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Autosomal dominant polycystic kidney disease (ADPKD) is a common monoallelic disorder associated with progressive cyst development and resulting in end stage renal failure (ESRD) in 50% of patients by 60y. However, there is considerable phenotypic variability, extending from in utero onset to patients with adequate renal function into old age. Autosomal dominant polycystic liver disease (ADPLD), as traditionally defined, results in PLD with minimal renal cysts. Classically there have been considered two ADPKD genes, PKD1 and PKD2, encoding PC1 and PC2, and two ADPLD genes, PRKCSH and SEC63, but in the past few years greater genetic heterogeneity has been described, with nine genes now implicated overall. Recent data also indicates an overlap in etiology and pathogenesis associated with ADPKD and ADPLD, with the efficient biogenesis and localization of the PC-complex central to both disorders. During the last funding period we identified a novel gene, GANAB, which is associated with both disorders, where the encoded protein, GII?? is involved in the maturation and trafficking of PC1. In this proposal we will take advantage of advances in next generation sequencing (NGS) methodologies, and large populations of ADPKD and ADPLD patients that have been assembled and screened for the classic genes, to hunt for novel genes for these disorders (Aim 1). The phenotype associated with these genes will be characterized (Aim 3) along with their mechanism of action (Aim 2). NGS methods will be perfected to screen the segmentally duplicated locus, PKD1, and to identify missed mutations at the known loci, including those present in just some cells due to mosaicism (Aim 1). The significance of many PKD1 nontruncating variants has been difficult to evaluate (classed as variants of unknown significance; VUS), but recently evidence that some are incompletely penetrant alleles partially explains phenotypic variability in PKD1 populations. In Aim 2 improved in silico predictions, in combination with machine learning, will improve the understanding of the pathogenicity and penetrance of VUS. A cellular assay of the biogenesis and trafficking of this PC-complex will also be employed to quantify the penetrance of VUS. The mechanism of pathogenesis will be explored in animal models with ultralow penetrant (ULP) Pkd1 or Pkd2 alleles. Employing the large clinically, imaging, and genetically well-defined populations phenotypic groupings of patients will be defined that will then be compared to the genic and PKD1 allelic groups (Aim 3). This iterative process will allow the Variant Score (VS) associated with each PKD1 VUS to be refined. In a separate population the revised VS, alone and in combination with clinical, functional, and imaging data, will be employed to generate a comprehensive, predictive algorithm for ADPKD (Aim 3). Disease modifiers to severe disease, via biallelic ADPKD, and due to alleles at other loci will also be identified and characterized in the cellular assay and in vivo in combination with the Pkd1 hypomorphic, RC model. The final aim will exploit the newly identified information that some PKD1 and PKD2 VUS are rescuable, folding mutations that in a maturation-fostering environment can traffic and function appropriately. A screening scheme based on the level of cell surface PC1 will be improved and new chaperone drugs specific for the PC complex will be sought in collaboration with Sanford Burnham Prebys. A second mutation group that will be explored therapeutically are nonsense mutations. A cellular assay for readthrough efficiency is being developed and will be used for screening. Identified chaperone or readthrough drugs will be tested in available mouse models. Overall this proposal will better explain the etiology and the genetic causes of phenotypic variability in ADPKD/ADPLD, develop better prognostic tools for individual selection of patients for treatment that are now becoming available, and explore allele based treatments for ADPKD.

The long term objective is to characterize key functionalities of the epithelial cells of the larval mosquito gut as these cellular functions influence and regulate the anionic basis of alkalinization of the gut lumen. A detailed understanding of how gut epithelial cells produce the remarkable and biologically unique pH extremes (i.e. > 10.5) that drive the digestive process will provide new avenues for the development of environmentally safe and specific larvacides. Two specific gene families have been targeted as they have central roles in anion production and transport in the gut alkalinization process: carbonic anhydrases and transmembrane anion transporters. This project will produce molecular and physiological characterizations of members of these two gene families. Their distributions in the tissue and specific roles in larval mosquito gut alkalinization will be defined. Specific cellular phenotypes throughout the gut will be defined and the role of each in the alkalinization process assessed. AIM 1 will examine the expression of multiple carbonic anhydrases. AIM 2 will define and characterize members of the anion transporter gene family. AIM 3 will define the cellular distributions of carbonic anhydrases and anion transporters in the gut and as functions of larval development. AIM 4 will produce a global analysis of gene expression in the specific functional domains of the larval mosquito gut identifying key functionalities which define the gut domains. AIM 5 will bring the localization of specific gene products together with physiological measurements of the activity of individual cells to produce a cell-specific and spatial analysis of anion dynamics in the gut epithelium. As mosquitoes are the number one threat to human health world wide and recognized as potential agents for bioterrorism, the development of new strategies for control based on unique aspects of their biology (i.e. gut alkalinization) has important potential. [unreadable] [unreadable] [unreadable]

Poly(ADP-ribose) polymerase (PARP) 1, whose primary role is initiation of DNA repair, is activated by damaged DNA and uses NAD+ to automodify itself and recruit other proteins involved in DNA repair. Due to its role in DNA repair, PARP-1 inhibition has been long targeted for treatment of different cancer types. By now there are already several different clinical APRP-1 inhibitors used in treatment of ovarian and breast cancers, and many others are under clinical trials for other types of cancer, such as prostate cancer, pancreatic cancer, blood cancer and others. PARP-1 inhibition has also been demonstrated to have promising effect for treatment of some cardiovascular conditions. Extensive DNA damage caused by number of cardiovascular conditions, such as a stroke or heart attack, can result in PARP-1's hyper-activation, leading to depletion of cellular NAD+ and subsequent cell death. It has been demonstrated that inhibition of PARP-1's activity using small molecules can prevent apoptosis and necrosis in such cells. Studies in animal models have indeed shown that inhibition of PARP-1 can have beneficiary effects for treatment of various cardiovascular conditions, such as ischemic stroke, cerebral ischemia, diabetic cardiomyopathy and others. Despite growing number of PARP-1 inhibitors, their molecular mechanism of action is not well understood. The overall objective of my project is to define the molecular mechanisms of activation and silencing of PARP-1. My central hypothesis is that the structural and dynamic changes occurring in PARP-1 upon DNA binding play key roles in the regulation of protein activation and dictate relative efficiency of PARP-1 inhibitors. Three specific aims are pursued in this project: 1. To define how PARP-1 is silenced through auto-modification and released from single-strand break (SSB) DNA, 2. To measure the effect of inhibitors on PARP1 structural dynamics for those that trap it at a SSB versus those that don't, 3. To define the organization and dynamics of the PARP- 1/nucleosome complex in conjunction with the housekeeping role of PARP-1 in transcriptional regulation. My proposed experiments will reveal key insights on the precise molecular mechanisms of PARP-1 activation and inhibition, aiding in the design of new PARP-1 inhibitors to improve outcomes in patients with various 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. Most organisms rely on an innate immune system as their first line of defense against infection. Within the innate immune system, the Toll-like receptors (TLRs), a family of evolutionarily ancient receptors found on the surface of many cell types, are critical for pathogen recognition outside the cell. About 12 TLRs recognize structures specific to pathogens, such as bacterial cell wall components, bacterial filament proteins, or certain types of nucleic acid. This recognition event initiates a signal inside the cell, which induces the rapid secretion of antimicrobial and inflammatory proteins. Inside the cell, the NOD proteins and RNA helicases such as MDA5 recognize similar pathogen-associated structures to those recognized by TLRs. Remarkably, given the structural diversity of the structures that they recognize, all TLRs and NODs rely on a "leucine-rich repeat" (LRR) domain to recognize pathogen-associated structures. The overall goal of our research program on innate immune sensors is to understand how they recognize conserved molecular patterns in pathogens, and how this recognition is translated into an innate immune response. Our structural approach will provide unique insights into these important processes. First, we aim to determine the structure of one or more TLR-ligand complexes, by X-ray crystallography. Alternative crystallization targets are NOD-ligand or helicase-RNA complexes. We propose to use novel protein expression techniques to maximize protein yields. Our structures will likely define novel principles of molecular recognition. By revealing the conformational changes associated with ligand binding, the structures will provide insight on how pathogen recognition is translated into a signal in the cell that elicits an immune response. Our work will also guide efforts to design synthetic agonists or antagonists with immunomodulatory properties. Such compounds would have a wide range of medical applications, particularly as vaccine adjuvants or anti-inflammatory therapeutics.

Given the potential pharmacological and physiological diversity arising from heterodimerization of opioid receptors, an important challenge in opioid research is the development of selective tools for the investigation of such phenotypic opioid receptors. Selective pharmacological tools that can span the divide between cultured cells and in vivo systems would clarify the functional roles and localization of heterodimeric opioid receptors in experimental animals. Thus, the broad, long-term objectives of this research are to develop ligands with selectivity for heterodimeric opioid receptors as tools to study the functional roles of physically associated opioid receptors in the central nervous system. The long-term goal is to use the information obtained from such studies to develop superior analgesics that are devoid of tolerance and dependence. [unreadable] [unreadable] The specific aims of the present application include the synthesis and biological evaluation of ligands that are selective for opioid receptor heterodimers. Based on reports of heterodimeric opioid receptors in cultured cells and on the large body of literature that implicates interaction between mu and kappa opioid receptors and mu and NK1, CCK2, ORL1, and CB1 receptors in vivo, a total of ten series of compounds will be synthesized. Eight of the proposed series are bivalent ligands that will include mu and kappa opioid pharmacophores or a mu agonist pharmacophore combined with NK1, CCK2, ORL1, or CB1 antagonist pharmacophores. The pharmacophores in each of these bivalent series will be linked to each other through spacers containing 12-22 atoms. The antagonist non-opioid pharmacophores were selected because interaction between mu opioid receptors and the above receptors have been reported to modulate antinociception, tolerance and/or dependence. The corresponding series of monovalent ligands with matching spacers and matching pharmacophores will be synthesized as controls. There will be 11 compounds in each of these 16 series. The remaining two series will be structurally related to 6'-GNTI which has been reported to produce analgesia in mice by selectively targeting spinal delta-kappa opioid receptor heterodimers. Because analgesia of 6'-GNTI is mediated spinally, such compounds should not possess the supraspinal side-effects generally associated with clinically employed analgesics. As a second approach to development of spinally-selective analgesics, the Pl/s library of ~1000 opiates will undergo Flexstation screening on cultured cells containing coexpressed and singly expressed delta and kappa opioid receptors. Target compounds and screening hits will be tested in cultured cells and in behavioral tests in mice that include evaluation of tolerance and physical dependence. [unreadable] [unreadable] [unreadable]

The overall goal of this project is to determine the role of bone marrow (BM) stem/progenitor cells in regulating fibrocyte homeostasis in the mammalian inner ear. It is well established that specific subpopulations of inner ear fibrocytes are actively involved in the generation of electrochemical gradients essential to normal auditory function. Injury to or loss of these cells resulting from ototoxins, noise-trauma, genetic defects and aging is associated with a range of hearing and balance disorders. These highly specialized fibrocytes undergo continuous replacement throughout life and their turnover rate has been shown to increase with injury and decrease with age. However, it is not known whether this self renewal is mediated by a resident population of adult stem/progenitor cells or through some other mechanism. New data provided here document that cells derived from BM have the capacity to engraft and differentiate towards ion transport fibrocyte phenotypes in the mouse inner ear. These data also suggest that BM may provide a continuous source of stem/progenitor cells for fibrocyte turnover in the inner ear but this remains unproven. Three Specific Aims are designed to address these important issues. Aim 1 will evaluate the relative ability of two highly purified populations of BM stem/progenitor cells to engraft and differentiate into fibrocytes in the normal inner ear. Aim 2 will investigate the potential of BM stem/progenitor cells to replace fibrocytes or other inner ear cell types damaged by chemical injury, genetic mutations or aging. Aim 3 seeks to develop and optimize procedures for the direct introduction of BM stem/progenitor cells into the inner ear. The experimental design incorporates mouse BM transplantation and parabiosis models employing donor cells that express high levels of EGFP, allowing their tracking with immunological and histochemical procedures. Given the increasing evidence that defects or injuries to inner ear fibrocytes are associated with a wide range of hearing and balance disorders, it is important to obtain further knowledge about the derivation of these cells and the mechanisms mediating their turnover and replacement in both the normal, traumatized and aging inner ear. Such knowledge will be essential in the design of therapeutic strategies for the treatment of inner ear disorders associated with injury or destruction of specialized populations of inner ear fibrocytes. [unreadable] [unreadable] [unreadable]

Control of neonatal pulmonary vascular and airway resistance remains poorly understood. Recently, two additional intrapulmonary control systems, pulmonary neuroendocrine cells (PNEC) and the pulmonary peptidergic innervation (PPI), have been described, which may influence pulmonary vascular and airway tone in the neonate. PNEC are granulated cells that line the airways of fetal, neonatal, and adult lungs. They are particularly numerous in the neonatal period and contain peptides (bombesin, calcitonin, leu-enkephalin) and amines (serotonin) known to affect smooth muscle tone in other organ systems. The PPI, composed of fibers containing substance-P and vasoactive intestinal peptide (VIP), have been described in association with pulmonary airways and vessels of all sizes. Both these peptides are known to profoundly influence smooth muscle tone in other organ systems. Limited indirect anatomic and physiologic evidence has suggested a role for both systems in altering intrapulmonary smooth muscle tone, particularly in neonates; however, basic knowledge of both systems is extremely limited. The aim of this research program is to provide some of the fundamental knowledge necessary to assess the role of both these systems in influencing neonatal pulmonary vascular and airway tone under normal and pathologic conditions. This aim will be accomplished in the following fashion: (1) Determination of the development and distribution of PNEC and PPI within the lung using immunohistochemical techniques. (2) Determination of the anatomic distribution and number of receptors for the above peptides and amines, and their relationship to receptors for the presently characterized intrapulmonary control mechanisms (adrenergic and cholinergic innervation) using redioautography. (3) Determination of pathologic alterations in the structure and distribution of PNEC, PPI, and adrenergic, cholinergic, and peptidergic receptors in (a) experimental asthma and (b) acute and chronic neonatal cardiopulmonary disease where alterations in vascular and airway tone are either known to play a significant role (hyaline membrane disease, bronchopulmonary dysplasia, congenital heart disease) or may play a significant role (sudden infant death syndrome, cystic fibrosis). Should these two systems, pulmonary neuroendocrine cells and the pulmonary peptidergic innervation, influence neonatal vascular and airway tone; understanding their normal function and their alterations in common neonatal cardiopulmonary pathologic states may lead to novel therapies for a number of currently untreatable, or poorly treatabale, neonatal cardiopulmonary diseases.

Most normal human cells undergo a limited number of cell divisions, eventually entering an irreversibly arrested state, through either senescence or differentiation. Both processes have major implications for human health; between them impacting birth defects, cancer and the degenerative effects of human aging. For example, defects in cell differentiation during embryo development result in human birth defects. Senescence and differentiation programs are both characterized by profound changes in chromatin structure, and, in both cases, this is thought to contribute to the altered cell phenotype. We are using senescence as a model system to study these changes in chromatin structure and their contribution to two hallmarks of both senescence and terminal differentiation, repression of proliferation-promoting genes and cell cycle exit. Recently, we showed that the chromatin regulatory protein, HIRA, and its physical binding partner, ASF1a, both play a key role in formation of a novel chromatin structure in senescent cells, called senescence associated heterochromatin foci (SAHF). SAHF is thought to silence genes that drive cell proliferation. HIRA and ASF1a drive SAHF formation, acting in concert with a subnuclear organelle, the PML body; and two chromatin associated proteins, HP1 and macroH2A. Preliminary data indicate that the HIRA/ASF1a pathway is activated by the key proliferation-regulating kinase, GSK3. To understand the physiological significance and molecular basis of SAHF formation and its mode of activation in presenescent cells, we will use cell and molecular biology techniques to: Specific Aim 1. Investigate the structure of SAHF, its mechanism of assembly by HIRA/ASF1a and PML nuclear bodies and identify its key growth suppressor components. Specific Aim 2. Investigate the function and mechanism of incorporation into SAHF of chromatin associated proteins, HP1 and macroH2A. Specific Aim 3. Investigate the role of GSK3 activity in localization of HIRA to PML bodies, formation of SAHF and onset of senescence.

Numerous clinically used agents contain the ester chemotype, a moiety frequently added to small molecules to improve their water solubility and bioavailability. However the inclusion of this function in these compounds makes them substrates for carboxylesterases (CEs), enzymes that can either inactivate or activate these agents. Typically examples include the anticancer agent CPT-11 (irinotecan, Camptosar) that is a prodrug of SN-38, a potent topoisomerase I poison, and the antiviral drug oseltamivir phosphate (Tamiflu) that requires hydrolysis to the carboxylate form to yield the active neuraminidase inhibitor. Hence, compounds that might inhibit the hydrolysis reactions would limit the efficacy of these drugs. We have identified a class of compounds (tanshinones) that are present within the Chinese herbal medicine Danshen. Extracts from this material can potently inhibit human CEs and modulate drug activity in vitro. Importantly however, the FDA has just approved the use of Danshen in clinical trials. Hence any esterified drug that is administered in conjunction with the herbal medicine might lead to reduced molecule hydrolysis, thereby mitigating the efficacy of the agent. We seek therefore, to evaluate the active component(s) in Danshen and to assess whether these molecules can modulate drug activity in defined animal models. The specific aims of this application are: 1) To determine the inhibitory compounds present within Danshen; 2) to assess the mechanism of enzyme inhibition by these compounds; 3) to assess the biological activity of these extracts in vitro; and 4) to determine the effect of such compounds/extracts on drug efficacy in animals models. We anticipate that compounds present within Danshen will inhibit the CEs in vivo, resulting in significantly reduced drug hydrolysis, and as a consequence, reduced drug efficacy. Since this material is currently in clinical trials, the information derived from these studies may identify novel drug:drug interactions that potentially would impact the effectiveness of clinically used esterified compounds. We envisage that the studies proposed here will validate this hypothesis and provide information concerning the use of such extracts in defined patient populations.

A marine/freshwater biomedical research and training core center is proposed. Its objectives will be to facilitate the development and use of aquatic animals as models of human disease processes of environmental significance. Principal efforts will be directed toward use of aquatic animals for mutagenesis and teratogenesis investigation with environmental pollutants such as hydrocarbons and metals as etiologic agents. The development of this center will foster training of environmental scientists in these interrelated fields.

Project Summary Postpartum cardiomyopathy (PPCM) is a disease of unknown etiology that arises as a complication of pregnancy in women with no prior heart disease. It occurs in 1:1800 to 1:3500 births in the United States and is characterized by an acute onset of heart failure during the last month of pregnancy or within five months postpartum. PPCM is a major cause of maternal morbidity and mortality with no PPCM-specific treatment options available. During pregnancy, pregnancy-associated hypertrophy initiates the activation of cardiomyocyte protective signaling pathways that block stress-mediated apoptosis. In PPCM patients there is an increase in cardiomyocyte apoptosis that leads to irreversible dysfunction and heart failure. The cellular mechanisms driving cardiomyocyte apoptosis are not fully understood. We have identified a gene PTRH2 (also called Bit-1) that is evolutionarily conserved, mediates integrin regulated cell survival and apoptosis, and mutations in this gene promote multisystem disease in humans. We hypothesize that PTRH2 is essential for cardioprotection from peripartum stresses in the maternal heart. To study this we developed a cardiomyocyte-specific deletion of PTRH2 (CKO). CKO male and never- pregnant female mice demonstrate no heart defects and live to old age. However, 100% of CKO pregnant female mice develop PPCM in a dose-dependent manner (CKO>HET). We will use cell and molecular biology and our CKO mice to determine how PTRH2 mediates cardioprotection, test whether PTRH2 associated proteins abrogate the PPCM phenotype in CKO pregnant mice, determine whether PTRH2 expression blocks hypertrophy and examine PPCM patient heart samples for PTRH2 gene mutations. The proposed project has the potential to identify an essential survival pathway that is activated in pregnancy-associated hypertrophy, test PTRH2 directed therapeutic strategies in a preclinical mouse model of PPCM and determine whether mutations in PTRH2 promote PPCM.

Neural crest cells (NCCs) are pluripotent cells that migrate from the developing neural tube to populate various tissues including craniofacial structures, neurons and glia of the peripheral nervous system, and pigment cells. Improper migration and development of NCCs can lead to a variety of birth defects collectively termed neurocristopathies. To become migratory, NCCs undergo epithelial to mesenchymal transition (EMT). EMTs at the wrong place and time are associated with cancer progression, invasion, and metastasis among other pathological events. Thus, it is critically important to have a complete understanding of the biology of EMT. While some work has focused on identifying signals that induce EMT, much of it was done in cells outside of their natural environment, which has a great effect on cell signaling and behavior. I have focused on the physical behaviors NCCs use to carry out EMT in vivo and this proposal will test how specific molecules, namely the GTPase Rho and Cadherin-6, control these behaviors. My specific aims are to 1.) Image the distribution and level of active Rho during NCC EMT 2.) Determine the effects of Rho manipulation on dynamic cell behavior and F-actin. 3.) Determine whether Rho and Cad-6 cooperate to promote NCC EMT. These experiments will begin to define molecular pathways that control EMT in vivo and have the potential inform therapies for treatment of pathologies involving abnormal cell migration and EMT. PUBLIC HEALTH RELEVANCE: Improper development of neural crest cell (NCC) derived structures, including craniofacial bone and cartilage, leads to a class of birth defects called neurocristopathies. To populate their targets NCCs must become migratory, which involves undergoing epithelial to mesenchymal transition (EMT). EMTs are important events in development that also drive pathologies such as fibrosis, chronic inflammation, and cancer metastasis. The experiments proposed here have the potential to explain how specific molecules control these critical events and inform therapies in diseases involving EMT and abnormal cell migration.

