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Genetic mutations in genes encoding presenilins (PS1 and PS2) are responsible for the majority of early-onset familial AIzheimer's disease (FAD). Extensive studies in the past few years focusing on the mechanisms by which mutant PS promote AD pathogenesis have demonstrated that PS1 is a key component of gamma-secretase complex, an unusual protease catalyzing the intramembrane cleavage of amyloid precursor protein (APP) and generating pathogenic small peptide Abeta. Nascent full length PS1 polypeptides undergo endoproteolytic cleavage to produce N-terminal and C-terminal fragments (NTF and CTF) which associate with each other to form functional heterodimer. Early studies about PS1 metabolism suggest that unknown cellular factors tightly regulate biogenesis of PS1 heterodimer. However, identity of these limiting factors as well as other members of gamma-secretase complex has remained elusive. Through biochemical and genetic approaches, recent discoveries of three essential proteins (Nct, PEN-2 and APH-1) for gamma-secretase activity, provide us candidates for those hypothesized factors. Indeed, three of them have been demonstrated to be components of high molecular weight gamma-secretase complex, while their precise functions are unclear. My preliminary results demonstrated the regulatory roles of PEN-2 and APH-1 in PS1 cleavage and Nct maturation. This proposal is designed to further investigate the biological functions of PEN-2 and APH-1 in regulating PS1/gamma-secretase complex formation via examining PEN-2 and APH-1 structure/function relationship (Specific Aim 1), their impact on protein trafficking (Specific Aim 2), as well as protein folding and intermolecular interactions (Specific Aim 3) of PS1 and Nct.

Neuronal nicotinic cholinergic receptors are expressed throughout the brain, in the spinal cord, and in the autonomic ganglia. Eleven neuronal nAChR subunit genes were cloned and sequenced in the 1980's. Some of these subunits closely resemble the a1 subunit that is included in the nAChR that is expressed at the neuromuscular junction (the so-called peripheral-type receptor). These a1-like subunits are called: a2, a3...a10 and, with the exception of a5, provide the binding site for nicotine and other nicotinic agonists. The three remaining subunits, [unreadable]2-[unreadable]4, are referred to as structural subunits. Enormous progress has been made towards understanding the structure and function of neuronal nAChRs using expression systems (cell lines, Xenopus laevis oocytes). For example, expression system studies have shown that subunit composition has profound effects on biophysical and pharmacological properties. The value of expression system studies has been limited, somewhat, because some of the more interesting subunits (e.g. a6, [unreadable]3) are not easily expressed in artificial systems. These, and other concerns, have prompted researchers to develop gene knockout (null mutant) mice for virtually every one of the known nAChR subunits. Transgenic mice have also been developed that express several gain of function mutations, or mutations that are associated with human diseases. These transgenic mice are being used to address questions such as: 1) What are the subunit compositions of naturally-occurring (i.e. native) nAChRs? 2) Where are these native receptors expressed? 3) What role do they play in modulating brain function (behavior?), and 4) What function do these receptors play in modulating addiction to nicotine, alcohol and other drugs? We have the world's most complete collection of nAChR mutant mice that are being used in several funded research projects that are centered at the University of Colorado. During the last 4 years we have built our colony from five mutant strains to 18 and have established a system where we have sent mice and/or breeding pairs to other researchers in the US and, more recently around the world (3 continents, to date). This application requests funds to support the continued maintenance and distribution of these mouse stocks.

Temporal and spatial expression of laminin (LN) isoforms is critical to glomerular development and contributes to altered cell function in diabetic nephropathy and other progressive renal diseases. Yet, little is known about the mechanisms that regulate LN isoform expression. We propose that insulin-like growth factor binding protein-5 (IGFBP-5) leads to formation of a filamin-based nuclear shuttle that binds transcription factors (e.g. sox9, GKLF, Sp1, Smad) and transcriptional co-activators (e.g., IGFBP-5 and LIM domain proteins such as FHL2) and regulates laminin (LN) isoform expression in mesangial cells (MC). This hypothesis suggests a novel mechanism whereby perturbations in the cell-matrix interface regulate gene expression. This hypothesis will be examined by: Specific Aim 1: To characterize the generation and composition of the filamin nuclear shuttle following treatment of MC with IGFBP-5. First, we will demonstrate that a fragment of filamin forms and translocates to the nucleus. Second, we will characterize the transcription factors that are recruited to the complex; and finally, we will evaluate the role of transcriptional co-activators, LIM domain protein FHL2 and IGFBP-5, in the complex. Specific Aim 2: To demonstrate that nuclear accumulation of filamin is required for IGFBP-5-mediated changes in LN gene expression. First, we will demonstrate that filamin is required for IGFBP-5-mediated effects on LN gene expression. Second, we will show that the filamin shuttle is recruited to LN gene loci where it specifically modifies LN gene transcription. Specific Aim 3: To define the mechanisms whereby the filamin nuclear shuttle activates LN gene transcription. First, we will determine if FLN interacts directly with the LN promoter and if it drives transcription. Second, we will define the elements in the shuttle that are required for transcriptional control. These studies will define the role of a filamin-based nuclear shuttle in mediating the effects of IGFBP-5 on laminin gene expression. Understanding transcriptional control of LN isoform expression will add new insights into the role that LN plays in normal glomerulogenesis and that becomes disordered in glomerular disease. [unreadable] [unreadable] [unreadable]

Chronic sinus disease affects over 31 million Americans, and surgery for this disease is performed over 200,000 times annually. Success in functional endoscopic sinus surgery (FESS) is frequently limited by poor wound healing and scar contracture. Sentrx Surgical, Inc., a University of Utah startup formed to commercialize in situ crosslinkable glycosaminoglycan hydrogels for ear, nose and throat and craniofacial surgery, proposes to establish the feasibility of using in situ crosslinkable hyaluronan gels to improve sinus surgery outcomes. Preliminary evidence demonstrates that HA gels can inhibit collagen contracture by a process we describe as "nanostenting". Commerically available HA products have been shown to promote inflammation and actually worsen scarring. The crosslinked HA gels to be studied herein show no evidence of inflammatory response in preliminary studies. In Phase I, Sentrx Surgical will partner with the University of Utah to establish the chemical and biological parameters that will provide feasibility for identifying the optimal nanostenting HA hydrogel to use in human clinical trails. Chemical modifications will be made to the HA gels to enhance their rigidity and nanostenting properties. The biomechanical properties of these gels will be measured. The nanostenting HA gels will then be tested in an established animal model by measuring their relative ability to prevent scar contracture. Histological analysis will be performed to ensure a continued absence of inflammation and promotion of normal wound healing. The results of these Phase I studies will determine the feasibility and the optimal chemical properties of a nanostenting HA gel for human trials.

A longstanding issue in environmental health is the need to understand the role the environment plays in human brain development. The brain of the neonate is particularly susceptible to disruption of the sensory environment, which can have profound effects on its physiology and morphology. Such susceptibility of the developing brain to environmental influence by sensory manipulation or to environmental toxicants is particularly pronounced during defined critical periods of postnatal life. On the one hand, this susceptibility makes the developing brain particularly vulnerable to toxic insults. On the other hand, the plasticity of the connections between neurons, or synapses, is critical for refining brain circuitry during postnatal development. Similar mechanisms for changing synapses are likely to serve the basis for learning in the adult. Our primary interest, therefore, has been to determine the molecular basis of long-lasting synaptic plasticity. Toward our goal of learning how neuronal activity can induce lasting modifications in neurons, we use a diverse collection of molecular, biochemical, electrophysiological, and imaging techniques. We use the hippocampal slice preparation from neonate and adult mice. The relatively simple laminar structure of the hippocampus, which itself plays an important role in learning and memory, allows electrophysiological studies to be performed easily. To measure synaptic plasticity, we use techniques that include whole-cell patch clamp recordings from hippocampal slices maintained in vitro, and stimulate them either electrically or pharmacologically to induce long-term potentiation (LTP) or long-term depression (LTD). To determine how transcription is regulated in different hippocampal subfields, we use molecular and biochemical methods with tissue harvested by laser capture microdissection (LCM). In addition, we now use in vivo recording and tracing techniques to address the role of the different hippocampal subregions in specific behaviors. One approach that we have taken to gain insight into the mechanisms of regulating synaptic plasticity has been to compare highly plastic brain areas, such as the CA1 area of hippocampus, with less plastic areas. From the expression pattern of some genes, we predicted and found that one area of the hippocampus, the CA2, would have a resistance to synaptic plasticity including LTP and LTD, even though we found that synaptic responses in CA2 were very similar to those in the neighboring CA1 and CA3 areas. We later established that dendritic spines in CA2 have very different calcium dynamics from spines in CA1 and CA3 in that both calcium buffering capacity and rates of calcium extrusion were higher in CA2 spines when compared with those in the neighboring regions. In addition, we have found that a regulator of G-protein signaling (RGS-14), and adenosine A1 receptors (A1Rs), which are highly enriched in CA2, are also negative regulators of plasticity in CA2. We have also found that the social neuropeptides oxytocin and vasopressin act to induce synaptic potentiation in CA2 pyramidal neurons in a way that closely resembled typical LTP. The vasopressin 1b receptor (avpr1b) is highly enriched in CA2 neurons over all other parts of the brain, suggesting that CA2 may play an important role in social behavior; recent work in other laboratories support this view. To gain further insight into the molecular profile of CA2 neurons, we used RNAseq to measure mRNA from specific cellular compartments of tissue isolated by LCM. These studies have revealed that CA2 neurons express an unusually large number of genes related to mitochondrial function. Using the information we have gained from studying CA2, we also aim to determine the nature of the developmental down-regulation of synaptic plasticity in the form of critical periods and how plasticity is modulated in different brain areas. Recently, we have found that a specialized extracellular matrix, called perineuronal nets (PNNs), play a role in limiting synaptic plasticity in area CA2. We found that in mouse CA2, PNNs surround pyramidal (excitatory) neurons and their excitatory synapses on dendritic spines. Importantly, we found that staining for PNNs increases during postnatal development and could be modified by early-life enrichment. Because PNNs had been previously implicated in limiting synaptic plasticity late in postnatal development, effectively ending critical periods for plasticity, we tested whether PNNs also play an important role in restricting synaptic potentiation of the normally plasticity-resistant excitatory CA2 synapses. We found that treatment of hippocampal slices with an enzyme that disrupts PNNs enables synaptic potentiation in CA2 pyramidal neurons. CA2 and its surrounding regions are anatomically and molecularly very similar, and as such, these findings may therefore lead to identification of other critical molecular components in the pathways necessary for developmental changes in synaptic plasticity and development of normal cognition. Prior to our work on synaptic plasticity in area CA2, few groups had appreciated that the area was in fact a distinct region of the hippocampus and as a result, nothing was known about the how neurons in area CA2 function during even the simplest of behaviors such as exploration of an open field. We therefore recorded neuronal activity of CA2 neurons in vivo in rats during exploration and social interactions and found that CA2 neurons are uniquely sensitive to changes in social context and other situations involving novelty, suggesting that neurons in this area may be providing downstream regions with information relevant to changes in context. We next sought to determine how CA2 activity may regulate hippocampal electrical oscillations, which arise from coordinated activity among distinct populations of neurons and are associated with cognitive functions. Using the controlled experimental system of excitatory and inhibitory Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) to manipulate CA2 neuronal activity in mice, we studied hippocampal-prefrontal cortical network oscillations. We found that modification of CA2 activity increased or decreased hippocampal and prefrontal cortical low-gamma oscillations with excitatory and inhibitory DREADDs, respectively, and that CA2 activity inversely modulated hippocampal ripple oscillations. These findings support a role for CA2 in low-gamma generation and ripple modulation within the hippocampus and underscore the importance of CA2 in extrahippocampal oscillations. As increasing evidence is implicating area CA2 in several types of rodent social behavior, including aggression, these findings have relevance to human psychiatric disorders such as autism and schizophrenia that manifest with deficits in social cognition. Together with our studies on the cellular mechanisms underlying synaptic plasticity, these studies will give us a better understanding of how experience during development shapes brain circuitry.

Establish the relationship between EEG, sleep, sympathetic tone and immune function, characterize the nocturnal secretary profile of lymphokines, neuroendocrine hormones and neurotransmitters, examine catecholamines in psychaitric patients with sleep disturbance, and test the link between sleep, neuroendocrine and sympathetic measures and immunologic responses using partial sleep deprivation.

Determining protein structure and function from genomic sequences and protein classification remains one of the most significant challenges in modern computational biology. Significant enhancement to the capacity of algorithms to predict protein shapes from sequences is proposed, focusing on major bottlenecks; e.g., the folding energy and the ability of making approximate matches. Algorithms to determine protein shapes from sequences have two major components: The first component (sampling) generates a set of plausible protein shapes; at least one of the sampled shapes is expected to be similar to the correct fold. The second component scores the different structures and decides on the best model. The radius of convergence of the energy function must be sufficiently large so that approximate matches will be detected as well (in threading approximate matches may include deletions and insertion). It is therefore clear that poor scoring functions (or energies), which are unable to identify the correct fold, are likely to diminish the capacity of the folding algorithm. At present, it is easy to generate a set of decoy (wrong) structures that will confuse existing energy functions. Mathematical programming and machine learning techniques (Support Vector Machines) will design enhanced folding and threading potentials. The training by these methods is automated and will lead to monotonic improvement in recognition as a function of the data size. To more effectively cover protein space, the goal is to learn 100 million data points in a single consistent potential with (at most) 10,000 parameters. The automated large scale learning is crucial at times in which the information on sequences and structures grows rapidly. A threading prediction server, based on the old and the new potentials, is and will be available, to the community at http://ser-loopp.tc.cornell.edu/Ioopp.html [unreadable] [unreadable]

The gastrointestinal epithelium functions as a dynamic barrier that serves as an interface between luminal contents and underlying tissue compartments, and is thus vital in maintaining mucosal homeostasis. Mucosal wounds have been observed following enteric infection, inflammatory bowel disease and ischemic insults. Disruption of the critical epithelial barrier allows access of luminal contents to immunologically privileged compartments thereby contributing to disease pathogenesis. In response to injury, intestinal epithelial cells (IEC) migrate and proliferate to rapidly cover denuded surfaces and re-establish the epithelia barrier. After identifying N-formyl peptide receptors (FPR1 and FPR2) in the intestinal epithelium, our studies suggest that FPR1 ligands including endogenous lipid/proteins and exogenous microbiota control intestinal epithelial homeostasis and repair. Thus, the proposed studies will further explore mechanisms by which these FPR ligands control restitution of the mucosal barrier. The proposed studies will not only provide a better understanding of basic mechanisms by which FPRs regulate epithelial repair, but will also aid in the development of new therapeutic strategies aimed at promoting healing of the injured mucosa.

Antinuclear antibodies are a central feature of SLE and related rheumatic diseases, and are thought to be directly involved in the pathogenesis of these disorders. The antigens recognized by these autoantibodies are generally components of large DNA-protein or RNA-protein particles. It has been shown that Th clones inducing the production of pathogenic anti-DNA antibodies are responsive to DNA-histone complexes, but not to either DNA or histones alone. We have found that autoantibody production and the activation of autoreactive T cells specific for a chromatin protein can be triggered by alterations in its quaternary structure induced by the binding of a viral protein. Thus, the quaternary structure of an autoantigen may be critical for the presentation of epitopes recognized by autoreactive T cells. In view of the importance of quaternary structure in generating autoreactive T cells, it seems worthwhile to characterize the interactions of autoantigenic chromatin proteins with other self and nonself antigens. This competitive renewal application will extend our previous work on the Ku (p70/p80) heterodimer, a nonhistone chromatin antigen recognized by autoantibodies found in the sera of certain patients with systemic lupus erythematosus (SLE), systemic sclerosis (SSc), and overlap syndromes. The basis for autoimmunity to this autoantigen in human disease will also be examined. We hypothesize that the binding of either foreign antigens or autoantibodies to certain functional domains of Ku or other antigens may trigger autoimmunity by enhancing the presentation of cryptic T cell epitopes of self to which tolerance is incomplete. In Specific Aim 1, the domains of Ku mediating p70-p80 dimerization and binding to a 350 kDa protein (p350) with DNA-dependent protein kinase activity will be determined using prokaryotic and eukaryotic expression systems and specific monoclonal antibodies generated previously in our laboratory. Parallel studies will address the question of whether the same domains are involved in interactions with foreign (viral) antigens that might potentially trigger autoimmunity. In Specific Aim 2, the importance of these functional domains as targets of autoantibodies in SLE, SSc and overlap syndrome will be determined. The HLA associations of autoantibodies to individual epitopes of Ku will be determined, and the basis for the association of anti-Ku, anti-Su, and anti-RNA polymerase II autoantibodies in many sera will be explored. Finally, the possibility that autoantibodies of particular specificities can spread autoimmunity from one antigen to another by altering antigen processing will be investigated. Although the latter studies are exploratory in nature, they may provide direct evidence that the binding of certain autoantibodies, like some foreign proteins, can trigger autoimmunity by altering the quaternary structure of an autoantigen.

The objective of this project is to provide an answer to a major question concerning the sarcoplasmic reticulum, namely what is the mechanism of calcium transport through the phospholipid bilayer. The role of protein through the membrane will be investigated in which multiple subunit aggregates of the Ca ion-activated ATPase mediate calcium transport. Protein aggregate formation will be determined by chemical crosslinking followed by analysis of crosslinked proteins by sodium dodecyl sulfate gel electrophoresis, and freeze-fracturing electron microscopy. These methods will be used to look for changes in the oligomeric state of membrane proteins as the Ca 2 ion-activated ATPase is interconverted between the different enzymatic states that accompany calcium accumulation. To determine the role of Calsequestrin and Calcium Binding Protein, possible interactions between these minor protein components and the Ca ion-activated ATPase in terminal cisternae-derived sarcoplasmic reticulum vesicles will be investigated. The calcium pumping activity of cardiac sarcoplasmic reticulum can be stimulated 3-fold by the action of a cAMP-dependent protein kinase. To determine whether this stimulation is mediated via protein-protein interactions, the relationship between the many proteins in cardiac sarcoplasmic reticulum and the Ca 2 ion-activated ATPase will be investigated as the extent of kinase-mediated phosphorylation is varied. In addition interactions between the protein phosphorylated by the kinase, Phospholamban, and the Ca 2 ion-activated ATPase will be looked for to determine the mechanism by which this phosphorylation can increase Ca 2 ion-activated ATPase activity. The goal of this project is to show whether ionic channels in membranes can be regulated by changes in monomer-oligomer equilibria of membrane proteins.

Project Summary The major objective of the Biostatistics and Bioinformatics Core (Core 3) is to provide centralized biostatistics, bioinformatics, and database support for all Projects and Cores. Core 3 will provide guidance in the design and conduct of clinical trials and other experiments that arise from the ongoing research of the SPORE, facilitate prospective collection, entry, quality control, and integration of data for the basic science, pre-clinical, and clinical studies, and provide bioinformatics data analysis of high-throughput and high-dimensional genomics data. We will provide innovative and tailored statistical modeling, simulation techniques, and data analyses for the main projects, developmental research and career enhancement projects, and other cores to achieve their specific aims. We will conduct data analyses and prepare statistical reports for all experiments within all projects, ensure that the results of all projects are appropriately interpreted, and assist all project investigators in the publication of scientific results. Core 3 will also be a resource for intra- and inter-SPORE collaborations, including study design and developing databases for multi-center clinical trials.

