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The purpose of these studies is to define the neurohormonal control of esophageal motility in order to provide an understanding of the pathogenesis of esophageal motor disorders. Opossum is used as an experimental model because of the similarity of opossum esophagus to that of man. We have shown that lower esophageal sphincter closure at rest is due to tonic intrinsic myogenic activity of the sphincter muscle. The main role of the vagus nerve is to cause sphincter inhibition. We are now examining the nature of the inhibitory transmitter. We have shown that the inhibitory transmitter is neither acetylcholine, nor a catecholamine. Dopamine, histamine and seratonin are excluded as possibilities. Now we plan to examine ATP and related purines, prostaglandins, peptides (VIP, neurotensin) and aminoacids as possible inhibitory transmitters of vagally mediated inhibition of the lower esophageal sphincter.

Despite recent advances, neurological diseases continue to be a major public health problem and are the source of significant morbidity and mortality not only in the United States, but world-wide. With the explosion of new knowledge in the neurosciences, the possibility of dramatic reductions in the overall neurological disease burden exists, but translation of new laboratory findings into new human treatments has lagged partially due to inefficiencies in the organization, funding, and execution of the necessary human clinical trials to prove the efficacy of new therapeutic approaches. This renewal for the Network for Excellence in Neuroscience Clinical Trials (NeuroNEXT) is proposed to help expedite the process of the conduct of early phase multi-center clinical trials to establish safety and dose information or to validate biomarkers and clinical outcomes in preparation for middle and late phase trials of efficacy. This application proposes the University of Virginia Health System be renewed as a Clinical Site for the NeuroNEXT network. The proposal describes the effective and efficient past performance of the University of Virginia as a NeuroNEXT site, the extraordinary experience and expertise of a multi-disciplinary cadre of clinical neurological investigators available to conduct NeuroNEXT trials, an already existing clinical outreach program that will afford access to rural minority and medically underserved patient populations, an outreach program with statewide neurology practices that will maximize trial recruitment throughout the catchment of the University of Virginia, the research infrastructure and leadership plan for successfully executing NeuroNEXT clinical trials at the University of Virginia, and our robust history and further plans to mentor future clinical researchers.

The long-term aim of this project is to determine the relationship between temporal processing deficits apparent in the brainstem response to complex acoustic cues and speech perception difficulties in noisy backgrounds. The specific objective of the proposed research is to characterize the brainstem encoding of speech and other complex stimuli in young and older adults. The proposed experiments are designed to compare younger and older listeners with near-normal audiometric hearing in order to understand how reduced fidelity of temporal processing affects encoding of the temporal components of speech and, consequently, speech perception. Aim 1 will employ auditory brainstem response measures elicited by speech as an index of periodicity coding. This study will test the primary hypothesis that the degree of periodicity coding measured at the level of the brainstem is dependent on stimulus duration. Aim 2 will model temporal deficits exhibited in the aging auditory system by estimating a measure of 'internal noise' for the older adults. This study will rely on an evaluation of the brainstem response elicited by speech stimuli that has been corrupted using a temporal jitter model. Aim 3 will relate the electrophysiological measures to psychophysical and speech perception measures in young and older adults. Both the psychophysical task (detection of dichotic low-rate frequency modulation) and the speech task (modulation masking release) are designed to capture aspects of temporal processing. The training program involves strong mentorship in research design, especially in the domain of signal processing (stimulus generation, response analysis, etc.) and data analysis essential to the proposed research. Results will contribute to a more refined understanding of sub-cortical processing of speech sounds and the effects of temporal processing deficits on speech processing. The translational emphasis of this project is to optimize the use of complex-ABR measures for the assessment of clinical populations who experience difficulty perceiving speech despite audiometrically normal hearing.

Abstract The objective of this proposal is to illuminate epigenetic transcriptional regulation during mouse embryogenesis and stem cells through investigations of the Polycomb group. The Polycomb group comprises a prominent set of histone modifiers that are essential for the execution of diverse developmental processes, including X- chromosome inactivation, self-renewal and differentiation of embryonic stem cells, cell and tissue specification, and body patterning in mammals. The Polycomb repressive complex 2 (PRC2) catalyzes histone H3K27me3 through the methyltransferases EZH2 and its paralog EZH1. H3K27me3 functions as a key epigenetic mark in development and is dysregulated in human diseases. Through much work in Drosophila, PRC2-catalyzed H3K27me3 has been shown to epigenetically maintain transcriptional silencing. Through preliminary investigations in early mouse embryos, we propose that PRC2 may also initiate transcriptional silencing. In Aim 1, we will therefore test the role of PRC2 in initiating epigenetic transcriptional silencing. We have further found the PRC2 protein EED can function independently of PRC complexes to execute epigenetic silencing in early embryos and in embryo embryo-derived stem cells. In Aim 2, we will define the non-PRC role of EED in epigenetic silencing in preimplantation mouse embryos and its derived stem cells. Finally, countering the prevailing dogma that H3K27me3 is deposited solely by PRC2 our results demonstrate an additional H3K27me3 catalyst. In Aim 3, we propose to identify and dissect the function of a novel H3K27me3 catalyst in extra-embryonic and embryonic stem cells. All three Aims utilize unbiased approaches to define novel functions and mechanisms of PRC2 proteins and H3K27me3 catalysis during mouse embryogenesis and in early embryo-derived stem cells. Our central hypothesis is that the mode of epigenetic regulation ascribed to the Polycomb group can occur via alternate mechanisms and proteins. The expected findings will increase our understanding of the epigenetic logic underlying embryonic development and how epigenetic dysregulation contributes to human disease.

The overall aim of Project 2 is to establish the physical basis for weakened cell-cell adhesion and enhanced motility in cancer metastasis and how mechanical tension regulates these functions. Tumor cells adhere to one another less well than their corresponding normal cell counterparts. The discovery of cell surface cadherin adhesive receptors and associated cytosolic catenins as key regulators of tissue architecture during development and adult tissue homeostasis provides molecular candidates that could explain altered cell adhesion in cancer. The disruption of the cadherin-catenin adhesive complex correlates with clinical features of tumor metastasis and poor patient prognosis. Thus, the cadherin-catenin adhesive complex is an important determinant of tumor progression by functioning to suppress tumor invasion and metastasis. Following disruption of the cadherin-catenin adhesive complex, tumor cells de-adhere from one another, invade the surrounding ECM, and migrate away from the primary tumor. How tumor cells invade the ECM may provide critical therapeutic targets for the prevention of cancer metastasis. In four inter-related aims we will test the hypotheses that cadherin-mediated adhesion forces on adjoining cells are regulated by cancer mutations in cytoplasmic proteins recruited to the cadherin adhesive receptor complex, which affect tumor intercellular adhesion, tumor invasion and metastasis and that once tumor cells de-adhere from one another, focal adhesion proteins regulate tumor cell invasion in the surrounding 3D matrix, the propulsive forces of migration, and mechanical tension sensing of the matrix. Linkage to PS-OC: The research aims of Project 2 fit the overarching theme of the Center of the role of forces in the metastatic cascade;Aims 1-4 are synergistically connected to Aims 1 and 2 in Project 1 and Aim 4 in Project 3 for further research integration of the Center;all Students and Fellows in the Project will be enrolled in the Center Training Program;this projed will make use of the resources provided by the Imaging Core and Microfabrication Core, as well as the Administrative Unit of the Center;cell lines and micromechanical methods will be the same as those used in all projects;computational efforts will be shared among all projects.

Otitis media is the most common significant bacterial infection of childhood affecting from 10-30 percent of infants and children under 5 years of age yearly. This leads to a cumulative incidence of otitis media of 80-90 percent by 5 to 6 1/2 years of age. This investigation will determine the humoral immune response in acute and convalescent serums and middle ear exudates of patient with otitis media proven by culture to be due to Diplococcus pneumoniae or Hemophilus influenzae. Antibodies will be qualified by radioimmunoassay and indirect hemagglutination. The immunoglobulin class of the antibodies will be determined with the use of fluorescent-tagged antibody to human immunoglobulins. Antibody responses will be correlated with serial cultures of the middle ear and the long-term clinical course of patients in an attempt to characterize the natural immune response to otitis media and to relate this response to protection of the patient from subsequent bouts and otitis media due to the same or other serotypes of these two bacteria. Since highly purified capsular polysaccharides from the pneumococcus and H. influenzae type b are presently under study as vaccines, characterization of the natural immune response to localized infection by these bacteria will serve as an important basis for comparison of the response to vaccination.

When endogenous repair fails, as is often the case with dense fibrous musculoskeletal tissues (like the knee meniscus), novel strategies and enabling technologies must be developed to enhance tissue regeneration. While numerous tissue engineering therapies have been developed for the engineering of fiber-reinforced tissues, one persistent limitation is the ability to fabricate scaffolds that maintain structural integrity and direct appropriate tissue architecture, while simultaneously promoting cellular infiltration throughout the regeneration period. In this proposal, we address these limitations with the production of a novel multi-polymer composite nanofibrous scaffold that provides a 3-dimensional micropattern for neo-tissue formation. These scaffolds, with fiber diameters on the order of the native extracellular matrix, can be produced with defined fiber anisotropies that mimic the structural arrangement of fiber-reinforced tissues. Further, by introducing flexibility in polymer properties via a library of photocrosslinkable macromers that exhibit a range of mechanical and degradation properties when polymerized, we propose to tailor temporal pore formation within the scaffold through the controlled degradation of individual polymer components. We hypothesize that these nanofibrous multipolymer photocrosslinked meshes will have controlled mechanical properties reflective of the stiffest and slowest degrading component and show a time dependent increase in void space while maintaining their overall mechanical properties. Further, we hypothesize that the controlled increase in void space within these scaffolds, via the erosion of sacrificial elements, will promote cellular infiltration into the multi-polymer mesh and will result in a more uniform functional tissue structure. Additionally, as the mechanical environment of the meniscus is paramount in its maturation and homeostasis, we hypothesize that tailored mechanical preconditioning regimens will likewise promote functional maturation of infiltrated meniscus constructs. To this end, a novel dynamic loading bioreactor that applies compression and tension is developed to promote tissue maturation. The first Aim of this proposal is to develop technology to electrospin multi-component nanofibrous scaffolds and compare properties to the native tissue using a predictive fiber-reinforced composite model. In the second Aim, the interaction and infiltration of cells and ultimate mechanical properties will be explored in single- and tri-polymer cell-laden nanofibrous scaffolds. The third Aim involves the development of a bioreactor for pre-conditioning scaffolds and evaluating the impact of mechanical stimulation on tissue formation in the short and long term. If successful, this innovative approach will provide several new enabling technologies for the functional regeneration of damaged fiber-reinforced musculoskeletal tissues. Public Health Relevance Statement (provided by applicant): This project develops a novel multi-polymer nanofiber fabrication system to exert control over polymer chemistry and overall scaffold mechanics and degradation with time to improve cellular infiltration and uniform tissue deposition in fibrous tissue-engineered constructs. If successful, this approach would surmount a major hurdle in the tissue engineering of dense structures of the musculoskeletal system and provide a mechanically functional, structurally anisotropic 3D micro-pattern for directed neo-tissue formation while promoting full cellular colonization and eventual replacement of the polymer structure after complete dissolution. This innovative approach, coupling scaffold fabrication, mechanical loading, and in vivo assessments, will aid in the development of tissue engineered therapies for fiber-reinforced musculoskeletal tissues such as the knee meniscus that otherwise fail to heal and have few clinically viable repair strategies.

To combat the high number of unintentional deaths and injuries sustained by children under age 14 who participate in recreational activities, Electronic Learning Facilitators will create an injury prevention kit to be incorporated into fourth grade health and safety curriculum. The primary goal of this program will be to increase safe recreational behavior among fourth graders. The kit will include a highly interactive, web-based multimedia program that will inform children about injuries associated with recreational activities, and how to prevent them; model safe and unsafe behaviors, allowing children to see the consequences of unsafe actions; and simulate recreational activities, giving children the opportunity to practice making safe decisions. The kit will also include a guide to help teachers incorporate the multimedia program into their larger safety curriculum. It will include lesson objectives and strategies for achieving them; methods for gaining parental support; activities for community involvement; motivational items, such as a poster for the classroom and stickers for the children; and information on how to create/change public policy. The multimedia program will comprise several modules, each pertaining to a different sport or activity. For the Phase I prototype we will complete one module, which will cover bicycling. PROPOSED COMMERCIAL APPLICATION: Safe and Fun: An Injury Prevention Kit will be a Web-based, interactive multimedia program for fourth graders designed for distribution through the classroom. The program kit also will include a teacher's guide; activities; motivational items; and ideas for gaining parental and community support, and changing public policy. Individual teachers, schools or school districts will be able to purchase the kit in order to gain access to the supplementary materials. PH5 6246-1 (Rev. 1198) Page 2

A vaccinia virus mutant which is resistant to the drug aphidicolin (AP) codes for an AP-resistant DNA polymerase. A change from a wild-type (WT) guanine to a thymine at position 2430 of the sequence of the DNA polymerase gene changes a leucine to a methionine in the polymerase and confers AP-resistance. The mutation was confirmed by site directed oligonucleotide mutagenesis of the WT DNA. To prepare the polymerase gene for the insertion of other mutations near position 2430 which might confer AP-resistance, a part of the WT gene, the Eco B segment, was cloned in a phagemid. Bgl I and Xho I restriction sites were inserted in the cloned Eco B segment at positions 2412 and 2514, respectively. Double-stranded oligonucleotides with sequences identical to the wild-type DNA except for single base changes can be inserted at the Bgl and Xho sites and the recombinant DNA tested for the ability to confer AP resistance. A phosphonoacetate resistance mutation was inserted at position 1537 to provide a means for selecting vaccinia virus with an altered B segment. Different mutagenic procedures were tested for their efficiency during the course of the work. The Bgl and Xho sites were inserted in the DNA by the Kunkel method which uses a bacterial strain that produces single-stranded template DNA containing uracil. This method, adapted for the phagemid system, had a 5% success rate for the creation of both the Bgl and Xho sites. When normal template DNA without uracil was used, the mutation frequency was tenfold less. The Eckstein method of mutagensis uses the restriction enzyme Nci, which will not cleave duplex DNA when one strand contains phosphorothioate nucleotides, but will nick a normal complementary strand. Initial results indicate a 90% success rate for the creation of the desired mutation. The conditions needed to increase the yield of the final DNA product used for the transformation reaction are under investigation.

We have described a group of cytokines that modulate mediator release from basophils and mast cells. Histamine releasing factors (HRF)-mediated activation of mast cells and basophils may represent a amplification mechanism of allergic process. The clinical importance of these cytokines can not be established until their chemical structure is better defined. We have purified one species of HRF (44 kD) by sequential HPLC. The objective of this grant is to purify this and other species of HRF to homogeneity and sequence them. In addition, we plan to raise monoclonal antibodies against HRF, develop immunoassays and affinity columns to aid purification. Crude supernatants containing HRF will be generated by culturing two cell lines, U937 and RPMI 8866, both of which produce HRF similar to that synthesized by mononuclear cells (MNC). Furthermore, MNC-derived HRF will be generated by culturing cells isolated from leukocytes concentrates obtained from Blood Bank donors. HRF will be purified by using sequential HPLC. We have recently demonstrated that a major fraction of HRF binds to heparin-agarose. We have also optimized the running conditions of a C4 reverse phase column, and recovered HRF from the column with satisfactory yield. The strategy for the purification of HRF is to apply sequential HPLC involving affinity, ion exchange and reverse phase columns. The homogeneity will be verified by one- and two-dimensional SDS-polyacrylamide gel electrophoresis. Purified HRF will be electroblotted onto Immobilon PVDF filters and used directly for sequencing. The latter will be accomplished using an updated Applied Bio-system Sequenator. Monoclonal antibodies will be raised in mice using purified HRF. Hybridoma cells will be grown in the peritoneum of pristane-treated mice. Purified antibody will be used to develop ELISA and/or RIA as well as affinity columns. Our final goal is to conduct a biochemical and immunological comparison of various species of HRF produced by different subpopulations of MNC. This will be accomplished by comparing the elution profile from HPLC columns and by testing the antigenic cross-reactivity among various species of HRF obtained from different sources.

Funds are requested to support three trainees per year over a five year period for training in the interface of behavioral and biomedical sciences. The core training faculty are from the Psychology Department and Neuroscience Program at Brandeis University. Affiliated faculty are from the Brandeis Life Sciences and adjunct faculty are from collaborating laboratories in the Greater Boston area. The faculty of this training program are firmly committed to educating students to study human behavioral health and development in terms of multi-directional, dynamic interactions among the biological building blocks of the body and brain and the emergent properties of whole individuals and groups of individuals. The training program we propose will be implemented within a Psychology Ph.D. program with strong, existing, intra- and inter-disciplinary research training, course work, and extra-curricular activities. Students in the training program will be required to fulfill the following requirements: 1) engage in rotations and research projects co-mentored by one or more faculty working in both psychological and biomedical disciplines; 2) take courses from menus of psychological, biomedical, and general quantitative categories; 3) attend a variety of integrative proseminars and small and large group colloquium/discussion series; 4) participate in short courses during the academic year and the summer designed to promote unity and to provide advanced training in biomedical or computational techniques. Non-trainee Ph.D. students will also be encouraged to fulfill these requirements. Full participation of Psychology students in biomedical laboratories is a challenge which will be met by systematic introduction of these curricular elements as well as by selection of qualified candidates. The adherence to and evaluation of the training program will be ensured by 1) annual internal reviews by the full core faculty of students' fulfillment of their goals as set out in a contract co-developed with the mentors; 2) annual reviews of the core faculty's guardianship by an internal steering committee and an external review committee; 3) feedback from students currently in the program and students who have graduated. Our general approach to recruitment of students will be through direct outreach and networking. To recruit and retain minority students, we will build upon the successful methods of our colleagues who are directing successful training grants in Psychology and Life Sciences at Brandeis Relevance: Integration of behavioral and biomedical sciences is essential for generating new knowledge about human mental health and disease. Training a new generation of scholars who have command of psychological, behavioral, and biomedical conceptual and methodological tools will help promote health and prevent and treat psychological and physical disorders

The major objectives of this research project are to determine the mechanisms responsible for volume regulation in dog regulation in dog erythrocytes. When dog erythrocytes are exposed to anastomotic media, the is an activation of either the Na/H antiporter or K-C1 cotransport systems to return the cell back to its normal volume. Studies in our laboratory have been designed to quantitate these pathways and determine the stoichimetry, inhibitors and activators of these transport systems. Recent studies have been design to determine the heterogeneity of cells have been assayed for their ion and water content as well as their specific density. Future studies will be designed to determine the transport properties of these cell fractions in order to explain the physiological mechanism responsible for these observations. It is anticipated that the results of these studies will allow us to determine the mechanisms responsible for cell volume regulation in vivo.

The major aims of the project continue to be a correlation between: 1. The chromosomal findings of early bladder lesions, particularly papillary tumors, with the cytologic picture and clinical course, and 2. The karyotypic picture in advanced carcinoma of the bladder with the response to therapy and clinical course, e.g., sensitivity to X-ray and/or chemotherapy, metabolic spread, recurrences, etc.

Given that mast cells increase increase at sites of inflammation and are located at surfaces where exposure to invading organisms may occur, we examined the Toll-like receptors expressed by cultured human mast cells and the ability to activate mast cells with TLR ligands. We found that viruses and polyI:C induced IFN-alpha and IFN-beta, in part through TLR-3. Human mast cells also expressed TLR-1, TLR-2, TLR-4, TLR-5, TLR-6, TLR-7, and TLR-9. The TLR ligands LPS, peptidoglycan, and flagellin induced the production of TNF, IL-1-beta, IL-5, and GM-CSF. IFN-alpha production involved nuclear factor-kappaB, p38; and C-Jun NH2-terminal kinase and mitogen-activated protein kinase. IFN-gamma treated human mast cells express FcgammaRI. Such mast cells when exposed to heat-aggregated IgG1 degranulate and subsequently generate PGD2 and LTC4; as well as release IL-3, IL-13, GM-CSF, and TNF-alpha. No mast cell activation was observed with the addition of heat-aggregated IgG2, IgG3, or IgG4. Simultaneous activation of human mast cells with aggregated IgG and C3a led to additive degranulation.