Massive immune hemolysis due to minor ABO incompatibility is an underappreciated, potentially fatal complication of allogeneic hematopoietic transplantation. The increased lymphoid content and rapid engraftment seen with peripheral blood stem cell (PBSC) transplants may increase the frequency and severity of this event. In addition, nonmyeloablative conditioning regimens favor rapid and vigorous donor-type immune reconstitution, relying on donor lymphocytes to mediate both an anti-tumor effect and durable myeloid engraftment. To further the graft versus tumor effect, antiproliferative agents such as methotrexate are frequently omitted from posttransplant anti-GVHD regimens. We observed abrupt, catastrophic hemolysis in the first NIH patient to receive a nonmyeloablative PBSC transplant involving minor ABO incompatibility. We established a protocol for close clinical and laboratory monitoring of the next nine consecutive minor ABO-incompatible, nonmyeloablative PBSC transplants performed on NHLBI and NCI services. Cyclosporine alone was employed to prevent GVHD in all nine cases. Two additional cases of massive immune hemolysis were detected. Hemolysis began 7 to 11 days following stem cell infusion. Both cases responded rapidly to vigorous hydration and prompt donor-compatible red cell transfusions, without adverse clinical consequences. All patients with hemolysis demonstrated a positive direct antiglobulin test (DAT), with eluate reactivity against the relevant recipient blood group (anti-A in two cases, anti-B in one). However, neither the intensity of the DAT nor the donor isohemagglutinin titer distinguished cases with from those without hemolysis. These results demonstrate that isohemagglutinins produced by donor passenger B lymphocytes in minor ABO incompatible, PBSC transplants utilizing cyclosporine alone for GVHD prophylaxis can mediate massive immune hemolysis in a considerable proportion of subjects at risk. In view of this high risk, anti-GVHD regimens in NHLBI protocols were changed to include mycophenolate mofetil (MMF), an antiproliferative agent. None of the next 10 consecutive minor ABO incompatible nonmyeloablative stem cell transplants was accompanied by significant immune hemolysis, although serologic abnormalities were seen. GVHD regimens continue to be modified to maximize graft anti-tumor immune effects while minimizing other immune complications of transplant, and MMF doses are being reduced in an effort to increase complete remission rates posttransplant. We continue to monitor daily blood counts and red cell serologic studies (DAT, IAT) during the period at risk (day 6 to 11 posttransplant) and to promptly administer donor-compatible red cell transfusions in these cases. Improved awareness can avert serious complications due to minor ABO incompatibility following stem cell transplant and should be practiced in all such cases.

The effect of inhibiting the parasympathetic nervous system on insulin, glucagon, and glucose will be examined in normal weight and obese men and women. Furthermore, the importance of early insulin release will be examined. Each subject will undergo 4 treatments: 1) saline infusion, 2) brief infusion 3) atropine infusion and 4) atropine and insulin infusion. During each of the treatments, subjects will ingest a mixed meal containing 600 kcal and will undergo a blood sampling protocol in which arterialized venous blood samples will be drawn over a 4 hour period of time.

Project Summary/Abstract. Group A streptococci (GAS; Streptococcus pyogenes) are remarkable for the wide range of diseases they cause in humans, their sole biological host. Yet, most infections are mild and involve one of two tissues - the epithelial surface of the throat or skin - giving rise to pharyngitis or impetigo, respectively. A long-term goal is to better understand the distinct pathogenic mechanisms leading to pharyngitis and impetigo. A primary focus of the proposal is the regulation of pili expression in GAS. Pilus-associated proteins mediate adherence to epithelial cells and enhance superficial infection at the skin. Pili correspond to the T-antigens of GAS. All strains examined have pilus genes, however, many natural GAS isolates lack T-antigen. The hypothesis to be tested in Aim 1 states that organisms recovered from a carrier state and/or invasive disease are significantly more likely to have defects in pilus production, as compared to isolates derived from cases of pharyngitis or impetigo. Aim 1 seeks to define the relationship between defects in pilus expression and disease. The nra/rofA locus encodes a [unreadable]stand alone[unreadable] response regulator that affects the transcription of pilus genes; nra and rofA denote discrete lineages of alleles. Both Nra and RofA can have positive or negative regulatory effects on pilus gene transcription, depending on the GAS isolate or strain. The hypothesis to be tested in Aim 2 states that there are strain-specific differences among modulators of pilus gene expression that lie in a pathway upstream of Nra/RofA. Aim 2 seeks to identify regulators of pilus gene transcription having a differential presence among strains. The distribution of Nra and RofA among GAS is strongly correlated with subpopulations of strains having a tendency to cause infection at either the throat or skin. Nra and RofA are global regulators of GAS gene transcription. Two hypotheses will be addressed in Aim 3: (i), that co-regulated non-pilus genes act in concert with pili to cause disease; and (ii), that Nra and RofA confer differential transcription of downstream genes. Aim 3 seeks to identify genes of the Nra and RofA regulons, and to test their role in virulence. Through a better understanding of the molecular mechanisms used by GAS to persist in their primary ecological niches - the throat and skin of the human host - will come new knowledge on how best to interfere with these vital processes. Effective control and prevention measures that disrupt the chain of transmission of GAS will result in a decreased burden of the more severe GAS diseases (toxic shock syndrome, rheumatic heart disease) which have a high morbidity and mortality for many people throughout the world.

Our main objective is to characterize the immunological properties and molecular nature of inappropriate antigens detected on SJL/J reticulum cell sarcoma (RCS) and to define the role of these antigens in tumorogenesis. We plan to demonstrate the presence of inappropriate alloantigens on spontaneous, transplantable and cultured RCS cells by cell mediated and complement cytotoxicity, and by immunofluorescence. Furthermore, biochemical analysis of inappropriate antigens will be examined by immune precipitation of NP40 lysed 35S methionine labeled tumor cells with specific alloantisera and characterization of the molecules by SDS gel electrophoresis. The role of inappropriate antigens in host stimulation will be investigated by examining the in vivo response to inappropriate alloantigenic specificities in both cellular and antibody mediated assays. The mechanism by which the immune response promotes tumor escape from immune destruction will be investigated including the role of antigen-antibody complexes in specific anti-tumor-antigen-reactive cell opsonization, modulation of tumor associated antigens by circulating antibody, direct tumor mediated suppression of antigen-reactive cells and activation of suppressor cells.

Focal Adhesion Kinase (FAK) is a 125 kDa tyrosine kinase that is localized to contact points between cells and their extracellular matrix. We have demonstrated that FAK overexpression and upregulation occurred in early stages of tumorigenesis. We have also shown that FAK overexpression suppresses apoptosis, thus providing a survival signal to human cancer cells. Furthermore, our data has recently shown that the amino-terminus of FAK can induce apoptosis in breast cancer cells and can bind to a death domain containing serine-threonine kinase, Receptor Interacting Protein, (RIP). In addition, we have identified peptides that bind to the carboxy-terminus of FAK and cause apoptosis. 1 of these peptides contained a sequence homologous to the vascular endothelial growth receptor 3 (VEGFR-3) protein that we have shown to bind FAK. This proposal focuses on the biology of FAK in the development of cancer and on the biological mechanism by which attenuation of amino-terminal and carboxy-terminal FAK signaling causes tumor cell apoptosis. The first specific aim is to define the critical components of the amino terminus of FAK (FAKNT) that are responsible for its pro-apoptotic properties. We will define the portion(s) of FAK-NT that induce loss of adhesion in breast cancer cells and bind to RIP. Next, we will perform functional analyses of different peptides that bind the carboxy-terminus of FAK (FAK-CD), inhibit FAK function, and induce apoptosis in breast cancer cells. We will define their biological effects in different tumor cell lines to determine if they can induce apoptosis, perform initial binding studies using NMR spectroscopy, and perform site directed mutagenesis to determine which amino acids are critical for interaction with FAK-CD. Finally, we will define the physical interaction between FAK and VEGFR-3, determine the mechanism of survival signaling mediated by this interaction, and examine the relationship between levels of expression of FAK and VEGFR-3 in translational studies of primary human breast cancers. By defining the mechanism of interaction between FAK and its critical binding partners, we will identify novel sites for molecular targeting to induce apoptosis in human breast cancer cells.

Body energy metabolism is a finely tuned system dependent on the interactions of numerous endocrine axes. Insulin is a key regulator of metabolism and growth that maintains serum glucose and other metabolites within a narrow, predefined range. While it has been traditionally thought that insulin activity is controlled solel by its abundance in circulation, modulation of tissue insulin sensitivity has been described during [instances of environmental stress (e.g. infection/inflammation and pregnancy). It is well known that inflammation can induce insulin resistance in multiple tissues and that this response may be important for the body's response to infection.] Developing an understanding of how [inflammation regulates] tissue insulin responsiveness is important as it could provide novel insights into [pathologies] associated with tissue insulin resistance, such as metabolic syndrome and Type 2 Diabetes Mellitus. Several groups have shown that inflammation can induce insulin resistance through [direct] post-translational modification of components of the insulin-signaling cascade [by inflammation activated kinases]. The existing model suggests that resistance should develop relatively rapidly in response to inflammation, consistent with the kinetics of post-translational modifications. Paradoxically, the observed resistance to insulin requires prolonged treatment with inflammatory cytokines, suggesting that inflammation may [induce a transcriptional program that results in insulin resistance.] The goal of my Ph.D. thesis, and this proposal, is to investigate the mechanisms of [chronic inflammation induced] insulin resistance. Based upon the observed kinetics and our preliminary data, we hypothesize that chronic inflammation induces a [transcriptional program that leads to impaired tissue responsiveness to insulin. Specifically, we hypothesize that TNF? induces/suppresses previously uncharacterized protein(s) that regulate insulin signaling.] Since the liver coordinates whole body metabolism and is known to change its responsiveness to insulin signaling under inflammatory conditions, we propose to investigate the role of chronic inflammation in regulating hepatic insulin signaling using [in vivo and ex vivo models, specifically focusing on the effect of chronic TNF? treatment]. In the first aim, we will investigate the effects of [ex vivo and in vivo] chronic TNF? on [hepatocyte insulin-signaling and functional response including gluconeogenesis, glycogen synthesis, and lip genesis]. The second aim will address the [role of differential gene expression] induced by chronic inflammation and identify the [gene(s) and mechanism(s)] responsible for altering insulin signaling. Our studies represent a novel approach to understanding the mechanisms that regulate insulin signaling. Ultimately, this may lead to the development of novel therapeutic targets for metabolic syndrome and Type 2 Diabetes Mellitus. PUBLIC HEALTH RELEVANCE: Altered insulin signaling in the liver is a key component of many chronic diseases affecting the population, including metabolic syndrome and type 2 diabetes. Inflammation represents a common, reversible, non- disease state that is associated with altered hepatic insulin signaling. Understanding inflammatory regulation of insulin signaling will provide insight into the mechanisms regulating insulin signaling, inform the treatment of diseases that result from altered insulin signaling, as well as identify potentially novel therapeutic targets.

The purpose of this contract is to provide support for a colony of rhesus macaques used in aging and diabetes research. The colony consists of both pre-diabetic and diabetic monkeys from the ages of 7 years to over 30 years. The colony is characterized extensively for parameters of aging and metabolism. These animals are made available to investigators outside of the University of Maryland through collaboration with or request to the P.I. Tissue and blood samples are also available through ODAAR's extensive tissue bank and prospective sampling.

Previous work on muscarinic subsensitivity of the ciliary muscle in monkeys is continued. Previous work on axoplasmic flow against pressure gradients will be extended to the optic nerve in vitro. Changes in the carbonic anhydrase content in eyes and other organs of animals chronically treated with carbonic anhydrase inhibitors will be studied. Attempts will be made to develop a technique for measurement of aqueous humor flow in conscious monkeys.

We propose to create the Stanford PharmacoGenetic Knowledge Base (PharmGKB), an integrated data resource to support the NIGMS Pharmacogenetic Research Network and Database Initiative. This initiative will focus on how genetic variation contributes to variation in the response to drugs, and will produce data from a wide range of sources. The PharmGKB will therefore interlink genomic, molecular, cellular and clinical information about gene systems important for modulating drug responses. The PharmGKB is based on a powerful hierarchical data representation system that allows the data model to change as new knowledge is learned, while ensuring the security and stability of the data with a relational database foundation. Our proposal defines an interactive process for defining a data model, creating automated systems for data submission, integrating the PharmGKB with other biological and clinical data resources, and creating a robust interface to the data and to the associated analytic tools. Finally, we outline a research plan that uses the PharmGKB to (1) address difficult data modeling challenges that arise in the course of building the resource, (2) study the user interface requirements of a database with such a wide range of information sources, and (3) model and analyze the structural variations of proteins to shed light on the molecular consequences of genetic variation. The PharmGKB will respect the absolute confidentiality of genetic information from individuals.

The ultimate objectives are (1) to place our understanding of the mode of action of the peptide antibiotics and hormones on a conformational basis and (2) to develop the spectroscopic approach to the study of conformation and conformational averaging in peptides. The experimental approach involves the combined use of (a) solid phase peptide synthesis (b) correlation, double resonance, and difference NMR spectroscopy (c) fluorescence and circular dichroism (d) thin film and equilibrium dialysis and (e) model building. In addition, we intend to investigate the interaction of the peptide antibiotics and hormones with a view to understanding (a) the sites for detergent and fluorescent probe binding (b) the rates and mechanism of cleavage by crystallographically defined proteases and (c) the structure and thermodynamics of peptide micelle formation. The availability of conformationally characterized peptides lends those studies uniqueness; they also have considerable importance for control and regulation of hormone levels--hormone homeostasis.

This renewal proposal continues our exploration of the vasa vasorum (the microvessels perfusing the arterial wall) in coronary artery disease. The long-term objective is to clarify the role of solute transport across the arterial wall in the initiation and/or progression of atherosclerosis. The relevance of this knowledge to the mission of NHLBI is that it should provide insight into possible early treatments to arrest or reverse arthrosclerosis and thereby reduce major public health problems resulting from advanced atherosclerosis causing coronary artery stenosis and myocardial infarction. This proposal is designed to test the overall hypothesis that the vasa vasorum can play an important role in the initiation of coronary atherogenesis by virtue of disturbed solute transport within the arterial wall occurring prior to cellular invasion and/or proliferation. So far the question of mere association of changes in vasa vasorum (W) with early atherogenesis has already been answered in the affirmative by our studies. What remains to be determined is to what extent the W actually play a primary or facilitating role in the initiation of the artherosclerotic process or merely respond to that process. Our approach is based primarily on use of micro-CT-based imaging methods including our novel, in-house developed, cryostatic micro-CT method and also by in-vitro characterization of isolated W contractile state. This will involve analysis of coronary arteries of pigs with diet-induced hypercholesterolemia or renal artery stenosis (stent-induced) arterial hypertension, both being risk factors for atherogenesis. AIM I: Explore individual W trees' perfusion territories', function in terms of location, shape, size and washin/washout characteristics. The 3D geometry and solute transport localized function of these territories will form the basis for understanding of lipid accumulation in the arterial wall. AIM II: Explore the role of endothelial permeability in the W and coronary artery using cryostatic micro-CT scans. Use micro embolism to cause acute occlusion of single W near their origin to evaluate the ability of W contiguous in perfusion territories, to compensate for the localized loss of perfusion. AIM III: Following chronic recovery from micro-embolization of some of the W, the relationship between location and size of coronary arterial wall perfusion-voids on the one hand and histologically localized 'lipid1 deposits on the other will be a direct test of the pathological consequences of alterations in solute transport characteristics of W. The specific significance of this proposal is that if W play an important role in the initiation or progression of atherosclerosis, a possible therapeutic benefit that may result from acquiring this new information is that either stimulation (or inhibition; depending on our findings) of vasculogenesis of W could retard the onset and/or severity of atherosclerosis.

The goal of this application is to develop a research network for "Harmonization of cross-national studies of aging to the Health and Retirement Study (HRS)." This network will support the development of new international studies with harmonized data, maintain harmonization and data development among existing comparable studies, and develop a cross-national research agenda. Harmonization of cross-national studies of aging to the HRS is one of the specific targeted emerging research areas identified under the PAR-09-233 Network Infrastructure Support for Emerging Behavioral and Social Research Areas in Aging. To foster cross-national studies of aging, we propose to develop a research network, bringing together leading scientists, data producers, and users ofthe HRS family of surveys. Through the proposed network support activities, we will accomplish the following five aims: (Aim 1) Organize an annual harmonization meeting, bringing together the principal investigators of all HRS-family surveys. The purpose of these meetings will be to maintain and improve harmonization among existing HRS-family surveys and to support development of new HRS-like surveys. (Aim 2) Document the comparability, prepare harmonized measures, and provide training on the HRS family of surveys. Using the HRS User Guides and the RAND HRS documentation as a model, we will prepare documentation about the concepts, measures, and questions in each HRS-family survey as well as on their comparability in seven specific domains (e.g,, labor force participation and retirement, chronic diseases). (Aim 3) Support small-scale pilots for establishing cross-nafional comparability. (Aim 4) Create network opportunities for users of the HRS family of surveys through biennial user conferences and the Internet discussion board, and provide technical support for users. (Aim 5) Disseminate the resources developed for harmonization (e.g., user manuals, conference proceedings) and the findings from cross national studies using the HRS family of surveys through public websites including those of RAND and each of the surveys. RELEVANCE (See instructions): The proposed research network activities will facilitate harmonization of the HRS family of surveys and cross-national studies on aging, health, and retirement. These network activities will provide immediate benefits for the producers and users of the HRS family of surveys and broader benefits for the entire research community.

Benign Childhood Epilepsy with CentroTemporal Spikes (BECTS), an extremely common type of childhood epilepsy, is traditionally assumed to have a benign course, but recent studies have shown that cognitive function, especially language, is often impaired in BECTS patients. However, it is not clear whether the seizures, the centrotemporal spikes (CTS), or other factors cause the negative cognitive consequences that may impact school performance and social interaction. BECTS patients have scattered seizures but very frequent CTS, and may be suffering with undiagnosed cognitive and language deficits. This suggests a causal role for CTS that has not yet been investigated in detail. This project will examine the impact of seizures and CTS on neurocognitive function in BECTS patients, at diagnosis and after one year. We will gather critical information regarding the effect of the antiepileptic medication levetiracetam on CTS, which will inform a future Phase III clinical trial aimed at eliminating CTS and improving long term outcome. This study will explore the interactions between CTS, seizures and neuropsychological outcomes using Functional MRI of language in order to decipher changes in neural circuitry that underlie language deficits found in children with BECTS. Using standardized neuropsychological testing and fMRI at the time of diagnosis, this study will first characterize the nature and incidence of language problems in children with BECTS, separating the effects of CTS and seizures. It is expected that children with BECTS will perform below normative standards on tests of language skill, accompanied by aberrations in the neural circuitry supporting language processing as tested with fMRI. These data will also make it possible to characterize which children with BECTS are most at risk for language problems, by taking into account contributing factors such as number of seizures, age of onset, and frequency and lateralization of CTS. The proposed exploratory clinical trial will also provide key information needed to properly design and conduct a future double blind Phase III randomized clinical trial (RCT) children aimed at improving language outcome through elimination of CTS. Using an open-label dose-ranging selection design and 1-year follow up, we will determine which dose of levetiracetam control seizures, eliminate CTS is well tolerated and should be used in the Phase III trial. We will also examine the extent of changes in language function and neural circuitry of language with 1-year follow-up neuropsychological testing and fMRI in LEV-treated and untreated BECTS compared to controls (which will document the natural history of neuropsychological function in untreated BECTS children and give additional information about the effect of levetiracetam). The future double blind RCT will compare levetiracetam to carbamazepine (the current standard of care that does not eliminate CTS) in BECTS. This future study would change clinical practice by demonstrating the need for AED treatment in all BECTS children to eliminate CTS, in turn improving long term language and cognitive outcome. PUBLIC HEALTH RELEVANCE: This project examines how seizures, and abnormal brain activity, affect language skill in children with Benign Childhood Epilepsy with Centro-Temporal Spikes (BECTS). BECTS is a common type of childhood epilepsy, and while BECTS patients stop having seizures by their late teenage years, many studies have shown that these children have language problems that may lead to academic and social difficulties. Using standardized language testing, monitoring of brain activity, and MRI brain imaging, this project aims to determine what particular combination of BECTS symptoms put children most at risk for language problems and what dose of the anti-epileptic medication levetiracetam may be helpful. Disclaimer: Please note that the following critiques were prepared by the reviewers prior to the Study Section meeting and are provided in an essentially unedited form. While there is opportunity for the reviewers to update or revise their written evaluation, based upon the group's discussion, there is no guarantee that individual critiques have been updated subsequent to the discussion at the meeting. Therefore, the critiques may not fully reflect the final opinions of the individual reviewers at the close of group discussion or the final majority opinion of the group. Thus the Resume and Summary of Discussion is the final word on what the reviewers actually considered critical at the meeting.

