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As an endpoint for many different types of cardiovascular disease, heart failure (HF) is a leading cause of mortality and morbidity in the U.S. Though some patients with HF have intact systolic function, virtually all patients with HF have abnormal diastolic function and an impaired ability to increase cardiac performance in response to physiologic stress, including exercise. During exercise, myocardial responses to increased heart rate and adrenergic stimulation normally involve augmentation of cardiac filling (requiring relaxation reserve) and enhanced ejection (requiring contractility reserve). At the cellular level, relaxation reserve, the focus of this application, requires faster decay of the intracellular calcium (Ca) transient and a decrease in myofilament Ca sensitivity. Ordinarily, both processes are enhanced by beta- adrenergic stimulation triggering PKA-mediated phosphorylation of key Ca regulatory and myofilament proteins. However, both relaxation reserved and adrenergic modulation of relaxation reserve are abnormal in failing hearts. Recognizing that Ca cycling dynamics are themselves abnormal in failing myocardium, the broad objective of the proposed studies is to examine beta-adrenergic/PKA-mediated modulation of relaxation reserve in failing human hearts in a manner that accounts for the defects in Ca cycling present in these hearts. Our working hypothesis is that adrenergic signaling defects result in an impaired beta-adrenergic augmentation of Ca uptake rates and a reduced ability to decrease myofilament Ca sensitivity. Mechanistically, we hypothesize that a reduced ability to phosphorylate phospholamban and troponin I cause an impaired ability of cAMP and PKA-dependent signaling to augment sarcoplasmic reticulum Ca uptake and reduce myofilament Ca sensitivity, respectively. Our specific aims are to: 1) examine beta-adrenergic modulation of relaxation reserve in patients with systolic an diastolic HF; 2) examine cAMP-induced modulation of relaxation reserve and Ca uptake in human myocardium; 3) examine PKA-dependent modulation of myofilament Ca sensitivity in human myocardium; and 4) determine whether Ca cycling defects or reduced targeting of PKA to troponin I limit PKA-dependent modulation of relaxation reserve. While defining mechanisms of impaired relaxation, these studies will help develop and validate dynamic noninvasive imaging strategies for clinical assessment of relation reserve in patients with HF.

This application embodies support for three research programs of the Radiation Therapy Center plus limited support for the core program. The largest program deals with the development of radiopharmaceuticals for tumor and organ delineation. Second is a project dealing with the effect of radiation on carcinoembryonic antigen titer. A final project is concerned with development of a tumor model for evaluating time-dose relationships.

Th long-term goal of this research is to determine the three-dimensional structures of M-components. The source of the immunoglobulins and immunoglobulin fragments, whose crystal structures will be determined by X-ray diffracton, will be patients with monoclonal gammopathies. Most often these will be malignancies involving myelomatosis, Waldenstrom's macroglobulinemia, or cryoglobulinemia. M-components associated with benign monoclonal gammopathies will be studied whenever possible also in order to compare them with proteins from cancer patients. A continuous search, now in progress, will be maintained to find proteins that yield good quality single crystals. The proposed research could lead to a better understanding of cryoprecipitation phenomena, and it could provide information concerning pathogenesis of M-components. It will also add to our knowledge of the molecular structures and the mechanisms of action of antibodies. BIBLIOGRAPHIC REFERENCE: "Angle Settings for Rotation Around the Diffraction Vector for Four-Circle Diffractometers," B.C. Wang, C.S. Yoo, J. Pletcher and M. Sax, Acta Cryst. A32, 918 (1976).

Post-absorptive changes of the plasma amino acids reflect the adequacy of the balance of the dietary amino acids. Previous studies conducted in our laboratory utilizing man or dogs as test subjects have been successful in developing an in vivo plasma amino acid (PAA) ratio procedure to assess the ideal intake of dietary amino acids. Subsequent studies adapted this PAA ratio procedure to the rat in order to make it possible to determine the amino acid requirements for this species. By using the rat, it will be possible to determine the validity of these requirement data by carrying out long-term growth and balance studies. During the initial 9 months of this program, the amino acid requirements for the one and three-month old rat have been determined by the PAA ratio procedure. The data show a lower amino acid need for lysine and the sulfur amino acids for the one-month old rat and for lysine, threonine, valine, and isoleucine for the three-month old rat than the requirement data published in the literature. Similar data will be determined for the six and twelve-month old rat. Then long-term growth and development studies will be carried out to determine whether these ideal dietary amino acid patterns as assessed by the PAA ratio technique are more exact in predicting amino acid needs than the requirements as published in the literature & obtained by the nitrogen balance procedure.

The objective of this project is to determine the effect that the neuropeptide fragment ACTH 4-10 has upon the cognitive abilities of young and elderly individuals. This drug has been suggested to facilitate the efficiency of a number of mental operations including arousal, selective attention, and memory retrieval. We will use the conceptual and methodological paradigms on the information processing model of experimental psychology to test for changes in these cognitive functions in subjects taking a synthetic form of ACTH 4-10. Every subject will take part in five testing sessions, each with a different dosage level of the drug. A double blind, complete crossover design will be used. The age of the subjects will be an important variable in as much as elderly individuals have been shown to have some problems in all of the cognitive functions to be tested, and thus will be more likely to show a substantial drug effect. Most of the tasks used will measure the effect of the drug on the speed with which the subject can carry out a given mental operation. We will examine the effect of ACTH A-10 on subjects' ability to: 1: use prior information to selectively attend to only the relevant cues in a visual search task: 2) use a warning cue to speed up their response in a disjunctive reaction time task; 3) retrieve information from long-term memory rapidly. In addition, we will examine whether the drug primarily affects subjects' ability to perceive a stimulus (sensitivity) or instead, whether it alters the criterion they use to make their decision. From these studies we hope to learn whether ACTH 4-10 affects certain specific mental operations, and whether this affect is greater in the elderly. If this drug does enhance cognitive performance in the elderly, it may be useful in treating some of the cognitive deficits which are often associated with advanced age.

The goal of this project is to cross-reference the functional information known about genes actively under study in model organisms with genetic maps of the human and mouse genomes. The public database of Expressed Sequence Tags (dbEST) will serve as the source of novel human transcribed sequences that will be searched using model organism protein sequences as queries. Human cDNA sequences from which the ESTs were derived will be mapped to human chromosomes and their positions determined precisely in the mouse genome. The comparative mapping information will directly link functional information from yeast, flies, worms, and other experimental organisms to mouse and human gene loci. Specific experimental aims are: 1. Search the EST database for human homologs of model organism genes. All yeast gene sequence information and gene sequences of interest to the scientific community from other organisms will be used to search the EST database on a monthly basis. Results will be communicated to participating laboratories via e-mail and will be available online via the World Wide Web. 2. Map ESTs on human and mouse chromosomes. ESTs will be assigned to specific human chromosomes using somatic cell hybrids and positionally mapped on mouse chromosomes using a highly informative interspecific hybrid backcross panel. 3. Continue to develop and improve XREFdb. The relational database that serves this project will be improved throughout the funding period to increase efficiency and maximize communication of information with the scientific community. 4. Develop methods for inter- and intra-organismal gene repertoire comparisons. As whole genome sequence data become available, protein sequences will be compared to determine similarities and differences within and across species. Mapping reagents generated by the Human Genome Project are facilitating positional mapping of mutations in genes responsible for mammalian mutant phenotypes including human disease. A wealth of biological information has been obtained about genes in yeast, flies, worms, and other experimental models for study of eukaryotic biology. This project will facilitate progress in the understanding of gene action in normal and disease processes in mammals.

Medicare payments to oncologists for chemotherapeutic agents have been a source of considerable controversy for several years. The Medicare Modernization Act (MMA) changed reimbursement methods, effectively reducing the markups oncologists receive, especially for expensive agents. In this work we propose a regression discontinuity design to study the effect of this change on the likelihood of prescribing chemotherapy to all cancer patients and, if chemotherapy is prescribed, the expensiveness of the agents used, and the setting in which chemotherapy is administered. If treatment changes are found, we will also consider the impact on indicators of untoward outcomes. In prior work we studied a similar question using data from before the MMA to assess the effect on treatment of variation in chemotherapy reimbursement to the same physician over time as well as across carriers. We found that more highly reimbursed oncologists were not more likely to prescribe chemotherapy to patients with metastatic cancer, but were more likely to use expensive agents. The complex nature of the recent reimbursement changes -- large reductions in payments for drugs but a significant increase in administrative fees -- makes predicting the MMA's effect from prior work difficult. Understanding its impact on chemotherapy treatment patterns has important implications not only for Medicare spending but also for the welfare of beneficiaries with cancer. [unreadable] [unreadable] [unreadable] [unreadable]

Project Summary Biomedical research increasingly involves examination of huge data sets. Introduction of new high- throughput sequencing technologies has led to production of new genome sequence for dozens of plants and animals and prompted major efforts to sequence individual human genomes. To take full advantage of these new large-scale data sets, researchers require flexible, user-friendly software that supports interactive, in-depth exploration of data at different levels of detail, ranging from chromosomes to individual base pairs. The Integrated Genome Browser (IGB) is a desktop, java-based genome browser implemented that implements innovative yet practical visualization techniques designed to help scientists explore genome-scale data sets, ultimately leading to better treatments for disease as scientists use IGB to achieve deeper insight into genes and genomes. This project will continue to develop the IGB software, adding new data integration and display features users have requested and which the IGB developers foresee will become increasingly important in the face of on-going and rapid technological change in genomics. Major improvements will include migrating the IGB graphical user interface to JavaFX, a toolkit released as part of Java 8 that replaces the Swing toolkit; speed and memory improvements for interactive display of large datasets; integration with cloud-based data storage resources; an all-new editing capability enabling users to annotate genomic scenes with new features inferred from data; and a new interactive alternative splicing visualization. The project will develop and harden the Integrated Genome Browser Application Programming Interface (API) for developers, transforming IGB into a flexible and easy-to-use framework for building and distributing all-new visualizations as IGB Apps, OSGi plug-ins that users will access via a new IGB App Store. To maximize project impact, we will accelerate user training and outreach, using social media, on-line training, and in-person workshops to help researchers develop deeper insight into bioinformatics analysis through visualization. This project will grow the IGB user community, which includes scientists working with data, programmers seeking to build powerful new tools, and bioinformatics analysts seeking easy ways to incorporate visualization into data analysis pipelines.

The intent of this application is to document the ability of Wills Eye Hospital's Glaucoma Service to participate as an effective and productive clinical center in the proposed multicenter clinical trial, entitled "Collaborative Initial Glaucoma Treatment Study" (CIGTS). This study's purpose is to evaluate whether initial treatment of open angle glaucoma is more effective by means of a stepped, medical regimen or by means of a surgical approach. The Wills Eye Hospital (WEH) Clinical Center will participate in the CIGTS by identifying and recruiting eligible patients, administering the treatment regimen as assigned by the CIGTS's Coordinating Center and defined within the study's Manual of Operations, following and retaining study patients, recording and transferring study data to the Coordinating Center, and contributing to reports that will present the CIGTS results. This application describes the ability of the Wills Eye Hospital's Glaucoma Service Clinical Center to enroll approximately 32 patients per year, based upon pilot studies of patient eligibility conducted during a three month period. Details are provided of our plans to identify, recruit, follow, and retain these patients, our intent to obtain consistently high quality data on the visual outcomes of treatment, and the personnel and facilities available within our center to carry out the provision of the CIGTS's Manual of Operations.

The quality of healthcare in the United States is inadequate and inequitable. There is a wide gap between the care people should receive and the care they do receive. Although much of the evidence for substandard quality comes from clinical care for adults, the limited data on children suggest that the quality of their care is comparably lacking. The 2009 Child Health Insurance Program Reauthorization Act (CHIPRA) calls for the establishment of a national Pediatric Quality Measures Program (PQMP) to convene experts, solicit public input, and build infrastructure to provide actionable measures for diverse settings and for a variety of purposes. As part of the PQMP initiative, we propose to establish the Children's Hospital Boston Center of Excellence for Quality Measurement (CoE) to develop, enhance, and test child quality measures, and ensure that they are ready for widespread use. The CoE will be a comprehensive quality measure development center that has the research expertise, clinical experience, and direct access to diverse healthcare delivery models and patient populations that are needed to transform quality measurement. In creating the CoE, we aim to: 1) Build a strong network of academic, clinical, payer, provider, and patient groups that jointly provide exceptional capacity in pediatric quality measure development, testing, implementation, and analysis; 2) Develop and enhance pediatric quality measures assigned by AHRQ following a public process; 3) Test measures in multiple settings across the clinical spectrum with a wide range of end-users and assure that measures are designed to identify disparities in care; and 4) Deliver to AHRQ actionable measures with detailed specifications that make them ready to use across the health care system. We will lead the development of innovative approaches to improve measurement, testing, and use, drawing on state-of-the-art health information technology (HIT) and statistical methods. We will propose improvements for meaningful use of electronic health records (EHR) and claims systems to enable automated data collection that is valid and reliable across all regions and settings. We will assess patient-level and practice-level disparities in care, and the value and cost-effectiveness of all measures developed. We have capacity in all 5 areas prioritized by CHIPRA for measures development, especially inpatient measures, health outcome measures, and measures of the most integrated health care settings. PUBLIC HEALTH RELEVANCE: We propose to create the Children's Hospital Boston Center of Excellence for Quality Measurement to develop, enhance, and test pediatric quality measures. We aim to build a strong network of academic, clinical, payer, provider, and patient groups that jointly provide exceptional capacity in pediatric quality measurement. We will assure that measures are designed to identify disparities in care.

Isozymes of heme oxygenase (HO) convert heme to biliverdin and provide the dominant heme-degradative mechanisms in tissues. HO-1 is the inducible, while HO-2 is the constitutive, isoform; HO-3 possesses trivial heme-degrading activity. The study of HO attracts considerable interest because of the recognized protective and vasorelaxant actions of HO in general, and the ready inducibility of HO-1, in particular. This R0-1 competitive renewal continues the study of HO-1 as a protective response against renal injury and pursues findings made in the present cycle: a novel stimulus for HO-1 was identified in the kidney, and one with critical relevance to renal vasoconstriction, namely, angiotensin II. Additionally, two novel pathways that may contribute to the cytoprotective actions of HO-1 were uncovered: the suppressive effect of HO-1 on monocyte chemoattractant protein-1 (MCP- 1), a chemokine incriminated in renal inflammation, and the inductive effect of HO-1 on p21Cip1.WAF1.SDII a cyclin-dependent kinase inhibitor which is also anti-apoptotic. Thus, induction of HO-1, on the one hand, may vitiate pathways of renal injury (angiotensin IT-induced vasoconstriction, the upregulation of MCP-1), while, on the other, HO-I induction may recruit p21-dependent pathways of protection. Because of the importance of angiotensin II, MCP-l, and p21 as determinants of renal injury, our renewal application pursues three specific aims. Aim I hypothesizes that the HO system is a countervailing one that opposes the systemic and renal vasoconstrictive actions of angiotensin II; studies in this aim alter the expression of the HO system by pharmacologic, antisense approaches, and gene delivery, and examine their effects on angiotensin TI-induced constriction. Aim II hypothesizes that the HO system suppresses renal expression of MCP-1. Using in vitro and in vivo models for lipopolysaccharide-induced MCP-l expression, this aim examines the regulatory role of the HO system on MCP-1 expression, focusing on the redox-regulatory effect of HO on NF-kB, the latter transcription factor critically controlling MCP-1 expression. Aim III hypothesizes that the HO system upregulates p21 which contributes to the protective actions of HO-1. This aim delineates the role of the cell cycle-inhibitory, anti-apoptotic molecule, p21, as a transducer of the cytoprotective effects of HO-1. Our proposals thus explore mechanisms by which HO interrupts major pathways of injury (Aims I and II) as well as recruits pathways that safeguard the kidney (Aim III).

Our long-term goal is to understand the molecular mechanism(s) that regulate(s) cytokinesis in mammalian cells, and to use this knowledge to develop novel cancer therapeutic strategies. Dysfunctional regulation of cytokinesis can lead to uncontrolled cell proliferation, a key characteristic of cancer. The integrity of cytokinesis relies on the precise coordinated regulation of the positioning and timing of contractile ring assembly. One of the prevalent models is that signals from the central spindle determine the positioning and timing of contractile ring assembly. Polo-like kinase 1 (Plk1) and aurora kinase B (aurora B) are two important mitotic kinases, which localize to the central spindle and coordinate with small GTPase signaling to regulate contractile ring assembly. The small GTPase proteins, such as RhoA, Rac1, and Cdc42, are activated by guanine nucleotide exchange factors (GEFs) and inactivated by GTPase-activating proteins (GAPs). Thus far, at least three GEFs, i.e. ECT2, GEF-H1, and MyoGEF, are implicated in regulating cytokinesis. However, it is still not clear why cells need three GEFs for cytokinesis and how they cooperate to regulate equatorial RhoA activation and localization. The overall objective of this grant application is to understand how MyoGEF regulates cytokinesis and why mammalian cells need more than one GEF for cytokinesis. Our central hypothesis is that Plk1 and aurora B coordinate to recruit MyoGEF to the central spindle and cleavage furrow, where MyoGEF cooperates with ECT2 and/or GEF-H1 to regulate RhoA activation and localization at the cleavage furrow. We propose two aims to test our central hypothesis, by using a combination of biochemistry, cell biology, and molecular biology, with mammalian cells as a model. In Specific Aim #1, we will elucidate how Plk1 and aurora B coordinate with MyoGEF in regulating cytokinesis. This aim will test our working hypothesis that, during anaphase, aurora B creates a Plk1 docking site in MyoGEF and allows Plk1 to bind and phosphorylate MyoGEF, in turn promoting the recruitments of MyoGEF to the central spindle, thus contributing to the regulation of equatorial RhoA activation and myosin contractile ring assembly. In Specific Aim #2, we will determine why mammalian cells need more than one GEF for cytokinesis. This aim will test our working hypothesis: 1) MyoGEF, ECT2, and/or GEF-H1 redundantly regulate cytokinesis in certain cells;2) MyoGEF, ECT2, and/or GEF-H1 are implicated in cell type-specific regulation of cytokinesis;3) MyoGEF, ECT2, and GEF-H1 coordinate the regulation of equatorial RhoA activation and localization.

This application is directed towards understanding the mechanisms involved in pathogenesis of Barth syndrome, a rare X-linked mitochondrial disorder, caused by mutations in the tafazzin (taz) gene and characterized by dilated cardiomyopathy, skeletal myopathy, chronic fatigue, exercise intolerance, cyclic neutropenia and organic aciduria. The taz gene encodes a mitochondrial protein with a high degree of homology to acyl-transferases. Mutations in taz gene result in reduction of cardiolipin, a tetraphospholipid, and accumulation of monolysocardiolipins (MLCL) in mitochondria, a diversification of cardiolipin species, which are normally dominated by tetra-linoleoyl cardiolipin (L4CL). Taz function has been studied in non-mammalian model organisms, such as yeast, flies and zebrafish. Although existing models of taz-deficiency provide valuable information about the role of taz in CL metabolism and mitochondrial function, these systems cannot be adequately used and manipulated to study the long range effects of gene deletion or mutation in the whole organism and do not allow development and testing of potential therapies. Therefore, we generated an inducible taz-knockdown mouse model by employing RNA- interference technology. We ascertained the efficiency of taz-silencing and analyzed its consequences, which we hypothesized would impact cardiac function and to alter the mitochondria of cardiac and skeletal muscles. Taz deficiency caused aberrant CL molecular speciation, reduction of L4CL and accumulation of MLCL in heart and skeletal muscle. Moreover taz deficiency resulted in severe cardiac malfunction, with ultrastructural defects and excessive autophagy in sarcomeric tissues. Based on our preliminary studies, we hypothesize that L4CL is essential for mitochondrial energy production, mitochondrial structural integrity, and cross-communication of mitochondria with other intracellular organelles. To explore our hypothesis, we propose three Specific Aims: (i) to characterize the detailed cardiac and skeletal muscle phenotype of the mouse tafazzin deficiency, (ii) To assess the extent of the permanent damage caused by cardiolipin deficiency and to estimate how much of this damage is reversible, (iii) To investigate mechanisms by which taz-deficiency impairs mitochondrial function. PUBLIC HEALTH RELEVANCE: The grant proposal entitled "A mouse model of Barth syndrome, a mitochondrial cardiolipin disorder " is designed to study molecular mechanisms of pathogenesis of Barth syndrome, a rare genetic pediatric disorder, caused by mutations in the tafazzin gene. We will utilize our newly-developed mouse model for tafazzin deficiency. We believe that the proposed study will define the roles of cardiolipin in mitochondrial function and will enable the development of efficient therapeutic strategies to treat Barth patients and related syndromes.