Green tea polyphenols appear to be chemopreventive against certain cancers, including oral cancer; but how cancer cells succumb while normal cells survive this polyphenol exposure is not known. Lack of this information prevented clinical uses of polyphenols for oral cancer chemoprevention or treatment. The long-term goal of this investigation is to elucidate the signal pathways and mechanisms by which green tea polyphenols differentially target normal and malignant cells to direct protective or apoptotic effects. Preliminary data from our laboratories have demonstrated that normal epithelial cells express p57 (KIP2) in response to green tea polyphenols in a dose- and time-dependent manner. We propose a novel concept, that green tea polyphenols are able to activate two pathways: 1) a p57-mediated survival pathway, and/or 2) a caspase 3-dependent apoptosis pathway. The hypotheses to be tested is that p57 induction by green tea polyphenols in normal epithelial cells may serve an anti-apoptotic function, absence of the p57 response in malignant cells may result in induction of caspase 3-dependent apoptosis. The immediate goal of this proposal is to identify the survival or apoptotic genes that are regulated by green tea polyphenols. In this proposed project, the survival/apoptosis gene expression profile will be determined following green tea polyphenol exposure, in normal human epidermal keratinocytes and in human oral squamous cell carcinoma cells. Specifically, the levels of p57 expression induced by the most potent green tea polyphenol, (-)- epigallocatechin-3-gallate (EGCG), in normal human epithelial cells will be determined. Using RT-PCR, mRNA stability assay, Northern and Western blot analyses, the relationship between transcription/translation levels of p57 induction and the time/dose of EGCG will be established. The RNA samples at specific time points will be subjeted to gene array analysis and profiling. Not only will the expression profile of those genes that are either activated or suppressed by EGCG in normal or tumor cells, but promising cellular targets for future chemotherapeutic intervention may be identified. Data generated from this proposal may reveal novel drug targets for treatment of head and neck cancer.

Clinical research projects in the University of Chicago Cancer Research Center can be grouped in two broad areas: disease-specific research and modality-specific research. In both cases, a group of investigators, usually representing multiple specialties, is formed to optimize patient care and clinical research. Over the last five years, the following programs have shown dramatic clinical and scientific growth: gastrointestinal oncology, head and neck cancer, concomitant chemoradiotherapy, bone marrow transplant and investigational chemotherapy. In addition, participation by UCCRC investigators in CALGB activities has grown substantially. During the next grant period, these programs will be further strengthened. In addition, we plan to further develop clinical research in breast cancer and neuro-oncology and to initiate new programs in prostate cancer and geriatric oncology. We hope to achieve these objectives by improving the research infrastructure within the institution, facilitating vertical integration of clinical programs with laboratory research and continuing to support multidisciplinary clinical research groups.

Specifically, the study described in this proposal will test the hypothesis that the increase in tuberculosis in San Francisco is largely due to "facilitated" transmission of M. tuberculosis in definable settings which have high tuberculosis prevalence and large numbers of individuals with impaired immunity. We will continue to use restriction fragment length polymorphism (RFLP) analysis, with which we have demonstrated the ability to identify individuals strains of M. tuberculosis (Daley, Small, et. al. N. Eng J. Med), to identify residents of San Francisco who are infected with the same strains. Detailed epidemiologic investigation of these individuals will be conducted to determine where they contracted tuberculosis, thus defining the specific environments in the city that foster tuberculosis transmission. Further investigation of the patients involved will be conducted to determine the modifiable risk factors for the acquisition of tuberculosis. Ultimately, this information will permit the design of more effective prevention strategies. RFLP analysis is a powerful epidemiologic tool. However, because culturing bacteria is a prerequisite for its use, it is limited to culture positive cases and results are not available for 4-6 weeks after a specimen is collected. In order to eliminate this delay we plan to design and validate a polymerase chain reaction (PCR) based fingerprinting system. This system will be optimized and validated on DNA, which will have been extracted and characterized in previous RFLP- based molecular epidemiologic studies.

Voltage-gated Ca2+ channels are important regulators of membrane potential in cardiac, skeletal and smooth muscle cells and cardiovascular neurons. Just as important is the Ca2+ the enters via these channels acts as a second messenger upon ion channels, C kinase, proteases, calmodulin, etc. The structure of Ca2+ channels in skeletal muscle is tetrameric consisting of alpha 1, alpha2, beta, and gamma subunits; elsewhere the subunit composition is unknown. We showed that this alpha1 subunit by itself can function as a Ca2+ channel and dihydropyridine receptor (Perez-Reyes et al., 1990). This was achieved by stably transfecting alpha1 into murine L cells which are devoid of all subunits and so not express Ca2+ currents. Mikami et al. (1990) showed that the alpha1 subunit from cardiac muscle expressed Ca2+ currents in Xenopus oocytes. Since oocytes have an endogenous Ca2+ current, the properties of alpha 1 were not specified as clearly. The molecular mechanisms of voltage sensitivity, divalent cation selectivity and regulation of Ca2+ channels are largely unknown. We will examine these properties for both alpha 1 subunits in combination with different skeletal muscle alpha 2, beta, gamma subunits and different cardiac muscle subunits as they become available. Expression will be studied in L cells and oocytes. Chimeras of the two alpha 1s will be used to determine the repeats responsible for the differences between skeletal and cardiac muscle Ca2+ currents. Chimeras with Na+ and K+ channels will be developed to determine the repeats important for gating and selectivity. The search for the essential parts will be narrowed by using segments from candidate repeats. These will be introduced into a K+ channel which has been genetically engineered for segment swapping. Sites for regulation by G proteins will be tested using similar approaches.

ABSTRACT Epilepsy is a family of chronic neurologic disorders characterized by periodic, unpredictable seizures. Current pharmacotherapy for epilepsy relies on ion channel inhibitors, GABAergics, and compounds of unknown mechanism. Following first-line or dual treatment with these drugs, more than 30% of patients continue to experience seizures. A critical barrier in the epilepsy field is the poor brain penetrance of many promising therapeutics. Indeed, there are a number of large or lipophobic compounds that do not readily enter the brain when given systemically, but which are well known in animal studies to show great promise for controlling seizures when they are administered through direct cannulation of the brain. In the proposed studies, we intend to focus on the development of a new technology that will use endogenous biological mechanisms to actively transport these compounds into the brain. We will focus our efforts on neuropeptide Y (NPY) and oxytocin (OT) because 1) these compounds do not readily enter the brain through passive diffusion, 2) there is evidence that these compounds control seizure activity, 3) we have generated exciting preliminary data supporting the use of our technology to transport these compounds into the brain, and 4) these compounds are excellent prototypes with which to conduct proof-of-concept feasibility studies. The long-term goal of this research program is to develop a safe and effective approach to the delivery of neuropeptide treatments for epilepsy and other neurological disorders to the central nervous system. The completion of these studies will provide a solid foundation for further studies to develop even more advanced formulations. These formulations would further refine our approach to increase the brain penetrance, sustain the release, or compartmentalize to the brain novel neuropeptide therapeutics. As such, this proposal will form the foundation of a new research program which we hope will support a new wave of therapeutics for epilepsy.

Many of the clinical research projects of the laser biophysics group involve the interaction of light with tissue (e.g., laser microsurgery, laser Doppler blood flow measurements, photo dynamic therapy of cancer, noninvasive platelet assessment). In order to more fully quantitate these techniques, we have undertaken theoretical modeling of light propagation in biological tissues and turbid media. Analytical equations have been devised characterizing various parameters of photons illuminating a tissue surface (probability of surface re-emission at a given distance, mean path before reemission, mean depth of penetration, probability of absorption with depth). These expressions have been used to interpret empirical measurements on living tissues and to quantify better a variety of clinical measurements (e.g. laser Doppler blood flow and volume measurements, dosimetry in PDT of cancer, and remote sensing of atherosclerotic plaque. Recently our theoretical predictions of path length distributions of re-emitted photons have been applied to temporal dispersion of picosecond laser pulses in brain and muscle. Such analysis allows absolute quantitation of hemoglobin and myoglobin oxygen saturation in vivo.

Our long term goal is to understand the structural and functional bases of the interaction of dantrolene with the intracellular RyR/Ca release channel. We have identified a sequence domain called DP1 (a. a. 590-609) on the skeletal muscle ryanodine receptor (RyR1) as a target for dantrolene, a region tightly linked to mutations resulting in susceptibility to malignant hyperthermia. Dantrolene appears to interact with RyR1 rather than cardiac RyR2 despite an identical DP1 sequence. The weak interaction of dantrolene with RyR2, therefore, may reflect conformational constraints imparted by the rest of the channel or associated macromolecules. Key question regarding the molecular basis of dantrolene interaction with RyR isoforms are: What are the contextual requirements, both domain-domain and protein-protein interactions that determine dantrolene binding and pharmacologic activity in RyR isoforms? The specific hypothesis for this proposal is that residues 590-609 on RyRldefine the major portion of the dantrolene binding site, and that conformation of this site determines the specific interaction of an RyR isoform with dantrolene and plays important role in the regulation of this Ca release channel. Two specific aims are designed to test this hypothesis: Aim 1, to define the structural and biochemical basis of dantrolene interaction with RyRI. Photoaffinity labeling with photoactive dantrolene congeners combined with mass spectrometry, and mutational analysis of identified sequences and functional analysis in Ca fluorescence measurements will be used. Aim 2, to explore the differential effects of dantrolene on RyR1 and RyR2 using pharmacological, immunological and molecular approaches. RyR1-RyR2 chimeras will be expressed in heterologous and homologous cells and their responses to dantrolene probed by Ca imaging and spectroscopy. The information gained will provide insight into the molecular mechanism by which dantrolene interferes with intracellular Ca release and lead to rational drug design for the therapy of Ca sensitive muscle diseases.

Enhancement of video images should enable the visual impaired greater access to TV. and other video sources. In previous years, contrast enhancement based on a band-pass filtering approach was developed and tested. Results were encouraging, showing improved performance in face recognition and in obtaining details from motion videos. Addition improvement will be gained by addressing other aspects of spatial vision beyond the loss of contrast sensitivity. We will study, in patients with central scotomas, the addition of wide-band feature markers improve the performance in localization, and in phase (shape) discrimination tasks, as suggested by studies carried out with normally sighted subjects. Using the results of these studies, we developed a novel extension of the previously used enhancement method, a wide-band enhancement that should have many advantages. The new method as a cartoon-like bar outlines (bipolar light and darkness) and increase the contrast of edge features to facilitate localization and discrimination. We will determine the optical parameters for such enhancement for patients with central scotomas, implement a software based approach to generate enhanced motion video segments and use such segments to measure the preference of visually impaired persons for the enhanced versions. We will continue to develop our visual model, and to use it in the development, evaluation and optimization of image enhancement for the visually impaired. The basic aspect of the study will examine changes in adaptation that occur in the peripheral visual processing with long term central visual loss. We will study performance differences on the number of visual tasks between patients with long term bilateral scotomas and with unilateral scotomas. The study of patients with monocular scotomas will provide better measure of the capability of the peripheral retina. The differences between monocular and bilateral scotomas patients performance will improve our understanding of adaptation to the force used of peripheral retina or form vision. Studies of long term facilitation low vision patients will be used in search for a measure of visual function that will explain and quantify deficit in peripheral retinal such as contour integration. The basic studies results will guide further fine tuning and optimization of the enhancement technique.

Project Summary Hutchinson-Gilford Progeria Syndrome (HGPS) is a rare, fatal autosomal dominant genetic condition characterized by many characteristics of accelerated aging in children. Children with HGPS die at an average age of fourteen, usually from heart disease. It has been known for some time that the syndrome is most commonly caused by a specific point mutation in the LMNA gene which normally codes for lamin A and its splice variant lamin C. The LMNA mutation commonly associated with HGPS leads to increased usage of a cryptic splice site which leads to the production of a truncated, farnesylated form of lamin A referred to as progerin. Quite interestingly, progerin is expressed at very low levels in healthy individuals and appears to play a role in the normal aging process. Lamin A normally serves as an important component of the nuclear lamina, a structure that provides mechanical support for the nucleus and regulates several critical cellular processes including a number of DNA repair pathways. In HGPS, the impact of overexpression of progerin on nuclear architecture and genomic stability is profound. One reported consequence of progerin expression is an accumulation of DNA double- strand breaks (DSBs). To our knowledge, however, the effect that expression of progerin or other mutated forms of lamin A has on the precise manner in which a DSB is repaired in the human genome has never been investigated in detail. This gap in our knowledge is significant since corruption of DNA repair is commonly viewed as playing a key role in the aging process. Over the past two decades, we have developed and productively used a model system for studying DSB repair, homologous recombination, and related DNA transactions in the chromosomes of human cells. We have engineered DNA constructs containing a mutated selectable marker gene and a closely linked sequence that can restore function to the marker through recombination. Constructs are stably transfected into cell lines of interest. A DSB can be introduced into the marker gene by transient expression of endonuclease I-SceI and so we can recover a variety of DSB repair events that restore marker function by applying genetic selection. We will apply our system to compare and contrast DSB repair events in normal cells versus cells that overexpress progerin or express a related variant form of lamin A. Our work will be the first in-depth investigation into how expression of progerin impacts DSB repair pathway choice as well as the nature of individual repair events at the nucleotide level. We will focus on the questions of whether expression of progerin reduces the stringency of recombinational repair, enabling genetic exchange between imperfectly matched sequences, and whether progerin inhibits precise joining of DNA ends. Either of these potential impacts of progerin expression would likely destabilize the genome. Our studies will provide novel information about the aging process, information that will enhance our understanding of progeria and normal aging.

Our goal is to enhance the efficacy of DNA vaccines using new bio-organic compounds to mediate oral deliver. Oral vaccination primarily protects an individual from oral pathogens, which include the AIDS retroviruses and opportunistic agents that accompany AIDS. Oral vaccination also confers some protection against pathogens that infect via other routes. Live oral vaccines have always been more effective than DNA vaccines in clinical trials, but recent technology allowing DNA delivery to specific cells provides new possibilities for DNA vaccines. Here we propose to test oral DNA vaccines for targeted delivery to dendritic cells (DC). using novel glyco-polyamines as the delivery vehicle. DC are the primary antigen-presenting cells and they express abundant cell surface carbohydrate receptors, such as mannose receptor and DC-SIGN. The cyclodextrin-based glyco-polyamines used in our studies consist of two functional domains: the multiple amino groups that bind and form complexes with DNA, and the sugar ligands that bind cell surface lectins and target the DNA complexes to DC. Enhancing the uptake of DNA by DC will enhance the immunogenicity of DNA vaccines. Cell culture studies have already demonstrated that mannosylated polyamines significantly enhance the uptake of plasmid DNA into DC. Here we propose to test the efficacy of targeted delivery in vivo. We hypothesize that DNA vaccines targeted to the cell surface carbohydrate receptors of dendritic cells will elicit enhanced cell-mediated responses. To test this hypothesis, we will employ the LCMV-infected murine model system that is known to elicit strong cell-mediated immune responses. We will have two specific aims. In aim 1, we will synthesize novel glycosylated cyclodextrinbased polyamines (CDPA) and test the uptake and expression of reporter genes in dendritic cell culture. Mannose and oligomannose ligands will be introduced into CDPA for targeting to mannose receptors and DC-SIGN on dendritic cells, respectively. In aim 2, we will test the magnitude of cell mediated immunity after oral inoculation with DNA vaccines in various formulations. Plasmid DNA encoding the LCMV NP gene will be orally delivered to mice as naked DNA or as DNA complexed with CDPA or glycosylated CDPA. Once we find a formulation that is optimum for eliciting cell-mediated immunity, we will replace the NP gene with DNA encoding an antigen associated with protective immunity in AIDS (p27 gag). We will determine, in the murine system, whether p27 DNA complexed with our compound can still elicit high cell-mediated immunity. These studies will lead to improved oral vaccines against AIDS and its attendant opportunistic infections. Once we have optimized the vaccine formulation in the murine model system, we will apply the targeted delivery system to monkey models and human clinic trials. [unreadable] [unreadable]

This study proposes new techniques for overcoming the two major obstacles to practical protein fractionation by ultrafiltration: (1) the diffuse pore-size distribution of the membrane, which allows only gross separation and (2) the buildup of retained proteins on the membrane surface, drastically decreasing selectivity and separation rate. Membranes having sharp size cut-off are formed by crosslinking proteins of appropriate size into the membrane pores they can penetrate. Then molecules larger than this protein can no longer permeate. Use of a pair of such membranes with different cut-offs would isolate a fraction of narrow size range. Pilot studies are described on blocking of pores with gamma-globulin. The technique could also be applied to chromatographic gels. The surface protein layer which builds up during ultrafiltration is kept at negligible thickness by pulsed electrophoresis, which frequently moves these proteins up into the stirred bulk solution. The combination of the two techniques would be of considerable help in research on blood biochemistry and could aid in large-scale fractionation of plasma for component therapy.

08: Neuroscience Using Yeast Cells To Define Mechanisms of Human Neurodegenerative Diseases The United States and other countries around the world are experiencing a demographic sea change owing to the rapidly growing elderly and 'Baby Boomer' populations. As our population continues to age, neurodegenerative disease will increase in prevalence and thus pose a daunting challenge to public health worldwide. These truly disastrous disorders include Alzheimer's, Huntington's, Parkinson's, amyotrophic lateral sclerosis and the frontal temporal dementias. Interestingly, though disparate in their pathophysiology, many of these diseases share a common theme manifest in the accumulation of insoluble protein aggregates in the brain. The long-term goal of my laboratory is to elucidate the mechanisms causing these proteins to misfold and aggregate, identify the genes and cellular pathways affected by misfolded human disease proteins, and understand their function in normal biology. We are taking an innovative approach to attacking this exceedingly difficult problem: harnessing the baker's yeast, Saccharomyces cerevisiae, as a model system to study the mechanisms underpinning protein-misfolding diseases. Surviving cellular stresses caused by misfolded proteins is an ancient problem that all cells struggle with and many of the mechanisms employed to deal with protein misfolding are conserved from yeast to man. We propose to create yeast models of human neurodegenerative diseases and to perform high-throughput genome-wide screens to elucidate the basic cellular mechanisms of toxicity. These yeast models will provide us with a unique opportunity to observe and understand protein folding and misfolding in real time as it occurs in a living cell and then to ask big questions on a genome- wide scale about the cellular pathways affected by the aberrant accumulation and/or function of human disease proteins. We hypothesize that the mechanisms identified by our studies will have broad applicability to multiple neurodegenerative diseases. The innovative aspect of our approach is not just that we are working in yeast, but that we are willing and able to use this system as a discovery tool, which we will validate in more relevant animal models. We have done this successfully in the past (via collaboration and on our own) and this will allow us to proceed with future experiments from a knowledgeable point of view, knowing the relative strengths of various organisms and methods.

Chronic lymphocytic leukemia (CLL) is susceptible to immune-mediated destruction as demonstrated by observations of the graft-versus-leukemia (GvL) effect after hematopoietic stem cell transplantation and donor lymphocyte infusion (DLI). Our preliminary studies demonstrate that DLI-treated CLL patients who developed GvL without apparent GvHD developed potent polyclonal B cell responses directed against antigens that are highly expressed in CLL and that mostly had perfect sequence identity between host and donor (and hence are not minor histocompatibility antigens). From these observations, we hypothesize that anti-CLL immunity can be further boosted by immunizing post-transplant patients with leukemia antigens combined with an adjuvant. Several unique characteristics of CLL support the rationale of developing this innovative therapeutic approach. Advanced CLL patients typically have high levels of circulating tumor, and thus tumor for vaccine generation is readily harvestable. As CLL patients are inherently immune-deficient, anti-tumor immunity is likely to be more effective after reconstitution of normal donor-derived immunity through allogeneic transplant. Finally, the potentially slow rate of disease progression in CLL patients should allow sufficient time for the development of immunity following transplant combined with several rounds of vaccination. Thus, we are actively recruiting patients to a newly opened phase I trial, in which we will administer irradiated autologous tumor cells as the source of leukemia antigens in combination with irradiated bystander cells expressing GM-CSF to patients with CLL following nonmyeloablative transplant. Studies at the DFCI have shown that local secretion of GM-CSF in the presence of irradiated tumor cells at the vaccination site can lead to destruction of tumor cells at distant sites of metastasis, reflecting development of anti-tumor B and T cell immunity. A recently engineered bystander cell line (called GM-K562 cells) will be used for this trial to secrete high and reproducible levels of GM-CSF at the site of vaccination, thus providing a consistent clinical reagent. In Aim 1, we describe the primary objective of the trial, which is to evaluate safety, toxicity and feasibility of the proposed treatment approach. As we anticipate the toxicity to be low based on previous GM- CSF trials, we will monitor the immunological effects of the vaccine by utilizing a set of assays that we have developed to measure antigen-specific B (Aim 2) and T cell (Aim 3) immunity against CLL-associated antigens. Novel aspects of this trial thus include: 1) vaccination of post-transplant patients with reconstituted immune systems;2) use of a novel engineered reagent for secretion of GM-CSF;3) detailed monitoring of anti-CLL immunity using a panel of CLL-associated antigens that we identified as targets of GvL;4) reduced intensity transplant as a platform for generating effective tumor immunity under acceptable conditions of toxicity. The studies will thus test an important hypothesis, provide a solid basis for further trials of tumor-based vaccines for CLL and further our long-term goal of generating curative, non-toxic immune-based treatment for CLL.

