thinking and dementia are disabling features of Parkinson disease that eventually affect 80% of patients. Successful completion of the proposed research will 1) define brain mechanisms that cause dementia, and 2) establish brain imaging methods to aid in the diagnosis of dementia in parkinsonian diseases. These advances will be important steps in devising improved treatments for mental impairments and dementia in Parkinson disease. [unreadable] [unreadable] [unreadable]

The identification of the Ian5 gene as the candidate Iddm2/lyp gene as has opened a new set of questions addressing the particular function of this gene in lymphopenia and its relation to the development of autoimmune diabetes. The knowledge about of Ian5, and of the other members of this gene family is still fragmentary, but seems to involve common themes, such as the ability to regulate apoptosis, to bind GTP, and to by associated with immune cells. It is not known how these emerging activities of Ian 5 are tied to the development of lymphopenia and type 1 diabetes, and whether both lymphopenia and autoimmune diabetes are merely epiphenomena or are indeed causally linked. Evolutionary divergence of the Ian gene family has further led to the presence of a paralog of Ian5, termed Ian4, in both humans and mice, but not in rats. Here, we investigate the specific function of the first identified candidate iddm2 gene, Ian5, in the development of immune diabetes. Specifically, we will utilize transgenic rescue of the lyp-phenotype in BB-DP rats to establish formal proof that the loss of Ian5 function is the sole cause of lymphopenia and autoimmune diabetic disease; Second, we will generate a mouse model of Ian5 deficiency and test the hypothesis that Ian5 prevents lymphopenia and autoimmune disease by inhibiting apoptosis of regulatory T cells induced by the ART2/Rt6-dependent activation of the P2X7 receptor by ribosylated nucleotides. Third, in order to gain first insights into the molecular interactions that couple Ian5 function to the inhibition of apoptosis and autoimmune disease, we will conduct an in vivo structure-function analysis of Ian5. Results from these experiments are expected to delineate the molecular and pathophysiological mechanism that control the survival of regulatory T cells and lead to autoimmune diabetes.

Advance technologies research field, such as genomics and bioinformatics, hold significant promise for developing new diagnostics and therapeutics to treat and prevent infectious and immune-related diseases. Deep sequencing technologies have the potential to significantly improve our understanding of the mechanisms by which these pathogens subvert host immunity, and aid in the development of novel vaccines and therapeutics.

Duchenne/Becker muscular dystrophy (DBMD) represents a spectrum of X-linked disorders that result from mutations in the dystrophin gene. Due to the large size of this gene, there is a high new mutation rate making eradication of DBMD unlikely. Comprehensive, population-based surveillance along with long-term follow-up of individuals with DBMD is needed to characterize the occurrence of this disorder and its complications and to evaluate methods of delivery of health care, specific medical and surgical interventions and psychosocial impact for affected individuals and their families. In 2002, the Iowa Registry for Congenital and Inherited Disorders (IRCID) was one of four charter awardees for the Muscular Dystrophy Surveillance, Tracking and Research Network (MD STARnet). As an MD STARnet site, we successfully integrated clinical, programmatic and referral resources provided by relevant stakeholders in Iowa with the resources of the IRCID to establish a statewide infrastructure for DBMD surveillance and to implement long-term follow-up of individuals with DBMD and their families. We also conducted a needs assessment among Iowa families to better understand the impact DBMD has on individuals and their families and to identify gaps in resources and barriers to accessing resources. In addition, we established and maintained the MD STARnet Data Coordination Center (DCC) for the Centers for Disease Control and Prevention (CDC) and participating sites. For the current agreement, we propose to successfully continue as an MD STARnet site and the MD STARnet DCC to: 1) generate population-based prevalence and incidence rates for DBMD in the United States with particular attention to differences in rates over time and by race/ethnicity; 2) identify the early signs and symptoms of DBMD to facilitate early diagnosis; 3) describe the medical and social services received and quality of life of families of patients with DBMD and whether these vary by race/ethnicity and socioeconomic status; and 4) investigate factors that may impact the severity or course of DBMD including medical management and genetic and environmental factors. Using this population-based approach, we will gain increased understanding of the epidemiology of this disorder and its complications, which will allow for development of improved guidelines for care for individuals with DBMD. [unreadable] [unreadable] [unreadable] [unreadable]

Linkage mapping has proved to be an extremely powerful approach for identification of functional genes in protozoan parasites (Toxoplasma, Plasmodium, Trypanosomes), allowing identification of genes underlying important biomedical traits such as host specificity, virulence and drug resistance. Using classical linkage mapping we have identified a strong quantitative trait locus (QTL) (LOD = 21) for Oxamniquine resistance on chr 6 providing proof-of-principal that linkage mapping is also feasible for Schistosoma mansoni. However, classical linkage mapping is extremely labor intensive, logistically challenging and expensive, because both phenotypes and genotypes must be measured in individual F2 generation parasites. New X-QTL (or linkage group selection) methods, developed by researchers working on rodent malaria and yeast, promise to further expand the power of linkage mapping, because many 1000s of progeny can be effectively analyzed. In X-QTL analysis large pools of drug selected (or unselected) F2 progeny are quantitatively genotyped to identify selected genome regions. As this method does not require phenotyping and can effectively examine 1000s of pooled progeny, we believe it is well suited to S. mansoni. Our central goal is (a) to develop and validate efficient X-QTL methods for linkage analysis of S. mansoni and (b) to use these methods to identify the genes underlying important biomedical traits in this pathogen. We focus on drug resistance traits as these traits have a genetic basis and are biomedically important. Reduced cure rate following treatment with praziquantel (PZQ), the mainstay of Schistosomiasis control, has been reported from multiple foci and has a genetic basis; furthermore resistance to the second line drug oxamniquine (OXA) also occurs in nature. Initially, we will focus on OXA. We will validate X-QTL methods by comparing the QTLs identified by X-QTL with those identified using classical linkage mapping. In addition, we will use X-QTL to fine map the genome region containing OXA resistance. In aim 2 we will apply X-QTL approaches to map genome regions that underlie resistance to PZQ, using next generation sequencing both to genotype parental parasites and to accurately measure genome wide allele frequencies in large pools of treated or untreated F2 progeny. This work will be conducted using reduced representation libraries generated using sequence capture methods. By focusing on just 5Mb of the 363Mb genome we will be able to sequence pools to high read depth, measuring allele frequencies and QTL location with great accuracy and at low cost. Successful identification of genes that underlie drug response will allow development of molecular tools for monitoring resistance spread, provide insights into the mode of drug action, allow development of modified compounds that can kill resistant parasites. Most importantly, this project will establish efficient X-QTL linkage mapping approaches in the S. mansoni toolkit and set the stage for identification of the genetic determinants of a wide range of biomedically important traits in the major helminth pathogen infecting humans.