Natural killer (NK) cells are nonimmune cytotoxic effector cells that have the capacity to lyse certain types of NK-sensitive tumor cells. NK cells are thought to participate in immune surveillance. Therefore, elucidating the NK lytic mechanism, as well as the mechanism whereby some tumor cells resist lysis by NK cells, may provide new ideas that can be applied to cancer therapy. Evidence indicates NK target cell lysis is mediated by natural killer cytotoxic factors (NKCF) released from the NK cell following contact with the target cell. Studies using NK-resistant variants derived from the initially NK-sensitive YAC-1 cell line have indicated there are at least four characteristics that determine NK sensitivity in a tumor cell. Those are: (1)\expression of the NK target structure; (2)\ability to stimulate release of NKCF; (3)\membrane receptors for NKCF; and (4)\sensitivity to lysis by NKCF. Based on these studies, we intend to analyze the role of NKCF receptors in the murine NK lytic mechanism using the NK-sensitive YAC-1 tumor cell as well as NK-resistant variants derived from YAC-1. Two of these variants (YAC-R15.1 and YAC-6-28) do not express NKCF receptors, whereas YAC-asc cells do express the receptor, although they are resistant to lysis. Monoclonal antibodies will be generated against the NKCF receptor and will be used to study the function of YAC-1 NKCF receptors as well as those expressed by the NK-resistant variants. Fluorescinated antibodies will be used to study the expression of NKCF receptors and to determine if the receptor may undergo endocytosis. Other studies will examine the morphological alterations both YAC-1 cells and the resistant variants undergo after exposure to NKCF. The effects of metabolic inhibitors will be tested to determine if NK-resistant variants engage in repair processes to counteract lysis by NKCF. Finally, techniques will be developed for the isolation and purification of NKCF receptors using the monoclonal antibodies in combination with conventional biochemical techniques. These studies will help to elucidate the NK lytic mechanism at the level of the NKCF-receptor interaction as well as to analyze mechanisms whereby tumor cells may resist lysis by NK cells. (CS)

The objective of this proposal is to provide increased understanding, at the molecular level, of the mechanisms of activation of intrinsic blood coagulation, kinin generation and fibrinolysis. Factor XII activation induced by negatively charged substances, as well as interactions among factors XII and XI, prekallikrein, high molecular weight kininogen and plasminogen, will be investigated. These studies will utilize fluorescence polarization, fluorescence resonance energy transfer, enzyme kinetics, and rapid reaction stopped-flow kinetic techniques. Quantitative data will be obtained for the stoichiometries, affinities and rates of the various interactions involved, using purified systems as well as normal and factor-deficient plasmas. It is anticipated that successful completion of this project will result in enhanced understanding of the molecular mechanisms of contact activation, as well as the relative importance of each factor XII-dependent pathway.

Kaposi's sarcoma-associated herpesvirus (KSHV) is the apparent etiological cause of Kaposi's sarcoma, a neoplasm of frequent occurrence in HIV infected individuals. The KSHV genome is present in the spindle cells of virtually all KS lesions of both HIV positive and negative cases, and there is an excellent correlation between seroconversion to KSHV and development of KS. This herpesvirus is also strongly linked to two specific lymphoproliferative disorders, Primary Effusion Lymphoma (PEL) and Castleman's disease (CD). Less certain is the suggested role of KSHV in multiple myeloma. The mode of KSHV transmission is unknown and the virus may not be ubiquitous. The viral genome, now completely sequenced, encodes homologues of cellular cytokines and growth factors. Since standard herpesvirus drug treatments (e.g.., acyclovir) are not effective against KSHV, there is heightened interest in developing new KSHV antivirals. One promising new antiviral target is the DNA polymerase (Pol-8) and Processivity Factor (PF-8) complex of KCHV. PFs associate with their cognate DNA Pols, enabling them to synthesize extended stretches of DNA without dissociating from template. The PF genes of certain other herpesviruses are known to be required for viral DNA synthesis and infection. Indeed, only a few dNTPs are incorporated into DNA by Pol-8 alone in vitro, but when combined with PF-8, thousands of dNTPs are incorporated. Moreover, Pol-8 complexes and functions with only PF-8 and not PFs of other herpesviruses. The specificity of the PF-8/Pol-8 interaction, which is necessary for DNA synthesis, predicts that it should be possible to identify antivirals which are capable of blocking KSHV infection without perturbing normal cellular activities. The goal is to elucidate the mechanism of PF-8/Pol-8 processive DNA synthesis and to develop and employ a novel high throughput screening method that can be used to identify inhibitors that specifically block DNA synthesis and KSHV infection by targeting PF-8/Pol-8. The aims are to determine the affinities, composition, and structures of PF-8 and Pol-8 both off and on DNA using analytical ultracetrifugation, surface plasmon resonance, mutagenesis, and crystallography. A high throughput assay to identify functional inhibitors of PF-8/Pol-8 will be developed and used to screen compounds from the NCI repository; blocking peptides with designs based on phage display and mutational analysis will also be tested. All of the inhibitors that block PF-8/Pol-8 DNA synthesis in vitro will be tested for their abilities to block KSHV infection.

The overall objective of this project is to identify the sequence of intracellular reactions which lead to the formation of hydrochloric acid by gastric mucosa and to identify the major controlling steps in this sequence. The project involves use of a combination of non-destructive optical techniques, conventional biochemical analysis and electron microscopy to investigate receptor mechanisms, cyclic nucleotide metabolism, energy metabolism and morphological changes as these parameters are modified during transitions of acid secretory state. The studies are performed on isolated intact gastric mucosa and isolated gastric cells. The goal of the project is to integrate these measurements so as to provide a more comprehensive view of the cellular events associated with acid formation.

Prior research has firmly established both that persons of lower socioeconomic status with systemic lupus erythematosus (SLE) experience poorer access to care, actual health care utilization, and outcomes due in part to the nature of their health insurance. The proposed project will assess the role that differences in quality of care play in explaining why persons with SLE from disadvantaged backgrounds have less access to care and poorer health outcomes. The project will draw upon the data from the Lupus Outcomes Study (LOS) to assess the effect of quality of care on SLE outcomes. The LOS currently includes 1,026 persons with SLE from a wide range of environments across the nation, most of whom have been followed for five years. The principal data in the LOS are data from medical charts and annual structured telephone interviews, supplemented by contextual data about the neighborhoods and medical markets of LOS participants from the Census and other publicly available data sources, matched to the survey responses by geocoding. The specific goals of the project are to: 1) describe the extent to which care for SLE adheres to measures of quality cross-sectionally and over time; 2) establish the relationship between quality of care and kind and extent of health care utilization; 3) document differences in quality of care by socioeconomic status, race/ethnicity, local communities, and the nature of medical markets; 4) establish the relationship among such features of the health care system as type of insurance (Medicaid, Medicare, or private), form of insurance (managed care or fee-for-service), and extent of cost sharing and quality of care, after taking personal SES and extent of health care infrastructure into account; and, 5) describe the role that differences in quality of care play in explaining outcome differences by SES, including traditional measures of outcome as well as costs. The project may help reduce disparities in access and outcomes by showing that adherence to standards for quality of care can redound to the disadvantaged with SLE either by assisting their physicians to provide such care or by spurring referral to high quality providers. PUBLIC HEALTH RELEVANCE: Systemic lupus erythematosus (SLE) is a severe autoimmune disease with a potential for significant morbidity due to impacts on many organ systems. Prior research has established that persons with SLE from lower socioeconomic backgrounds have poorer access to care and long-term outcomes. The proposed project will use a cohort of over 1,000 persons with SLE followed for up to ten years to determine the extent to which the poorer outcomes of those of low socioeconomic status is because they are less likely to receive health care that meets minimal criteria for quality. The project could help persons with SLE from low socioeconomic backgrounds by showing that referral to physicians providing high quality care will reduce disparities in outcomes. [unreadable] [unreadable] [unreadable]

The purpose of this contract is to assure and provide the services of a consignee and quarantine agent for New World primates imported into the United States through purchase or negotiation by the Interagency Research Animal Committee from or with the Pan American Health Organization or sovereign governments of Central and South America. An additional requirement is to develop approaches and recommend methodologies to acquire new clinical, husbandry and/or physiological data regarding the species entering the United States via this project.

The first specific aim of this project is to provide COBRE Investigators and collaborators access to state of the art high throughput genetic analysis platforms, other sophisticated equipment, and experimental capabilities. Genetic discoveries and emerging technologies are radically changing biomedical research and the methods by which our COBRE investigators unravel the complex genomic architecture of autoimmune diseases. The Genomics Core serves as the central resource for COBRE investigators to access cuttingedge genomic technologies that allow their research programs to develop and test their hypotheses, therefore, the Genomics Core is highly significant and vital to the success of our Center investigators. The second specific aim of this project is to transition the Genomics Core from a COBRE-subsidized facility to a self-sustaining business entity supported by users both internal to OMRF as well as external. The capabilities of the Genomics Core make it particularly attractive to those researchers planning research projects that incorporate genomics. As such, researchers have utilized and can be expected to continue utilizing the Core as a simple fee-for-service unit or in a sub-contractual setting in funded grants. With our services in high demand from both COBRE and external investigators, the Core is poised to pursue multiple lines of funding through various mechanisms in our move towards overall financial self-sufficiency in Phase III. Furthermore, with the embrace of exome and whole genome sequencing of human patients, the Core could expand its local impact and open an entirely new revenue stream by providing medical diagnostic tests in addition to our central focus of supporting research. It is anticipated that the expansion into clinical genomics, coupled with increasing grant support from other mechanisms, will lead to the Core becoming completely self-sufficient at the end of 5 years.

A deep understanding of visual system function will require both a knowledge of the physiological properties of individual neurons and a detailed knowledge of the patterns by which these units are interconnected. Recently anatomical methods have been extraordinarily successful in determining which regions and subregions of the brain are interconnected and have provided, in essence, excellent "block diagrams." So far, however, very little is known about the detailed "wiring" of any region of the visual system. Although it is known, for example, that synapses from the lateral geniculate terminate in layers I, IV, and VI of cortical area 17, it is not known which of the many cell types there receive the input, nor whether the input is on the cell bodies, proximal or distal dendrites -- all questions of grave physiological consequence. We propose to answer such questions by constructing with serial section cinematography detailed "wiring diagrams" of the cat retina, visual cortex (layer IV of area 17), and superior colliculus (superficial gray). Our procedure is to photograph long series of thin sections in the electron microscope. Successive negatives are aligned and rephotographed to produce a high resolution 35 mm "movie." The detailed synaptic connections are reconstructed by viewing the movie frame-by-frame through a microscope. Reconstructions are carried out on experimental material with particular cells and afferents labelled by orthograde degeneration, horse radish peroxidase, and radioisotopes. The resulting wiring diagrams should provide a firmer basis for understanding the physiology of the neurons and also the changes in synaptic organization that occur during development and in pathologic states.

Undertreatment of pain in hospitalized patients has been associated with increased length of stay and adverse clinical outcomes such as higher complication rates, slowed would healing, and reduced quality of life. Minority populations have been shown to experience a higher likelihood of poor pain management (Cleeland, Gonin et al., 1994; Bates, Edwards, & Anderson, 1993). Specific aims are to: 1) describe the perceptions and experiences of Hispanics regarding experience of acute pain and pain management, 2) adapt and translate into Spanish the American Pain Society's Patient Outcome Questionnaire (APS-POQ) to measure outcomes of acute pain management in Hispanics, 3) evaluate the psychometric performance of the modified Spanish language APS-POQ in Hispanics, 4) analyze the factors influencing relationships among demographic variables, selected characteristics of pain, and satisfaction with pain management in an Hispanic sample, and 5) describe outcomes of acute pain management in Hispanics. The project will be conducted in two one-year phases. The purpose of Phase 1 is to understand the experience of acute pain and its management in adult Hispanics. Focus groups will be used to elicit information from Hispanics regarding acute pain experiences, beliefs, expectations related to pain experience and pain relief, and approaches used to manage pain. Data will be gathered on perceptions of satisfaction with pain management and health care providers. Qualitative techniques will be used to analyze the data for themes, in order to develop items for the Beliefs, Interference and Satisfaction subscales of the APS tool. The modified tool will be evaluated for content and cultural validity. The tool will be translated, back-translated and pilot-tested for equivalence of Spanish and English versions. Correlational analysis will be used to test for equivalence; test-retest procedures will examine stability. Phase 2 purposes are to: 1) test the modified APS-POQ for reliability and validity; 2) determine Hispanic satisfaction with overall pain management and with the responses of health care providers; and 3) gather descriptive data regarding the reported effectiveness of pain management strategies, including non-pharmacologic or alternative strategies, in Hispanics. In Phase 2, a cross-sectional study design will test the modified Spanish APS-POQ for internal consistency reliability, construct and convergent validity. In addition to the modified tool, the Brief Pain Inventory will be administered to assess convergent validity, and the Pain Management Index will be computed from analgesic regimen information in the medical record; these scores will be correlated with APS-POQ subscale scores and demographic data. Data from the modified APS tool will be analyzed using factor analysis procedures to establish construct validity. The study will produce a culturally sensitive, reliable and valid tool for determining outcomes of pain management in Hispanics.

Congenital cardiac defects affect nearly 1% of all live births and are the most common cause of infant mortality in the United States. Among the most severe forms of congenital heart defects are those involved in outflow tract and interventricular septation, whereas valve anomalies are the most common form of congenital heart defect. The long-term goal of the proposed studies is to define the molecular mechanisms and pathways that control cardiovascular development and underscore these congenital cardiac anomalies. Specifically, this application is focused on the identification of transcriptional pathways and mechanisms that function during the development of the second heart field (SHF) and its derivatives in the OFT and RV. The LIM-homeodomain transcription factor Isl1 and the MADS domain transcription factor MEF2C are essential regulators of SHF development. Recent work has established that the Mef2c gene is a direct transcriptional target of Isl1 in the SHF, forming the basis of a transcriptional pathway in the AHF. However, the upstream transcriptional regulators of the Isl1 gene in the SHF remain undefined in spite of the early, essential role for Isl1 in that lineage. Similarly, the downstream targets of MEF2C are not known, even though Mef2c is required for heart development, and its function is required specifically in the SHF for proper OFT alignment and endocardial cushion remodeling. This proposal will therefore test the hypothesis that MEF2C is required in the SHF to control cell number or migration of SHF-derived cells into the OFT endocardial cushions and that MEF2C is a direct transcriptional activator of Nfatc1 in the endocardial cushions. This is significant since the transcription factor NFATc1 is required for endocardial cushion remodeling and heart valve maturation. This proposal will also test the hypothesis that the expression of the Isl1 gene in the SHF is regulated via a novel modular enhancer that integrates upstream signals to direct expression in cardiovascular progenitors in the anterior lateral mesoderm. Two specific aims are proposed. Specific aim one will determine the requirement of MEF2C in OFT alignment and endocardial cushion development by examining the requirement of Mef2c for migration of SHF-derived cells into the heart and for cell proliferation and survival using a conditional knockout approach in mice. Specific Aim 1 will also determine if Nfatc1 is a direct transcriptional target of MEF2C in the outflow tract endocardial cushions via a conserved, consensus MEF2 site present in the Nfatc1 endocardial- specific enhancer using a transgenic mouse approach. Specific Aim 2 will identify regulators of Isl1 transcription in the SHF by defining upstream regulators of a novel SHF-specific Isl1 transcriptional enhancer. The goal is to place Isl1 into a transcriptional and signaling pathway, which will provide essential information about the early events controlling heart development and Isl1+ progenitor self-renewal and differentiation. PUBLIC HEALTH RELEVANCE: Heart defects are the most common class of birth defect in the United States, affecting nearly 1% of all newborns. Among the most serious of these defects are the cyanotic lesions, which affect septation of the ventricles and outflow tracts and allow oxygenated and deoxygenated blood to mix. The proposed studies will contribute to the understanding of the molecular pathways and the biochemical and genetic mechanisms controlling heart development. Defining these pathways is essential to determine how to reactivate the genetic programs controlling the development of heart tissue for the purpose of regeneration and repair, tissue engineering, directed differentiation of induced pluripotent cells, and diagnosis and intervention in cardiovascular birth defects.

Cyclosporine, an endecapeptide fungal product with specific anti-T-cell characteristics, will be administered to patients with sight-threatening ocular inflammatory disease of noninfectious origin who have failed on either corticosteroid or cytotoxic agent therapy. This will be done to test cyclosporine's efficacy in the treatment of uveitis. Within the context of these ongoing studies, the effect of hydergine in reversing cyclosporine-induced nephrotoxicity is being evaluated in a randomized, masked, crossover study. In addition, selected patients whose uveitis has been well controlled on cyclosporine for 1 year or more are undergoing kidney biopsies to evaluate the long-term effects of this agent. A phase I/II randomized trial using cyclosporine A and cyclosporine G has begun.

Substance use during pregnancy is a major public health problem. Motivating pregnant drug-abusing women to attend outpatient treatment, however, is a significant problem. Behavioral incentives have proven highly successful in reducing illicit drug use during outpatient treatment. The proposed research will determine the effectiveness of behavioral incentives in motivating pregnant drug-abusing women to attend outpatient treatment. It is hypothesized that improved attendance will result in greater exposure of patients to treatment services, which in turn will result in better outcomes including improved retention and reduced illicit drug use. The incentives will be material rewards made available in the form of certificates that can be exchanged for a variety of goods and services in the local community. The research subjects will be low income pregnant drug-abusing women who are in need of a myriad of treatment services (drug abuse counseling, obstetric, gynecological). Nearly all of the subjects abuse cocaine and over 50% are dependent on opiates and are maintained on methadone during treatment. The research will be conducted in a comprehensive treatment program that was established specifically for treating such women. The initial research will study the effects of basic variables that are expected to be important in the use of behavioral incentives to reinforce program attendance. The effectiveness of different magnitudes of reinforcement (Study 1) and different durations of reinforcement (Study 2) will be examined. Based on data obtained in these two studies, a clinical trial will then be conducted (Study 3) to directly compare the effectiveness of a behavioral strategy that targets program attendance to that which targets drug abstinence (negative urinalysis) in the outpatient treatment of drug abuse. The research will also determine if combining both strategies will be more effective that applying either strategy alone. Outcome measures will include effects of the behavioral interventions on program participation and retention, illicit drug use, alcohol use, psychosocial functioning, and maternal and fetal/infant outcomes (e.g., birthweight, obstetrical complications, hospitalization in the neonatal intensive care unit). The results of the research are expected to provide clinically useful information on the most appropriate behaviors to target in the behavioral treatment of drug abuse, as well as benchmark data on the effectiveness of using behavioral incentives in the treatment of pregnant drug-abusing women.

ABSTRACT The objectives of the Kidney Precision Medicine Project (KPMP) project are to create a kidney tissue atlas, define disease subgroups, and identify critical cells, pathways and targets for novel therapies by obtaining and evaluating kidney biopsies from participants with acute kidney injury (AKI) and chronic kidney disease (CKD). As part of this effort, there is a need for new technologies that improve the safety of the human kidney biopsy as well as the quality and diagnostic yield so that the biopsy tissue yields useful research or clinical information. Ultrasound-guided kidney biopsy is a critically important tool in the evaluation and management of renal pathologies. The goal of this Phase I SBIR proposal is to develop a virtual simulator to train clinicians to improve procedural skill competence in real-time ultrasound-guided renal biopsy. Although improvements in needle design and ultrasound imaging have been helpful, excellent biopsy technique and skill are essential to consistently obtaining high yield renal biopsy samples. Furthermore, poor renal biopsy technique can potentially lead to significant complications and adverse lethal outcomes. The simulator proposed in this project will accelerate the training of ultrasound-guided renal biopsy in a risk-free environment, ultimately improving the safety of kidney biopsy and ensuring that the biopsy procedure yields high quality specimen that can provide useful clinical information. The proposed system will benefit radiologists, nephrologists, and interventional radiologists in two ways: (a) Improved three dimensional and dynamic ultrasound imaging interpretation specific to performing a needle biopsy and (b) Improved manual dexterity in needle handling with the aid of tactile feedback. This project is a collaboration between Kitware Inc. and Children's National Medical Center. Kitware is a leader in the creation and support of state-of-the-art software and technology with more than 15 years of experience in image-guided interventions. Children's National Medical Center (CNMC) is an internationally recognized pediatric hospital with extensive expertise in the clinical management of renal pathologies. The two institutions have already collaborated successfully on several projects including image guided biopsy projects. The aims of the Phase I project are to build the virtual simulator system and evaluate it by conducting a study with pediatric radiologists, nephrologists, and interventional radiologists at CNMC. The successful completion of the Phase I work will result in a proof of concept of our simulator system and lay the groundwork for further system development and validation in Phase II.