Benign urological diseases create a substantial burden on the US healthcare system because of their prevalence, impact on quality of life and cost. Collectively, congenital genitourinary anomalies, kidney stones, urinary incontinence, erectile dysfunction, lower urinary tract symptoms, benign prostatic hyperplasia, pelvic pain and urethral strictures affect millions of persons and account for over 3.6 billion dollars in annual healthcare expenditures.1 Urinary tract infections contribute an additional 2.47 billion dollars in direct costs. Unmeasured costs related to over-the counter-incontinence products, prescription drugs, and lost productivity due to kidney stones and UTI are exponentially greater. Diabetes, obesity and inactivity, injury, and nutritional choices amplify the prevalence and severity of thes urological disorders. The daunting challenges to effective prevention, control, and treatment require new research paradigms and innovative solutions that harness advances in technology, information and thinking. Thus, this competing renewal creates a multidisciplinary research training program for M.D. and Ph.D. postdoctoral research fellows that addresses benign urological diseases across the entire lifespan. Our long term goal is to train the next generation of urological researchers who can substantiate the first goal of Healthy People 2020, namely to attain high quality longer lives. Two training slots per year will be tailored to our main constituents: M.D. and Ph.D. trainees seeking a two- year fellowship after completion of graduate medical education or graduate school respectively. These trainees will be recruited from our own fellow candidates in Pediatric Urology and Trauma and Reconstruction; from other clinical Departments at UW including Medicine, OB/GYN, Pathology and Radiology; from the graduate programs of our mentors; and from peer organizations nationally. Our program emphasizes robust research projects over methodology. Innovation in research will be central to the program and will be catalyzed by the interplay of mentors from basic science, epidemiology, engineering and patient oriented research disciplines. Each trainee will be linked to an experienced NIH funded research mentor and a practicing urologist with relevant clinical expertise. Multidisciplinary Training in Benign Urology leverages strengths at the University of Washington including a longstanding tradition of urological education, cutting edge collaborative learning environments, and world-class researchers within and outside of traditional areas of urological research. PUBLIC HEALTH RELEVANCE: Multidisciplinary Training in Benign Urology leverages strengths at the University of Washington to address the critical need for Training in benign urological diseases including congenital genitourinary disorders, kidney stones, urinary incontinence, erectile dysfunction, benign prostatic hyperplasia, pelvic pain and urinary tract infections. Collectively these disorders account for over 6 billion dollars in annual health care costs, and their burden will be amplified by diabetes, obesity, aging and inactivity. Our training program creates small multidisciplinary teams that include M.D. and Ph.D. researchers from outside the field, linked to a practicing urologist to focus from the outset on the potential clinial impact of each project.

. The investigators propose to determine the complete genome sequence of the extreme thermophile Thermus flavus and assign gene functions to the sequence in order to identify commercially important activities. ThermoGen's chromosomal integration system will be used to verify functional predictions and study phenotypes of ORFs lacking homology with known genes. In Phase I they will optimize sequencing techniques to generate inexpensive and rapid sequence data for this high-GC genome. They expect to sequence 150- 200 kb of the Thermus genome and analyze it by the `Magpie' automated genome investigating environment to find ORFs with potentially important functions. The functions of these ORFs will be verified experimentally. In Phase II they will complete the sequence of the organism and assign functions for most of Thermus genes. Phase III will proceed with commercializing the enzymes useful in human therapeutics and industrial applications.

Currently in the U.S., 1 in 4 children are at risk for becoming overweight, and even higher rates are found in minority children. Despite the social, health, and economic costs that accompanies obesity, few causal risk and protective factors have been identified and prevention programs have had limited success in influencing children's weight gain. In response, investigators now emphasize the need for integrated theoretical frameworks to better identify these factors and design cost-effective prevention programs. Specifically, researchers have cited ecological, behavioral, and developmental models. Together these models help to understand the etiology of childhood obesity over time in diverse populations. The current research proposal outlines a training plan with the long-term goals of fulfilling this need in obesity research and prevention. The specific aims are: (1) to train in developmental, behavioral, and ecological models related to the etiology of childhood obesity within diverse populations, (2) to train in advanced methodological approaches, (3) to employ theoretical models and sophisticated methodologies to gain expertise in investigating correlate and [unreadable] causal risk and protective factors, and (4) to utilize the aforementioned training to develop expertise in [unreadable] preventive intervention design, implementation, and evaluation. To fulfill these aims, the proposed activities include graduate coursework and research training in theoretical concepts, methodology, prevention, basic research, and obesity development. PUBLIC HEALTH RELEVANCE: Through the proposed training, the applicant will promote pubic health by (1) preparing manuscripts that improve current knowledge on obesity development and effective prevention strategies, and (2) acquiring the skills necessary to later conduct effective obesity prevention research within ethnically-diverse populations. This training plan addresses the agendas of both the NICHD and NHLBI by its focus on identifying risk and protective factors for childhood obesity and designing effective prevention programs. It is also in agreement with the goals of the NIH Strategic Plan for Obesity Research: i.e., to promote research in preventing and treating obesity through lifestyle modification, and understanding health disparities in obesity. [unreadable] [unreadable] [unreadable] [unreadable]

An immune response occurs only if a T lymphocyte has been able to recognize the foreign antigen in association with a self molecule encoded by the major histocompatibility complex (MHC). These self molecules expressed on antigen-presenting cells are the polymorphic class II MHC antigens. Most foreign antigens must be processed in order to bind to MHC molecules. Antigen processing for presentation by class II molecules generally involves endocytosis of antigen into an acidic compartment through which newly synthesized class II molecules travel on their way to the cell surface. Once at the cell surface the foreign peptide/MHC class II complex interacts with the T cell receptor and the CD4 molecule expressed on the antigen-specific T cell. The aim of this project is to define the function of human class II molecules in their interaction with T cells and the requirements for class II-restricted processing and presentation of viral antigens to CD4-positive T cells. It was demonstrated that several endogenous processing pathways lead to the presentation of cytosolic antigen to class II-restricted T cells. Most interestingly, one of the processing pathways was different from the one utilized for presentation by class I molecules. This unsuspected presentation of endogenous proteins by class II molecules has important implications on the T cell repertoire selection, on T cell tolerance, and on autoimmunity. Several staphylococcal toxins bind to class II molecules and have a strong mitogenic effect on T cells by stimulating entire families of T cells with particular Vbeta chains in their TCR. These toxins are representative of the newly described superantigens that do not require processing for presentation. the invariant (Ii) chain, bound to newly synthesized class II molecules, serves as a chaperon for the transport of class II molecules to an endocytic compartment where it dissociates from class II prior to the binding of immunogenic peptides and the cell surface appearance of peptide/class II complexes. It had been suggested that superantigens utilized the Ii chain binding site of class II molecules. However, it was shown that the binding site of HLA-DR for the 7 toxins tested was distinct from that for the HLA-DR-associated Ii chain. In addition, cell surface class II/Ii chain complexes were still able to stimulate T cells by superantigen. An important implication from this finding is that endogenous superantigens encoded by pathogens may bind to class II molecules intracellularly prior to the dissociation of the Ii chain.

DESCRIPTION (Applicant's abstract): This is the second revision of an application for funds to conduct a controlled clinical trial of an innovative psychosocial treatment designed to improve the workplace skills of seriously and persistently mentally ill (SPMI) individuals. The initial development and limited testing of the treatment has been funded by a grant Developing a Workplace Fundamentals Training Module (MR 53466); the proposed protect will determine the effects of the treatment on job retention. The project will recruit a total of 48 SPMI individuals who will be randomly assigned to either Individual Placement and Support (IPS; Drake & Becker, 1996) vocational services or to IPS plus participation in the module. All 48 will be selected based on having a history of difficulty retaining competitive employment and all will have either begun or will begin employment within a 3-week "window" of their enrollment in the project. The effects of the module on job retention will be monitored for 18 months after participants' enrollment.

A recently identified family of intracellular proteins termed Smads, are key components of TGFbeta signaling pathways. Phosphorylation of Smad proteins by activated TGFbeta-type I receptors lead to their translocation to the nucleus where they act as transcriptional regulators. Different Smad proteins act as downstream effectors in response to specific TGFbeta-related ligands. Thus Smad1 mediates the effects of Bone Morphogenetic Proteins, whereas Smad2 is activated in response to TGFbeta and activin. This project aims to gain further insight into the functional activities provided by Smad1 and Smad2 proteins during early mammalian development. A null mutation in the mouse Smad2 gene results in early embryonic lethality. I propose to characterize the primary tissue defects in Smad2 deficient embryos. I plan to isolate Smad2 deficient cell lines. Their abilities to respond to a variety of TGFbeta molecules will be analyzed. To evaluate the requirement of the Smad2 pathway in mesoderm formation, the differentiation abilities of Smad2 deficient ES cell lines will be tested in vitro and in chimeras. Additionally I plan to create Smad1 deficient embryos using gene targeting techniques. Their phenotype will be compared with that of the Smad2 mutants. Overall, results of these experiments will further our understanding of the diverse roles played by distinct Smad pathways during establishment of the embryonic body plan.

During the last decade, many characteristics of OHC motility have been garnered through the in vitro (isolated cell) approach, including voltage dependence, subcellular site, nonlinearity, sensitivity, and phase. Despite such progress, it remains a difficult task to envision exactly how OHC motility might induce sharp tuning in the basilar membrane (BM), IHCs and single units. It is entirely possible that characteristics of OHC motility observed in vitro may differ from those within the normal in vivo milieu. A determination of in vivo characteristics in requisite for an accurate assessment of the influence of OHC motility on BM mechanics. OHC motility in contributing something special to peripheral auditory processing, but exactly what and how remain key questions which certainly require in vivo investigations. In this regard, it in proposed to analyze aspects of OHC motility in the intact cochlea. 1) Is it possible to measure the effects of the electrically evoked motility of a single OHC on basilar membrane notion? The in vivo measurement of BM motion during current injection in a single OHC may unequivocally demonstrate that OHC can influence the BH, and may permit the determination of the in vivo voltage-to-movement (V-M) function. 2) What in the voltage dependence of OHC nonlinear capacitance in vivo? OHCs possess a voltage dependent capacitance. Measures of nonlinear capacitance will provide insight into the voltage dependence of OHC motility in vivo, and should compliment data on the in vivo V-M function. 3) Does transient hypoxia alter the voltage dependence of the in vivo motility and nonlinear capacitance functions? It is clear that metabolic insult in vivo can dramatically and reversibly interfere with the normal functioning of the organ of Corti. A concomitant change in these OHC characteristics may provide evidence for OHC motility involvement. 4) Are OHCs coupled via gap junctions to supporting calls? A sensitive capacitive technique will be used to confirm the results of Zwislocki et al., which indicate that coupling exists. Communication between these cell types may provide an interesting mechanism for controlling OHC function. In sum, this type of in vivo analysis may help to understand how the OHC performs its duties, and further, may provide insight into the auditory pathologies which have as their basis OHC dysfunction.

Pilot study work was completed, and nationwide enrollment began in October 2004. A total of 50,000 sisters of women who had breast cancer will be enrolled. At enrollment, the participants answer questions about life history, health, jobs and environment; and give samples of blood, urine, toenails, and housedust. The participants will be followed for at least 10 years. Data and samples from women who develop breast cancer will be compared to those for the majority who won't. Analyses will look for environmental associations with risk. Contract is on budget, and contractor is performing satisfactorily. See Annual Report for Dale Sandler for full progress report. Keywords breast cancer; sister; prospective epidemiologic study; environment; genes; exposures; blood; toenails; occupation; dust; urine; questionnaires

The coordinated regulation of gene expression is a fundamental process in biology. In this proposal, we explore the role of poly (ADP-ribose) polymerase-1 (PARP-1) and its associated factors in the chromatin- dependent control of transcription in response to cellular signaling by a steroid hormone, estrogen, and a cytokine, tumor necrosis factor alpha (TNF). PARP-1 is an abundant, chromatin-associated enzyme that catalyzes the polymerization of poly (ADP-ribose) (PAR) chains on target proteins from donor nicotinamide adenine dinucleotide (NAD+) molecules. PARP-1 plays key roles in a variety of physiological and pathological systems, including hormonal signaling, metabolism, inflammation, differentiation, adipogenesis, carcinogenesis, development, and aging. The long-term objective of these studies is to achieve a better understanding of the chromatin-dependent molecular mechanisms underlying signal-regulated transcription by PARP-1 and its associated factors. Our broad hypothesis is that the gene regulatory activities of PARP-1 are determined by: (1) the local chromatin environment (e.g., chromatin composition, histone modifications), (2) physical and functional interactions among PARP-1, transcription factors (e.g., ER, NF-?B), histone-modifying coregulators, and components of chromatin, (3) the targets of PARP-1 enzymatic activity, and (4) the availability of NAD+ in the nucleus. We will test this hypothesis as it relates to estrogen- and TNF-dependent gene regulation by using an integrated approach that combines a complementary set of tools from biochemistry, molecular biology, cell biology, chemical biology, and genomics. Specifically, we will determine (1) the chromatin-dependent molecular mechanisms of PARP-1 localization and activity at target gene promoters, including the role of the histone variants H2A.Z and H3.3; (2) the effects of PARP-1-dependent site- specific PARylation on the activity and function of chromatin-regulating proteins, including the histone demethylase KDM5B and the histone methyltransferase Ezh2; and (3) the mechanisms by which NAD+ production by the nuclear NAD+ synthase nicotinamide mononucleotide adenylyltransferase-1 (NMNAT-1) regulates PARP-1 enzymatic activity at target gene promoters. Collectively, these studies will provide new insights into the molecular mechanisms of PARP-1's gene regulatory activities in the context of chromatin, especially in relation to estrogen and TNF signaling, and nuclear NAD+ biosynthesis and availability. Given the important role of PARP-1 in human disease, as well as its potential drugability, our studies could lead to new ways to exploit these factors as therapeutic targets.

The majority of hormones and neurotransmitters communicate information to cells via G protein-coupled receptors (GPCRs), and GPCRs represent the largest group of targets for drug development. The 22 adrenoceptor (22AR) has been one of the most extensively characterized members of the GPCR family. It responds to the catecholamine neurotransmitters epinephrine, norepinephrine and dopamine; and much is known about its agonist binding and G protein coupling domains from extensive mutagenesis studies. The coupling of the 22AR to Gs, the stimulatory protein for adenylyl cyclase, was one of the first hormone activated signaling pathways to be discovered and serves as a paradigm of GPCR signaling. During the past several years the Kobilka lab has made significant progress towards characterizing the structural changes associated with agonist activation, and has recently obtained a crystal structure of the wild type 22AR in complex with a Fab fragment, as well as a crystal structure of a 22AR that has been modified to improve its structural stability. The Sunahara lab recently developed recombinant HDL phospholipid particles as an ideal biochemical system for studying structural interactions between the 22AR and Gs. This proposal represents a close collaboration that combines the expertise of these two labs to characterize the structural interactions between the 22AR and Gs. The findings will likely apply to the large number of closely related monoamine receptors and to GPCRs in general. Moreover, the methodologies developed for characterizing 22AR coupling to Gs will be applicable to other GPCRs. A better understanding of the structure and mechanism of activation of the 22AR-Gs complex will further the potential for structure-based drug design and in silico screening for GPCR targets, leading to more rapid development of highly selective and effective drugs. the specific aims are: aim 1. characterize the structural dynamics of 22ar coupling to Gs. We will use biophysical approaches to examine the structural basis of functional cooperativity observed in the 22AR-Gs complex. We will examine the effect of Gs on the structure of the 22AR, and the effect of the 22AR on the structure of Gs. We will examine coupling of Gs to 22AR monomers and oligomers. In addition, we will study the effects of ligands having different efficacies on interactions between the 22AR and Gs. Aim 2. Determine the structure of the 22ar-gs complex. We will take several complementary approaches to obtain a high-resolution crystal structure of the 22AR-Gs complex. We will also use single particle imaging by cryoelectron microscopy to study the structure of the 22AR-Gs complex in a native lipid environment. [unreadable] [unreadable] Public Health Relevance: The goal of this proposal is to determine the mechanism by which G protein coupled receptors (GPCRs) respond to hormones and neurotransmitters, and modify the function of cells. This information will facilitate the process of drug discovery for GPCRs, which are the largest family of membrane proteins in the human genome. Drugs acting on GPCRs can have an impact on a broad spectrum of diseases including: cardiovascular disease, pulmonary disease, inflammation, diabetes and obesity, behavioral disorders and Alzheimer's disease. [unreadable] [unreadable] [unreadable]

Chronic kidney diseases are major health problems that affect over 30 million Americans and the incidence is increasing at an alarming 16% in the last decade. The kidney is an intricate organ that requires coordinated regulation by distinct cell types and complex networks of genes to ensure its proper development and function. Kidney diseases are thus multifactorial and complex. The underlying causes to progression to kidney failure and the treatment and prevention of kidney diseases remain a challenge. We discovered that COUP-TFII, a transcription factor that regulates cell fate determination, embryonic development and adult organ function, plays a role in kidney development, function and disease. Several major cell types in the kidney express COUP-TFII. At the onset of kidney development (E10.5), COUP-TFII is expressed in the metanephric blastema, the urogenital ridge, and the metanephric mesenchyme (MM). Upon ureteric bud outgrowth, COUP-TFII is expressed in the condensed mesenchyme surrounding the ureteric buds and in the renal vesicle. At the nephrongenesis stage (E13.5), COUP-TFII becomes regionalized with high expression in the distal tubules and the glomeruli (podocytes and Bowman's capsule), but not detected in the proximal tubules. When COUP-TFII is conditionally ablated early, the MM cannot form properly and no kidney is formed. When COUP-TFII is knocked out at a later stage, very few nephrons are apparent and there is no detectable distal tubule, suggesting that COUP-TFII is critical for the formation and patterning of the nephron. Furthermore, adult mice with the loss of one COUP-TFII allele display polycystic kidneys, glomerulosclerosis (FSGS) and loss of kidney function, phenotypes resembling human kidney diseases. Preliminary results suggest that COUP-TFII regulates the expression of Angiopoietin 1, WT1, PKD1, TGF and many inflammatory genes, raising the possibility that COUP-TFII functions to protect the kidney from fibrosis, inflammation and from diabetic complications. To further define the defects of COUP-TFII mutants and dissect the underlying mechanism of COUP-TFII action, our specific aims in the next five years are: 1) Delineate the role of COUP-TFII in kidney development and its underlying mechanism; 2) Determine the role of COUP-TFII in kidney function and diseases; and 3) Determine the role of COUP-TFII in diabetic nephropathy. Understanding the precise role of COUP-TFII in these diseases will provide timely insights that could be used in therapeutic strategies for the treatment and intervention of kidney diseases.

ABSTRACT CORE B: Immunology Core Core Director: Roberto Calcedo, Ph.D. We are entering a new era for treating inherited genetic disorders where gene therapy holds great promise for their treatment. After several decades of research and development in viral vectors, we finally have the technology to efficiently transfer genes into cells. Unfortunately, several studies have shown that immune responses to the viral vector and transgene heavily influence in vivo gene therapy performance. The Immunology Core focuses its efforts on studying the natural existing T and B cell immunity to viral vectors used in gene therapy, Adenovirus (AdV) and Adeno-associated virus (AAV), in human and nonhuman primates (NHPs). We have also centered our studies on vector and transgene-specific T and B cell responses after systemic and local administration of various rAAV and rAdV serotypes carrying different transgenes in NHPs and in subjects from several ongoing clinical trials. This will help us to determine if rAAV and rAdV administration can re-activate existing or induce new T cell responses to the vector or the transgene and if these have any effect on the outcome of the gene transfer and the health of the patient. We are also interested in T cell responses to self-antigens especially in those subjects with genetic diseases where a significant portion of the gene is missing. Introduction of the correct copy of the gene in these subjects could induce an immune response to the transgene and compromise the treatment. We are evaluating these responses in CF subjects and found low to high levels of self-reactive CFTR-specific T cells directed to the mutated region of the CFTR. In all these studies, we isolated lymphocytes from blood, liver, intestine, primary and secondary lymphoid organs and characterized the T cell response by ex vivo IFN? ELISPOT, lymphoproliferation assay, in vivo CTL assay, and polychromatic flow cytometric analysis of antigen specific T cells. PHS 398/2590 (Rev. 09/04, Reissued 4/2006) Page Continuation Format Page

To prepare transition state models of the peptide bond formation step of the protein synthesis. According to the current views, the biosynthesis of a peptide bond proceeds via a ribosomal complex of aminoacyl and peptidyl tRNA. It is proposed to prepare bifunctional analogs of both tRNA's with covalently linked terminal adenosine residue and investigate the potential transfer of peptide chain to aminoacyl residue in non-ribosomal and ribosomal systems. Simplified bifunctional analog - 2'(3')-O-acetyl-1',2-bis-(adenosin-N6-yl. ethane was prepared.