Infection with a subset of human papillomaviruses (HPV) is the major risk factor for cervical cancer. The HPV E6 and E7 genes are selectively retained and expressed in most malignant tumors, and persistent infection with HPV and immortalization of cervical cells are important events in cervical carcinogenesis. The majority of HPV infections are eliminated by the host immune response. However, women with acquired immune deficiency syndrome (AIDS) develop persistent HPV infections that progress to cervical intraepithelial neoplasia or cancer. AIDS patients also respond poorly to conventional therapy and their disease is likely to recur. Thus, women with AIDS would benefit from chemoprevention or therapy targeted to molecular pathways that are important for cervical carcinogenesis. The epidermal growth factor receptor (EGF-R) is a relevant target. The EGF- R is over expressed in cervical dysplasias and carcinomas, and patients with high EGF-R levels in their tumor have a poor prognosis. Our preliminary results indicate that inhibition of the EGF-R blocks an important step in cervical carcinogenesis;immortalization of cervical cells by HPV-16. Our long term goal is to determine whether the EGF-R is an effective target for chemoprevention or therapy of cervical cancer in AIDS patients. The objectives of this proposal are to (1) confirm that EGF-R inhibition prevents immortalization of cervical cells by HPV-16, and (2) identify the mechanism by which immortalization is inhibited. We will examine these questions using cultures of human epithelial cells derived from the cervical transformation zone, the site where most cervical cancers originate. Experiments will use Erlotinib (Tarceva), a small molecule EGF-R tyrosine kinase inhibitor that has been approved for therapy of human cancer. Studies will determine whether Erlotinib prevents immortalization of normal cervical cells by HPV-16 or inhibits growth of cervical cancer cells. Experiments will also determine whether Erlotinib prevents immortalization by (1) increasing susceptibility of E6/E7-expressing cells to apoptosis, (2) stimulating premature senescence, or (3) decreasing expression of HPV-16 DNA. Our results will clarify how EGF-R inhibition targets a novel pathway that is potentially important for chemoprevention and therapy of cervical cancer. Women with AIDS are particularly susceptible to cervical cancer. They respond poorly to conventional therapy and their cancers are likely to recur. Our results describe a novel target for chemoprevention or chemotherapy of cervical cancer that is relevant to AIDS patients or immune suppressed individuals. This involves prevention of immortalization by papillomaviruses by inhibition of the epidermal growth factor receptor. Since drugs that inhibit this receptor have already been approved to treat lung and colorectal cancer, it is reasonable to test whether they also inhibit cervical carcinogenesis.

Project Summary/Abstract The piperidine heterocycle is a privileged motif that appears in countless bioactive molecules, including natural products and drugs. As the most prevalent nitrogen-containing heterocycle in chemical therapeutics, the piperidine motif is featured in a plethora of blockbuster drugs. Due to the prevalence of piperidines, new and innovative methodologies to access this motif remain highly sought after. Strained alkyne intermediates such as benzyne and piperidyne have proven to be useful intermediates towards this end, however cycloadditions to these scaffolds typically lead to the introduction of sp2 centers. The use of highly reactive cyclic allenes introduces an added layer of complexity by building an sp3 center and vicinal stereocenters. The proposed research aims to develop the first 3,4-azacyclohexylallne Diels-Alder reaction capable of accessing decorated piperidines that possess quaternary centers. This new strategy will then be employed to complete a concise total synthesis of the manzamine alkaloid Acantholactone.

New approaches to enhance drug delivery across the blood-tumor barrier are being investigated. We have demonsterated that the short-acting nitric oxide (NO) donor Proli/NO selectively opens the blood-tumor barrier in rat brain tumors, but not in normal brain, to various-sized radiotracers (up to 70 kD) and to an adenoviral vector after intracarotid infusion and does so without significant systemic effects. Proli-NO has been evaluated for its ability to selectively increase the permeability of the blood-tumor barrier to molecules of a wide range of molecular weights over a wide range of doses and infusion regimens. Further studies will focus on 1) the mechanism of selective action of nitric oxide in tumor endothelial cells, the reason for its selectivity for tumor blood vessels and 2) the determination of the active compound(s) which induce the permeability changes. Our group is also studying the NO effect on other vascular physiology, such as cerebral blood flow. Furthermore, we plan to study the pathophysiology of the capillary permeability differences between tumor vasculature and normal brain vasculature as part of an effort to develop approaches that enhance microvessel permeability to increase delivery and therapeutic efficacy of drug therapy. Treatment studies using NO donors in combination with water-soluble drugs and other therapeutic agents, such as viral vectors, in rodents are planned. Our results clearly indicate that this approach might be an effective new way to opening the BBB in patients with brain tumors to enhance delivery of biologically-active macromolecules, including chemotherapuetic agents, genetic vectors etc., to malignant brain tumors for therapy. A clinical study using PET with a small group of patients with malignant brain tumors should determine whether the tumor-selective blood-brain barrier opening to tracer molecules which has been seen in the rodent model can be reproduced in humans.