Influenza A and B viruses cause a highly contagious respiratory disease in humans. Influenza B viruses infect only humans, whereas influenza A viruses infect many avian and mammalian species. Influenza A viruses are responsible for the periodic wide-spread pandemics that result in high mortality rates. The 1918 pandemic was the most devastating. Highly pathogenic H5N1 influenza A viruses are prime candidates for causing the next pandemic. The NS1 proteins of influenza A virus (NS1A protein) and of influenza B virus (NS1B protein) are multifunctional proteins that suppress cellular antiviral responses and are virulence factors. Many of the functions of the NS1A protein differ from those of the NS1B protein. The overall aim is to elucidate how multiple functions of the NS1A and NS1B proteins play important roles during infection. The NS1A protein has a binding site for double-stranded RNA (dsRNA) and binding sites for several cellular proteins, including CPSF30, a cellular factor required for the processing of cellular pre-mRNAs. CPSF30 binding, which results in the inhibition of the production of all cellular mRNAs, including interferon-2 (IFN-2) mRNA, is the primary, if not the only, mechanism by which the NS1A protein of a human influenza A virus (isolated in 1972) inhibits IFN-2 mRNA production. One aim is to determine whether this mechanism is shared by all other human influenza A viruses, including H5N1 and 1918 viruses. This research will entail the generation of multiple recombinant viruses expressing NS1A proteins containing specific amino acid changes. Recent structural results on the dsRNA- and CPSF30-binding sites of the NS1A protein described in this grant application may lead to the development of new antivirals directed at these binding sites and also to the development of better live attenuated virus vaccines against H5N1 viruses. For the latter purpose, one aim is to generate viruses whose attenuation is fine-tuned by site-specific amino acid changes in these two NS1A binding sites. Motivated by genetic experiments that showed an unexpected functional interaction between the NS1A protein and the viral polymerase, another aim is to use biochemical approaches to elucidate the molecular mechanisms underlying this interaction. The IFN-induced ISG15 protein, which is conjugated to multiple cellular proteins, has antiviral activity against both influenza A and B viruses. One aim is to identify the viral and/or cellular proteins whose conjugation to ISG15 accounts for the antiviral action of ISG15 conjugation against influenza A virus. Because the NS1B protein, unlike the NS1A protein, binds ISG15 and prevents its conjugation to target proteins, research on the antiviral function of ISG15 conjugation against influenza B virus will analyze the defects in replication of a recombinant influenza B virus that expresses a NS1B protein lacking an ISG15 binding site. As an integral part of this research, the role of the dsRNA-binding activity and other functions of the NS1B protein will also be determined. Elucidation of the molecular mechanisms by which ISG15 conjugation inhibits influenza A and B viruses may lead to therapies that exploit these mechanisms.

The long-term goal of the proposed research is to understand molecular characteristics of ion movement through transmembrane channels. This goal will be pursued through studies on voltage-dependent sodium channels that are incorporated into planar lipid bilayers of defined composition. Ion permeation, pharmacological modification, and voltage activation (gating), will be examined, with special emphasis on the role of fixed charges at the extra- and intracellular surface of the protein and host bilayer in modulating channel function. The mechanism of ion entry into the channels will be studied to determine whether negative charges close to the channel entrance have a physiological function as guides for ion entry. Ion permeability and block of channels modified by batrachotoxin, veratradine, pyrethroid insecticides, and group-specific modification will be compared in an attempt to clarify why channels that have a decreased single-channel conductance have a decreased ion selectivity. Group-specific modification and proteolytic cleavage will be used to modify the channel entrance and examine the relation between the guanidinium toxin binding site and the extracellular channel entrance. The kinetics of toxin-induced channel closures will be examined to determine whether the guanidinium toxin-induced channel closures occur as a two-step event, where the channel is closed thorough a conformational change subsequent to toxin binding. The stationary voltage-activation of single channels will be examined to define to what extent gating behavior is affected by lipid surface charges, and to further define the asymmetry in the apparent surface charge density at the extra- and intracellular surfaces of the channel. The slow "mode changes" that affect gating will be studied. The aim is to characterize some of the stationary conformational fluctuations that occur in an integral membrane protein, as well as to determine whether the mode changes can be fully accounted for by discrete shifts in the midpoint potential of the activation curves.

This proposal aims to genetically determine the involvement of excess N-methyl-D-aspartate receptor (NMDAR) activity in the pathogenesis of early cognitive deficits of Down syndrome. Oligomeric Ap peptides are known to be generated very early in Down syndrome because of the extra copy of the APP (amyloid precursor protein) gene on chromosome 21 (the trisomic chromosome in humans with Down syndrome). We propose that the early synaptic damage that is known to occur in Down syndrome brains may, at least in part, be due to excessive levels of Ap peptides. One possible mechanism by which excessive AB may cause synaptic loss and neuronal injury is via NMDAR activation, since it is known that AB increases glutamate release from astrocytes. NR3A subunits inhibit NMDAR activity and act as neuroprotective molecules that mitigate neuronal injury caused by various experimental paradigms. Our recent data indicate that AB induces greater synaptic injury to NR3A-knockout (KO) neurons compared to wild-type (WT) neurons. In addition, we have identified a large gene family that we named takusan (meaning many in Japanese), which regulates synaptic activity. Based on the properties of takusan proteins, we postulated that this molecule might also protect synapses. In this proposal, we will genetically cross Ts65Dn mice, a mouse model of Down syndrome, with mice mutant in NR3A or takusan genes. We will then evaluate the consequences of these crosses for early neurological and cognitive deficits, as well as dendritic spine loss and electrophysiological abnormalities that have been observed in Ts65Dn mice. These experiments will determine whether NR3A and/or takusan play mechanistic roles in possible protection from the synaptic damage that occurs in Down syndrome. Our Specific Aims are as follows: (1) To determine the potential ill effect of NRSA KO of the NMDAR in the pathogenesis of cognitive deficits in Down syndrome. (2) To determine the potential beneficial effect of expression of the NR3A subunit of the NMDAR on the pathogenesis of cognitive deficits in Down syndrome. (3) To identify the effect of takusan in protecting from AB-induced synaptic spine loss.

DESCRIPTION (Applicant's Description): Under conditions of glaucoma, the cells of the retina begin to die, leading to progressive vision loss. This cell death may be due to a pressure-induced, excessive release of the neurotransmitter glutamate. While glutamate is a neurotransmitter normally used by retinal neurons, when it is present at high levels for a prolonged period of time, it is toxic to the cells, causing their death. The objectives of this study are to 1) Localize the source of excess glutamate, 2) Determine whether the amount of glutamate measured will lead to retinal cell death, and 3) Evaluate cannabinoids as possible drugs to prevent the retinal cell death.

This proposal represents the second competing renewal of Vanderbilt's GI SPORE. This SPORE continues to focus on colorectal cancer, the second leading cause of cancer deaths in the US, where it affects more men and women than all other gastrointestinal malignancies combined. Over the last four years, Vanderbilt's GI SPORE has made discoveries and advances that hold great promise toward improvement of the management of individuals with colorectal neoplasia. These include 1) discovery of a novel Wnt antagonist (pyrvinium) and its target (casein kinase 1alpha ) , 2) discovery of a biologically-based, prognostic gene signature for colorectal cancer, 3) evidence that p120 acts as a tumor suppressor in colorectal cancer, 4) development, biological validation and clinical implementation of novel molecular imaging modalities to predict early response to treatment and 5) further development of a unique biorepository of colorectal adenomas, as well as matched normal rectal mucosa and bodily fluids (serum and urine). Our potential for continued success is high based on 1) productivity during the past funding cycles, 2) strong and highly integrative institutional support, 3) recruitment of talented investigators to the field of GI cancer through career development and pilot project funding, 4) access to unparalleled resources for proteomics, drug discovery and small animal imaging, 5) a team of highly interactive clinical investigators and basic scientists working together in a collegial environment and 6) strong Inter-SPORE, pharmaceutical, national and international collaborations. After a rigorous internal and external review process followed by consultation with NCI SPORE administrators, we propose three new projects and continuation of one project. Project 1. Multimodal Imaging & Targeted Therapeutics of Stem Cell-Derived Colon Cancer Project 2. Targeting K-RAS in Colorectal Cancer Project 3. Molecular Markers of Colorectal Cancer Recurrence Project 4. Genetic & Epigenetic Markers of Colorectal Adenoma Recurrence Core A. Administrative Core B. Translational Pathology & Imaging Core C. Biostatistics & Bioinformatics Career Development Program Developmental Research Program

The proposed demonstration project will test the effectiveness of a new, intensive multiphasic intervention model for chronically homeless veterans with alcohol and/ or drug problems. The proposed Case Managed Residential Care (CMRC) program will provide five days of intensive hospital "detox" followed by a graduated step down therapy featuring intensive case management during the CMRC program and during a six nth aftercare period. It is hypothesized that the CMRC program will reduce the costs associated with the treatment of homeless substance abusers to the VA and to society in general. It is also hypothesized that clients, after participating in CMRC, will demonstrate a decrease in their alcohol and/or drug use, an increase in their residential stability and improvement in their economic/employment status and mental health status. The study will be a three year prospective experiment involving approximately 270 homeless male veterans. The majority of subjects will be Black with an average age of 42-43 and a range of 25-70. All will have DSM III R diagnosis of substance abuse/dependency; and a significant number (70%) will also have co-existing mental disorders. After the five day detox phase, subjects will be randomized into two groups: 1) the experimental group which will enter the CMRC program and 2) the control group which will receive the usual treatment of the current VA substance abuse unit. Structured interviews with subjects will occur at entry into the study, at discharge, six mouths post admission, and 12 months post admission. In the analyses, the two groups will be compared on abstinence, psychiatric, social and vocational functioning and the utilization of medical and mental health services. A cost benefit analysis will determine whether the costs of the experimental intervention are offset by the reduction in the use of other services.

With an estimated total cost of $90 billion ($12.5 Billion for medical care alone) and an overall prevalence of about 6%, diabetes has an impressive impact on the health care system. Between 90 and 95% of all diabetes cases consist of type 2 diabetes. the prevalence of type 2 diabetes among African-American women is particularly high and is estimated to be at least 30% for those over the age of 55. African-Americans are not only at greater risk of developing type 2 diabetes, but experience twice the rate of nephropathy, retinopathy, amputation, and in-hospital mortality related to amputations. Exercise, diet and weight control represent known and potentially modifiable risk factors for the management of complications related to type 2 diabetes. For many type 2 diabetes patients, however, moderate weight is very difficult to maintain and interventions to reduce weight have had limited success. Theoretically, exercise can improve metabolic control independent of weight loss, and a few small intervention studies have confirmed that moderate-intensity exercise (55 to 70& of maximum heart rate) can improve HbA1c by at least 15% within 8 to 12 weeks with little or no weight loss. in addition, exercise holds great importance for cardiovascular health and overall quality of life. The vast majority (i.e. at least 80%) of persons with type 2 diabetes, however, do not exercise. for this reason, physician referral for exercise has been widely recommended. Its effectiveness has rarely been tested, but a small unpublished study using physician-initiated referral to a carefully designed group-based exercise program produced a 36% adoption rate among 22 African-American women over 50 years of age, and 7 of the 8 who adopted have participated for over 18 months. The primary aim of this study is to implement, support and evaluate a pragmatic physician-initiated exercise referral program for up to 24 months in three separate community based primary care clinics. The primary outcomes will be rates of group-based exercise adoption and maintenance and changes in overall physical activity level. The secondary aims will be to identify baseline predictors of exercise adoption and maintenance and overall physical activity level over the course of the intervention period and assess the effect of exercise on health and clinical outcomes. Measures for secondary aims include HbA1c, insulin and oral hypoglycemic dosages, blood pressure and heart rate at rest and at set workloads during progressive exercise testing, weight, anthropometrics, skinfold thicknesses, self-rated health and function and exercise self-efficacy.

The Scientific Core exists to facilitate the scientific projects within the program. The services, materials and reagents provided by the Scientific Core are purified proteins from natural and recombinant sources and infectious prions, synthetic peptides, DNA sequencing, PrP antibodies, transgenic mouse screening and a chemical and biochemical analytical service. This core will be directed by Drs. Michael Baldwin and Giuseppe Legname. Dr. Baldwin, an Adjunct Professor, is an analytical chemist with extensive experience of working with peptides and proteins, and has been associated with Dr. Prusiner's prion research group for 14 years. Dr. Legname has been at UCSF for 4 years and is an Assistant Adjunct Professor with expertise in molecular biology, immunology, protein expression and biochemistry. They are already directing the Scientific Cores of other program grants in Dr. Prusiner's laboratory. Dr Giuseppe Legname will direct the preparation of recombinant PrP variants and recombinant antibodies that will be expressed in cell cultures and purified for use in the various projects, assisted by Mr. Patrick Culhane. The preparation of purified prions has been carried out in this laboratory for many years and for more than a decade has been supervised by Ms. Hana Serban, recently with assistance from Ms. Nadia Kovaleeva. The purification of infectious prions will be essential for Project 1. Ms. Serban will also supervise the production of PrP antibodies required for Projects 3 and 4 and Core C. The screening of the transgenic mice is essential for Projects 3 and 4 and will be carried by Ms. He-Ying Lin under the supervision of Dr Kurt Giles, Assistant Adjunct Professor. Peptide synthesis will be conducted by a newly appointed Assistant Adjunct Professor, who will also assist Dr. Baldwin with chemical and biochemical analysis, which will benefit all the projects. All of these activities are currently being carried out very successfully in this laboratory through the efforts of the highly experienced personnel referred to above.

N-(4-(5-Nitro-2-furyl)-2-thiazolyl) formamide (FANFT) is a potent and highly specific carcinogen for the bladder of rat and provides a potential model for the study of human bladder cancer. The metabolism of FANFT may include deformylation, ring hydroxylation and nitroreduction. Its activation may involve the formation of hydroxylaminofuran, conjugation of this metabolite with glucuronic acid, and transportation of this conjugate to the bladder where free hydroxylaminofuran is regenerated and acts on the urothelial cells. The detoxication mechanism may involve the conjugation of hydroxylaminofuran with thiols, ring hydroxylation of nitrofuran, and synthesis and transformation of aminofuran. Based on this hypothesis, we propose to investigate tumorigenesis by FANFT and its prevention. To approach these: (1) the urinary metabolites of FANFT will be isolated and identified and their bacterial mutagenic activities ascertained; (2) rats will be fed FANFT and allopurinol, phenothiazine, cysteine, or 2,5- di-O-acetyl-D-glycosacharo-(1 yields 4) (6 yields 3) dilactone (SLA). Urine will be collected, metabolites of FANFT analyzed, and beta- glucoronidase activity determined. The preventive effect of these dietary supplements on the induction of bladder tumors will be evaluated. The correlations of tumorigenesis with urinary metabolites of FANFT and beta-glucuronidase activity will be determined; (3) in vitro experiments will be done with rat organs, particularly liver and bladder, for the activation and interaction of FANFT with macromolecules. These explorations will contribute to the understanding of bladder tumorigenesis and prevention in experimental animals, and may be relevant to bladder carcinogens in the human environment.

The mechanism or mechanisms by which the local blood flow is regulated according to the needs of the tissues remains uncertain. Metabolic, nervous, myogenic, and humoral factors may all be involved. An understanding of this control system is essential to rational treatment and prevention of many diseases. We intend to test the proposition that there are several control mechanisms with separate inputs and separate pathways, and that in different muscles or tissues, in different species, and under certain conditions one mechanism may predominate and thus lend itself to analysis. Accordingly, we will use a variety of preparations from several species, but primarily auto-perfused, isolated, soleus (red) and gracilis (white) skeletal muscle of the cat. A number of parameters will be measured and/or controlled including tissue PO2, perfusion pressure, flow, and the content of gasses and other substances in the perfusion media. In innervated and denervated preparations we will look for autoregulatory responses as blood composition and metabolic demand are varied. Local responses seen in the visualized microcirculation of the gracilis preparation will be compared with the overall responses of the same muscle. To delineate mechanisms we will, in the visualized preparation, produce localized variations in the chemical environment combined with recordings of PO2 (intra and extra cellular) and other possible significant parameters. As mechanisms become apparent, or norms established, we will study animals in various pathological states, especially shock and hypertension, and look for significant differences.

Pollen is a ubiquitous allergen that affects a large population with allergic diseases. However, the mechanisms leading to resolution of pollen allergen-induced inflammation remain poorly understood, a knowledge gap preventing us from developing new targeted therapies to cure allergic diseases. We have recently uncovered a novel pollen/TLR4 innate immunity pathway where short ragweed (SRW) pollen triggers allergic inflammation via TLR4-dependent innate signaling by mucosal epithelium that produces proallergic cytokine thymic stromal lymphopoietin (TSLP). We thus extended our investigation to another epithelial proallergic cytokine interleukine (IL) 33 and other major innate immune cells that may respond to pollen allergen. We hypothesize that TLR4-dependent innate immunity by ocular mucosal epithelia, dendritic cells and macrophages in response to pollen allergen, initiates Th2-dominant allergic inflammation via two stimulated allergic pathways, TSLP/OX40L/OX40 and IL-33/ST2, with an inhibited protective signaling IL-27/IL-10. The long-term goal of this project is to discover new molecular mechanisms and novel therapeutic targets for treating allergic diseases. Four Specific Aims are proposed to fulfill this novel project for public health. Aim 1 is to confirm the hypothesis that SRW pollen stimulates production of two pro-allergic cytokines TSLP and IL-33 by ocular epithelium via activating TLR4/MyD88/NF-?B innate immunity pathway; Aim 2 is to investigate the hypothesis that TLR4-dependent innate immune responses by dendritic cells amplify Th2-dominant inflammation via autocrine activation of TSLP/OX40L and IL-33/ST2 signaling with inhibitory regulation of IL-27 in response to SRW pollen allergen; Aim 3 is to explore the hypothesis that SRW pollen allergen primes macrophage polarization toward an alternatively activated (M2) phenotype to promote Th2- inducing cytokines (TSLP, IL-33 and OX40L) and Th2-attracting chemokines (CCL17 and CCL22) via TLR4-dependent innate immunity; and Aim 4 is to test the hypothesis that pollen/TLR4 concept may create a novel TLR4-targeted therapy for pollen-triggered allergic diseases using TLR4 antagonists and/or agonists. At the conclusion of this project, we will uncover a novel phenomenon and molecular mechanism by which pollen allergen triggers Th2-dominant allergic inflammation via TLR4-dependent innate immune response that activates TSLP/OX40L/OX40 and IL-33/ST2 allergic pathways but suppresses IL-27/IL-10 protective signaling in mucosal innate immunity system. The end product of this project will be a fundamental new understanding and potential TLR4-targeted therapeutic strategies, a new hope to prevent and cure allergic disease.