The long term objective of this application is to further develop two lead compounds, MAP 30 and GAP 31, which have shown three activities that may be critical to the antiviral action. These include RNA N-glycosidase, DNA topoinactivation,and HIV-integrase which are critical target in selective antiviral therapy. In the revised application Dr. Lee-Huang proposes to study the followings: Peptide fragmentation and activity mapping of MAP 30 and GAP 31 to define the domains responsible for anti-HIV activity, ribosome inactivation, DNA tipoinactivation and HIV-integrase inhibition; recombinant expression and site-specific mutagenesis to facilitate rational development of effective anti-HIV mimetic. The identification of domains responsible for these activities of MAP 30 and GAP 31 will be important in: revealing the requirements for the structure and function of these proteins; defining the extent to which these activities, or whether in all of them, contribute to the anti-HIV action; and identifying particular peptide sequences or fragment that confer anti-HIV activity. This may provide ideal therapeutic tools for the treatment of AIDS. Such defined peptides may be conveniently synthesized and easily modulated for improved efficiency. Many AIDS organizations and AIDS patient groups have expressed strong support of her work. This work may provides insight into the mechanism underlying the antiviral activity of these compounds, and will hopefully contribute to the development of clinically useful drugs.

DESCRIPTION: (Applicant's Abstract) The aims are to explore behavioral effects of some abused drug mixtures, to use recently developed methods to analyze discriminative effects of single drugs with multiple effects, and to explore the potential of Occasion Setting in relation to drug action. Both mixtures of drugs and single substances with multiple effects will be analyzed as compound interoceptive stimuli, bringing to drug action the theoretical framework of associative learning that is more commonly used in studies of compound exteroceptive stimuli. The emphasis will be upon mixtures of which one component is an opioid, notably mixtures of heroin plus cocaine ("speedballs") and of pentazocine plus tripelennamine ("T's & blues"). Experiments will use methods developed previously in the project: [1] the AND-discrimination procedure will provide sensitive measures for detecting interactions between drugs in mixtures and [2] the AND-OR discrimination procedure will be used to enhance the pharmacological specificity of stimuli produced by mixtures. The first experiments on Occasion Setting with drugs in two-lever paradigms will be carried out. The specificity of discriminative stimuli produced by mixtures of abused substances such as heroin plus cocaine will be evaluated in tests with pharmacologically similar drugs and drug mixtures, and with compounds from other pharmacological classes. Similarities and differences between different abused and non-abused mixtures will be determined to identify the key drug actions responsible for the characteristic effects of the mixtures. These techniques will be used to analyze in greater depth than previously the discriminative effects of single opioid drugs with multiple effects; the mixed agonist-antagonist opioids cyclazocine, pentazocine, buprenorphine and butorphanol may produce their characteristic effects and patterns of abuse through different profiles of effects across m- and k-opioid, NMDA and s receptors. Cyclazocine will be a reference drug and experiments with it will be primarily to standardize methods; the other mixed agonist-antagonists have different, less well-understood effects and neuropharmacological actions, but are of greater present-day clinical significance. Profiles of effects of these drugs will be examined for their intrinsic importance and to inform studies on their actions in mixtures with tripelennamine and cocaine.

Methamphetamine [(+)]methamphetamine, or (+)METH] has been a serious drug problem in the United States since the middle 1980s. It is by far the most prevalent synthetic controlled substance clandestine manufactured in the United States and it is abused throughout the world. Although there has been good success in the development of therapeutic drugs to treat some types of drug abuse, at this time there is no pharmacological treatment of (+)METH abuse that has clinical acceptance. This project will use antibody-based therapies as "pharmacological antagonists" of (+)METH abuse. (+)METH abuse will be studied using two animal models, drug self-administration and drug discrimination. Drug self-administration is generally considered to be a model of the reinforcing properties of (+)METH and drug discrimination is considered to be a model of some of the subjective effects of (+)METH and drug discrimination is considered to be a model of some of the subjective effects of (+)METH that presumably contribute to its abuse. Anti-(+)METH antibodies will be screened for their ability to reduce (+)METH self-administration and to block the discriminative stimulus effects of (+)METH in rats. The most promising antibodies will be subjected to more rigorous testing to determine dose-response relationships, duration of action, specificity for blocking the effects of other (+)METH-like drugs, ability to prevent relapse to (+) METH abuse, and interaction with behavioral treatments for (+)METH abuse. Experiments on the effects of the most promising anti-(+)METH antibody on (+)METH self administration will be repeated in squirrel monkeys to develop a preclinical data base for the testing of these antibodies as pharmacotherapy for (+)METH abuse in humans.

Corneal infection with herpes simplex virus-1 (HSV-1) causes herpetic stromal keratitis (HSK), a leading cause of infection-induced corneal blindness worldwide. Despite intensive research and substantial progress in understanding the pathogenesis of HSK, the management of this condition continues to be challenging. One significant hurdle is our poor understanding of the role of neuropeptides, secreted by abundant corneal nerve fibers, in the development of HSK lesions. Lack of such knowledge is an important problem because neuropeptides are well known to regulate both inflammation and tissue repair. The objective of this application is to determine what regulates substance P (SP) neuropeptide levels in the cornea after ocular HSV-1 infection, and how SP interactions with its receptor NK1R control the viral load and affects the development of HSK lesions. We will approach these questions in a mouse model by using SP-/- and NK1R-/- mice. We recently reported that blocking SP-NK1R interactions in the absence of replicating virus (clinical period) significantly reduced the development of HSK lesions. However, lack of SP-NK1R interactions during active viral replication in the cornea (pre-clinical period), enhances the severity of HSK lesions, as noted in SP- /- and NK1R-/- mice. Our preliminary results showed delayed viral clearance and reduced levels of epidermal growth factor (EGF), a corneal wound healing factor, in NK1R-/- mice. On the basis of our preliminary results, we hypothesize that SP, produced in HSV-1 infected corneas by macrophages and corneal nerves, promotes corneal tissue repair and regulates the influx, survival, and function of innate immune cells involved in viral clearance therefore, enhancing SP-mediated effects during active viral replication should reduce the development of severe HSK lesions. Three aims are proposed to address our hypothesis. In aim 1, experiments will be carried out to determine the role of corneal macrophages and sensory neurons in regulating the levels of SP peptide in HSV-1 infected corneas during the pre-clinical and clinical periods of HSK, respectively. In aim 2, experiments will be carried out t determine whether SP-NK1R interactions regulate viral load in the cornea by promoting the migration, survival, and functions of dendritic cells (DCs), inflammatory monocytes (IM) and natural killer (NK) cells in HSV-1 infected cornea. In aim 3, we will determine whether enhancing SP-mediated effects during the active viral replication period through topical application of EGF alone, or in combination with subconjunctival administration of SP, modulates the development of severe HSK lesions. The information generated by this study could aid in the development of novel pharmaceutical strategies to reduce HSV-1 induced chronic inflammation by promoting viral clearance and corneal tissue repair.

Spotted-glass microarrays and Serial Analysis of Gene Expression (SAGE) have been combined successfully to identify downstream targets of FIGLA. Taking advantage of Figla null mice and the NIA 22K microarray that contains elements enriched for expression in oocyte and early development, ovarian gene expression was profiled at four embryonic time points from E12.5 to newborn. However, microarrays are limited by the elements spotted on glass during their fabrication. Therefore, to broaden the search for potential direct or indirect downstream gene targets, SAGE libraries were constructed from poly(A)+ RNA isolated from newborn normal and Figla null ovaries. A 10 base tag immediately adjacent to the 3 prime most Sau3A1 restriction enzyme cleavage site was used to identify both up-regulated and down-regulated transcripts. The further characterization of the genes that are differentially regulated by FIGLA should prove useful in defining developmental pathways that affect the postnatal female germ cell. These targets represent not only genes that affect folliculogenesis and fertilization, but also maternal effect genes required for successful completion of early mouse development.[unreadable] [unreadable] In an alternative approach to identify additional downstream targets of FIGLA, two-dimensional gel electrophoresis was used to compare the protein expression profiles of newborn ovaries isolated from normal and Figla null mice. Differentially expressed proteins were identified by microscale mass spectrometry and differences were confirmed at a transcriptional level by quantitative RT-PCR. Unexpectedly, twenty testis-specific proteins were over-expressed in Figla null newborn ovaries suggesting that FIGLA is not only an activator of oocyte-specific genes, but also a repressor of testis-specific genes, during female gonadogenesis. To further investigate this hypothesis, transgenic mouse lines ectopically expressing FIGLA in male germ cells were established. Although fertile, transgenic male mice expressing FIGLA had decreased fecundity and as mice aged, vacuoles were sporadically observed within seminiferous tubules consistent with patches of germ cell apoptosis. Collectively, these data indicate that FIGLA plays a critical role as the transcriptional activating of oocyte-specific and repressor of testis-specific genes necessary for successful development of the female gonad.

The overall objective of our work is to understand the mechanisms of glomerular endothelial (GEn) cell differentiation. The current proposal explores the role of podocytes in this process, podocytes being the immediate neighbors of GEn cells in vivo. In mature glomeruli, podocytes express extraordinarily high levels of the angiogenic growth factor VEGF, yet GEn cells in vivo ordinarily do not proliferate and do not undergo angiogenesis. indeed, even in diabetic patients with retJnopathy and nephropathy, retinal angiogenesis is stimulated but glomerular angiogenesis is not observed. We propose that podoctye derived signals are instrumental in locking the GEn cells into a terminally differentiated state. We find that in vitro GEn-podocyte co-culture using monolayer and trans-filter assays, results in the inhibition of GEn migration, and strong protection of GEn cells from apoptosis. Hence, podocytes, in co-culture with GEn cells produce soluble anti-apoptotic, anti-chemokinetic mediators. We know that podocytes produce two relatively endothelial cell specific survival factors; angiopoietin-1 and VEGF-A. We will therefore explore the hypothesis that angiopoietin-1 and/or VEGF-A serve survival and differentiation functions for GEn cells in vitro and in vivo. Both, angiopoietin-1 and VEGF stimulate endothelial cell migration in vitro, but differentiated podocytes inhibit GEn migration. Inhibition of VEGF- stimulated migration by endothelial cells is typically seen when metalloproteinase activity is blocked. In this regard, we find that GEn ceils express abundant collagen XVIII, and generate soluble endostatin-Ilike collagen XVlli NCl domain cleavage products, likely through the actin of matrix metalloproteinases. Podocyte co-culture completely abrogated production of the endostatinlike collagen XVill fragments. We therefore propose to test the hypothesis that podocytes inhibit matrix meta[ioproteinase activity, and consequently suppress the angiogenic phenotype of GEn cells. We propose to use GEn cells and podocytes in culture to define the potential role of three putative podocyte derived mediators, VEGF-A, angiopoietin-1 and metalloproteinase inhibitors in GEn differentiation. In addition, using conventional light and electron microscopy, as well as intravitai microscopy, we will explore the role of these podocyte derived stimuli in the process of glomerular capillary differentiation in neonatal mice in vivo.

Exercise stress testing with imaging is widely used to detect heart disease, but current stress imaging systems suffer from false positives that may lead to unnecessary invasive testing, and false negatives that may miss disease detection until a serious event occurs. Thus, consistently accurate stress imaging remains an important target for technology development. Cardiac magnetic resonance (CMR) provides superior imaging of the heart without ionizing radiation, but technology has not been developed to allow CMR immediately following maximal exercise stress. A revolutionary change to the current landscape of cardiac stress testing could result by adding exercise testing to CMR, thus establishing a one-stop imaging modality for accurately assessing heart disease in a single examination. This project focuses on the research and commercialization of an MRI-based system for enhanced exercise stress testing for patients with suspected cardiovascular disease. The key enabling technology is an innovative non-ferromagnetic treadmill that enables convenient placement immediately adjacent to the MRI machine. Standard treadmills would have to be placed far from the MRI magnet outside the MRI room, leading to critical delays between exercise and image acquisition, as well as safety concerns for compromised patients who must, in a fatigued and stressed state, traverse the distance from the treadmill to the MRI table. Our innovative design overcomes these problems and thus enables image acquisition immediately post-exercise. The resulting high resolution images are expected to be superior to nuclear single photon emission tomography (SPECT) and ultrasound, and clearly show stress wall motion, stress perfusion, and viability. The design, implementation, and feasibility testing of this new technology has been completed (Phase I), and this proposed comparative effectiveness study (Phase II) is designed to evaluate how improved test accuracy translates into downstream cost savings. The anticipated positive outcome of this study is expected to lead to a sizeable commercial opportunity for the manufacture and sale of MRI stress testing equipment. Phase II of this project will be accomplished by meeting the following specific aims: Aim 1. Demonstrate that the technical advantages of treadmill stress CMR over treadmill stress SPECT result in a superior ability to measure the extent and severity of anatomic disease, and equivalent or superior diagnostic and prognostic accuracy. Aim 2. Demonstrate superior cost-effectiveness of treadmill stress CMR vs. treadmill stress SPECT in a prospective, randomized trial. Successful achievement of the aims of this Phase II project is expected to lead to successful introduction of the treadmill CMR system as a commercial product. Treadmill exercise stress CMR could greatly enhance our understanding of CAD, and enable earlier diagnosis and more effective treatment strategies.

To develop rabbit as a model system for HIV-associated neuropathogenesis and to determine if HIV-1,br (an isolate from the brain of an AIDS dementia patient, Virology, 1989;168:79-89) has unique neuropathic characteristics, a 36-day-old rabbit was inoculated intraperitoneally with cell-free HIV-1,br. The rabbit was treated with thioglycollate four days prior to inoculation and was then bled for serum and lymphocytes approximately at biweekly intervals. The rabbit was sacrificed 90-days after inoculation and its brain, spleen, thymus and adrenals were removed. Collected lymphocytes and tissues were tested for markers of infection and sera were tested for the presence of antibodies to HIV-1. Lymphocytes were negative for the presence of HIV-1 in cocultivation assays. Of the tissues tested, only the brain was positive for the presence of HIV-1 in cocultivation, although spleen, thymus and adrenals were weakly positive by PCR. The brain had the highest level of HIV-1 DNA. RNA-PCR showed the presence of viral RNA in the brain samples. The serum samples were negative for the presence of HIV-1 specific antibodies both by the ELISA and Western blot methods. Amplified gag gene fragment was molecularly cloned and sequenced which proved unequivocally that HIV-1 was present in the brain. In conclusion, HIV-1,br established infection in the brain in the absence of detectable seroconversion. Currently, studies are planned to investigate the incidence of brain infection with HIV-1,br and also to examine the relative predilection of HIV-1,br for the rabbit brain as compared to the other HIV-1 isolates.

Heavy alcohol consumption in an HIV-infected person may accelerate HIV disease progression and end organ disease with one leading explanatory pathway being via enhanced microbial translocation and inflammation/altered coagulation. Heavy alcohol consumption and HIV infection are both causes of microbial translocation, the process by which bacterial products leak across the gastrointestinal membrane with resultant destructive immune activation. Alcohol can lead to microbial translocation directly through zinc deficiency. Low zinc levels, affecting 30-50% of alcohol dependent persons, are associated w ith reduced immune function and HIV disease progression. Among HIV-infected people, high levels of microbial translocation (as measured by soluble CD14) and inflammation/altered coagulation (as measured by D- dimer) are each associated with an increased risk of death. Of importance, among HIV-infected persons, heavy drinking is also significantly associated with higher levels of D-dimer in cross-sectional studies. Of note, initiation of antiretroviral therapy (ART) is associated with a reduction in D-dimer levels. Yet the following is not known: is there a longitudinal relationship between alcohol consumption and these biomarkers independent of ART; and does zinc supplementation improve biomarker levels? Answering these questions requires a setting with prevalent HIV infection, heavy alcohol use and limited current ART use. As Russia has a relatively young, expanding HIV epidemic, a growing HIV treatment infrastructure and enormous per capita alcohol consumption, St. Petersburg is a setting where this research is possibi e. Thus, as part of the Uganda, Russia, Boston Alcohol Network for Alcohol Research Collaboration on HIV/AIDS (URBAN ARCH) Consortium, we seek to create the Russia ARCH cohort from participants of a recently completed NIAAA-funded randomized controlled trial (RCT) of HIV-infected Russian heavy drinkers and perform the following: [1] an assessment of the longitudinal association between alcohol consumption and biomarkers of microbial translocation (sCD14) and inflammation/altered coagulation (D-dimer); [2] implementation of the Zinc Intervention Nested in a Cohort (ZINC) study, a double-blinded, placebo controlled, RCT to determine if short term zinc supplementation results in lower biomarker levels and whether the effects differ based on alcohol consum ption. These studies will clarify the association between alcohol and key biomarkers overtime and evaluate a pragmatic treatment, zinc supplementation, as a therapy in HIV-infected heavy drinkers.

Inhibition of intestinal mucosal growth occurs commonly in various critical surgical disorders such as trauma, shock, and massive surgical operations. Because our deficient understanding of the mechanism underlying this critical pathological process, effective therapies to maintain the mucosal epithelial integrity in patients with critical surgical illnesses are limited, leading to mucosal atrophy, delayed healing, impaired barrier function, and bacterial translocation. Recently, the essential contribution of posttranscriptional events, particularly altered mRNA turnover and translation, in the control of gene expression program in the GI mucosa is becoming increasingly recognized, but little is known about their importance in the pathogenesis of mucosal growth inhibition in critical surgical conditions. Based on our significant progress during the previous funding period and exciting preliminary studies, experiments proposed in this competitive renewal application are to test the HYPOTHESIS that the RNA-binding protein CUGBP1 plays an important role in the regulation of intestinal mucosal growth by altering expression of its target mRNAs encoding growth-regulatory proteins and its effect is regulated by given microRNAs and cellular polyamines. Three specific aims are proposed to test the hypothesis. 1) To determine the exact role of CUGBP1 in the regulation of gut mucosal growth in critical surgical conditions and to further identify its target mRNAs. 2) To determine whether microRNAs and CUGBP1 jointly regulate the stability and translation of target mRNAs in response to surgical stress. 3) To define the mechanisms by which polyamines regulate CUGBP1 expression and microRNA biogenesis. Completion of these specific aims will uncover novel functions of CUGBP1 and microRNAs in the pathogenesis of gut mucosal growth inhibition and will impact upon efforts to improve therapeutical approaches for patients suffering gut mucosal atrophy. It is hoped that these studies will identify potential therapeutic targets and agents which could be used in the future to maintain intestinal mucosal integrity in patients with critical surgical illnesses.

There is substantial evidence that aggressive and/or agitated behavior, whether externally or self-directed is associated with low indices of serotonin function, suggesting dysfunction of the central serotonin system. Decreased functioning of central serotonin systems may lead to dysregulated and disruptive behavior in the AD patient. The technique of tryptophan depletion (TD) allows a rapid, but short-lived lowering of brain serotonin levels. TD is performed by administering an amino acid mixture which lacks tryptophan. This mixture induces liver protein synthesis, which rapidly depletes circulating tryptophan (>80% in 5 hours), which in turn depletes brain tryptophan. Eight (8) AD patients in the early stages of illness (GDS stage 2-3) will be administered a tryptophan-deficient amino acid mixture early in the morning. Their behavior will be monitored throughout that day and through tryptophan repletion until the next morning. A control (no depletion) day will also be performed. If significant behavioral changes are seen with tryptophan depletion, this will suggest that further attempts to use this technique to understand the etiology and phenomenology of behavioral disturbances in this disorder are justified. We have run 8 Alzheimer's disease patients in this protocol. Data is currently being analyzed for this study.

The overarching goal of this Senior Scientist Award (K05) application is to provide the candidate with a period of stable funding in which to conduct full-time programmatic research on the developmental patterning of familial transmission of biobehavioral factors that contribute to the transgenerational manifestations of conduct deviation and substance use disorders (SUD). The emerging discipline of developmental psychopathology attempts to elucidate the etiology, course, and sequelae of conditions such as SUD, Conduct Disorder (CD), and Antisocial Personality Disorder (ASPD) through the integration of knowledge from multiple disciplines such as psychology, psychiatry, genetics, epidemiology and neuroscience within a developmental orientation. Basic researchers and theorists have posited that critical developmental periods exist when particular familial or unique environmental factors are most influential in determining an adverse psychopathological phenotype. Limited research has been conducted on the presence and timing of critical developmental periods in the etiology of CD, ASPD and SUD, yet such information is vital to the efficacious timing and nature of targeted drug abuse prevention interventions. The proposed program of investigation aims to undertake systematic prospective research into the nature and predictors of transmissible and non-transmissible developmental risk for conduct deviancy, substance use and SUDs beginning in the prepubertal period and continuing across the transition from childhood to adolescence and young adulthood. The research will also examine sex heterogeneity of transmissible influences. The existence of sex heterogeneity is plausible since differential transmission effects have been described between same- sex-parent to child transmission for externalizing behaviors. The results have important implications for drug abuse prevention.

Interviews were conducted with 284 mothers and their teen-age children from three ethnic groups in an urban ghetto community (Grant No. DA 01103-01/02). The 284 mothers had 403 adolescents ages 13-17 (138 white children, 141 American blacks, and 124 British West Indian blacks). The interviews focused on socialization patterns associated with experimentation with drugs. The analysis to date of the extensive data set reveals strong and consistent patterns relevant to a variety of basic themes. In addition to demographic and social data a large number of scales have been obtained from parents and teen-agers on important socialization themes. This proposal requests support to return to these households in order to (1) resolve problems of interpretation of cross-sectional data which a panel analysis can facilitate; (2) to obtain selected information relevant to specific themes under investigation; and (3) to estimate the reliability of selected variables significant for the interpretation of findings, particularly self-reports on drug use. The time-lag of two years from the first wave (summer 1974) will result in a considerable increase in the use of drugs, and especially "harder" drugs as the teen-age sample will be 15-19, adding considerable power to the analysis of the antecedents of drug-taking behavior.