Project Summary - Ventricular arrhythmias remain the single most important cause of sudden cardiac death (SCO) among adults living in industrialized nations. Great progress has been made in identifying genes underlying various Mendelian disorders associated with inherited arrhythmia susceptibility as models for understanding more common causes of SCO. The best studied familial arrhythmia syndrome is the congenital long QT syndrome (LQTS). The observation that not all mutation carriers have equal risk for experiencing the clinical manifestations of LQTS (i.e., syncope, sudden death) has motivated the hypothesis that genetic factors other than the primary disease-associated mutation can modify the risk for disease- related morbidity and mortality. This proposal is the first competing renewal of R01-HL68880 which has funded a multi-national translational research collaboration to identify and characterize clinical predictors and candidate genetic modifiers in a large, unique LQTS founder population in South Africa (SA-LQTS). We have hypothesized the existence of two types of modifier genes: genes which affect myocardial repolarization and produce an arrhythmia-prone substrate, and genes which affect the propensity for triggering events acting through the autonomic nervous system. Specific Aim 1 describes our ongoing efforts to collect detailed information regarding the phenotype of the SA-LQTS population. We plan to continue our current focus on major clinical outcomes but will also explore more deeply for intermediate phenotypes related to abnormal myocardial repolarization and autonomic tone. For independent testing of candidate modifier gene hypotheses, we will also use a second large LQTS founder population ascertained in Finland (Fin-LQTS; 80 families, > 600 mutation carriers) that is associated with a different KCNQ1 mutation. The addition of this second LQTS founder population coupled with an important new collaboration with genetic epidemiologists at Columbia University will ensure that our observations are valid and have broad implications. In Specific Aim 2, we will examine the molecular mechanisms responsible for genetic and autonomic influences on the major repolarizing myocardial ionic currents. Finally, in Specific Aim 3 we will study genetic and epigenetic mechanisms to explain variation in the transcription of KCNQ1 and other genes that may act to modify the clinical expression of LQTS. The goals of this study are consistent with the mission of NHLBI. Relevance to Public Health - Identification of long QT syndrome (LQTS) modifiers will enhance our understanding of the pathophysiology of an inherited cause of sudden cardiac death (SCO), provide valuable new information to promote more accurate risk counseling for LQTS families, and will contribute to our understanding of more common arrhythmia syndromes associated with highly prevalent cardiac diseases (e.g. ischemic heart disease and congestive heart failure) that are burdened by a high incidence of SCO. [unreadable] [unreadable] [unreadable]

The potential value of HIV-infected human peripheral blood mononuclear cells (PMB) cultured in vitro with phytohemagglutinin (PHA) in the anti-HIV evaluation of nucleoside analogs was examined. 2',3'-Dideoxy-3'-azidothymidine (AZT) exhibited 100-fold higher potency against HIV replication than did 2',3'-dideoxyinosine (ddI) in this system, an observation which does not readily correlate with clinical observations. Studies on metabolism showed that after 12 hr exposure to PHA, cellular AZT was preferentially phosphorylated to its mono-, di-, and triphosphates with 110, 30, and 40-fold increase of the pool sizes, respectively, whereas 2',3'-dideoxycytidine was only moderately phosphorylated, and ddl as well as 2',3'-dideoxyadenosine was poorly phosphorylated. Thymidine kinase activity was found to be the most sensitive to induction among all the cellular kinases regarding stimulation by PHA, and to be responsible for the extreme potency of AZT in this system. Sensitivity of cellular kinases to PHA activation was in the order: thymidine kinase is greater than uridine kinase is greater than deoxycytidine kinase is greater than adenosine kinase. The ratio of ddATP, the active form of ddI, vs. dATP was more than 10-fold higher in resting PBM cells than activated cells. Since the majority of lymphocytes in vivo are in the resting stage and HIV-1 proviral DNA synthesis can be efficiently initiated in resting cells, ddl may exert a disproportionate effect on resting cells rather than activated cells. These data suggest that PHA-stimulated PBM should not be used alone in the screening and evaluation of nucleoside analogues for possible use in the treatment of HIV infections.

US biomedical and health services researchers are increasingly asked to adopt a more pronounced patient- centered orientation in their work. Such a shift requires a more active engagement of the public (e.g., community representatives) in the research process. As we know from decades of experience with Community Based Participatory Research, active community participation in healthcare research has the potential to make science more transparent; improve the relevance of interventions to patients' needs; enhance the level of cultural sensitivity of healthcare research; boost public engagement in, and use of, research; and facilitate policy efforts to increase healthcare quality and reduce health disparities. At the same time, active research engagement of new stakeholders calls for revisiting some aspects of the scientific process that may traditionally have been just assumed among academics. The meaning and practice of research integrity (RI) is one such topic that is worth revisiting. In the proposed project, we argue that the increasing prominence and promise of partnered work can inform the development of a more thorough understanding of RI by exploring (1) how academic and community research partners conceptualize and maintain RI and (2) how partnered processes might support or undermine RI in a range of research approaches that aim to be patient-centered. First, we will gather a wide range of academic and community perspectives on RI in partnered research to broaden our understanding of RI, identify threats to RI, and explore strategies used to overcome them. We will do so by conducting semi-structured interviews with community and academic partners, asking them to delineate specific strategies that support the RI in partnered work, such as those used to ensure against bias, maintain trust and transparency, safeguard data security, and guarantee subject autonomy. Second, we will explore the extent to which the lessons learned about RI in partnered research, including dimensions of RI, threats to RI, and strategies to overcome them, are relevant and applicable to a range of scientific models, such as bench, translational, comparative effectiveness, and implementation research. We will use an innovative online iterative panel process called ExpertLens to catalogue the viewpoints of a diverse group of researchers and community research leaders working in these scientific fields. We will use these results to generate a set of preliminary guidelines for maintaining RI in research that strives to engage diverse stakeholders. The findings will have high significance because they will improve our understanding of the features of the internal and external environment that can support or undermine responsible research behavior in community-engaged research, and because they will generate recommendations for conducting patient- centered outcomes research with high degree of RI. Results will also form the basis of an R01 application that will empirically assess the impact of partnering on RI across a broad range of scientific projects.

Summary/Abstract (from Parent R01AA025848) A recent review of trajectory research found that there is a great deal of research aimed at better understanding transitions in alcohol and other drug (AOD) use patterns from early to late adolescence and late adolescence to emerging adulthood. However, no studies to date have: a) 13 assessments of AOD use from age 10-24 across all developmental periods (middle school, high school, and emerging adulthood); b) a large sample with substantial racial and ethnic diversity, particularly among Hispanic and Asian youth; c) in-depth coverage of 10 areas of functioning across three key domains; d) subjective and objective neighborhood data; and e) the capacity to examine developmental trajectories for more than one substance. The current proposal continues R01AA016577 and R01AA020883 (PI: D?Amico), which together have assessed AOD use across nine waves of data from age 10 to age 19. The proposed study capitalizes on the longitudinal data on protective and risk factors we have collected since age 10 in a diverse cohort (44% Hispanic, 21% Asian, 21% white; 2% Black, 12% multiethnic/other; 54% female) by continuing to annually assess these youth in order to capture important transitions to emerging adulthood. Our specific aims are to: Aim 1a: Identify trajectories of alcohol, tobacco, marijuana, and other drug use from age 10 into emerging adulthood. Assess differences by race/ethnicity. Aim 1b: Examine individual (e.g., resistance self-efficacy), peer (e.g., peer use) and family (e.g., parent disapproval of use) factors that may impact these trajectories. Test for similarities/differences in these associations across racial/ethnic groups. Aim 2a: Merge the survey data with the largest existing database of neighborhood measures to examine effects of neighborhood characteristics (economic, social, demographic, alcohol and marijuana outlet density) on AOD trajectories. Aim 2b: Test for similarities/differences in these associations across racial/ethnic groups. Aim 3a: Examine how AOD use trajectories during middle and high school predict outcomes through age 24 in three core domains: 1) risk behaviors (e.g., AOD use, risky sexual behavior, delinquency), 2) health related quality of life (e.g., mental, physical, social), and 3) adult role functioning and transitions (e.g., education, relationships, life satisfaction). Aim 3b: Test for similarities/differences in these associations across racial/ethnic groups. Aim 4a: Using new data gathered annually from ages 20-24, examine how adult role functioning and transitions longitudinally predict subsequent changes in AOD use and other risk behaviors, as well as health-related quality of life, in emerging adulthood. Aim 4b: Test for similarities/differences in these associations across racial/ethnic groups. By advancing the epidemiology of alcohol use during adolescence and emerging adulthood, our findings can impact prevention and intervention programming for young people and address public health policy. 1

Procedures such as exercise testing, pulmonary compliance, and work of breathing have been found successful for evaluating pulmonary function. By exercising a patient on a treadmill and gradually increasing the workload (i.e., speed and incline), the physician can better assess cardio-pulmonary disease, which in its early stages generally does not manifest itself except under physical exertion. In order to help the physician perform these procedures more effectively, a microcomputer system has been developed to enable automated real-time collection, analysis and display of pulmonary compliance data. Work is in progress to complete an automated exercise procedure as well. Data is stored in a local disk data base for future reference.

Anterior cruciate ligament (ACL) injury creates considerable disability and predisposes the knee to the early onset of osteoarthritis. This is a concern because the past two decades has seen an increased participation of women in sports and with this has come a concomitant increase in ACL injuries. The incidence rate of ACL tears is 2 to 9 times greater amongst females in comparison to males; however, there is limited information with regard to the demographic, injury history, hormonal history, anatomic, neuromuscular, and environmental variables that individually or collectively contribute to the risk of suffering an ACL injury. Most of what is known has come from risk factor studies that have focused on selected variables in isolation (e.g. femoral intra-condylar notch width, A-P knee laxity, or phase of menstrual cycle) and consequently it is not surprising that a comprehensive ACL injury risk model has not been established. Our prior research has revealed that the risk model for ACL injury is probably different for females compared to males and this serves as the rational for our investigation, the objective of which is to find the unique combination of risk factors that identify females and males at increased risk of suffering an ACL injury. The hypothesis of our study is: A combination of demographic, injury history, hormonal history, anatomic, neuromuscular, and environmental variables can be used to identify females at increased risk of non-contact ACL trauma, and that a separate combination of these variables can be used to identify males at increased risk of ACL trauma. Initially, we will establish that examiners can measure the potential risk factors in a reliable manner. We will assemble a cohort of collegiate and pre-collegiate athletes and follow them to prospectively accrue ACL injury cases. We will then perform a nested case-control study to identify risk factors for ACL injury. Three controls for each injury case will selected at the time of injury from among teammates who have not been injured at or prior to that time. For subjects that suffer an ACL tear, the potential risk factors that are not modified by the injury will be measured from the injured limb while the factors that are modified by the injury will be measured from the contra-lateral, normal, limb and used to represent the pre-injury condition of the ACL-deficient limb. The same measurements will be obtained on the matched controls. This investigation is significant because it will determine the putative risk factors that identify females and males at increased risk for ACL injury and allow future intervention studies that reduce the incidence of this debilitating injury.

Tissue specific gene expression is central to the development of complex eukaryotes. This project addresses the long term goal of understanding the molecular mechanisms regulating the tissue-specific expression of the zein family of maize seed storage protein genes. Molecular and genetic analyses have shown that the developmental and tissue-specific expression of zeins is dependent on the trans-acting regulatory locus, opaque-2 (o2). Our research has demonstrated that the product of this locus is a regulatory protein belonging to the basic leucine zipper (bZIP) class of eukaryotic transcription factors. We have shown that O2 affects the expression of only certain members of this gene family through recognition of a specific binding site (the O2 box) in promoters of 22-kD zein genes. From characterizations of mutant o2 alleles, analyses of DN4 binding properties, and assays of transcriptional activation in yeast and maize cells, it appears that the capacity of O2 to bind to specific zein promoters in vivo is influenced by interactions with other endosperm proteins. As part of our efforts to understand how these proteins may affect the activity of O2, we have cloned the cDNAs encoding two proteins that, like O2, bind the O2 box in a sequence specific manner. One of these encodes a bZIP protein that forms DNA-binding heterodimers with O2. We intend to characterize these proteins as to their possible interaction with O2 in regulating zein gene expression. In addition, the question will be addressed as to what regulates the expression and activity of O2 in maize endosperm. We have determined that O2 is multiply phosphorylated in vivo, with phosphorylation affecting DNA binding. We will determine if this phosphorylation is influenced by the carbon-nitrogen balance or by availability of nitrogen or sulfur. This study may establish an important link between nutrient availability and storage protein gene activation. These studies will also include an analysis of O2 promoter activity in transgenic plants to identify regulatory elements important to the developmental expression of O2. Finally, new mutant alleles of O2 will be generated by transposon mediated mutagenesis to identify domains in this regulatory protein. The knowledge gained from these studies will not only contribute to our basic understanding of the mechanisms underlying seed storage protein gene expression, but should also provide insight into the general mechanism by which eukaryotes control the process of tissue specific gene expression.

The purpose of this proposal is to elucidate the mechanism through which the reaction of antigen with cell-bound IgE antibody leads to the release of chemical mediators from mast cells. Research plan is based on our recent findings which indicated that bridging of IgE-receptors by divalent anti-receptor antibody stimulated phospholipid methylation at the membrane, and that this process set a stage for opening Ca ion-channels and subsequent histamine release. We shall study 1) as to whether the phospholipid methylation is controlled by intracellular cyclic AMP level, and 2) whether methyltransferases are the first enzymes to be activated by receptor bridging. 3) Possible role of methyltransferases in the process of "desensitization" will be studied. Another question to be asked is how phospholipid methylation leads to opening of Ca-channels. 4) Considering possible role of phospholipase A2 in the process of mediator release, we shall investigate pharmacological effect of glucocorticoid in the biochemical process.

Herpesviruses are important pathogens of both humans and animals, and information on the molecular aspects of herpesvirus replication is useful in the diagnosis and treatment of these infections. Overall goals are to employ our models of equine herpesvirus 1 (EHV) cytocidal and persistent infection to understand herpesvirus gene regulation in terms of structure/function relationships of the viral regulatory proteins and to ascertain whether EHV regulatory polypeptides interact with cellular proteins to mediate the varied outcomes of infection. Aims for years #39 to #43 focus on the functions of EHV regulatory proteins with emphasis on the immediate-early protein (IEP): To characterize the transactivation domain (TAD) with aa#3-89 of the EHV IEP by assessing mutant forms of the TAD both as GAL4- fusion constructs and in the contest of the virus for transactivation ability. To use our panels of GST-EHV fusion proteins to identify the domains of EHV IEP, ICP22, ICP27 and ICP0 regulatory proteins that mediate their protein-protein interactions (by protein crosslinking assays) and enhance IEP binding to specific sequences within EHV regulatory proteins by the approaches of the two hybrid system, coimmunoprecipitation assays, and affinity chromatography. Mutant constructs and EHV-1 mutants that express specific domains of the auxiliary regulatory proteins, especially ICP22, will be used in transient transfection assays and experiments to monitor EHV gene programming, respectively, to define the roles of these proteins in EHV replication. Lastly, focused efforts will address the functions of the ICP22-ICP27 hybrid protein (HYB.) encoded by EHV DI particles (DIP) that mediate persistent infection. Since the HYB. retards expression of specific EHV promoters in initial transfection assays, our constructs, cell lines, and recombinant virus that express the HYB. will be employed to define viral promoters affected by the HYB. and ascertain whether HAB.. expression by a recombinant EHV alters viral gene programming, possibly mediates persistent infection, and is essential for DIP to establish persistent infection. If warranted by these data, the interaction of the HAB.. with EHV regulatory proteins and cellular factors will investigated by the approaches described above.

Pseudomonas aeruginosa (PA) is an opportunistic gram-negative pathogen responsible for a large number of nosocomial infections. Dr. Joanne Engel and her group have shown that PA103 induces apoptotic-like cell death in epithelial cells and macrophages by a Type III Secretion System (TTSS)-dependent mechanism. Consistent with the results of others, They have found this to be a Fas ligand- and Fas receptor-dependent process. We propose that PA-induced apoptosis occurs either by (A) direct translocation of a bacterial effector into the host cell (PAF, for Pseudomonas Apoptosis Factor) by the TTSS, resulting in activation of Fas-dependent apoptosis or by (B) direct induction of Fas-dependent apoptosis by one or more components of the type III translocation apparatus itself. We will identify the mechanism by which PAl03 induces TTS-dependent apoptotic-like cell death in eukaryotic cells. We will initially use genetic and proteomic approaches to identify a putative apoptosis-inducing type III secreted effector molecule. We may also test the alternative hypothesis that the TTSS itself is responsible for the induction of apoptosis by PA. We will express the PA TTSS in a Yersinia strain that lacks the plasmid encoded TTSS and test for restoration of apoptosis-inducing activity.

In Vivo Induced Antigen Technology (IVIAT) has been well documented as a sensitive, fast, and inexpensive method for identifying novel genes of pathogenic bacteria that are specifically expressed during an actual infectious process. However, the use of IVIAT is limited to analysis of diseases where the pathogen infects a host that is capable of mounting an antibody response. In this application, we describe a modification of IVIAT called Change Mediated Antigen Technology (CMAT) that allows identification of both pathogen and host genes specifically expressed during infection. Proof of principle has been accomplished using Xanthomonas campestris infection of bean plants. In general, CMAT is potentially capable of identifying any gene that is expressed by any cell when it undergoes any sort of change. We intend to expand the application of CMAT to use it as a tool to study genes that are specifically expressed during oncogenesis in colorectal cancer. Genes that are discovered will potentially serve as excellent biomarkers for studying the efficacy of therapy of this disease and provide novel targets for new diagnostic and vaccine strategies. There are 3 Specific Aims. In Specific Aim 1, phage display libraries of human genomic DNA and cDNA from colorectal cancer tissue samples of subjects will be constructed in bacteriophage T7. Sufficient independent clones will be obtained to assure complete coverage of these libraries. In Specific Aim 2, a CMAT IgY probe will be created by immunizing chickens with colorectal cancer tissue samples from subjects. Antibodies produced in response to the immunogens will be purified from eggs and adsorbed with lysates made from healthy tissue of the same subjects. In Specific Aim 3, the CMAT IgY probe will be used to biopan the T7 libraries to enrich for clones expressing proteins made by colorectal cancer cells that are not made by healthy cells. A verification step employing Western blotting will be used to eliminate false positives. The cloned inserts in verified clones will be sequenced and the results subjected to genomic and proteomic analyses to generate a list of genes and their proteins that are expressed by transformed cells during different stages of colorectal cancer and not by healthy cells. This list will provide the starting material for the Phase II work, which will entail screening of the expressed proteins for their potential to serve as biomarkers in diagnosis of colorectal cancer and as possible targets for early diagnosis and vaccine strategies. Change Mediated Antigen Technology (CMAT) is a new method for identifying genes that are expressed when a cell undergoes a change. This project will use CMAT to identify proteins expressed by colorectal cancer cells that are not expressed by healthy cells. Such proteins are likely to be useful in monitoring treatment, diagnosing this disease, and in preventing it. [unreadable] [unreadable] [unreadable]

With the cooperation of the Director of the centers, specific procedures will be established to identify the patients and assure that their care conforms to specific standards for diagnosis treatment and management that we have identified in year 1 of the project. Two sets of activities will be conducted during the second year of this grant. First, a descriptive field study using approximately 60 diabetic and 60 hypertensive patients will be conducted in order to: 1. Describe the interrelationships among the following patient contributions to care; a) patients knowledge of disease and therapy. b) patients beliefs regarding the severity of the disease, the benefits of therapy and the barriers to implementing therapy. c) patient compliance with prescribed therapy. 2. To produce a prediction model of clinical health states using knowledge, beliefs, compliance and intake severity of disease. 3. To produce a prediction model of psychosocial health states using knowledge, beliefs, compliance and intake severity of disease. 4. To classify patients according to the pattern of scores on measures of knowledge, beliefs, and compliance. Second, based on the results of this descriptive phase, protocols will be developed for the experimental nursing intervention phase of the study. Nurse interviewers will be trained for the experimental phase of the study during the latter part of this second year of funding. In addition, patient interviewers and record auditors will be trained to conduct these functions necessary for the conduct of the experiment.