The Aging Research Center Laboratory Animal Core will operate as a shared resource, to facilitate and coordinate animal model studies for all five projects in this proposal. This core will build upon existing colonies from two different project members (Granholm and Rohrer) to develop disease-free, carefully phenotyped colonies of conventional, aged and growth factor knockout mice, in order to provide a common animal stock and therefore to reduce variables that could compromise data analysis between projects. This continuity will permit a more complete evaluation and will facilitate the consolidation of data for an accurate model of aging of the dopaminergic system. Aim #1. The first specific aim if this Core is to provide specialized maintenance of knockout and control mice, both as young and aged individuals;to provide sufficient breeding pairs and animals of particular age groups to the individual projects;to provide genotyping services as part of the ongoing effort to develop and maintain these specialized colonies;and to coordinate the sharing of animal resources. Aim #2 The second specific aim is to provide regular comprehensive health evaluations for all animals in the Core facility. This will include regular serological tests to maintain a disease-free status of all animals, diagnostic gross and histopathological examinations for all experimental animals to provide baseline levels for all parameters tested, as well as final evaluations at the completion of the experiments. Aim #3. A third specific aim is to provide a centralized facility to examine dopamine levels and metabolites for animals from all projects. Aim #4. The fourth aim is to centralize and standardize treatment paradigms, which increases the reproducibility of experiments between different projects. Aim #5. And in a final and fifth specific aim, Core personnel will maintain the database, which will include monitoring and tracking of all project animals, and which will be accessible via Web-interphase by all project members. Core members will input genotypes and all animal health and final pathological evaluations at necropsy into the database, and project members will input all animal-specific behavioral, physiological, neurochemical, cell-biological and molecular data. Well-maintained and centralized data records will facilitate post hoc comparisons and cross-correlation-based data analysis between all the data generated in the individual projects.

This project is designed to study local and systemic, humoral and cellular immunological responses in the lung of the rabbit following inhalation of antigen. Immunogenic requirements for hypersensitivity lung disease will be investigated as will immmunologic accompaniments of disease and of defense. Antigen-specific antibodies of individual immunoglobulin classes will be determine by an amplified enzyme-linked immunosorbent assay (ELISA) and by precipitin quantitation. Localization of immunoglobulins and antibodies will be studied by immunofluorescence techniques in lung, lymph nodes and other tissues. Cellular responses will include antigen-specific release of lymphokines (e.g. MIF), cytotoxicity, and chemotactic assays. These studies are expected to increase understanding of immunogenic requirements, pathogenesis and protective mechanisms in this model and potentially in human inhalational lung diseases.

Dehydration is a frequent cause of morbidity and mortality in the elderly. Identifying at risk individuals and maintaining adequate fluid balance is an essential component of health care in aging population. Currently, there is no effective tool available to measure hydration status and distinguish those at risk. The reliability and validity o current hydration assessment methods and criteria such as thirst, skin turgor, blood pressure, pulse, urine output and specific gravity, MRI, dilution methods and bioimpedance, are limited; the methods are inaccurate or expensive. Given that dehydration is both preventable and reversible, the need for an easy-to-perform method for the detection of water imbalance is of the utmost clinical importance. The goal of this project is to develop and commercialize an inexpensive and easy-to-use device that monitors changes in hydration status and predicts at risk in home-bound and institutionalized elderly. The Hydration Monitor (HM) which is being developed under this grant can objectively quantify changes in the body water content and hydration status. The proprietary patented method implemented in the device is based on the experimental fact that ultrasound velocity through soft tissue is a linear function of the tissue water content. Because muscle provides the largest body reservoir for water, the assessment of water imbalance is conducted by measuring speed of ultrasound in muscle. In the course of Phase I studies we designed and assembled HM -prototype, extensively tested it on tissue phantoms and excised animal tissues. Based on the results of these tests we designed the HM -prototype for Phase II clinical studies. The aims of this Phase II SBIR project include building and bench-testing eight HM -prototypes and providing the prototypes to three sites for clinical validation. The objectives of human studies include: to determine normal daily variation of the individual hydration level in longitudinal study on elderly in assisted living facility; to assess range between individual hydration baselines for elderly in a normal physiologically hydrated state; to establish the ability of the HM to assess initial and corrected hydration status in a population of elderly patients admitted to the hospital for hypernatremic dehydration; to determine euhydration and dehydration thresholds when employing ultrasound velocity measures in parallel with conventional tests and to detect changes in the hydration status of young healthy adults before and after undergoing 3% acute dehydration; and to test the hypothesis that body dehydration is a generalized physiological process equally affected all body muscles by comparing the data obtained on calf and biceps muscles. The Phase II activities will culminate with developing a pre-production HM prototype and preparing comprehensive engineering documentation. The device will aim to comply with respective FDA Quality System Regulations, Good Manufacturing Practices and regulations which are incorporated into Artann Laboratories quality systems.

Malaria is one of the most widespread infectious diseases of man and threatens almost half the world's population. Currently, there are an estimated 350+ million acute cases worldwide each year and approximately (sic) 1-2 million infants and young children die in Africa alone because of the disease. Traditional methods of controlling malaria with insecticides and drugs have been inadequate. Thus, increased attention has focused on developing a safe and effective vaccine against the parasite. In Phase I of the project, our objective is to assess the protective efficacy of immunization with recombinant mycobacterial (e.g. BCG) vaccines containing the gene from the major surface protein of malaria sporozities with the goal of inducing protective cell mediated immunity. We will utilize a P. berghei rodent malaria model since no reliable animal model is currently available for the human malaria parasites, P. falciparum and P. vivax. Data from this study will be used to establish principles that can be applied to the development of novel vaccine candidates for the human malaria parasites. This approach is of special interest because BCG is currently used for vaccination in the WHO/Expanded Program for Immunization in developing countries where malaria is endemic.

Nira Pollock, MD, PhD, is currently an instructor of medicine and infectious diseases (ID) at Beth Israel Deaconess Medical Center (BIDMC). The candidate's long-term goal is to develop an independent patient-oriented research career focused on the development and evaluation of new diagnostic tests for infectious diseases. Dr. Pollock's interest in diagnostics emerged from her strong combined basic science and clinical training and was focused by her ID specialization. Recognizing the rise of global tuberculosis (TB) case rates in the era of HIV and the limitations placed on TB control efforts by the weaknesses of currently available TB diagnostics, the candidate has chosen to focus her research and clinical efforts on TB. Under the guidance of two senior Harvard-affiliated TB researchers, Dr. Antonio Campos-Neto and Dr. Edward Nardell, she has initiated a promising TB diagnostic development project and has prepared a multi-disciplinary career development plan which will allow her to develop into an independent translational researcher with an exceptionally strong grounding in both ID and clinical microbiology. For her project, Dr. Pollock will work to develop a new urine antigen detection test for diagnosis of active TB disease. Her goal is to create a fully non-invasive, point-of-care, rapid test which would be useful in resource-limited settings, thereby making a substantial contribution to patient care and TB control. After completing pre-clinical development, she will evaluate the new test by integrating testing into an established clinical study of TB in Peru, run by Partners in Health. This will allow her to assess test performance for routine diagnosis of active pulmonary TB in adults, as well as to gain preliminary data about test utility for diagnosis of TB in children and for monitoring treatment efficacy. Dr. Pollock will combine her research program with a structured, comprehensive didactic curriculum in clinical research methodology at the Harvard School of Public Health. Finally, she will continue to develop her expertise in clinical microbiology through further practical experience in the BIDMC microbiology laboratory, recognizing that the clinical microbiology laboratory provides an ideal setting for applied infectious diseases diagnostics research. RELEVANCE (See instructions): The goal of this project is to develop a new, rapid, simple, and highly accurate urine test for diagnosis of active tuberculosis (TB). This test could dramatically change how TB is diagnosed world-wide, and thus could help prevent the spread of this highly contagious and often deadly disease.

It has been proposed that the basal ganglia and the supplementary motor area (SMA), to which much of the output of motor areas of the basal ganglia is directed, play special roles in the control of movements that are based on internally stored information. It also has been proposed that information from SMA and the postarcuate premotor cortex (APA), which is hypothesized to play an important role in movement guided by externally derived sensory signals, send information to separate neurons in the basal ganglia. To test these hypotheses, the proposed experiments will: 1) compare the activity of individual pallidal (and SMA) neurons during kinematically similar movements made under behavioral conditions that do or do not require memory of target location or sequence, 2) determine whether different groups of pallidal neurons receive input from different motor and premotor cortical areas, and 3) determine the changes produced in motor performance (and SMA neuronal activity) on internally and externally guided tasks when normal pallidal output is disrupted. These experiments will extend our understanding of the control of normal targeted limb movements and will provide information that can be used to assess, and perhaps rehabilitate, individuals with motor disabilities due to conditions such as Parkinson's disease, Huntington's chorea, slowly growing brain tumors and vascular or traumatic brain injury.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. We started studying lipid-protein interactions using WALP and KALP peptides. These alpha-helical transmembrane peptides, with a hydrophobic sequence of leucine and alanine of varying length, are flanked at both ends by two tryptophans or lysines as membrane anchors. WALP: Ac-GWW(LA)nLWWA, for example for WALP23 n=8, and KALP: Ac-GKK(LA)nLKKA. These peptides, unlike natural peptides previously studied in our group, can be synthesized in a variety of alpha-helical lengths. It will allow us to engage in a systematic study of the role of hydrophobic mismatch between the peptide length and the lipid bilayer thickness. Studying lipid-protein interactions from the standpoint of hydrophobic matching/mismatching is a familiar approach for our group;see for example our study of Gramicidin A in a variety of lipid phases. Use of several of peptides with different alpha-helical length in the same lipid phase will allow us to separate specific effects of hydrophobic mismatch from effects of lipid environment, in general. Our primary interest is the effect of hydrophobic mismatch on helical tilt and aggregation number WALP/KALP peptides in model membranes.

Use of the amphetamine MDMA (ecstasy, 3, 4-methylenedioxymethamphetamine) can result in life- threatening medical complications including fatal hyperthermia and cardiac arrhythmias. These complications result in part from simultaneous activation of the sympathetic nervous system, the hypothalamic-pituitary- adrenal axis (HPA) and somatic motor systems. It is the long-term objective of the principal investigator to identify the key brain regions and mechanisms through which MDMA activates these systems. The objective of this application is to determine the mechanism by which MDMA activates neurons in the dorsomedial hypothalamus (DMH) - a site involved in the responses evoked by MDMA - and to subsequently determine how this activation stimulates other brain regions involved in sympathetic, somatic motor and neuroendocrine responses. In the DMH, neuronal activity appears to depend on the balance between inhibitory and excitatory inputs from two key brain regions: inhibitory (GABAergic) projections from the medial preoptic area (mPOA) and excitatory (glutamatergic) projections from the lateral/dorsolateral periaqueductal gray (l/dlPAG). Once activated, neurons in the DMH then send excitatory signals to brain regions involved in activating the HPA axis (paraventricular nucleus, PVN) and sympathetic cardiovascular and temperature responses (medullary raphe pallidus, rPa). The activity of neurons within these areas (the DMH, mPOA, l/dlPAG, PVN and rPa) appears to be governed by glutamatergic and GABAergic neurotransmission. The specific aims of this grant will define the roles that these brain regions play in MDMA-induced responses. These studies will be performed in freely moving conscious rats using the following three complimentary experimental techniques: (1) immunohistochemical techniques to identify the putative GABAergic and glutamatergic neurons projecting to and from the DMH that are involved in MDMA-evoked responses; (2) microinjection techniques to determine the effects of inhibiting or exciting the regions of interest on MDMA-evoked responses; and (3) microdialysis techniques to determine how inhibiting or exciting the regions of interest affect GABAergic and glutamatergic neurotransmission in the DMH. The proposed research is significant because understanding the central pathways and mechanisms responsible for toxicity associated with the use of substituted amphetamines provides the necessary framework for rational treatment and prevention strategies. In addition these results may lead to treatments for medical conditions that, like MDMA, involve pathophysiologic activation of sympathetic, somatic motor and neuroendocrine systems: serotonin syndrome; cocaine toxicity, neuroleptic malignant syndrome; dysautonomia from brain injury, and heat stroke.

We request partial support for the 2014 Lipoprotein Metabolism Gordon Research Conference (GRC) and Gordon Research Seminar (GRS) to be held at Waterville Valley Resort, NH on June 16-20, 2014. The Co- Chairs, Drs. Robert Farese, Jr. and Joachim Herz, in consultation with past chairs, the 2016 vice chairs, and leaders in the field, have planned nine oral sessions for the GRC: Session 1 Lipases Session 2 Hepatic & Intestinal Lipid Metabolism Session 3 HDL & Reverse Cholesterol Transport Session 4 Lipoprotein Receptors and Vascular Wall Session 5 CNS & Lipids Session 6 Intracellular Lipid Metabolism Session 7 SHORT TALKS FROM ABSTRACTS AND Keynote Talk on Lipid and Lipoprotein Metabolism Session 8 Genetics of Lipid and Lipoprotein Metabolism Session 9 Therapeutics of Lipid and Cardiovascular Disease The GRC will be preceded by the Lipoprotein Metabolism GRS, an international forum for the presentation and discussion of frontier research by graduate students and postdoctoral fellows. Our conference satisfies all aspects of the NHLBI Mission. First, we bring together international leaders in lipoprotein metabolism, a physiological process that accounts for more than 30% of global cardiovascular risk burden. Second, gender-related issues of genetics, biology, biochemistry, and clinical manifestations of lipoproteins have been identified, so our conference satisfies the NHLBI responsibility for addressing these issues in women. Also, three Discussion Leaders are women, and two leaders are from Europe originally and one from Canada. Dr. Helen Hobbs, an internationally recognized human geneticist, will be a keynote speaker. Third, our conference has been carefully coordinated to include a diversity of research that is related to the causes, prevention, diagnosis and treatment of dyslipidemias and cardiovascular disease. Fourth, the NHLBI has traditionally supported our meeting and we have been honored to invite selected members of NHLBI administration as attendees. Fifth, we share the NHLBI's priority on prevention; indeed key aspects of primary and secondary prevention of cardiovascular disease that are currently in widespread routine clinical use (e.g. statin drugs) were first reported at our past meetings. Sixth, the co-Chairs have placed the highest priority on participation by individuals early in their training and early in their career development.

Over the past decade, investigations using genetically-engineered mice have led to new insights into the genetic control of embryonic vascular development, which has also had a major impact on our understanding of neovascularization in many human diseases including cancer, atherosclerosis and diabetes. Micro-imaging methods such as ultrasound biomicroscopy (UBM) and magnetic resonance micro-imaging (micro-MRI) can play an important role in this research, enabling direct in utero visualization of the developing mouse embryo. To date, there has been relatively little progress in the area of molecular imaging with ultrasound and MRI, especially in the area of vascular development. UBM is a real time imaging method enabling noninvasive in vivo analysis of mouse embryonic cardiovascular anatomy and hemodynamics, and can also be applied for image-guided intravascular injection of contrast agents. Micro-MRI provides better 3D resolution and more flexibility than UBM in manipulating cellular/tissue contrast, including more available contrast agents and approaches for cell-targeted imaging, but requires longer acquisition times, and has only recently been demonstrated for effective in utero imaging of mouse embryos. Several reports have recently demonstrated that biotinylation of cell surfaces can be achieved, allowing cell-targeted imaging with avidin-conjugated contrast agents, which are now available for both ultrasound and MRI. This is an attractive option for imaging vascular endothelial cells (VECs), since contrast agents can be delivered to the cells of interest via intravascular injection, even at embryonic stages of development. Moreover, the binding between avidin and biotin is the strongest found in nature, which should make it possible to label vascular cells even in the face of high wall shear rates associated with arterial blood flow. The specific aims of this project are: 1) To optimize the micro-MRI protocols required for in utero analysis of cardiovascular development; 2) To produce transgenic mice designed for targeted imaging of VECs with UBM and micro-MRI; and 3) To establish VEC-targeted micro-MRI approaches for improved analyses of embryonic vasculature. The approaches developed in this project will provide powerful new tools for direct analysis of vascular development in living mouse embryos. Significantly, these new molecular imaging methods will provide, for the first time, the ability to detect vascular gene expression in utero in normal and genetically-engineered mice. PUBLIC HEALTH RELEVANCE: This project aims to move ultrasound and MRI vascular micro-imaging methods beyond measures of anatomy and function, to the level of in vivo molecular imaging in genetically-engineered mouse models of a wide range of cardiovascular disease. These new molecular imaging approaches will revolutionize mouse genetics, enabling new studies linking gene expression to vascular morphology and physiological function in the best characterized mammalian model of human development and disease.

Genalyte will demonstrate the feasibility of our bio-sensing instrument platform for rapid sensitive high- throughput and low cost multiplexed panels to identify and monitor immune status for Type 1 Diabetes (T1D) auto-antibodies. The key to this capability is a proprietary label-free sensor based on silicon photonics chip technology. This sensor array is low cost, compact, mass manufacturable, and sensitive. Coupled with a real-time scanning instrumentation, specimens of serum (typically 25L) are sampled from a 96 well plate and introduced to the chip where they are read immediately, with simultaneous results for up to 128 analytes. The goal of this application is to devise and generate a novel multiplex assay for the detection and profiling of autoantibody responses associated with T1D. A two tier approach will involve first creating a basic multiplex assay to detect autoimmune response for known targets for T1D. Second, we will explore the analytical capability of the system with an expanded multiplexing to epitope map antibody binding across regions of the antigen as well as developing methods to assess autoantibody affinity. The ability to profile autoantibody response by multiple criteria will enhance our ability to detect and monitor disease development. PUBLIC HEALTH RELEVANCE: Project Narrative Genalyte has developed a novel bio-sensing instrument platform for rapid, sensitive, high-throughput, low cost, analysis of blood proteins, requiring minimal sample preparation. We propose to use this to generate a novel, multiplexed assay for T1D that will measure autoantibody load, target specificity, and affinity, as a means to profile immune response through the course of disease progression.

High resolution images of molecular oxygenation images can provide crucial guides to the delivery and monitoring of cancer therapy. EPR imaging (EPRI) of oxygen provides a unique combination of spatial resolution, oxygen resolution, and uniform sensitivity with depth in tissue. This Center focuses on the optimization of In Vivo EPRI oxygen imaging anticipating human images. It is a consortium between the Universities of Chicago, Maryland and Denver. Successful images require coordinated development of instrumentation to obtain the images, spin probes to sample and report the tissue fluid environment and imaging strategies to optimally sample and analyze the information obtained. The Center is built on multidisciplinary effort from engineers, biologists, radiation oncologists, medical physicists, physical and organic chemists, statisticians and imaging mathematicians. In the past 21/2 years the Center 1) validated EPRI oxygen images point by point, 2) developed co-registration of oxygen with anatomy and other physiology, 3) increased animal tumor size imaged, 4) produced the first electron spin echo oxygen image nearly an order of magnitude faster than continuous wave (CW) acquisition, 5) developed a source of narrow line trityl spin probe capable of bulk synthesis, 6) synthesized persistent intracellular nitroxides and 7) radically altered reconstruction of images from projections allowing partial projections in even dimensions. These achievements directly improve spatial, oxygenation and/or temporal image resolution. Physiologically relevant resolution goals mandates improvement in resolution of EPRI oxygen images for small animals. This improvement will be necessary to scale to imaging larger volumes of tissue and tumor. We propose to: 1) Implement multiple techniques for CW acquisition to increase projection acquisition rate, select regions of interest and construct novel resonators 2) For pulsed image acquisition increase bandwidth and bandwidth uniformity as well as novel resonators and techniques to select regions of interest 3) Develop novel, even narrower line spin probes to with higher oxygen sensitivity 4) Build on the image acquisition and analysis progress. We hope to acquire a human image in the 5th year of support.

This proposal is for Phase I, or, the "Treatment of Mild Hypertension Study" (TOMHS). Men and women with mild hypertension (DBP 90-99 mmHg) not on drugs, and DBP less than 95 mmHg on drugs, who are 45-69 years of age and free of evidence of end-organ damage related to their hypertension, will be recruited and randomized intothe "Phase I" study. Randomization will be carried ut to one of four possible treatment conditions: 1) placebo 2) chlorthalidone 3) atenolol 4) prazosin. The study is a double-blind controlled trial. All groups will be simultaneously given advice on non-pharmacologic methods designed to lower blood pressure including weigh control, dietary sodium reduction, and reduction of alcohol intake. The non-pharmacologic intervention will be initiated at the second eligibility visit (of three). Clinic visits will be conducted at a minimum of every three months and annually on all participants. Standard measures will be made at baseline and at subsequent clinic visits and at visits of record of blood pressure (random-zero), weight, overnight urine for sodium, potassium and creatinine, blood chemistries, ECG, Holter monitors, and echocardiograms. Four clinical centers will recruit 125 participants each. The study will be coordinated and administered centrally by the University of Minnesota, School of Public Health, Divisions of Epidemiology and Biometry. The major endpoint in Phase I is blood pressure change from baseline and for Phase II is hypertension related mortality and morbidity, including evidence of end-organ damage. The Phase I feasibility study inclues six months of planning, two months of training twelve months of recruitment, and average follow-up of approximately twenty months, six months of analysis and renewal preparation, and nine months for transition to Phase II of the study.