The Ohio State University College of Optometry proposes to establish an institutional training program for clinician-scientists in primarily patient-based research, with one lead mentor available for a trainee interested in molecular biology. At the outset, two trainees will enroll in the program. They will have Doctor of Optometry (OD) degrees and will pursue PhD degrees in Vision Science. Trainees will complete coursework and mentored research to end up with a key skill set. They will emerge with knowledge and experience in vision science (optics, binocular vision, monocular sensory processes, ocular anatomy and physiology), molecular biology (cell biology, immunology, genetics), epidemiology, biostatistics, clinical study design, ethics in biomedical research, scientific writing, and the dissemination of scientific results. Trainees will complete intensive coursework in molecular biology, cellular biology, and genetics to facilitate training in translational research. Karla Zadnik, OD PhD, Glenn A. Fry Professor in Optometry and Physiological Optics, will serve as the Program Director. Since 1999, Dr. Zadnik has served in the same role on the OSU College of Optometry's K30 grant. "Clinical Research Curriculum," funded through the National Heart, Lung, and Blood Institute. The Program Advisory Committee will consist of Ronald Jones, OD PhD (chair of the Program Advisory Committee and Chair of the Graduate Studies Committee at the College of Optometry); P. Sarita Soni, OD MS (Director of the Borish Clinical Research Center at the Indiana University School of Optometry and Program Director of the new K12 program there); Michael Robinson, PhD, Assistant Professor at Children's Hospital, OSU College of Medicine and Public Health; and Melvin L. Moeschberger, Professor, Division of Epidemiology and Biometrics, School of Public Health, OSU College of Medicine and Public Health. Dr. Zadnik will serve as an ex officio member of the Program Advisory Committee. Potential lead mentors in the Graduate Program in Vision Science will include faculty members who have graduate faculty status within the OSU Graduate School that allows them to supervise a PhD student and who also have federal grant funds (listed below). Specific training programs will be developed by trainee applicants and their lead mentors. The first trainee will be Michael D. Twa, OD MS, and one slot will be left open to recruit a trainee at the time of funding.

Addiction to ethanol is a significant problem in our society and is a leading cause of morbidity and mortality in the population. Major changes in neurochemistry, such as in the brain levels of the opiate peptide methionine enkephalin Met-En k , have been associated with exposure to ethanol. It is our long term goal to elucidate the mechanisms and significance of these changes in Met-Enk and to design therapeutic interventions directed at their reversal. The level of Met-Enk in the brain has been linked to ethanol ingestion and withdrawal seizures while treatment with enkephalins, their analogs, or blockers of their enzymatic degradation can decrease ethanol drinking and prevent ethanol withdrawal seizures. The level of Met-Enk in the brain is determined by a balance of synthesis, release, degradation, and transport across the blood-brain barrier BB by PTS-1, which carries Met-Enk from the brain to the blood. Our work has confirmed that PTS-1 is a major regulator of the level of Met-Enk in the brain of ethanol-naive mice and that during physical dependence to and withdrawal from ethanol the control of the level of Met-Enk in the brain is post-translational and mediated at least partially by PTS-1. In the requested funding period, we will determine the relative importance during physical dependence to and withdrawal from ethanol of PTS-1 to that of other post-translational mechanisms that affect Met-Enk levels in the brain.

PROJECT SUMMARY Up to 50% of peripubertal youth with anxiety have unmet clinical needs, leaving these youth at high risk for suicide, depression and substance abuse across adolescence. In accord with the NIMH strategic plan, we aim to deepen mechanistic understanding of anxiety during the sensitive period of peripuberty to inform novel treatments and reduce health risks. This proposal focuses on negative overgeneralization, which is a core dimension of anxiety that is poorly understood, and refers to the tendency to generalize aversive responses from one context (house fire) to other contexts (camp-fire) that share features. Amygdala activity, induced by heightened emotional arousal, enhances plasticity in associative learning mechanisms, facilitating the binding of contextual features in memory that are only loosely related. We posit that sleep plays a critical role in negative overgeneralization. Specifically, we draw from basic neuroscience to propose a model by which heightened amygdala reactivity during wakefulness, induced by increased emotional arousal, facilitates replay of negative memories during sleep. This facilitated replay leads to the stabilization and integration (consolidation) of negative memories into long-term memory networks via slow wave oscillatory events during NREM sleep. We further propose that facilitated replay of negative memories during sleep promotes generalization by influencing underlying neurocomputational mechanisms (i.e., pattern completion ? a computational process that makes neural representations similar). Finally, we propose that sleep-dependent consolidation is malleable, such that Targeted Memory Reactivation (TMR) of positive memories during sleep can competitively displace consolidation of negative memories, and reduce negative overgeneralization. The current proposal tests this model using a novel multi-method approach combining neuroimaging, polysomnography, and a memory task that captures behavioral generalization and its underlying neural mechanisms (i.e. pattern completion). Aim 1 examines 200 peripubertal youth (ages 10-13 years) across a full continuum of anxious symptoms in a randomized sleep (n=140) versus wake (n=60) design to demonstrate sleep-dependent effects on behavioral and neural mechanisms of negative overgeneralization. Aim 2 focuses on the 140 youth in the sleep condition to evaluate amygdala reactivity at encoding and sleep neurophysiology during post-encoding sleep as mediators between anxiety and negative overgeneralization. Aim 3 uses the same design as the sleep condition but recruits a new sample of youth with elevated anxiety (n=60) to enroll in a randomized trial in which positive memories are cued during sleep (TMR, n=30), or sham cues are presented during sleep (n=30), to examine malleability of sleep-dependent mechanisms of negative overgeneralization. This project will set the stage for the long-term goal of developing novel interventions that manipulate sleep (e.g. via TMR) not only to improve existing symptoms, but also to positively shape neurodevelopment and reduce risk in the sensitive period of peripuberty.

Abstract In everyday experience, we effortlessly recognize faces, objects and scenes, yet the mechanisms underlying how we perceive the world remain elusive. Moreover, it is unclear how cognitive processes such as selective attention and memory interact with object recognition. The conference on Object Perception Attention and Memory (OPAM) is a one-day conference that showcases young investigators (i.e. undergraduate, graduate and post-doctoral trainees) and their theoretical and empirical work on object processing in terms of attention, perception and memory. In addition, this conference highlights different methodological approaches to understand object processing including psychophysics, neuroimaging and neuropsychology. The goal of OPAM is three-fold: (1) to foster scientific research from junior investigators, (2) to facilitate interactions between junior and senior investigators, and (3) to provide a venue for a concentrated discussion on the role of attention and memory on object perception.

In vertebrates, there are over 15 different myosin II isoforms, each of which contains different myosin II heavy chains (MHC IIs). MHC II isoform diversity is generated by multiple genes as well as by alternative splicing of pre-mRNA. Previous studies have demonstrated cell type-specific expression of MHC II isoforms as well as changes in MHC II isoforms during muscle and neural tissue development. This research program has investigated the regulatory mechanisms responsible for tissue-dependent alternative splicing of two nonmuscle MHC II (NMHC II) genes, NMHC II-B and NMHC II-C. Our past study revealed that an RNA binding protein family, the Rbfox family, plays a critical role for neuron-specific alternative splicing of NMHC II-B pre-mRNA. In this report, we focus on function of Rbfox proteins. Rbfox proteins contain a single conserved RNA recognition motif (RRM) in the central region of the molecule and bind specifically to an RNA penta(hexa)nucleotide (U)GCAUG. There are three genes for Rbfox family proteins in mammals, Rbfox1, Rbfox2 and Rbfox3. Rbfox1 is expressed in brain and striated muscles whereas Rbfox2 is expressed in various tissues including brain and muscles. Notably, Rbfox3 expression is restricted to neural tissues. Biochemical analyses of brain cells sorted by Rbfox antibody staining and histological analyses demonstrated that the expression level of the neuron-specific splice variant of NMHC II-B mRNA correlated better with the level of Rbfox-3 expression rather than with that of Rbfox1 or Rbfox2 expression. These observations suggest that Rbfox3 contributes to neuron-specific splicing of NMHC II-B mRNA, although brain expresses all three Rbfox proteins. Next we extended our research to study the biological function of Rbfox3 using two model systems. First we made use of mouse embryonic carcinoma P19 cells which are capable of differentiating into neuronal cells following retinoic acid treatment. Neuronal differentiation of P19 cells can be monitored by outgrowth of a long axon-like extension which contains an axonal marker, phosphorylated neurofilaments. During neuronal differentiation, expression of Rbfox3 is induced whereas undifferentiated P19 cells do not express Rbfox3. Rbfox1 is barely detected under both undifferentiated and differentiated conditions and the Rbfox2 expression level is unchanged before and after differentiation. The shRNA-mediated knock-down of Rbfox3 results in a decrease in axon-like extensions and an almost complete elimination of phosphorylated neurofilaments. These results indicate that Rbfox3 is required for neuronal differentiation of P19 cells. To understand the molecular mechanism for Rbfox3 function in neuronal differentiation of P19 cells, we carried out Photoactivatable-Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation (PAR-CLIP) to identify transcriptome-wide target RNAs of Rbfox3 in neuronally differentiated P19 cells. Unexpectedly, we found that Rbfox3 interacts with a much broader range of RNA sequences other than UGCAUG, and that pri-miRNAs are over-represented in Rbfox3 targets. Rbfox3 binds specifically to a subset of pri-miRNAs and functions as either a positive or negative regulator in pri-miRNA processing. Second we used the chicken embryonic spinal cord to study a role for Rbfox3 in neuronal development in vivo. SiRNA-mediated loss of function and rescue experiments showed that Rbfox3-regulated alternative splicing of Numb, which encodes a signaling adapter protein and plays an essential role in differentiation of motor neurons and interneurons during spinal cord development. During characterization of chicken Rbfox3, we also found that the early chicken embryo expresses two Rbfox3 splice isoforms, the full-length Rbfox3 (Rbfox-full) and the 31 amino acid-deleted Rbfox3 (Rbfox3-d31). The latter has a defective RRM and lacks the RNA-binding activity. Rbfox3-d31 mRNA is highly expressed during the early developmental stages of the chicken embryo, while Rbfox3-d31 protein is barely detected during the same stage due to its rapid degradation mediated by the ubiquitin-proteasome pathway. Importantly, this degradation is specific to the Rbfox3-d31 isoform and it does not occur with Rbfox3-full. These results suggest that Rbfox3 protein level is regulated at multiple levels including transcription, alternative pre-mRNA splicing and protein degradation in vivo.

The goal of this research is to investigate the biological function of endogenous bombesin-like peptides in oat cell carcinoma. Bombesin-like peptides are present in the normal lung in low densities; however, in human small (oat) cell lung cancer (SCLC) biopsy tissue and cells in culture the density of immunoreactive bombesin (BN) is elevated by up to three orders of magnitude. In particular, 32 of 34 SCLC cell lines had measurable levels of BN (94%). Of the 32 positive cell lines, BN levels ranged over 180-fold (0.1-18.3 pmol/mg protein). In contrast, immunoreactive BN was not detectable (less than\0.1) in seven non-SCLC lung cancer, one hypernephroma, two melanoma and three breast cell lines. Therefore, BN-like peptides may serve as unique biochemical markers to monitor the progression of SCLC. These peptides may be stored in the dense core neurosecretory granules present in SCLC cells. When these granules undergo exocytosis, peptides may be secreted into the extracellular fluids. In vitro, immunoreactive BN is secreted from SCLC cell lines into the tissue culture medium by elevating the extracellular K+ or intracellular cAMP concentrations. In vivo, the normal level of immunoreactive BN present in human plasma is 50 fmol/ml. In patients with extensive SCLC, however, the levels may be elevated 40-fold to 2 pmol/ml. Hypersecretion of BN-like peptides from oat cells would likely account for some paraneoplastic syndromes associated with SCLC such as impaired ability to thermoregulate and anorexis. Because BN may function as an important regulatory agent in SCLC, the presence of receptors or BN-like peptides was investigated. In particular, SCLC cell line NCI-H446 had a high density of sites (2000/cell) which bound BN with high affinity (Kd, 0.5 nM). These cell surface receptors, when activated by endogenous BN0-like peptides, may mediate the growth of oat cell carcinoma.

Addiction to opioid drugs such as morphine is a major public health concern. The complex pathology of opioid addiction can be initiated by activation of specific drug targets in the brain, and the main target of abused drugs is the mu- opioid receptor (MOR), a member of the G protein-coupled receptor (GPCR) family. Activation of GPCRs elicits a sequence of events that results in regulated receptor removal from the cell surface by endocytosis. In the case of MOR, receptor endocytosis controls the de-sensitization and re-sensitization of the neuronal response to MOR signaling, and affects the long-term cellular changes that lead to the development of drug tolerance and dependence. While the traditional view is that regulation of receptor endocytosis is achieved by controlling receptor interaction with the endocytic machinery, my recent studies have identified a novel mechanism by which GPCRs, including MOR, specifically modulate their own endocytosis by controlling the local endocytic machinery. This suggests a novel and unanticipated facet of opioid regulation. The proposed studies seek to identify the mechanistic basis of this regulation and to investigate its functional significance to the effects of clinically relevant opioid drugs. Specifically, this proposal aims to: 1) identify structural determinants on MOR that mediate regulation of the endocytic machinery; 2) establish its mechanistic basis by identifying endocytic regulatory proteins; 3) determine the effect of different opioid drugs on this regulation in physiologically relevant neurons; and 4) define the functional consequences of this regulation on MOR signaling. CANDIDATE: The applicant has prior training in cell biology and biochemistry, and is committed to pursuing independent research in the cellular neurobiology of neuropsychiatric disorders and drug addiction. He will be mentored by Dr. Mark von Zastrow in the pharmacology, molecular genetics, and neurobiology of signaling receptors implicated in these disorders. RELEVANCE: The results of these studies will provide insight into a novel mode of regulation of a key opioid receptor, and improve our understanding of the development of drug tolerance and dependence. Thus they have the potential to serve as a platform for designing better and more informed therapeutic strategies against neuropsychiatric disorders and drug addiction. Further, the general principles defined will likely have broad implications to signaling events underlying a variety of pathological conditions. [unreadable] [unreadable] [unreadable]

The understanding of cancer has evolved rapidly over the last decade, particularly with discoveries regarding the role of physical factors, such as extracellular matrix (ECM) stiffness and cellular forces, in carcinogenesis. This research has shown that altered ECM stiffness is not just a symptom of tumors, but is now known to trigger the actual onset of and progression of malignancy. Another key finding established by the Co-Investigator of this grant, is that cellular traction stresses increase with increasing metastatic potential, suggesting that cell traction forces could be a biomarker for the likelihood of metastasis. Additionally, it has been found that (2D) collective behavior of cell populations can be significantly different from that of isolated cancer cells, and that cell migratory behavior in 3D matrices is significantly different migration on 2D surfaces. Although this has motivated the adoption of 3D microenvironments in cancer mechanobiology research, current imaging methods to quantify ECM mechanical properties and local cellular forces only provide 2D imaging, or when they do support 3D imaging, they do not provide long-range volumetric measurements of collective mechanical behavior with cellular resolution. The central objective of this proposal is to demonstrate the feasibility of traction force optical coherence elastography (TF-OCT) for volumetric time-lapse imaging of cellular forces over distances that are long with respect to a single cell. We will utilize this to map local mechanical forces associated with individual and collective cancer cell migration in physiologically relevant assays for studying local invasion and early cancer metastasis in vitro. Aim 1 will develop and demonstrate the advantageous use of aberrated optical systems combined with computational image formation methods in OCT to enable cellular-resolution imaging over millimeter-scale volumes without having to scan the focus in depth. This aim will also develop 3D bead- tracking algorithms for OCT-based TFT. Aim 2 will apply this new imaging capability to image cell forces associated with single and collective cell migration in 3D microenvironments. These experiments will take advantage of a physiologically-relevant tumor spheroid assay developed by Co-Investigator Dr. Reinhart-King, and be patterned after these recent cell migration and ECM remodeling studies, but will now provide the volumetric time-lapse imaging of cellular forces. Imaging of the spatiotemporal forces associated with invasion dynamics of single/isolated highly metastatic and invasion-compromised breast adenocarcinoma cells, will be compared to collective invasion behavior at the boundary of tumor spheroid assays. This will include 3D force measurements of the ?leader-follower? cell behaviour recently observed by Dr. Reinhart-King?s group. This new volumetric time-lapse imaging capability could lead to a deeper understanding of potential physical (mechanical) hallmarks of cancer, that can be used in the future to design and test new ?mechano therapies? that target/modulate the mechanical properties of the ECM. The clinical compatibility of an OCT-based imaging will greatly enhance future efforts to translate cancer mechanobiology research to the clinic.

The purpose of this study is to understand how treatment for breast cancer affects the well-being of the person with cancer, her partner, and their relationship because treatment for cancer can be physically and emotionally demanding. I am free to decide not to take part in the study. My decision whether or not to take part in the study will not affect the medical treatment of myself or my partner at the Universityof Pennsylvania Medical Center.

Beta thalassemia is one of the most prevalent forms of heritable blood disorders in the world. It is caused by mutations in human beta globin genes that result in reduced or abolished beta globin synthesis. Without beta globin chains to pair with, excess alpha globin chains are susceptible to oxidation to hemichromes, precipitate and damage red blood cell precursors as well as mature red blood cells, leading to ineffective erythropoiesis and profound anemia. Patients afflicted with the most severe forms of beta thalassemia require lifelong blood transfusions and iron chelation treatment. The only cure at present is BM transplantation with histocompatible donor cells, a limited option for many adult patients. The effort proposed here aim to ultimately, significantly improve the clinical picture in all patients with no transplant option by identifying an optimal targeted genetic engineering approach to do so. We will explore, side-by-side, three promising approaches alone and/or in certain combinations: 1) to reactivate developmentally silent gamma globin to pair with excess alpha globin (SA#1); 2) to downregulate alpha globin synthesis (SA#2); and 3) to drive targeted insertion of a therapeutic gamma globin gene into the beta globin locus (SA#3). For this purpose we plan to utilize an open-source targeted genome engineering platform, TALE effector nucleases (TALENs) to edit the relevant genomic loci of primary normal or patient mobilized peripheral blood (MPB) hCD34+ cells. Our intended genetic targets include a) potent gamma globin repressors BCL11A and its enhancer, as well as KLF1 (3 target sites), b) putative gamma globin repressor binding sites within the beta globin locus (3 target sites), c) the binding sites f alpha globin transactivator KLF4 in alpha globin promoters (3 target sites), d) alpha+ thalassemia- associated genomic sites in alpha globin locus (4 target sites), and d) two putative sites within beta globin locus for the insertion of a therapeutic gamma globin gene (2 target sites). The efficiency of editing and general effects on globin expression, erythropoiesis, and other potential side-effects will first be evaluated using normal MPB hCD34+ cells. The durability of editing and globin modulation will be studied by transplantation of edited normal hCD34+ cells into immunodeficient NOD/SCID IL2?null mice. The most promising approaches based on the evaluation of normal hCD34+ cells will be applied to beta thalassemic hCD34+ cells where the improvement in erythroid parameters both in vitro, and in vivo, will be examined. Our preliminary data on efficient ex-vivo editing of 3 selected genomic loci in normal and 1 in beta thalassemic MPB hCD34+ cells, robust gamma globin reactivation, and durable editing and gamma reactivation in exogeneic recipients are compelling and suggest that this novel approach has the potential to be developed into curative therapies for beta thalassemia.

In the developing brain, finely specified cell interactions regulate the extensive cell migrations, patterns of cell association, and subsequent synaptic connections which establish the complex neural network. This research proposal concerns reconstruction of brain tissue from single cells in vitro for detailed investigation of these critical interactions and the spatial, temporal and informational requirements for expression of organotypic patterning. Earlier preliminary studies provided a background of histogenetic data for such investigations and culminated in the isolation of cell recognition factors from mouse cerebrum cells which markedly and specifically promoted histogenesis of homologous cerebrum cells (Garber and Moscona, 1972a,b). The present objective is further purification and characterization of these cerebrum factors and the preparation of additional specific histogenetic factors from a variety of brain cell types. Localization of the functional activity of these factors in the cell, presumably at the cell surface, will be examined by studying the interaction of dissociated cells with labeled antisera prepared against purified factors. Also, analytical studies of the enhancement process (including uptake of factors, their metabolic dependencies, competitive behavior with defined molecular moieties, and effect on neural differentiation) will be designed to clarify basic mechanisms of cell association and neural patterning in the brain. The ultimate goal is the complexing of these specific cell recognition factors to collagen or synthetic matrices to construct coded pre-neural pathways and elicit directed migration of nerve cells according to pre-determined patterns.