The inherited bone marrow failure syndromes (IBMFS) are a heterogeneous group of disorders characterized by marrow failure, congenital anomalies, and predisposition to myelodysplastic syndromes (MDS). Studies of IBMFS to date have largely focused on pediatric patients, but these are increasingly recognized in adults presenting with cytopenia(s). There are no paradigms defining the optimal care of adult patients with IBMFS. A significant subset of patients fail to fall within the known categories of IBMFS. The diagnosis and medical care of IBMFS patients are limited by our lack of knowledge regarding genetic causes. We will pursue complementary bidirectional studies moving between the pediatric/adult clinics and the laboratory to investigate the clinical features, genetic etiology, and pathophysiology of IBMFS. We will also exploit recent technological advances as a platform to develop novel diagnostic tests for these syndromes. The identification of molecular pathways contributing to marrow failure should provide insights into global molecular pathways regulating hematopoiesis as well as inform our understanding of acquired marrow failure and MDS in the general population. PUBLIC HEALTH RELEVANCE: Understanding the genetic pathways contributing to marrow failure will allow the development of new diagnostic tests and rationally designed medical therapies. Elucidating the molecular mechanisms underlying inherited marrow failure will provide insights into marrow failure arising in the general population.

The Massachusetts Institute of Technology proposes that support for the High Field NMR Resource (Grant no. RR00995-13), located at the Francis Bitter National Magnet Laboratory (FBNML), be continued for five years and that its scope be enlarged to support ongoing activities in in-vivo spectroscopy and imaging of animal models and human subjects at the M.I.T. MRI Facility. We thus propose to create and operate a new entity called the Comprehensive NMR Center for Biomedical Research. This center will be comprised of two division; (1) the existing High Field NMR Resource, now in its 13th year of operation, and (2) the In-vivo MNR/MRI Facility, a research laboratory developed in parallel during the past six years at M.I.T. almost exclusively with private funds. The major new technologies which we propose to introduce are: (1) a capability to perform high-resolution 3D-NMR: (2) an enhanced capability for NMR microscopic imaging and spectroscopy; (3) the use of M.I.T.'s unique actively shielded pulsed gradient coils for NMR microscopy and in- vivo spectroscopy and imaging; (4) a high performance spectrometer for dynamic nuclear polarization studies of crystalline proteins; (5) a high- resolution multinuclear 600 MHz spectrometer; (6) a whole body human in- vivo spectroscopy/imaging system utilizing a 1.5T/120 cm magnet of our own design and construction; (7) enhanced computer facilities for acquisition, data processing and display of 2D- and 3D-NMR data sets and for in-vivo spectroscopy and imaging. Our objective during the next five years is to establish an integrated, comprehensive capability which will allow study by NMR of all the diverse biological systems -- macro-molecules, subcellular organelles, microorganisms, cells, mammalian tissues, intact organs, animals and humans. Experimental capabilities will exist for NMR microscopy, NMR spectroscopy (solutions, solids, in-vivo tissues) and NMR imaging. The comprehensive nature of the Center will allow for research projects initiated at the High Field NMR Division to migrate naturally to the In- Vivo MRS/MRI Division. Thus, a given project begun at the cellular or small animal level can be easily broadened in scope to include investigations of larger animals and humans. Such advanced NMR capabilities, which permit long-term, in-depth, health-related investigations and to which researchers have ready, equitable access do not exist at a single site in the greater Boston/Cambridge area.

The retinal vasculature is prone to damages, leading to serious ocular diseases including loss of vision. While angiogenic therapies are being explored as potential treatments, a significant hurdle is our inability to control the three dimensional organization of the vascular network induced by angiogenic factors to ensure proper functioning of pharmacologically induced retinal vasculature. Thus, our long term objective is to unravel mechanisms that control the spatial organization of retinal vascular beds, and in particular to investigate how components of the hypoxia signaling pathway act as spatial cues to determine the direction and position of vascular growth in the retina. These studies will be carried out in three specific aims. Aim 1. Investigate roles of prolyl hydroxylase domain proteins in controlling retinal vascular pattern formation. PHDs negatively regulate the abundance of hypoxia inducible factors (HIFs), the latter of which are essential for angiogenesis. We hypothesize that the level of PHD activity in a tissue microenvironment determines the activity and directionality of vascular growth in its vicinity, and will test this hypothesis by generating chimeric retinas that contain micro tissue domains with PHD deficiency or overexpression. Aim 2. Determine if HIF-la accumulation in a micro tissue domain controls the position and direction of vascular growth in nearby tissues. Aim 3. Explore the role of VEGFR-l in defining vessel to vessel distances. VEGFR-l is produced by endothelial cells and forms tight complex with VEGF-A, a key angiogenic molecule induced by hypoxia. We propose that VEGF-A/VEGFR-1 interaction diminishes bioavailable VEGF-A near the source of VEGFR-l expression and therefore disallows the growth of more microvessels within a certain distance from an existing microvessel. This hypothesis will be tested by creating chimeric retinas that contain micro tissue domains overexpressing VEGFR-1, and assessing vascular density near such tissues. The objective of these studies is to facilitate the development of effective therapies aimed at repairing damaged retinal vascular beds and is highly consistent with the mission of the National Eye Institute (NEI).

Visual stimuli that are spatially removed from a neuron's classic reception field and which they elicit no response can strongly affect the magnitude of a response to stimuli within the classic field. It is thought that lateral connections among neurons mediate the interaction. Behaviorally, the presence of surround stimuli can profoundly affect the ability to perform fine spatial discriminations between similar center patches. In the proposed research, we test recent findings from the neuroscience literature in the psychophysical domain. An understanding of the normal mechanisms governing these lateral interactions is critical for our understanding of normal visual processing. For example, excitatory contextual modulation is thought to reflect the operation of contour mechanisms that mediate reading, whereas inhibitory interactions over space may be involved in segregating parts of a scene. It is also of import to provide normal behavioral data as a baseline in clinical applications. Deficits shown in amblyopia and strabismus include both hyperacuity tasks and lateral interactions. Here, we use psychophysical discrimination tasks to ask how these mechanisms integrate or segregate information from different regions of space, and how the spatial arrangement of stimuli in one part of the visual field affects performance in another. Specifically, we test several hypotheses: (1) that the spatial extent of regions of psychophysically-determined summation and inhibition in discrimination depends upon the contrast; (2) that there are spatial asymmetries in contextual modulation of mechanisms mediating fine spatial discriminations; (3) that the modulation process can be characterized by at least one of three quantitative models of gain control; (4) that apparently contradictory results in the psychophysical literature can be resolved within the context of a relatively simple neural model of contextual modulation; and (5) that contextual influences are both qualitatively and quantitatively altered by changes in apparent figure-ground relationships. We propose to begin to characterize these properties quantitatively.

Benign essential blepharospasm (BEB) and spasmodic torticollis (ST) are progressive forms of focal dystonia characterized by their unique presentation in upper facial musculature and cervical musculature, respectively. Little is known about the role of the cerebral cortex despite the fact that emotional and stress-related events, behaviors regulated by the cerebral cortex, often trigger and exacerbate these disorders. Furthermore, cortical control of lower motor neurons innervating muscles implicated in these movement disorders is poorly understood. This study is designed to isolate for the first time cortical neural systems that directly innervate facial motor neurons selectively engage in BEB and spinal accessory motor neurons selectively engage in ST. Once identified, synaptic interactions of these neuronal assemblies will be anatomically characterized. The major goals of this project are to examine the corticobulbar projection from the face/neck representation of the cingulate motor cortex (M3 or Area 24c) to the facial and spinal accessory nuclei. The investigators will determine if the M3 projection: 1) targets lower motor neurons innervating the orbicularis oculi, corrugator supercilia, frontalis, sternocleidomastoid and trapezius; 2) makes direct contact with these motor neurons; 3) is excitatory or inhibitory. Their studies are designed to test the hypothesis that the cerebral cortex plays a role in the physical expression of BEB and ST. It is further hypothesized that recruitment of musculature in BEB and ST patient is a consequence of neuroplastic alterations, such as local sprouting. This project will lead to an increased understanding of cortical systems governing upper facial expression and cervical torsion. These data will be used to design a cortical model of focal dystonia and provide guidance in developing new approaches to surgical treatment of intractable cranial-cervical dyskinesias.

We have cloned a 9.5-kbp fragment of an oncogene present in a human fibrosarcoma cell line (HT-1080). The cloned fragment is present in all tested HT-1080-derived transformants, and is different from another fragment of the same oncogene (14 kbp) previously isolated. When both fragments are put together, the construction shows a molecular structure similar to that of the N-ras oncogene present in human neuroblastoma cell lines.

The applicant's long-term goal is to direct a basic science research program exploring the role of endothelial cells and their progenitors in organ fibrosis and tumor progression. The applicant has been trained in somatic stem cell biology during her thesis work and was later able to apply this knowledge in cardiovascular research where she was additionally trained in embryonic stem cell biology. During the first part of a postdoctoral fellowship, the applicant expanded her knowledge on stem cell biology and gained new insights into embryonic development. During these studies, the applicant became interested in the cellular plasticity that is displayed by endothelial cells both during embryonic development and also in adult disease states. The applicant is now an Instructor in Medicine in the Kalluri laboratory, where both tumor angiogenesis and organ fibrosis are major research foci, and thus it was a natural extension of the applicant's interests to study endothelial cell plasticity in the setting of the tumor microenvironment. The applicant's previous studies for the first time demonstrated that in cardiac fibrosis endothelial cells contribute to the pool of fibroblasts via an endothelial transition and that inhibition of EndMT can lead to decreased fibrosis and improved cardiac function. The research project proposed here centers on testing the hypothesis that EndMT also plays a role in tumor fibrogenesis. This hypothesis is based on immunohistochemical staining which revealed cells that are double positive for both endothelial and fibroblasts in several tumor types, indicating that endothelial cells can undergo EndMT. The primary aim of the proposal is to provide genetic evidence that endothelial cells contribute to the fibroblast population in the tumor microenvironment by undergoing EndMT. For this purpose, endothelial lineage tracing by TielCre;R26RstoplacZ double transgenic mice, in which all cells of endothelial origin are irreversibly marked with lacZ, will be used. Second, the impact of EndMT on tumor progression will be evaluated by creating transgenic mice in which fibroblasts of endothelial origin will be selectively ablated. If the tested hypothesis is valid, EndMT might represent an important target for the treatment of cancer.

This project continues and extends work carried out on the relationship between structure and function of glycogen phosphorylase. The amino acid sequence of the enzyme is being determined; approximately 30% of its structure has been resolved, including the structure of two of its functional sites. A second aspect of this project relates to the regulation of glycogen breakdown in which phosphorylase is involved. While much is known about the reactions that trigger this metabolic process, the way in which the activated enzymes are returned to their inactive (resting) state is still obscure. The properties of one such enzyme, phosphorylase phosphatase, are being investigated. A third purpose of this research is to attempt to define how regulatory mechanisms arose in early species and evolved with time. Work has been initiated on the control of carbohydrate metabolism in the Pacific dogfish which has separated from the main line of evolution leading to mammals 450 million years ago, and on yeast which is of intermediate complexity between bacteria and higher fungi.

The general goals for the proposed Career Development project are twofold. They are (a) to aid cancer prevention by developing smoking-cessation research tools applicable to people in/from low- and middle-income countries and (b) to develop the skills necessary to become an independent investigator of smoking cessation. The proposed career enhancement program will take four years to complete, and will be based at Stanford University and the University of California-San Francisco. This dual-institution design will permit the candidate to take advantage of training opportunities unique to affiliation with each university. The proposed research is the centerpiece of the applicant's four-year career development program. Every aspect of the research study will draw upon and serve as a real-life testing ground for the project's more formal didactic components. The research is designed to respond to the global public health mandate that greater amounts of analytical resources and personnel from high-income countries go to help people in/from low- and middle-income countries acquire the research tools needed so that they may mount more effective tobacco-control initiatives. In particular, the study will examine whether research instruments based on a well-known U.S. framework for analyzing smoking and smoking-cessation (the Transtheoretical Model of Behavior Change) can be successfully revised and validated for smokers and former smokers of Chinese ancestry. The proposed project will be sited in China's southwest city of Kunming, and it will be conducted in collaboration with the leading university in the region, Yunnan University. The project will draw upon both qualitative and quantitative methods, and it will make extensive use of the applicant's expertise conducting China-based research. At minimum, it is hoped that the research will have the following significance: (a) expand understanding of an important theoretical framework's cultural malleability, (b) serve as the foundation upon which, in the future, the candidate will continue researching smoking and smoking cessation, and (c) provide data of assistance to public health personnel interested in developing interventions for smokers of Chinese descent living in the PRC and elsewhere, including the United States. I

The Section on Cellular Biophotonics, of the Laboratory of Molecular Physiology, NIAAA, was established in April 2003. The principle aim of this section is to use imaging techniques to study how protein complexes, with special emphasis on complexes comprised of integral membrane proteins, are formed and maintained in living cells. Membrane proteins, such as receptors, channels, and their regulatory partners, are typically synthesized by ribosome?s associated with the endoplasmic reticulum, but ultimately function at the cell surface. Thus, the logistics of their production, assembly, transport, and exocytotic insertion into the cell surface is key to understanding both normal physiological function, and abnormal physiological states associated with human maladies. Furthermore, the delivery of membrane protein complexes to cell surface micro-domains must involve the coordination of exocytotic and endocytotic reactions. Lesions in exocytosis have been tied to some forms of muscular dystrophy, and may also be involved in alcohol induced myopathy. The section is comprised of Drs. Steven Vogel (Chief), Srinagesh Koushik (Research Fellow), Christopher Thaler (Postdoctoral IRTA), and Jose Fernando Covian-Nares (Visiting Fellow). The sections initial efforts have been in building and testing a laser scanning microscope specifically designed for studying protein-protein interactions in living cells. The microscope is a fully functional laser scanning two-photon microscope, with the additional capabilities of measuring florescent emission spectra (spectral imaging), fluorescent lifetime decays (FLIM), and fluorescent anisotropy lifetime decays (rFLIM). These added capabilities make it specifically useful for monitoring Forster?s Resonance Energy Transfer (FRET) between either dissimilar (Hetero-FRET) or similar (Homo-FRET) fluorophores. We have used this microscope to develop a new, photon efficient method for measuring FRET in living cells based on spectral imaging. This study has been accepted for publication in the Biophysical Journal (due out in October 2005) and will be highlighted on the journals cover. A second manuscript, evaluating how quantitative spectral imaging is for discriminating proteins labeled with either blue (CFP) or yellow (YFP) variants of green fluorescent protein (GFP) has been submitted and is currently under review. We are currently working on 5 projects in the lab. Three of them are methodological, and the last two utilize our microscopes unique capabilities to answer biological questions: 1. We are in the final steps of data collection for a study that describes the generation and characterization of a set of FRET standards to aid in the interpretation of FRET analysis. In the course of this study we have identified two important potential artifacts that can complicate the proper interpretation of FRET analysis. 2. We are also in the final data collection stage for a study describing the generation of Homo-FRET standards that will be used in interpreting anisotropy decay experiments. This relatively new method has the potential for monitoring how proteins form multimers, and their stoichiometry in living cells. The generation of fluorescent protein anisotropy standards required the generation of a mutant variant of GFP that has the same fundamental structure as GFP but is not capable of participating in FRET reactions. 3. The third methodological project investigates how FRET efficiencies change when multiple acceptors are present. This type of problem is important, because many proteins form multi-meric complexes, and the assembly and disassembly of these complexes might play an important role in regulating cell functions. The accepted mathematical formalism for dealing with this class of FRET problems assumes that the rates of energy transfer (to single acceptors) sum linearly when multiple acceptors are present. Our preliminary data suggests that this treatment underestimates the true FRET efficiency. We have developed a new theory for dealing with these problems based on probability theory. We are currently testing this new formalism. 4. The fourth project, in collaboration with Dr. Dave Lovinger?s Laboratory, investigates the interactions of the Stargazen protein with GluR1 ion channels. GluR1 has been shown to play a decisive role in synaptic efficacy, as activity causes GluR1 to migrate to dendritic spines in hippocampal CA1 neurons. Stargazin, a member of the transmembrane AMPA receptor regulatory protein family has also been shown to affect the trafficking of GluR1 as well as its kinetics. We are using spectral analysis, FLIM analysis, and anisotropy analysis to investigate the interactions of stargazing with GluR1, as well as their multi-meric structure. 5. Compensatory endocytosis is a distinct form of endocytosis that compensates for exocytotic activity. It plays an essential role in the maintenance of cell surface homeostasis. In our fifth project we have developed a new imaging based assay for testing the effects of either over expression of exogenous proteins, or down regulation of endogenous proteins, on compensatory endocytosis in developing sea urchin embryos. Using this assay we find that over expression of Src kinase, an oncogene, inhibits compensatory endocytosis. Inhibitors of tyrosine phosphatase had a similar effect, suggesting that the balance of tyrosine kinase and phosphatase activity plays a key role in how cells regulate their cell surface area. Cell proliferation, as observed in tumors, requires a constant increase in cell surface area to support multiple rounds of cell division. Our finding suggests that the increase in Src kinase activity observed in many tumors might disrupt cell surface homeostasis by inhibiting compensatory endocytosis.

Huntington disease (HD) belongs to a neurodegeneration disease family in which selective neuronal loss is caused by misfolded proteins. In HD, a polyglutamine (polyQ) expansion (>37 glutamines) in the N-terminal region of Huntington (htt) causes N-terminal htt fragments to misfold or aggregate, conferring neuropathology. Consistently, N-terminal htt fragments containing an expanded polyQ are able to interact with a number of proteins to mediate multiple pathological pathways. Thus, an important therapy approach is to inhibit the expression of mutant Huntington (htt) or its activity by blocking its abnormal interactions with other important proteins. Intracellular antibody (intrabody) provides a promising approach to achieve this goal, as intrabody is able to interact with an antigen intracellular to block its activity ortoxicity. However, normal htt is required for cell survival, and deletion of htt causes cell degeneration. Thus, a challenge for using the intrabody therapy for HD is to identify an intrabody that can selectively inhibit the toxicity of mutant htt but not interfere with the pivotal function of normal htt. Using the same antigen for an antibody (EM48) that reacts preferentially with misfolded and aggregated htt, we have established a hybridoma cell line that generates a monoclonal antibody (mEM48), which also preferentially reacts with mutant htt. By isolating the gene encoding mEM48 from this cell line, we have generated an intrabody that selectively binds mutant htt. We propose to characterize the protective effect of this intrabody on htt's toxicity in HD cellular models. We will also modify this intrabody to increase its expression level and stability. Finally, we will deliver this intrabody to HD mouse brain via viral vectors and to examine its protection against the neuropathology in HD brain. This proof of principle study will have a broad implication for the treatment of other neurodegeneration diseases that are also caused by misfolded proteins.

Single Walled Carbon NanoTubes (SWCNT) are new materials of emerging technological importance. Their manufacturing requires iron resulting in its high content in SWCNT. Because iron is a catalyst of oxidative stress, iron-containing SWCNT are likely more toxic than iron free SWCNT. Our central hypothesis is that SWCNT are toxic to the lung and the toxicity is dependent on their content of iron. The major toxicity mechanisms include inflammatory response synergistically enhanced by oxidative stress exacerbated by iron. SWCNT toxic effects are further augmented by microbially-induced inflammation. The apoptotic/necrotic target cell death ratio dependent on the WCNT iron is also a regulator of SWCNT toxicity via production of anti-/pro-inflammatory cytokines, respectively. Specific Aim 1 is to establish the extent to which SWCNT alone are pro-inflammatory to lung cells and tissue and characterize the role of iron in these effects using genetically manipulated cells and animals as well as antioxidant interventions. Specific Aim 2 is to determine the potential for SWCNT and microbial stimuli to synergistically interact to promote macrophage activation, oxidative stress, and lung inflammation. Specific Aim 3 is to reveal the extent to which SWCNT are effective in inducing apoptosis and whether apoptotic cells exert their macrophage-dependent anti-inflammatory potential during in vitro and in vivo SWCNT exposure. The project involves a team of interdisciplinary investigators with unique expertise in redox chemistry/biochemistry (V. Kagan), cell and molecular biology of inflammation (L. Ortiz) and its interactions with microbial agents (J. Fabisiak), pulmonary toxicology of (nano)particles (V, Castranova, A. Shvedova). Based on our results, mechanism-based interventions, such as specific antioxidants, new means to control iron content (using non-toxic chelators) as well as biotechnological approaches (phosphatidylserine liposomes and/or apoptotic cells down-regulating inflammatory response) may be developed to decrease toxicity of (iron-containing) SWCNT. [unreadable] [unreadable]

Recent work with cultured mammalian cells indicates that the capability for active Na/K transport increases in response to partial inhibition of this transport system. I propose to combine the approaches of ion transport physiology and somatic cell genetics to investigate the changes in Na/K transport in cultured Chinese hamster ovary cells (CHO) exposed to low (K) medium. I will study the transport properties of cell lineswhich differ in their ability to recover Na/K transport capacity during a 72 h low (K) treatment. Tracer flux measurements will be used to investigate the Na:K coupling ratio and the kinetic parameters of K influx. Also, changes in the synthesis of Na/K pumps will be estimated by measuring the amount of metabolically labelled Na, K-ATPase in the cell membranes. Additional low (K) resistant variants will be isolated and utilized to explore the variety of mechanisms which can produce this phonotype. I will attempt to isolate a variant which is temperature sensitive for ability to recover transport capacity during low (K) treatment. This type of variant will be used to investigate further into the metabolic mechanisms involved in this compensatory response.