Chronic infection with the hepatitis C virus (HCV) is a major cause of cirrhosis, decompensated liver disease and hepatocellular carcinoma (HCC) worldwide. Globally there are an estimated 170 million persons with HCV. In the United States (U.S.), there are 4.1 million persons who are anti-HCV positive, 3.2 million of whom have chronic infection based on the detection of HCV RNA in serum. In 2001, chronic liver disease was the 12th leading cause of death in the U.S. These figures underscore the magnitude and impact that chronic HCV infection has on global and US public health.[unreadable] The natural history of chronic HCV infection is poorly understood. The protracted and silent course of infection, the absence of large cohorts of persons known to be infected, and the wide variability in outcome are major obstacles to natural history studies. Five to twenty-five percent of HCV-infected persons will develop cirrhosis over a 25-30 year period but some patients remain asymptomatic, without significant liver disease for many decades if not for life. Knowledge of the rate of progression among individuals who have not developed cirrhosis is unknown. An equally important and related issue is the clinical assessment of disease severity. Unfortunately, there are no good laboratory markers of disease severity and liver biopsy, the accepted gold standard for assessing disease severity is imperfect. Non-invasive methods to assess disease severity are highly desirable for the clinicians diagnostic toolbox.[unreadable] The optimal treatment for chronic HCV infections results in sustained eradication of the virus in 54% to 56% of persons. It is evident that a large number of persons do not respond to therapy. The current state of therapy is inadequate, expensive, and cannot be applied to a substantial proportion of affected individuals due to problematic side effects. Therapeutic options for non-responder patients and persons who do not qualify for interferon-based regimens are limited. Thus newer, safer and more effective therapies are urgently needed for chronic HCV infection.[unreadable] [unreadable] Hypotheses/problems addressed:[unreadable] [unreadable] 1) Define the host, viral and environmental factors that determine the natural history and outcome of HCV infection.[unreadable] [unreadable] To address this problem, we have created a large database of untreated patients with chronic HCV (n700) and have analyzed this database to identify factors that affect the natural history of chronic HCV infection. Using this database, we have already reported on rates of fibrosis progression in untreated patients with chronic HCV infection and that hepatic steatosis does not appear to be a risk factor for progression of fibrosis in patients with chronic HCV infection. More recently, the database was used to search for candidate genes that were associated with fibrosis progression using a genetic haplotype approach. Mx1-CAGT and PKR-TGATT haplotypes were independently associated with less severe hepatic fibrosis (defined as Ishak fibrosis stage &#8804;3).[unreadable] To further define factors associated with fibrosis progression and clinical outcome of chronic hepatitis C, we have analyzed a large, well characterized cohort of over 1000 patients participating in a randomized, controlled, multi-center trial of long-term peginterferon versus no therapy for patients with advanced HCV infection (HALT-C-see below). This analysis revealed that higher AST/ALT ratio and total serum bilirubin and lower serum albumin and platelet count were associated with a higher likelihood of developing a clinical outcome in patients with Ishak stage &#8805;3 fibrosis over a period of 3.8 years.[unreadable] Finally, we are validating the usefulness of a new technology, ultrasound elastography (Fibroscan), to non-invasively assess hepatic fibrosis. Results of the Fibroscan will be compared to liver biopsy, MRI elastography and plasma will be stored for future proteomic analysis. Our goal is to develop a series of blood and imaging test that will obviate the need for liver biopsy in most patients with chronic HCV infection.[unreadable] [unreadable] 2) Develop and evaluate novel, safer and more effective therapies for chronic viral hepatitis.[unreadable] [unreadable] Though therapy of chronic HCV infection has improved over the last decade, it remains sub-optimal. Recent studies suggest that monitoring viral kinetics can be used to tailor therapy for individual patients. Response to early viral kinetics can be used to shorten the course of therapy and to discontinue therapy early in patients who are unlikely to respond. In study 02-DK-0065, we are examining the effect of two different regimens of pegylated interferon alfa 2a plus ribavirin compared to the standard regimen on viral kinetics in patients with chronic HCV infection. We are studying the effect of ribavirin only as well as twice weekly versus once weekly dosing of peginterferon alfa-2a on viral kinetics. In addition we are studying the viral kinetics in special populations of patients with chronic HCV infection, namely those with renal failure. These studies are in progress.[unreadable] Therapy of non-responders with advanced hepatitis C is problematic. One approach to prevent disease progression in non-responder patients with chronic HCV infection is to administer a therapy with anti-fibrotic effects long-term. Limited clinical and in vitro data suggest that interferon alfa may slow or arrest progression of injury-related fibrosis, even in non-responders who fail to clear virus. The LDB was one of 10 sites participating in a large NIH sponsored multicenter study, the HALT-C Trial: a randomized controlled trial to evaluate the safety and efficacy of long-term peginterferon alfa-2a for treatment of chronic HCV infection in patients who fail to respond to previous interferon therapy. This study revealed that long-term therapy with low dose peginterferon was ineffective at preventing clinical decompensation and fibrosis progression in patients with CHC with advanced liver fibrosis and therefore should not be recommended to patients. [unreadable] Ribavirin, a guanosine nucleoside analogue is critically important for the success of therapy for CHC. Several post-hoc analyses and one pilot study have suggested that higher doses of ribavirin may be associated with higher rates of viral clearance. Accordingly, we have initiated an open label study to evaluate the safety and efficacy of high dose ribavirin in combination with standard dose peginterferon alfa-2a for non-responder and relapser patients with CHC. Enrollment is scheduled to begin shortly.[unreadable] [unreadable] 3. Elucidate the viral pathogenesis of chronic HCV infection and mechanisms of action of anti-viral therapy[unreadable] [unreadable] The conduct of clinical trials over the last 30 years has allowed the LDB to acquire invaluable clinical material (patient serum, liver tissue and lymphophocytes) to which state of the art laboratory techniques can be applied to address issues of the pathogenesis of HCV infection and the mechanisms of action of antiviral therapy. The mechanism of action of ribavirin is unknown. Error catastrophe and lethal mutagenesis due to an increase in the viral mutation rate has been shown to be a possible mechanism of ribavirin in poliovirus, bovine diarrhea virus and GBV-B, viruses very similar to HCV. We have previously shown that ribavirin use is associated with an early, transient increase in the mutation rate of HCV but this effect was not observed at later time points. This suggests that lethal mutagenesis and error catastrophe is unlikely to be the sole mechanism of action of ribavirin during therapy for chronic HCV infection. Our plan is to extend this investigation, examining the effects of higher doses of ribavirin and the effects of the combination of ribavirin and interferon on viral mutation rate.