Our goal is to elucidate some of the presynaptic mechanisms that control synaptic efficacy. By regulating neurotransmitter release, the activity of specific neuronal pathways can be adapted according to prior activity in the pathway and according to other neuronal and hormonal signals. These processes, we believe, are bases for autonomy in the autonomic nervous system and for functions such as learning and memory in the brain. This project will be a study of nicotinic synaptic transmission in the superior cervical sympathetic ganglion. A particularly fascinating control of synaptic efficacy is the hours-long potentiation of nicotinic transmission that follows a brief burst of preganglionic activity. This "long-term potentiation" is due to an augmentation of acetylcholine (ACh) release. Catecholamines and adenosine are stress-related signal molecules that also regulate nicotinic synaptic efficacy, apparently through regulation of ACh release. It is apparent that presynaptic mechanisms are prominent in the control of synaptic efficacy. To determine what those mechanisms are, isolated nerve terminals are needed. Synaptosomes will be isolated from the rat superior cervical ganglion and will be used in a multidisciplinary approach including electrophysiological and neurochemical measures of synaptic efficacy and ACh release in the intact ganglion. The regulation of ACh metabolism and release in the synaptosomes will be compared to that in the ganglion, providing a critical test of synaptosomes as models for the nerve terminals of the intact tissue. Finally, the synaptosomes will be used to determine the roles of second messengers and ion permeability in controlling ACh release. The superior cervical ganglion offers special advantages for electrophysiological and neurochemical approaches in the study of synaptic transmission. Synaptosomes will add another dimension and will be part of a powerful model system. To further increase that power, liposomes will be investigated as a means of "injecting" impermeant biochemical tools into synaptosomes and nerve terminals. It will then be possible to dissect presynaptic metabol c control mechanisms in ways that were previously impossible.

Diabetic foot, venous stasis and pressure ulcers are examples of chronic non healing wounds that are refractory to standard therapy and represent a major health care problem especially for the increasing elderly population. Polypeptide growth factors are a class of biological mediators which have been proposed as possible vulnerary agents. The combination of platelet-derived growth factor (PDGF) and epidermal growth factor (EGF) has been shown to be more potent that individual growth factors in stimulating the healing of partial thickness skin wounds in healthy swine. In preparation for testing this composition on diabetic mice of PDGF-BB, EGF or the combination project will examine the dose dependent efficacy on wounds in diabetic mice PDGF-BB, EGC or the combination of PDGF-BB/EGF delivered in a 2% methylcellulose gel vehicle. Specifically, investigators will evaluate the healing of full thickness wounds on genetically diabetic mice (C57BL/KsJ-db/db) following the repeated application of: 1) 4.0 mug PDGF-BB, 15mug PDGF-BB and 50mug EGF per cm wound surface are, 2) 4.0mug PDGF-BB, 3)4.0mug PDGF-BB 60mug EGF or 4.0 PDGF-BB and 60mug EGF per cm wound surface area. Control wounds will receive vehicle alone. Gel treatments will be applied immediately following surgery and 2, 4, and 6 days, thereafter. At the time of surgery and at days 8, 10, 12, 14 and as needed thereafter, standardized photographs will be taken of each wound for wound closure analysis. Complete wound biopsies will be harvested on day 14 for histologic and hydroxyproline analyses. Statistical analyses will be performed for wound closure, histological and hydroxyproline data using Analysis of Variance (ANOVA), Kruskal-Wallis test and Student's t-test, respectively. The results from these studies will elucidate appropriate dose levels for initial evaluation in human clinical trials in chronic wounds of diabetic patients.

Project Summary/Abstract Scandium-44 (44Sc) is a positron emitting radionuclide with a half-life of 3.97 h that is well-suited for use in positron emission tomography (PET) imaging. Scandium-47 (47Sc) is a beta emitter with a half-life of 3.345 d appropriate for targeted radiotherapy. When combined, 44Sc and 47Sc present an ideal radioisotope pair for use in theranostic agents meant to both diagnosis and treat disease. As radiometals, both isotopes of Sc require the use of a bifunctional chelator to attach the radioactive metal cation to a targeting vector that dictates the specific localization of the drug conjugate. Since 44Sc and 47Sc are both up and coming radionuclides for medical applications, there is a need for further chelator development for Sc-based radiopharmaceuticals. The most widely used chelator for Sc(III) is 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA); however as a macrocylic chelator, DOTA requires harsh radiolabeling conditions (high temperatures and long reaction times) to form the Sc-DOTA complex that are incompatible with sensitive biological molecules often used as targeting vectors in the drug conjugates. This research aims to investigate and develop an acyclic chelator for stably complexing Sc(III) under biologically compatible radiolabeling conditions to facilitate the use of Sc radioisotopes in targeted radiopharmaceuticals. To achieve these goals, this proposal aims to investigate the chelation chemistry of Sc(III) through the synthesis of small peptides as model systems to probe the effects of denticity, cavity size, and binding moiety on chelate stability, both experimentally and computationally. A series of tripeptides and tetrapeptides comprised of natural and non-natural amino acids will be synthesized and complexed with non-radioactive Sc(III). Selected Sc-peptide complexes will be further studied using density functional theory (DFT) calculations to probe the structure and geometry of the complexes. The trends observed with the peptide systems will inform the selection of promising acyclic chelators to be complexed with non-radioactive Sc and radioactive 44Sc. The acyclic chelators will be assessed based on their ability to readily chelate Sc under mild conditions with high purity, and on complex stability. The lead radiolabeled Sc-chelates will then be evaluated in vivo in mouse models through a collaboration with Memorial Sloan Kettering Cancer Center. PET imaging and biodistribution studies will be carried out to determine the in vivo stability and utility of the acyclic chelators in comparison with the current gold standard DOTA chelator. 44Sc-chelator complexes will be studied in healthy mice while 44Sc-chelator-octreotate (Y3-TATE) bioconjugates will be studied in tumor-bearing mice to monitor targeting to the somatostatin subtype 2 receptor (SSTr2) that are upregulated in neuroendocrine tumors. The development of acyclic chelators for rapid complexation of Sc radionuclides under mild conditions will lead to the development of a novel class of Sc-based radiopharmaceuticals for imaging and treating cancer.

This request is for funds to purchase a Zeiss 510 meta confocal microscope. This instrument will be housed on the 10th floor of Salk Hall in the University of Pittsburgh School of Pharmacy. Basic and clinical research within the School of Pharmacy has grown tremendously over the past six years. During the past four years, the School of Pharmacy has been rated among the top 10 in NIH funding among all Schools of Pharmacy in the nation. Despite this success, the School is relatively equipment poor and is forced in many cases to rely on major equipment in other schools. Not only does this create difficulties for existing personnel, but also presents obstacles in recruiting highly qualified research faculty. The purpose of this request is to provide the School of Pharmacy with its own confocal instrument to meet the critical needs of its research faculty. Specifically, the School currently is in need of a four-laser, spectral, confocal microscope, equipped for live cell imaging. Six NIH-funded investigators within the School have identified a need for such equipment. Most of this work falls within the realm of new drug development (particularly the development of anti-cancer agents and gene therapy methodologies) and focuses on following the uptake and trafficking of molecules and drugs within cells and tissues. Currently, the School of Pharmacy has no confocal instrument. Instruments are available elsewhere within the University, primarily at the University's Center for Biologic Imaging (CBI). Unfortunately, CBI is not able to provide sufficient access to accommodate our needs. CBI resources are in very high demand, and School of Pharmacy personnel have the lowest priority with regard to access. For example, School of Pharmacy personnel have no access on evenings or weekends, and are limited to 3-hour time slots, when slots are available. Often School of Pharmacy personnel must wait 1-2 weeks before getting access to an instrument, and during the access time available are not able to analyze and collect all of their data. This lack of available instrument time is extremely frustrating and limits the pace of our research. Acquisition of the requested instrument will eliminate this problem, and will provide the School of Pharmacy with a greatly needed resource. [unreadable] [unreadable] [unreadable]

Ionizing radiation is capable of producing several kinds of biological end results, including mutation and cell death. In spite of these possible detrimental effects on biological systems, radiation is being used both as a diagnostic tool in medicine and as a therapeutic tool in cancer treatment. The goal of this project is to further understand the interaction of ionizing radiation with the DNA molecule. This molecule is chosen for study because of its central and critical role in maintaining the genetic integrity of the cell. The research is designed to study the physical damage in DNA as expressed by scission of the DNA strand, and to correlate this response with damage to the biological activity of the DNA molecule. A replicative intermediate in the synthesis of bacteriophage phi X174 DNA will be used in this research. Two ideal properties of this molecule are, first, it is a supercoiled molecule and thus allows sensitive analysis for strand breaks, and second, it is a complete genome and has biological activity, thus allowing analysis for biological damage. The biological damage is undoubtedly a result of physicochemical changes induced in the DNA by energy released from the ionizing radiation. Because strand breaks represent a type of physicochemical damage, and can be measured with a minimum of manipulation following irradiation and without chemical treatment (which could aggravate the radiation damage in an unidentifiable fashion), the production of strand breaks will be used as the standard measure for physiocochemical damage. Irradiation will be in a controlled environment so that secondary factors affecting radiation response can be evaluated. Appropriate radical scavengers will be used in order to elucidate the role of water radicals in radiation damage to DNA. Radiation sensitive mutants of the bacterial cells for the biological assay will be used in order to ascertain which biological repair systems are effective in the recovery of the DNA from biological damage. The eventual goal of this project is to determine the fundamentals of radiation action of DNA with particular emphasis on the mechanisms of radiation induced biological damage in the DNA.

Calcium (Ca) is a critical regulator of cardiomyocyte contractile function. Cardiac excitation contraction (EC) coupling requires activation of the Ca release channel/ryanodine receptor (RyR2) in the sarcoplasmic reticulum (SR) by signals transmitted via the voltage-gated Ca channel/dihydropyridine receptor (DHPR) in the transverse (T) tubule. Altered a homeostasis due to an as yet unidentified defect in EC coupling has been proposed as a possible contributory factor to the pathogenesis of heart failure. In the failing heart a defect in Ca signaling could result from a number of abnormalities including one or more of the following: 1) changes in the expression of Ca handling proteins; 2) post-translational modifications of Ca handling proteins; 3) anatomical changes in cardiomyocytes and/or extracellular matrix; 4) decreased myofilament responsiveness to Ca. To establish that a defect in any of the above is causal in the pathogenesis of heart failure, it is necessary to demonstrate that introducing the defect in Ca signaling can cause heart failure or correcting the defect can restore normal cardiac function. The goal of this proposal is to identify specific molecular defect(s) in Ca handling that contribute to the pathogenesis of heart failure. The investigators have access to a unique human model of heart failure: human myocardium from pre- (dysfunctional) and post- (normal function) left ventricular assist device (LVAD) implantation patients. Animal models of heart failure that are physiologically relevant to human heart failure have been developed to test hypothesis regarding the etiology of contractile dysfunction in the failing heart. The investigators propose: Aim 1 to use pre- (failing) and post-LVAD (normalized function) myocardial samples to obtain data comparing Ca signaling and EC coupling in normalized and failing myocardium obtained from the same patient; Aim 2 to use well defined animal models of heart failure to analyze Ca signaling and EC coupling during the progression of heart failure at four levels: 1) isolated functional systems (including RyR2 single channel properties), 2) integrated cellular function (including Ca transients), 3) biochemical studies examining the levels of expression (mRNA and protein) of Ca handling molecules; Aim 3 to test the hypothesis that a defect in activation of RyR2 is present in cardiomyocytes from failing hearts compared to cardiomyocytes from normal hearts and to induce defects in EC coupling in cardiomyocytes from normal hearts and to induce defects in EC coupling in cardiomyocytes by transfecting mutant RyR2 channels and by overexpressing IP3 receptors and to determine whether specific molecular defects can cause altered Ca handling, defective EC coupling and heart failure in transgenic mice.

The complex and dynamic communities of microbes that are present on and within the human body (the human microbiota) are thought to profoundly influence human health in a variety of ways, through effects on human physiology, nutrition, immunity, and development. Studies on humans and vertebrate animal models have generated evidence that this is the case for some specific diseases and suggest that further studies in this area may be vital for the understanding, prevention, and treatment of many human diseases, as well as the maintenance of homeostasis. It is currently a challenge to even identify comprehensively the components of the human microbiota, although genomic approaches have greatly improved the feasibility of doing so. The study of the collective DNA (the human microbiome) of community members has been spurred by recent advances in DNA sequencing and other technologies; such technologies have created the new field of metagenomics (determining the DNA sequence of genomes from a mixed community of organisms). One issue associated with metagenomic studies is that analysis of mixed populations is extremely difficult due to the highly heterogeneous nature of the sample. To avoid this issue, efforts to isolate individual organisms from these mixed populations have been used. Unfortunately, obtaining sufficient quantities of pure, individual isolates is also a challenge. Growth of bulk cultures and purification of DNA for analysis is tedious and labor intensive, and many isolates are difficult, if not impossible, to culture. The utilization of whole-genome amplification methods on single-cell isolates to provide sufficient DNA for comprehensive downstream testing is a solution to this problem. To date, reliable whole-genome DNA amplification methods have failed to provide DNA suitable for analysis from trace samples due to reagent contamination, method sensitivity issues, and the generation of chimeric products that confuse analysis. As a collaborative effort between GE and the Broad Institute (a world leader in the implementation of new technologies to generate DNA sequence), the methodology developed will provide a process that can be used by high throughput sequencing facilities to completely characterize individual microbes using DNA sequencing, furthering the knowledge, and stimulating work, in this area. PUBLIC HEALTH RELEVANCE: There is a growing desire to understand more about the relationship human beings have with the microorganisms growing in and on their bodies (the human microbiome). DNA sequencing of the entire genome of each organism is one method being used to gather precise information about these microorganisms. The team plans to develop a whole-genome DNA amplification method with single cell sensitivity that will enable high-throughput DNA sequencing of entire genomes to be performed from single cells, eliminating the requirement to purify and culture each isolate.

The Section is the statistical and data management component of the clinical oncology Program (COP). The Section provides statistical leadership and data management consultation for major activities of the Program, and is involved in the design, conduct, monitoring, and statistical analyses of intramural and national multicenter clinical trials of experimental treatments for cancer, and intramural clinical trials for treatment of AIDS. other major collaborative efforts include studies to identify important prognostic and treatment selection factors, evaluate diagnostic procedures, develop improved staging systems, and assist investigators in the design, execution, and analyses of major in vitro drug testing studies. The Section develops new statistical designs and biometric methods related to the development and evaluation of new cancer and AIDS treatments. The Section maintains computerized data collection systems for intramural and national multicenter clinical protocols, and it works closely with interested branches to improve data recording and retrieval. The Section is working to develop specialized clinical data bases for individual branches within the COP as well as a unified system for tracking basic information on all patients registered on COP treatment protocols.

The main purpose of this study is to determine the efficacy of radio- immunotherapy using a monoclonal anti-CD20 antibody labeled with 131-I in patients with B-cell lymphoma who have relapsed after chemotherapy or who have not been previously treated. Our hypothesis is that this treatment may be superior to chemotherapy and that this can be assessed using molecular markers of minimal residual disease.

The urinary excretion of the renal cortical enzyme kallikrein is abnormal in various human and animal hypertensive states, including essential hypertension. These findings, as well as the relationship between the renal kallikrein-kinin system and adrenal sodium-retaining steroid activity, suggest that this system could be involved in renal function and the control of systemic blood pressure. The proposed research will continue to determine if maneuvers or disease which alter adrenal-renal function and/or blood pressure affect the activity of components of the renal kallikrein-kinin system and conversely, if altered kallikrein or kinin activity affect renal function and blood pressure. Studies of the renal kallikrein-kinin system will continue in epidemiological surveys, clinical investigation, animal model experimentation and cellular biochemistry. Normal volunteers and patients with hypertensive diseases and hypertensive animal models will continue to be used. The objectives are: 1) to continue to define factors which influence kallikrein excretion in studies at different levels of biological organization; 2) to determine relationships between altered kallikrein excretion and the activity of the renal kallikrein-kinin system; and 3) to assess the role of this system in hypertensive disease states. BIBLIOGRAPHIC REFERENCES: Geller, R.G., H.S. Margolius, J.J. Pisano and H.R. Keiser: Effect of Altered Sodium Intake on Urinary Kallikrein Excretion in Spontaneously Hypertensive and Wistar-Kyoto Rats. Circ. Res., Suppl. I, 36: 103, 1975. Kass, E.H., H.S. Margolius and S.H. Zinner: Urinary Kallikrein and Blood Pressure in Childhood. In The Epidemiology and Control of Hypertension, Vol. II, O. Paul, ed., Stratton Medical Publishers, Miami, Fla., 1975.

Since interstitial cystitis is likely caused by or exacerbated by defects in the bladder's permeabllity barrier, an understanding of the bladder's barrier function may be critical to understanding the pathogenesis of this disorder. The proposed studies seek to examine, at a fundamental level, the characteristics of this permeability barrier, its molecular basis, and the mechanisms used by bladder epithelial cells to maintain the barrier in the face of the large changes in bladder surface area resulting from filling and emptying the bladder. The first aim is to characterize the permeability properties of intact rabbit bladder epithelial cells to water, protons, and small nonelectrolytes. Rabbit bladders will be stripped of their underlying musculature and mounted in an Ussing chamber where they will undergo measurements of transepithelial voltage, short circuit current, and capacitance. Fluxes of water and small nonelectrolytes such as urea will be measured using isotopes, and proton and NH3 fluxes will be measured using a pH stat. All fluxes will be corrected for unstirred layer effects and normalized to the surface area estimates provided by the capacitance measurements. These results will provide the first direct measurements of the permeability properties of the bladder permeability barrier, and will set the stage for future studies examining the effects of various potential toxins on this tissue. The second aim is to characterize the permeability properties of apical membrane endosomes (AME) which shuttle membrane into and out of the apical surface. Using methods similar to those developed by the PI for studies of toad urinary bladder endosomes, fluorescein derivatives will be entrapped in AME and these vesicles partially purified under conditions in which only AME contain the fluorophore. Using this partially purified preparation, permeabilities to water, small nonelectrolytes, protons, and NH3 will be determined by means of stopped flow fluorimetry. Functional AME will then be purified to homogeneity using a combination of flow cytometry, free flow electrophoresis, and phase separation techniques. These purified AME will be subjected to analysis of lipid composition and extracted lipids from AME will be reconstituted into liposomes to determine if the permeability properties are preserved. These studies will define how lipid composition and bilayer structure determine the permeability properties of the bladder permeability barrier. The third aim is to examine mechanisms by which the trafficking of AME into and out of the apical membrane might be governed. AME will be purified to homogeneity using density shift and flow cytometry approaches and their protein composition determined to identify proteins likely involved in the regulation of AME trafficking. Studies with the flow cytometer will help define whether there are more than one population of AME and will determine the characteristics of each. These studies will provide some initial clues as to mechanisms regulating the insertion and removal of AME from the apical membrane.

Acinetobacter baumannii is an emerging pathogen responsible for a significant percentage of nosocomial infections. It has also been problematic for the military in Iraq and Afghanistan, where it is the cause of softtissue infections associated with war-related trauma. Two properties of A. baumannii Xhaf contribute to its persistence as a pathogen are its propensity to accumulate drug resistance genes and a striking ability to tolerate biocides and desiccation. This project will use RNA-seq analysis to identify and define the functions of sRNA regulators (sRNAs) whose expression is associated with seven clinically relevant resistance traits of A. baumannii. These include resistance to four antibiotics, two biocides and desiccation. sRNAs are expressed in response to specific environmental signals, they are synthesized rapidly and they act quickly to control gene expression at the translational level. We hypothesize that sRNAs help A. baumannii io cope with cell envelope and metabolic stresses associated with antibiotic, biocide and desiccation exposure. Our results will open avenues for the development of new lines of defense against Acinetobacter infections.