This proposal concerns two fundamental aspects of development: the creation of cell diversity and the directed movements of differentiating cells. The experimental organism is the simple eukaryote Dictyostelium discoideum. During the developmental phase of its life cycle, two precursor cell types diverge from undifferentiated amoebae. Early in development, the differentiating cells move, or sort out, resulting in spatially distinct spore and stalk "tissues." The technique of density gradient centrifugation provides a means of separating the precursor populations. It will therefore be possible to make detailed biochemical comparisons of the proteins and messenger RNA composition of developing pre-spore and pre-stalk cells. Differences will be sought particularly with respect to molecules residing on the cell surface and those previously implicated in slime mold chemotaxis and early differentiation. The effects of possible physiological regulators on the relative proportions of the two cell types will be investigated. By mixing together radioactively and/or genetically labelled amoebae of different developmental tendencies, the sorting out of like cells within the aggregate can be observed. In this way the timing and the developmental significance of sorting out will be explored. It is assumed that knowledge gained in this simple system will be in large measure applicable to developmental questions in more complex organisms.

DMA topoisomerase I (Topi) plays important roles in DMAreplication, transcription and recombination and is also the target of camptothecin (CRT), FDA approved analogs of which are effective new agents in the treatment of human cancers. CRT poisons Topi by reversibly stabilizing a covalent enzyme-DNA complex. During S-phase, the collision of replication forks with CPT-Top1-DNA adducts produces DMAlesions that signal cell cycle arrest and cell death. Although it is generally accepted that Topi targeted drugs induce DNA damage in S-phase, it is clear that signaling pathways activated in response to damage ultimately dictate cellular fate. Using yeast as a model system, conserved components of the replication machinery, CDC45 and DPB11(TopBP1), protect cells from Topi damage. Rapamycin-sensitive TOR signaling also protects yeast cells from cytotoxic DNA lesions during S-phase. Our data support a model whereby TOR acts as a survival pathway in response to genotoxic stress by maintaining replication fork stability and the dNTP pools necessary for error-prone translesion DNA polymerases. Thus, TOR-dependent cell survival in response to DNA damaging agents coincides with increased mutation rates, which may contribute to the acquisition of drug resistance. Three specific aims are proposed to investigate conserved aspects of the replication machinery and TOR signaling that maintain cell survival in response to cytoxic agents, suca at CRT. In Aim 1, a combination of yeast genetics and chromatin immunoprecipitates to query high-density tiling arrays (ChlP-chip experiments) will investigate the mechanism by which rapamycin-sensitive TOR signaling maintains replication fork stability and regulates DNA damage-induced mutagenesis. Aim 2 proposes to determine if rapamycin- sensitive mTOR signaling regulates human cell sensitivity to cyotoxic chemotherapeutics and the acquisition of drug resistance. A DNA fiber labeling strategy will determine if rapamycin treatment affects replication fork progression and stability in the presence of DNA damage, while the extent of DNA damage induced will be defined by yH2AX staining. siRNA-based approaches will determine if S-phase checkpoint function is required for the protective function of mTOR. In Aim 3, an analysis of synthetic lethal interactions will define pathway interactions of the conserved human DNA replication proteins, CDC45 and TopBPI, in regulating cell sensitivity to CRT and rapamycin. These studies will provide critical insights into the function of the TOR pathway in modulating cellular responses to DNA damage, while will impact the clinical development of rapamycin in combination with topoisomerase l-targeted therapeutics. The potential to block drug-induced mutations that confer resistance represents a unique application of rapamycins with clinical importance for the treatment of pediatric malignancies.

Project Summary: Previous research has shown that abnormal electrical conduction in the infarct border zone is important in the pathogenesis of ventricular arrhythmias in the setting of coronary artery disease. These abnormalities are due to remodeling in tissue architecture (fibrosis) and of connexins in gap junctions. Similarly, structural remodeling from congestive heart failure (CHF) leads to diffuse fibrosis and altered connexin expression that thus predispose to arrhythmias. This proposal seeks to investigate therapeutics that target these distinct features of remodeling (abnormal connexin function/distribution and fibrosis) in order to suppress ventricular arrhythmias that can lead to sudden cardiac death. Pirfenidone (PIRF) is an anti-fibrosis agent that has been shown to significantly diminish and possibly reverse collagen formation in various animal models, likely through inhibition of transforming growth factor-61. Using canine models of CHF and healed myocardial infarction (Ml), we will assess the effects of pirfenidone on ventricular fibrosis and test the hypothesis that attenuation of such ventricular fibrosis will decrease adverse electrophysiologic remodeling and thus ameliorate the arrhythmogenic potential of ventricular remodeling. We will correlate various measures of fibrosis to electrophysiologic parameters, such as conduction heterogeneity, dispersion of repolarization, and ventricular arrhythmia susceptibility. Rotigaptide (ZP123) is a novel hexapeptide that promotes cell-cell coupling through improved gap junction conductance without affecting membrane currents. We will use the same canine models of healed Ml and CHF to study the effects of rotigaptide on ventricular arrhythmia susceptibility and connexin remodeling using optical mapping, molecular, immunohistochemistry, electrophysiologic and histological studies. The results of these studies may add supporting evidence for pirfenidone and rotigaptide as potential therapeutic agents for the treatment of arrhythmias, particularly those that can lead to sudden cardiac death. Relevance: Nearly half a million people die from sudden cardiac death annually in the United States, with the vast majority of cases due to fatal cardiac arrhythmias. Current drug therapy is marginally effective, and we propose to study two drugs with potential anti-arrhythmic effects: rotigaptide, which improves connections between heart cells, and pirfenidone, which reverses adverse structural changes in the heart. We plan to show that the actions of these drugs will decrease fatal cardiac arrhythmias in animal models of congestive heart failure and myocardial infarction.

Chronic renal disease (CKD) is closely related with poor cardiovascular outcomes. Our recent experimental data indicate that Na/K-ATPase reduction may be related with renal insufficiency-induced cardiac dysfunction. First, we have observed that 5/6th partial nephrectomy (PNx) induces a time-dependent decrease of Na/K-ATPase in the mouse heart along with maladaptive cardiac remodeling and deterioration in heart function. The left ventricle initially shows hypertrophic growth and then dilation, which correlates with changes in Na/K-ATPase expression. Second, we have new evidence demonstrating that reduction in Na/K-ATPase potentiates cardiotonic steroid (CTS)-induced cardiac cell death in vitro, and dilated cardiomyopathy in vivo. In transgenic mice, genetic reduction of Na/K-ATPase stimulates the expression of pro-apoptotic proteins and potentiates CTS-induced cardiac cell death. It also causes decreased contractile function in these mice. Third, our data have revealed that in normal cardiac cells there exists a self-protection mechanism preserving the membrane abundance of Na/K-ATPase and protecting cells from death. Reduction of Na/K-ATPase attenuates the signaling function that is related with this self-protection. Furthermore, our work demonstrates that endogenous CTS are elevated in animals and humans with renal diseases. These studies lead us to hypothesize that reduction of Na/K-ATPase together with a sustained increase in CTS potentiates myocyte apoptosis and results in cardiac dysfunctions in renal insufficiency. To test this hypothesis we will conduct experiments in a well established PNx animal model, and use transgenic mice and newly developed tools to assess the pathways that are related with myocyte death. In parallel, we will perform the human study to link renal function and CTS release to cardiac dysfunction. PUBLIC HEALTH RELEVANCE: The overall goal of this project is to test the hypothesis that in renal insufficiency reduction of Na/K-ATPase and sustained increase of cardiotonic steroids may cause cardiac cell death and heart chamber dilation. It will also test the role of signaling Na/K-ATPase complex in this process. The validation of these hypotheses will establish a mechanistic link between renal disease and poor cardiovascular outcomes and creates a new therapeutic target for renal insufficiency-related cardiac dysfunction.

Hunter College of the City University of New York is a large, urban, diverse campus with over 50% minority students. The Hunter SCORE program supports research in 16 of the 50 research labs in the School of Arts and Sciences, which in 2002 published 145 peer-reviewed papers based on research of 35 post-docs, 150 PhD students, and 110 undergraduates. Funded since 2000, SCORE has achieved most of its measurable objectives including a 25% increase in publications, a 78% increase in post-docs, a 50% increase in undergraduate researchers, and an unanticipated 75% increase in doctoral students, including increased number and percentage of undergraduates and PhD students from under-represented minorities. In addition, we exceeded our objective of 4 new sources of major independent research funding: SCORE faculty garnered 9 new awards and 7 renewals so far, including NSF Career Awards to three junior faculty, and an R01 award to a former MARC student. These outcomes result from direct research support, support of research seminar programs, and institution of two major activities: meetings of small SCORE mentoring/interest groups called Scientific Research Interest Groups (SRIGs), and grantsmanship activities including workshops and structured mandatory internal and external critiquing and editing of proposals. In this supplement we seek to extend these successes to additional Hunter research faculty. We ask support for 4 full and 4 pilot proposals, mostly from new Assistant Professors and Associate Professors. Funding of these proposals will strengthen Hunter's research in bioinformatics, quantitative biology, and neuroscience and behavior. The Hunter SCORE program will support research in these labs, as well as expanded grantsmanship training, seminar support, SRIG meetings, and facilitation of junior faculty travel to scientific meetings and to collaborators. These activities will lead to achievement of the following objectives: *increase by one third the number of refereed publications by SCORE participants; *a further 50% increase in the number of post-docs in SCORE labs; *six new grants and six renewals among SCORE participants; *a 50% increase in the number of doctoral students in SCORE labs. [unreadable] [unreadable]

A Bridges to the Doctorate program between the two major Hispanic Serving Institutions (HSI) in California's Central Valley is proposed. This program will assist under-represented minority (URM) students from the California State University, Fresno with completing a Master's program in the biomedical sciences before continuing and completing their Ph.D. program at the University of California, Davis. This proposal will build on major research themes, activities and facilities including Research Infrastructure in Minority Institutions Program (RIMI) at Fresno State, the UC Davis Comprehensive Cancer Center, and the Clinical and Translational Science Center at UC Davis. Faculty members from the Colleges of Science and Mathematics at Fresno State and biomedical research focused graduate groups at UC Davis will create a highly interactive training program. The overall aim of the Fresno State Bridges to Doctorate program is to increase the number of URM graduate students entering and earning doctoral degrees in biomedical sciences. The program will recruit five graduate students each year with potential interests in pursuing research as a career and provide individually designed academic and research training plans to each. The major aims include: Refine professional skill sets through multiple opportunities to practice their oral and written skills and to network with other peers; actively participate and present at national/international research conferences to improve identification as a researcher and increase self-efficacy and science identity; Develop an understanding of and appreciation for their responsible conduct in research through supplemental responsible conduct in research (RCR) training course as well as for instructions on enhancing scientific reproducibility; and Provide summer research at research-intensive labs at UC Davis for the B2D students to integrate research experience that mirrors what they will experience in graduate school. The short- and long-range objectives are: To ensure that 90% of the B2D Scholars submit a graduate school application, with half the students being accepted into graduate Ph.D. programs in biomedical sciences in the first five years of the grant (short-range); Over the next decade we expect to maximize (80%) our B2D Scholars to join a Ph.D. program and pursue a career in biomedical research (long-range); and Over the next ten years, create a culture to expand and enhance the biomedical science curriculum developed by the B2D-Scholars program by the institutionalization of these practices (long-range). The Fresno-Davis B2D program will support the Scholars and lay the essential groundwork required to face the rigors of graduate school education in a highly competitive atmosphere. The Program Directors from both campuses have gathered a team of Faculty Mentors, an Advisory Committee and staff members, who are committed to the successful development of the program. All components of the Fresno-Davis Bridges program will be rigorously evaluated by an ongoing evaluation process to assess the success of the program.

PROJECT SUMMARY This application seeks partial support for the upcoming Federation of American Societies of Experimental Biology (FASEB) conference entitled, ?Skeletal Muscle Satellite Cells and Regeneration?, to be held July 24-29, 2016 in Keystone, CO. This will be the 9th meeting on satellite cells and the 8th sponsored by FASEB. We expect approximately 200 attendees from around the world, whom we expect ~40% will be female and ~55% will be junior researchers. The overall objectives of this biennial meeting include the following: 1) to provide a comprehensive analysis and integration of recent discoveries in the field with the goal of understanding the regulatory mechanisms controlling normal and abnormal functions of muscle stem cells in regeneration, muscle homeostasis, hypertrophy, aging and myopathic states; 2) to create and foster an interactive environment for the exchange of ideas and unpublished data to hasten discoveries and facilitate new and existing collaborations; 3) to provide opportunities for students and junior investigators to present their work and network with senior investigators to foster career development; 4) to facilitate career development of women and other underrepresented groups at all levels by ensuring appropriate representation in the planning, execution and participation in the conference program. No other scientific meeting has a primary focus on muscle satellite and stem cells. The need for a meeting with this focus is evidenced by the steady increase in attendance since the meeting?s inception and the consistently excellent post-meeting evaluations by meeting attendees. In addition, this meeting attracts essentially all of the leading muscle stem cell researchers from around the world, further demonstrating the value of this meeting for established and future leaders in the field. The meeting will consist of eight platform sessions with a total of at least 44 speakers. A minimum of 10 speakers will be chosen from submitted abstracts based on the quality and impact of the work within the major themes of the conference, with inclusion of junior investigators being a priority in the selection process. The conference will also include three poster sessions, providing ample opportunity for students and junior investigators to present their work. Major themes will include intracellular and extracellular signaling regulating satellite cell activity; roles of chromatin and transcription factors including MRF & PAX family members; biology of non-limb satellite cells and non-satellite stem cells; satellite cells in the contexts of disease and aging; and work in satellite cell biology performed in translational and industry settings. Collectively, this conference will provide a venue to present cutting edge research and promote collaborative interactions among all participants with the goal of advancing a fundamental understanding of muscle stem cell biology, and ultimately, translating this knowledge to the clinic.

Project Summary/Abstract________________________ Induced pluripotent stem cells (iPSCs) hold great promise in the areas of disease modeling and regenerative medicine and as a result have unique commercial potential. There are currently more than 300 ongoing clinical trials in stem cell therapies including the first-in-human trial using human iPSC- derived retinal pigment epithelial cells to treat macular degeneration. Another set of recent studies have shown direct reprogramming of target cells (e.g. fibroblasts) into neural or cardiac cell types via delivery of genes encoding tissue-specific transcription factors. These scientific advances may lead to new avenues for treatment of human diseases. These potential therapeutic applications, in addition to the potential of reprogrammed human cells as unique research tools, frame a substantial commercial opportunity. The initial and currently most commonly used methods of cellular reprogramming use retroviral vectors to deliver reprogramming factors into the cells. This method results in random integration of reprogramming factors into the genome, which is highly problematic for clinical use. Non- integrative methods of delivering reprogramming factors include the use of episomal DNA or Sendai Virus. These methods work well for a variety of cell types; however, they require monitoring for vector or viral clearance, resulting in a longer workflow and increased labor and reagent costs. There is a need to develop commercial tools for safe, efficient and adaptable cellular reprogramming, with a particular emphasis on non-viral approaches for research and therapy. A promising avenue for reprogramming cells involves delivering mRNA that encodes reprogramming factors. Using mRNA is especially attractive, as it dramatically reduces the risk of genomic integration, and reduces the time required for clearance of the reprogramming factors. The goal of this proposed Phase I Project is to develop an mRNA delivery system utilizing mineral coated microparticles and apply this innovative system for the generation of foot-print free iPSCs from clinically relevant cell types. Specific aim 1 will utilize our mineral coated microparticles to deliver mRNA encoding fluorescent reporter proteins for the optimization of transfection conditions to fibroblasts and blood derived cells. Specific aim 2 will use the optimized conditions to deliver mRNA encoding reprogramming factors to fibroblasts and blood derived cells to generate iPSCs which will be characterized for pluripotency, expansion capability and karyotype stability. This work is significant as it will improve the work flow for mRNA reprogramming in fibroblasts and will enable derivation of iPSCs from patient blood cells utilizing mRNA reprogramming. This work is innovative as it will improve mRNA delivery methods for these and other cell types, which will lead to further utilization of this technology in direct reprogramming, differentiation and in vivo applications.

The objective of this study is to test the effectiveness of an evidence-based, Spanish language prescription drug (Rx) labeling strategy to improve patient understanding and use. The primary aims of this study are to: 1) Test the efficacy of enhanced Spanish Rx labeling (container label, leaflet) to improve Spanish-speaking patients'understanding of instructions compared to two current standard labeling strategies, and 2) Evaluate the effectiveness of the enhanced Rx labeling to improve Spanish-speaking patients'understanding and actual use of their own medicine compared to a current labeling standard. Research activities will be conducted in three phases: i) Preparation, ii) Comprehension Testing, and iii) "Actual Use" Assessment. During a brief Preparation phase, an evidence-based Spanish language Rx container label prototype and accompanying 'enhanced print'leaflets will be refined and pilot tested among Spanish-speaking adults recruited from three diverse settings. In the Comprehension Testing phase, Spanish-speaking patients'ability to comprehend the instructions for use on the enhanced Rx labeling will be compared against two standard labeling practices representing current pharmacy practices. For Aim 2, the proposed research study takes advantage of a rare opportunity within a pharmacy practice to evaluate the effectiveness of this evidence-based, Spanish Rx labeling strategy in actual use. A randomized trial will be conducted among diabetic Spanish-speaking patients at safety-net clinics affiliated with a central-fill pharmacy. Correct understanding and self-reported adherence (3-month follow-up) will be compared between Spanish-speaking patients who receive the enhanced Rx labeling and those receiving current standard labeling for all of their prescribed medicines. Pharmacy and medical records data will be used in exploratory analyses to examine label differences in adherence (refill), health care use, and outcome (HBA1C) at one year. We will also conduct a 'post-use'formative assessment of patients'perspectives regarding the clarity and utility of enhanced Rx labeling, and gather patient data pertaining to specific cultural factors to examine the association between health beliefs and practices on medication understanding and use. Findings gathered will inform anticipated next steps for a dissemination strategy.

A common feature of abused drugs such as heroin and cocaine is that their use by humans tends to become habit forming. Similar behavior has also been observed in rats and mice, who will self-administer opiates or cocaine repeatedly if given the opportunity. The repeated self- administration of these drugs is thought to be primarily due to their strong positive reinforcing properties. Efforts to identify the neuroanatomical substrates that may be involved in an animal's repeated administration of heroin or cocaine has focused on lesioning of specific brain nuclei, microdialysis and neuropharmacological studies. The current interpretation of this literature is that mesocortical-mesolimbic dopamine neurons are responsible for mediating the positive reinforcing properties of these drugs. Originating in the ventral tegmental area (VTA), these neurons project primarily to the frontal cortex, olfactory tubercle, amygdala, septum and nucleus accumbens. Although each of these brain areas may be important with respect to some aspect of drug action, it is the VTA's dopaminergic input to the nucleus accumbens that has attracted the most attention. This interest is founded in the profound effects that both lesioning and neuropharmacological manipulations of dopamine in the nucleus accumbens which have on the animal's behavioral response to opiates and cocaine. Since dopamine's effects are thought to be mediated by a family of G protein-coupled receptors, collectively referred to as D1-like and D2-like, and the dopamine transporter, there is considerable interest in elucidating the role that each of these proteins may play in drug reward. Unfortunately, nothing is known about the relative contributions that each of the three D2-like receptors, D2, D3 and D4 make to an animal's overall behavior. Therefore, to examine the relative participation of each of the three D2-like receptors in behaviors related to drug abuse we propose to develop an in vivo model system. Through the use of gene targeting by homologous recombination in embryonic stem cells and transgenic mouse technology we are attempting to develop mice deficient in dopamine D2 and D4 receptors. Once these strains have been established they will be crossed in an attempt to generate a third strain expressing only D3 receptors (D2rec-/ D4rec-). The successful targeting of the dopamine D2 and D4 receptor genes in the transgenic mice will initially be evaluated, with respect to wild type mice, four ways. First, in situ hybridization analysis of D2 and D4 receptor mRNA expression will be performed on pre- and postnatal rec- mice. Second, competition binding assays will be performed on brain membranes from the D2 and D4 rec- mice. Next, tissues derived from these animals will be used in second messenger assays. Finally, stereotypic behavior, conditioned place preference and locomotor activity of the rec- animals will be assessed relative to controls following drug treatment and lesioning. The availability of these mice will be a valuable resource for many different in vivo studies of D2-like receptor involvement in drug sensitivity and dependence.