The study is comparing the efficacy and safety of heater probe or injection therapy for ulcers (actively bleeding, non-bleeding vivible vessel, or adherent clots) versus medical-surgical treatment. This is a randomized controlled trial of endoscopic treatments for bleeding peptic ulcers and long term follow-up of patients.

Research conducted by this laboratory focuses on the investigation of modified, new or alternative procedures for determining the safety and potency of a number of biological products. Information obtained from new methods is compared to the current and historical data provided from existing methods to establish equivalency. Suitable standards and reference preparations must be developed and prepared if studies reveal that current materials are not appropriate. Particular emphasis is placed on alternative procedures that reduce the number of animals required for product testing and provide comparable results in less time than existing methods. In Vitro Potency Assay for Diphtheria Antibody Response. Production of a specific quantity of protective antibodies capable of neutralizing active toxin following immunization with vaccines containing a Diphtheria component is required for release of the vaccine lot. Currently, a second animal test is employed to evaluate the efficacy of the animal response to the test vaccine. This research has focused on replacing this second animal test with an in-vitro cell test. Results of the six laboratory collaborative study were presented in December, 1997 at the FDA Science Forum. These data were also reviewed in several international meetings, and plans to develop and implement a collaborative study continue to be discussed. Discussions about modifications of the STS proposed method are continuing. These discussions involve the amount of toxin used in the in-vitro assay, and the use of a hyper-immune horse serum vs. sera from a single immunization of guinea pigs. Development of an In Vitro Potency Assay for Tetanus Antibody Response. This project relates to the investigation of an in vitro ELISA potency assay to replace the guinea pig death test portion of the official release test for the tetanus component in vaccines. Reports of an ELISA method in the literature, and the use of this method in the laboratory are dependent on a source of purified Tetanus Toxin. Preliminary data and proposals for an international collaborative study were discussed at a meeting in June 2000. Problems related to the quantitiation of a response and the correlation between animal and laboratory tests continue to be discussed. Investigation of the Microstar Rapid Sterility Test Method. For biological products claimed to be sterile, testing requires a 14 day incubation period, with observations based on visual examination of product-inoculated media, to conclude with reasonable confidence that the product is free from extraneous bacterial or fungal contamination. A group of newly emerging "rapid" methods suggests that such bacterial contamination can be detected in less than 14 days by other methods. The Microstar system, made by Millipore Corporation, utilizing bioluminescence to detect viable contaminating organisms, was selected for evaluation based on the laboratory's current use of existing Millipore Steritest equipment and supplies. Prior incubation in USP and CFR specified sterility test media appears to enhance detection of multiple types of organisms; using the MicroStar filters, individual CFU's can be transferred and cultured for subsequent identification using selective media or enzyme based analysis. The MicroStar system has shown the capacity for early detection of small quantities of environmental contaminants in conventional, adsorbed and cell based products. This year, efforts to detect bacterial contaminants in whole blood products resulted in the development of a lysing procedure to negate background activity of blood cells. Work continues in this area. Comparison of LAL Test with the Rabbit Pyrogen Test. Determination of pyrogen or endotoxin content may be measured by the Rabbit Pyrogen test as described in 21 CFR 610.13 or, if validated as an equivalent method, use of the Limulus Amebocyte Lysate (LAL) as described in the USP 26 Bacterial Endotoxins Test (BET). Allowable endotoxin content for a number biological products can be found in the "FDA Guideline On Validation Of The Limulus Amebocyte Lysate Test As An Endproduct Test For Human and Animal Parental Drugs, Biological Products, And Medical Devices"; alternatively, product or manufacturer specific limits are established in conjunction with licensing. Chromogenic, Turbidimetric and gel clot methods for the in-vitro BET test are commercially available; the gel clot method is considered definitive in the absence of enhancement or inhibition. In general, advantages of BET methods over the Rabbit test include requiring less sample and the ability to perform repeat or confirmatory tests quickly. This year, several manufacturers of blood products have provided data to support conversion from Rabbit testing to BET. Studies continue to expand BET testing to additional biological products. This project incorporates FY2002 projects 1Z01BR003003-04, 1Z01BR003004-15, 1Z01BR003005-12, and 1Z01BR003006-03.

The long-term project objective is the commercialization of a safe, effective, easy to use, and painless polynucleotide vaccine delivery system that can be used in polynucleotide vaccines for biodefense against NIAID Category A, B and C Pathogens. Polynucleotide vaccines are on the forefront of vaccine BIOHAZARD development. They are important because of the fast development times possible and because cell mediated immune responses can be induced. The delivery system proposed here would be effective for most polynucleotide vaccines. This delivery system specifically addresses the requirement as presented in the NIAID Strategic Plan for Biodefense Research, February 2002, page 8. In addition to Biodefense, this system will provide effective polynucleotide vaccine delivery for less lethal viruses, some cancers and some third world diseases. The defense and commercial applications are extensive. The polynucleotide vaccine delivery system described here uses a microneedle array with the polynucleotide coated right on the needle in the array. There are hundreds of needles each about 0.15 mm long. This array is inserted into the skin with the needle penetrating to about the basal lamina. After insertion the polynucleotide leaves the needle surface and an electric field is used to permeabilize dendritic and epithelial cell membranes to permit the polynucleotide to enter the cell. The system will be tested with the WRAIR/Cyto Pulse dengue DNA vaccine, which will be used as a model for hemorrhagic fever viruses. The specific aims of this project are to design and develop to FDA QSR Standards the vaccine delivery system prototype, test for safety and efficacy in mice, and to test for safety in a Phase I human trial.

The adult dentate gyrus continuously generates granule cells (DGCs) that are needed for specific learning and memory tasks, but the precise contribution of new neurons to information processing in the hippocampal circuitry remains unknown. In the past, we have demonstrated that key developmental processes occurring in the perinatal brain such as maturation of excitability, afferent synaptogenesis and function are all recapitulated during adult neurogenesis. We now propose the central hypothesis that newly born cells establish functional outputs as they develop, and the population of postsynaptic target cells contacted by young neurons is predominantly inhibitory, therefore different from the mixed excitatory/inhibitory network activated by mature granule cells. Thus, there would be a time window in which young DGCs primarily activate feedforward and/or feedback inhibitory circuits without exciting pyramidal cells, exerting a tight inhibitory control over the dentate gyrus output. In Aim 1 we will build a spatio-temporal map of target activation by young developing neurons of the adult dentate gyrus. We will use retroviral transduction to express the light- activated cation channel Channelrhodopsin-2 in newborn DGCs of young-adult female mice (C57Bl6/J). We will sacrifice the animals at different times and prepare acute brain slices to carry out electrophysiological recordings. By stimulating the whole hippocampal slice with brief light pulses, all retrovirally transduced neurons will spike. We will search randomly for active postsynaptic target cells throughout the hilus and CA3 regions, and identify and characterize responsive neurons by combining loose patch and whole-cell recordings. In Aim 2 we will investigate the functional maturation of new mossy fiber synapses made onto GABAergic and glutamatergic targets. We plan to utilize whole-cell recordings to test whether synapses forming onto GABAergic interneuron targets mature faster than those made onto pyramidal cells, as suggested by our previous structural studies. We will also study presynaptic mechanisms of short- and long-term plasticity that will shape both activity-dependent competition and activation of postsynaptic circuits. This project will address fundamental questions about connectivity and activation (spiking) of newborn cells that will contribute to understanding the precise impact of adult neurogenesis in the preexisting hippocampal network and the rules of neuronal connectivity in the adult brain. Identifying the rules by which neurons integrate in the existing network in a manner that is both safe and functionally relevant is also crucial for developing future brain repair therapies. Novel retroviral tools will be developed in collaboration with the Gage lab to enhance Channelrhodopsin-2 expression, thus improving the capability of light activation of newborn cells. In turn, experimental data obtained here will be used to feed into the theoretical model being developed by the Gage lab on the role of immature neurons in signal processing. The success of the proposed project relies on strengthening the close interaction between the Schinder laboratory at the Leloir Institute (Buenos Aires) and the Gage laboratory at the Salk Institute of La Jolla. Members of the Schinder lab will have the opportunity to train at Salk on the generation and characterization of novel retroviral vectors, improving the capabilities to develop advanced molecular tools at the foreign institution. We anticipate that capacity building leading to the design, generation and use of novel retroviral tools at the Leloir Institute will have an enormous impact on the local scientific community. This collaborative effort will therefore serve as a driving force to increase the critical mass of Argentine investigators that incorporate competitive technologies for the study of neurodegenerative disorders within their research focus. An increase in the number of laboratories carrying out regeneration- related projects will certainly enhance awareness to these and related problems to our community.

The principal aim of this proposal is to further development of new methods for analyzing observational data bases and randomized trials of HIV-infected persons and the application of these methods to data obtained in randomized and observational studies in an attempt to help answer important open substantive questions concerning the treatment and course of HlV-related disease. The proposed approaches are based either on (i) the estimation of new classes of causal models which include structural nested models, marginal structural models (MSMs), direct effect structural nested models, continuous time structural nested models, and optimail regime structural models (SNMs). Many of the new methods are fundamentally epidemiologic in that they require data on time-dependent confounding factors, that is, risk factors for outcomes that also predict subsequent treatment with the drug or cofactor under study. In particular, we plan to further develop optimal regime SNMs and dynamic MSMs to help detemnine the optimal times to start HAART therapy and to change HAART regimens as a function of a subject's CD4 count, HIV RNA, clinical history, and, where available, results of genot^lc or phenotypic resistance testing. Our methods will be developed with the goal of directing analyzes and reanalyzes, with collaborators, of data from the HIV Causal Colioboration at HSPH . the Multicenler AIDS Cohort Study, The Women's Interagency HIV Study, The Swiss HIV Cohort Study, The Study of The Consequences of Protease Inhibitor Era (SCOPE), Pediatric Late Outcomes Protocol (PACTG 219) and the ALLRT study. RELEVANCE (See instructions): Observational methods are used to answer pressing causal questions that cannot be or have not yet been studied in randomized trials. In particular we are developing methods that are the best available to determine the optimal CD4 and HIV RNA levels at which to initiate HAAART therapy in HIV infected subjects and the optimal time to change therapy once resistance to a initial HAART regime has developed.

Clostridium difficile infection (CDI) is an important source of morbidity and mortality among U.S. Military Veterans. The primary virulence factors are TcdA and TcdB, toxins that induce diarrhea, inflammation, and significant damage within the colon. This proposal is designed around the hypothesis that inhibition of toxin activity represents a therapeutic approach that can impact clinical treatment and outcome for individuals suffering from CDI. TcdA and TcdB are homologous glucosyltransferases that modify and inactivate Rho family GTPases. The glucosyltransferase activity of the toxins has been linked to a `cytopathic' disruption of the acti cytoskeleton and is important for systemic toxicity in a mouse intoxication model. The X-ray crystal structures of small molecule glucosyltransferase inhibitors bound to the TcdA and TcdB glucosyltransferase domains (Aim 1) will provide a foundation for structure-guided design of molecules with enhanced potency. In addition to the cytopathic effects, TcdB is a potent cytotoxin that causes necrotic damage in cells and tissue. The cytotoxicity results from TcdB-induced assembly of the epithelial cell NADPH oxidase (NOX) complex. The assembly results in the production of reactive oxygen species (ROS), which cause necrosis. Preliminary data indicate that a Nox1 knockout mouse is protected from CDI tissue damage and underscores the hypothesis that inhibition of the NOX1 pathway will protect against the colonic tissue damage observed in severe cases of CDI. A high-throughput screen has led to the identification of 176 small molecules that inhibit TcdB-induced necrosis. Experiments in Aim 2 will categorize these compounds according to their mechanism of action and result in the identification of lead compounds for further analysis. In Aim 3, the efficacy of N- acetylcysteine, an FDA-approved antioxidant, along with lead compounds from Aims 1 and 2 will be evaluated in a mouse model of CDI. Spore challenge with an epidemic M7404 strain and a panel of variants with defined mutations in one or both toxins will permit dissection of the specific effect each compound has on TcdA- and TcdB-mediated events. These are the key studies needed to advance small molecule inhibitors of the C. difficile toxins into clinical practice.

The focus of the proposed research is on structural plasticity and large-scale dynamics as determinants of protein function. Detailed and highly specific characterization of functionally important transient non-native protein states will rely on development of experimental strategies combining hydrogen/deuterium exchange in solution (HDX), electrospray ipnization (ESI MS) and gas phase protein ion fragmentation. The new strategies will provide conformer-specific structural and dynamic information by using HDX in combination with (i) simultaneous fragmentation of several ionic species representing a single protein state and (ii) covalent trapping of native and non-native protein states. A new method to probe stability of protein cores by monitoring HDX selectively at polar side chains will also be developed. Multi-stage protein ion fragmentation in both positive and negative ion modes in combination with solution-phase HDX will be evaluated as a means of characterizing local dynamics within large (>30 kDa) proteins with multiple disulfide bonds. These new strategies will be used to further our understanding of transient disorder in cellular retinoic acid binding proteins I and II (CRABP I and II) as a critical factor controlling binding and release of retinoic acid (RA). Structural characterization of non-native protein states through which ligand binding occurs will be completed, followed by investigation of the dynamic events within CRABP II induced by its binding to a retinoic acid receptor (RAR), which facilitate ligand transfer from the transport protein to the receptor. The new experimental strategies will also be applied to further our understanding of metal delivery to cells via the transferrin cycle by examining dynamics and metal-binding properties of serum transferrin (Tf) and their modulation by transferrin receptor (TfR). Understanding the molecular mechanisms of RAR/CRABP and Tf/TfR interactions will be invaluable for the enhancing of a variety of therapeutic strategies exploiting the pleiotropic properties of retinoic acid and the ability of Tf to deliver cytotoxic ligands to malignant cells.

Transforming simple achiral molecules into stereochemically complex molecules remains a central challenge in organic synthesis. Asymmetric metal catalysis provides an avenue to meet this challenge. However, the majority of asymmetric organometallic catalysts mediates one type of reaction and allows one transformation. Therefore, traditionally complex organic molecules are often built transformation by transformation. This process is tedious and expensive. This proposal describes an innovative approach to asymmetric catalysis, asymmetric tandem organocatalysis, which uses one catalyst to mediate multiple transformations. The utility of tandem organocatalysis will be demonstrated in a very concise synthesis of Eiseniachloride A, a molecule of potential great medicinal interest. The completion of this proposed research would show the concept of tandem organocatalysis is valid and superior to the traditional one catalyst-one transformation organometallic catalysis. More importantly, the realization of asymmetric tandem organocatalysis would provide large quantities of potential drug candidates that can not be obtained otherwise.

To determine the safety and immunogenicity of vaccinia-derived HIV-1 recombinant envelope glycoprotein in asymptomatic HIV-infected adult volunteers, and to compare safety and immunogenicity of two different schedules of gp160 administration, and examine the effects of gp160 or hepatitis B vaccine on plasma P24 and/or other markers of viral load and on selected immune parameters.

A widely held hypothesis of sudden infant death syndrome (SIDS) is that it represents fatal sleep apnea due to a maturational defect in brainstem neural circuits controlling respiration during sleep. This hypothesis is based upon the consistent association of silent death to sleep periods in SIDS victims. Developmental mechanisms are implied by the unique age range of SIDS, with a peak at 2 - 4 months, a critical time in the maturation of respiratory and sleep patterns. Yet, proof of a brainstem defect is not forthcoming, because qualitative abnormalities have not been detected and quantitative abnormalities have not been consistent. We propose that the developmental brainstem defect in SIDS is quantitative and requires sophisticated morphometrics for its proof. Our aim is to test this hypothesis through the rigorous quantitation of morphological and neurochemical parameters of astrocyte and neuron maturation. We will use quantitative cytoarchitectonics to assess the number of reactive astrocytes and number and size of neuron subclasses in brainstem nuclei related and unrelated to the control of respiration and sleep. Formalin-fixed paraffin embedded brainstems, primarily from the Valdes-Dapena/Tildon collaborative study of the seven tissue markers of chronic hypoxemia, will be used in these studies. Sophisticated computer programs for quantitation and analysis of the number and size of cell subtypes in serial sections in 2- and 3-dimensions will be employed. We will use quantitative autoradiography to map the distribution of postsynaptic receptors as a probe of neurochemical development for neurotransmitters postulated to play a major role in respiratory and sleep regulation. Frozen brainstem specimens collected by us from a network of donating pathologists and medical examiners will be used in these studies. Autoradiographic and computer methodologies will be used for quantifying and analysing the anatomical distribution of receptor densities in 2- and 3-dimensions and correlating these findings with the results from the quantitative cytoarchitectonics. The rigorous quantitative methods proposed here have the potential to resolve many of the controversies concerning claims of morphologic and neurochemical abnormalities in the SIDS brainstem. While not directly explaining the cause of SIDS, a rigorously verified brainstem abnormality would provide a crucial lead. The establishment of normative standards about human brainstem maturation based upon the control specimens form a significant part of this study.

The regulation of proteolytic activity within the central nervous system (CNS) is essential in both normal and pathological processes. However, the precise role that proteolysis plays in the CNS is not well established. Recent work from many laboratories has indicated that the serine protease tissue-type plasminogen activator (tPA) is involved in a number of important processes both during development and in the adult brain. These include events associated with synaptic plasticity such as long term potentiation (LTP), motor learning and anxiety-like behavior, all processes which are though to involve synaptic remodeling. TPA has also been implicated in neuronal death following excitotoxic injury, seizure and stroke. The primary inhibitor of tPA within the CNS is the serine protease inhibitor (serpin) neuroserpin. The tissue distribution of neuroserpin indicates that it is predominantly expressed in neurons in areas where either tPA message or activity has also been localized. Neuroserpin is also found in areas known to have the highest susceptibility to ischemic injury, and neuroserpin is neuroprotective in stroke and seizure. Like tPA, neuroserpin has also been suggested to regulate anxiety-like behavior since both over-expression or complete deficiency of neuroserpin in mice leads to increased anxiety and neophobia. Thus, this proposal will focus on understanding the basic biology of neuroserpin and on its role in regulating tPA activity within the CNS. By using a combination of biochemical, molecular and genetic approaches both in vitro and in vivo, and building on previous studies of neuroserpin, and tPA, a number of important activities will be characterized and hypotheses tested. We will characterize the cell biology of neuroserpin and tPA within the CNS and test the hypothesis that tPA activity regulates neuroserpin release from neurons at the synapse. Sensitive assays will be used to localize neuroserpin and tPA and to monitor their trafficking and activity in primary neuronal cultures. We will also investigate the biochemical mechanisms of neuroserpin's regulation of tPA, and test the hypothesis that the inhibition of tPA by neuroserpin is regulated by pH. The role(s) of neuroserpin and tPA during normal and pathologic physiology such as anxiety and seizure will be examined in vivo, and we will test the hypothesis that neuroserpin and tPA regulate activity dependent changes in synaptic structure. Together these studies will give a better understanding of the roles of tPA and neuroserpin both normal and pathological processes, and may provide the basis for improved therapies for the treatment of CNS disorders. [unreadable] [unreadable] [unreadable]

The objective of these analyses is to identify interacting influences of family relationships and conditions that enter into the child's early experience. Of special interest are the mutual contributions of mother and child to relationships that damage or facilitate the child's development. Outcomes of children with early secure and insecure attachments were significantly modified by the other coexisting stressful or nonstressful relationships. In stressed contexts of maternal psychopathology and losses of significant persons, secure attachment was not a sufficient buffer against the child's later development of affective problems. Losses of significant persons had a main effect on children's subsequent depressed affect and disruptive behavior. When relationships between severely depressed mothers and their young children were characterized by very high mutual investment and dependence on each other, children were more likely to exhibit depressed affect in later years.

Anatomical, physiological, biochemical and behavioral studies will be carried out on the peripheral auditory system of bats of the genus Pteronotus. These bats emit intense ultrasonic pulses that contain a harmonic series of frequency modulated and constant frequency components. The comparative structure of their pulses will be examined with a new phase-locked loop device capable of resolving instantaneous ultrasonic frequency shifts as small as 20 HZ. The comparative acoustic behavior of these bats will be studied under natural conditions, especially during insect pursuit and capture. Cochlear microphonic potentials will be recorded from flying animals with the aid of miniaturized telemetry systems. The comparative fine structure of the cochleae will be examined with the aid of TEM, SEM and light microscopy. Particular attention will be directed toward: (1) the identifcation, analysis and description of an unusual, thick lining of the scala tympani in Pteronotus parnellii; (2) ultrastructural studies of different cell types in the organ of Corti with attention directed to specialized regions of the cochlea; (3) investigation of evidence indicating that some of the cellular elements of the organ of Corti may contain contractile proteins; (4) the possible physiological significance of these contractile proteins; and (5) the effects of ototoxic drugs on the audiogram, acoustic behavior and ultrastructure of the sharply tuned ear of Pteronotus parnellii.