Abstract Embolic stroke (or cerebral embolism) accounts for approximately 25% of all strokes. It occurs when an embolus formed elsewhere in the body, lodges in a cerebral blood vessel, suddenly obstructing blood flow. Cerebral emboli most commonly arise from atherosclerotic plaques formed in the aorta, carotid artery, vertebral artery or from the heart. It has been generally accepted that the lodging of an embolus in a brain blood vessel depends on its size, frequency of embolism and the presence of collateral flow. However, increasing evidence now pinpoints brain inflammation as a contributing causative factor for stroke. Platelets are often present in embolic material of both vascular and cardiac origin and they have the ability to interact with activated endothelial cells. This opens the possibility that cerebral emboli may also lodge in brain vessels because of adhesive interactions between platelets in the emboli and inflammation-activated brain endothelial cells. Thus, the proposed study is aimed at determining whether and how endothelial cell activation during brain inflammation affects the lodging of emboli in the cerebrovasculature. Specifically, we will test hypothesis that brain inflammation contributes in part to embolic stroke occurrence by promoting adhesion of platelets from emboli to the brain endothelial cells activated by the brain inflammation through JAM-A dependent mechanisms. JAM-A is a brain endothelial tight junction protein that becomes an adhesion molecule during inflammation. The hypothesis will be addressed in the following aims: a) to examine the role of inflammation- activated brain endothelial cells in the pathogenesis of embolic stroke in mice (Specific Aim 1) and b) to investigate the involvement of JAM-A in the lodging of platelet rich emboli in inflamed brain and assess its potential as a drug target (Specific Aim 2). Collectively, the results from this project will lead to a better understanding of the mechanisms that underpin embolic stroke and will help define new strategies to prevent such stroke and improve outcomes.

Choline is necessary for the normal functioning of the mammalian organism. Hepatic steatosis associated with the use of total parenteral nutrition (TPN) is caused by choline deficiency. The aim of this study is to confirm that choline-supplemented TPN causes a decrease or resolution of hepatic steatosis in long term TPN patients with choline deficiency manifested by hepatic steatosis, and to determine whether recurrence can be prevented by continued choline supplementation. Subjects will be randomized to receive choline in their TPN or placebo.

The specific aims of this proposal are to identify clinical criteria, including specific diagnostic tests, which best identify those patients who would profit from corpus callosum section; to measure the callosotomy; to develop improved criteria to select patients for each of these procedures; and to determine the immediate and long-term clinical, neuropsychological, and electroencephalographic consequences of corpus callosotomy. Corpus callosotomy is a promising therapeutic modality for the approximately one million people with epilepsy in the United States who are medically intractable. All too many patients do not have a discrete epileptiform focus in a surgically resectable region, but do suffer from rapid secondary generalization and frequent injuries from the resultant falling seizures or loss of awareness. We have performed 10 complete corpus callosotomies to date with highly significant reductions in both post operative generalized epileptiform discharges and falling seizures. We expect 30 patients to be candidates for the corpus callosotomy procedure during the three year study period. We propose to perform 10 more complete sections to identify those critical EEG, clinical, and neuronpsychological criteria to allow us to perform 20 partial callosotomies in the remaining two years of the study. Success of therapy will be measured by reduction of seizure frequency, ability to simplify antiepileptic medication regimens, and evidence of increased independence in daily living. Neuropsyhological effects will be studied in a separate proposal. Appropriate statistical analysis will be used as outlined in detail in the proposal.

This protocol will evaluate the utility of the two GFR markers, creatinine and cystatin C, in comparison with measured glomerular filtration rate (GFR) by iohexol clearance in 375 Black and White adult participants of the population-based Multi-ethnic Study of Atherosclerosis (MESA) cohort from the Johns Hopkins clinical center. Although GFR estimation by creatinine has improved over the past decade, there has been inadequate inclusion of persons over 65 years of age, and no study of GFR measurement has occurred within a population-based sample of Blacks and Whites in the United States. To advance the detection and definition of CKD across a broad age range of diverse adults,, we must focus on the higher ranges of GFR, and we must identify the optimal strategy for GFR estimation. The first Aim of this cross-sectional study will be to determine the influence of Black race-ethnicity on serum levels of filtration markers (creatinine and cystatin C) in the general population, and to modify current GFR equations to capture optimally the influence of Black race. The second Aim will be: to evaluate age-related decline in GFR; to compare the relative ability of the filtration markers to capture declining GFR with age; and, to modify current GFR equations to incorporate the influence of age. In executing these Aims, we will determine the optimal biomarker(s) for GFR estimation ? creatinine, cystatin C, or both. In addition, blood specimens will be stored with the potential for examination of future filtration markers. To accomplish our objectives, we will measure iohexol-GFR in approximately 375 Black and White MESA participants, spanning a target age range of 55-95. This study could be a major contributor to the National Institute of Health?s efforts to address the persistent racial disparities in the burden of kidney disease in the United States.

The studies proposed concern the synthesis of new membrane proteins after T5-bacteriophage infection of Escherichia coli and the role of these proteins in DNA transport through the membrane, host DNA breakdown, the control of protein synthesis, and the maintenance of energy producing systems. Experiments on membrane protein synthesis and membrane function will be conducted in both a system in which T5 can replicate normally and in the presence of the colicin Ib factor, a plasmid which causes infection of T5 to be abortive. The abortive infection results from the interaction of a plasmid protein and a phage protein and we believe this to cause aberrant membrane function. Included in the experiments will be a purification of the T5-induced Al protein solubilized from the membranes of infected cells, a study of the association of this protein with and its effect upon cell membrane components and phage and host DNA, a determination of the genes controlling the synthesis of three other T5-induced membrane proteins, a study of membrane function during the abortive infection of T5 in the presence of the colocin Ib factor, and experiments to determine the mechanism and genes involved in the inactivation of the cell surface receptors for T5 after infection.

Project Summary Tinnitus or ?ringing in the ears? is a hearing disorder that disproportionately impacts those who are or have served in the military. There is currently no effective cure for tinnitus. Patients with tinnitus exhibit broad changes in brain activity in the auditory system and elsewhere. One of the fundamental characteristics of tinnitus is a dysregulation in the excitatory/inhibitory balance in the central auditory system (CAS) leading to neuronal hyperexcitability and synchrony. The large conductance calcium-activated potassium (BK) channel is implicated in other neuronal excitability disorders like temporal lobe epilepsy, tonic-clonic seizures and alcohol withdrawal seizures. Gated by both voltage and intracellular calcium, and expressed throughout the peripheral and central auditory system, the BK channel is able to modulate auditory neuronal signaling across a wide variety of conditions. Recently, Lobarinas et al. found that two BK channel openers, Maxipost and its enantiomer, reduced behavioral evidence of salicylate-induced tinnitus in rats. Though Maxipost was originally developed as a BK channel opener, in fact it is a more potent KCNQ channel opener. Nonetheless, these findings provided the impetus for our preliminary studies showing that BMS-191011, a more specific BK channel opener, reduces behavioral manifestations of tinnitus in two mouse models. Consistent with a mechanistic aim of counteracting hyperactivity in the CAS, our preliminary data and other?s shows that BK channel openers reduce neuronal activity in the auditory midbrain. The overall aim of the proposed studies is to test whether treatment with a class of BK channel openers, exemplified by BMS-191011, can reduce behavioral and neurophysiological manifestations of chronic tinnitus. Behavioral assays will probe whether treatment with the BK channel opener modifies responses that can be linked to CAS function. Both invasive and non-invasive neurophysiological recordings in vivo will characterize treatment effects on neural correlates of tinnitus in the CAS to 1) provide cross-methodological confirmation for the behavioral effects, 2) facilitate preclinical target validation and engagement studies, and 3) allow translation to clinically-measurable markers of tinnitus. The studies will employ a mouse model of acoustic trauma-induced tinnitus that matches the etiology of a substantial portion of the patient population. The use of mice will enable a longitudinal study design in which treatment begins ~2-3 months after acoustic trauma. Many mammals preclude long duration studies or higher usage rates, but the CBA/CaJ mouse is both affordable and now considered a reliable model of tinnitus following noise trauma. Together, the proposed preclinical studies will explore whether administration of a class of BK channel openers exemplified by BMS-191011 is a valid strategy to counteract maladaptive CAS function underlying stable tinnitus. This therapeutic approach clinically would allow a lapse in time following noise trauma before treatment onset, in line with the practical needs of at-risk members of the Armed Forces who may develop tinnitus while deployed, and the many patients who already have acoustic trauma-related tinnitus.

The long-term goal of this work is to implement novel gene-based therapeutics to reverse contractile performance deficits in the failing human heart in vivo. This proposal is focused on troponin I (TnI), the inhibitory subunit of the troponin complex and molecular switch to cardiac contraction. The focus of this proposal on advances in troponin function in the diseased heart has considerable health relevance and, accordingly, well serves the mission of the NIH. Novel preliminary evidence is presented on troponin-modified myofilaments dictating myocyte Ca2+ handling through a direct effect on sarcoplasmic reticulum (SR) Ca2+ load. The overarching hypothesis that guides this proposal is myofilament function, by virtue of unique properties of histidine modified TnI at codon 164, uniquely and directly tunes SR Ca2+ content independent of effects on canonical regulators of SR Ca2+ load, including SERCA2a -dependent rapid Ca2+ sequestration. We further posit that novel bio-engineered adeno-associated viral vectors, together with our new high fidelity hypoxia responsive gene switch system, will permit translation of this proposal's molecular/cellular advances (Aim1) directly to the beating heart in vivo in pre-clinical studies in living mammals (Aim 2). The Specific Aims are: Aim 1. To determine the molecular mechanism underlying optimization of global cardiac myocyte Ca2+ handling between the myofilaments and sarcoplasmic reticulum in vitro and in vivo by single histidine-modified troponin. Hypothesis: Real time sensing of the patho-physiological biochemical milieu of the healthy and diseased myocyte by the troponin modified sarcomeres will have a dominant effect to directly decrease the Ca2+ load requirements of the SR, including conditions of accelerated SR Ca2+ influx by an increased SERCA2a/PLN ratio. Aim 2. To implement a novel evolutionary-directed bio-engineered AAV vector and double oxygen genetic sensing system for optimization of efficient systemic gene delivery of modified troponins in the diseased heart in vivo. Hypothesis: Compared to wild-type striated muscle tropic rAAV serotypes AAV1, pseudo-typed AAV6, AAV8 and AAV9, the novel bio-engineered rAAV capsid M41 with O2 sensing genetic elements will lead to higher cardiac tropism and greater efficiency resulting in hypoxia/ischemia sensitive long-term constitutive expression of modified TnIs in the cardiac sarcomeres of small mammals. PUBLIC HEALTH RELEVANCE: Ischemic cardiomyopathy is a major problem in the United States. This proposal focuses on a modified heart muscle protein troponin to enhance heart pump performance in the ischemic heart. Therefore the health relevance of the proposal is substantial and highly significant to the mission of the National Institutes of Health.

Ras oncogenes are part of a large family of related proteins. Many of these Ras-related proteins are suspected of playing a role in tumorigenic transformation. Indeed, certain Ras related proteins appear to serve as downstream components of Ras signaling and transformation. We have focussed on those proteins which not only share structural homology to Ras, but also undergo the same type of lipid processing as Ras, farnesylation. Drugs directed at inhibiting farnesylation (Farnesyl transferase inhibitors or FTIs), which is essentail for Ras function, are currently in clinical trials at the NIH. Consequently, understanding the overall importance of other farnesylated proteins in growth and development may have considerable clinical relevance. We have found that the novel Ras-related proteins Rho6 and Rho7 are also farnesylated and appear to act as inhibitors of certain aspects of Ras transformation. Moreover, the inhibition is dependent upon Rho6/7 farnesylation. Studies are underway to determine if these proteins may play a role as tumor suppressors. If so, the long term use of FTIs may serve to inadvertantly inactivate tumor suppresors. We are also examining a novel Ras related protein sub-family identified by data base searching which we have designated Rig. There are three members, Rig1, Rig2 and Noey2. These proteins mediate cell death and tumor inhibition in a farnesylation dependent manner and are frequerntly down-regulkated during tumor development. We are currently attempting to define a mechanism of action as well as charactrizing a further two novel Rig related proteins. These studies have been facilitated by a successful 2-hybrid screen to identify Rig interacting proteins. We have also initiated experiments in a zebra fish backgound to determine the role of these proteins in development.

[unreadable] [unreadable] The specific aim of the Phase II project is to complete the design and develop a full interactive virtual experience that will reduce the risk faced by First Responders when dealing with incidents involving hazardous materials. If First Responders, particularly fire fighters, follow a systematic approach to handling emergency incidents, their safety and health and that of any potential casualties will be better protected. [unreadable] [unreadable] The title of the proposed training tool is "Responding to HazMat Incidents - a Virtual Experience." It will be targeted toward First Responder organizations, such as the International Association of Fire Fighters, to be used in their regularly scheduled instructor-led training programs. The training tool will be developed for the Digital Versatile Disc (DVD) format. DVD will enable MetaMedia to incorporate compelling and realistic interactive video simulations of hazardous materials incidents, instructive computer animations, and engaging team exercises to produce an effective training tool that successfully transfers knowledge. The aim of the HazMat Incidents DVD is to create a training tool that [unreadable] can be easily incorporated into existing training programs to improve the readiness of First Responder organizations to safely handle emergency incidents. [unreadable] [unreadable] The DVD training program will represent a technology innovation in instructor-led training. MetaMedia's application of DVD technology will take advantage of the unique features of DVD, such as high-quality motion video, interactive branching, and low-cost delivery systems to bring real-world simulations into the classroom. During Phase I of the project, MetaMedia and the International Association of Fire Fighters (IAFF) determined the training needs of the target audience, designed a prototype training tool to meet those needs, and produced a working prototype of the product that will was evaluated in an actual training session. Our task is to expand on the successful Phase I prototype and create a visually [unreadable] exciting training solution that will achieve this goal. [unreadable] [unreadable] The deliverables for Phase II will be a completed DVD program providing up to 4 hours of content. The Phase I program will be expanded to include additional components of the interactive experience, facilitator's and student guides, a complete new module on "Awareness of HazMat Dangers," and Web Links to OSHA and FEMA websites. [unreadable] [unreadable] [unreadable] [unreadable]

This proposal is for an Underrepresented Minority Supplement for Wilmarie Morales- Soto, a second year predoctoral student in the Neuroscience Graduate Program, to the funded parent RO1 (RO1DK103723). Wilmarie?s research project will build upon studies described in specific aim 1 of the parent grant. Aim 1 is designed to identify how the activation of glial signaling pathways that couple to fluxes in intracellular Ca2+ contributes to reactive gliosis and how reactive gliosis contributes to neuroinflammation in the gut. These studies test the hypothesis that glial Ca2+ responses driven by ADP cause reactive gliosis, neuron death and gut dysfunction. Ongoing studies performed by the candidate have discovered that communication between nociceptors, enteric neurons, and enteric glia is required for the development of neuroinflammation in the gut. Further, her preliminary studies indicate that proinflammatory mediators act on glial release mechanism to alter intercellular communication between glia and neurons. These findings are important because they identify novel mechanisms that contribute to neuroplasticity in the gut and these mechanisms could be targeted by therapies to improve gut motility and visceral pain. This body of work will be the focus of her dissertation and she will complete this work over the two years of requested supplemental funding. She will also develop and submit her own F31 fellowship as part of her training under this supplemental support and will transition to this funding if successful. Wilmarie has already begun studies that utilize GFAP::hM3Dq mice to drive glial Ca2+ signaling as described in specific aim 1a to study how this affects nociceptor activity. She aims to complete these studies during the first year of support. She is also working to complete studies that test how inflammatory mediators affect gliotransmitter release mechanisms and aims to complete these experiments in the next year as well. These experiments build upon the cellular physiology studies described in specific aims 1a and 1b. The abstract of the funded parent grant is pasted below for reference: Reflex behaviors of the intestine including peristalsis are orchestrated by the enteric nervous system (ENS); a complex neural network embedded in the gut wall. Inflammation profoundly alters ENS circuits controlling motility by promoting enteric ganglionitis; an inflammatory neuropathy characterized by the death of enteric neurons. Neuropathy is increasingly recognized as a trigger for persistent gut dysfunction in gastrointestinal (GI) motility and functional bowel disorders but the mechanisms that regulate neuropathy are not understood. This proposal investigates the role of enteric glial cells, astrocyte-like cells that surround neurons in the ENS, in the regulation of enteric neuropathy. The proposed studies will use in vivo models of GI inflammation, transgenic mice, immunohistochemistry, live-cell imaging with fluorescent probes, biosensing assays and functional tests to study neuron-glia interactions. The central hypothesis is that purinergic activation of enteric glial cells differentially regulates neuron survival depending on glial activation by ADP or adenosine. There are 2 specific aims in this proposal, each with three sub-aims. Each aim will link in vitro mechanistic studies in tissue from humans and mice with in vivo functional studies in transgenic mice. Aim 1 will test the hypothesis that glial Ca2+ responses driven by ADP cause reactive gliosis, neuron death and gut dysfunction. Specific aim 1A will test how activation of glial Ca2+ responses in GFAP::hM3Dq mice or human tissue transduced with glial-specific vectors affects the induction of reactive gliosis and neuron death. Aim 1B will test whether glial cells directly drive neuron death by releasing neurotoxic substances or if glial-driven neuron death requires immune cell recruitment. Mice with an inducible ablation of connexin-43 or MHC-II in glia will be used to specifically interfere with gliotransmitter release or immune cell recruitment, respectively. Aim 1C will test how manipulation of gliosis using the transgenic mice listed above affects in vivo and ex vivo intestinal function. Aim 2 will test the hypothesis that adenosine inhibits reactive gliosis and stimulates protective mechanisms in glia to preserve ENS function. Aim 2A will use drugs and CD73 null mice to test if activation of glial adenosine receptors is necessary and/or sufficient to reverse reactive gliosis. Aim 2B will test whether the neuroprotective actions of glial A2BR activation are mediated by altering the release of glial mediators or by decreasing the inflammatory infiltrate following in vivo inflammation in CD73 null mice. Aim 2C will use in vivo inflammation, drugs and CD73 null mice to determine how manipulation of glial adenosine signaling impacts in vivo and ex vivo assays of gut function following acute inflammation. Significance: Intestinal inflammation can drive enteric neuropathy, leading to persistent gut dysfunction in GI motility disorders. Understanding how glial mechanisms both promote, and limit enteric neuropathies is important because it could lead to the discovery of novel therapeutic targets and a common causative mechanism of neuron death in GI motility disorders, functional bowel disorders and inflammatory bowel disease.

The proposed investigation will include the identification of the metabolic pathways of oxalate metabolism in man and other mammals and the isolation, purfication and characterization of the enzymes involved. The contribution of each pathway and oxalate precursor will be evaluated in the light of potential regulation and control of the biosynthesis of oxalate in man: Each enzyme will be characterized with respect to their individual contribution to oxalate synthesis, location in tissues, specific inhibitors and activators, and potential for regulation or control. The ultimate goal is the prevention of Primary hyperoxaluria, kidney stones, and ethylene glycol toxicosis. BIBLIOGRAPHIC REFERENCES: Richardson, K.E. and Chou, J.Y. 1977. Effect of pyrazole and 4-methylprazole on ethylene glycol toxicity and metabolism. Fed. Proc. 36: 413. Fry, D.W. and Richardson, K.E. 1977. Isolation and characterization of glycolic acid dehydrogenase from human liver. Fed. Proc. 36: 717.