The goal of this project is to identify the genes (and their respective sequence variants) that play a role in conferring susceptibility to obesity in humans. Knowledge of the nature of these genes, and of the biological mechanism(s) of their actions, would contribute enormously to diagnosis, treatment and prevention of obesity and its allied disorders such as diabetes, cardiovascular disease and certain cancers. 1.) Novel candidate genes influencing adiposity and energy homeostasis will be identified by ongoing surveillance of the literature and by studying large-scale differential gene expression in rodent and human models of altered energy expenditure using cDNA expression microarrays. 2.) Highly sensitive techniques for detecting DNA variants in both coding and predicted regulatory sequences for these candidate genes will be applied to a collection of 2,000 subjects selected for extreme and/or early onset obesity. Allele frequencies of these DNA variants will be determined in four major ethnic groups. Based upon these analyses, phenotypically well-characterized subjects of suitable ethnicity will be selected for association studies from over 88,000 individuals available through 28 collaborating investigators around the world. Statistical analysis will include joint tests of linkage and association using transmission disequilibrium testing, new methods of genomic control, and haplotype analysis. We are also developing new methods to analyze pooled data from multiple populations and to model multiple loci with epistatic interactions. 3.) For variants with positive association results, assessments of possible functional significance will be made using computer modeling and predictions of function, as well as in vitro and in vivo (transgenic) functional assays. In cases in which the alleles associated with increased adiposity have no apparent functional consequence, the entire intronic, exonic, and 5' and 3' regions of the gene will be sequenced in 20 (15 obese; 5 lean) selected subjects to find non-coding variants that account for the phenotypic variation. Coding and non coding sequence variations will be examined for functional effects using in vitro expression techniques and transgenic mice with graded/inducible expression and human allele substitution. Finally, technologies will be developed to rapidly and inexpensively genotype large numbers of subjects for a panel of obesity candidate gene SNPs. Twenty five new genes will be studied over a 5 year period. A great strength of the proposed approach is access to large numbers of phenotypically well characterized subjects of varying ethnicity. These numbers will likely be required to detect sequence variants with specific effects on particular subphenotypes, small phenotypic effects, ethnically-specific genotypes or epistatic interactions among genes.

One major long term objective is to develop and have available for extensive use, a preschool cognitive screening test which will more effectively identify four and five - year - old children who, absent any educational intervention, will experience academic failure, develop emotional and/or motivational disturbances, and be classified as either mildly retarded or learning disabled. The early identification of these mildly cognitively impaired children can be expected to at least reduce their cognitive impairment and thus eliminate or severely ameliorate the negative impact that continuous academic failure has on their mental state. In this proposal, the psychometric properties of a cognitive screening test will be evaluated by administering the screening test to both an experimental and cross validation sample, using scores to predict outcome (special education or not) and then validating those predictions based on the educational classification of those students in third grade. We will collect data which will allow us to determine the test's concurrent validity, predictive validity, construct validity, test-retest reliability and inter-rater reliability. We will use special education outcome to streamline the test to maximize its validity. In addition, the second goal of this project will be maintained in this proposal. Same age normally achieving, learning disabled and mildly retarded children will to be compared on a broad array of cognitive tasks in order to understand better how similar or different the two impaired groups are from each other and from the two normal groups. In addition, a normal, but low, achieving group will be added to help delineate the specific cognitive deficits associated with the LD group. These data have implications for decisions about the best conditions under which to provide special educational services for the mildly impaired groups.

The genome of dengue virus and other members of the flavivirus group consists of a positive strand 42S RNA 10-12,000 bases in length. A 42S mRNA produced in infected cells codes for the three structural proteins V1, V2, and V3 which are also designated the envelope, core and matrix proteins, respectively. The remaining approximately 70% of the viral genome encodes a number of as yet unidentified non-structural proteins. The locations on the genome coding for the dengue structural and non-structural proteins have not been mapped and the functional role that each of these proteins plays remains to be determined. Extension of cloned DNA by "genome walking" has thus far yielded dengue type 4 cDNA of 9 kilobases in 5 overlapping segments. The 5 clones are currently being extended in order to complete our cloning of the dengue genome. The clones are also being sequenced in order to study the gene organization of dengue virus. We plan to approach the mapping and protein analysis of dengue virus by using the recently developed baculovirus vector-host cell system for expressing cloned DNA segments. Expressed polypeptides will be used to immunize animals for preparation of antisera and the antisera will in turn be used for identifying viral proteins present in infected cells. In this manner, the expressed gene product will identify the viral structural or non-structural protein each gene encodes and allow us to map the position of each gene on the dengue genome. In addition, the antisera produced from expressed DNA segements should be useful for identifying the antigenic determinants of dengue virus.

FepA, an 81 kD protein of gram-negative bacteria, transports iron across the outer membrane in a manner that requires a number of accessory proteins in the bacterial cell envelope. For this reason, it is desirable to study protein dynamics in the intact cell. Spin-label binding sites were introduced into the cell-surface region of FepA by site-directed mutagenesis. Reaction conditions are being sought that will allow selective labeling of these sites in line, intact E. coli.

Specific areas of research include: Coupled transcription and translation in vitro with negative strand viruses; transcriptional mapping of (-)RNA viruses by UV survival curve analysis; mechanism of action of interferon --- role of oligonucleotide inhibitor and of dsRNA and ATP activated endoribonucleasease; regulation of vaccinia virus gene expression; translation discrimination by initiation factors; translational control (tc)RNA; cDNA of purified myoglobin mRNA; stored mRNPs; kinetics of viral protein synthesis and degradation; cellular location and function of viral proteins; amino acid analogue and viral-induced selective enhancement of cell proteins; inhibition of cellular protein synthesis by viruses; translation of viral mRNAs in vitro; viral functions required for cell killing and protein synthesis inhibition; ribosome modification by virus infection; processing of polyproteins; Zn ions inhibition of protein cleavage; viral-induced phosphorylation of ribosomal proteins; role of viral DNA-protein linkages in replication and transformation; adenovirus terminal protein; post translational maturation of viral proteins; adaptive response of cells to the carcinogenic mutagen MNNG; role of MNNG-adaptation to DNA repair processes and protection against carcinogens; mechanism of interferon induction by single molecules of dsRNA; inhibition of viral transcription by interferon action; dsRNase induction by dsRNA. Viruses and virus mutants used include: adeno-, mengo-, rubella, vaccinia, reo-, Newcastle disease, Sindbis, vesicular stomatitis, and poliovirus.

IDENTIFY BIOCHEMICAL INDICATORS FOR TOXICITY

Our aim is to investigate the role of nociceptive neurons in the peripheral and central nervous system in mediating cutaneous pain, itch and hyperalgesia. Peripheral neural mechanisms of chemogenic pain and itch will be studied by obtaining psychophysical measures in humans of the threshold, magnitude and duration of sensation during and after a superficial intradermal injection of capsaicin (pain) or histamine (itch). After injection of capsaicin, heat and mechanical stimuli will be applied directly to (or outside of) the injection site in order to determine the time course and magnitude of primary (and secondary) hyperalgesia. We will record, in the anesthetized monkey, the evoked responses in single A-fiber and C-fiber nociceptors when the same stimuli are applied within or near their cutaneous receptive fields. Results will determine the chemical sensitivity of nociceptors in relation to chemogenic pain, itch and hyperalgesia. The central neural contributions to heat pain, hyperalgesia after heat injury or intradermal injection of capsaicin, and itch after intradermal injection of histamine will be studied by recording evoked responses from single "nociceptive specific" and "wide dynamic range" neurons within the ventral posterior lateral thalamus in awake monkeys trained to escape or tolerate and detect heat stimuli used in the psychophysical experiments in humans. The results will be useful in identifying those central neurons, at the level of the thalamus, that process sensory information about pain, itch and hyperalgesia. Both the peripheral and central studies are prerequisites to developing specific pharmacologic agents that act peripherally to prevent endogenous chemicals from sensitizing nociceptors or act centrally to block sensory processing in nociceptive neurons.

PROJECT SUMMARY/ABSTRACT US healthcare policy, practice and research leaders have expressed increased interest in the vision of the ?learning healthcare system,? a set of organizational goals and arrangements in which healthcare delivery processes are continuously studied and improved, largely by applying formal research methods to data and experience generated during routine care delivery activities. A core element and requirement for a successful, productive learning healthcare system is robust research and analytical capacity and infrastructure. ?Embedded? or ?partnership? research programs are research teams located within healthcare systems that design and conduct studies that aim to contribute to health system performance and improved patient care while simultaneously developing new scientific knowledge. Such programs contribute to the realization of the learning healthcare system vision and are found in selected health systems, including the US Department of Veterans Affairs, Kaiser Permanente Southern California, and a small number of additional public, private and academic health systems. The expert meeting proposed in this application will convene approximately 50 policy, practice and research leaders to (1) review and discuss the vision and desired features of embedded research programs, (2) describe and carefully examine existing examples and models for embedded research programs, assessing their strengths and weaknesses and diverse features, and (3) produce guidance and a set of recommendations for policy, practice and research stakeholder activities that will facilitate significant growth in the number, size and performance of embedded research programs in the U.S. Based on the highly successful Department of Veterans Affairs ?State of the Art Conference? model, the proposed activity will include extensive pre-meeting preparation, a highly structured two-day meeting involving a small number of brief lectures and a series of small-group working sessions, and extensive post-meeting dissemination and follow-up activities to operationalize the recommendations and facilitate their implementation. The overall goal of this initiative is to contribute to more rapid realization of the vision of the learning healthcare system in which research is conducted on an ongoing basis to guide continuous improvements in healthcare delivery quality, safety, equity and value.

This study will focus on the fundamental question of whether reactive metabolites of dopamine, may be contributing to the pathogenesis of neurodegenerative disorders such as Parkinson's disease. Dopamine has been shown to be toxic to cells both in vitro and in vivo. However, the exact mechanism associated with the toxicity is not known. Because mitochondria play a critical role in mechanisms of cell death, the proposed studies are designed to increase our understanding of the interplay between reactive metabolites of dopamine and mitochondrial function, and their ability to enhance the vulnerability of dopaminergic neurons to injury. In Aim 1, we will characterize the temporal relationship between loss of mitochondrial function and cell death following exposure to dopamine. In Aim 2, using striatonigral organotypic cultures, we will examine whether dopaminergic neurons exhibit an increased vulnerability to mitochondrial inhibition and whether dopamine contributes to this effect. Finally, in Aim 3, we will determine the identity of critical proteins modified and inactivated by dopamine quinones using mass spectrometry, with a focus on mitochondrial proteins. The outcome of these studies has potential for identifying new therapeutic targets for stopping and preventing the neurodegenerative process in Parkinson's disease.

Objectives are 1) to inventory the perceived and measurable effects of Federal health grants on the administrative procedures and program priorities of local recipients, public and private; and 2) to assess the significance of these findings for grants policy, comprehensive planning, and regulation of health providers. Procedure will be to interview 250-300 administrative officials and health providers in Philadelphia and the Delaware Valley region and to follow interviews with a questionnaire. The study should prove useful in judging the effectiveness of Federal grants administration, in identifying obstacles to organizational and regional cooperation, and in assessing the feasibility of regulatory strategies.

The Comprehensive Sickle Cell Center at The Children's Hospital of Philadelphia (CHOP) is in its fourth year of operation. In the past four years, we have made much progress in all the major components of the Center. Although much has been learned about sickle cell disease at all levels, effective therapy which takes advantage of this knowledge is still elusive. Even if such therapy were to become available soon, there is a need for accurate diagnosis and counseling; diagnosis and management of serious complications such as stroke; and education of those affected by the disease and those who manage it. These broad needs are addressed in this application. The main objective of our proposal is to seek new information about sickle cell disease and to translate this information into treatment programs which will improve the health and quality of life of sickle cell patients. The laboratory research includes studies on the expression of fetal hemoglobin, interaction of Hb S and mutagenized globin chains, mRNA structure and stability, experiments with transgenic mice, and in red cell deformability using image analysis system. Clinical projects address nutrition, energy metabolism and growth, the development of cerebrovascular disease, and a limited multi-center trial of hydroxyurea in children with sickle cell disease. In our diagnosis and counseling projects, we will develop methods for specific identification of the common beta-gene mutations, and another project determines if a newborn screening program for sickle cell disease can be designed and implemented in West Africa. Educational projects include an extension of the computer-based education on sickle cell disease, a program to improve educational outcome for young children with sickle cell disease and employment opportunities for adolescent patients. A third educational project takes educational programs developed and evaluated at this Center into regional programs of professional education about sickle cell disease. These projects are supported by a central administrative core, a clinical research unit, and a laboratory core which includes a state-of-the-art nucleic acid and protein chemistry laboratory service. In addition to investigators from the Division of Hematology of CHOP, investigators from other Divisions of CHOP and from other departments of the University of Pennsylvania are participants in the Center. We continue our collaborative arrangement with one investigator from Thomas Jefferson University Hospital. The Center proposes to maintain its strong links with the community.

The overall goal of this proposal is to test whether targeting individual cell cycle proteins represents a highly selective therapeutic strategy in different types of human breast cancer. The proliferation of mammalian cells is driven by the core cell cycle machinery operating in cell nucleus. The key components of this machinery are proteins called cyclins, which bind, activate and provide substrate specificity to their associated cyclin-dependent kinases (CDKs). These cyclin-CDK complexes phosphorylate cellular proteins, thereby driving cell proliferation. Mouse gene knockout experiments demonstrated that individual cyclins and CDKs are dispensable for development and for normal proliferation of the majority of cell types. In contrast, these proteins are essential for the initiation and for maintenance of specific cancer types, depending on the genetic lesion they carry. Relevant for this application, our laboratory recently demonstrated that an ubiquitous, global shutdown of cyclin 01 in mice bearing MMTV-Erb82 (HER2) driven breast cancers blocked tumor cell proliferation and triggered tumor cell senescence, without having any obvious impact on animals' physiology. Importantly, administration of an inhibitor of CDK4 and CDK6 (PD 0332991) to tumor bearing animals had the same effect, namely it caused senescence of Erb82-driven breast cancer cells. These observations suggest that inhibition of CDK4/6 kinase activity may represent a very effective therapeutic strategy in women with HER2-positive (HER2+) breast cancers. In the work proposed in Aim 1, we will extend our analyses to human HER2+ breast cancers. We will take advantage of a very large collection of human breast cancer cell lines (including several HER2+) assembled by Dr. Polyak. We will also use xenografts of primary HER2+ breast cancers, to test the effect of CDK4/6 inhibition on human mammary carcinomas. Lastly, we will elucidate how human HER2+ breast cancer cells develop resistance to CDK4/6 inhibition. In Aim 2, we will extend our approach to triple-negative breast cancers, where very few therapeutic options are available. We will test our hypothesis that this specific cancer type depends of cyclin E-CDK1 and/or A-CDK1 kinase. The Specific Aims are: Aim 1. To determine the response of human Erb82-positive (HER2+) breast cancers to cyclin D-CDK4/6 inhibition; Aim 2. To study the requirement for CDK1 function in triple-negative breast cancers

Rationale and Relevance: Infectious diseases still account for significant morbidity and mortality in the developing world. Vaccines are one of the most cost effective advances in medical science, and if appropriately used, are potent instruments in the control of infectious diseases. Successful vaccine development requires an integrative scientific and public health approach, and requires input from basic scientists, epidemiologists, public health officials, clinicians, institutional and regulatory agencies, and industry, among others. The development of a safe and effective vaccine most often requires a firm understanding of the underlying pathogenesis of the infectious process, as well as host-pathogen interactions and immune responses. Objectives: The main objective of this training program is to build sustainable infectious disease research capacity in the field of vaccine development at the ICDDR.B: International Centre for Health and Population Research, Dhaka, Bangladesh. The focus would be the training of individuals in the fundamental development and evaluation of vaccines against enteric infections,focusing on the development and evaluation of vaccines against Vibrio cholerae, the cause of cholera. Special emphasis would include the role of high throughput genomic and proteomic technologies in V. cholerae vaccine development. Individuals trained in these areas would complement the expertise and skills already present at the ICDDR.B, and would help create a synergistic core group of researchers at the ICDDR.B able to meet current and ongoing infectious disease threats globally and in the developing world. Design: We propose a five year Global Infectious Disease Research Training Program in vaccine development between the Massachusetts General Hospital-Harvard University and the ICDDR.B. The training program would build upon the strengths of both institutions and a number of on-going collaborations, and would include both intensive and long-term training of a small group of individuals (one year at the MGH- Harvard; two years at the ICDDR.B), as well as shorter and more focused training of a larger group of individuals through workshops and seminars (at the ICDDR.B) on topics pertinent to vaccine development.

During my career I have worked on several projects creating software (FASTLINK, CASPAR, rh_tsp_map,[unreadable] PedHunter, etc.) and a database (Anabaptist Genealogy database) for genetic studies.[unreadable] My association with this software and a past track record of effective collaboration[unreadable] with wet lab scientists leads to more and more such collaborations.[unreadable] Two highlights from the past year are:[unreadable] -- publication in the September 2006 issue of Genome Research of a paper showing that[unreadable] a large deletion disrupting the LIX1 gene causes spinal muscular atrophy in cats.;[unreadable] -- the discovery of the gene mutated in some cases of severe congenital neutropenia[unreadable] including those in the original family published by Kostmann in 1956.[unreadable] [unreadable] The cat SMA work is part of a longstanding collaboration with Dr. Marilyn Menotti-Raymond (NCI).[unreadable] The pedigree in this particular study was developed and phenotyped by Prof. John Fyfe;[unreadable] I have also been collaborating with Prof. Fyfe on some dog genetic[unreadable] studies.[unreadable] [unreadable] The neutropenia work is part of a longstanding collaboration with Dr. Bodo Grimbacher[unreadable] (U. Freiburg, GERMANY) on human immunodeficiencies. Our work on neutropenia, including[unreadable] a paper on Hermansky-Pudlak syndrome type 2 published int he July 1 issue of[unreadable] Blood, is led by Prof. Christoph Klein (Hannover Medical School, GERMANY).

The helical growth system of Bacillus subtilis is being investigated. An examination of over 50 films illustrating the manner in which helical macrofibers are built suggests that the folding cycle and its associated physical movements are a direct reflection of the individual cell cycles taking place within the macrofibers. The geometry of helix hand orientation and macrofibers requires that folding be driven by the equivalent of a negative supercoil, yet the films indicate always only a single direction of helical turning with growth, a direction which if overwound would lead to positive supercoiling. Pertinent here are new findings concerning the change of helix hand discovered earlier. Now both temperature and ionic environment have been found to influence helix hand selection. A theoretical examination of helical geometry on cylinder surfaces has led to the discovery of a clock and cell measurer based upon helical geometry. The physics of this clock are currently being explored both theoretically and experimentally. The model predicts that cells divide in the opposite helix hand from that used for the assembly of their cell cylindrical surfaces. Helix hand inversion apparently represents the use of division orientation for growth and vice-versa. The observed symmetry is now understandable.