CRISPR-based gene editing of the brain has the potential to revolutionize the treatment of neurological diseases. A large number of incurable brain diseases, such as Huntington's, Alzheimer's and Parkinson's disease, are caused by the over-expression of pathogenic proteins and could be treated with CRISPR based therapeutics. However, despite its potential, developing CRISPR based therapeutics for the brain has been challenging because of delivery problems. In particular, two key challenges need to be solved before gene editing in the brains of large animals and in humans is possible. First, strategies for efficiently and safely delivering Cas9 and gRNA into neurons, after an intracranial injection, need to be developed. Second, strategies that can enable a large volume of brain tissue (> 1 cm) to be transfected after an intracranial injection of CRISPR reagents also need to be developed. The central objective of this proposal is to develop a delivery strategy for gene editing the brains of large animals after an intracranial injection, termed convection-enhanced CRISPR (C-CRISPR). C-CRISPR is based on using convection-enhanced delivery (CED) to deliver an engineered Cas9 RNP, which has been fused to multiple nuclear localization signals (NLS), and has been encapsulated in PEGylated block copolymers. C-CRISPR addresses the key translational bottlenecks that have prevented CRISPR from having a translational impact in the brain. In particular, because it delivers the Cas9 RNP directly, it avoids the toxicity problems of viruses and the manufacturing challenges of using mRNA, and consequently has great translational potential. In addition, C-CRISPR uses CED to distribute the Cas9 RNP across centimeters of brain tissue, and therefore has the potential to edit the brains of large animals. C-CRISPR is based on our preliminary data demonstrating that the Cas9 RNP fused to multiple NLS signals can edit genes in murine brains after an intracranial injection, and that Cas9 RNP complexed to PEG-block copolymers can be delivered to centimeters of brain tissue, in the striatum, after delivery via CED. CED of engineered Cas9 RNP complexed to PEG block copolymers, therefore, has the potential to edit genes in human patients. We propose therefore the following aims/milestones: UG3 Specific Aim 1. Develop C-CRISPR formulations that distribute throughout the striatum of rats UG3 Specific Aim 2. Develop C-CRISPR formulations that edit centimeters of brain tissue UH3 Specific Aim 1. Develop C-CRISPR formulations that edit centimeters of tissue in pig brains The experiments in this proposal are significant because, if successful, C-CRISPR will be the first example of a non-viral delivery strategy that can edit genes in the brains of large animals. The experiments in this proposal are innovative because C-CRISPR is the first example of a delivery strategy that effectively integrates 3 complementary technologies, (1) engineered Cas9 RNPs (2) PEGylation and (3) convective enhanced diffusion, and will provide a roadmap for developing strategies for gene editing in higher animals.

PROJECT SUMMARY/ABSTRACT Tendon and ligament injuries account for nearly half of all musculoskeletal injuries each year in the United States. Even with considerable improvements, surgical repairs do not consistently restore physical function of injured tissues. As a result, there are lengthy delays or failures to return to pre-injury activities, thus posing a substantial clinical, economic and social burden. Poor surgical outcomes primarily result from excessive inflammation and insufficient regeneration of repaired tissues. Therefore, the long-term goal of this study is to develop biologic therapies to enhance tendon repair. With recent advances in regenerative medicine and extracellular vesicle (EV) research, the proposed project has been designed to investigate the mechanistic role and clinically relevant application of stem cell EVs in tendon repair. EVs are membrane-enclosed nanoparticles that mediate intercellular communication by transferring bioactive molecules (proteins, RNAs, etc.) from cell to cell. Although EVs from adipose-derived stem cells (ASCs) have been found to modulate tissue inflammatory response and to promote regeneration of some soft tissues, their role in tendon repairs has not been directly explored. Aim 1 will use co-culture models to determine the role and underlying molecular mechanism of ASC EVs in regulating macrophage inflammatory response and tenocyte activity and function in the context of tendon injury and repair. Aim 2 will use a clinically relevant mouse Achilles tendon injury and repair model to determine the therapeutic efficacy of ASC EVs in reducing inflammation, increasing tendon matrix regeneration and restoring structure and strength of injured tendons. Results from Aim 1 will reveal new insights into the mechanism of ASC EVs in regulating tendon healing process and provide guidance for further engineering of defined components within ASC EVs to provide improved therapeutic efficacy and safety. Positive outcomes from Aim 2 will prove the concept of ASC EV-based therapy for tendon injury as well as other related disorders and lead to clinical studies, thus contributing to the improvement of musculoskeletal health.

Personality disorders are common in the general population and are particularly prevalent among psychiatric patients. Of concern, personality disorders are associated with a number of adverse outcomes, including suicidality, attenuated treatment response, increased utilization of health care services, and significant functional impairment. It is therefore crucial that clinicians and researchers have tools available to efficiently assess this key aspect of psychological functioning. Unfortunately, many common measures of personality pathology are quite long and inefficient and/or must be administered and scored by professional staff. Moreover, a number of categorical and dimensional models of personality pathology have been proposed, but the lack of integration and comprehensiveness among these models has led to confusion in health care and research settings and has resulted in a decrease in assessment of personality pathology. Thus, in the proposed research, our general aims are to (a) identify a comprehensive and integrative set of dimensions relevant to personality pathology, and (b) develop an efficient computerized adaptive method the CAT-PD to measure these dimensions. Computerized adaptive tests (CATs) are rooted in modern psychometric methods and are designed to select items for administration that are tailored individually to each patient, which typically leads to significant gains in measurement efficiency with little or no cost to test reliability or validity. To accomplish our general goals, we plan a five-phase project in which we (Phase 1) identify the content domains to be assessed and develop the initial item pool based on the public domain International Personality Item Pool (IPIP), (Phase 2) collect self-report item responses from two samples each of psychiatric patients (total N = 500) and community-dwelling adults (total N = 500) and conduct analyses of those responses to develop and cross-validate the CAT-PD scales, (Phase 3) calibrate the CAT-PD items using item response theory (IRT) and conduct CAT simulations to guide construction of the live CAT-PD software, (Phase 4) develop CAT-PD software based on the results of all previous phases, and (Phase 5) conduct a live-testing study of the CAT-PD in a new sample of psychiatric patients (N = 300) to study its efficiency and construct validity and establish cut points and scoring rules designed to increase the tool's practical utility across a wide variety of applied and research settings. It is our hope that the proposed research will help to integrate the numerous dimensional models of personality pathology, yield a comprehensive set and efficient measure of personality disorder dimensions, and contribute to advancements in personality disorder theory, research, diagnosis, and intervention. PUBLIC HEALTH RELEVANCE: Because personality disorders are common in the general population and are associated with a number of adverse outcomes, including suicidality, attenuated treatment response, increased utilization of health care services, and significant functional impairment, the proposed project is relevant to public health. It is crucial that clinicians and researchers have tools available to efficiently assess this key aspect of psychological functioning. The proposed research will help to integrate the numerous models of personality pathology, yield a comprehensive set and efficient measure of personality disorder dimensions, and contribute to advancements in personality disorder theory, research, diagnosis, and intervention.

Sudden cardiac arrest (SCA) is a major public health concern, accounting for over 400,000 deaths in the US each year. While environmental factors clearly contribute to the determinants of SCA, familial aggregation studies and advances in the molecular genetics of inherited arrhythmias suggest that genetic factors confer susceptibility to SCA in the general population. Identifying these genetic factors will not only provide insight into the mechanisms of SCA, it will also have significant public health implications for risk stratification and prevention of SCA. We propose to systematically investigate the association of SCA and intermediate phenotypes with variations in biologically important molecular pathways involved in arrhythmogenesis using complete sequence information and haplotype analyses. Specifically, we will study the association of SCA risk with variation in candidate genes (Aim 1) that are proximal determinants of electrogenesis and propagation (e.g. cardiac ion channel genes and connexins) and genes in pathways that influence these proximal determinants (e.g. neurohumoral modulators). We will explore these associations in the context of the genetic and environmental milieu, investigating interactions with medications (Aim 2a), exercise (Aim 2b), and other genetic factors (Aim 2c). We will also examine main effects of genetic variation on intermediate quantitative phenotypes, such as ECG QT interval, heart rate, and heart rate variability (Aim 3). This application represents a multi-center collaborative effort to efficiently combine advances in genomics with material from four on-going studies that enable rigorous identification of the SCA phenotype or relevant intermediate phenotypes. Specifically, we will use material from a large population-based repository of SCA cases (Cardiac Arrest Blood Repository), two case-control studies (Cardiac Arrest Blood Study and Group Health Cooperative study), and a cohort study (Cardiovascular Health Study):

We are requesting partial support for the next three Molecular and Cellular Cognition (MCC) Meetings. The Conference will be held in the San Diego Convention Center on November 1st and 2nd, 2007, right before the annual meeting of the Society for Neuroscience. The MCC meeting brings together junior and senior scientists that combine molecular (pharmacology, genetics, transgenics, viral approaches, etc) and physiological (electrophysiology, optical physiology) and other cellular approaches to study behavior, including learning and memory. The general goal of these studies is to derive explanations of cognitive processes that integrate molecular, cellular, and behavioral mechanisms, as well as to use this information and related animal models in the search for treatments for cognitive, psychiatric and neurological disorders in children, adults and the elderly. These meetings have been organized under the sponsorship and leadership of the Molecular and Cellular Cognition Society (MCCS) (www.molcellcog.org), a relatively new group whose main function is to organize meetings and promote interaction and collaborations among laboratories working in this general area. Although there are a few learning and memory meetings in the USA and abroad, the Molecular and Cellular Cognition meeting is unique because it brings together individuals that integrate molecular, physiological and behavioral approaches in studies of memory and learning related disorders. Although the molecular and cellular cognition field is relatively new, it has already had a profound impact on neuroscience research. Currently, this is the only periodic meeting in the field, an invaluable opportunity to exchange information, and develop this young field. The 2005 and 2006 meetings were highly successful, attracting each year a diverse group of approximately 400 participants from North America, Europe, and Asia, and we have every reason to believe that the 2007-2009 meetings will be equally successful. (Summaries at http://www.molcellcog.org/meetings.htm). The Molecular and Cellular Cognition Society meeting brings together junior and senior scientists that combine molecular (pharmacology, genetics, transgenics, viral approaches, etc) and physiological (electrophysiology, optical physiology) and other cellular approaches to study behavior, including learning and memory. The general goal of these studies is to derive explanations of cognitive processes that integrate molecular, cellular, and behavioral mechanisms, as well as to use this information and related animal models in the search for treatments for cognitive, psychiatric and neurological disorders in children, adults and the elderly.

[unreadable] During the first phase of the AMDCC our group at TJU studied the decorin KO diabetic mouse. Our hypothesis was that decorin acts as an endogenous protective factor by inhibiting active TGF-b. We found that decorin is indeed protective, as decorin KO mice had accelerated kidney disease and surprisingly increased mortality. Based on our results, the AMDCC investigators as a group chose the decorin KO diabetic mouse as a leading success during the 1st funding period. Of major mechanistic interest, we found that decorin KO diabetic mice that died exhibited evidence of renal insufficiency and low plasma adiponectin levels, months prior to mortality. This is similar to the human clinical condition. Low adiponectin levels are a powerful biomarker of increased cardiovascular morbidity and mortality in patients with kidney disease. Additionally, the decorin KO mice had increased NADPH oxidase (Nox4) expression in the kidney which may contribute to more severe nephropathy. In the next phase of the AMDCC, we propose test the following hypotheses. 1. Deficiency of adiponectin in combination with lack of decorin leads to enhanced lethality and diabetic nephropathy and 2. Increased Nox4 in vascular smooth muscle cells enhances diabetic nephropathy and the vascular complications of diabetic kidney disease. We propose to generate new diabetic mouse models by crossing adiponectin KO mice with decorin KO mice and by generating mice transgenic for smooth muscle Nox4 using the SM22 promoter. Diabetes will be induced by crossing with Akita mice. These mice will be characterized for diabetic nephropathy and cardiovascular disease. Both new models will be fully characterized for diabetic nephropathy. More importantly, we propose a series of interventional studies to test the causal role of TGF-b, adiponectin, and Nox4 in the pathogenesis of accelerated diabetic nephropathy. Key features we will focus on include matrix accumulation, renal function, autoregulation, and mortality. The proposed studies are directly related to the human condition, in which TGF-(, adiponectin, and Nox4 are key biomarkers for worse disease, but their pathogenetic role is unproven. Our findings will advance our mechanistic understanding of diabetic nephropathy and vascular disease and may lead to better biomarkers and novel treatments. [unreadable] [unreadable]

Cholesterol and bile acids have been implicated in playing important roles in several major diseases of "Western Society" including: arteriosclerosis, cholesterol gallstone formation, cholestatic liver disease and colon carcinogenesis. The overall goal of this renewal application is aimed at a more detailed understanding of the role bile acids play in the regulation of cholesterol and bile acid homeostasis, liver/intestinal physiology, and cholesterol gallstone disease. The overall goal will be accomplished through the following specific aims: [1] a) Determine which isoform(s) of protein kinase C is involved in the regulation of cholesterol 7 alpha-hydroxylase; b) Determine the mechanism of activation of protein kinase C isoforms by bile acids; c) Define protein kinase C and the bile acid responsive element of the cholesterol 7 alpha-hydroxylase promoter; d) Determine if bile acids activate protein kinase C isoforms in vivo in liver and ileum (Vlahcevic, Stravitz, Heuman, Hylemon); [2] a) Quantify adsorption of bile salts to model membranes; b) Develop and validate a general quantitative model of bile salt-membrane adsorption; c) Determine the effects of lecithin and cholesterol on toxicity of bile salts toward membranes; d) Determine the effect of biliary lipid composition on biliary bile salt toxicity in animal models of bile salt induced liver injury; e) Determine if protein kinase C activation by bile salts are consequences of the accumulation of bile salts on the membrane surface that can be predicted by quantitative modeling of bile salt-membrane adsorption (Heuman, Stravitz, Valhcevic ; [3] a) Selective overexpression of cholesterol 7 alpha- hydroxylase, sterol 27-hydroxylase, cholesterol ester hydrolase and acyl CoA:cholesterol acyltransferase in vitro (Hep G2 and Chinese hamster ovary cells); b) Assess the role each enzyme plays in maintaining cellular and whole body cholesterol homeostasis using recombinant adenovirus vectors in vivo (hamsters); c) Investigate the regulation of cholesterol ester hydrolase and acyl CoA:cholesterol acyltransferase in primary rat hepatocyte cultures and in vivo by bile acids, cholesterol and hormones (Pandak, Vlahcevic); [4] a) Complete the cloning, sequencing and analysis of a large bile acid inducible operon (bai) from the intestinal Eubacterium sp. VPI 12708; b) Determine the function that each gene product encoded by this operon plays inbile acid 7 alpha/beta-dehydroxylation; c) Isolate, characterize and identify bile acid 7 alpha-dehydroxylating bacteria from cholesterol gallstone patients having high (>30%) levels of deoxycholic acid and determine if these patients are colonized by unique 7 alpha-dehydroxylating bacterial species (Hylemon, Berr).

Project Summary/Abstract Concussion, a traumatically induced brain injury, is described by the Centers for Disease Control as a serious public health problem. Currently, management protocols are based on consensus and expert opinion, rather than high quality evidence, potentially resulting in difficulty in diagnosis and identification of physiologic recovery. The use of objective and quantitative measurements to better understand the etiology of concussion symptoms and the efficacy of intervention protocols may allow investigators to improve post-concussion care. Symptoms such as headache, dizziness, or cognitive difficulties commonly occur after concussion and likely relate to cerebrovascular dysfunction. Cerebral autoregulation, cerebral reactivity, and neurovascular coupling represent three areas of cerebrovascular function that are impaired after a concussion and relate to the presence of specific concussion symptoms. Cerebrovascular functioning may also relate to higher order cognitive functions, such as executive functioning. Combined gait and cognitive tests (dual-tasks) can probe executive function abilities, and can also objectively reveal post-concussion deficits. Thus, improved understanding of how concussion symptomology, cerebrovascular function, and gait function relate to one another will help in the objective monitoring of recovery. Additionally, rehabilitative strategies in concussion care have remained relatively ineffective, particularly when initiated soon after injury. Sub-maximal aerobic exercise is one potential rehabilitative strategy to improve cerebrovascular function, locomotor performance, and executive functioning, in addition to alleviating post-concussion symptoms. Therefore, we seek to examine 52 symptomatic adolescents within two weeks of a concussion on measures of symptom severity, cerebrovascular function, gait, and executive functioning. Participants will be pseudo-randomized into an 8- week sub-maximal aerobic exercise intervention or usual care, and physical activity intensity and duration will be objectively monitored with a heart rate monitor. Accordingly, we will 1) determine the relationship between symptoms, cerebrovascular function, gait, and executive function soon after a concussion, and 2) investigate the effect of an aerobic exercise intervention protocol on concussion recovery. We believe this represents an important line of research as few studies have used quantitative and objective measures to examine the efficacy of concussion treatments, and our study design will allow us to observe how post-concussion aerobic exercise initiated soon after injury affects recovery trajectories. Thus, improved understanding of the role of physical activity after concussion may eventually pave the way to develop better therapeutic interventions, and eliminate ?one therapy for all? treatments.

Cytochrome P-450 is a family of isozymes that are present in most tissues of the body and are involved in the metabolism and activity of steroids, prostaglandins, leucotrienes, fatty acids, drugs and environmental chemicals. It has recently been shown that several of these isozymes turnover at different rates, but the basis for these findings is not understood. In this study we have found that during the catalytic turnover of rat liver microsomal cytochrome P-450 in vitro that a selective degradation of the isozymes occurred. Most striking were the findings of covalently bound heme fragments to apoprotein and high molecular weight derivatives of the apoproteins. We believe that this may represent the selective "tagging" of the apocytochrome P-450 by heme fragments or other cellular components that signals the catabolic enzymes to hydrolyze this group of enzymes.

Increasingly, advances in skin biology depend on cross-disciplinary, collaborative approaches. Through systems biology, multiple disciplines can now be integrated into a cohesive approach to skin biology. The broad, long-term objective of the UC Irvine Skin Biology Resource-based Center is to promote cross- fertilization between multiple disciplines that allows an innovative, integrated approach to regulatory mechanisms in skin biology and disease. This will be achieved through the establishment of resource cores in genomics-bioinformatics, imaging and systems biology. The interactions between investigators and cores, and between cores, will be coordinated through the Administrative Core, which will also be responsible for an enrichment program that promotes goals of the Center. The Specific Aims are: 1. To foster multidisciplinary, collaborative science between skin researchers and experts in genomics, advanced imaging, and biologically- oriented computation; and to integrate these approaches through systems biology. 2. To develop and adapt new cutting-edge technologies in genomics, imaging and biologically-oriented computation to advance skin biology research and its translation to disease. 3. To train skin researchers in advanced imaging, genomics, bioinformatics and system biology. 4. To recruit and support junior investigators in research in biology and diseases of the skin. 5. To produce and make available resources useful to the skin research community at large. The establishment of the Center will have strong impact on the well-funded, innovative research community, leading to significant discoveries relevant to regulatory mechanisms in skin biology. The Center will also draw investigators from other fields into NIAMS-funded research and promote entry of junior investigators into skin biology research.

Evaluate quantum efficiency of CsI photocathodes on the front end of an imaging photon detector (IPD) for 8 KeV x-rays.

One of the fascinating problems in biochemistry is to determine how subtle changes in related molecules cause drastic changes in their biochemical behavior. An example of this structure function problem is the role of similar high=valent porphyrin intermediates in the different reactions catalyzed by hydroperoxidate and cytochrome p-450 enzymes.Hydroperoxidases are hemoproteins with essential roles in the catabolism of toxic peroxides. Cytochrome p-450 is a monooxygenase with essential metabolic functions dealing with drug metabolism, chemical carcinogenesis, membrane detoxification and biosynthesis. The short term goal of this project is to encourage students to pursue graduate studies in the health sciences by providing a summer research opportunity to the investigators undergraduate biochemistry majors. A long term goal of this project is to increase the investigators fundamental chemical understanding of the role of high-valent porpyhrins in biological systems. This project involves the use of novel techniques in quantum chemistry to study the magnetic and electronic structure of high-valent porphyrin n- cation radicals found at the active site of oxygen activation enzymes. The primary goal of this research is to understand the nature of spin-coupling in these systems, and to explore how these couplings influence their observed spectral and catalytic properties. This will be accomplished by calculations on Heisenberg coupling constants, Mossbauer and magnetic resonance parameters (including g and hyperfine tensors), electronic transition dipoles, and charge densities as a function of systematic variations in active site geometries.All calculations will use Xalpha or local spin density effective potentials. Systems to be studied include: (a) ferryl oxygen intermediates which function as the active catalytic agent in peroxidase, catalase, and cytochrome p-450 enzymes; and (b) planar and ruffled synthetic tetraphenyl porphyrins which serve as models for spin coupling in hemoproteins. Models for these systems will be used to interpret experimental magnetic measurements on these systems.