The rapidly growing world population and the high rate of unintended pregnancies make contraception a need and a priority for any public health program. While several contraceptive methods for women are currently available, a more comprehensive approach to birth control requires extending contraception to males. However, a safe, effective and reversible contraceptive for men is still unavailable. An attractive approach t develop male contraceptives consists in targeting proteins that are specifically expressed in sperm and are required for sperm fertility. Evidence from our laboratory has shown that Na,K-ATPase ?4, a sperm specific plasma membrane transporter, which exchanges cytoplasmic Na+ for extracellular K+ is one of these attractive targets for male contraception. Na,K-ATPase ?4 is only expressed in male germ cells of the testis after meiosis and abundant in the sperm flagellum. Importantly, ?4 has functional characteristics that are highly unique and critical for sperm function. Activity of ?4 is essential for maintaining sperm intracellular Na+ levels ([Na+]i) and several vital sperm parameters, including membrane potential (Vm), intracellular Ca+2 ([Ca2+]i) and pH. Importantly, ?4 is crucial for sperm motility and hyperactivation, a key event associated with sperm capacitation. Accordingly, knockout of ?4 in mice results in complete male infertility. From a biochemical standpoint, ?4 has a particularly high affinity for the cardenolide ouabain. We have developed a series of synthetic compounds with steroidal and non-steroidal backbone structure, which, by targeting the ouabain binding site of ?4, selectively inhibit ?4 and affect sperm motility. These results provide strong evidence for the suitability of ?4 as a pharmacological target for the control of male fertility. The key to targeting Na,K-ATPase ?4 is that inhibition of this protein will allow to specifically interfere with sperm function, without affecting spermatogenesis and providing temporary and reversible contraception. In addition, the lack of ?4 in somatic cells reduces the chances that its inactivation will produce side effects. However, before the ?4 inhibitors can be moved forward into their application as male contraceptives, it is necessary that their efficacy, drug-target interaction, biomarkers for their in vitro and vivo specificity, side effects and mechanisms of action are identified and optimized for future clinical use. We will test this here by determining the efficacy and selectivity of action of our inhibitors on Na,K-ATPase ?4 activity, on sperm motility and on sperm parameters that are controlled by Na,K-ATPase ?4 activity, including sperm [Na+]i, [Ca2+]i, pH, Vm, sperm hyperactivation, motility and fertility. Finally, we will determine the pharmacokinetic parameters, safety and contraceptive effectiveness of Na,K-ATPase ?4 inhibitors in mating trials in mice. The rationale for the research is that once the suitability of cardenolides as targets of Na,K-ATPase ?4 is validated, both in vitro and in vivo, they can be advanced as agents for the reversible control of male fertility.

The goal of this proposal is to use morphological and biochemical approaches to analyze insulin receptor regulation. Ultrastructural studies on insulin-sensitive cells have shown significant cell-specific heterogeneity in the organization, distribution and mobility of insulin receptors. These morphological observations correlate with cell-specific differences found in biochemical binding studies on the same cells. Recent evidence suggests that cell-to-cell variability may exist in the processes involved in insulin receptor internalization and recycling. The specific aims of this proposal are: (1) to qualitatively and quantitatively determine and compare insulin receptor organization and distribution on different cell types. Relationships, if any, will be established between occupied insulin receptor and specific cell surface structures and components. (2) to analyze the post-occupancy mobility of insulin receptors on cells that demonstrate occupancy-induced changes in receptor organization or distribution. Various chemical reagents will be used in attempts to block receptor microaggregation in order to determine the mechanisms involved and relationships to insulin action. (3) to determine the routes and subcellular structures involved in the internalization, degradation or processing, and recycling of insulin and insulin receptors. In addition to using monomeric ferritin-insulin for morphological studies, immunocytochemistry using both anti-insulin receptor antibody and biotinyl-labeled insulin binding to insulin receptors followed by antibody or avidin conjugated colloidal gold particles and/or peroxidase will be used to demonstrate the insulin receptors in intact as well as permeabilized cells. This laboratory is in a unique position to accomplish this proposal because of our proven capability to perform both quantitative morphological and correlative biochemical studies. These studies will contribute to our understanding of insulin action and of possible sites for post-binding defects in insulin resistant states.

An in-hospital study involving 11 patients with chronic stable angina pectoris was undertaken to determine the effects of verapamil given alone and combined with propranolol. Patients improved exercise capacity with verapamil compared to placebo or propranolol and increased exercise time even further with the combination of verapamil and propranolol.

Older driver safety is a complex phenomenon that extends beyond the person level to multiple systems (e.g., regulatory, policy-making, societal and health care). Although a myriad of predictors for safe driving have been identified in the existing literature, they are not integrated into a meaningful causative model. Unless we use an integrated approach, grounded in a unifying public health model, we will not have taken the adequate steps to understand how behavioral, ecological, health education, administrative, policy and regulatory strategies may promote safe elderly driving. Continued neglect of these needs, accompanied with the "graying of America", could sharply increase the number of older people killed in crashes, and leave many more injured and disabled. The work I propose will develop a unifying public health model for safe elder driving. The Precede-Proceed Model of Health Promotion will provide the theoretical framework to: 1) review the current literature systematically by applying the Cochrane Collaborative Descriptive Methodology; 2) test the fit of existing population-based a) quantitative data, using epidemiological methods and structural equation modeling (SEM), and b) qualitative data using content analysis; and 3) refine the model to develop an intervention plan for safe elderly driving. This career development plan, vital to enhance my understanding of promoting safe elderly driving, includes: 1) supervised training in systematic reviews, data base analyses (SEM), model refinement and application; 2) research projects producing a narrative synthesis and meta-analysis of the older driver literature, (dis)confirmation of the Precede-Proceed model of health promotion for the older driver, and developing a pilot plan for safe elderly driving; and 3) formal didactic coursework to culminate fulfilling the requirements of this proposal. This period of supervised research and training, with the guidance from the College of Public Health and Health Professions and the College of Medicine's Departments of Epidemiology and Health Policy Research, and Biostatistics, combined with the specific supervised research proposed in this plan, will allow me to obtain novel data for application of a NIH R-03 award. This step will facilitate my transition to an independent federally funded investigator, and is directly related to my long-term goal of testing a public health model to guide a plan of assessment, prediction and promotion of safe elderly driving.

The objective of this project is to develop the existing gas target neutron source to the point where it is capable of being utilized for patient treatment. Our goal is to provide clinically useful dt neutron beam of 20 rads per minute at 125 cm SSD. To achieve this goal the present source output must be doubled and the system operation modified to increase the unit reliability. The installation of a suitable neutron shield will be followed by construction and installation of a continuously variable collimator. We plan to utilize only a fixed horizontal beam. Pertinent measurements will be made of the resulting neutron beam. The thrust of these measurements will be to form a basis permitting delivery of a radiotherapeutic neutron dose. Characteristic depth dose and isodose curves from the beam will be obtained as well as measurements of the effect source diameter and SSD of the source. Measurements will also be made of the gamma ray contamination and the strength of the neutron source as a function of operating time. Following these physical measurements experiments will be conducted to characterize the radiobiological characteristics of the beam and to permit a comparison with the cyclotron neutrons currently in use. Radiobiology studies will also establish normal tissue tolerance for this particular neutron mean and collimation system. BIBLIOGRAPHIC REFERENCE: DeLuca, P.M., Torti, R., Chenevert, G.M., Tesmer, J.R., and Kelsey, C.A., Radiation Protection Aspects of a High Flux, Fast Neutron Generator, in Ninth Midyear Topical Symposium on Operational Health Physics (Denver, Colorado) 1976, p. 568.

The ability of vertebrate skeletal muscle to regulate intracellular pH (pHi) has received very little attention, despite the fact that changes in pHi can have a marked effect on muscle contraction and metabolism. Only in two muscles, the frog semitendinosus and the mouse soleus, have the membrane transport systems responsible for pH regulation been described. In the proposed research, three questions will be addressed with the aim of defining more fully the relationship between muscle cell function and pHi regulation. i) Are the pH-regulating systems in frog muscle (Na/H and (Na+HCO3)/C1 exchangers) localized to the surface membrane, the T tubular membrane or both? Localization of the transporters will be achieved by studying: pH recover (with glass electrodes) in glycerol-shocked fibers (whose T tubules vesiculate and are no longer in contact with the external medium); pH recovery in large vesicles made from surface membrane; Na uptake into small vesicles made from either surface or T tubular membrane; and acid extrusion into the T tubular lumen, using impermeable pH-sensitive fluorescent dyes. ii) Does acidification of unstirred layers, especially the T tubular lumen, limit the rate and extent of pHi recovery? The effect of unstirred layers will be assessed by measuring recovery in single vs. multicellular preparations and in fibers exposed to varying external buffer concentrations. The effect of acid extrusion on the pH in unstirred layers will be directly determined using pH-sensitive electrodes close to the fiber surface and using pH-sensitive fluorescent dyes in the T tubular lumen. iii) How does the ability to regulate pH differ in red vs. white muscle fibers? The buffering power, the rate and extent of pHi recovery, and the properties of the transport systems responsible for recovery will be compared in mouse soleus (red) and EDL (white) muscles. These studies will represent the first localization of a carrier system in muscle membrane, the first direct measurements of pH in the T tubular lumen and the first correlation of pH-regulating capabilities with cellular metabolic and contractile properties. Beyond the basic understanding of pH regulation gained in these studies, detailed knowledge of pH regulation may shed light on the etiology and/or symptoms of the muscular dystrophies and myotonias. One of the primary lesions of these diseases is an alteration of membrane properties which results in altered contractile and metabolic processes. The role that changes in pHi regulating ability may play in such pathology remains to be studied.

Cell cycle-dependent securin proteins regulate sister chromatid separation by inhibiting separin function. We isolated and have characterized pituitary tumor transforming gene (PTTG) from rat pituitary tumor cells, and PTTG is functionally homologous to yeast securin. PTTG is overexpressed in several tumor types, and also in some normal replicating tissues (including testis, lympocytes). When the PTTG gene was deleted, resultant knockout mice were viable, fertile and exhibited splenic and testicular hypoplasia and thrombocytopenia. Surprisingly, after 6 months, male PTTG -/- mice do not gain weight, develop profound hyperglycemia, hypo-insulinemia, and hypo-leptinemia with intact insulin sensitivity. In preliminary experiments, pancreatic beta cells appear hypoplastic with diminished islet insulin immunoreactivity, and no evidence for autoimmune islet involvement. This proposal aims to study the role of mammalian securin in pancreatic beta cell function by assessing insulin transcription, secretion and action, regulation of adipocyte hormones and assessment of pancreatic beta cell development and replication, and pancreatic regeneration. As securin-deficient diabetes is restricted to male mice, intact or gonadectomized PTTG -/- animals will be treated with sex steroids, and their impact on glycemia and pancreatic function assessed. These studies highlight the role of a cell cycle-regulating protein in pancreatic beta cell development and function. In the context of this unique genetic background, these experiments identify securin as a critical factor for pancreatic cell function and provide insights into a novel monogenic cause of diabetes. [unreadable] [unreadable]

Many pathogens use the intestinal mucosa as the primary site of entry. The intestinal mucosa must induce a rapid and strong immune response to defend against such pathogenic invasion, yet an excessive immune response to commensal flora leads to chronic inflammation like inflammatory bowel disease. While it is evident that mucosal antigen-presenting cells (APCs) play a key role in orchestrating the effector response to pathogens, it is unclear how APCs maintain homeostasis with commensals. At the initiation of an immune response, mucosal APCs sense pathogens by using toll- like receptors (TLRs) that in turn signal through the adapter molecules MyD88 (myeloid differentiation factor 88) or TRIF (Toll/interleukin-1 receptor domain-containing adapter inducing IFN-). Most research has focused on the MyD88 pathway because it is used by almost all TLRs and it leads to a robust NF-B response. However, strong NF-B activation may also destroy host tissues. By contrast, the TRIF pathway is only induced by TLR4 or TLR3 and supports regulatory responses through induction of IFN. Therefore, there must be unique roles of TRIF pathway in intestinal immune regulations, but how this pathway contributes to host defense against intestinal bacterial infection has not been studied. Our ultimate goal is to protect the intestinal mucosa from infection and chronic inflammation through the future development of vaccines and immunotherapies. But fundamental research is needed to understand specific aspects of TLR signaling in the intestinal mucosa to achieve this goal. The objective of this application is to understand how APCs regulate intestinal immune responses through TRIF signaling. We wish to test the hypothesis that TRIF signaling in APCs regulates protective immune responses in the intestine. Using Yersinia enterocolitica as a model enteric pathogen, we found that TRIF-deficient mice had a significant defect in macrophage bactericidal activity. They succumb to infection due to systemic Y. enterocolitica dissemination. These findings lead us to examine how TRIF signaling in APCs regulates intestinal immune responses during bacterial infection. Once the mechanism of TRIF-mediated immune defense is elucidated, this signaling can be pharmacologically manipulated leading to innovative approaches in the management of inflammatory and infectious diseases affecting the intestine. Targeting TRIF is advantageous because a clinically tested TLR3 ligand is available.

Renal dysplasia/hypoplasia is a leading cause of renal failure in children, leading to significant morbidity and mortality associated with transplan and dialysis. The risk of chronic kidney disease is linked to decreased renal reserve as a result of the formation of fewer and/or abnormal nephrons during kidney development. While much is known about the genetic control of nephron development, very little is known about the role of microRNAs (miRNAs), small, non-coding RNA molecules that negatively regulate gene expression. Our laboratory has data demonstrating that the miR-17~92 miRNA cluster is crucial to regulating nephron number and formation. Conditional loss of miR-17~92 in nephron progenitors results in renal hypodysplasia, glomerular injury and renal dysfunction in adult mice. Moreover, we observe an intermediate phenotype in animals with heterozygous loss of miR-17~92 in nephron progenitors, suggesting that the gene dosage of miR- 17~92 is key. Heterozygous mutations in the orthologous human gene (MIR17HG) results in the first known developmental defects associated with a miRNA mutation in humans, including renal anomalies. We hypothesize that loss of the miR-17~92 cluster in nephron progenitors results in an intrinsic nephron progenitor defect, and therefore abnormal nephron number and pattern during kidney development. Aim 1. Define the role of miR-17~92 gene dosage in establishing nephron number and pattern. Aim 2. Characterize the intrinsic defect in miR-17~92 null nephron progenitors. Aim 3. Validate downstream miR-17~92 targets to elucidate mechanism(s) by which the miR-17~92 cluster regulates nephron number and patterning.

To isolate and characterize the cell surface glycoproteins and other complex cargohydrates (glycosaminoglycans, glycolipids) produced by human mammary carcinoma cells.

Neutrophils play an important role in the host defense against infection and in a number of allergic and nonallergic tissue-damaging inflammatory reactions. They migrate from blood to the site of inflammation through the process known as chemotaxis, in response to agents liberated at the site of inflammation termed "chemotactic factors". Calcium, cAMP and phospholipids and their derivatives have been considered as the possible intracellular messengers for the chemotactic activation of neutrophis. But it is not clear through what molecular mechanism the signals for the many observed metabolic alterations are achieved. An essential part for the inducing and regulating process in the functioning and responses for a variety of cells is protein phosphorylation. It is proposed to study the relation between neutrophil chemotactic activation and protein phosphorylation. (1) To define the varieties of protein kinases and their endogenous substrates in various subcellular fractions of neutrophil; (2) To define the phosphoproteins whose phosphorylation level are regulated by various chemotactic factors (f-Met-Leu-Phe, C5a and Leukotriene B4) in intact cell and to correlate the physiological responses of neutrophil to the phosphorylation level of endogenous phosphoproteins; (3) To identify the physiologically important phosphorylation systems in the neutrophil activation; (4) To search for possible regulation of the f-Met-Leu-Phe receptor protein phosphorylation; and (5) To study the possible role of vimentin phosphorylation in neutrophil chemotactic activation.

This application extends follow-up of old and very-old persons who have received cognitive interventions as part of ACTIVE (Advanced Cognitive Training for independent and Vital Elderly). Findings at five years indicate the effects of the intervention on cognitive abilities are durable and that these intervention effects nave transferred to maintenance of IADL function as hypothesized. The Specific Aims of this extended follow-up are to: 1) To determine if the cognitive interventions continue to have protective effects up to 10 years after initial training: a) basic cognitive abilities of memory, reasoning, and speed or processing; b) self- reported and performance-based instrumental activities of daily living; and c) health-related quality of life. 2) To determine if the cognitive interventions have beneficial effects on the distal outcomes of driving safety, personal care activities of daily living, health service utilization, and mortality. 3) To examine heath, genetic and cognitive moderators in individual response to training, including such factors as low cognitive function, cardiovascular status, engagement, and ApoE genotype. 4) To estimate and project the effects of ACTIVE training to the general population of older adults by linking the measures and outcomes of ACTIVE to the Health and Retirement Study. This application affords a unique opportunity to prospectively study lagged effects of three types of cognitive interventions on functional decline in this well-characterized cohort that, given its advancing age, is now at imminent risk for both IADL and ADL decline. We will be able to test the basic question in this study: For older adults exposed to these cognitive interventions, can disability in the performance of key everyday activities be delayed and independence maintained as subjects age into their 80s. We estimate power to detect effect sizes of 0.2 over 10 years for the cognitive abilities, everyday problem solving, and everyday speed and an effect size of 0.4 for everyday functioning. The ACTIVE study will determine if cognitive training can help older adults to continue functioning and living independently for a longer period of time. The ACTIVE study will determine if cognitive training can help older adults to continue functioning and living independently for a longer period of time. [unreadable] [unreadable] This application is a renewal of the application rifled "ACTIVE Phase II: UAB Field Site". This application is for the Field Site at the University of Alabama at Birmingham. Phase I of ACTIVE (Advanced Cognitive Training for Independent and Vital Elderly) was a randomized controlled trial of three cognitive intervention arms, addressing the question of whether improving basic cognition aided in maintaining functional independence in elders. As to be reported in JAMA (11/12/02), Phase I found strong, broad and durable cognitive ability-specific training effects. The effect sizes were comparable to or greater than the amount of cognitive decline observed in other longitudinal studies, suggesting that the interventions have the potential to reverse age-related decline. There was minimal transfer of training effects to everyday activities (i.e., functional competence). However, it should be noted that through the two- year followup, there was no evidence of a significant decline in ADL and IADL status. Therefore, to adequately understand the cognitive transfer effects of the training interventions, a longer followup period is required, particularly to see whether there is a separation of the change trajectories for everyday activities of trained and untrained participants over time. Phase II of ACTIVE is proposed as a followup study focused on measuring the long-term impact of training effects on cognitive function and cognitively demanding everyday activities. The Phase II followup will consist of one assessment to include the Phase I post-test battery and a clinical assessment. The ACTIVE cohort (n = 2832) is a special sample, containing substantial oversampling of African American, socioeconomically poor, and very old adults. The Specific Aims of Phase II of ACTIVE are: 1) to determine whether the cognitive interventions (as initial treatment or as a consequence of repeated boosters) have long-term protective effects on functional outcomes; 2) to document any delayed transfer of the cognitive training to secondary outcomes; and 3) to identify individual factors that affect response to intervention. As in Phase I, the primary analytical approach to detecting treatment effects on both cognitive and functional abilities will be a repeated-measures, mixed-effects model incorporating all design features as fixed effects and individual-level variability as random effects. Other multivariate analyses including lagged and cross-lagged analyses of change using latent change analysis, structural equation modeling, and growth curve analyses will also be used as appropriate to characterize relationships between individual difference factors and change in functional competence. Retention is projected conservatively at 72% with 65% of the cohort providing full data and another 7% providing partial data at year 5. Power analysis shows that extending the study will make it possible to observe effect sizes on the order of 0.05-0.10 with excellent power, in the range of at least 80-90%. [unreadable] [unreadable] [unreadable]

CRISPR-based Modular Therapy for Precision Medicine 3 CTR 6 MCB Project Summary Efforts to treat genetic diseases have suffered from insufficiently transformative clinical technologies. The recent development of CRISPR genome editing tools is paving the way for new innovations in precision medicine. In 2013, we spearheaded the first successful correction of an inherited liver disease mutation in an adult mammal using the CRISPR-Cas9 system. However, in vivo delivery of CRISPR components for disease treatment remains a challenge as we strive to reduce off target effects, provide transient editing systems and increase the rates of gene correction. In this application, we propose to develop an innovative modular delivery strategy to help address these issues. Our programmable modular approach will allow us target a large number of genetic mutations in mouse models of human disease. The modules comprise: (1) Cas9, (2) single-guide RNA (sgRNA), and (3) a DNA repair template. By breaking CRISPR into smaller components, we can use both adeno-associated virus (AAV) and non-viral vehicles to achieve transient therapeutic delivery to various organs. This new method will advance precision medicine research for an array of genetic diseases. Specifically, we will develop a Cas9 module using lipid nanoparticle delivery of Cas9 mRNA and AAV delivery of self-targeting Cas9 in order to improve the precision of genome editing. We will fine-tune DNA repair pathways in vivo to increase the rate of gene correction and explore whether inhibiting non-homologous end joining (NHEJ) can promote homology-directed repair (HDR) in non- dividing somatic cells such as pancreatic cells and neurons. We will also develop platforms for CRISPR-based modular therapy for a panel of genetic disease, especially those which require a high rate of gene correction such as alpha-1 antitrypsin (AAT) deficiency. These investigations will form the basis of a clinically-relevant platform capable of precisely targeting a wide range of disease gene mutations in somatic organs.