The long-term goal of the proposed research is to use the fruit fly, Drosophila melanogaster, as an animal model to unravel the mechanisms through which insects respond to sensory cues, ranging from changes in temperature to insect repellents. These questions are of potential relevance to the control of insect pests, since mosquitoes that spread diseases are attracted to humans through thermosensory, visual and chemical cues. Aversive temperatures and chemical repellents deter insects. Therefore, understanding the mechanisms underlying avoidance behavior may provide important insights into insect pest control. A key group of receptor proteins that sense environmental stimuli are Transient Receptor Potential (TRP) cation channels. Among the 13 Drosophila members, TRPA1 is of particular note as it is a detector for a wide array of noxious sensory inputs, including slightly warm or hot temperatures, insect repellents, and excessive light. Here, we propose to dissect the molecular, cellular and behavioral mechanisms through which TRPA1 allows larvae and adult flies to elude aversive stimuli. To accomplish our goals, we propose to employ a multidisciplinary approach, using a combination of molecular genetics, biochemistry, cell biology, electrophysiology and behavioral approaches. Aim 1 will test the hypothesis that bright light, which larvae avoid, activates TRPA1 through a novel mechanism of light detection that is independent of a cell surface protein. In Aim 2 we propose to test the hypothesis that TRPA1 functions as a detector that allow flies to use the daily changes in temperature to set circadian cycles of locomotor activity. The experiments proposed in Aim 3 will test hypotheses concerning the function and mechanism by which TRPA1 is activated via a thermosensory signaling cascade. This cascade represents a new mode of activation of TRP channels in contrast to the well-known direct activation of thermoTRPs by changes in temperature. Aim 4 is an outgrowth of the observation that TRPA1 is required for flies to avoid the insect repellent, citronellal, and this response occurs through both direct and indirect mechanisms in both mosquitoes and fruit flies. In the first part of this aim we will test the hypothesis that an additional TRPA channel functions in the aversive responses to other insect repellents (geraniol and camphor). The second part of aim 4 tests the hypothesis that G-protein coupled receptors detect repellents directly, and initiate signaling cascades that lead to indirect activation of TRP channels. The proposed studies ultimately could lead to the identification of a new generation of safer and more effective repellents to control insect-borne disease by identifying and characterizing molecular targets for such compounds.

The present project is concerned with an investigation of the genetic basis of DNA repair and mutagenesis in the complex eucaryote, Drosophila melanogaster. The experimental approach, patterned after recent procaryotic studies, seeks to identify genes involved with repair and mutagenesis on the basis of mutagen sensitivity. Variations of standard Drosophila methods have led to the isolation of forty-nine X-linked mutants which display an increased sensitivity to killing by the alkylating mutagen, methyl methanesulfonate (MMS). Analysis of twenty-eight of these strains has resulted in the identification of six complementation groups and these complementation groups have been localized to six separable loci. Mutants at four of these loci confer enhanced sensitivity to MMS, X-rays and ultraviolet light while mutants at the other two loci yield increased sensitivity to MMS and X-rays. Female sterility appears to be associated with many of the mutants and preliminary tests indicated associated meiotic difficulties. One of these mutants has been shown to exhibit mitotic chromosome instability and to be defective in postreplication repair. Current experiments are concerned with multiple mutant analyses for determinations of hypothetical repair pathways and large-scale mutagenesis experiments to determine the effect of the various mutations on spontaneous and induced mutagenic events. BIBLIOGRAPHIC REFERENCES: Smith, P. Dennis. 1976. Mutagen sensitivity of Drosophila melanogaster. III. X-linked loci governing sensitivity to methyl methanesulfonate. Molecular and General Genetics 149: 73-85. Baker, Bruce S. inter alia P. Dennis Smith. 1976. Genetic controls of meiotic recombination and somatic DNA metabolism in Drosophila melanogaster. Proc. Natl. Acad. Sci., U.S.A. 73: 4140-4144.

DESCRIPTION: (Adapted from the application) Cerebral amyloid angiopathy (CAA) is a major cause of primary intracerebral hemorrhage in the elderly and a disease of growing importance in the aging population. CAA also represents an intriguing model system for exploring the pathogenesis of Alzheimer's disease (AD). The research proposal at the core of this training program is a multidisciplinary epidemiologic study aimed at understanding the molecular pathogenesis of CAA. Several potential molecular risk factors, including the apolipoprotein E e4 allele, cerebrospinal fluid B-amyloid peptide, and serum cholesterol, will be assessed for their effects on the presence and progression of CAA. A major goal of this research program is to lay the groundwork for future interventional drug trials in CAA. Towards this end, the technique of gradient-echo MRI will be evaluated as a possible clinical marker for extent of disease. In addition, a preliminary pharmacologic study will be performed to determine whether activation of a specific signaling pathway might decrease production of B-amyloid peptide in patients with CAA. These research projects will be performed in collaboration with and under the supervision of Drs. John Growdon and Bradley Hyman, two distinguished investigators who have applied similar batteries of approaches to the understanding of AD. The quality of training will be further enhanced by the candidate's use of the diversity of clinical and scientific expertise available in the Harvard Medical School community. The proposed training program will include advanced epidemiologic and statistical coursework at the Harvard School of Public Health. Building on the candidate's strong background in molecular research and substantial early progress in clinical studies, the proposed training will assure the candidate's development into a fully independent clinical investigator.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The goal of this proposal is to obtain funding to purchase a Leica Digital Inverted Fluorescent compound Microscope or equivalent to use in biomedical research and teaching. Because imaging is central to advances in a wide range of biomedical fields, 4 SWOSU PI's are submitting this application. All PIs have experience with conventional fluorescent microscopy from traveling to other institutions and all PIs plan to carry out protein localization studies or wish to image fluorescent markers in living specimens. Currently, we have no fluorescent compound microscope at SWOSU, so many scientist would be aided if this technology was available on campus. The equipments requested will also dramatically enhance training of student and researchers. These trainees and also the PIs will be able to pursue proiects that are currentlv set aside owing to inconvenience and expense.