A new and successful Gordon Research Conference (GRC) on Cell Polarity Signaling was held in 2014, 2016, and 2018 and filled a very important but unmet need, as prior to the initiation of this GRC there were no regular conferences on the broad topic of cell polarity, either in the US or elsewhere. Based on highly positive feedback from the first three GRCs we are organizing a fourth meeting for 2020 to continue an ongoing forum for the very dynamic and diverse cell polarity field. Polarity is fundamental to all of biology, with profound implications for cell and tissue organization in normal development and homeostasis, and in numerous diseases including cancer. For these reasons, cell polarity has become an important area of research that has caught the imagination of many hundreds of investigators. To bring new participants and topics to the meeting, the 2020 GRC is focusing on the ?Dynamics of Cell Polarity?, and will feature many invited speakers who are bringing high-resolution imaging and image analysis methods to the field. A Gordon Research Seminar (GRS), organized by two postdoctoral trainees and subtitled ?Cell Polarity Across Developmental Time and Space,? will precede the GRC. The GRS is expected to attract ~50 trainees to present and discuss their research. The GRS will foster networking and collaborations to help trainees develop their careers. It will also provide a valuable learning experience for those involved in organizing the GRS. Several senior investigators in the cell polarity field will be invited to the GRS to discuss their research and provide mentoring and feedback. Sessions at the GRS and GRC will range from basic cell biology and development of model organisms to translational studies in cancer biology. Many talks at the GRC will focus on the meeting subtitle ? ?Dynamics of Cell Polarity.? The invited speakers for 2020 will be different from that of the 2018 conference, and 70% have never been invited as speakers to any previous Cell Polarity GRC. These new additions to the meeting, as well as the new focus on ?Dynamics of Cell Polarity?, will keep the meeting fresh and relevant and will broaden the number of investigators who can present their research. Invited speakers include established investigators from disciplines both within and outside the cell polarity field. Among the 30 invited speakers from multiple countries, 50% are women, 20% are junior PIs, and one to our knowledge is a URM as defined by the NIH. 20 short talks will be selected from submitted abstracts. Interactive poster presentations will take place on 4 afternoons, and several poster presenters will be selected to present poster preview talks to advertise their poster. The program includes a ?Power Hour? to discuss the challenges women face in academia. We will promote diversity in the programs for the GRC and GRS, through GRC minority fellowship funding, and through focused advertising via scientific societies. We envision that the 2020 GRC/GRS will facilitate discussion of cutting edge research in cell polarity and will foster collaborations that will fuel the field forward.

HIV-infected individuals who abuse heroin are at greater risk for HAND. The synaptodendritic injury that underlies HAND is largely mediated by infected and/or activated microglia and astroglia as neurons are rarely, if ever, infected. CNS neurons and glia are derived from neuroepithelial precursors in the subventricular zone (SVZ). In development, undifferentiated, common neural progenitor cells (NPCs) give rise to cells that form neurons, astroglia, and oligodendroglia on a precise schedule of proliferation, migration, and differentiation regulated by complex intra- and extracellular signals. In the mature CNS, neurogenesis is largely limited to specific regions (dentate gyrus and SVZ of lateral ventricles), while gliogenesis also occurs in the parenchyma. During the 1st funding period, we showed that HIV-1 and HIV proteins diminished NPC ability to differentiate and populate the striatum, in mice exposed both perinatally and as adults, and in vitro. In general, HIV R5 (culture) and HIV-1 Tat (in vivo stereology; culture) reduced Sox2+ NPCs and slowed proliferation. HIV-1 Tat in vivo also seemed to redirect cell fate as mature cell populations were changed ( ? oligodendroglia, ? astroglia) while total cell numbers and volume were normal. Co-exposure to morphine exacerbated some outcomes. Thus, both HIV and Tat interfere with NPC development via mechanisms that opiates can amplify. Data demonstrate that R5 or chronic Tat exposure distort normal NPC ? microglia interactions, resulting in abnormal CNS populations. Imbalances (? inflammatory ? astroglia, myelinating OLs) likely contribute to HIV-related deficits in CNS function. We hypothesize that HIV by itself, and more potently with morphine, overactivate microglia, causing aberrant signaling to NPCs through NADPH oxidase (NOX2) and iNOS/nitric oxide. The inappropriate signaling misdirects NPC lineage/differentiation, ultimately altering CNS cell populations. This theory is explored in vivo (mouse/HIV-1 Tat model) and in human brain cultures (HIV infective model) in 2 related aims. Aim 1 tests if microglia play a central rol in the aberrant proliferation/lineage of NPCs after exposure to HIV-1 Tat opiates. Studies use a macrophage/ microglia deficient transgenic model (HIV-1 Tat x CSF1op/op mice), with chronic Tat induction at both adolescent (high risk for HIV and opiate exposure through exploratory behaviors) and adult stages. Hippocampus and striatum are studied; motor and cognitive behaviors correlated to population changes. NPCs are also examined in HIV+/HIV- autopsy tissue. Aim 2 tests the roles of microglial NOX2 and iNOS signaling as causal in dysregulated NPC dynamics, using 3-dimensional aggregate cultures from human brain and real-time tracking of individual human NPCsHIVopiatesspecific inhibitors of iNOS, NOX2 and R5 binding. Amnis flow-single cell imaging used to identify infected cells.

Myosin V functions as an actin-dependent ATPase. In Drosophila it is enriched in the brain. Identification of a mutant bearing a P-lacW insertion 600 bp from the ATG initiation site in the myosin V gene of Drosophila melanogaster has enabled us to study the effects of limited myosin V on the developing fly. Maternally contributed mRNA may support the survival of the homozygote mutant larva into the later larval stages since there are no apparent behavioral or developmental defects in either embryos or in early instar larvae. Neurological problems are first evident in the 3rd instar, where the distal portion of the larva is paralyzed, preventing them from righting themselves when turned on their dorsal side, and also decreasing the force and propagation of the peristaltic wave which propels them forward. These neurological defects are magnified in the adult stages in which mutants are slow moving and unable to jump or fly. Their wings are held at their side and are largely immobile. These phenomena are probably neurological, since they have normal muscle physiology as test by polarized light microscopy for birefringence. They also exhibit a general tremor of their head and legs. This chorea progresses rapidly, with newly hatched flies tending to move around at an almost normal rate, with movement becoming slower and less coordinated until death at 1 week. The mutants attempt grooming behaviors, but their movements are clumsy and slow. Additionally, no courtship behaviors are observed. Mobilization of the P-element has restored the wild type phenotype by precise excision. - unconventional myosin V; Drosophila; P-element; GST-fusion protein;

Lifetime exposure to sex steroids is a major contributor to breast cancer risk. The risk-benefit ratio of prevention strategies that target the endocrine axis would be significantly improved if it were possible to assess risk for hormone sensitive breast cancer. Nipple fluid hormone levels are attractive candidate markers of breast cancer risk. Our pilot studies provide preliminary data that nipple aspirate fluid (NAF) levels of sex steroids (estradiol and progesterone) are potential biomarkers of breast cancer risk in both pre and postmenopausal women. We show that these markers are significantly higher in NAF than in serum, are stable over time, and are biologically relevant, as reflected by the strong correlations of NAF estradiol levels with the content of estrogen response proteins in NAF. Additionally, we show that the hormone content of NAF reflects the systemic environment, since hormone levels rise with the use of hormone replacement therapy, are low in oral contraceptive users, and rise after cessation of oral contraceptives. Finally, preliminary analyses show, for the first time, a correlation of NAF estradiol and progesterone with estimated Gail risk. The primary hypothesis to be examined in this proposal is that NAF estradiol levels are markers of risk for hormone sensitive breast cancer. To accomplish this, we are proposing a case-control study of 600 women: 300 newly diagnosed breast cancer cases aged 40 to 60 from the Lynn Sage Breast Center, and 300 controls from the adjacent Mammography Screening Center of Northwestern. Cases and controls will be matched for age and menopausal status. We will obtain NAF, blood samples, and mammographic density measurements. NAF assays will include sex steroids (estradiol and progesterone), their precursors (estrone, estrone sulphate, androstenedione dehydroepiandrosterone and testosterone) and related proteins (epidermal growth factor, cathepsin D, pS2, and prostate specific antigen). We will perform sub-analyses to examine the effect of specific breast cancer risk factors, menopausal status, and race on the association of NAF hormones and breast cancer risk. We will seek corroborating evidence for the biological importance of NAF hormone levels by examining the NAF content of estrogen and progesterone related proteins. By examining the hormone receptor (HR) status of the cancers among cases, we will test the hypothesis that NAF hormone levels are more strongly related to HR positive breast cancers. Finally, we will assess the degree to which NAF hormones explain mammographic density in cases and controls. This project, evaluating breast intracrinology, mammographic density, and cancer risk will provide data on the validity of NAF hormones as new breast cancer risk markers, which can potentially be modulated by preventive interventions and therefore may serve as surrogate endpoints to assess their effects.

NIH investigators are gathering terabyte- to petabyte-scale datasets generated using state-of-the-art biomedical technologies. To properly manage these large datasets, researchers need to keep them secure from unauthorized access and from data loss, while still being able to share the data with collaborators, and maintain these best practices over long periods of time. Although Stanford investigators have access to petabyte-scale High Performance Storage (HPS) systems, these systems are designed for high performance computation and not long-term protected storage, and they do not have granular secure file sharing. We request funding for a storage appliance known as an object store that will provide a robust mechanism for long-term protected data storage and secure data sharing. Additionally, our proposed storage appliance will be usable by multiple researchers simultaneously and be simple for users and administrators to manage. ?Need for data sharing?: Data sharing is essential for expedited translation of research results into knowledge, products, and procedures to improve human health, so researchers are under pressure from a variety of institutions to make their data available. This appliance will enable investigators to put up a lightweight webpage for data-sharing (e.g., laboratory website, wiki, or github) while storing the bulk of the data on this appliance. ?Need for data security?: Raw biomedical data are often shared with strict security and privacy requirements. Our proposed appliance supports file access management by supporting authentication, authorization, and access controls with Stanford?s user database. Its fine-grained approach to authentication will allow files to be shared with limited sets of users, which is valuable when exchanging data with collaborators. ?Need for data protection?: The traditional RAID scheme for data protection used by HPS systems to provide high throughput is not resilient enough for long-term data protection in the age of high capacity disks (8TB/hard drive). Our proposed object storage uses an erasure-coding scheme, a method that provides better availability at lower overhead and cost when compared with RAID. An appliance like this object store will make a fundamental impact to 100s of researchers at Stanford by providing them with a flexible and cost-effective resource for long-term protected data storage and secure file sharing.

This application requests support for a program of basic and clinical research on speech perception and spoken word recognition. The primary objective of this project is to understand how spoken words are recognized and how acoustic-phonetic and indexical information in the speech signal interact with other knowledge sources to support robust spoken language understanding. The proposed research will involve behavioral studies of speech perception and spoken word recognition as well as computational analyses of the sound patterns of word-forms in the mental lexicon to study global organization and connectivity patterns of spoken words. Four specific aims will be studied: (1) lexical knowledge and organization, (2) perceptual learning and adaptation, (3) speech perception under adverse listening conditions, and (4) individual differences in working memory dynamics (capacity and speed) in hearing-impaired listeners with cochlear implants (CIs). The research findings will provide a much stronger conceptual and theoretical basis for explaining the core underlying factors that are responsible for the variability and individual differences observed in speech and language processing in normal-hearing typical-developing listeners. The results from this project will also have important direct clinical implications for understanding individual differences in speech and language outcomes in hearing-impaired children and adults who use CIs.

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. The overall goal of the project is to develop a statistical multi-modal population brain atlas construction framework incorporating all the available imaging modalities. The imaging modalities that the framework will support are T1, T2, various diffusion weighted protocols such as DWI and DSI as well as arterial spin labeling perfusion MRI. The atlas construction framework will be used to study the anatomy of the aging brain and impact of various diseases on the aging process. In particular, the generation of probabilistic brain atlases will improve power to identify systematic brain differences between diseases and aging brains by utilizing information of local brain variability. Moreover, a probabilistic brain atlas that includes jointly information from all imaging modalities will allow more comprehensive investigations in brain alterations than conventional methods that use a single image modality or multiple modalities separately. Moreover, the mapping of multimodal image data into a common 3D coordinate space is a prerequisite for many imaging analysis projects of this P41, as it supplies a quantitative spatial reference system in which brain data from multiple subjects and modalities can be compared and correlated.

BioCARS Station 14 BM-D is established as a facility capable of producing the data needed to achieve the aims of this project to determine the structure of proteins in a timely fashion through single, effective experiments. It is the station of choice because of familiarity with the equipment and procedures of the station.

My long range goals are to develop an understanding of how neural activity regulates gene expression, and to ultimately understand how these processes contribute to environmental and drug induced stable/adaptive changes in the nervous system. A better understanding of theses processes will help us understand the adaptive physiologic changes underlying drug addiction, tolerance, withdrawal, and drug-seeking behaviors. The molecular mechanisms underlying learning, memory, long-term effects of addictive drugs, and other stable/adaptive changes in the nervous system are largely unknown. With the discovery that neurons transduce environmental inputs into changes in gene expression, the hypothesis has emerged that trans-synaptic regulation of gene expression may induce the biochemical changes underlying many forms of neural signaling plasticity. To understand these processes it is necessary to identify both the pathways and mechanisms mediating the effects of learning, psychotropic drugs, and other synaptically mediated processes on gene expression. Neural regulation of the opioid precursor, proenkephalin, is an excellent model system to study mechanisms underlying synaptic regulation of gene expression because: i) transcription is both activated and repressed by synaptic signals, ii) in the brain, events such as electrical stimulation which induce LTP also induce dramatic and long lasting changes in proenkephalin gene expression, iii) the gene products, enkephalins, are important signaling molecules regulating diverse neural pathways including analgesia, reward and motivational states, and hormone release. The primary objective of this research proposal is to identify and characterize nucleoprotein complexes which regulate proenkephalin gene expression and to determine how these nucleoprotein complexes mediate synaptic regulation of proenkephalin transcription via their interaction with a well characterized second messenger inducible DNA enhancer. In the period covered by this proposal, studies will focus on defining components of ENKTF-1 and Fos/Jun nucleoprotein complexes which mediate synaptic regulation of proenkephalin transcription. In addition their functional and biochemical interactions with the proenkephalin inducible DNA enhancer and signals transmitted through intracellular signaling pathways will be investigated. Because the transcription factors described above are activated by both neurotransmitters and pharmacologic agents they can be considered to be important targets of drug action. Progress in understanding these mechanisms should provide insight into the pathogenesis and treatment of many disorders affecting the nervous system.

Circadian rhythms are often disrupted with advancing age and under conditions of stress, including social stress. Breakdown in human circadian rhythms are believed to occur most often downstream of the central circadian oscillator. Our lab has developed electric fish as a powerful non-mammalian vertebrate model system in which controlled changes in the social environment alter predictably the magnitude of two circadian rhythms in electric signal waveform parameters. Our system makes a valuable model because we have identified multiple social conditions that modulate circadian rhythm expression in multiple ways. As with mammalian models, situations promising social upheaval disrupt the circadian outputs. Social isolation, however, causes a progressive diminution of these rhythms, as though the coupling between the central oscillator and the peripheral effector had been weakened or broken. Restoration of favorable social conditions restores the strength of the rhythms. We have made significant progress in identifying the neurochemical components of the circadian output pathway. Two neurochemical messengers, 5-HT and ACTH, modulate the behavioral outputs, ACTH at the level of the peripheral effector organ. Results with males indicate that the circadian rhythms are regulated somewhere downstream of serotonin. We also have found that non-aromatizable androgens can enhance the amplitude of these circadian rhythms in a manner resembling certain favorable social manipulations. Building on our progress to date, we propose specific aims (1) to better understand the behavioral conditions that regulate circadian rhythms in the electric waveform, (2) to elucidate the roles of androgens and glucocorticoids in regulating the circadian rhythms, and (3) to identify which hormones of the hypothalamus-pituitary-adrenal axis are in direct control of the circadian outputs. Because the neuroanatomy and neurochemistry of the circadian control pathway have been conserved across vertebrate evolution, these studies will lead directly to testable hypotheses about mechanisms !underlying circadian rhythm pathologies in mammals, including humans.

Many anticancer and antiviral agents are analogs of purines or pyrimidines that are thought to act by being incorporated into DNA. Most of the studies performed to date concerning the mechanism by which these drugs act have used DNA into which the analog has been randomly incorporated. For one such analog, 6-thioguanine (TG), we have performed experiments that suggest that the action of this drug may depend on its being incorporated into a specific DNA sequence. The overall objective of the proposed work is to determine if such "sequence-specific incorporation" is necessary for the action of Tg, and also of three other analogs which are believed to act via incorporation into DNA. This objective will be met by achieving the following specific goals: 1. Construction of specifically substituted viral DNA molecules. The eukaryotic virus SV40 will be the model system for these studies. SV40 DNA contains a sequence at its origin of replication whose integrity has been shown to be critical for initiation of viral DNA synthesis. Analog substitutions will be performed either collectively within this sequence or in other regions of the viral genome known to be insensitive to mutation. In addition to TG, substitutions will be performed using ara-C, ara-A or bromodeoxyuridine. 2. Testing the biological effects of sequence-specific substitution. Once the contructed molecules are in hand their ability to function will be tested in two systems. First, infectivity of these species will be tested by DEAE-dextran mediated transfection. Second, the ability of the substituted molecules to function as templates in a recently reported cell-free system will be assayed. 3. Testing the protein binding properties of substituted DNA. The virally-coded "T-antigen" (T-ag), whose function is critical for initiation of replication, binds to the site within the origin where specific substitutions will be made. The affinity of analog-substituted monlecules for T-ag will be measured by means of an immunoprecipitation procedure using an anti-T-ag monoclonal antibody.

Recently, Adeno-Associated Virus (AAV)-based vectors have emerged as promising gene delivery vehicles for a wide array of diseases, including cardiovascular disorders. Despite early encouraging results, the CUPID (Calcium Upregulation by Percutaneous Administration of Gene Therapy in Cardiac Disease) trial using an AAV serotype 1 vector encoding the sarcoplasmatic calcium ATPase SERCA2a failed to meet both its primary and secondary endpoints. These results were surprising because in porcine models of HF AAV1.SERCA2a improved cardiac function. Preliminary results suggest that the disappointing outcome was due to a failure of AAV1.SERCA2a to deliver efficiently the SERCA2a gene. One possible explanation for the poor gene delivery is that neutralizing antibodies (NAbs) against AAV1 were not detected with the in vitro NAb assay used in the CUPID trial, but that these NAbs prevented transduction. In Aim 1 of this application we will test in a porcine HF model the hypothesis that extremely low levels of NAbs, which can only be detected by a more sensitive in vivo NAb assay, can prevent transduction and therapeutic efficacy of AAV1.SERCA2a. Conversely, if in vitro NAb assays are sufficiently sensitive, we will determine the maximal NAb levels that are still compatible with efficient transduction and therapeutic efficacy of AAV1.SERCA2a (in pigs). An alternative explanation for the negative results of the CUPID trial is that AAV1 displays specie-specific tropism, i.e. that AAV1 can efficiently transduce pig but not human cardiomyocytes. To bring cardiac AAV gene therapy to the clinic, it will be critical to isolate AAV variants that 1) Can efficiently transduce human cardiac cells and 2) Show increased resistance to NAbs. The isolation of such variants is the goal of Aim 2. Unfortunately, it seems unlikely that AAV variants that can efficiently transduce human cardiomyocytes and that are also resistant to very high levels of NAbs against the naturally occurring AAV serotypes can be isolated. Therefore, in Aim 3, we will test an approach to deplete NAbs from the blood by plasmapheresis coupled with immunadsorption with columns with immobilized AAV particles. With the successful completion of this proposal, we will have established whether an in vitro NAb assay is sensitive enough to serve as an exclusion criterion for cardiac AAV gene therapy trials where AAV is delivered by intracoronary infusion, or if a more sensitive in vivo assay must be used. We will have isolated novel AAV variants with tropism for human cardiomyocytes and increased resistance to NAbs. Finally, we will have established whether plasmapheresis coupled with immunadsorption on AAV columns can be used to deplete NAbs from blood. These parameters will be critical in the design and execution of future gene therapy trials for cardiovascular diseases.

Provide isolated housing and care for approximately 500 nonhuman primates of several genera, 1,000 small rodents (mice, rats, hamsters), 20 rabbits or guinea pigs, and 20 dogs or cats. Provide inoculation of pregnant, newborn, juvenile, and adult monkeys with various infectious agents. Inoculated animals will be maintained and samples collected as indicated in each protocol. Furnish suitable rooms for animal examining and surgical preparation, necropsy, surgery, and x-ray examinations. A secure storage area of approximately 500 square feet will be required.