ANTICIPATED IMPACTS ON VETERAN?S HEALTHCARE: Knowledge generated by this proposal will inform VHA about: 1) the prevalence (and incidence) of post-colonoscopy colorectal cancer (PCCRC) in Veterans; 2) determine whether and the extent to which patient outcomes are affected relative to detected colorectal cancer (DCRC); 3) identify patient-, endoscopist-, and facility- / system-specific factors associated with PCCRC, in patients who do and who do not have one or more index polyps identified and removed. Identifying remediable factors associated with PCCRC will lead to interventions to improve colonoscopy performance and adherence to appropriate surveillance intervals, aligning with recent VHA directives for high-quality colonoscopy. Deployment of these interventions will help ensure that Veterans receive colonoscopy of the highest quality. BACKGROUND: Colorectal cancer (CRC) that occurs after a colonoscopy showing no CRC but prior to the recommended interval for follow-up colonoscopy is referred to as ?post-colonoscopy CRC? (PCCRC). PCCRC results from missed colorectal lesions, incompletely resected lesions, or from de novo, fast-growing lesions. A robust, but heterogeneous literature shows that 3-9% of all CRCs are ICCs. More limited studies show an inconsistent effect of PCCRC on patient outcomes as compared to DCRC, and attribute PCCRC to specific colonoscopy-related factors and to polyp characteristics. As the prevalence of PCCRC, its associated factors, and effect on patient outcomes have not been well-studied within VHA, we propose the following specific aims: PROJECT AIMS: 1) Quantify the a) prevalence and incidence, and b) outcomes of PCCRC in Veterans, as compared with DCRC; 2) Assess the role of colonoscopy-related factors, polyp characteristics, patient factors, and facility factors for the risk for CRC after colonoscopy a) with polypectomy, and; b) without polypectomy METHODS: Using VA electronic databases (VA Central Cancer Registry, Corporate Data Warehouse, VA- CMS data repository, VA Informatics and Computer Infrastructure, VA Vital Status File, and others), we will perform a retrospective cross-sectional study (for prevalence), a retrospective cohort study (for incidence and outcomes) and nested case-control studies (to identify risk factors). The retrospective cross-sectional study will quantify prevalence of PCCRC, using definitions consistent with the published literature and experience from other large healthcare systems in order to facilitate comparison of PCCRC prevalence with those other systems for the interval 1/1/06-12/31/2011. From all patients undergoing colonoscopy during this interval, we will calculate PCCRC incidence for Veterans with non-advanced neoplasia and no neoplasia for whom a 5- year and 10-year surveillance / rescreening interval, respectively, is recommended. Incidence and prevalence estimates will be adjusted for diagnostic-error rates, which will be based on manual medical record review. We will conduct a retrospective cohort study to compare Veterans aged 50-85 years diagnosed with PCCRC to those diagnosed with DCRC between 1/1/2006 and 12/31/2011, examining the primary outcome of 5-year overall survival and secondary outcomes of urgent hospitalization, disease stage, surgery, and 30-day post- operative mortality. Multivariate analysis will include adjustment for covariates including age, sex, rurality, comorbidity, and cancer site. For all CRC diagnosed between 2004 and 2011, we will use a case-control study (CCS) design to identify risk factors for PCCRC among Veterans ages 50-85 years who did or did not have polypectomy. Cases will be Veterans with PCCRC either following polypectomy (CCS-1) or not (CCS-2). For both CCSs, controls will be Veterans who do not have PCCRC during the same timeframe as that of the cases. Exposure variables will be procedure-related (extent of exam, preparation quality, others), endoscopist-related (specialty, level of training, others), and institution-related (volume, mechanisms for ensuring follow-up, complexity, others). Odds ratios and attributable (etiologic) fractions will be derived using multiple logistic regression and Greenland?s method for logistic regression, respectively.

This application is in response to the NIH Research Project Grant (Parent R01) (PA-10-067). As individuals age, muscle weakness and the development of neuromuscular disorders have the potential to severely diminish the quality of one's life. The larynx is part of a complex sensorimotor system that serves as both a vibratory source for phonation and as a regulatory valving mechanism that protects the airway from the incursion of foreign bodies and food. In the elderly, age-related changes in the laryngeal muscles may compromise voice quality, impairing the ability of individuals to communicate and diminishing their ability to remain socially active and engaged. More significantly, laryngeal muscle dysfunction may contribute to the emergence of dysphagia and subsequent increases in the risk of aspiration, factors that contribute to higher rates of mortality and morbidity. Our preliminary findings indicate that the intrinsic laryngeal muscles have a unique phenotype that is significantly altered by age. More specifically, therapies for certain voice disorders contend to use common principles of skeletal muscle rehabilitation in the hope of increasing muscle mass, strength, and endurance. However, the rationale and applicability of limb muscle rehabilitation concepts to the laryngeal muscles has not been empirically tested. Therefore, this proposal has three principle objectives:(1) to use chronic electrical stimulation as a fictive endurance "exercise" program in aging rat laryngeal muscle;(2) to characterize the morphological changes consequent to the fictive exercise model;and (3) to provide the background data to eventually extend these findings to clinical treatments with humans using vocal exercises. We will test the central hypothesis that the physiology of aged laryngeal muscle can be modified as a function of chronically-induced activity using the Fisher 344-Brown Norway F1 hybrid rat model of aging. We will examine to what extent chronic electrical stimulation remodels the aging posterior cricoarytenoid (PCA, vocal fold abductor) and thyoarytenoid (TA, vocal fold adductor) laryngeal muscles. Specific Aim I will examine the effects of chronic electrical stimulation on muscle morphology in aging PCA and TA. Specific Aim 2 will test whether chronic stimulation modifies the metabolic capacity in the aging PCA and TA muscles. Specific Aim 3 will determine to what extent chronic nerve stimulation will reverse functional denervation in aging laryngeal muscles. These studies will be a first test of the applicability of limb muscle rehabilitation interventions intended to effectively alleviate, reverse and/or compensate for age-related dysfunction in the laryngeal muscles. This project will generate novel data to assist in the development of interventions aimed at preventing, reversing, and/or compensating for age-related dysfunction in the human larynx, providing a first step toward testing the efficacy and legitimacy of current voice disorder interventions. PUBLIC HEALTH RELEVANCE: In older individuals, changes in the laryngeal muscles may compromise voice quality, impairing the ability to communicate and diminishing the ability to remain socially active and engaged. This project examines the muscle biophysiological changes that occur as a function of exercise and training using a well-accepted animal model for aging. Understanding the consequences of these changes is deemed important for eventually developing preventative and rehabilitative strategies for laryngeal muscle-related dysfunction in the older human population.

This proposal is intended to identify brain regions and neurotransmitter systems involved in ethanol-induced place preference. Given that the conditioned place preference paradigm has been considered a model of drug-seeking behavior, studying the neural systems involved in mediating this behavior in rodents may lead to a better understanding of drug-seeking behavior in humans. We propose to use immunohistochemistry to identify regions activated by stimuli associated with ethanol. We will then perform site-specific microinfusions of agonists and antagonists into these regions to determine if such treatment alters expression of ethanol-induced place preference.

Dyspnea or shortness of breath is the most common disabling symptom in patients with chronic obstructive pulmonary disease (COPD). The increased availability and growing acceptance of emerging technologies as a channel for health communication has opened up opportunities to provide convenient and seamless support for self-management of dyspnea and related symptoms to greater numbers of individuals. The overall goal of this proposal is to compare the impact of a new Internet dyspnea self-management program (i-DSMP) with a face-to-face program (f-DSMP) that has been shown to be effective in patients with COPD. It is hypothesized that there will be no significant differences between the i-DSMP and the f-DSMP in the primary outcomes of dyspnea with ADL, pulmonary exacerbations, exercise performance and adherence secondary outcomes of perception of social support, self-efficacy for managing dyspnea exercise, health resource utilization, and satisfaction with program at 3,6, and 12 months. Secondary aims are to relate subject usage patterns and satisfaction to changes in outcomes and to describe changes in symptom clusters and exercise patterns before, during and after acute exacerbations. Subjects with COPD who are currently using the Internet (N= 200) will be randomized to i-DSMP, f-DSMP, or Attention Control. Guided by physiological concepts, Social Cognitive Theory, and the Transtheoretical Model, the programs include: 1) initial face-to-face dyspnea interview and exercise consultation, 2) dyspnea education, 3) individualized exercise program, 4) self monitoring of exercise and symptoms, and 5) reminders and reinforcement of exercise. The i-DSMP will use online interactive learning modules, text chats, and bulletin board for communication and education and a wireless device (PDA) for monitoring symptoms, exercise, and to receive reinforcement for exercise. The f-DSMP will meet face-to-face, use written logs, and receive telephone reinforcement. If this i-DSMP is as effective as the f-DSMP, it will represent a first step in establishing an evidence-base for widespread implementation of self-management programs for people with symptoms of chronic illness using the Internet and mobile environments.

Recurrent aphthous stomatitis is characterized by painful, recurring ulcerations of the oral mucose. It is the most common ulcerative disease in many parts of the world, yet it is poorly understood. The ulcers are localized in the oral cavity and may be oral manifestations of systemic disease. The object is to test the efficacy of application of a 5% strength Amlexalox paste applied topically in healing the ulcers.

PROJECT SUMMARY This UO1 application is a response to the NIMH Program Announcement intended to accelerate the development of a high priority therapeutic agent by establishing its dose-related pharmacodynamic effects on biomarkers designed to inform subse- quent clinical development. Dopamine D1 receptor (D1R) agonism is among the most highly prioritized adjunctive treatment mechanisms for schizophrenia. Currently, all D1R agonists are also D5R agonists. D1R/D5R agonists have pro-cognitive and antipsychotic-like effects in preclinical studies, reflecting their ability to stabilize prefrontal cortical network activity in the face of distractors, and to enhance the precision of spatial working memory (sWM) by enhancing inhibitory tuning of prefrontal cortical (PFC) functional connectivity (FC). Yet, dose-related benefits of D1R/D5R agonism in patients could not be demonstrated in prior pilot studies. This application proposes that the testing of D1R/D5R agonists requires both a more direct translational/computational neuroscience framework (i.e., the most appropriate biomarkers) and a precision medicine strategy (i.e., the appropriate subpopulation of patients). To accelerate the selection of an optimal dose, we propose a multi- center study that densely maps the dose-related effects of the D1R/D5R partial agonist, PF-06412562 immediate release (IR), on three informative translational functional neuroimaging (fMRI) biomarkers as primary outcome measures: i) sWM-related activation; ii) task-based FC; and iii) resting-state FC in early course schizophrenia patients. Primary Aim 1 will apply a mul- tivariate analytic strategy to these three outcome measures (sWM-related activation, task-based FC and resting-state FC) to test if PF-06412562 produces a dose-related effect. This multivariate translational neural marker is designed and powered to inform a clear Go/No-Go decision with regards to proceeding to a full-scale clinical trial. A Go decision will be indicated if there is a significant dose-related drug effect on the neural signal measured via the multivariate combination of task-evoked activation and FC during the sWM task and FC during rest. Conversely, a No-Go decision will be reached if there is an absence of a dose-related effect on the multivariate index. Secondary Aim 2 will quantify dose-related drug effects on sWM precision based on behavioral data collected during fMRI. Exploratory Aim 3 will model the biophysical properties of PF- 06412562 in a cortical circuit model capable of sWM simulations, which will simulate hypothesized molecular mechanisms governing pro-cognitive PF-06412562 effects on sWM. In turn, we will will test if the dose-related pattern of PF-06412562 effects on resting FC in patients maps onto D1R and D5R receptor transcriptomic profiles in humans derived from from Allen Human Brain Atlas. Finally, Exploratory Aim 4 will study potential clinical predictors and moderators of PF-06412562 ef- fects on neuroimaging biomarkers. Collectively, this translational biomarker study informs the highest priority experimental treatment mechanism identified by the NIMH MATRICS Initiative using a precision medicine strategy that targets a specific subpopulation of early course schizophrenia patients who may pro-cognitively respond to D1R/D5R agonism.

Further study of human transitional cell cancer would be facilitated by propagation and positive identification of benign and malignant urothelial cells in vitro. Recent experience indicates that this is now feasible. Explant and dispersion cultures of human malignant (primary and metastatic) and non-malignant transitional tissue cells obtained at surgery will be established to identify the optimal conditions for cell growth and propagation. Microdissection techniques will be used to insure that the starting material has little adherent fibromuscular tissue. Various dispersion techniques and support media will be examined. The cell populations established will be monitored visually to insure that the morphology of the recovered cells is not that of non-malignant supporting stroma. Colony characteristics of the recovered cells will be examined both in soft agar and on chick chorio- allantoic membrane. As mammalian transitional epithelium is characterized by an asymmetric unit membrane with specialized subsurface discoid vesicles, ultrastructure studies will be conducted on these cultures to determine the persistence of this specific morphologic marker. Cloning and karotyping will be conducted as appropriate. The primary effort will be (a) to define the optimum in vitro environment for the initiation, maintainance and propagation of malignant and non- malignant urothelium, (b) to develop methodology for the positive identification of the cultured cell as urothelial in origin, and (c) to provide malignant and non-malignant urothelium and their support media for the comparative morphologic, biochemical and immunologic studies of non-malignant and malignant transitional epithelial cells.

After stroke, two opportunities to improve the patient's outcome are 1) to preserve the non-infarcted tissue in the ischemic penumbra and 2) to reperfuse severely ischemic tissue without producing further injury. To help devise useful protection strategies, we propose to investigate the factors that contribute to ischemic brain injury and propose the following hypotheses. 1) In partially ischemic tissue, electrical activity ceases but neuronal metabolism continues. With low O2, glycolysis increases to maintain ATP levels, producing lactic acid which accumulates because flow is reduced. Since ATP production by glycolysis cannot fully compensate for oxidative phosphorylation, AMP and purine levels increase so that tissue adenylates are irreversibly lost, either enzymatically or through clearance by blood. Stimulation of glutamate receptors and Ca++ influx accelerate these metabolic processes and increase tissue injury. 2) Upon reperfusion of severely ischemic tissue, depletion of tissue adenylates, through clearance by blood or enzyme activity, and propagation of oxygen free radicals, initiated during severe ischemia, add to rapid shifts of intracellular pH, Ca++ and osmolarity to worsen tissue injury. To test these hypotheses, a basic set of measurements will be made in vitro using two brain slice models of ischemia. To model the ischemic penumbra, we will use well-oxygenated 1000micro thick hippocampal brain slices. To create a model of reperfusion injury, 500micro and 1000micro slices will be exposed transiently to anoxia, unmodified or with 0 Ca++, lactic acidosis or both, and then returned to normal buffer. For both models we will 1) assess tissue injury histologically, 2) measure brain slice glucose utilization with 14C-2-deoxyglucose, 3) measure Ca++ influx with 45CaCl2, and 4) measure tissue lactate, ATP, ADP, AMP, adenosine, inosine, hypoxanthine, xanthine and uric acid. 5) We will also measure tissue hydroxyl free radicals after transient anoxia. There are four Specific Aims. 1) To test the hypothesis that persistent neuronal metabolic activity contributes to tissue injury in the ischemic penumbra, we will study well-oxygenated 1000micro and 500micro hippocampal slices during hypothermia or in the presence of NMDA, AMPA or metabotropic glutamate receptor antagonists for up to 4 hours in vitro. 2) To test the hypothesis that loss of tissue adenylates contributes to injury of the ischemic penumbra, we will study 1000micro and 500micro thick hippocampal slices in the presence of adenosine, the adenosine deaminase inhibitor deoxycoformycin (DCF) or adenosine with CDF for up to 4 hours in vitro. 3A) To test the hypothesis that reperfusion increases tissue injury after severe ischemia, we will expose 500micro and 1000micro brain slices to varying periods of anoxia to define a period of anoxia that consistently injures 50% of CA1 neurons and then study the effect of cooling slices to 33 degrees C prior to reoxygenation to determine whether the duration of anoxia or the reperfusion plays the major role in tissue injury. 3B) To further test this hypothesis and to define the relative roles of anoxia, low extracellular Ca++ and acidosis during severe ischemia, we will transiently expose 500micro and 1000micro hippocampal brain slices to anoxia, alone or with 0 Ca++, pH 6.4 or both, and determine whether these buffer modifications worsen the outcome seen with unmodified anoxia. 4) To test the hypothesis that depletion of adenylates during severe ischemia contributes to tissue reperfusion injury, we will expose 500micro and 1000micro brain slices to anoxia simultaneously with adenosine, DCF or adenosine with DCF.

Completion of the HGP (human genome program) has opened the 'Genomic Era' in biomedical research and enables functional genomics analysis of various gene elements. The long-term goal of the ENCODE Project is to identify and functionally characterize all of the sequence-based genomic elements. Functional elements that have been studied in ENCODE include transcribed sequences, regulators of transcription, and regulators of RNA transcripts themselves. The function of more than 50% of transcribed gene sequences is unknown. Most of them are expressed in specialized cells and thus their functions can only be studied in a biologically relevant context (i.e. primary cells, in vivo). In this propoal we plan to develop and commercialize a new RNAi platform that enables gene studies in a biologically relevant context with a long term goal of building a functional map of human genome. Introduction of small interfering RNAs (siRNAs) into cells with transfection reagents results in efficient gene silencing. siRNA-based functional genomics is widely used in established cell lines in vitro, but its applicability to primary cells and in vivo target validatin has been limited because of lack of efficient and non-toxic delivery systems. In collaboration with RXi pharmaceuticals we have developed a novel class of RNAi compounds - self deliverable RNAs. These small, asymmetric, hydrophobically modified RNA compounds enter cells and tissues without requirement for delivery formulation and efficiently silence genes in vitro and in vivo, enabling functional genomics studies in primary cells, embryonic cells, tissues, ex vivo, and in vivo. The major technical hurdle, which impedes wide spread use of this platform by scientific community is the complex and costly process of compound identification, synthesis and validation. The focus of this fast-track proposal is optimization of compound discovery process (Phase I) and development of a panel of 200-1000 functionally validated sdRNAs (Phase II), Completion of this proposal will build a commercially available product platform that will revolutionize functional genomics studies in biologically relevant systems such as primary cells, stem cells, tissue and organ models and eventually in vivo.

Stage I - testing the effect of an oral application of Succinylcholine on a volunteer to demonstrate the safety of the procedure and to determine the effect on taste. The starting dose was 2.5 mg in 5 ml water. It was increased to 20 mg in 5 ml water. There was no significant effect on taste sensation at these doses. The project is now pending reassessment of protocol and approval to use larger doses of the test drug.

The translocation of protons in biomolecular systems is a phenomenon of fundamental importance to such biological processes as ATP synthesis, enzyme catalysis, the maintenance of pH gradients, proton pumping, and bioenergetics. From the computational point of view the modeling of proton translocation represents a particularly difficult challenge most notably because of the many complex interactions involved, the fact that bonding topologies are continually evolving due to the Grotthuss proton shutting between water molecules and also possibly with other molecular groups, the interplay of charge migration via proton shuttling and classical ion diffusion, and the overall structural complexity of the target biomolecular systems. In most instances, the primary question is the way in which Nature utilizes the proton shuttling characteristics of hydrogen bonded water chains within proteins, as well as how specific molecular groups within the protein participate in the proton translocation process via electrostatic interactions and possibly even via direct participation in the proton shuttling mechanism itself. In this project the continued development and application of a unique and powerful computer simulation methodology is described for the study of proton translocation in several key classes of proton translocating biomolecular systems, including channels (M2 proton channels of influenza A and B, mutated aquaporin channels, and chloride/proton antiporter channels), enzymes (carbonic anhydrase), and proton pumps (cytochrome c oxidase). The overall research plan is made possible by a novel Molecular Dynamics simulation approach that allows for the study of explicit proton transport through water molecules and ionizable molecular groups in hydrogen bonded networks. A primary target in the research will be to reveal the underlying microscopic biomolecular interactions which influence proton translocation in the above mentioned systems, as well as the way in which structural and chemical modifications of the proteins can affect this important property. These studies will be carried out in collaboration with several key experimentalists, while adding a new dimension to the field of biomolecular computer simulation as a whole. 1The project concerns computer simulation studies of proton translocation in several key biomolecular systems. Proton translocation is important to understanding numerous aspects of human health, including influenza viral replication, neurodegeneration, glaucoma, metabolism, and aging.