The proposed study is designed to improve assessment techniques currently available in the substance abuse field by establishing the psychometric properties of screeners for trauma history and for trauma-related psychological symptomatology, specifically post-traumatic stress disorder (PTSD). It will assess the utility of these screening measures for predicting treatment retention and functioning and further expand our knowledge of the relationship of trauma to the current functioning of methadone clients. The specific aims of this study are: (1) to test the reliability and criterion validity of screening questions for assessing history of traumatic events in a methadone treatment population; (2) to establish the reliability and criterion validity of a self- report screening instrument for PTSD in a methadone treatment population; and (3) to examine the predictive validity of these screeners for determining current functioning, treatment retention, and treatment outcome in a methadone treatment population. Reliability of the screeners will be examined in two ways. First, internal consistency reliability will be assessed using coefficient alphas computed from the baseline assessments of a sample of 200 methadone treatment clients. Second, stability over time will be assessed by computing test- retest reliability coefficients, i.e., the correlation between the scores on the screeners at baseline and at a second administration 7 to 14 days later. Validity of the screeners will also be examined in two ways. To assess criterion validity, a sample of 120 TEP participants will be selected to undergo a Gold Standard Diagnostic Battery following their routine baseline assessment. This battery, a semi-structured clinical interview that will be administered by trained clinicians, will contain "gold standard" assessments of lifetime exposure to trauma and a PTSD diagnostic assessment. Criterion validity will be assessed as the correspondence between the screeners and their respective gold standards in terms of sensitivity, specificity, positive and negative predictive power, and the Kappa statistic. To assess predictive validity, scores on the screeners will be correlated with measures of treatment needs, client functioning, and retention in treatment to determine whether the screeners are related to these other important indices. The ability of the screeners to predict retention, and to predict client functioning in a variety of domains, is important to their ultimate utility in treatment settings. Retention and quarterly follow-up data that are routinely collected as part of TEP will be used in these analyses.

This is a continuation to develop and expand upon data collected in a retrospective longitudinal study exploring the relationships among a variety of sexual experiences - including sexual socialization, voluntary sexual practices, and sexual abuse - and their effect on women's psychological well-being. Previous research has tended to examine these areas separately from one another and most studies have focused on restricted samples, such as college studies. As a result, there is little available to mental health professionals that identifies factors contributing to the occurrence of sexual abuse and its effects on psychological and sexual functioning, especially as these pertain to ethnic minority women. The purpose of the continuation is to develop a more comprehensive model of these complex relationships that will have utility for women from a variety of cultural backgrounds. The method of sampling involved a multi-stage stratified probability sample using quotas representative of young adult women, ages 18 to 36, in Los Angeles County. Random-digit dialing was used to recruit participants, and in-depth face-to-face interviews were conducted with 126 Afro-American and 122 White women. In order to allow for between-group as well as within group comparisons, women from the two ethnic groups were matched on the basis of age, education, marital status, and the presence of children. The study has provided extensive descriptive information - spanning childhood, adolescence and adulthood - concerning voluntary and abusive sexual experiences. Analyses conducted thus far have identified the following association: environmental and familial characteristics contributing to the risk of sexual abuse, the effects of child sexual abuse on adult sexual and psychological functioning; and the influence of family life and sexual socialization on adolescent sexual activity. Proposed secondary analyses will first involve further data reduction to develop and refine indicators of key constructs. Path analyses and latent-variable structural equation modeling will then be employed to test the relationships between constructs. These statistical methods permit the separation of measurement error from true score variance. Thus, the structural models will be quite powerful and the resulting relationships substantially larger than those obtained from standard methods. The ultimate goal of the continuation is to develop a theoretical model or models than can then be tested on other samples.

Linguistic and psychological theory coupled with empirical studies on the heritability of individual differences in language development and language disorders support the hypothesis that the capacity for language development in humans is genetically influenced. The proposed research will provide for further understanding of this genetic influence on language by studying individuals with specific language impairment (SLI) who have poor language development despite normal linguistic experience, sensory abilities, and nonverbal intelligence. Individuals with SLI provide an excellent means of identifying quantitative trait genetic loci that are likely to contribute to the full range of individual differences in language development and use. The proposed research will use two complementary strategies. First, it will take advantage of two individuals (TB) with severe developmental speech and language impairment, who also have a chromosomal translocation that appears to involve the FOXP2 gene. The FOXP2 gene is known to be a regulatory gene implicated in developmental speech and language disorder. This strategy will contrast the neuroanatomical, neurophysiological, and neuropsychological characteristics of these two individuals with their unaffected family members. This work will emphasize the procedural and declarative learning systems and vocabulary and sentence use. Thus, this strategy moves from gene to language phenotype to understand the neurologic and cognitive pathways between gene and phenotype. The second strategy will use a large epidemiologic sample of young adults with SLI along with their siblings who have participated in a 10-year longitudinal study. The language status, as well as associated conditions of these individuals, has been studied extensively. Furthermore, DNA samples have already been obtained from the SLI probands, siblings, and their parents. Thus, a valuable data bank of Doth tissue and phenotypes is available to identify genes by means of high density genome-wide screens using sib-pair linkage methods and follow-up fine mapping methods using transmission disequilibrium tests (TDT). Additionally, ongoing fine mapping of candidate genes and regions of interest informed by findings from our laboratories and other laboratories will be carried out along with microdeletion studies. Additional phenotyping of the SLI probands and siblings will be conducted to obtain procedural and declarative learning measures and additional language measures that parallel the data to be obtained from the TB family. The learning measures are to serve as endophyenotypes that may be more sensitive and specific to genetic influence. The phenotypes and endophenotypes from these individuals will then be used in conjunction with the existing genotyping for additional genome-wide screens and further follow-up fine mapping studies.

DESCRIPTION (provided by investigator): The early years between ages 3 -7 have been described as a critical period in the development of obesity. The importance of the family on the development of eating and physical activity behaviors in young children has been emphasized by experts, yet to date no obesity prevention interventions incorporating a strong family focus with emphasis on improved parenting practices have been developed. The work proposed here will address this gap by testing the efficacy of a 12-mo parent-focused intervention designed to change multiple aspects of children's home environments (both social and physical), which in turn will reduced percent body fat (%BF) in children. Subjects will be 280 parent-child pairs recruited through child care settings that cater to lower income families. Following baseline measures, pairs will be randomized into intervention or control. The intervention will first focus on teaching more effective parenting skills (time management, communication, etc.); then showing how specific parenting practices can influence a child's physical and social environment and encourage healthier behaviors. The intervention will be delivered through group sessions and home-based telephone calls (group chats and tailored calls) facilitated by a certified parent educator. Group sessions will use multiple teaching strategies such as "Parentela Novellas" (soap opera-like videos which provides models for skills practice). Sessions will also provide separate child activities that build off the coercion hypothesis and prepare children to receive new parenting practices. Group phone chats will build peer support among parents and tailored counseling calls will use motivational interviewing techniques to help parents overcome barriers to implementing new behaviors. The intervention is guided by Darling and Steinberg's Integrative Model of parenting (1993) which highlights the importance of parenting values, style and practices in child socialization. In addition, Self-Determination Theory (Ryan, 2000) is used to create a program that fosters parents' autonomous motivation to adopt new parenting style and practices. The result is a intervention that will help parents learn skills to reduce their parenting-related stress and create a home environment that supports the development of healthy-weight behaviors. The primary outcome, %BF in children, will be assessed using anthropometry and a validated prediction equation (Denzenberg, 1999). Analyses will include baseline measurements of the outcome as a covariate. Secondary measures include child and mother diet and physical activity, parenting style and practices, parenting stress, and home environment. Maintenance of intervention effect will be assessed following a 12-mo no-intervention period. This intervention will create the intensive exposure necessary to make life-altering changes in parent and child behavior that will result in reduced body fat. Plans are based on extensive formative work, and the research will be lead by a group of very experienced investigators. [unreadable] [unreadable] PUBLIC HEALTH RELEVANCE: Efforts are needed to assure that young children develop healthy eating and physical activity behaviors for healthy weight development. Parent behavior affects both the social and physical home environment, creating the context within which these behaviors develop. Few programs exist to prevent obesity and fewer still have used a family-focused approach. This application describes an innovative, theory-driven intervention designed to help parents implement more effective parenting behaviors and reduce their parenting-related stress, while also creating a home environment that supports the development of healthy weight practices in their preschool children. [unreadable] [unreadable] [unreadable]

Completion of sequencing of the human and of bacterial genomes has made it possible to apply the principle of oligonucleotide hybridization competition to the inhibition of nefarious genic expression. We shall focus our efforts on three targets: l) M. tuberculosis, with its uniquely constructed mycocerosic outer cell wall; 2) Cystic Fibrosis, in which for our novel oligonucleotide insertion technique we must pin down the extent of insertion. Present estimates, as a result of over thousand sequencings, are that insertion occurs in 10-25 percent of delta508 mRNA molecules; 3) Huntington's Disease, requiring further studies to firm up our finding of inhibition of expression of Huntington protein in tissue cultures from patients with this disease, using derivatives of antisense oligonucleotides.

The structure of tandem chromosomal duplications of the tryptophan operon will be studied in S. typhimurium. Factors which affect the stability of the duplications will be studied including the effects of interactions of mutant supX, recA, recB and recC alleles with temperature, UV and photo reactivating light. Studies of the effects of chromosomal insertions on the cotransduction linkage of nearby markers will be used to further characterize the process of viral encapsulation of host cell DNA. The bacterial growth inhibiting effects of normal metabolites such as indole and riboflavin will be analyzed.

My career goal is to become an academic psychiatrist and independent investigator in the areas of schizophrenia focusing on its neurobiology and therapeutics, applying molecular mechanism-based research to prospective clinical studies. Specifically, I propose to investigate the importance of neurosteroids in schizophrenia. Neurosteroids are differentially expressed in males and females, modulate GABAA and NMDA receptors, regulate neuronal cytoarchitecture, demonstrate neuroprotective effects, play a role in neurodevelopment, and possess memory-enhancing effects. Neurosteroids are therefore logical candidates of investigation to elucidate schizophrenia pathophysiology, since they are potential modulators of schizophrenia gender differences, GABAergic and glutamatergic dysregulation, neurodevelopmental insults associated with increased schizophrenia risk, cytoarchitectural abnormalities in postmortem specimens from patients with schizophrenia, and cognitive disturbances in the disorder. The laboratory has demonstrated that neurosteroids protect embryonic cerebral cortical neurons against anoxia, a neurodevelopmental insult associated with increased schizophrenia risk. We have also demonstrated that acute olanzapine and clozapine administration alters cerebral cortical neurosteroids in rodents. The investigators hypothesize that neurosteroids are important modulators of schizophrenia pathophysiology (including the pronounced gender differences of the disorder) and antipsychotic drug action. We also propose that compounds affecting neurosteroid expression or neurosteroids themselves may be developed as novel therapeutic agents in the treatment of schizophrenia. To test this hypothesis, three investigational strategies are proposed: 1.) A preclinical study examining the effects of antipsychotics on cerebral cortical and serum neurosteroid levels in rodents, 2.) A postmortem study determining neurosteroid levels in parietal cortex and posterior cingulate specimens provided by the Stanley Foundation from patients with schizophrenia compared to control subjects, and 3.) A clinical study examining neurosteroid levels in subjects with schizophrenia from two UNC clinical trials (Dr. Lieberman, PI) to determine if serum or CSF neurosteroid alterations are correlated with antipsychotic efficacy, neurocognitive changes, and/or structural changes on MRI. Results from these preliminary investigations will inform the design of future prospective clinical studies to confirm initial findings and target neurosteroids as therapeutic agents in schizophrenia. To achieve these goals, the candidate will receive training through formal coursework in neuropharmacology, clinical trials design, drug development, and biostatistics. She will also learn highly sensitive and specific gas chromatography mass spectrometry (GC/MS) and other state-of-the-art neurosteroid detection methods. The mentorship of Drs. Jeffrey Lieberman and Leslie Morrow will be critical to the overarching goal of this proposal, the successful translation of exciting preclinical neurosteroid findings to prospective clinical studies examining neurosteroids in schizophrenia pathophysiology and therapeutics.

Osteoarthritis (OA), which affects millions of Americans, is a debilitating condition characterized by the loss of articular cartilage at the join surfaces. Osteoarthritis is a promising candidate for regenerative stem cell therapy. However, despite decades of investigation, the cells and molecular pathways that repair cartilage remain undefined. Recently, we and another group (Irving Weissman's group at Stanford University), have independently identified a distinct population of skeletal stem cells in adult bone marrow. These cells live near the surfaces of the joints, are capable of self-renewal, and give rise to osteoblasts, cartilage cells (chondrocytes) and reticular cells during adulthood, and participate i the repair of fractures. The isolation of OCR cells raises a crucial question regarding the pathogenesis and treatment of OA. Do these cells contribute to tissue repair in OA? Could their function be augmented to assist in OA repair? In this proposal, we wish to investigate the origins of cartilage repair in vivo, test which mouse stem/progenitor populations repair cartilage, and define molecular pathways involved in cartilage repair. We will use a well-established model of OA in mice: transection of the medial menisco-tibial ligament, resulting in wear and tear degeneration of the articular cartilage that recapitulates the anatomical pathology of human osteoarthritis, including cartilage loss and bony sequelae, 4-8 weeks after injury. We wish to ask the following questions: What is the cellular origin of cartilage repair in OA? We will use lineage tracing from chondrocytes, OCR stem cells and MSCs in an OA model to determine the origin of cartilage repair. What are the best cells for repairing articular cartilage? We will assess if injecting these populations into the knee during OA reduces the cartilage damage. What are the targetable signaling pathways that promote OA repair? We will investigate and therapeutically utilize Bmp and VEGF signaling in cartilage differentiation and repair. We will define other signaling pathways relevant to chondrogenesis from OCR cells.

Dental caries is one of the most prevalent chronic infections in humans. It is caused by acid production from fermentation conducted by acidogenic bacteria that colonize the tooth surface. Intake of fermentable carbon by the host promotes acid generation, resulting in tooth demineralization, which is symptomatic of dental caries. Establishment of the acid-producing biofilm that leads to caries is initiated by Streptococcus mutans, which produces adhesive proteins and glucans required for biofilm development. Although it contributes to pH reduction, S. mutans is sensitive to extremes of pH. Such extremes are rarely encountered in the oral environment, as normal saliva has a pH range from 6.0 to 7.8, with stimulated saliva flow having a pH from 7.4- 7.8. While much research has been devoted to understanding how S. mutans withstands low pH, there are few studies that have targeted the response of S. mutans to alkaline conditions. Quite unexpectedly, a deletion of the adhC gene, encoding the lipoylated E2 subunit of acetoin dehydrogenase (Adh), confers acute sensitivity to pH of ~7.5, a pH value commonly observed in human saliva. Mutations that render defective production of the other adh operon products (E1, E3, and LplA, the lipoyl ligase) also confer sensitivity to modest elevations in pH (pH 7.2-7.6). The adh/lplA mutants also exhibit defects in carbohydrate uptake and/or consumption and the adhD null mutation confers a severe defect in biofilm formation when sucrose is present. The finding raises the possibility of targeting specific functions in S. mutans in order to sensitize the bacterium to the ambient pH of the human oral cavity while also compromising metabolic operations within the oral pathogen. The exploratory, hypothesis-generating project proposed herein will identify the factors associated with Adh- dependent alkaline tolerance. Suppressor mutations that overcome the alkaline sensitivity of the adhC mutation have been isolated and will continue to be uncovered. Such mutations will identify genes that potentially operate within the network that Adh functions to render cells resistant to elevated pH. The lipoyl cofactor attachment sites of encoded in adhC and adhD will be mutationally inactivated and the effect on alkaline sensitivity will be tested to assess the importance of Adh redox chemistry in alkaline tolerance. Micromolar Zn2+ concentrations, which inhibit 2-oxo acid dehydrogenases, will be used to examine test the Adh catalytic requirement for pH tolerance. A genomic Tn-seq experiment will be undertaken to identify genetic loci that function in alkaline tolerance. These mutations will be combined with the adhC suppressor mutations to determine if the identified genes? functions are related to that of Adh-dependent alkali resistance. The adh mutants and those identified in the mutant screens will be tested for fitness in mixed cultures with commensal, arginolytic competing species, S. gordonii or S. sanguinis, which are known to cause pH elevation in plaque biofilms. These experiments will be performed with planktonic cultures and mixed-species biofilms. The project will generate potential targets for interfering with S. mutans proliferation in the human oral environment.

We employ several approaches to attempting to understand the etiology and pathogenesis of multiple sclerosis. We will continue attempts to recover a virus or identify virus antigen in brain tissue of patients with MS. We will use human tissues in culture and animal model systems in vivo and in vitro to study the behavior of known neurotropic viruses, virus latency, and attempt to improve virus detection systems. We will study immunologic alterations associated with MS, with particular attention to compartmentalization of the immune response within the nervous system as compared with non-nervous system tissues. We will continue to focus on the oligodendrocyte as a possible target cell in MS, and employ isolated oligodendrocytes for immunologic and virologic studies related to MS.

The purpose of this agreement is to provide support to the Center for Substance Abuse Treatment[unreadable]s (CSAT) initiative with the Washington Circle to advance the science of performance and quality measurement of addiction services.

Work will continue on an investigation by small-angle x-ray scattering of the structure of E. coli ribosomes in solution. Radius of gyration measurements will be made on the 50S ribosome in H2O and D2O. The measurements will be made on samples prepared by Engleman and Moore of Yale University for comparison with their measurements by neutron scattering on the same samples. Experiments will be done to elucidate the nature of the 50S to 45S transition on addition of 0.5 M NH4Cl to 50S ribosomal subunits. The reversibility of the transition will be investigated in terms of the sedimentation coefficient, the radius of gyration and the diffusion coefficient.

The key features of the Molecular Biology Core of the Skin Disease Research Center are its[unreadable] comprehensive range of services designed to support the needs of both proposed and future SDRC[unreadable] members, and its objective of training young investigators in the area of its expertise. This Core[unreadable] provides assistance in three essential areas of molecular biology central to virtually any studies[unreadable] ranging from the investigation of the genetic basis of human disease to cell biology experiments.[unreadable] These include 1) Disease-gene regulation; 2) In Situ labeling of genetic alterations; and 3) Real-[unreadable] Time PCR, automated sequencing and cloning of DNA constructs. The core will assist center[unreadable] investigators in identifying the epigenetic mechanisms of a particular skin disease by promoter and[unreadable] mutagenesis studies and siRNA knockdown models. The Core will assist investigators in automated[unreadable] DNA sequence analysis to identify mutations implicated in skin disease pathogenesis and[unreadable] subsequent histological detection of target cells harboring these genetic mutations. The Core will[unreadable] also assist investigators with the design and optimization of Real-time PCR analysis for candidate[unreadable] disease-genes and developing a plan for ascertaining their functional consequence, and the way in[unreadable] which it elicits a clinical phenotype. The Core will also provide instrumentation, general resources[unreadable] and technical expertise in the use of automated DNA sequencing and DNA cloning. By offering[unreadable] these routinely used services, the Molecular Biology Core will interact closely with the other two[unreadable] cores. Further, the expertise offered in this Core will be almost universally utilized and provide a[unreadable] valuable service to most, if not all, of the Pilot and Feasibility studies, as well as other investigators[unreadable] within the Department.