Proper brain function requires the establishment and maintenance of effective neural circuits. Neural circuit development entails axon guidance, synaptic target selection, synapse formation, and synaptic growth and remodeling in response to developmental and environmental cues. Large protein complexes in both the pre- and post-synaptic cells act to coordinate synapse maturation, which involves clustering and aligning of the pre-synaptic neurotransmitter-releasing machinery with neurotransmitter receptors and signaling proteins. Evidence suggests that once formed, synapses are maintained over the lifetime of an organism. While mechanisms of axon guidance and synapse development have been well studied, little is known about the mechanisms involved in axon and synapse maintenance. Our lab is using genetic screens in Drosophila to uncover the molecular mechanisms that form and maintain synapse integrity. One of the most interesting mutations we've uncovered in the type of screen mentioned above is in With No Lysine [K] (wnk), a conserved serine-threonine kinase that has been implicated in a number of human diseases including hypertension, hereditary neuropathy and cancer. Although Wnk is highly expressed in the nervous system, its function there has not been described. We found that wnk mutant flies have defects in axon transport of synaptotagmin, a protein involved in calcium-dependent vesicle release. Interestingly, Wnk mutants also show significant synaptic retraction, indicating that mechanisms of synapse stability are compromised. In addition, we uncovered a novel interaction between Wnk and the Rab3-GEF, an activator of the Rab3 GTPase involved in vesicle and protein trafficking at the synapse. We aim to define the mechanism by which WNK controls axon and synapse maintenance using Drosophila as a model. We will identify the domains that are critical for WNK function at the synapse and determine the sub-cellular localization of WNK. We will also define the novel interaction between WNK and Rab3-GEF that we've discovered and identify proteins in the WNK signaling pathway. In addition, we aim to extend our findings to mammalian sensory neurons by using mouse dorsal root ganglion (DRG) cultures to study axon integrity and synapse formation in the absence of WNK. Results from these studies will lead to novel insights into how WNK shapes the development and maintenance of the synapse. In addition, because the mammalian orthologs of WNK have been implicated in hereditary sensory and autonomic neuropathy type II (HSANII), study of the WNK signaling pathway may also yield novel insights into this and other neuropathic disease, including identification of therapeutic candidates. PUBLIC HEALTH RELEVANCE: Axon and synapse development are crucial for establishing and maintaining neuronal circuits and brain function throughout life. Defining the molecular mechanisms that control these processes is therefore critical to our understanding or normal brain function as well as how dysfunction occurs. We plan to characterize the role of the serine/threonine kinase Wnk and delineate its role in regulating axon and synapse development and maintenance. The results of these experiments will not only provide novel and fundamental insights into mechanisms regulating the control of synaptic maintenance, but also lead to possible therapeutic targets for human diseases, including neuropathies such as hereditary sensory and autonomic neuropathy type II (HSANII).

The primary purpose of this program is to develop services for alcoholics and their families. The goal is to induce and maintain sobriety for the alcoholic and provide comprehensive services to him and his family through effective relationship with other ongoing programs such as health services, vocational rehabilitation, employment and welfare assistance. Program objectives are: (1) Provide counseling and referral services to approximately 300 clients and their families, (2) to refer clients to the various supportive community services dealing with the illness, alcoholism, i.e. Alcoholics Anonymous, National Council on Alcoholism, Mental Health Clinics, etc., (3) to provide for physical, medical, psychological and diagnostic & evaluation treatment, (4) provide for vocational counseling and/or training focused specifically on the problems of the unemployed alcoholic, (5) provide community education, (6) provide ongoing education and training of alcohol program staff, (7) provide in-service training to the Community Action Council areas, thus enabling them to render services to the alcoholic as an integral part of their social service activities, (8) follow-up on all cases, (9) provide evaluation for the program, (10) begin a system for collecting data, (11) initiate AA meetings within the Centers, (12) provide a plan to involve clients in group rap sessions, (13) initiate a plan to provide for a Vocational Counselor.

Grant Title:Whitaker-Dowling Pitt - Collaborative Biomedical Grant

The research proposed compares several methods of evaluating level of habitual physical activity (PA) and fitness in athletic and 100 nonathletic men and women age, 20 to 59. This research will determine the reliability and validity of a variety of evaluation tools for assessing PA and fitness. PA will be assessed repeatedly by the comprehensive Minnesota Leisure Time PA questionnaire and shorter questionnaires; the results of these questionnaires will be compared with those from 24 hour PA and food diary records and mechanical motion detectors (Caltrac). The PA and diet diary and the motion detectors data will be collected 21 times per year (3 times for each day of the week). Physical fitness will be assessed by treadmill exercise testing using the Bruce Protocol and the Beckman Metabolic Measurement Cart for determining maximal oxygen uptak (VO2 max). The results will be compared with estimated VO2 max. values obtained with simpler submaximal step and bicycle ergometer tests; and anthrophometric measurements, including relative body weight, multiple skinfold thickness, body circumferences, and underwater weighing determination of body density and percent body fat. Two years will be required to complete the assessments of the 100 subjects (50 per year). The ultimate goal is to provide researchers in epidemiologic studies and clinical trials guidelines about the relative merit of various procedures for assessing PA and fitness in terms of reliability, validity, practicality, costs and implementation requirements.

Parkinson's disease is a devastating, universally fatal, incurable neurodegenerative disorder, which is debilitating and inflicts terrible suffering on the patient's mind and body. Human pluripotent cells, including embryonic stem cells, have been identified as a potential alternative cellular substrate for treatment, but to date, have not been tested for long-term therapeutic efficacy in disease relevant animal models (MPTP lesioned monkeys). Our long-term goal is to assess the safety and temporal efficacy of HESC-DA cell therapy in PD patients. The current objective is to determine if grafted HESC-DA cells are a potent long-lasting therapy that structurally integrate within the dopamine depleted primate brain, as well as reverse functional deficits associated with PD. Our central hypothesis is that intrastriatal delivery of HESC-DA cells will reverse motor disability in MPTP treated monkeys and this effect will be potentiated by co-treating with AAV-neurturin. The rationale for the proposed research is if HESC-DA grafts ameliorate functional deficits in parkinsonian primates, we can pursue large-scale, long-term safety and tolerability studies as the next logical step for clinical translation. Guided by preliminary data, this hypothesis will test the following Specific Aim, that intrastriatal grafting of HESC-DA cells, or HESC-DA cells + AAV2-Neurturin will provide structural and functional recovery in MPTP monkeys. Utilizing a novel floor-plate based, dopaminergic neuralization paradigm that allows HESC to be efficiently differentiated into midbrain specific dopaminergic neurons, and we will determine the effects of grafted HESC-DA cells on host anatomical structure and function and correlate these results to overall quality of life (clinical rating scale). In the proposed Aim, immunohistochemical analysis and behavioral testing will be used to complement in vivo imaging in an effort to bridge a critical gap in knowledge for PD therapeutics. This research is innovative, as it 1) focuses on well characterized, functionally active, midbrain specific HESC- DA neurons as an alternative renewable source for cellular transplantation 2) advances our knowledge of the functional efficacy of this approach by utilizing a clinically relevant non-human primate model of PD, and 3) vertically advances and bridges our present studies with the addition of AAV-2 neurturin neurotrophic support. This proposal is significant as rigorously testing HESC-DA therapies in parkinsonian monkeys more accurately depicts human PD, and therefore, the proposed experiments are more likely to provide quality translatable results to the clinic. The outcomes are expected to vertically advance the field of PD therapy through the blending of neurosurgery and stem cell biology. The knowledge obtained here has the potential to provide a therapeutic option that will reduce the terrible symptoms and enhance overall quality of life in PD. PUBLIC HEALTH RELEVANCE: The proposed research is relevant to public health and the NIH-NINDS's mission because the discovery of human pluripotent stem cell based treatments that successfully slow or reverse progressive neurodegeneration and behavioral deficits will help develop a desperately needed treatment for PD patients. Furthermore, pre-clinical development of human stem cell therapies are needed now more than ever with the advent of exciting new technologies centered on patient-derived induced neural cells. Thus, the proposed research is relevant to the part of NIH's mission that pertains to the application of knowledge that enhances health and reduces the overall burden of illness.

Determine the relative efficacy of 3 treatment approaches to reduce myocardial ischemia in individuals with stable angina and documented coronary artery disease, ischemia on treadmill testing, and ischemia on ambulatory ECG (AECG) monitoring. Patients were randomized to angina-guided therapy, angina-guided plus AECG-guided or revascularization and followed for one year.

Influenza B viruses have been represented in human populations in recent years by two divergent strains, B/Victoria/2/87-like and B/Yamagata/16/88- like viruses.Drift in the B/Yamagata-like viruses has been identified by a panel of ferret antisera during the past year and has raised the question of need for further alterations in vaccine composition.In order to determine human responses to new B/Yamagata-like variants, sera from 23 children and 30 adults who were immunized with vaccines containing the antigens of influenza virus B/Yamagata/16/88 were tested.Against B/Hong Kong/22/89 (an immunologically distinguishable variant of B/Yamagata), post-immunization geometric mean titers (GMT) of neu-tralizing antibodies were only approximately half those against the vaccine virus (reciprocal GMT = 344 for B/Yamagata vs. 157 for B/Hong Kong). Among the subset of children who were immunologically unprimed the disparity was more marked (GMT = 473 for B/Yamagata vs. 141 for B/Hong Kong). Against a panel of variants of B/Yamagata-like viruses that have drifted even further immunologically than B/Hong Kong (Panama/45/90, Bangkok/163/90, Illinois 2005, and Texas VC-1 1037), adult postimmunization sera tested by hemagglutination inhibition (with detergent split virus as antigen) showed no difference in GMT (218 for B/Yamagata, 218 for B/Panama, 218 for B/Illinois, 230 for B/Texas, and 179 for B/Bangkok). These results suggest that adults would be adequately protected against the newly drifted influenza B strains related to B/Yamagata, but that children, particularly those with little or no previous exposure to influenza B antigens, might be suboptimal for protection against infection. The data suggest that vaccine strategies for adults may be inadequate for children.

This FIRST award proposal investigates alterations in intracellular signal transduction as molecular mechanisms in sepsis and endotoxemia. Gram- negative sepsis is a leading cause of death worldwide. Treatment remains supportive. New and more effective therapies will require defining the pathogenesis of sepsis at molecular levels within the cell. The central hypothesis of this proposal is that specific intracellular signal transduction pathways are altered in macrophages upon repetitive endotoxin (LPS) stimulation. The activation states of these pathways ultimately protect or harm the host by exerting translational control of cytokine gene expression. The specific aims of this proposal focus on distinct LPS-signaling pathways which diverge at inositol phosphate turnover. The pathway LPS chooses to take is proposed to determine the cellular LPS response. Using molecular biology tools applied to cell culture and animal models of LPS presensitization (tolerance), we propose to confirm or exclude the following: 1) Altered inositol phosphate signaling limits LPS responses; 2) LPS signaling is linked to translational control of cytokine gene expression through phosphorylation of specific cytoplasmic proteins; 3) Upon repetitive LPS stimulation, cell signaling shifts away from these pathways allowing the cell and host to regulate its response while confronting the bacterial threat. In a clinically relevant model of experimental sepsis, we propose to pharmacologically redirect cellular signaling and provide a protective survival benefit. Preliminary data suggest the central hypothesis is correct, and moreover, point to an unexpected link between LPS and insulin as extracellular ligands: co-utilization of signaling pathways coupling ligand binding with the translational control of cellular protein synthesis.

In this randomized, double-blinded multicenter trial, sponsored by ISIS Pharmaceuticals, a novel approach for the treatment of condyloma acuminata using an "antisense" compound (ISIS 2105) is being evaluated for safety and efficacy.

Clinical studies have shown that estrogen (E2) can ameliorate symptoms of mood disorders in women, including severe depression. The presence of symptoms may represent an abnormal response to normal hormonal changes, thereby implicating contextual factors in the brain in the etiology of these symptoms. Some of these factors include second messenger systems that lead to the production of neurotrophic agents, including brain-derived neurotrophic factor (BDNF). Chronic stress results in deleterious effects on neurons and synapses which, in turn, may be related to alterations in affective behavior. Because of its involvement in cellular and synaptic growth and/or function, BDNF may counteract these negative effects and restore the appropriate behavioral responses. The gene transcription factor cyclic AMP response element-binding protein (CREB), has been shown to be a target of antidepressant and E2 action. Activation of CREB can lead to transcription of the BDNF gene. We propose that the Ca2+/calmodulin-dependent protein kinase IV (CaMK IV) pathway, "CaMK IV - CREB - phosphorylated CREB (pCREB) - BDNF," mediates some of the effects of long-term E2 treatment on behavior. Long-term E2 treatment results in the persistence of these messengers, even after two weeks. E2 may regulate CaMK IV and BDNF via alterations in their mRNAs; we will perform in situ hybridization to address the hypothesis that E2 regulates levels of CaMK IV mRNA and BDNF mRNA. We will also determine if E2 decreases relevant phosphatases, including protein phosphatase 2A and the calcineurin pathway, negative regulators of CaMK IV and pCREB, respectively. To determine if CaMK IV, CREB and/or BDNF mediate E2 effects in the forced swim test, a test for the efficacy of antidepressants in rodents, antisense oligodeoxynucleotides (ODNs) to CaMK IV, CREB or BDNF will be infused into the amygdala or hippocampus and animals will be subjected to the test conditions. Infusion of these antisense ODNs may interfere with the positive effects of E2 on forced swim. We will also determine if infusions of BDNF protein with the antisense ODNs to CaMK IV and CREB reverse the actions of the ODNs on behavior. These experiments will give insight to the molecular effects of E2 in areas of the brain related to affective processing and will uncover mechanisms that may allow hormonal intervention for the amelioration of female-related mood disorders.

Dietary restriction (DR) is defined as a 25-50% decrease in calorie intake without a decrease in essential nutrients. Such undernutrition without malnutrition 1) increases average and maximal survival times of rodents, 2)\prolongs aspects of normal organ function, and 3) delays the onset of several late life diseases. For example, it has been observed that 11 month old lenses of mice on restricted diets show enhanced solubility of gamma crystallins as compared with normally fed age matched control animals. This is significant since insolubization of damaged or altered crystallins (particularly gamma) is associated with lens aging and cataractogenesis in many species including humans. It is the objective of this research to determine if dietary restricted mice have delayed onset- or diminished incidence of cataract. In order to pursue these studies we will use the Emory mouse since in chronology of development, patterns of protein aggregation, and histological appearance, Emory cataracts resemble human senile cataract. Furthermore 100% of mice in our colony are expected to develop cataract in less than a year. It is generally agreed that altered proteins are more rapidly degraded than native proteins by cellular proteases. Recent work from this laboratory demonstrated that with increasing age the lens proteases are inactivated. It is plausable that this decrement in proteolytic capability results in the accumulation and eventual precipitation in cataractous opacities of damaged lens proteins. By comparison of the incidence of cataract in DR groups of Emory and CFW (control) mice with normally fed mice we will ascertain the effect of DR on lens function. Furthermore by analysis of protein profiles and endo- and exoprotease activities before, at the time of, and subsequent to the onset of cataract, we will determine if protease inactivation is preceeded by, is synchronous with, or follows age related protease inactivation.

DESCRIPTION (provided by applicant: Cholera is a waterborne diarrheal disease caused by Vibrio cholerae of serogroup O1 and O139. The serogroup O1 can be divided in two biotypes: classical and El Tor. The current and seventh cholera pandemic is characterized by the predominance of the El Tor biotype. Recently, hybrid V. cholerae O1 clinical isolates (the Hybrid O1 and the El Tor variant) with attributes of both biotypes and enhanced virulence have been identified. Lateral gene transfer, transcriptional silencing of xenogenic DNA and gain of function through anti-silencing could play an important role in biotype evolution. The histone-like nucleoid structuring protein (H-NS) is a global regulator known to silence the transcription of laterally- acquired genes. We have shown that H-NS and quorum sensing differentially regulates the transcription of the vieSAB operon encoding a three-component regulatory system in V. cholerae biotypes. The VieSAB regulatory system is expressed in classical biotype V. cholerae where it regulates the expression of 10 % of the genome. Genes regulated by this system include those required to make cholera toxin and biofilm development. Thus, the VieSAB regulatory system is a major contributor to biotype-specific phenotypic differences impacting V. cholerae pathogenesis, environmental fitness and transmission. We found that H-NS binds with similar affinity to the promoter of classical and El Tor biotype vieSAB. However, H-NS was found to function as weaker repressor of vieSAB in the classical biotype. In this application we propose to characterize the regulation of vieSAB transcription and establish the basis for its differential expression in V. cholerae biotypes. In aim 1, we will determine the molecular basis for the differential transcriptional silencing of vieSAB by H-NS in classical and El Tor biotype V. cholerae. In aim 2, we will determine the connection between quorum sensing and vieSAB transcription in the El Tor biotype. Our study will likely identify new regulators that modulate V. cholerae pathogenesis by acting through the VieSAB regulatory system. Further, a better understanding of the molecular determinants of biotype-specific behavior could support the development of more effective cholera management strategies and perhaps even predict the emergence of new pathogenic variants.

The overall aim of this study is to determine risk factors for the development of high intraocular pressure (IOP) and glaucoma visual field defects (GVFD). These factors include systemic hypertension, antihypertensive therapy, cardiovascular risk factors, family history, demographic and othe variables. Previous studies have provided suggestive but inconclusive evidence that systemic blood pressure levels are involved in glaucoma pathogenesis. Because blood pressure and IOP levels may be related, hypertensives could be at high risk of developing ocular hypertension (elevated IOP). In addition, hypertensives may be at increased risk of GVFD afte receiving antihypertensive drugs. The first objective of this study is to evaluate the relationships between hypertension, its treatment and open-angle glaucoma risk. This information is important for patient management and for an increased understanding of glaucoma pathogenesis as well. The study will: 1) determine whether hypertension and antihypertensive treatment are associated with an increased risk of GVFD, and 2) determine whether hypertension increases the risk of ocular hypertension. The second objective is to evaluate the independent contribution of cardiovascular risk factors, familial, socioeconomic, occupational and other environmental variables to the risk of GVFD and/or ocular hypertension. The study will determine which of these factors discriminate between ocular hypertensive and glaucoma patients. This information will be obtained by a case-control study which will consist of private ophthalmologic patients, 45-74 years, to be divided in three age-sex matched groups of 200 patients each: I. Glaucoma patients with GVFD and high IOP, II. Ocular hypertensive patients with high IOP, but no evidence of optic nerve damage and III. Control patients with normal IOP and no GVFD. All patients will be interviewed and will have blood pressure measurements, tonometry, visual field testing and fundus photography. Information on past medical history, antihypertensive therapy and other factors will be obtained by patient interview and confirmed by the treating physician. Groups will be compared regarding the frequency of the variables under investigation. Estimates of the magnitude of the risk involved will be obtained from these comparisons.

Brain corticotropin-releasing factor (CRF) systems play an essential role in coordinating stress-induced endocrine, autonomic, behavioral, and immune responses. In the amygdala, CRF peptide and receptors are located in nuclei implicated in fear and anxiety. Importantly, pathophysiology of CRF systems and the amygdala is associated with mental illness including anxiety disorders, depression, and drug abuse. Thus, the long-term goal of this research program is to understand how CRF systems contribute to psychopathology. The overall aim of this proposal is to provide an in-depth analysis on the functional significance of stress-induced central amygdala CRF secretion and its putative actions on basolateral CRF1 receptors to activate fearful memories and promote anxiety. We hypothesize that stress-induced CRF secretion from the central amygdala acts on the high affinity CRF1 receptor located in the basolateral amygdala to facilitate the acquisition and retrieval of conditioned fear. We further hypothesize that stress-induced central amygdala CRF secretion plays a key role in promoting anxiety behavior by acting on basolateral CRF1 receptors. To test these hypotheses, behavioral studies in rodents will be performed that combine site-specific CRF receptor antagonist microinfusions, ex vivo receptor binding, in vivo microdialysis, and gene disruption techniques. Establishing the role of CRF peptide and receptor subtypes located in specific amygdala nuclei will provide the foundation for future studies aimed at determining the downstream cellular and molecular mechanisms underlying the effects of stress on fear conditioning and anxiety. Knowledge of amygdala CRF biology may yield valuable insights into the neural basis of emotional predispositions and the pathogenesis of psychiatric disorders.