The ribosomal DNA (rDNA) from the yeast Saccharomyces cerevisiae is an interesting and versatile system for studying gene structure and expression, chromosome organization and chromosome replication in a simple eucaryote. A basic knowledge of the eucaryotic cellular regulatory mechanisms which govern gene expression and DNA replication is essential in understanding normal cell growth and alterations which occur in pathologic states such as infection, cancer, inherited metabolic errors, etc. Our experiments concern specific aspects of these topics and are divided into three main sections. The first section includes experiments which will examine the chromosomal location and the organization of the reiterated ribosomal RNA (rRNA) genes both genetically and biochemically. The discovery of a yeast strain which contains rDNA with an EcoRI restriction endonuclease pattern different from that usually found enables us to follow the segregation of rDNA in a genetic cross and to examine its linkage to other genetic markers. Biochemical experiments will include analysis of high molecular weight DNA containing rDNA by electron microscopy and restriction endonuclease digestion, as well as the cloning and characterization of DNA adjacent to rDNA. The second section involves an examination of the fine structure of individual rDNA repeating units from different yeasts. By noting the degree and pattern of evolutionary divergence among these rDNAs, we expect to gain information regarding the functional roles of different parts of the rDNA units. The final section of our experimental plan includes experiments to determine the timing of rDNA replication during the yeast cell cycle and to examine the fine structure of replicating chromosomes which contain rDNA.

One of the key challenges in biomedical research and late-stage drug discovery is the lack of appropriate pre-clinical animal models of human disease. The conference Animal Models and Drug Discovery will be jointly presented by Imperial College London, King's College London, and the New York Academy of Sciences (NYAS) at the NYAS Conference Center in New York on September 15-16, 2011, and will critically examine the traditional role of animal models in drug discovery, how these models most effectively contribute to translational medicine, changes needed to increase the predictive power of models for drug efficacy in humans, and ways in which to further refine, reduce, and replace animal models in translational biomedical research. The multidisciplinary scientific organizing committee comprises experienced scientific researchers and clinicians from diverse fields that employ pre-clinical animal models for therapeutic development, including the PI for this grant: Garret A. FitzGerald, MD, Professor of Medicine and Pharmacology;McNeil Professor in Translational Medicine and Therapeutics;Associate Dean for Translational Research;Chair, Department of Pharmacology;Director, Institute for Translational Medicine and Therapeutics, The University of Pennsylvania. The 2-day program, anticipated to attract up to 250 attendees across a wide span of medicine and health, will be especially timely and exciting given the recent explosion of broadly applicable new technologies in bioimaging, biosimulation, and bioinformatics;the generation of genetically modified animals and emerging non-rodent models;and the use of embryonic stem cells, patient-specific induced pluripotent stem cells, and humanized animal models. Thematically, the conference goals align well with NCRR's mission to provide clinical and translational researchers with the training and tools they need to transform basic discoveries into improved human health. This program will also showcase research tools and techniques that serve as the foundation for disease diagnosis, treatment, and prevention, thus also fitting the mission of NIGMS. Lectures, workshops, interactive discussions, networking breaks, and a conference reception and dinner will provide a neutral forum for interaction among multidisciplinary participants who may not otherwise interact at a single scientific meeting and ensure that recent improved pre-clinical animal models with good predictive validity readily become available to the pharmaceutical industry and clinical researchers so as to aid the discovery and development of new disease treatments as quickly as possible. Poster sessions and travel awards will encourage participation of junior/minority investigators and trainees. We expect that the discussions originating from the conference and their dissemination via an open access online multimedia report will ultimately foster advancement in the diagnosis, management, and treatment of a multitude of human diseases. PUBLIC HEALTH RELEVANCE (provided by applicant): This 2-day scientific conference, jointly presented by Imperial College London, King's College London, and the New York Academy of Sciences, will provide a neutral forum to critically examine the traditional role of pre-clinical animal models in drug discovery, how these models most effectively contribute to translational medicine and drug discovery, changes needed to increase the predictive power of various models for drug efficacy in humans, and ways in which to further refine, reduce, and replace animal models in biomedical research. By convening multi-disciplinary clinical and basic science investigators we hope to identify common hurdles and pathways forward to improving model systems for the evaluation of therapeutic efficacy in the areas of metabolic and cardiovascular disease, inflammation, and pain, among others. Sessions on the recent explosion of broadly applicable new technologies in bioimaging, biosimulation, bioinformatics, generating genetically modified animals and phenotype screening along with emerging non- rodent models, the use of embryonic stem cells, patient-specific induced pluripotent stem cells, and humanized animal models will ensure that recent advances in basic science knowledge gained from improved pre- clinical animal models with good predictive validity readily become available to the pharmaceutical industry and clinical researchers so as to aid the discovery and development of new disease treatments as quickly as possible.