Huntington's disease (HD) is a late-onset neurodegenerative genetic disorder that produces uncontrolled physical movements, devastates intellectual faculties and triggers emotional disturbances. Those suffering from HD typically die within 10 years after symptoms surface and there is currently no cure. There is an emerging link between HD and clathrin-mediated endocytosis through Huntingtin-interacting protein 1 (HIP1). The purpose of this renewal application is to elucidate how the interaction between clathrin-coated vesicles and HIP1 (and HIP1R, R for related) is regulated in healthy cells to gain insights into the role of clathrin- mediated endocytosis in HD. A closely related purpose of this proposal is to find what structural determinants mediate the binding of HIP1 to Huntingtin-interacting protein 1-protein interactor (HIPPI), an accessory protein that modifies the activity of HIP1 in Huntington's disease. The first specific Aim is to determine what destabilizes the opened region of HIP1 to understand if molecular flexibility in the coiled-coil of this protein is important for its function. Site-directed mutagenesis will target specific positions in the coiled-coil to see if the opened region of HIP1 can be stabilized. Deletion mapping studies will be performed to pinpoint the location of "hard-wired" structure in the opened region that was found to be highly resistant to temperatures that would inactivate other proteins in the body. The powerful methods of X-ray crystallography and Nuclear Magnetic Resonance (NMR) will be used to study the static and dynamic properties of the stable sub-structure unit to assess if this feature is significant for HIP1 function. The second specific Aim is to determine if the binding dynamics of clathrin to HIP1 and HIP1R are regulated through molecular flexibility. First, the surfaces of HIP1 and HIP1R that make contact with the light chain subunit of clathrin will be fully characterized. Second, two binding models will be evaluated to determine if the binding determinants for clathrin light chain are spread between the two helices of dimeric HIP1 or if the determinants are contained entirely in one of the two helices. Third, a mutagenesis approach will determine if clathrin light chain and HIP proteins bind in a "head-to-head" or "head-to-tail" direction to deepen our understanding of how clathrin-coated vesicles build up in cells. The third specific Aim is to finish the 3.7 E crystal structure of the clathrin trimer domain to understand if this domain can interact productively with the N-terminus of HIP1. The fourth specific Aim is to investigate what structural determinants control the formation of the HIPPI/HIP1 complex that triggers the caspase-3/caspase-8 apoptotic pathway in Huntington's disease. Planned mutagenesis and in vivo studies will focus on two highly charged regions in the opened region of HIP1. The sum total of the information that will flow from the proposed research will deepen our understanding of how HIP proteins function in clathrin-mediated endocytosis and how HIP1, specifically, can potentiate the progress of Huntington's disease.

Abstract: QDS-Web(tm) Interview Administering System PEW surveys report 73% of American adults (147 million) use the Internet and 20% indicate it greatly improves getting health information, including drug and disease treatment facts. Thus, it is also an increasingly popular, cost-effective survey research environment. Web surveys facilitate health researchers rapidly and efficiently reaching very large numbers of targeted individuals. Web interviews can collect knowledge, attitudes, and behaviors related to disease prevention, health promotion, and clinical trial and/or health services delivery participation. However, to effectively conduct Internet research requires an easy, intuitive and rapid survey development and deployment tool designed to accommodate complexities of research interviews. Most web- based survey development tools are designed for simple, linear market research and customer satisfaction surveys. Few are designed for the researcher conducting multifaceted research and/or surveillance survey studies and still fewer meet Federal web survey requirements. NOVA's proposed Questionnaire Development System (QDS(tm)) module for Internet administration (QDS-Web(tm)) is such a tool. It uses Windows-based designs to easily generate survey files that meet Federal Web display (Sec. 508) and DHHS/FDA data security/audit trail requirements, an administrator's tool to quickly deploy secure Web-based complex research surveys, and a Web-based database to securely store and manage interviewee responses. Targeted users are researchers conducting behavioral surveys, clinical trials, health services research, and disease surveillance. QDS-Web(tm) supplements NOVA's commercial QDS(tm) Suite, with a growing base of more than 1,500 users worldwide. Phase II involves (1) enhancing code to handle survey control file functionality, including response types, skip patterns, range edits/consistency checks, logic expressions, and randomization algorithms; (2) creating web options allowing user-designed cascading style sheets and Web-based reports; (3) providing Cyrillic, Arabic, and Asian scripting and capability to select character formats (font, style, size); (4) implementing secure data transmission and storage, audit trails, and data transfer to SAS/SPSS using format libraries; (5) providing support for additional databases, middleware languages, and application servers; and (6) developing online documentation. During Phase II, individual and small group evaluations, in-depth qualitative interviews, and standardized usability surveys will be conducted with diverse sets of researchers. Beta testing will evaluate usability and functionality on comprehensiveness of features, ease of survey development/deployment, general usefulness, and overall satisfaction with using the QDS-Web(tm) module. The end product, QDS-Web(tm), as an added module to the QDS(tm) Suite, will be the only U.S.-made survey development integrated product enabling researchers to conduct computer-based surveys in all current modes of data collection from one design specification and securely manage data in one Warehouse Manager. QDS-Web(tm) Interview Administering System Questionnaire Development System Web-based data collection administration module (QDS-Web(tm)) is an added module to NOVA's commercial QDS(tm) Suite that enables public health researchers to conduct knowledge, attitude and behavior data collection for pubic health research and surveillance for general and sensitive health information. While most web-based survey research software is designed for simple, linear market research surveys and only supports web surveying, the QDS(tm) Suite, when including QDS-Web(tm) will permit researchers to easily, quickly, and cost-effectively conduct complex research surveys in all existing modes of survey administration from one integrated set of survey specifications that meet DHHS data transmission and storage security and audit trail requirements for behavioral, health services, and clinical trials research. Web surveying facilitates health researchers reaching large numbers of targeted populations, remote and hidden populations, and otherwise difficult to interview individuals not visiting face-to-face interviewing venues, and individuals not wanting to be personally identified. [unreadable] [unreadable] [unreadable]

This proposal presents a four-year research career development award for Dr. Reza Yaesoubi, an Assistant Professor in the Department of Health Policy and Management at the Yale School of Public Health (YSPH). His proposal aims to address important policy questions to control the ongoing spread of multidrug-resistant tuberculosis (MDR-TB) strains, which are resistant to essential antibiotics included in the first-line TB treatment regimen. While global efforts to scale up the diagnosis and treatment of MDR-TB and confront the growing problem of drug resistance have been slow moving, the recent introduction of new tools to control the threat of MDR-TB offer promise to accelerate the response. These new tools include rapid diagnostic tests for drug resistance and the novel anti-TB drugs. This proposal describes a rigorous, model-based investigation to identify strategies that can maximize the beneficial impact of these novel tools for TB and MDR-TB control. To study the overall impact of different control policies on MDR-TB spread, the candidate will develop detailed mathematical and simulation models of TB epidemics and calibrate them using data from two high MDR-TB prevalence countries, Peru and The Republic of Moldova (Aim 1). The candidate will then use these models to i) identify cost-effective, adaptive policies which use updated epidemiological information to guide targeted screening for TB and MDR-TB (Aim 2); and ii) identify drug-introduction policies that maximize the societal benefits of novel anti-T drugs and regimens while minimizing the probability of the emergence of antibiotic resistance (Aim 3). The proposed work will be conducted under the co-mentorship of Dr. Ted Cohen, MD, MPH, DrPH, Associate Professor of Epidemiology of Microbial Diseases at YSPH, and Dr. A. David Paltiel, PhD, MBA, Professor of Public Health, and Health Policy and Management at YSPH, and will be done in collaboration with the Center for Interdisciplinary Research on AIDS, the Center for Infectious Disease Modeling at YSPH, and the Center for Communicable Disease Dynamics at the Harvard School of Public Health (HSPH).The candidate will also be advised by Dr. Marc Lipsitch, DPhill, Professor of Epidemiology at HSPH and Dr. Joshua Salomon, PhD, Professor of Global Health at HSPH. Dr. Yaesoubi's training goals are (1) to develop a solid understanding of the dynamics of infectious diseases and the biological mechanisms governing the emergence of resistant bacteria, in particular MDR-TB, (2) to become an expert in methods for modeling complex multi-agent systems and optimizing the overall performance of these systems through the efficient allocation of available resources, and (3) to improve his mentoring, management, and collaboration skills that are essential for his successful transition to independence. The candidate's long-term goal is to become an independent scientist developing and applying decision science techniques to improve policies for the management and eradication of infectious diseases.

Melatonin is a hormone produced by the pineal gland at night that may be involved in the natural regulation of circadian rhythms. Animal studies have shown that exogenous melatonin administration can synchronize "free- running" circadian rhythms and can accelerate the rate of adaptation to a new rest/activity cycle. In studies currently in progress, we administered melatonin orally to five totally blind subjects with free-running endogenous melatonin rhythms in an attempt to entrain them to a 24-hour day. Although entrainment did not result, melatonin treatment produced large phase advances in the endogenous melatonin rhythms. These advances are much more robust than when melatonin is given to sighted people, possibly because the competing time cues from the light-dark cycle are not present in the blind subjects. In this project, we will evaluate the constraining effects of the light- dark cycle by giving melatonin to both blind and sighted subjects. We will define the parameters that produce the maximal phase-advancing effects in both groups by varying the timing, dose and duration of melatonin administration. We will attempt to enhance the phase-advancing effects of exogenous melatonin administration by suppressing endogenous melatonin production with the drug atenolol will test the effects of melatonin in sighted subjects who are adapting to an abrupt advance of their sleep-wake cycle (a laboratory model of shift work adaptation). We will evaluate the phase-advancing effects of exogenous melatonin administration by measuring its effect on the timing (phase) of endogenous melatonin production by the pineal gland. Phase will be assessed by obtaining serial blood samples and measuring plasma melatonin concentrations to determine the precise "onset" of active production. An RIA will be employed for efficiently processing most of the samples but critical values around the time of the melatonin onset will be verified with a highly specific gas chromatographic negative ion mass spectrometric assay. These studies may lead to the use of melatonin as a way to treat both blind and sighted patients with abnormal circadian rhythms.

Phosphoinositide-specific phospholipase C (PI-PLC) isozymes play a central role in receptor-mediated signal transduction in mammalian cells, cleaving the membrane lipid phosphatidylinositol 4,5-biphosphate to generate two second messengers, inositol 1,4,5-triphosphate and diacylglycerol. PI-PLCs are a large family of enzymes, and are also produced by parasitic protozoa including Trypanosoma brucei and the malaria-causing Plasmodium falciparum, by Drosophila, yeast, and plants. The smallest PI-PLCs are phosphatidylinositol-specific, i.e. only unphosphorylated PI is cleaved. They are secreted by the Gram plus bacteria Bacillus cereus and the nearly identical B. thuringiensis, by Staphylococcus aureus, which causes toxic-shock syndrome and related illnesses in humans, and by Listeria monocytogenes, responsible for bacterial infections in pregnant women, newborn children, and immunocompromised adults. The bacterial PI-PLCs are implicated virulence factors in these diseases. Bacterial PI-PLCs share with T. brucei PI-PLC the ability to cleave glycosylphosphatidylinositol (GPI) anchors, and are widely used to release mammalian GPI-anchored proteins. This laboratory has previously cloned, overexpressed, and solved the crystal structure of the B. cereus PI-PLC in informal collaboration with others in the Institute, and, in collaboration with Dr. John F. W. Keana, initiated the synthesis of inhibitors and substrate analogs. We now plan to complete mapping the active site region of B. cereus PI-PLC, to use this enzyme as a model system for the catalytic domain of mammalian PI-PLCs, and to examine related bacterial PI-PLCs. Specifically, we plan to complete the identification of residues of B. cereus PI-PLC important in substrate binding and catalysis through a combination of site-directed mutagenesis, crystallography, and enzyme kinetics. New fluorgenic substrate analogs will be synthesized in collaboration with Dr. Keana, to provide a sensitive continuous assay for the kinetic studies. Inhibitors, already available through this collaboration, will be used to identify which residues interact with the phosphoinositol, glycerol, and lipid groups of the substrate and to determine the effect on the pKa of catalytically critical residues by NMR. To exploit the fact that B. cereus PI-PLC closely resembles the catalytic domain of mammalian PI-PLC delta 1, the bacterial isozyme will be used as a model system, substituting critical mammalian segments into the enzyme to alter its substrate specificity. Finally, the new substrate analogs and inhibitors will be used in a comparative study of the PI-PLC virulence indicators of S. aureus and L. monocytogenes.

We have examined the role of raf and myc family oncogenes in naturally occurring human tumors and in the mouse, and have determined their transforming properties and likely mechanisms of action. In the mouse, raf tumor induction was studied using a series of recombinant murine retroviruses. Viruses carrying only raf family oncogenes, including constructs with the cellular homolog of v-raf, c-raf-l, and a raf-related gene, A-raf-1, induce predominantly fibrosarcoma and erythroid hyperplasia. The J-2 virus, which contains both v-raf and v-myc, demonstrated a synergism of these two oncogenes in inducing hemopoietic and epithelial neoplasias in newborn mice with a greatly reduced latency period compared to disease induction by raf or myc viruses. Murine retroviruses containing only v-myc (J-3 and J-5) predominantly induce lymphomas (of both T and B lineage) but transform other hemopoietic and epithelial lineage cells as well, with a latency period which depends upon the replication efficiency of the pseudotyping MuLV. raf-induced neoplasias require specific interleukins (IL) for culturing in vitro, whereas cell lines established from J-2, J-3, or J-5 neoplasias grow in culture without specific growth factor supplements. A function for v-myc in synergism with v-raf has been established by experiments demonstrating that v-myc can abrogate growth factor requirements of IL-3- and IL-2-dependent cells and v-myc-infected fibroblasts grow in medium depleted of PDGF. A function for raf in the signal transmission pathway of growth factors has been established in studies involving raf transformation of Ki-ras-resistant cells and in microinjection analyses of ras and raf antibodies into normal, ras- and raf-transformed mouse fibroblasts. These studies suggest that raf is located downstream of ras in signal transmission. Finally, even tumors induced by the dual oncogene virus J-2 are clonal, suggesting that additional events may be required in the maintenance of the transformed phenotype. In an in vitro-derived J-2-infected myeloid cell line, we have identified a likely alteration in the c-myb proto-oncogene, a proviral insertion common to an in vivo-derived tumor.

Salivary gland malignancies are rare and heterogeneous cancers of the head and neck. Mucoepidermoid and adenoid cystic carcinoma are the most common and demonstrate an unpredictable clinical course with locoregional failure and distant metastasis. Treatment is primarily surgical followed by adjuvant radiotherapy based on certain pathologic features. The role of chemotherapy, including targeted therapy, is investigational. The five year survival for high grade salivary carcinomas is approximately 40%. Given the rarity and diversity of these tumors, comprehensive genetic analysis has not been performed to date. However, there is a need for novel clinical approaches and new molecular targets are critically important for therapy development in patients with salivary gland malignancies. By performing whole genome sequencing on high grade mucoepidermoid and adenoid cystic carcinoma, this project will identify potential new avenues for therapeutic, diagnostic and prognostic intervention in salivary gland cancer. For mutational screening, we will employ whole genome sequencing on clinically annotated 20 high grade mucoepidermoid carcinoma and 20 adenoid cystic carcinoma samples. All mutations will be examined in normal DNA from the same patient to identify and confirm somatic mutations and rearrangements. The point mutations and rearrangements that are identified will then be queried in the plasma and saliva to identify their roles as biomarkers. Identification of these genetic alterations and biomarkers will likely provide potential prognostic, diagnostic and therapeutic strategies for salivary gland malignancies.

This project will be devoted to the development and evaluation of a crossed beam Doppler ultrasound concept for improved detection of atherosclerosis.

Bacterial endospores derive much of their longevity and resistance properties from therelative dehydration of their protoplasts. This dehydration is maintained by a surrounding peptidoglycan structure, the spore cortex. A structural modification unique to,the spore cortex is removal of all or part of the peptide side chains from the majority of the muramic acid residues andconversion of 50% of the muramic acid to muramic lactam. A mutation in the cwLD gene of Bacillus subtilis, predicted to encode a muramoyl-L-alanine amidase, results in the production of spores containing no muramic lactam. These spores have normally dehydrated protoplasts but are unable to complete the germination/outgrowth process to produce viable cells. Our objective is a detailed understanding of formation and maintenance of a dormant, resistant bacterial (Bacillus subtilis) spore. The aims are: a) analyze the sequence, regulation and function of additional genes encoding penicillin-binding proteins (PBP); b) with strains lacking one or more PBPs, analyze the cortex structure and heat resistance of their spores; and c) identify the enzyme(s) initiating cortex hydrolysis during spore germination. Work being carried out with B. suhtilis should yield knowledge applicable to other Gram-positive spore formers. The latter organisms are significant agents of food spoilage and food-borne human disease, largely because of the resistant nature of spores. Thus knowledge gained herein could have significant applications. Muropeptide structures are determined by amino acid/amino sugar analysis and NLAJ-DI-TOF MS.

More than half a million cholecystectomies are performed annually in the United States, and 10-20% of these patients present afterwards with continuing or recurrent pains. Patients with normal imaging and laboratory findings are often suspected of suffering from Sphincter of Oddi dysfunction Type III (SOD III), but are difficult to evaluate. Sphincter of Oddi manometry (SOM) is used for diagnosis, but is not widely available, not completely accurate, and can cause pancreatitis. The usual treatment for SOD III (endoscopic ablation of the biliary and/or pancreatic sphincters) has complications which can be life-threatening. Endoscopic injection of Botulinum toxin (Botox) into the sphincter zone reduces the pressure for up to 6 months, and may be a good therapeutic trial. The objective of this R03 Planning Grant is to provide support to carry out the organization and grant-writing activities needed to prepare an R01 NIH application for a major Phase III Randomized Multicenter Clinical Trial in patients with suspected SOD III. Funding is requested to support the following activities: finalize the protocol, case report forms, Data Management Plan, and Manual of Operations; recruit and certify additional study sites; prepare training schedules and materials; develop the Digestive Disease Pain-Burden Questionnaire (DDPBQ) and finalize the planning for the validation of the instrument; establish membership of committees and identify independent medical monitor; and write the NIH R01 grant for the SOD III Multicenter Trial. The properties associated with SOD III render it uniquely suited for such an investigation. The Phase III SOD Randomized Multicenter Clinical Trial will determine: 1) if SOD III really exists, 2) if SOD III can be detected by either clinical parameters, the temporary response to intra-sphincteric injection of Botox, SOM, or a possible combination, 3) which strategy for detection of SOD III is most cost-effective, and 4) if the new Digestive Disease Pain-Burden Questionnaire (DDPBQ) is useful in evaluating these patients, and their responses to treatment. This trial will answer several of the important questions raised by the expert panel at the NIH State of the Science of ERCP Conference, held in January.

Macrophages and neutrophils play major roles in host defense against microbial infections. In order to perform this function, these cell types must ingest and destroy pathogens, generally in phagosomes, as well as secrete a number of products that signal other immune cells to respond. Generation of low organellar pH is primarily driven by the V-ATPases, proton pumps that use cytoplasmic ATP to load H+ into the organelle. Alongside the pumps are various channels that shunt the transmembrane potential generated by movement of protons; in different organelles these comprise H+ channels, K+ channels and Cl- channels. Nevertheless, the contribution of these pathways to maintenance of intraorganellar pH is poorly studied. Recently, we demonstrated that murine alveolar macrophages (AMs) but not neutrophils employ the CFTR (Cystic Fibrosis Transmembrane conductance Regulator) Cl- channel as a major shunt mechanism. Lysosomes and phagosomes in murine cftr/- AMs failed to acidify and the cells were deficient in bacterial killing compared to wild-type controls. We have also shown that AMs lacking CFTR are deficient in stimulus-induced secretion. Here we propose to extend these observations by investigating the role of Cl- flux in a common set of phagocyte core functions namely, organellar acidification, granule secretion, and microbicidal activity. We will compare the phagocytic and secretory activities of alveolar and peritoneal macrophages as well as neutrophils and the anion channels that are involved in the regulation of these activities. CFTR and ClC-3 chloride channels are the most reasonable channels with which to begin our studies given our preliminary data on CFTR in murine AMs and that of others in human and mouse neutrophils showing a differential functional dependence on either ClC-3 or CFTR depending upon the species. Utilizing primary cells obtained from normal, Cftr-deficient (or mutant) and ClC3-deficient mice as well as human cells from non-CF and CF patients, we will use a variety of molecular, immunochemical, microscopic and electrophysiological techniques, well-established in our laboratories, to provide a multi-faceted systems approach to the problem. PUBLIC HEALTH RELEVANCE: Patients with CF are highly susceptible to chronic bacterial infection. To date, lung dysfunction in CF has been largely attributed to depletion of the liquid layer covering the upper airway epithelium with a consequent accumulation of mucus that is thought to contribute to the persistence of bacteria in the airway tree. We propose that an additional defect may be attributable to a failure of CFTR-deficient alveolar macrophages to exhibit vigorous bactericidal activity. Defects in the behavior of the innate immune system could have important consequences for microbial defense in CF patients. [unreadable] [unreadable] [unreadable]