Hearing loss is a leading public health concern, with about 17% of American adults reporting some degree of impairment. Of those, fewer than 20% seek help for their condition for a variety of reasons. To address this issue, the National Institue of Deafness and Other Communication Disorders (NIDCD) is seeking solutions to improve hearing healthcare access and lower its cost (see RFA-DC-12-004). Solutions requested include enabling currently available commodities (telephone, Internet, and smartphones) to conduct surveillance of hearing loss, Internet screening systems, and automated audiometry to be used beyond traditional clinical and research environments. One key challenge to implementing these solutions is the need to generate calibrated tones and to ensure a low noise environment. Creare proposes to complete development of a hearing threshold screening device that performs automated audiometry and is controlled through a mobile platform. Our device is designed for use at point-of- care (POC) locations with very limited personnel and testing facility resources; it can also be used for self-assessment. Background noise attenuation is provided with innovative, integrated high quality noise attenuating ear cups. The resulting device will provide a solution to the problem of hearing healthcare access by allowing quality screening at POC locations, in community centers, pharmacies, and big box stores (which are now making low-cost hearing aids available). In addition, because it is designed for use with mobile platforms and is calibrated to applicable standards, the proposed device will enhance the development of quality audiology applications for use with smartphones, tablets, and the Internet. During Phase I, we demonstrated the feasibility of our innovation by building a benchtop prototype of the device and testing its performance using standard audiometry. We showed that thresholds measured with our prototype were equivalent to thresholds measured with a commercial audiometer on a pilot human study that included 16 subjects. During Phase II, we will refine the design of our device, build several integrated prototypes, and demonstrate their use and performance with several human subject studies in target locations and populations. The Phase II human study will have two objectives: (1) compare the test results and test/retest variability using the new device to what can be achieved with existing devices, and (2) compare the performance of our device in a representative noisy environment such as POC locations to its performance in a quiet sound booth. The Creare team is extremely well qualified to successfully develop and commercialize this hearing screening device for automated and remote applications. Our system is made possible by leveraging hardware and software that Creare has previously developed for hearing assessment in varied settings including remote assessment in developing countries, Otoacoustic Emission (OAE) measurement in noisy environments, and advanced hearing protection.

Over 100 million Americans consider themselves to be regular drinkers, and approximately 20 percent of these individuals will eventually develop alcohol-related problems. Despite the staggering socio-economic cost of alcoholism, little is known about either the physiological factors the predispose an individual to this disease or about the molecular mechanisms that mediate the intoxicating actions of alcohol. We hypothesize that a better understanding of the molecular targets of alcohol and the physiological mechanisms that regulate ethanol sensitivity at these sites will facilitate the development of more effective treatment strategies. The overall aim of this research project is to characterize the synaptic mechanisms underlying ethanol potentiation of GABAA receptor-gated inhibitory synaptic transmission. The GABAA receptor mediates the majority of fast inhibitory synaptic transmission in the mammalian CNS, and a considerable volume of evidence suggests that these receptors may be one of the major CNS mediators of ethanol intoxication. However this interaction has not been studied, in detail, at the level of the synapse. Preliminary evidence from our laboratory indicates that ethanol may enhance GABAA receptor-mediated synaptic transmission via both a postsynaptic effect (possibly reflecting a direct interaction with GABAA receptors) and a previously uncharacterized presynaptic facilitation of GABAA receptor-mediated synaptic transmission. The first set of experiments will use the whole cell patch clamp technique to record from hippocampal CA1 pyramidal neurons in brain slices from mature rats. By characterizing the effects of ethanol (1-100 mM) on spontaneous and miniature GABAA receptor- mediated synaptic currents and currents evoked by somatic application of low concentrations of GABA, the specific mechanisms through which ethanol enhances presynaptic and postsynaptic components of GABAA receptor-gated synaptic transmission at a central mammalian synapse will be elucidated. The second series of experiments will use similar electrophysiological methods to characterize the mechanisms through which antagonism of GABAB receptors and elevation of basal PKC activity facilitate ethanol enhancement of GABAA receptor-mediated synaptic transmission. The results of these experiments will clarify the pharmacological relevance of presynaptic and postsynaptic ethanol actions to its facilitatory effect on GABAA receptor-gated synaptic transmission and identify mechanisms underlying the physiological regulation of ethanol sensitivity at these sites.

Nephrocalcin (NC) is a calcium-binding glycoprotein that has been isolated from human urine and human kidney tissue culture, and that inhibits growth of the calcium oxalate crystals in vitro. Molecular abnormalities have been found in NC isolated from the pooled urine of calcium stone formers, which decrease its affinity for crystal surface, and thus its ability to slow crystal growth. This abnormality includes a deficiency of gamma-carboxglutamic acid (GLA). It is unknown whether abnormalities in the structure and function of NC contribute to kidney stone formation in certain individuals. The goal of this proposal is to begin to study the role of NC in stone-formation in individual calcium stone-formers, and to study the factors that regulate production of the protein. In previous studies by the PI, antibodies to the protein have been used to estimate excretion rates of NC in normal men and women, and to correlate this with the ability of the urine to inhibit calcium oxalate crystal growth in a seeded crystal growth system. Our studies have also shown that Vitamin D may play a role in regulation of NC excretion. Finally a similar protein has been isolated from kidney cortical tissue culture supernatants, providing a model for in vitro studies of regulation of protein production. To investigate the possibility that some stone-formers make an abnormal NC molecule, decreased either in amount or in ability to inhibit crystal growth, and to learn more about the abnormalities themselves, three types of studies are proposed; I) Calcium stone formers with known metabolic defects and stone composition will be studied using ELISA assay to measure NC excretion, and assays of crystal growth to detect abnormalities in inhibitory activity. Isolation and study of abnormal proteins will be done II) Patients taking coumadin will also be studied. If lack of GLA is the crucial defect, these patients may display abnormalities similar to those of stone-formers. III)Studies of regulation of protein production will be carried out in cell culture. Effect of vitamin D, PTH, medium calcium, vitamin K deficiency, etc. can be assessed. It is likely that inhibitor abnormalities contribute to the pathogenesis of stones in a subset of stone-formers. Knowledge about the specific abnormalities that lead to impaired ability to inhibit crystal growth and of the factors that regulate production of this protein may lead to effective therapeutic interventions.

A general understanding of the interaction of (monoclonal) antibodies with their homologous saccharides is important in order to a) gain an insight into the immunogenicity of oligo- and polysaccharides, and b) gain insights into the general nature of the interaction of proteins (including e.g. receptors, enzymes) with saccharide haptens (it has been shown that enzymes show common hapten-binding idiotypi with anti-hapten antibodies: Strickland et al. J. Immunol. 34, 1053, 1985). Of the three projects in the Section, two (A and B) are intimately tied to the above approach, while the third (C) is associated with it also: A. Study of the molecular interaction of monoclonal antibodies with polysaccharide antigens. B. The preparation and characterization of immunodiagnostic antibodies to saccharide determinants occurring on glycoproteins (GP) and glycolipids (GL). C. Chemistry of the antigens of the pathogenic fungus Cryptococcus neoformans, and their interaction with antibodies.

[unreadable] [unreadable] Our mission is to make available human brain and other body parts specimens from neuropsychiatric diseases and disorders, and normal controls. The Human Brain and Spinal Fluid Resource Center (Bank), established since 1961 is a repository of blood, cerebrospinal fluid (CSF) and cryopreserved postmortem nervous system tissue, available for research. Located at West Los Angeles VA Healthcare Center, our Bank functions as a dependable bridge between patients, clinicians and scientists. Volunteer patients donate specimens through our Gift of Hope donor program. Specimens are collected during clinical course and after death, and cryopreserved in our Bank. To provide the highest possible quality of research specimens, we have developed specialized quick-freeze preservation technique for whole coronal sections of brains. Therefore, corresponding structures can be studied in left and right brain from the same patient. We dissect regions on demand and histologically evaluate to confirm the requested details. To validate specific findings, inclusion of control specimens in any biomedical research project is indispensable. We provide control specimens of the user's choice. Banked neuropsychiatric and control specimens are used by scientists to search for the cause of diseases, design diagnostic tests, find markers of disease activity and find targets for treatment. In the last 5 years we have delivered over 10,000 dissections of tissues, 1,816 CSF specimens, 1,888 serum specimens and 151 urine specimens to researchers working on a variety of disease and disorders. More than 800 publications (580 in refereed journals, cited over 26,000 times) were published from 1985 to June 2005 utilizing the Bank specimens. Research results generated from our specimens may lead to new drug discoveries, which are then used by clinicians in the treatment regimen. Therefore, by making high quality research specimens readily available, our Bank helps research scientists help clinicians who help patients. The Bank's collection is an invaluable resource for human research. [unreadable] [unreadable] [unreadable]

The generation of energy in mammalian mitochondria is accomplished by a series of oligomeric complexes in the inner membrane. The synthesis and assembly of these complexes requires genetic information ;contained in both the nuclear and mitochondrial genomes. Limited information is available on the mechanism by which the mitochondrially-encoded components in these complexes are synthesized and assembled into the large oligomeric complexes in the inner membrane. The overall objective of this research is to investigate the mechanism of protein biosynthesis in mammalian mitochondria and its integration into the macromolecular metabolism of this organelle. The first objective is focused on polypeptide chain initiation and is designed to provide an understanding of the roles and mechanism of action of two translational initiation factors required for protein biosynthesis in mammalian mitochondria. Studies will be carried out to examine their binding sites on ribosomes and to investigate their interactions with other components of the protein biosynthetic machinery. In addition, the secondary structure of the RNA near the start sites in mitochondrial mRNAs will be probed. The second major objective focuses on the properties of the translation elongation factor (EF-Tumt) that promotes the binding of aminoacyl-tRNA to the A-site of the ribosome. A collaborative project has been established with the laboratory of Dr. Soren Thirup (Univ. Aarhus, Denmark) to determine the structures of the ternary complex (EF-Tumt:GTP:aa-tRNA) and several other components of the translational cycle in mammalian mitochondria. These structural insights will lay the foundation for understanding the properties of the unusual tRNAs found in mammalian mitochondria. The third major aim of this project is to examine the interaction of the mitochondrial ribosome with the inner membrane. All of the products of mammalian mitochondrial protein biosynthesis are hydrophobic membrane proteins, which are thought to be synthesized while the ribosome is associated with the inner membrane. The interaction of the ribosome with the transmembrane protein Oxal will be studied and the parameters governing the binding of the ribosome to the inner membrane will be investigated. Well over 100 disease states in humans are associated with alterations in mitochondrial function and gene expression. Understanding protein synthesis in this organelle is essential for developing strategies to combat these diseases.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Alzheimer disease (AD) is a chronic disorder that progressively leads to the loss of cognitive functions, with synaptic loss appearing as a critical element in the development of the disease. Neurochemical studies have suggested that the cholinergic neurons in the neocortex and hippocampus are those predominantly affected in AD (Bartus et al., 1982;Dunnet et al., 1993;Weinstock, 1997), a fact that likely accounts for the strong decrease in neurotransmitter acetylcholine (ACh) levels in AD victims'brains. Symptomatic treatment has therefore, so far, focused upon restoring cholinergic neurotransmission. Acetylcholinesterase (AChE) is the enzyme responsible for ACh hydrolysis, hence it is the target of most currently used anti-Alzheimer drugs.

Epstein-Barr virus (EBV) causes infectious mononucleosis in adolescents and malignant B lymphocyte proliferation in AIDS patients and patients undergoing immune suppression for organ transplantation. EBV is etiologically associated with African Burkitt's lymphoma and nasopharyngeal carcinoma. In vitro, EBV transformed, latently infected B lymphocytes contain EBV episomes and nine virus encoded proteins. Six are nuclear proteins (EBNAs) and three are the integral membrane proteins, LMP1, LMP2A, and LMP2B. These nine proteins are presumed to mediate latent virus infection or B lymphocyte proliferation and thus are under intense investigation. Besides EBNA1, which is required for episome maintenance, LMP1, LMP2A, and LMP2B are the latently expressed proteins consistently detected in EBV related malignancies, and the LMP2A message is the most readily detected EBV-specific message detected in PCR analysis of B lymphocytes from individuals harboring latent EBV infections. Our previous studies have shown that LMP2A is essential for down modulation of cell surface receptor mediated signal transduction in B lymphocytes infected with EBV. By down modulating cell surface signal transduction, LMP2A is important for maintaining EBV latent infection in vitro. Currently, studies designed to elucidate LMPA function in vitro are hampered by the need to use continuous cell lines or EBV transformed lymphoblastoid cell lines (LCLs) which do not reflect the unactivated B lymphocyte in which EBV is latent in the human host. To begin to understand the function of LMP2A in latent infection, we have developed a murine transgenic model system which targets LMP 2A expression to B lymphocytes thereby mimicking in vivo latent infection in humans. The model has proved useful in characterizing the alteration of normal B cell function by LMP2A and has proved invaluable in investigating the requirements for LMP2A function in latent infection. In the current proposal, we will continue our analysis of LMP2A function in our transgenic mice using both in vitro and in vivo techniques. An understanding of LMP2A function may provide insight for the development of novel therapeutics for the treatment or eradication of EBV latent infections in the human host, thereby preventing the development of AIDS-associated lymphoproliferative disease.

Infectious diseases remain a global threat to human health that significantly contributes to mortality and morbidity in developing and industrialized countries. While many can recall the fear of polio, now nearly eradicated, the presence of new diseases and reemerging diseases that strike individuals and populations has returned. Today, the specter of bioterrorism permeates the national research agenda. Effective long- term success in meeting the infectious diseases-based demands on our medical and public health systems depends on training a qualified workforce capable of addressing such research challenges. The predoctoral PhD training program in Infectious Diseases and Immunity at the University of California, Berkeley was established 7 years ago to meet the unique and challenging needs for modern research training of individuals with a focus on infectious disease agents. The issues surrounding infectious diseases are touched upon by many disciplines, ranging from the most basic to the most applied. An interdisciplinary approach must therefore be applied in relevant research, training, practice, and prevention programs, with advances in basic research providing the essential platform. Consistent with the NIAID mission, the goal of the Graduate Program in Infectious Diseases and Immunity is to address these multifaceted issues by training research scientists to extend their knowledge across traditional disciplinary boundaries. This interdisciplinary training will give students the tools to develop an integrated and comprehensive approach to the nascent health challenges posed by infectious disease. The nexus of the predoctoral training program is an intensive curriculum representing infectious disease biology, immunology, biochemistry, molecular genetics, cell biology, epidemiology and biostatistics. Training in experimental design and methods, critical thinking and research ethics complements the curriculum. This then provides the basis for laboratory-based training in independent research of infectious agents that cause human diseases. We initially request predoctoral training support for 4 students per year with an addition of 2 students in years 4 and 5 consistent with the demonstrated growth trajectory of the Program. Most students enter the program with a bachelors-level degree and the normative time to completion of the degree is 5.5 years [unreadable] [unreadable] [unreadable] [unreadable]

This is a new project, just commencing. Extensive studies of Ewing's sarcoma and related round cell tumors of childhood have served to distinguish most from one another on the basis of positive reactivity with one or more antibodies. No antibody specific for Ewing's sarcoma, or restricted to a few tumors including Ewing's, has been identified to date. It is the aim of the present study to produce a battery of monoclonal antibodies against selected, well-characterized Ewing's sarcoma lines established in this laboratory. They will then be studied and compared in their reactivity with other monoclonal antibodies previously reported. Any antibody found to be strongly reactive with Ewing's sarcoma cells will be further characterized, with the eventual intention to use same as a potential diagnostic, imaging, and therapeutic tool.

A series of 22 Substance P analogs, characterized by elongation or shortening of the peptide chain or by replacement of some amino acids, will be synthesized and pharmacologically analyzed in order to elucidate the chemical and biological reactivity of Substance P, and to support and expedite the establishment of the clinical benefits of Substance P in treating disease. The availability of analogs will open up the possibility to study the agonist-antagonist interaction in more detail, and to determine which part of the molecule is important for affinity and activity, respectively, for Substance P. Of the eight different amino acids in the undecapeptide sequence of Substance P, Arg1-, Lys3-, Phe7-, and Phe8- appear to be the four crucial amino acids. The ARg1- and Lys3- moieties, in protonated form, could be very important for activity and potency because they contribute an ionic nature to the molecule. The Phe7-Phe8-moiety could contribute unique structural specificity to the entire molecule because of possible pi-pi bond interaction with a receptor site. Modification of these amino acids by elimination or substitution, as well as modification of both termini of the peptide chain by substitution, elimination, or elongation, will allow us to design Substance P analogs in order to (1) study the immunochemical affinity and reactivity of Substance P, (2) act as an inhibitor of Substance P, and/or (3) prolong the activity of Substance P to make it more clinically effective. The analogs will be synthesized by the solid phase technique on a Beckman 990 automatic peptide synthesizer, purified, and appropriately characterized by thin layer chromatography, amino acid analyses, electrophoresis, and optical rotation. The pharmacological studies will be carried out by Dr. Bengt Pernow of the Clinical Physiology Department of the Karolinska Institute, Stockholm, Seden, through a continuing cooperative agreement with Dr. Folkers.

Project summary: Ants are social insects that can be developed as experimentally tractable organisms for probing the dynamic changes in the epigenetic programs that control an array of processes, including aging. Aging is a process of progressive decline in intrinsic physiological functions. There is a well-established trade-off between lifespan and reproduction as higher reproductive activity in females is associated with shorter lifespan. However, in social insects, the reproductive queen has up to 10X longer lifespan than non-reproductive workers. In the ant Harpegnathos saltator, adult individuals that are not exposed to queen pheromones can undergo a reversible switch from non-reproductive workers to reproductive pseudo-queens (gamergates) that exhibit fully developed ovary and, importantly, a 5X increase in lifespan, showing that aging is reversible. Lifespan is shortened again when gamergates are reverted to workers (revertants). Thus, Harpegnathos provides an effective system to study epigenetic regulation of aging and rejuvenation given the adult plasticity that allows switching between castes. We have performed transcriptome analysis of the longevity-regulatory tissues: the fat body, ovary and brain, in workers vs. gamergates vs. revertants. We have identified a group of differentially expressed genes (DEGs), some of which have been implicated in the regulation of longevity, e.g. IIS (insulin and IGF signaling) pathway components. To further analyze the genetic and epigenetic regulation of longevity in ants, we will first compare physiology, lifespan, transcriptome and histone modifications in gamergates derived from young vs. old workers to ascertain the epigenetic regulation of aging and, most intriguingly, rejuvenation. The cellular localization and functions of important DEGs will be further analyzed. Second, we will utilize our newly established genetic tool ? CRISPR in ants ? to generate knockout (KO) ants in the two DEGs in the ovary, Hs- IMPL2 and Hs-ALS, which likely act as inhibitors of IIS in ants. These KO ants will be used to characterize the role of IIS in the dramatically extended lifespan in gamergates. Third, Hs-ILP1 (insulin-like peptide 1) is differentially expressed in the brain and Hs-ILP2 in the ovary. While both Hs-ILP1 and Hs-ILP2 are strongly increased in gamergates compared to workers, IIS is decreased in the fat body and ovary. To address this paradox, we will analyze (a) the role of Hs-ILP1 and -ILP2 in regulating the two branches of IIS: AKT and MAPK; (b) the role of Hs-ImpL2 and Hs-ALS in regulating activity of Hs-ILPs; and (c) how two insulin receptors (InRs) mediate the role of Hs-ILPs in differentially regulating IIS.