The objectives of this project are to understand and modulate the processes of kindling and quenching. Kindling involves the development of convulsions following repeated, intermittent administration of a subconvulsant stimulation. Quenching is a procedure developed in our laboratory to inhibit the development and expression of kindled seizures by increasing the seizure and/or afterdischarge thresholds. Both models involve long-term changes in the nervous system; with kindling lasting possibly for the entire lifetime of the animal and quenching lasting for weeks to months after discontinuation of the procedure. The effects of various anticonvulsants on kindling have been examined in relation to stage of kindled seizure development (e.g., development vs. completed vs. spontaneous) and type of kindling stimulation (e.g., pharmacological vs. electrical), indicating the importance of both parameters in relation to anticonvulsant responsivity. Agents with specific biochemical target systems have been used to elucidate the mechanisms of action of anticonvulsants, and studies have also been conducted to determine mechanisms of amygdala kindling and quenching. Significant findings to date include demonstration of the following. 1) Distinct patterns of anticonvulsant responsivity occur based on the stage and type of kindling stimulation; e.g., carbamazepine is an effective anticonvulsant during the completed phase of amygdala kindling, but not during seizure development, and chronic, but not acute, carbamazepine blocks the development, but not expression, of local-anesthetic-kindled seizures (and their associated lethality). 2) The cholinergic system is involved in local anesthetic kindling and is distinct for procaine and cocaine compared with lidocaine. Atropine blocks seizures induced by procaine and cocaine and potentiates seizures induced by lidocaine. Physostigmine attenuates lidocaine kindling. 3) In amygdala-kindled rats, time off from seizures leads to a diminished anticon-vulsant response upon subsequent testing and a decrease in seizure threshold (i.e., increased seizure susceptibility), indicating a functional role for seizure-induced endogenous anticon-vulsant adaptations, which appear to be transient and to facilitate response to exogenous anticonvulsant agents. 4) TRH is one of the hypothesized endogenous anticonvulsant adaptations: following intrahippocampal administration, TRH dose-dependently attenuated the afterdischarge and seizure duration in amygdala-kindled rats. 5) The mRNA expression for a number of immediate early genes, trophic factors, and peptides is increased in a regionally selective manner during kindling development and after completed or spontaneous seizures. Some of these regional effects are dependent upon the length of the elicited afterdischarge; others are dependent on where in the amygdala the stimulation is occurring. 6) Quenching paradigms have been developed using very low levels (LL) of direct current (DC) with or without concurrent low frequency stimulation which produce a long-lasting increase in afterdischarge and seizure threshold, and an inhibition of kindling development and seizure expression in fully kindled animals. These threshold effects persisted for weeks to months after quenching stimulation was discontinued. 7) Quenching was associated with increases in benzodiazepine receptor binding in the entorhinal and perirhinal cortices, but did not produce increases in mRNA expression for a number of immediate early genes and TRH. 8) DC quenching is associated with local increases in the mRNA for glial acid fibulary protein and a strip of silver deposition, raising the question of whether a microscopic lesion is being produced by the LL-DC and of its ultimate clinical relevance. 9) DC has also been utilized in the amygdala slice preparation wherein it induces homosynaptic but not heterosynaptic inhibition that can be overridden by increasing intensity of stimulation.

The goal of the proposed research is to understand the process of intracellular proteolysis in bacteria. Already available peptidase mutants of Salmonella typhimurium and E. coli will be further characterized and new types will be obtained. The important peptidases and proteases will be purified and characterized biochemically. The effects of peptidase and protease mutations on protein modification and maturation and on protein degradation will be studied.

It is widely accepted that the hepatotoxicity of CCl4 results from the metabolism of CCl4 to the trichloromethyl free radical by cytochrome P- 450. Earlier studies reported that sublethally CCl4-treated rabbits were 1`0 times more susceptible than normal rabbits to the lethal effect of bacterial endotoxin. Endotoxin absorbed from the gut becomes involved in hepatotoxicity by its interaction with peritoneal and splenic macrophages and Kupffer cells. These cells, when stimulated, produce reactive mediators, including oxygen-derived free radicals, tumor necrosis factor- (TNF-alpha, leukotrienes, and nitric oxide (NO), thereby causing cellular damage. Upon CCl4 administration, Kupffer cells increase in number and peripheral monocytes/ macrophages are recruited to the liver. Both Kupffer cells and hepatocytes respond to cytokines and endotoxin by expressing an inducible isoform of nitric oxide synthase (NOS). We published data showing that, upon immunologic activation with endotoxin, NO was produced synergistically in whole blood and livers of rats that had been treated previously with CCl4. Nitrosyl complexes formed by NO binding to the hemoproteins, hemoglobin (whole blood) and cytochrome P-450 (liver biopsies) were measured with EPR spectroscopy. Decreased nitrosyl hemoprotein complex formation occurred in livers following treatment with either an inhibitor of macrophage activation (GdCl3), an inhibitor of cytokine responses (dexamethasone), or a ~NO synthase inhibitor (N-monomethyl-L-arginine, L-NMA). GdCl3 or dexamethasone treatment decreased TNF-alpha levels, while L-NMA treatment increased them. TNF-alpha levels, but not nitric oxide hemoprotein complex concentrations, correlated with increased hepatic damage as indicated by release of hepatic enzymes into the serum. In fact, the L- NMA NOS inhibitor actually increased hepatic damage. It appears that NO may play a protective role, and TNF-alpha, which is found at high concentrations, may be associated with pathophysiological responses.

Multiple Sclerosis (MS) is a common neurological disorder characterized by immune attack on myelin and mylein forming cells of the central nervous system. Current treatments are directed at reducing the immune-mediated inflammatory damage to myelin. Recent observations from neuroimaging and pathological research demonstrate, however, that axonal pathology may also play a large role in the clinical deficits of MS patients, suggesting that the protection of axons may be a potentially important addition to MS therapies. The long-term objective of this proposal is to test the hypothesis that the protection of axons through the inhibition of calcium-activated proteases, or calpains, will ameliorate the clinical deficits in a mouse model of MS. This hypothesis is based on a large volume of data demonstrating the importance of calpain activation in axonal degeneration, and our published experience with preventing axonal degeneration and clinical disease in another animal model of neurological disease. In Aim 1 we will use the model of experimental autoimmune encephalomyelitis (EAE) to investigate the relationship of calpain activation to axonal degeneration and clinical disease. We will measured calpain activation using calpain activity assays and immunocytochemical and immunoblot methods to identify calpain-specific spectrin-breakdown products in tissues. In Aim 2 we will treat animals with EAE with our novel ketoamide calpain inhibitor AK295 and measure attenuation of disease by clinical and patholgical methods. In Aim 3 we will test the ability of AK295 to cross the blood-brain barrier (BBB), since any effective calpain inhibitor will need to get into the brain in sufficient quantities. We will also test the BBB permeability of 10 other novel calpain inhibitor compounds taken from our library of compounds, and compare them to AK295 for BBB permeability. Finally, we will attempt to increase BBB permeability by chemically modifying AK295 and other promising compounds by adding choline and nucleosides in order to take advantage of the inherent BBB transporter systems. These studies of calpain inhibition in a model of MS represent a novel approach to the treatment of this common and devastating neurological disorder. Combined with immunomodulatory therapy, axonal protection by calpain inhibition may significantly change the course of disease for people with MS. [unreadable] [unreadable]

Botulinum neurotoxins (BoNTs) represent one of the most serious bioterror threats to our national security. Secreted by spore forming bacteria Clostridium botulinum, baratii, and butyricium, BoNTs are responsible for the flaccid paralysis associated with botulism. Listed as category A biothreats by the CDC, BoNTs are the most toxic of biological toxins, are easily produced, and may be delivered by various routes including food spiking (solid and liquid) and aerosol dispersion. There are no therapeutics to counter BoNT induced paralysis once the enzyme has entered the neuronal cytosol - at this time the only treatment option is impractical mechanical ventilation in a critical care setting. The long-term objective of this project is to address this public health threat by developing potent small molecule, non-peptidic, inhibitors (SMNPIs) of BoNTs as therapeutics that will serve as both rescue agents and prophylactics. Specific aims 1 - 3 of the proposal focus on translating our lead uM range SMNPIs of BoNT serotype A into low nM range SMNPIs that are effective in an animal model. Specific aim 4 concentrates on identifying and developing SMNPIs of BoNT serotype B that are effective in a neuronal assay, and specific aim 5 covers the identification of SMNPIs of BoNT serotype E. Finally, under specific aim 6, we propose examining all identified and developed SMNPIs for broad spectrum inhibitory activity against more than one BoNT serotype. To achieve the project goals we combine three-dimensional pharmacophore-based methods and synthetic organic chemistry strategies. Specifically, such methods combine X-ray crystallography, in vitro and in vivo biological testing, molecular modeling and 3-D database mining, and organic chemistry to guide SMNPI discovery and optimization (i.e., good in vitro potencies, good ADMET profiles, reduced toxicities, and enhanced in vivo efficacies in an animal model). RELEVANCE (See instructions): Botulinum neurotoxins (BoNTs) are the most potent toxins on the planet and are capable of being used as bioterror devises. There is currently no therapeutic to counter the inevitable suffocation and certain death resulting from the paralysis induced by BoNT poisoning. We propose the use of therapeutically viable chemicals as drugs. Our proposal includes molecules that already possess activity against critical components of the BoNT intoxication process for two serotypes.

Human leishmaniasis is caused by the Leishmania spp. In this protozoan parasite, initiation of transcription is not a major mode of gene regulation. Consequently, post-transcriptional pathways for controlling protein expression, which is critical for virulence, are of utmost importance. Untranslated regions of mature mRNAs can have major effects on several aspects of mRNA metabolism in other organisms. Our long-term goals are to determine general principles by which initiation-codon proximal sequences influence translation and/or mRNA stability in Leishmania. We performed bioinformatic analysis of two hundred Leishmania genes, and discovered hexamer nucleotides that were popular in regions close to translation initiation codons. Further analysis revealed that the hexamers were components of five extended sequence motifs that might be Leishmania equivalents of "post-transcriptional operons" recently describe, vertebrates. We hypothesize that the Leishmania sequence motifs coordinate expression of genes af, naturation of pre-mRNAs. To test the theories (above) representative sequence motifs will be cloned upstream of reporter genes, electroporated into Leishmania, and stable cell lines of parasites selected. The influence of the motifs on mRNA stability and/or translation will be determined in promastigote, metacyclic, and amastigote developmental stages. These studies are likely to lead to discovery of novel regulators of mRNA metabolism, thereby furthering our understanding of post-transcriptional gene regulation in Leishmania. [unreadable] [unreadable]

SUMMARY The spatial and temporal organization of intracellular components by the microtubule cytoskeleton is required for cell division, many aspects of development, and neuronal function. Defects in the microtubule cytoskeleton cause neurological diseases in humans. The long-term goal of this research program is to determine how the multiple components of this transport system?the motors and their tracks, cargos, cargo adaptors, and regulators?work together. Microtubules are dynamic polar structures, with ?plus? ends usually located near the cell periphery and ?minus? ends embedded in internal microtubule organizing centers. Dyneins move towards the minus ends of microtubules, whereas most kinesins move in the opposite direction. In humans, a single dynein (cytoplasmic dynein-1) and ~15 kinesins are responsible for the interphase transport of organelles, proteins, and mRNAs. Viruses hijack these same motors. How does a small subset of motors transport such a large and diverse set of cargos? Understanding this is one of the major frontiers in the transport field. The goal of this proposal is to determine how cargo specificity is achieved for cytoplasmic dynein-1, the major minus- end-directed microtubule-based motor in eukaryotic cells. Traditional biochemical approaches have yielded surprisingly little information about these mechanisms. To solve this problem, we are using both genetic and proteomic discovery approaches to identify motor-cargo interactions and to determine how they are regulated. Genetic approach: Using a forward genetic screen in the filamentous fungus Aspergillus nidulans we co- discovered that peroxisomes ?hitchhike? on early endosomes to achieve motility. Previously, the paradigm was that each cargo directly recruited the transport machinery. We will investigate the mechanism of hitchhiking and determine if it is widely used for organelle motility across eukaryotes. Proteomic approach: Mammalian dynein requires the dynactin complex and a coiled coil containing ?activator? to achieve processive motility. Using proximity-dependent biotinylation we identified the components of the human dynein proteome in human interphase embryonic kidney cells, including novel dynein activators. We will use these proteomic approaches to identify additional novel dynein activators in different cell types and at different stages of the cell cycle. We will use these novel dynein activators as ?stepping stones? to identify their proteomes and determine which cargos they transport.

The effects of ryanodine on left ventricular (LV) function and hemodynamics were studied in 16 conscious dogs, chronically instrumented for measurements of LV pressures and dimensions. Systemic infusion of ryanodine (0.5 - 4 fg/kg i.v.) resulted in a dose-dependent depression of cardiac contraction. For example, ryanodine, 4.0 g/kg i.v., decreased LV fractional shortening by 30.5q4.1%, LV dP/dt by 41.5q4.0% and Vcfc by 37.8q4.1%, while increasing the isovolumic relaxation time constant, tau, from 23.1q1.4 to 34.1q3.6 msec without a major effect on preload or afterload. Ryanodine also depressed (p<0.05) the plateau phase of the mechanical restitution and postextrasystolic potentiation responses, indicating a direct effect on excitation-contraction coupling. The heart rate dependent positive staircase ("Treppe") was significantly enhanced (p<0.05) after ryanodine infusion, i.e. LV dP/dt rose by 43.1q4.7% with an increase in heart rate from 150 to 240 beats/min in the presence of ryanodine 4 fg/kg, but by only 7.5q2.1% without ryanodine. The more pronounced "Treppe" in the conscious dog under the condition of impaired SR calcium release caused by ryanodine, supports the concept that the classical Bowditch "Treppe"reflects either a state of myocardial depression due to alteration in SR calcium handling, or enhanced availability of trans-sarcolemmal Ca2+ influx. This finding may help to understand the discrepancy in the importance of the "Treppe" between conscious animals and more isolated preparations.

The effects of hypoxia on contraction and relaxation in chick embryo ventricular cells cultured as confluent, spontaneously contracting monolayers will be studied. Inotropic effects are detected by measurement of changes in the amplitude and velocity of individual cell wall motion. Similar changes in cell wall motion during diastole can be measured, and can be analyzed to detect the influence of hypoxia on parameters of relaxation. Negative inotropic effects of hypoxia will be correlated with changes in calcium contents and rapid Ca influx and efflux measured using 45Ca as a tracer. The effects of hypoxia on monovalent cation active transport, and on cellular contents of Na and K, will also be determined using radioactive isotopes of Na and K. These effects of hypoxia will be correlated with the effects of this intervention on cellular contents of ATP and creatine phosphate. Our overall goal is to determine the mechanism(s) of the negative inotropic effects of hypoxia and to investigate the cause of abnormal relaxation during this insult.

Extreme changes in cellular architecture are experienced by germ cells during spermatogenesis. The most extensive of these changes take place after meiosis, when the chromatin is condensed, the nuclei are dramatically reshaped, the mitochondria fuse into a single giant organelle, and extensive reorganization of the cytoplasm must be accomplished. Correspondingly, the majority of male-sterile mutations in Drosophila appear to affect postmeiotic steps in spermatogenesis. The reorganization of spermatid nuclei in particular may be a critical determinant of male fertility, as several studies argue for a clear correlation between sperm nuclear morphology and fertility. While a large number of Drosophila genes are mutable to "late" male-sterile phenotypes, in which highly elongated bundles of sperm fail to be properly matured, most of these mutations appear to lie within genes whose functions are required in other tissues and stages of development. Genes whose functions are required only in spermiogenesis may be of considerable interest for the understanding of the genetic logic by which spermiogenesis proceeds, and for the development of chemotherapeutic strategies that will specifically target this process with a minimum of effects on other tissues and physiological pathways. Preliminary evidence suggests that a previously undescribed Drosophila gene, sad sack, is a hotspot for mutations affecting spermatid maturation, is required only for male fertility, and may play a role in the nuclear reorganization of spermatids. The detailed spermatogenic defect of sad sack mutants will be determined through a combination of fluorescence and electron microscopy, with particular attention to the structure, composition, and dynamics of the nuclear envelope. The sad sack gene, currently localized to a chromosomal region of 320 kb, will be mapped to a fine-structure level by male recombination and/or strategies based on the generation of small overlapping deletions throughout the region. Candidate gene(s) identified through this approach will be analyzed in more detail by examining their expression throughout development, screening for molecular lesions in candidate genes, and tested for genetic rescue of sac mutants by germline transformation. For localization of the sac gene product within developing sperm, antibodies will be generated against synthetic peptides of amino acid sequence unique to sac or bacterially-expressed sac protein.

To improve the effectiveness of medical treatment for people with lung disease produced by cigarette smoking. Chronic obstructive pulmonary disease (COPD) is a disabling disorder and inability to exercise is a foremost problem. Focus will be on strategies predicted to improve the muscle's ability to tolerate exercise. Strategies include administering testosterone to COPD patients to increase muscle mass and initiating a conditioning program consisting of a varied group of weight lifting exercises to improve exercise tolerance.

In adult dogs after pneumonectomy (PNX), mechanical forces on the remaining lung are thought to stimulate compensatory lung growth. Following moderate (42% of total) resection, lobar growth is non-uniform. As resection increases (58-70%), alveolar growth is stimulated in all remaining lobes probably due to mechanical forces exceeding a threshold for cellular stimulation. Supplementation with all trans-retinoic acid (RA), a promoter of cell growth and differentiation, enhanced some aspects of alveolar-capillary response following 58% but not 42% resection, suggesting a relationship between the intensity of endogenous stimuli and the capacity for pharmacological growth enhancement. Lobar expansion explains ~40-70% of post-PNX compensation, presumably via increased septal strain/shear; residual compensation might be explained by elevated lobar perfusion via increased strain/shear on alveolar capillaries. Lobar expansion may also alter and interact with regional perfusion. Relationship of in vivo tissue mechanical stress to cellular response has not been established, but the hypoxia-inducible factor-erythropoietin-vascular endothelial growth factor (HIF-EPO- VEGF) axis is implicated. Our hypotheses are: 1) Regional compensatory lung growth varies directly with the intensity of regional mechanical stimuli, and 2) Growth factor supplementation amplifies natural response to post-PNX mechanical stimuli, but cannot initiate de novo compensatory lung growth in the absence of sufficient endogenous stimuli. In Aim 1, we will quantify post-PNX regional lung strain and shear using bronchovascular landmarks imaged at different transpulmonary pressures by high resolution computed tomography (HRCT) and correlate strain/shear with regional cellular response in HIF-EPO-VEGF axis. In Aim 2, we will test the effects of altering lobar perfusion on post-PNX compensation by banding one lobar pulmonary artery, which restrict its perfusion while exaggerating post-PNX perfusion to unbanded lobes. Regional perfusion will be measured by fluorescent microspheres. The remaining lobes will be assessed for cell signaling and for alveolar ultrastructure by morphometry. In Aim 3, we will establish cause-effect mechanisms of mechanical stresses on growth- related signaling in vitro using cultured lung epithelial cells subjected to mechanical strain and endothelial cells subjected to strain and fluid shear. Mechano-stress response of HIF-EPO-VEGF axis will be compared to its O2-sensitive response. In vitro signaling events will be related to parallel in vivo stress response. Epithelial- endothelial co-cultures will test for diffusible mediators of mechanotransduction. In Aim 4, we will amplify EPO/EPO-R signaling post-PNX by nebulization of recombinant human erythropoietin (rhEPO) to determine its local effect on angiogenesis, growth and function in relation to in vivo regional mechanical stresses. These studies define fundamental mechanical stimuli-response relationships in a robust model of compensatory lung growth, and explore a potential intervention for augmenting the endogenous response. Results have important basic and clinical implications for the re-initiation of lung growth in human chronic lung disease.

During the early development of D. melanogaster a single egg cell is converted into a multicellular organism (the first instar larva) which contains in a defined spatial arrangement a variety of specialize organs, tissues, and cell types. The steps in embryogenesis of D. melanogaster include: differential cell divisions, cellular movements, the establishment of cell-cell interactions, and the formation f specialized tissues and cell types. The research described in this proposal will focus on one aspect of embryogenesis which is particularly amenable to study in D. melanogaster - the role of, and the mechanisms controlling differential gene expression during embyrogenesis. Two questions are of special interest: 1. Is there a temporal coordination between a "program" of gene expression and the differentiation of specific tissues or cell types during embryogenesis? and 2. If so what are the mechanisms involved that result in coordinate gene expression?