The H+, K+-ATPase of the gastric mucosa is the enzyme responsible for secretion of acid into the stomach. It is of considerable medical importance because disturbances in acid output appear to play a role in gastrointestinal diseases such as duodenal ulcer. Medical therapy for these diseases often involve treatments that alter acid secretion either by a direct inhibition of the pump or by influencing the regulation of its activity. Our long term objectives are to define the structural and functional domains of the gastric ATPase and to describe the number, sequence organization and regulation of its genes. During the past year, we isolated and characterized cDNAs containing the entire coding region of the rat stomach H+, K+- ATPase. Using the rat cDNA as a probe, we will isolate the gene(s) encoding the human gastric ATPase. The gene(s) will be characterized by restriction mapping and blot hybridization analysis and then the entire sequence will be determined. In addition to providing basic information about the structure of the gene, this study will provide cloned DNA from the human H+, K+- ATPase locus that could be useful in future studies as molecular probes in the analysis and diagnosis of disease states involving abnormalities in acid secretion. Using Northern blot and S1 analyses, we will examine the tissue distribution of the H+, K+- ATPase in the rat and analyze the levels of its expression during development. In order to study the structural and fucntional domains of the enzyme, we will develop expression systems which will be used to analyze the activity of the enzyme before and after modification of various domains. Full length cDNAs for the H+, K+-ATPase and the Na+, K+-ATPase will be used to construct a series of hybrid coding regions in vectors that allow expression in mammalian cells, in yeast or in an in vitro transcription/translation system. The high degree of sequence similarity between the two proteins and perfect amino acid alignment for almost 950 amino acids suggests that functional fusion proteins can be expressed. This will allow the identification and examination of the domains involved in cation specificity, energy transduction and the binding of drugs such as omeprazole. As information is acquired from these fusion protein experiments, a much finer structure/function analysis, at the level of individual amino acids, will be conducted using site- directed mutagenesis techniques.

It is the central hypothesis of this grant that the protein kinase, glycogen synthase kinase-3b (GSK-3b) is a negative regulator of hypertrophic growth of the heart. Over the past several years we have presented evidence to support that hypothesis. However, virtually all of the work in this area has relied upon over-expression of GSK-3b, and this can cause spurious results. This reliance on over-expression strategies has been necessitated by the embryonic lethality caused by deletion of the GSK-3b gene. Herein, we propose to definitively determine the role of this kinase and its isoform, GSK-3a, in the hypertrophic response using genetic loss of function approaches. This is now possible due to the recent creation by our long-time collaborator, Dr. James Woodgett, of mice in which either GSK-3b or GSK-3a can be conditionally deleted, selectively in the heart. Thus we propose to determine the role of GSK-3b in the hypertrophic response to three clinically relevant models: 1) banding of the aorta, mimicking aortic valve disease, 2) myocardial infarction, and 3) the "two-kidney one clip" model of systemic hypertension. We will also determine what role, if any, GSK-3a plays in hypertrophic responses- an area that is, to our knowledge, totally unexplored. Finally, we will make use of a mouse we have recently created that conditionally expresses an inhibitory mutant of lymphocyte enhancer factor-1 (Lef-1), a transcription factor that is an obligate partner of b-catenin, a key downstream target of GSK-3b that we have recently found regulates the hypertrophic response. We will employ this mouse to attempt to identify novel targets of b-catenin that are expressed following pressure overload and that, therefore, might play a role in the genetic re-programming that is necessary for hypertrophy. We believe the studies outlined herein could identify novel targets, inhibition of which could retard or reverse the process of hypertrophy, a major risk factor for heart failure, stroke, and myocardial infarction. [unreadable]

DNA segments carrying linked genes from the genome of transformable B. subtilis are fractionated by agarose gel electrophoresis following cleavage by restriction endonucleases. The selected segments are then ligated to plasmid vectors and amplified in a recombination deficient strain of B. subtilis. The cloned gene sequence will then be used as donor DNA for transformation. Thus these molecules are homogenous with regard to size, sequence, ends and genetic constitution. Biochemical alteration of these molecules during entry into the cell and their physical incorporation into the resident genome are to be analyzed. Several recombination deficient mutants will be employed to pinpoint the blocked steps in recombination. In addition, the role of defective phage and cryptic genes' expression in B. subtilis will be elucidated. Recently we showed one such gene produces a DNA methylase during transformation stage of the cell.

The broad goal of this program is to develop a more complete understanding of the mechanisms responsible for the vascular changes in hypertension and in the sequelae of hypertension. The studies proposed are based on results of our first research period, on important recent findings in hypertension research throughout the world and on the particular expertise of local investigators in the field. Our strategy is one of monitoring all relevant variables during the development of hypertension in order to define the complete sequence of events leading from the introduction of an intervention to the resultant vascular change that causes the elevation of arterial pressure. The overall problem will be studied at three levels: 1. Vascular smooth muscle contraction, with emphasis on cell membrane permeability, 2. Hemodynamics and local circulation, and 3. Endocrine and nervous control. At all levels special attention will be given to the role played by sodium.

For serious infections caused by Bacillus and Clostridia species the contagion is often the spore not the vegetative bacillus and represents an intervention point for controlling the infectious cycle. Germination is an essential early event for numerous infections including: anthrax (B. anthracis), C. difficile- associated infections (CDI), wound and GI infections, gas gangrene, tetanus, forms of botulism and other serious diseases. Germination can be rapid, occurring soon after spores enter the body, or can linger dormant for extended periods in the body before actual germination and onset of disease. Spores are hardy, not easily cleared from the body by natural means and are not susceptible to antibiotics. Their robust physical properties necessitate prolonged periods of antibiotic treatment with a high risk of developing serious drug intolerances and, in many instances (e.g., CDI and anthrax), residual spores cause re-infections after initial treatments are stopped. We hypothesize that blocking or delaying germination after exposure will be useful to slow the growth cycle and provide another line of defense in the instance of multi-drug resistance. It is also desirable to be able to induce germination, rendering the bacterium vulnerable to traditional antibiotics provided on a shorter treatment schedule. Research progress in anthrax spore germination are now to the level required to directly examine the germination processes and machineries, and develop means for real countermeasures. Using B. anthracis as the model system, this proposal uses validated, cross-species-conserved, genes required for bacterial spore germination to determine membrane topologies, protein interactions, complex formation and molecular mechanisms of the proteins responsible for this process. These studies will provide a solid basis for future translational work towards the goal of controlling germination as a means of medical countermeasures. PUBLIC HEALTH RELEVANCE: Spore-forming bacteria are of great concern in biodefense [e.g., anthrax] and emerging infections [e.g., CDI]. Detailed mechanistic understanding of spore germination will identify molecular targets for future small compound control, allowing either prevention or enhanced initiation of germination that result in bacterial forms far more susceptible to medical countermeasures.