The commonest gastrointestinal (Gl) carcinoids occur in the small intestine and appendix. Both are derived from the enterochromaffin (EC) cell, but their malignancy and propensity to metastasize vary widely. Small intestinal carcinoids (SICs) are aggressive and exhibit hepatic and lymph node (LN) metastasis; appendiceal carcinoids (APCs) are slow-growing and rarely metastasize. Gastric carcinoids (GCs) [enterochromaffin-like (ECL) cells], are slow-growing and rarely metastasize unless gastrin-independent. No biological explanation for the variable malignancy of Gl carcinoids exists. We hypothesize that a gene expression-based definition of these tumors will: 1) allow prediction of malignancy and metastasis, and 2) provide accurate staging and facilitate rational therapy. Utilizing: i) GeneChip analysis, ii) quantitative real-time PCR (Q RT-PCR), and iii) carcinoid tissue microarray (TMA) immunostaining/quantitation, we evaluated gene and protein expression in Gl carcinoids. In SICs and their metastases, the neuroendocrine marker chromogranin A (CgA); NAP1L1 (mitosis regulator); MAGE-D2 (liver metastatic predictor), and MTA1 (metastasis, migration), are over- expressed. In malignant APCs and GCs, CgA, MAGE-D2, MTA1, and NAP1L1 are all significantly over- expressed. In 30% of histologically-negative lymph nodes, molecular analysis detected CgA transcript and protein, making sensitive molecular staging of carcinoid tumors possible. This proposal seeks, using Q RT- PCR of frozen and paraffin specimens and carcinoid TMA analysis, to define the malignant potential of Gl carcinoids. The aims are: 1) Confirm the specificity of the neuroendocrine cell marker gene, CgA, in identifying Gl carcinoids, 2) Determine whether increased levels of the mitotic and metastasis-associated genes, NAP1L1, MAGE-D2 and MTA1, characterize malignant Gl carcinoids, 3) Determine whether benign and malignant appendiceal carcinoids show predictive differential gene expression, and 4) Identify occult metastasis in histologically normal lymph node or liver biopsies using Q RT-PCR. We propose that delineation and quantification of gene expression will: 1) define malignancy of an individual carcinoid and 2) identify occult metastasis. This will provide novel biological information and provide molecular data to identify occult metastasis predict spread and facilitate preemptive appropriate therapy. [unreadable] [unreadable] [unreadable]

Cellular membranes are not simple barriers, and the appropriate membrane composition is essential to ensure proper function of the membrane. In fact, dysfunction as a result of altered membrane composition has been observed in a wide range of diseases including cancers, neurodegenerative and age-related diseases. The composition of the membrane is known to impact its key properties including permeability, curvature, and fluidity;however, the lipid processing pathways and regulators that affect these composition changes have not yet been identified. This is largely due to the fact that in vivo studies of membrane dynamics have been limited, because they require high levels of isotope enrichment to obtain the resolution required to analyze membrane lipids in detail. In the Van Gilst lab, I developed stable isotope enrichment strategies in C. elegans that allow for the quantification of dietary carbon into the fatty acids of the animal. These isotope feeding strategies provide the enrichment levels required for flux analysis of phospholipid membrane dynamics. In this proposal, I will expand the scope of these stable isotope tracer methods to assay the turnover and synthesis of the acyl chains and phospholipid head groups in membranes via gas chromatography/mass spectrometry (GC/MS) and liquid chromatography/mass spectometry (LC/MS), respectively. In doing so, I hope to define many of the pathways that impact membrane composition and its ability to respond to stress and ultimately contribute to the overall understanding of membrane biology and dynamics. One of the most dramatic impacts of membrane composition is in aging, where the phospholipids begin to contain drastically more saturated fatty acids, ultimately making the membranes much more rigid and negatively impacting their function. As examples, the increased saturation index in the aged membrane can affect diffusion properties, transporter function, vesicle fusion, and even signaling. The progressive accumulation of saturated fat in the membranes over aging has been theorized as a major contributor to aging and aging-related dysfunction. The genetic tools of C. elegans will allow us to determine the relative contribution of membrane turnover on the overall membrane composition in young and old animals. The goal of this proposal is to define the genetic regulators and pathways that influence membrane aging and, in doing so, contribute to the understanding of how membrane biology impacts the aging process. PUBLIC HEALTH RELEVANCE: The phospholipid composition of the membrane has a dramatic impact on the membrane's ability to function properly, and altered membrane composition has been associated with a wide array of diseases including cancers, neurodegenerative and age-related diseases. However, the mechanisms that are responsible for the aberrant changes in membrane composition have not yet been identified. Here, I will perform an in vivo study of membrane dynamics and maintenance in C. elegans to define the pathways that regulate membrane composition and how those pathways are altered during aging.

In addition to examining in more detail the influence of antineoplastic agents upon the metabolism, distribution, and binding of drugs and their toxicity, the metabolic fate of selected antineoplastic agents will be studied. Carcinolytic agents will be employed as model substrates for the in vitro measurements of microsomal enzyme activity. The biliary excretion model will be extended to study excretion of antineoplastic agents and their metabolites with subsequent identification by mass spectral techniques.

Better understanding of the etiologic roles of family history, prenatal environmental factors, and potential biologic mechanisms, such as epigenetic changes, in autism spectrum disorders (ASD) are research priorities identified in the Autism Coordinating Committee 2011 Strategic Plan for Autism Spectrum Disorder Research, but rapid progress is hampered by the challenges of acquiring relevant data in large epidemiologic samples. The goals of the current proposal are to examine: (1) fundamental controversies concerning familial and environmental contributions to risk for ASD; (2) transmission of risk across generations; (3) investigate pregnancy-related environmental factors in ASD, and (4) the potential role of epigenetic changes in those factors. We will build on an existing research network leveraging established population-based epidemiologic resources from seven countries (USA-California, Australia, Denmark, Finland, Israel, Norway, Sweden) that include individual-level perinatal medical, and demographic information and archived biospecimens. Study data will be based on over 4.5 million births (1998-2007), over 20,000 cases of ASD, and family linkages over three generations (grandparents, parents/aunts/uncles, siblings/cousins). Using this unparalleled resource, we propose a novel multigenerational perspective in ASD risk across four integrated aims: Aim 1: Model familial recurrence risk and the contributions of shared environmental factors to ASD liability, building on advanced modeling approaches using extended family relations. Aim 2: Determine if parental components of ASD risk are transmitted across generations, specifically, advancing parental/grandparental age and parental/grandparental immigration of minority groups. Aim 3: Examine ASD risk from prenatal exposure to medications with potential adverse neurodevelopmental effects: a) valproate; b) 2-adrenergic receptor agonists; c) selective serotonin reuptake inhibitors; or d) antibiotics that impede folate metabolism, i.e., sulfonamides and Trimethoprim. Aim 4: Using genomic DNA extracted from archived neonatal blood-spot samples, examine epigenetic changes in children with ASD exposed prenatally to the maternal medications in the previous aim. The resource established by the MINERvA Network will allow more accurate and precise determination of the contributions of familial and environmental factors to the etiology of autism, in particular if medications for maternal chronic and acute conditions prescribed in pregnancy contribute to ASD risk, and whether epigenetic processes underlie a biological abnormality linked to autism. From a public health perspective the study will accelerate the characterization of high risk groups, modifiable risk factors and the elucidation of mechanisms in autism etiology that could ultimately contribute to preventive measures or interventions and treatments.

The goal of our research is to identify and characterize the machinery involved in eukaryotic DNA replication. We focus on the isolation of the individual proteins and protein complexes required for DNA synthesis and define their role in this critical macromolecular process. All growing cells must duplicate their DNA accurately and perform this feat once and only once per cell cycle. Events that interfere with these requirements can lead to cell death or altered proliferation that result in cancers. At present, many steps involved in supporting replication and its control are either unknown or poorly characterized. [unreadable] [unreadable] We plan to extend our previous work on various key replication proteins and their complexes that support the activation of initiation complexes required for the elongation of DNA chains. For this purpose, we will study: a) the role of a complex, containing Cdc45, the MCM2-7 complex and GINS (CMG) which contains DNA helicase activity and may contribute important to the unwinding reaction at the replication fork; b) the interaction between GINS and the Pol 1-primase complex which markedly stimulates its DNA polymerase activity; c) the properties of cloned Pol5 and its interaction with PCNA required for its processive elongation of primed DNA templates. We have also observed functional interactions of GINS with Pol5 as well as Pol4. We plan to examine these effects further and evaluate whether the CMG complex does the same. Coupled replication and unwinding reactions will be carried out which may contribute to strand selection; d) We plan to explore the role of the Cdc7 kinase on the activity and formation of the CMG complex. In vivo, this kinase was shown to influence the interaction of Cdc45 with the MCM2-7 complex. We will further characterize a novel interaction recently detected between the Cdc7 kinase complex and the cohesin loader complex Scc2/Scc4. The requirements for this interaction and its consequences will be investigated. All cells must make identical copies of their DNA to survive. Our studies are directed at how these copies are made and regulated. We plan to isolate the proteins involved in copying DNA and determine how they function. Results from these studies will have a direct impact on genetic diseases and cancer. [unreadable] [unreadable] [unreadable]

Case Western University's Begun Center for Violence Prevention Research and Education, Kent State University's Institute for the Study and Prevention of Violence, and the Partnership for a Safer Cleveland will collaborate to address youth violence prevention information dissemination and research through a joint effort of existing academic centers and community agencies in the Cleveland, Ohio area. More specifically, over a three year period, an academic-community partnership will be created in the form of a consortium for the purpose of information sharing and research collaborations in the area of youth violence prevention, The Greater Cleveland Consortium for Youth Violence Prevention. The proposed collaboration has three aims: 1) to identify/assess the community's interests in addressing youth violence prevention; 2) to provide health education to the community in the area of youth violence prevention; and 3) to develop and establish Memorandums of Understanding (MOUs) with community organizations that will support the conduct of Community-Based Participatory Research (CBPR) and establish The Greater Cleveland Consortium for Youth Violence Prevention. The means for achieving these aims will be provided by:1) conducting community forums on youth violence to solicit the community's understanding of the problem; 2) identifying organizations interested in CBPR participation/board membership; 3) disseminating information on youth violence, violence prevention, and the related health disparities for Cleveland's youths; 4) sponsoring workshops and symposia on youth violence, violence prevention and related health disparities; 5) developing a research agenda addressing the unique needs of Cleveland's youths for violence prevention; 6) developing formal partnerships with community representatives to conduct community-based research.

The studies proposed in this application are designed to 1. determine whether the circadian rhythm of IOP reflects a circadian rhythm of aqueous formation, 2. explore the neural and humoral pathways which mediate regulation of this rhythm of IOP, and 3. identify biochemical mechanisms which underlie the local, ocular mediation of the circadian rhythm of IOP in this animal model. The section on Glaucoma and Aqueous Dynamics of this department is committed to the long term goal of understanding the anatomic, physiologic and biochemical basis for regulation and maintenance of IOP and aqueous flow. The circadian rhythm of IOP represents a non-invasive technique for manipulating aqueous dynamics, and, as such, provides a "handle" for studying mechanisms involved in variation of IOP. Better understanding of these mechanisms which mediate the circadian rhythm of IOP may further our understanding of the mechanisms which maintain and regulate the steady state of IOP. IOP in humans is recognized to show a daily rhythm which can be exaggerated in glaucoma patients. This spontaneous variation of IOP in open-angle glaucoma remains one of the most important and least understood enigmas of glaucoma, and a better understanding of the mechanisms underlying this phenomenon may permit exploitation of normal physiologic mechanisms to achieve control of IOP in glaucoma patients. This animal model for the circadian rhythm of IOP may also prove to be a useful non-invasive model for evaluating potential drugs for glaucoma therapy.

Urinary tract infections (UTIs) result in considerable morbidity and health care costs. The most common cause is the Gram-negative bacteria, uropathogenic Escherichia coli (UPEC). Human infections are characterized by bacteriuria and the release of cytokines, exfoliated cells, and polymorphonuclear leukocytes (PMN) into the urine. These clinical manifestations of disease are all seen in mice. Using qRT-PCR, microarray analyses, microscopy, and immunohistochemistry, we have further described the response to UPEC infection. We have demonstrated that type 1 pili-mediated UPEC binding to and invasion of bladder epithelial cells potentiates toll-like receptor (TLR) 4-mediated signaling and results in rapid upregulation of genes involved in cell differentiation, proliferative and immediate-early responses, pro-inflammatory responses, apoptosis, stress responses, signal transduction, cell-cell contacts, and metabolic changes. Further, we have shown that TLR4-mediated signaling on both stromal and hematopoietic cells is required for efficient clearance of bacteria. Hematopoietic cells include macrophages (M), dendritic cells (DC), granulocytes, and B and T cells. In this proposal we describe experiments to build on our previous work to understand host processes important in determining the outcome of an encounter between pathogens and the normally sterile host urinary tract. We will delineate the host response to UPEC, including the kinetics of cytokine production within the infected bladder, the hierarchy of infiltration of immune cells, and real time visualization of early interactions between immune cells and infecting bacteria. We will also broaden our understanding of UTIs and host response by characterizing the host response to two Gram-positive bacteria that also cause UTIs in humans, Staphylococcus saprophyticus and Enterococcus faecalis. Differing host response mechanisms and bacterial tropisms between Gram-negative and Gram- positive UTIs may have implications for disease symptoms, progression, and treatment. Using mouse mutant backgrounds and cell and cytokine depletion experiments, we will examine the roles of host immune cells and defense molecules in inflammation, bacterial clearance, reservoir maintenance, and epithelial exfoliation and regeneration. A detailed understanding of the host response to UTIs may lead to elucidation of new and better ways to evaluate, treat, and prevent these common infections. PUBLIC HEALTH RELEVANCE: UTIs occur frequently in otherwise healthy women and result in an estimated ~$2.5 billion annually in health care costs. In this proposal we describe experiments to broaden our understanding of UTIs by characterizing the soluble mediators and host immune cells involved in the host response to the most common cause of UTI (uropathogenic Escherichia coli) and two Gram-positive bacteria (Staphylococcus saprophyticus and Enterococcus faecalis). A better understanding of the host factors involved in determining the outcome of infection is needed to better diagnosis, treat and prevent this common disease. [unreadable] [unreadable] [unreadable]

This project involves developing imaging agents which will allow the asessment of dopaminergic and serotonergic activity in the brain non-invasively using positron emission tomography (PET). Short-lived positron emitting isotopes, 18F (t+=110 min.) and 11C (t+=20 min.), are introduced into amino acid analogs which are substrates of the rate limiting enzymes in the biosynthesis of dopamine and serotonin. Non-radioactive samples of these compounds are first sythesized and characterized by multinuclear NMR (1H, 11C, 19F). In addition, both the precursors and enzyme products of the desired compounds are prepared and characterized by NMR. These precursors and enzyme products are used in the synthesis of the desired compounds and their metabolic characterization, respectively. Radiolabeled compounds are then evaluated, in vitro and in vivo, as imaging agents for use in the study of neuropsychiatric disorders such as Parkinson's disease, affective disorders and depression.

Children with Attention Deficit/Hyperactivity Disorder (ADHD) are at risk for serious adverse developmental outcomes, including criminal behavior, depression, and suicidality. Parenting and parental psychopathology are robust predictors of long-term developmental outcomes in this population. For instance, early maternal positive parenting predicts fewer conduct problems over time among children with ADHD, whereas maternal depression predicts higher levels of future conduct problems. Little is known about biological markers which may be associated with individual differences in maternal parenting in families of children with ADHD. One such biological marker previously associated with parenting in non-ADHD samples is the hypothalamic- pituitary adrenal (HPA) axis and its byproduct, cortisol. Neurobiological models of parenting suggest that cortisol may be associated with variations in parenting such that elevated levels of maternal cortisol following a stressful task are associated with negative parenting in mothers of toddlers with difficult temperaments. Existing studies however, fail to examine the relationship between maternal cortisol reactivity and parenting in school-age children. Additionally, existing studies do not address maternal cortisol reactivity and parenting in mothers of children with ADHD. In non-ADHD samples, difficulty regulating stress responses is related to variations in maternal parenting and poor maternal adjustment, both of which have been found to predict poor developmental outcomes among children with ADHD. Furthermore, mothers' regulation of physiological stress responses may be particularly important for families of children with ADHD, as parenting a child with chronically challenging behaviors represents a persistent environmental stressor. The proposed study seeks to extend the existing literature by providing an empirical examination of the relationship between individual differences in maternal cortisol reactivity following a stressful task and observed parenting behavior in mothers of children with ADHD. This investigation can have implications for understanding biological mechanisms of adaptive maternal parenting among families of children at risk for adverse developmental outcomes.

[unreadable] The purpose of this competing continuation is to continue our training program in the human behavioral pharmacology of drug dependence. Our decision to seek to continue this training program is driven by our training successes. For example, during the past ten years we have trained 14 pre-doctoral student of whom 12 (86%) remain involved in research or academics (teaching) and 23 postdoctoral students of whom 22 (96%) remain involved in research or academics. We are very proud to note that among our 23 postdoctoral trainees, 13 (57%) currently hold university faculty positions. Collectively, our trainees have authored over 500 publications. The value of continuing our training program in human behavioral pharmacology relates to the unique niche that it fills. Human behavioral pharmacology of drug dependence is concerned with factors that affect the development, maintenance, and cessation of drug abuse in humans. In our view, individuals trained in human behavioral pharmacology are at the crossroads of basic and clinical research and as such provide a crucial link between these two research domains. In this competing continuation, we propose to continue our training program that consists of 4 pre-doctoral psychology students, and 4 postdoctoral (M.D. or Ph.D.) fellows in the behavioral pharmacology of human drug dependence. The 6 core faculty consist of 5 Ph.D.s and 1 M.D. who have joint appointments in Psychiatry, Psychology and Family Practice. These core faculty have 14 NIDA and 1 drug company grants. Research opportunities range from laboratory studies of human drug self-administration to outpatient studies of behavioral and pharmacological treatments of cocaine, heroin, nicotine and marijuana dependence. Training is conducted at the Human Behavioral Pharmacology Laboratory (HBPL), Substance Abuse Treatment Center (SATC), or Treatment Research Center (TRC). Trainees are selected on the basis of excellence in their scholastic record and by their commitment to a career in drug abuse research. Pre-doctoral psychology students are required to take psychology courses including those developed specifically for this training program, and complete masters and doctoral theses. Medical students are enrolled in a summer internship program that provides didactic training as well as 'hands on1 experience. Postdoctoral students have the opportunity to further enhance their education via course-work as well as conduct independent research. The training period will generally be 4 to 5 years for Ph.D. candidates and 2 to 3 years for postdoctoral fellows. Each trainee has a primary advisor from the core faculty. An annual off-campus retreat permits trainees to present their research and discuss research issues. Additionally, trainees attend colloquia in the Psychology and Psychiatry Departments. The proposed project will continue to train human behavioral pharmacologists to empirically examine behavioral and pharmacological factors contributing to drug use, the consequences of drug use, and how that basic understanding translates to novel treatments for drug dependence. Moreover, this training program will continue to train qualified individuals to be successful independent researchers in the human behavioral pharmacology of drug dependence. [unreadable] [unreadable] [unreadable] [unreadable]

Angiotensin ll-infused rats exhibit increases in renal angiotensinogen (AGT) mRNA and protein, and this may be one mechanism by which low levels of circulating angiotensin II induce progressive hypertension. Using an interesting transgenic mouse model in which human AGT is expressed only in the kidney, it is possible to enhance angiotensin II content only in the kidney without altering circulating levels of angiotensin II. The goal of this research project is to define and characterize the mechanisms responsible for the intrarenal enhancement of AGT expression by angiotensin II. My hypothesis is that the augmented intrarenal AGT expression by angiotensin II involves angiotensin II type 1 (AT1) receptor-dependent and c-Src/MAPK-, ROS-, or Rho-kinase/NFkB-dependent pathways. In accord with this hypothesis, the following specific aims are targeted for the proposed period of support: 1) To demonstrate that chronic overproduction of angiotensin II only in the kidney elicited by stimulating human AGT expression in the presence of human renin will cause increases in endogenous mouse AGT mRNA expression as well as endogenous mouse AGT protein levels in proximal tubular cells leading to slowly progressive hypertension in gene-targeted male mice. 2) To demonstrate that subacute overproduction of angiotensin II only in the kidney elicited by stimulating human AGT expression in the presence of human renin will cause increases in endogenous mouse AGT mRNA expression as well as endogenous mouse AGT protein levels in proximal tubular cells leading to progressive hypertension in gene-targeted female mice. 3) To demonstrate that the enhancement of the endogenous mouse AGT production in proximal tubular cells involves AT1 receptor-mediated mechanisms in gene- targeted female mice. 4) To determine if AT1 receptor-dependent AGT enhancement by angiotensin II involves c-Src/MAPK-, ROS-, or Rho-kinase/NFkB-dependent mechanisms in primary-cultured mouse proximal tubular cells. 5) To examine whether the 5'-upstream promoter region of mouse AGT gene has positive responsive element(s) by angiotensin II. The results obtained from the proposed studies will provide important information regarding the intrarenal augmentation of AGT expression by angiotensin II, which may help to develop a novel concept to treat angiotensin ll-dependent hypertensive subjects and to establish the importance of the tubular renin-angiotensin system in the genesis of angiotensin ll-dependent hypertension.