[unreadable] An important role for the balance between pro-inflammatory and anti-inflammatory cytokines in determining the severity and progression of rheumatoid arthritis (RA) is now well established. A key role for the inflammatory cytokines tumor necrosis factor (TNF) and interleukin-1 (IL-1) has been confirmed by the efficacy of new biological therapies that block TNF and IL-1 activity. RA synovitis is also characterized by the expression of the potent antiinflammatory cytokine IL-10. A key activity of IL-10 is the suppression of TNF and IL-1 production and activity. However, it is clear that, at least during active disease, synovial IL-10 is insufficient to suppress TNF and IL-1 to the point where synovitis diminishes or resolves. The presence of autoantibodies and immune complexes in RA synovium was discovered over 25 years ago, and a prominent role for immune complexes in RA pathogenesis has been proposed. Recently, there has been a resurgence of interest in the role of humoral immunity, immune complexes, and downstream effector pathways mediated by Fc receptors for IgG (Fc?Rs) and complement in RA pathogenesis. [unreadable] [unreadable] In this application, we propose to link the emerging ideas and evidence on the importance of autoantibodies and immune complexes in RA with the established paradigm concerning the importance of balance between pro- and antiinflammatory cytokines. Our hypothesis is that ligation of Fc?Rs by immune complexes in RA synovium shifts cytokine balance toward increased production of pro-inflammatory cytokines. This hypothesis is based upon our preliminary data that Fc?R ligation suppresses IL-10 signaling, Stat3 activation, and bioactivity, thus compromising the ability of IL-10 to suppress TNF production. We have found that IL-10 signaling is blocked in RA synovial macrophages. In addition, low levels of IFN7 (which activates Statl), such as those found in RA synovium, modulate Fc?TR expression and function to more strongly inhibit IL-10 signaling, with concomitant increases in TNF production. We propose that greater understanding of the molecular mechanisms that regulate IL-10 signaling in RA, and underlie the cross talk between Fc? Rs, IL-10, and IFN 7 will yield insight into RA pathogenesis and may serve as the basis for novel therapeutic approaches. Our specific aims are to: 1. Delineate the molecular basis and pathophysiological significance of the suppression of IL-10 signaling in RA. 2. Identify mechanisms by which FcRs regulate cytokine balance in inflammatory arthritis using the K/BxN serum-induced arthritis murine model. 3. Characterize the effects of IFN7 and STATs on immune complex-induced, Fc?R-dependent arthritis, and explore underlying mechanisms. [unreadable] [unreadable]

A wide variety of marine organisms--the majority originating from the ocean off the coast of California and the rest from the Indian and Pacific Oceans--are being examined for the occurrence of novel natural products with major emphasis on steroids and terpenoids. Separation techniques employed are primarily gas chromatography (analytical as well as preparative) and high pressure liquid chromatography, while mass spectrometry (both light and low resolution) and nuclear magnetic resonance spectrometry (both proton and carbon C-13) are two of the principal physical methods used in the structure elucidation work. Synthetic studies center on methods for generating stereospecifically some of the novel side chains (e.g., cyclopropane-containing) which are encountered in marine sterols.

Accurate assessment of optic nerve and nerve fiber layer (NFL) structure is important to the early detection and timely treatment of glaucoma. The specific aims of this revised application are to: 1) develop novel structural measures of the optic nerve and NFL which are sensitive and specific for early and progressive glaucomatous optic nerve damage; 2) determine the diagnostic precision of existing and novel measures to detect the presence of early structural damage; 3) determine the diagnostic precision of existing and novel measures to detect the progression of structural damage; and 4) identify and validate methods that provide the most sensitive and specific measures of early glaucomatous structural damage. The techniques to be tested include but are not limited to: qualitative and quantitative assessment of color optic disc stereophotographs, qualitative and quantitative assessment of monochromatic NFL photographs, and scanning confocal laser image analysis of the optic nerve head and NFL. Cross-sectional analysis in age-matched normal and glaucomatous eyes will determine which techniques best detect the presence of glaucoma defined by early, reproducible visual field loss. A longitudinal study in progress will continue to follow normal subjects, ocular hypertensives and glaucoma patients to determine the sensitivity and specificity of various measures to identify change. Sensitivity and specificity will be determined for each measure and comparisons facilitated with receiver operating characteristic curves. Discriminant analysis and neural networks will be used to test the effectiveness of combined measures, and to estimate the probability that damage is present in individual eyes. Long-term follow-up of a well established cohort of subjects will enable early changes in structure to be validated by the subsequent development or worsening of visual field loss. This study will provide appropriately controlled, quantitative information to identify the most sensitive and specific methods to detect and monitor glaucoma damage in practice, and to define the most appropriate outcome measures for clinical trials and studies of glaucoma care.

Description: This project proposes two outreach programs: 1) outreach to the "Community" through an effort with people in Mexico and 2) an outreach to "Industry" through an effort in economic development and technology/information transfer. Both programs are in progress but at reduced levels because of the lack of specific persons assigned to those programs. With the passage of the North American Free Trade Agreement (NAFTA), a great deal of national attention has been directed to problems in environmental health and environmental quality at the U.S.-Mexico border region. It is clear that environmental concerns have not received the same attention in Mexico as they have in the United States but that is not because of a lack of concern by the people or the government. A major part of this problem is that there have not been any institutions or centers of environmental study which have the knowledge to implement environmental regulations similar to those found in the United States. The objective in past activities has been to encourage the development of environmental toxicology in Mexico. The proposal is to extend these initiatives in the following manner: 1) To jointly sponsor conferences with Mexican colleagues which address problems in hazardous waste and their dumpsites, particularly in the border region. 2) To maintain a directory of persons in the border region who are involved with hazardous waste research, management, risk assessment, risk communication and public policy from government, academia, industry and the general population. 3) To encourage the development of environmental toxicology degree programs, research centers and expertise at universities or research institutes in Mexico. 4) To recruit graduate students and faculty from Mexico to enter degree programs or take courses while doing research at the University of Arizona. Outreach to Industry is part of a larger outreach effort initiated by the Governor of Arizona, Fife Symington, to stimulate industrial development in Arizona. The proposal is to create a cooperative working relationship between members of Environmental Technology Industry Committee (ETIC), a private industry association, who are interested in hazardous waste risk assessment and remediation with members of the University of Arizona Hazardous Waste Basic Research Program; and to focus this interaction on addressing environmental problems along the US- Mexico border.

Natural and synthetic derivatives of vitamin A (retinoids), are active in cancer therapy and chemoprevention. Mechanisms involved in these clinical effects and why retinoid resistance occurs need to be better understood. This is the objective of this revised NIH R01-CA87546 competing renewal application that examines comprehensively, use of classical and non-classical retinoids as tools to uncover chemopreventive mechanisms. Work from the prior finding cycle revealed that all- trans-retinoic acid (RA) prevented tobacco-carcinogenic transformation of human bronchial epithelial (HBE) cells through novel mechanisms. These involved proteasomal degradation of cyclin D1 and transcriptional repression of the epidermal growth factor receptor (EGFR) that each repressed cyclin D1. The resulting G1 arrest permitted repair of carcinogenic damage to DNA and thereby conferred chemoprevention. Cyclin D1 was a molecular target for both pathways. Notably, these pathways were each deregulated in RA-resistant HBE cells that we derived and found to exhibit retinoic acid receptor-beta (RARbeta) silencing, as often occurs in lung carcinogenesis. A novel RARbeta isoform was also discovered that could account for clinical retinoid resistance. Notably, retinoid X receptor (RXR) agonists (rexinoids) by-passed this RARbeta3 block and activated similar down-stream pathways as classical retinoids to repress cyclin D1. The specific aims will: (1) comprehensively explore using cultured HBE and lung cancer cells that are sensitive or resistant to all-trans-retinoic acid (RA) whether transcriptional or post-transcriptional chemopreventive mechanisms engaged by classical or non-classical retinoids confer D-type cyclin repression. Function of the novel RARbeta isoform will be studied. Whether cooperation with demethylation agents restores RARbeta expression and retinoid response and whether repressing D-type cyclins by siRNA targeting or EGFR inhibition enhances retinoid or rexinoid response will be learned. (2) Studies that target for chemoprevention D-type cyclins in carcinogen-induced and transgenic mouse models will be conducted. (3) Findings will be translated to the clinic using unique tissues harvested from proof of principle trials that target D-type cyclins. These trials are yielding exciting preliminary findings that validate D-type cyclins as chemopreventive targets. Unique pre-clinical and clinical models and resources are available. Successful achievement of these aims will improve understanding how classical or non-classical retinoids exert chemopreventive effects. These findings would translate this work from the bench to the bedside to advance a mechanism-based approach to cancer chemoprevention. [unreadable] [unreadable]

Numerous infants born to women consuming foods contaminated with organic mercury during pregnancy have exhibited severe neurologic sequelae after birth. Because of similarity to the human with respect to reproductive parameters, fetal maturation and placental transport, monkeys (Macaca mulatta and M. arctoides) will be used for the following studies. 1) Localization of methylmercury and sites of damage in the macaque brain are being compared in the fetal and adult monkey. 2) The teratogenic potential of methylmercury in the embryonic macaque will be evaluated. 3) Potential therapeutic measures will be examined where a differentiating organism is involved. 4) Women of child-bearing age also exposed to phenylmercury and mercury vapor. Thus, measurements of placental transfer and fetal toxicity following maternal administration of these substances are to be carried out.

Morbidity in schistosomiasis is caused by the host response to schistosome eggs which are deposited in the venous system and carried to the tissues. Pathology is proportional to the number of eggs laid, and it is thus important to understand factors underlying worm fecundity and the extent to which fecundity is reflected by eggs passed in the feces, the measurable indicator of infection intensity in humans. More detailed examination of the reported decrease in the fecundity of S. mansoni in immunodeficient scid mice shows that worms are delayed in maturation, but that when mature, they lay normal numbers of eggs.

PROJECT SUMMARY Follicle-stimulating hormone (FSH) is essential for follicular development and fertility in women. Since the FSH? gene was recently reported to be associated with Polycystic Ovary Syndrome (PCOS) and age of menopause in genome-wide association studies (GWAS), we hypothesize that dysregulation of FSH? transcription contributes to female infertility including PCOS and Premature Ovarian Failure (POF). The goal of this proposal is to understand the contributions of FSH to the pathophysiology of the female hypothala- mic-pituitary-ovarian axis utilizing mechanistic analyses in immortalized gonadotrope cells, a sophisticated pri- mary pituitary perifusion system, novel genetic mouse models of PCOS and POF, and a new mouse model of PCOS. Aim 1 will focus on the impact of androgen excess on FSH? transcription and secretion in females. First, we will use primary pituitary cells in an innovative perifusion system to directly test the actions of andro- gens on basal, pulsatile GnRH- and activin-induced regulation of FSH secretion and FSH? gene expression. Then, female mice lacking AR selectively in gonadotropes will be studied for altered reproductive function including FSH? transcription and FSH secretion in response to excess androgens using a letrozole-induced PCOS mouse model. Finally, we will investigate whether decreased FSH production in our letrozole PCOS mouse model is dependent on GnRH or due to changes in the activin autocrine feedback loop. Aim 2 will char- acterize the functions of single nucleotide polymorphisms (SNPs) found within the regulatory sequences of the FSH? gene that are associated with PCOS, age at menopause, and altered LH/FSH ratios in GWAS studies. We have shown that one of these SNPs interferes with LHX3 binding to the proximal FSH? promoter and reduces FSH? gene expression. These SNPs will be analyzed for molecular mechanisms altering FSH? transcription. We will then use CRISPR/Cas9 mutagenesis to create mouse models to determine the effects of these SNP alterations on fertility and FSH secretion in vivo and in primary pituitary cells. Aim 3 will investigate the role of FOXL2 in FSH? regulation and POF. We have shown that FOXL2 interacts with SMAD proteins for activin induction of FSH? gene expression, with cJun for synergy between GnRH and activin, and with proges- terone receptor for synergy between activin and progesterone. First, we will investigate the molecular basis for these interactions and their roles in hormonal regulation of FSH?. Next, FOXL2 mutations found in POF will be studied for their effects on FSH? transcription in coordination with activin, GnRH, and steroid hormones. Lastly, these mutations will be modeled in mice using CRISPR/Cas9 mutagenesis to study effects on fertility and reproductive senescence in vivo and on FSH? gene regulation in primary pituitary cells. Together, these aims will delineate the mechanisms by which androgens and SNP mutations within the FSH? regulatory region alter FSH? gene expression and contribute to PCOS, and how dysregulation of FSH? by mutant FOXL2 proteins contributes to POF. These studies may provide insight for diagnosis and treatment of female infertility.

DESCRIPTION (not provided by the applicant): [unreadable] [unreadable] [unreadable] [unreadable]

We have studied the application of specimen pooling to DNA from case-parent triads when the genotyping assay counts the number of variant alleles in a pooled specimen. Our procedure partitions a sample of triads into small subsets of, say, two triads each, and, for each subset, constructs three pooled DNA specimens: one each from mothers, from fathers, and from offspring. Treating the individual genotypes that comprise each pool as missing data, our log-linear-modeling approach uses the expectation-maximization algorithm to estimate relative risk parameters for inherited alleles, maternal alleles, or parent-of-origin effects, something other DNA pooling approaches cannot do. We see little loss of power compared to genotyping individuals when genotypes are measured without error, but power declines as genotyping error rates increase. For sufficiently accurate assays, our approach promises to reduce genotyping costs with minimal loss of power. Genome-wide association studies usually focus on variation in autosomal genes; however, less studied genetic mechanisms may also influence risk for complex disease. We have studied three mechanisms that can induce differential risk in maternal versus paternal lineages of affected individuals. These include mitochondrial variants (inherited from the mother), imprinted genes (where the effect of variants depends on the parent of origin), and maternal genes (that influence risk through modulation of the prenatal environment). Asymmetries in risk can be measured by inter-lineage relative risks or relative odds. We define the relative risks of interest; and, under certain simplifying assumptions, we derived algebraic expressions for the inter-lineage relative risks predicated on commonly used risk models. We find that careful analyses of family history data provide clues about whether these mechanisms contribute to risk but dissecting which mechanism may be operating requires genotype data on family members. When using case-parent triads to study the association of single nucleotide polymorphisms with disease, the phenotype of the child is used but the phenotypes of the parents are ignored but all three family members are genotyped. When parental phenotypes for the disease under study are available, including them in the analysis should bring more information to bear on disease-gene associations. We are in the process of developing and evaluating new approaches for using parental phenotypes together with the usual data from case-parents studies to increase power for detecting associations. (see also Z01 ES040007 BB; PI Clare Weinberg.)

The specific aims of the project are (1) to determine the types of impairments of emotional expression and perception that are found in autism, and (2) to investigate the neuropsychological bases of these impairments. Measures will be taken on three groups of subjects: autistic children ranging from 6 to 18 years of age, and developmentally-matched normal and mentally retarded children. These three groups will be compared in terms of their ability to perceptually discriminate and categorize facial expressions, and in terms of their spontaneous expression of various emotions. Measures of spontaneous emotional behavior will be taken in both a controlled laboratory setting and in naturalistic school situations. Parents will also provide information about their child's emotional behavior in a questionnaire The above measures will be used to investigate whether individual differences in affective and social ability are related to specific patterns of frontal and parietal lobe functioning. Electroencephalographic (EEG) recordings of right and left frontal and parietal activity will be taken in response to a variety of affective and cognitive stimuli. EEG will be subjected to power spectrum analysis to yield measures of alpha band power frequency (8-13 Hz). Measures of right and left parietal and frontal integrated alpha will be used to infer differential cortical activation during affective and cognitive processing. Comparisons of patterns of brain activity will be made among autistic, and matched mentally retarded and normal subjects. Specific predictions regarding the pattern of brain dysfunction expected in autistic subjects are based on current neuropsychological models of emotion. The long term objectives of the research program are to shed light on the neuropsychological bases of early infantile autism and to use this information to develop more effective diagnostic and treatment methods for autistic persons.

Human neonates use vision to guide behavior, form caregiver attachments, and build more complex visual and cognitive abilities. Defining the mechanisms by which vision comes 'online' in time for birth is thus essential to understand the development of normal and pathological visual processing. Visual perception requires the development of organized receptive fields as well as the development of cortical states that generate visual alertness and attention. Animal models have provided a mechanistic understanding of the former, but insight into the mechanisms by which alert visual response dynamics develop has been limited by a paucity of unanesthetized animal models with demonstrated homology to fetal and perinatal human cortical activity. To overcome this limitation, my collaborators and I have made a significant investment in the characterization of a behaving infant rat model that recapitulates early human cortical activity development. We propose to use this model to identify the network mechanisms that drive the development of cortical alertness, and characterize their role in the perinatal functional maturation of the visua response. In prior studies we identified a rapid maturation of visual cortical activity occurring 23 weeks before term (birth) in humans and 1-2 days before eye opening in rats. Before this switch cortical activity is dominated by network silence, interrupted by infrequent, large amplitude oscillatory bursts that occur both spontaneously and in response to light. The electrographic signature of alertness typical of the adult waking state, namely the activated or desynchronized state in the EEG, is not observed at these ages even when the infant is clearly awake. This period ends suddenly when oscillatory bursts are replaced by fast visual responses superimposed on an activated cortical state during wakefulness, which we call visual alertness. We will test the hypothesis the emergence of visual alertness is the result of rapid development of feed-forward cortical inhibition and a surge in norephinephrine (NE) release occurring just before eye opening. Our results will provide a novel understanding of how changing cortical network properties influence development of vision, and how ontogenetic control of the timing of these properties is achieved. We expect this information will inform the diagnosis and treatment of central visual and attention deficits prevalent in pre-term and other at risk infants.

Action of Fra-2: FRA-2/FOSL2 is a basic region-leucine zipper motif transcription factor that is widely expressed in mammalian tissues. The functional repertoire of this factor is unclear, partly due to a lack of knowledge of genomic sequences that are targeted. Here, we identified novel, functional FRA-2 targets across the genome through expression profile analysis in a knockdown transgenic rat. In this model, a nocturnal rhythm of pineal gland FRA-2 is suppressed by a genetically encoded, dominant negative mutant protein. Bioinformatic analysis of validated sets of FRA-2-regulated and -nonregulated genes revealed that the FRA-2 regulon is limited by genomic target selection rules that, in general, transcend core cis-sequence identity. However, one variant AP-1-related (AP-1R) sequence was common to a subset of regulated genes. The functional activity and protein binding partners of a candidate AP-1R sequence were determined for a novel FRA-2-repressed gene, Rgs4. FRA-2 protein preferentially associated with a proximal Rgs4 AP-1R sequence as demonstrated by ex vivo ChIP and in vitro EMSA analysis; moreover, transcriptional repression was blocked by mutation of the AP-1R sequence, whereas mutation of an upstream consensus AP-1 family sequence did not affect Rgs4 expression. Nocturnal changes in protein complexes at the Rgs4 AP-1R sequence are associated with FRA-2-dependent dismissal of the co-activator, CBP; this provides a mechanistic basis for Rgs4 gene repression. These studies have also provided functional insight into selective genomic targeting by FRA-2, highlighting discordance between predicted and actual targets. Future studies should address FRA-2-Rgs4 interactions in other systems, including the brain, where FRA-2 function is poorly understood. (From Davies et al 2011) cAMP control of AANAT transcription: Arylalkylamine N-acetyltransferase (AANAT) is the key regulatory enzyme controlling the daily rhythm of melatonin biosynthesis. In chicken retinal photoreceptor cells, Aanat transcription and AANAT activity are regulated in part by cAMP-dependent mechanisms. The purpose of this study was to identify regulatory elements within the chicken Aanat promoter responsible for cAMP-dependent induction. Photoreceptor-enriched retinal cell cultures were transfected with a luciferase reporter construct containing up to 4 kb of 5'-flanking region and the first exon of Aanat. Forskolin treatment stimulated luciferase activity driven by the 4 kb promoter construct and by all 5'-deletion constructs except the smallest, Aanat (-217 to +120)luc. Maximal basal and forskolin-stimulated expression levels were generated by the Aanat (-484 to +120)luc construct. This construct lacks a canonical cyclic AMP-response element (CRE), but contains two other potentially important elements in its sequence: an eight times TTATT repeat (TTATT(8) ) and a CRE-like sequence. Electrophoretic mobility shift assays, luciferase reporter assays, chromatin immunoprecipitation, and siRNA experiments provide evidence that these elements bind c-Fos, JunD, and CREB to enhance basal and forskolin-stimulated Aanat transcription. We propose that the CRE-like sequence and TTATT(8) elements in the 484 bp proximal promoter interact to mediate cAMP-dependent transcriptional regulation of Aanat. (From Haque et al 2011)3)