Scientific Abstract Idiopathic inflammatory myositis (IIM) is a heterogeneous group of systemic disorders characterized by inflammation in the muscle and other organs. Unfortunately some patients are refractory to currently available treatment options. This has led to the exploration of novel therapies for IIM. Several lines of evidence support a role for B cells in the pathogenesis of IIM and because high levels of the B cell survival cytokine BAFF (B cell activating factor) are found in the serum and muscle of affected patients, BAFF inhibition is considered a relevant therapeutic approach. The goal of this proposal is to characterize the mechanism of action of belimumab, a potential new therapeutic for myositis, in patients being treated in the setting of a funded multicenter randomized placebo controlled clinical trial. The ancillary studies proposed here are based on the hypotheses that belimumab will target nave B cells and IFN driven autoimmune plasmablast responses and will, either directly or indirectly decrease systemic inflammation and T cell and monocyte activation. Technologies will include detailed phenotyping of immune cells including B cells, T cells and myeloid cells, analysis of immunoglobulin gene repertories, multiplex analyses of cytokines, effects of drug on the interferon signature and transcriptional profiling of selected cell subsets. Each subject will act as their own control and samples will be collected at intervals throughout the trial. Results will also be compared to those of similar assays we are performing in SLE patients to determine whether there are differences in responses to belimumab between the two diseases. These studies should shed new light on the mechanism of action of belimumab in the setting of muscle inflammation and may help us to understand which myositis patients may benefit from this therapy.

PROJECT SUMMARY This renewal application requests funding for the Brown School?s Training Program in Mental Health Services Research at Washington University in St. Louis (Wash U). Our program will prepare 2 pre- and 2 post-doctoral trainees per year to acquire advanced mental health services research skills to address challenges faced by the most vulnerable populations in our nation. Specifically, persons living with mental disorders who seek care at the intersection of multiple services sectors, experience high needs for care, and are the least likely to secure needed services or to obtain high quality care. Our training program includes five knowledge domains: mental health services research, research with vulnerable populations, intervention research, implementation science, and advanced research methods (e.g., systems science, mixed methods). A team of 30 highly talented mentors led by Dr. Leopoldo J. Cabassa, Associate Professor at the Brown School, supports our training program. Our faculty are drawn from 4 Schools and 6 Departments across Wash U. They have a distinguished record of accomplishment in mentoring and active NIH-funded research in the critical knowledge domains of our program. An exceptional transdisciplinary training environment including 17 research centers at the Brown School, the Institute of Public Health, and the Institute for Clinical and Translational Science supports our program. Building on our 25-year history of successfully training mental health services researchers, our program continues to innovate with the following enhancements. We strengthened our methodological training by adding new courses and faculty mentors conducting cutting-edge research in systems science, health disparities research, implementation science, and advanced data analytics. We added a Steering Committee (SC) composed of leaders in health and mental health services research from inside and outside Wash U and new training opportunities for our faculty to enhance their mentoring skills. We enhanced trainees? scientific networking by requiring a learning site visit to existing NIMH-funded mental health services studies across the U.S. We added workshops to improve trainees? science communication skills to better disseminate their work to a broader audience. The overall mission of our program is for our trainees to acquire mental health services research knowledge and skills to meet the most pressing needs in the field of social work and to advance the public health significance and impact of NIMH-services research.

Seeinstructions): Pneumonic plague is highly lethal in humans, and the agent of this disease, Y. pestis, is considered a major threat as a biological weapon. Current strategies to prevent pneumonic plague, such as the use of vaccines or antibiotics, are non-effective or could be bypassed by genetic manipulation of the pathogen. A better understanding of Y. peso's pathogenesis is needed to guide the development of novel countermeasures. Y. pestis is a facultative intracellular pathogen that can survive and replicate in macrophages. Survival of Y. pestis in macrophages plays an important role in pathogenesis, most likely during the early stages of infection. Although much remains to be learned about the mechanism of Y. peso's survival in macrophages, we recently identified several bacterial genes important for intracellular survival, have determined that Y. pestis inhabits a novel phagosomal compartment in macrophages, called the Yers/n/a-containing vacuole (YCV), and shown the ability of Y. pestis to survive in activated macrophages. The YCV acquires markers of late endosomes and autophagosomes, but fails to acidify to the normal levels of a phagolysosome. We hypothesize 1) that Y. pestis actively prevents acidification of the YCV, possibly by direct inactivation of the vacuolar ATPase (vATPase); and 2) that blocking acidification is essential for YCV formation and survival of Y. pestis in macrophages. To further elucidate the mechanism of Y. pestis survival in macrophages, the following aims are proposed. 1) Genetic approaches will be used to identify Y. pestis genes that are required for intracellular proliferation (rip)and for blocking YCV acidification in macrophages. The proteins encoded by rip genes will be studied using molecular, biochemical and immunological techniques to reveal the underlying bacterial mechanism for YCV formation. 2) The cellular basis for YCV formation will be investigated using immunological or chemical probes on infected macrophages or purified YCVs, to better characterize the environment of YCV, and to determine how the activity of the vATPase is modulated. The contribution of host proteins to YCV formation will be explored by the use of gene deficient macrophages. 3) mice will be infected by the pulmonary route with rip mutants generated in Aim 1. Mouse survival and bacterial growth in organs will be measured to determine how intracellular survival contributes to virulence. Conditional expression of rip genes will be used to test the hypothesis that survival of Y. pestis in macrophages is critical for virulence at early stages of pneumonic plague. The studies proposed here will foster the development of new strategies to prevent or treat pneumonic plague in humans. RELEVANCE (See instructions): Plague is a highly virulent disease in humans and is considered a major threat as a biological weapon. Current strategies to prevent pneumonic plague, such as the use of vaccines or antibiotics, are non-effective or could be bypassed by intentional genetic manipulation of the pathogen. The studies proposed here will foster the development of new strategies to prevent or treat pneumonic plague in the human population.