The proposed study, the Mexican Health and Aging Study-II (MHAS-II), aims to design and field the 3rd and 4th waves of survey data collection in Mexico. The two waves will be fielded in 2012 and 2014, eleven years after the two previous waves funded by NIA/NIH were fielded in 2001 and 2003. Funds are sought also to archive, document, and disseminate for public use the 3rd and 4th rounds as well as the resulting integrated data base containing all four waves. The previous study (MHAS-I) aimed to create a longitudinal prospective study of Mexican aging, starting with a national sample of persons aged 50 and older, using study protocols and survey instruments that were highly comparable to the U.S. Health and Retirement Study. In addition, the study design anticipated the need to examine long term implications for health and aging of the massive migration flows across the Mexico-U.S. border. Thus, the sample design included an over-sample in 6 states of Mexico with historically high levels of migration to the United States. MHAS-I had these and other unique features that MHAS-II will replicate and improve in the 3rd and 4th waves. New emphasis areas will be the study of aging in a mixed infectious-chronic epidemiological regime;assessment of the quality of self-report;the continuous culture of Mexico-U.S. migration and its consequences for aging;and the impact of a recent health sector reform in Mexico. This application has the following aims: 1) To carry out a third and fourth waves retaining the original substance of MHAS and adding the new content, following the survivors of waves 1 and 2 and refreshing the sample to yield again (as of wave 3) a representative cross section of the Mexican population aged 50 and over. 2) To enhance data distribution, dissemination, and outreach activities, to expand the knowledge about and use of the data sets and products of the four waves of the Mexican Health and Aging Study. User-friendly web-based platforms that have become available in the last ten years will be employed to provide enhanced public access to the data and project documentation. While the previous MHAS-I provided two waves of data spanning over two years, the analytical significance of the MHAS-II data will be much higher, forming a unique longitudinal study of four waves that span over thirteen years. The new data will enable enhanced research on aging and related population changes: of physical and mental health and disability, health behaviors and health care use, family support, aging and the life course, wealth, income, labor and retirement, migration and old age, and mortality, in a developing country aging fast with limited institutional support for individuals in old age. The data will enable cross-period and cross-cohort analyses of health and aging, and will be highly comparable with other similar studies in developed and developing countries, in particular the United States, enhancing the study of aging and health with a cross-national perspective. PUBLIC HEALTH RELEVANCE: This study extends an existing national longitudinal study of aging and health in Mexico, with two additional waves of data collection, eleven years after fielding the initial two waves. The resulting public use database will have 4 waves extending over 13 years. Highly comparable to similar studies in the U.S. and other countries, the study will allow enhanced research on aging and health in a rapidly aging developing country with limited institutional support for the elderly, and with health, economic, and social processes tightly linked to the U.S.

To study the effect of Type C RNA viruses on hematopoiesis with particular emphasis being placed on the spontaneous disease in AKR mice. Major emphasis will be placed on the preleukemic phase of the disease, i.e., hematopoietic changes occurring during the first 3 months of life. To contrast animal data in Rauscher virus leukemia and AKR leukemia with similar studies being conducted in human beings with illnesses that mimic the virus diseases in mice, i.e., human acute leukemia; the myeloproliferative diseases, idiopathic thrombocytopenic purpura, and immunohemolytic anemia. To study the effect of specific drugs on the preleukemic phase of the disease in experimental animals to ascertain whether therapy at this point might abort the ultimate development of the leukemic state.

I propose to investigate the role of calcium and cyclic nucleotides in the physiology of the vertebrate rod photoreceptor. It is well established that these substances are involved in generating the rod's electrical response to light. However, the difficulty of observing the light regulation of calcium and cyclic GMP in the intact rod has precluded a clear understanding of their functional roles. I will address this problem by measuring light evoked changes in extracellular calcium concentration in the space surrounding the rod outer segments, utilizing a planar calcium-sensitive membrane electrode. Extracellular calcium measurements provide the only method, at present, for studying the light regulation of calcium in the intact cell. Specifically, I will investigate the relationship between changes in extracellular calcium and the rod's electrical response, to infer the role that intracellular calcium and the rod's electrical response, to infer the role that intracellular calcium plays in generating this reponse. I will investigate these phenomena during light and dark adaptation in order to learn the role of calcium in adaptation. Finally, I will study the effects of altered cyclic GMP levels on the extracellular calcium changes and electrical response to infer the role that cyclic GMP has in the cellular mechanisms of visual transduction and adaptation in the retina.

This is an open label, Phase II, multicenter study. Patients will receive Atragen 140mg/m2-three times weekly. The drug will be given on Monday, Wednesday and Friday over a course of 28 days. Patients will continue to receive therapy for a minimum of six months, unless evidence of disease progression. The primary objectives of this study are to assess the efficacy of Atragen in the treatment of patients with androgen-independent, metastatic cancer of the prostate and to evaluate the safety of Atragen in this patient population.

An isolated anterior echo-free space is generally regarded as a false-positive echocardiographic finding for pericardial effusion. Even when an anterior echo-free space is accompanied by a posterior echo-free space, the echo-free spaces have been occasionally reported to be falsely positive for pericardial effusion, principally in patients with cardiac neoplasms. The basis for these findings has never been adequately explained. In the present study, evaluation by computed tomographic imaging or necropsy examination of 5 patients in whom there was either an anterior or posterior echo-free space or both demonstrated that subepicardial adipose tissue is the echocardiographic imitator of pericardial effusion.

The primary aim of this study is to further examine the effects of hypokalemia and hypernatremia on the blood perfused working heart of the dog. In some animals the changes will be produced locally by interposing a blood dialyzer in the left common coronary artery. Experiments already completed indicate that local hypokalemia initially increases both myocardial contractile force and coronary vascular resistance and that local hypokalemia enhances the increased force seen with local hypernatremia. In an attempt to shed light on mechanism, these responses will be examined before and after the administration of propranolol. In other animals, the time course of the contractile force, coronary blood flow, coronary sinus PO2, cardiac oxygen consumption, and ionic flux responses to local hypokalemia and to local hypokalemia plus hypernatremia will be examined by prolonging exposure time to two hours. In still others, hypokalemia and hypokalemia plus hypernatremia will be generalized throughout all of the blood by transferring the dialyzer to the femoral vessels and cardiac response will be examined by monitoring the same variables as in the local studies. Since there is evidence that low potassium concentration eventually leads to decreased contractility and a waning of the increase in coronary resistance, tissue levels of high energy phosphates and venous concentration of adenosine will be examined in the guinea pig heart. Secondary aims of this study are to examine cardiac responses to localized and generalized hyponatremia, hypocalcemia and hypomagnesemia. In addition to providing basic information on the role of cations in function of the intact blood perfused working heart, these studies may be relevant to certain experimental cardiomyopathies and to disease states in man which produce hypokalemia, hypernatremia and other electrolyte abnormalities. BIBLIOGRAPHIC REFERENCES: Brace, R.A., J. B. Scott, W. T. Chen, D. K. Anderson and F. J. Haddy. Direct effects of hypoosmolality on vascular resistance and myocardial contractile force. Proc. Soc. Exptl. Biol. and Med. 148:578, 1975. Bunger, R., F. J. Haddy and E. Gerlach. An isolated guinea pig heart preparation with in vivo-like features. Pflugers Arch. 353:317, 1975. drg*

Building on our success as world leaders in acute lymphoblastic leukemia (ALL) and as leaders in the Pharmacogenomics Research Network (PGRN), we have formed a Center for Precision Medicine in Leukemia. Our goal is to identify the mechanisms underlying interpatient variability in response to antileukemia medications. We will integrate state-of-the-art genomic, transcriptomic, and epigenomic interrogation of somatic cell ALL tumor cells, host germline DNA variation, and comprehensive assessment of treatment variables and non-genetic features in children and adults with ALL, coupled with laboratory mechanistic studies, to identify sources and mechanisms of interpatient variation in response. Many ALL medications are also used to treat other pediatric and adult cancers and also nonmalignant diseases: for example, glucocorticoids, methotrexate, and thiopurines are commonly used for nonmalignant diseases such as asthma, autoimmune, and inflammatory diseases, and so ALL can serve as a model for how to optimize use of therapy that will have broad implications beyond ALL. Our investigators include leaders in adult and childhood ALL, pharmacogenomics, human genomics, clinical pharmacology, computational biology, bioinformatics, and biostatistics. We have three major aims, addressed in three highly integrated projects (all of which capitalize on front-line ALL clinical trials) and three Center cores. In Proect 1, we will define the landscape of genome variation among ALL subtypes and identify the inherited and somatically acquired genomic variation, along with other clinical features, that are associated with ALL treatment response in patients. In Project 2, we will identify the genome variation associated with de novo and acquired drug resistance in primary ALL cells from patients at diagnosis and at relapse, and will elucidate mechanisms by which genomic variation influences drug resistance and treatment response. In Project 3, we will identify genomic variation associated with specific serious adverse effects of antileukemic agents (osteonecrosis, hepatotoxicity, pancreatitis, and neuropathy), establishing mechanisms and testing interventions for the phenotype of osteonecrosis. Synergies in the Center stem from the substantial overlap in patients and genomic data among the three projects; the complementary expertise and prior collaborations among investigators; the leadership of our Administrative Core with extensive ties to existing pharmacogenomic, genomic, and clinical cancer networks; and uniform approaches to data analysis, management, integration, and deposition provided by our Cores. Our Center's overarching aim is to use the knowledge gained from the research in the three projects to build a comprehensive precision medicine approach to minimize relapse while also minimizing adverse effects. This Center will allow us to engage additional adult ALL and genomics collaborators, accelerate progress in the discovery and translation of genomics into more effective and less toxic treatments, and provide a paradigm for other diseases for the integration of genomic methods and knowledge into precision medicine.

Sensory inputs are not represented faithfully in the brain. Rather, they are integrated with the information provided by the animal's internal states that are affected by factors including arousal, attention, prediction and experience. This integration process is impaired in psychiatric disorders such as schizophrenia and attention deficit hyperactivity disorder. Despite the scientific and clinical importance, our knowledge is limited as to the neural circuit mechanisms of how external sensory (`bottom-up') information and internally-generated (`top-down') information are integrated and how these processes are affected with experience and learning. We propose to combine cutting-edge techniques to gain a mechanistic insight into the dynamics and regulation of bottom-up and top-down inputs onto the primary visual cortex (V1) of mice. We will study how these processes are influenced by passive sensory experience and learning. Towards this goal, we have developed a visually-guided active avoidance task for head-fixed mice. In Aim 1, we will use chronic two-photon calcium imaging to record the activity of V1 L2/3 excitatory neurons and the sources of their bottom-up and top-down inputs during passive experience and association learning over days. We hypothesize that 1) sensory experience increases the weight of top-down inputs and decreases that of bottom-up inputs and 2) association learning induces L2/3 neurons to signal the potential timing of the associated event and this information is contained in top-down input activity. In Aim 2, we will test how the activity of local inhibitory circuits is influenced by visal experience. Bottom- up inputs to L2/3 neurons arrive at their perisomatic regions while top-down inputs arrive at their distal dendrites. Therefore, dendrite-targeting, somatostatin-expressing inhibitory neurons (SOM-INs) could regulate top-down inputs, and perisomatically-targeting, parvalbumin-positive inhibitory neurons (PV- INs) could control bottom-up inputs. We hypothesize that learning causes a reduction in SOM-IN activity and an increase in PV-IN activity. Such changes could accommodate the shift in the balance of bottom-up and top-down influences on V1 L2/3 neurons. In Aim 3, we will perform manipulation experiments to test some of the predictions of our model. We will test whether the learning-induced shift of activity timing of V1 L2/3 neurons requires 1) top-down inputs from higher cortical areas and 2) the reduction of SOM-IN activity. We will test these possibilities by 1) inactivating higher areas and 2) activating SOM-INs after learning on a trial-by-trial basis using optogenetics while monitoring the activity of V1 L2/3 neurons with calcium imaging. These experiments will reveal fine-scale circuit mechanisms governing dynamic sensory representations and also establish a paradigm to combine cutting-edge technologies that can be applied to other forms of learning and behaviors in the future.

The major focus of this project is the development of a vaccine to prevent or control infections caused by Chlamydia trachomatis. The chlamydial major outer membrane protein (MOMP) is considered to be important in the development of protective immunity to chlamydial infection. Thus, current vaccine strategies are focused on the development of recombinant or synthetic peptide MOMP vaccines that can be target to induce mucosal immunity. We have undertaken an extensive effort aimed at characterizing the antigenic properties of the MOMP at the molecular level to identify immunologically relevant structures of this protein that may have utility for the rational design of a chlamydial vaccine. Previous efforts were focused on characterizing immunodominant neutralizing B-cell epitopes of the MOMP. Serovar-specific and broadly cross reactive subspecies-specific neutralizing sites were mapped to contiguous epitopes located in within the variable domains (VD) of the protein. In this year, we have undertaken studies to characterize T-helper cell antigenic determinants of the MOMP that function in inducing B-cells to produce antibody specific to neutralizing B-cell epitopes. Two regions of the MOMP were shown to contain Th-cell epitopes capable of directing B-cell clones to produce antibody specific to immunodominant neutralizing sites. Chimeric peptides containing both Th-and B-cell epitopes were synthesized and shown to be highly immunogenic. Immunization of mice with the chimeric peptides induced high titered antibodies that reacted with the native MOMP and were neutralizing in vitro. The Th-cell epitopes under study are apparently "promiscuous" since strains of congeneic mice differing at H-2 are capable of responding to the chimeric peptide immunogens. Evaluation of the immunogenicity and vaccine efficacy of the chimeric peptides in sub-human primates will be initiated in the upcoming year. We are also working on the construction of infectious vectored chlamydial vaccines. Towards these goals we have expressed the complete MOMP protein in vaccinia virus and have evaluated the immunogenicity of the recombinant virus in mice. The results are promising; mice infected with recombinant vaccinia virus expressing the MOMP induced high titered antibodies specific to the protein. We plan to evaluate the protective efficacy of the recombinant virus in animal models of chlamydial infection in the upcoming year.

PROJECT SUMMARY: Clinical research must enroll individuals from diverse backgrounds to yield generalizable knowledge and ensure that the benefits and burdens of research are fairly distributed. Under- representation of diverse groups may: (1) lead to poor enrollment leading to delays and/or study failure, (2) prevent diverse groups from experiencing the benefits of research and (3) result in investigators missing important diversity based differences in illness and treatment. The pediatric literature has not produced consensus regarding under/over enrollment of diverse groups in research and there is inadequate understanding as to why disparities may exist. Enrollment in emergency and critical care (ECC) settings presents unique challenges. These factors, coupled with knowledge that health outcomes are strongly tied to evidence supporting medical interventions, motivate the proposed research. The goal of this proposal is to characterize research participation by children at risk for health disparities (CRHDs) in pediatric ECC research and to determine the degree to which modifiable and potentially modifiable factors relate to participation. The central hypothesis?that CRHDs are underrepresented (losing research benefit) and overrepresented (risking exploitation) in others and that modifiable factors are the primary drivers of differential study enrollment?will be evaluated in a mixed-methods study with 3 interrelated but distinct aims: Aim 1: To prospectively characterize enrollment by CRHDs in pediatric ECC research and to identify factors associated with enrollment and retention. Aim 2: To elucidate qualitative insights into modifiable and non-modifiable factors impacting enrollment/retention in pediatric ECC research. Aim 3: To determine which factors are most associated with successful enrollment of CRHDs. Aim 1 will be addressed through prospective tracking of study enrollment/retention data in an anticipated 18 studies at a single site and factors associated with enrollment. Aim 2 will be accomplished through semi-structured interviews of parents and study team staff. Aim 3 combines prospective survey and interview methods to develop a priority order for the most important factors related to parental decisions to participate to plan future intervention studies. This project will advance the candidate?s long-term goal to engage in interdisciplinary research and policy development using evidence-based, stakeholder-informed, and ethically-sensitive practices to reduce health disparities, address bias, and promote health equity for children. Mentors with expertise in pediatric ethics, disparities, clinical trials, decision-making, and health communication augmented by essential institutional support will permit the candidate to expand knowledge of research methods relevant to decision- making, develop expertise in health disparities research, and cultivate dissemination and implementation skills to effectively improve the evidence base relied upon to treat the sickest children. The proposed plan is instrumental to a successful transition to independence and becoming a leader in the field.

PROJECT 1: RSV bronchiolitis and early childhood asthma are the most common, serious, acute, and chronic conditions of infancy and childhood, respectively, and diseases that disproportionately burden vulnerable populations. Opportunity and Impact. This project draws together three important elements in understanding the role of RSV on recurrent wheezing and asthma inception - RSV infection severity, host response and susceptibility. In studying the association of RSV with asthma inception, studies have overwhelmingly focused on the 3-5% of RSV-infected infants requiring hospitalization, while the vast majority of RSV infections are mild. Whether mild infection confers intermediate risk or has a protective effect is an important question. The answer will influence proposals for primary asthma prevention strategies. The proposed series of investigations will aid in our understanding of the role and mechanisms through which RSV may both lead to chronic lung disease, and may protect from chronic lung disease. Approach. Utilizing the ReSPIRA cohort, established for this investigation and described in Core B, we will investigate the relationship between infant RSV infection, host response to infection, and genetic determinants of recurrent wheezing, asthma and allergic disease development following RSV infection. Our specific aims are to: (1) Establish the association between RSV LRTI, RSV URI/exposure, and no RSV infection in the first 6 months of life on the risk of recurrent wheezing and asthma, (2) Define whether host immune response and/or airway injury biomarkers assessed during infant RSV infection are associated with recurrent wheezing, atopic disease or early childhood asthma, and (3) Identify the genetic determinants of the phenotype of recurrent wheezing, early childhood asthma and atopy following infant RSV infection. Utilizing the ReSPIRA cohort which includes 2000 infants followed from early infancy through early childhood, and established through this U19 grant, this project will answer the following questions: (1) how does RSV cause asthma, (2) does mild RSV infection during infancy increase or decrease the risk of asthma, and (3) what host factors are important in the progression from infant RSV infection to early childhood asthma. Answering these questions will allow us to develop preventive interventions for chronic lung disease in children, and ultimately improve the health of infants and children with bronchiolitis and asthma in the U.S. and worldwide.

The NICHD's Contraceptive Clinical Trials network conducts Phase I, II and III clinical trials of a wide variety of new female and male contraceptive methods. The objective of this contract is for CCTN contractors to conduct female contraceptive clinical trials, conduct treatment trials of gynecologic conditions, and to develop research protocols on a task Order basis under an Indefinite Delivery/Indefinite Quantity (IDIQ) contract. To provide assistance in protocol review, protocol development and activities associated with past, present, and future CCTN activities. To assist CCTN with Protocol review, protocol development, regulatory approval, informal consultation, and query resolution.

Abdominal Aortic Aneurysm (AAA) is a common and devastating disease which is increasing in incidence. Although easy and inexpensive to detect by ultrasound, most aneurysms are small when detected and there is currently no medical regimen which will inhibit their growth. There is an increasing body of evidence implicating a family of matrix degrading enzymes, the matrix metalloproteinases (MMPs) in AAA. Although both MMP-9 and MMP-12 may have a role in AAA, we have identified a significant increase in total MMP-2 in AAA. Importantly, a larger proportion of the MMP-2 in AAA tissue is in the active form and is directly bound to the matrix suggesting ongoing proteolysis. In addition, we have demonstrated that AAA tissue contains increased levels of membrane type 1 MMP, the activator of MMP-2. We have also shown that doxycycline inhibits MMP-2 production by aortic smooth muscle cells in culture. We hypothesize that MMP-2, through its increased activation, has a central role in aneurysm formation and that this could be inhibited by doxycycline. This hypothesis will be examined through the following specific aims: 1. Determine the effects of individual MMPs implicated in AAA including MMP-2, MT1-MMP, MMP-9 and MMP-12 on the size and rate of aneurysm formation in a murine AAA model. 2. Determine the effects of doxycycline on the size and rate of aneurysm formation and progression in a murine model and correlate these effects with serum doxycycline levels. 3. Determine the mechanisms by which doxycycline down regulates MMPs in human aortic smooth muscle cells. Specific aim 1 will be accomplished using a mouse model of AAA characterized in our laboratory with four different knock-out mice, including MMP-2, MMP-9, MMP-12 and a TIMP-2 knock-out mouse in which activation of MMP-2 does not occur. Specific aim 2 will be accomplished by using doxycycline treatment in our murine model of AAA and correlating effects on aortic MMP expression, aneurysm size and growth rate with serum doxycycline concentrations. Specific aim 3 will be accomplished by determining MMP- 2 mRNA levels, mRNA half life, rate of mRNA transcription and identifying the doxycycline responsive elements in the MMP-2 promotor. The long term goal of this work is to develop pharmacologic therapies which specifically target MMPs important in aneurysm pathogenesis and progression.