Over 200 human tumors of various histopathologic types and anatomic sources have been evaluated using surface marker assays designed to detect and quantitate lymphoreticular cells, particularly macrophages, within the tumor mass. Few of the tumors contained significant numbers of lymphocytes or granulocytes, but a high proportion of those same tumors contained significant numbers of macrophages (0-80% of total tumor associated cells). That the numbers obtained on single cell suspensions represented an accurate picture of macrophages in intact tumors was demonstrated by immunohistologic quantitation of infiltration of tumor by IgGFc receptor positive cells. Those studies also established that the macrophages had infiltrated the entire tumor in most cases. Studies on the tumor associated cellular response were performed on primary and passaged 3-methylcholanthrene induced murine fibrosarcomas. Those studies were designed to assess the contribution of T lymphocytes, granulocytes and macrophages to the total tumor from the initial appearance throughout tumor progression. The results to date demonstrate that a) T lymphocytes completely infiltrate the tumor in very high numbers when the tumors are small but their relative contribution decreases dramatically with tumor progression, b) all tumors are completely infiltrated by large numbers of macrophages, and c) macrophage function as measured by phagocytic capacity decreases in parallel with decreases in T cells. At present, it remains unclear whether the cellular infiltrate represents a specific immune response to tumor antigens or a non-specific inflammatory response.

We have generated continuously prpagatable T cell clones to study the biochemical basis of specific T cell functions. All Ly2+ clones mediate suppressor activity and all Lyl+2- clones mediate helper activity; each type (helper or suppressor) secretes a characteristic pattern of polypeptides. We have purified one antigen-specific suppressor protein (70,000 daltons) from a glycophorin-specific Ly2+ T-cell clone. We have also purified a 45,000 dalton protein from h supernatant of Lyl clones and this 45K protein activated B-cells to secrete immunoglobulins. The aims of this project are: (1) purification of large amounts of 70K proteins from h supernatant of SRBC-specific and TNP-specific Ly2+ clones in order to study their structural and functional properties, with particular emphasis on the structure of constant (45K) and variable (24K) domains of the 70K protein (peptide mappig and sequencing); (2) modulation of the synthesis of proteins by Lyl TH clones exposed to the 70K suppressive protein; (3) generation of serological reagents that bind to the 70K Ts molecule; (4) generation of cDNA probes from mRNA coding for 70K proteins after translation of mRNA from Ly23 cloned cells, to study the genetic basis of T cell recognition of the antigen.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. This highly integrated, multidisciplinary application is focused on developing Japanese Macaque Encephalomyelitis (JME), a disease that occurs spontaneously in Japanese macaques and which mimics multiple sclerosis in humans, as a pathophysiogical and genetic model of MS whose etiology and progression more closely resemble MS in humans than other EAE and viral models in non-human primates and rodents. We aim to use this model to better understand how gamma-herpesviruses trigger MS;whether polymorphisms in gene loci that have been linked to MS in humans also predispose JMs to JME;to evaluate the lesions in both symptomatic and subclincal animals to determine if they model numerous aspects of human MS lesions;and to test if gamma-herpesvirus infection directly influences astrogliosis and the accumulation of factors, such as hyaluronan, that can inhibit remyelination in demyelinated lesions. Our long-term goal is to develop JME as a model for testing immunological and neurobiological process underyling MS, and to use this model in pre-clinical screens of novel agents with the potential to inhibit MS attacks and to promote remyelination and regeneration.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Mesenchymal stem cells or marrow stromal cells (MSCs) are a subset of adult stem cells from bone marrow. These cells are of medical and therapeutic interest because they have been shown to differentiate into osteoblasts, adipocytes, chondrocytes, myocytes, astrocytes, oligodendrocytes and neurons. Due to their inherent plasticity, these cells have the potential to be useful for the treatment of a large number of genetic diseases. Dr. Bunnell has successfully defined the requirements for the expansion and characterization of rhesus macaque MSCs. The Stem Cell Production Core Facility (SCPC) focuses on generation, maintenance and distribution of nonhuman primate MSCs. We routinely prepare MSCs from rhesus macaque bone marrow and adipose tissue samples. We presently have a bank pf MSC cell lines generated from over 100 rhesus macaques of varying age prepared and ready for distribution. The SCPC presently has a large impact not only on the Gene Therapy Program, but on other divisions such as Comparative Pathology within the TNPRC, but also Departments and Centers within the Tulane Health Sciences Center and the Pennington Biomedical Research Center and other research labs nationally. Produce rhesus macaque stem cell preparations for collaborative research projects. For this we plan to use the techniques developed over the last funding period to isolate stem cells (MSCs, NSPCs and ESCs) to expand the bank of various stem cell types isolated from animals of various ages (fetal, newborn and adults) and sexes of animals. The cells will be used to generate large cell banks for use internally or externally by collaborating scientists. We have generated an extensive bank of stem cells for use by the scientific community for various ages of animals. Continue the characterization of nonhuman primate stem cells. Our goal in these studies will be to delineate the cellular and molecular events that take place during maturation and/or differentiation of the various stem cell populations. Scientists in the division will continue to compare the biologic properties of the various stem cells in vitro. Assays mediating in vitro differentiation specific to rhesus stem cells that will maximize the differentiation and proliferation of MSCs and their progeny will be generated. One focus of the in vitro characterization will be the differentiation along neural lineages for all of the stem cells described. In order to accomplish the treatment of Krabbe's disease, stem cells will need to undergo differentiation into oligodendrocytes. A significant effort will be made towards the development and application of differentiation protocols to accomplish this goal. Over the past year, we have collaborated with investigators form LSU on the characterization of the frequency and biologic properties of MSCs isolated form rhesus macaques exposed to chronic alcohol and infected with SIV to assess the effects on the composition bone marrow cavity and stem cells.