We propose to assess the robustness of a high-throughput mass spectrometric (MS) technology platform for quantitative measurement of multiple candidate biomarker proteins in complex samples such as human plasma. Specific assays will be developed for 300 candidate biomarker proteins drawn from published literature, pathway analysis, microarray, and proteomics discovery efforts. These assays will be used to measure biomarker levels in 200 selected cancer and 200 control plasma samples at three different laboratory sites using two variant MS platforms. The results will characterize the quantitative reproducibility of the assay methodology within- and between-laboratories and over time. A series of standardized reagents will be developed allowing the assays to be implemented in other laboratories using similar standardized instrumentation. Our measurement platform makes use of well-established quantitative MS techniques used in the routine measurement of small molecules: so-called "multiple reaction monitoring" (or MRM) assays and stable isotope labeled internal standards for quantitation, here in the form of synthetic, labeled peptides. By measuring both the labeled and unlabeled (sample-derived) peptides by MS, the method provides a quantitative measure of the relative amounts of the signature peptide and therefore the protein that it is derived from. In order to access lower abundance biomarkers (those in the lower 5 orders of magnitude of the overall 10 orders of magnitude plasma abundance scale) we will employ specific enrichment techniques using immobilized anti-peptide antibodies. After extensive optimization and characterization, our assays will be deployed on clinical plasma samples from breast cancer cases and controls. Key outcomes of this project will be to demonstrate 1) that we can make sensitive and specific assays quickly and inexpensively;2) that the assays can be highly multiplexed, greatly reducing the cost-per-analyte;and 3) that the protocols and technology can be standardized and distributed. Several members of our CPTAC team are currently involved in proteomic consortia aimed at MS-based discovery of candidate breast cancer biomarkers in human samples and mouse models. These efforts are generating robust datasets that will also be leveraged by this CPTAC team to inform selection of candidates for assay development and ultimate validation.

This project will test the underlying hypothesis that hospital behavior and performance are influenced by the spatial configuration of the local hospital market. Hospital performance has typically been examined with cross-sectional approaches that assume each hospital is independent. Physicians and hospital administrators, however, are acutely aware of the locations of alternative institutions. The spatial configuration of institutional providers has been studied by medical geographers, but their focus has been on time and distance within a market area. This project will combine the cross section and spatial configuration approaches to explicitly include hospital interdependencies in the analysis of hospital behavior. The spatial framework will be applied to three broad analytic questions: (1) What factors influence the acquisition, availability, and use of specialized clinical services and facilities; (2) What factors underlie the observed negative relation between hospital volume of specific patients and mortality; and (3) What factors influence the effectiveness of regulatory strategies in altering the distribution of services among hospitals. The spatial framework methodology will also be used to test the concept that some services and facilities are used by hospitals in a competitive fashion, while others serve as complements. The project draws upon research in economics, sociology, geography, quality assessment, hospital administration and organizational behavior. Multivariate regression models with data from all U.S. community hospitals will be used to examine factors that influence the distribution and use of specialized clinical services in 1972 and 1982. These data will be linked to information on the number of patients with selected procedures or medical diagnoses and their outcomes. The validity of the results of these studies will be tested with a series of localized case studies.

Intravenous drug abuse continues to be a major contributor in the perpetuation of the AIDS epidemic. Primarily, this situation relates to the needle-sharing and sexual practices of addicts. However, there is also ample evidence to suggest that the drugs themselves are an integral part of the problem through their immunomodulatory properties. Since intravenous drug addiction is accompanied by many factors, including drugs, that are capable of modulating immune responses, determination of the potential of drugs to alter immunity and influence contraction and outcome of HIV1 infection cannot be effectively addressed solely by studying drug addicts themselves. Because of this situation, efforts in this renewal proposal are directed toward investigation of in vitro effects of drugs of abuse on specific immunological properties of T-cell lymphocytes and their ability to cope with HIV infection. These studies maintain relevance to the human circumstance by focusing on immune properties of T-cells which have already been shown to be modulated in drug addicts as well as being of known relevance to HIV1 infection and AIDS. Using a kinetic marker modulation assay developed in this laboratory, and a newly defined, tandem, direct-staining assay, investigations of the ability of morphine, cocaine and other abused drugs to modulate expression of CD2, CD4 and CD8 surface antigenic markers of T-cells are to continue. By exploring the biological, physiological and pharmacological bases for modulation of markers in these assays, a better understanding of the means by which opiates modulate immune function and T-cell susceptibility to HIV1 infection ought to be achieved. Signal transduction mechanisms related to the function of guanine nucleotide binding (G) proteins and to ion channel activities are proving to be a particularly important target of these efforts which are now supported by new electrophysiology and cytofluorometry laboratories. Ultimately, the plan aims to link basic, mechanistic studies with analyses of the ability of the drugs of concern to modulate the functional capacity of T-cells associated with activation of cell division through the 'alternate', CD2, replicative pathway and with susceptibility of T-cells to HIV1 infection via CD4. Taken together, these efforts should yield important clues to the role of abused drugs in AIDS.

Substance use disorders (SUDs) are chronic, recurring disorders of major cost to society at all stages of the life cycle. They are of particula concern in Ukraine, whose rate of alcohol use is 5th highest in the world and whose lifetime rate of illicit drug use is already 21% by age 16 (WHO, 2011). At the same time, because of long periods of political turmoil and limited national resources, intervention programming to address these issues is primarily focused on chronic, end stage disorder, and concomitant professional training and addiction science have a similar focus. Both developmental/etiologic science and the intervention programming derivative from that are absent, although a small subset of researchers familiar with developments over the past decade in western countries have been appreciative of this changed perspective, and have been looking for ways to import it. Working in collaboration with two nationally visible physician scientists (MD/PhDs) from this cadre, and drawing on the experience gleaned from a successful program of substance abuse research infrastructure development in other Eastern European countries over the last 10 years, this project would implement a set of substance abuse research training experiences at several different levels of specificity, duration, and focus. The content goal is for transfer of knowledge pertaining to the developmental etiology of SUDs and implementation of intervention techniques that build upon that scientific base. In the latter years of the program, we would mentor and collaborate in design and pilot testing of an early screening and intervention program that ultimately will have the potential for national dissemination. The proposed program involves collaboration among faculty from 16 disciplines at the University of Michigan (UM), Department of Psychiatry (Addiction Research Center), at the Ukrainian Scientific Research Institute of Social, Forensic Psychiatry and Narcology (USRISFPN), and at the National University of Kyiv- Mohyla Academy (NUKMA). A set of interlocking training experiences at different levels of intensity and career status will include the following: (a) Yearly workshops (~ 35 attendees) held in various sites in Ukraine, for all levels of post-doctoral substance abuse scientists interested n the developmental psychopathology of SUD, research methodology, and SUD prevention and intervention strategies in non-clinical or community-based settings. (b) Year-long fellowships at UM, for 5 early-career post-doctoral fellows to learn more research methodology and work with mentors in areas of common interest, (c) Home-country research projects that these fellows develop while in the U.S., (d) 3 intermediate-term fellowships at UM for mid-career substance abuse researchers. (e) 3 short-term fellowships for senior research faculty to consult at UM and NIH about future program directions. (f) Creation of a Research/Intervention Program working group to develop a clinical trial on SUD screening and early intervention in targeted communities; and (g) Support of increased scientific collaboration between Ukrainian substance abuse researchers and other Eastern European researchers.

Project Summary (Abstract) Obstructive sleep apnea (OSA) is a common sleep disorder affecting a significant portion of adult population in United States. OSA results in poor quality of sleep and is associated with increased cardiovascular risk, including hypertension. Blood pressure (BP) is known to increase with each OSA event, although the degree and frequency of BP increase will vary from individual to individual. Measuring BP during sleep studies could provide valuable information concerning cardiovascular risk in patients with OSA. However, BP is currently not measured during sleep evaluation because of the lack of a suitable method that is not intrusive to sleep. Pulse transit time (PTT) is a noninvasive measure that can be obtained from patient monitors in present use in polysomnography and has been suggested as a potential tool for BP estimation. However, PTT has rarely been studied for the purpose of evaluating BP during sleep. Analysis of PTT patterns has also been suggested as a tool to detect subtle arousals that accompany acute BP increase events. Therefore, PTT by itself may be an important measure of OSA's cardiovascular effect. Despite this potential, PTT has not been adopted as a routine sleep measure due to unresolved technical limitations, such as susceptibility to artifacts and the need for repeated calibrations, as well as lack of studies informing its clinical utility in predicting CV outcomes. In this proposal, we will test and validate an internally-developed novel artifact-robust PTT-based BP estimation algorithm using existing hospital-based data. The benefit of this dynamically-calibrated adaptive algorithm is that, unlike all other PTT-based BP estimation algorithms, it can accurately estimate BP over time without recalibration. Subsequently, we will apply this algorithm to construct BP patterns in existing sleep studies obtained as a part of a large observational study (MESA Sleep cohort). This cohort study has a wealth of information about participants' cardiovascular risks. We will test the utility of the characteristics of BP and PTT in predicting cardiovascular risks using the MESA data. This will be achieved by examining their associations with measures, such as heart mass and aortic stiffness, that are well- established markers of cardiovascular risk. We will also examine the association with the prevalence of cardiovascular disease. We anticipate that adding these novel measures (BP and PTT characteristics) to conventional measures of OSA will improve the identification of higher- risk groups of patients with OSA. Our findings will introduce a useful novel method that is compatible with routinely performed sleep studies and will facilitate better assessment of OSA.

Studies in this laboratory focus upon an analysis of the neural control of cardiac dynamics and rhythm in behaviorally conditioned, unanesthetized animals. Previous studies have indicated that post-myocardial infarction cardiac arrhythmias occur principally within a specified range of heart rates (the "critical HR range" hypothesis). Rhesus monkeys will be operantly conditioned to control their heart rates at levels which are below, within, and above the probable critical range. They will then be subject to experimental coronary occlusion and tested in the operant conditioning paradigm to assess the validity of the "critical HR range" hypothesis. In addition, baboons will be operantly conditioned to elevate their arterial blood pressure by 30 mm Hg for minute long intervals. The behavior of individual segments of myocardium will be assessed using ultra-sonic "segment-length" gauges during periods of rest and blood pressure elevation both before and after coronary arterial occlusion. Finally, the cardiovascular responses of chronically instrumented dogs to acute coronary arterial occlusion. Finally, the cardiovascular responses of chronically instrumented dogs to acute coronary occlusion will be examined in normal animals and animals subject to surgical cardiac denervation. Each of the above projects is designed to elucidate a specific facet of the neural control of the normal and ischemic heart of intact, non-anesthetized animals.

The objective of this project is to provide information on metabolic changes occurring in nuclei of pancreatic tissue exposed to carcinogens. The initial studies indicated that exposure to 7,12-dimethylbenzanthracene (DMBA) resulted in alterations in content of DNA and RNA for periods up to 35 hours after exposure. Protein synthesis was also depressed during this same time period. During this reporting period we measured the rate of RNA synthesis as reflected by the uptake of radiolabeled uridine. These studies demonstrated that RNA synthesis was altered in a biphasic response, in parallel with the changes in RNA content, and that changes in synthesis preceded the maxima and minima of RNA content. Preliminary studies with benzopyrene (BP) indicated that only the initial change in synthesis of RNA was observed following BP treatment. The reason for the difference between carcinogens is presently unknown. We have measured changes in the DNA-dependent RNA polymerase enzyme activity during the same period of time and observed that changes in rates of polymerase enzyme activity were in parallel with the changes in uridine uptake. Ribonuclease enzyme activity and ribonuclease enzyme inhibitor activity were measured concurrently with the polymerase enzyme activity. The enzyme and inhibitor activities demonstrated changes that were in parallel with ribonuclease activity which suggested that the RNA content was not significantly affected by ribonuclease enzyme activity. These studies showed that DMBA significantly affected RNA synthesis, but they could not distinguish between changes in the polymerase enzyme content, an effect on DNA template, or an effect on some rate-limiting enzyme or cofactor. There are reports in the literature which demonstrate the rate-limiting effect of ornithine decarboxylase on RNA synthesis. Studies measurng the change in ornithine decarboxylase following DMBA treatment are underway.

Instrumentation is in place to capture images for morphometry of neurons developing in the absence of electrical activity. Redesign of multicompartment chambers for the study of activity effects on synapse stabilization / elimination improves optical resolution and axonal growth. Antibodies against the vesicular acetylcholine transporter label a subpopulation of neurons and synaptic terminals, and may allow the immunohistochemical identification of cholinergic neurons as early as one week after plating. Antibodies against the homeobox gene (Hoxa7) product label neuronal and not glial nuclei.

Data from the initial funding period of this grant have substantially informed us regarding the interaction between CVD and AD pathologies as they relate to the transitions between normal aging, MCI and dementia. These data suggest that CVD affects cognition through impairment of executive function, which in turn, adversely affects episodic memory performance. Anatomical evidence from these data strongly suggests that CVD influences executive function through white matter injury along specific tracts connecting parietal and frontal lobes and that the AD process also injures parietal subcortical white matter integrity, in addition to the more commonly recognized neuronal pathology. This information serves as the basis for a hypothetical disease model whereby CVD may act alone to cause impaired executive function (and possibly episodic memory impairment) or acts to amplify the cognitive effects of AD pathology. In addition, we hypothesize that both CVD and AD disease processes affect parietal-frontal connections through different mechanisms, and when they both occur in a given individual, will significantly increase the likelihood of future dementia. An important principal to our research is the recognition that both AD and CVD pathologies cause cognitive impairment through neuronal death or dysfunction. We also recognize that both diseases cause overlapping structural brain changes. For example, there is evidence that brain atrophy and WMH are the consequence of both AD and CVD pathologies, whereas MRI infarctions are strictly CVD and hippocampal atrophy is a strong, but not entirely pure indicator of AD. Unfortunately, until recently, it was impossible to dissociate these two disease processes on the basis of neuroimaging tools alone. A major advantage of our current study design is that-through use of PiB imaging-we can determine the presence or absence of cerebral amyloid, thereby allowing us to test the joint contributions of the AD and CVD processes on cognition without relying solely on MRI to make specific inferences regarding which structural brain changes relate to AD and which relate to CVD. The significance of our proposal continues to rest on the fact that CVD is currently the only treatable disease associated with cognitive impairment. We hope further understanding of how CVD impacts cognition will soon be used to develop clinical therapeutic trials that may reduce the incidence of cognitive impairment and dementia among our aging population. PUBLIC HEALTH RELEVANCE: This project seeks to extend current work evaluating the heterogeneous causes of mild cognitive impairment (MCI). Current work strongly suggests that MCI, including the amnestic subtype, results from the additive effects of cerebrovascular and Alzheimer's pathologies. This project is designed to identify the unique contributions of both pathologies to cognitive aging, MCI and risk for dementia.

This is the first renewal of a Program Project that has been in effect for two years. It was originally submitted in order to study the molecular related to a number of devastating human conditions which have as their central feature an association with the with the major histocompatibility complex. Three investigators who have worked on both independent and collaborating projects relate to both clinical manifestations and such as insulin dependent diabetes mellitus and allergies as well as to basic 1-1. of T cell recognition and cellular proliferation wish to continue their integrated approach to these various human diseases. As previously, central core facilities are requested in order to centralize resources and technology and to more efficiently utilize personnel and funds. Each of the individual projects follows directly from the laboratory to the clinic and back to the research laboratory and should provide important insights into the pathogenesis of human disease.

Transcripts of the cellular c-Ha-ras are increased during liver regeneration and at certain stages in diet-induced hepatocarcinogenesis in rats. C-Ha-ras is the cellular homologue of the transforming gene of Harvey murine sarcoma virus. In liver regeneration, induced by either partial hepatectomy or carbon tetrachloride injury, a 2-4 fold increase in the levels of c-Ha-ras mRNA coincides with the activation of DNA synthesis followed by rapid (within 24 hours) return to normal levels. In prenoeplastic livers of rats fed a diet deficient in choline and containing 0.1% ethionine there is a dramatic increase in the level of c-Ha-ras mRNA at 4-6 weeks followed by an equally rapid decrease by 8 weeks although the levels remain higher than normal. Since we have identified two cases where transcripts of a cellular oncogene are transiently elevated, we have a good experimental system to: a) define the expression of both members of the ras family (c-Ki-ras and c-Ha-ras) and to ask which of the multiple copies of these genes are normally transcribed and which are transiently activated; b) determine if chemicals which act as liver carcinogens, only if given during regeneration, modify the pattern of expression of the ras genes during liver regeneration in the rat; c) determine if the transient elevation of ras transcripts can be correlated with changes in methylation patterns and DNase I hypersensitivity of the ras locus; d) determine if DNA from normal, regenerating, MNU-treated regenerating and 4-6 week preneoplastic rat liver contain transforming DNA which can be identified by the NIH 3T3 transfection assay.

LIUNA Training and Education Fund (LIUNA Training) is applying for the Hazardous Materials Worker Health and Safety Training cooperative agreement, RFA-ES-09-004 to conduct a Hazardous Waste Worker Training Program (HWWTP - $18,154,084) and a Hazmat Disaster Preparedness Training Program (HDPTP - $1,902,370), at a total cost of $20,056,454 over the five year program. LIUNA Training will train approximately 25,504 trainees under the HWWTP and 11,700 trainees under the HDPTP. Twenty-six training sites located nationally will be used to train workers who are, or have the potential to be, employed on hazardous waste sites, who are at risk of exposure to hazardous waste on the job, or who may provide assistance as Skilled Support Personnel (SSP) on disaster sites. Trained SSP will learn how an emergency response operation functions, their roles and those of other participating responders, and will obtain an awareness of potential problems and appropriate solutions. LIUNA Training's courses will help protect worker health and safety during the entire remediation or emergency response. Training includes hazardous waste worker (exceeding OSHA and NIEHS Minimum Criteria requirements) and other environmental remediation courses, health and safety training and specific job skills courses required to meet regional and job site needs. Training incorporates hands-on exercises; activity based instruction, and advanced training technologies. This program targets Construction Craft Laborers, other building trades and environmental contractor and government personnel. Special outreach and training efforts will focus on recruitment and retention of minority and non- or limited-English speaking workers. General Construction and Employability Skills training along with social support services provided by community-based organizations will help disadvantaged workers succeed in training and job retention.: The LIUNA Training HWWTP/HDPTP teaches workers how to protect themselves and others while working on hazardous waste and disaster sites. They learn how to handle, remediate, and remove contaminated materials safely and in a manner that protects the environment and the general public. Training helps participants identify and address potential hazards on the job, at home and in their communities. Hazardous Waste Worker Training Program (HWWTP)

The purpose of this study is to interrelate biochemical activity and cytoanatomic structure of the leukemic lymphocyte to its in vitro and in vivo response to antineoplastic agents.

DESCRIPTION: (adapted from the application): This application proposes a three year continuation of Dr. David Wise s academic leadership award on the economics of aging. The initial award was made in April, 1994 and approved through March, 1997. The purpose of the initial award was to extend the National Bureau of Economic Research (NBER s) ongoing research program on the economics of aging to more comprehensively address issues in health, health care, and health policy for older Americans. Progress has been made in achieving this objective, first in developing extensive resources for health care research, second in assembling a team of scholars to work on health and aging issues, and third in initiating and coordinating a series of new projects on health and health care among the elderly. The purpose of the continuation award is to support the continued leadership of David Wise in coordinating the NBER aging project, and in developing new research initiatives on aging issues. A large part of the NBER s research agenda on aging and health care issues would proceed without the continuation award. However, this award will enable Dr. Wise to coordinate the many otherwise independent projects and activities into an integrated and cohesive effort, and will enable him to interact more extensively with other investigators in developing new research.