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This project will investigate the impacts of the 2004 Indian Ocean tsunami on economic well-being in Sumatra, Indonesia using multivariate methods and data from a unique longitudinal household survey. The 2004 Indian Ocean tsunami was one of the most devastating natural disasters in recent human history, claiming over 200 thousand lives in ten countries and destroying the livelihood-supporting resources of many affected households. Previous studies of the human impacts of natural disasters have largely relied on small scale and non-representative data sources due to the difficulty in anticipating natural disasters and in collecting survey data in their wake. This project draws on a longitudinal survey of 10,000 households in Sumatra, Indonesia who were interviewed prior to the tsunami and each year since. I will use this dataset and a series of multivariate analyses to examine the impacts of the tsunami on multiple measures of economic well-being and to describe the pattern of recovery in assets, income and consumption. Specifically, this project will investigate: (1) the vulnerability of individuals and households to economic losses due to the tsunami;(2) economic coping strategies in the wake of the tsunami;and (3) trajectories of economic recovery in the context of widespread reconstruction efforts. The results of these analyses will inform important debates regarding the resilience of households to natural disasters and the role of post disaster reconstruction in economic recovery. This research will draw on my previous training and experience in human-environment geography and social demography and on my current research using this dataset to investigate post-tsunami human displacement. To make this research possible, I will conduct fieldwork in Indonesia, be mentored by four prominent population scientists, and receive significant additional training in development economics and econometrics in Duke University's Department of Economics. This training and research experience will prepare me to independently investigate a broad array of questions in population, environment and development, and to develop an R01 proposal to initiate a large-scale longitudinal data collection in Ecuador incorporating population-environment approaches. PUBLIC HEALTH RELEVANCE: The 2004 Indian Ocean tsunami caused widespread devastation of coastal areas in Sumatra, Indonesia and destroyed the livelihood-supporting resources of many households. The proposed research will use a large survey dataset to investigate how the tsunami affected the material well-being of surviving households and individuals in Sumatra and to examine their recoveries over time.

Hepatitis B virus (HBV) is a major cause of chronic viral hepatitis that increases dramatically the risk of liver cancer and other end-stage liver diseases such as cirrhosis. HBV belongs to the Hepadnaviridae, a family of para-retroviruses that have a small DNA genome and replicate through an RNA intermediate (the pregenomic RNA, or pgRNA), by a unique reverse transcription pathway. The initiation of both viral DNA synthesis and nucleocapsid assembly is triggered by the assembly of a specific ribonucleoprotein (RNP) complex between the viral reverse transcriptase (RT) and a specific RNA signal located at the 5' end of pgRNA termed epsilon. RNP formation is, in turn, facilitated by specific host cell factors, some of which have been identified in vitro, and furthermore, can be inhibited by unknown cellular factors under specific conditions. Following DNA synthesis, the mature, DNA-containing nucleocapsids are selectively enveloped and secreted as extracellular virions. The long-term goal of this application is to elucidate the viral and host factors that positively or negatively modulate viral replication and assembly, with a focus on the early step of initiation of reverse transcription and nucleocapsid assembly and the later step of nucleocapsid envelopment and virion secretion. Four specific aims are proposed. First, the role of the chaperone heat shock protein 90 (Hsp90), shown to be important for RT-epsilon interaction and protein priming in vitro using the duck HBV (DHBV) model system, to viral replication in vivo will be tested in host cells, through the use of specific pharmacological inhibitors, dominant negative mutants, and the RNA interference approach. Second, the viral and host requirements for RT-epsilon interaction in HBV will be determined using a recently developed RNA gel-mobility shift assay in vitro and verified in vivo. Third, cellular factors that can inhibit RNP formation will be isolated using recently established in vitro assay conditions and their role in cytokine-induced anti-HBV defense will be explored. Fourth, the state of post-translational modifications of the DHBV capsid protein at different stages of reverse transcription will be assessed through detailed biochemical and biophysical analyses of purified nucleocapsids at different stages of maturation; host factors responsible for mediating these modifications will be identified; and the role of these modifications and the relevant host factors in different steps of viral DNA synthesis and assembly will be determined. The proposed studies should provide important insights into the mechanisms of virus-host interactions and also facilitate the development of novel and effective anti-HBVstrategies.

The Center for Clinical Epidemiology and Biostatistics (CCEB) of the University of Pennsylvania (Penn) School of Medicine (SOM) resubmits this proposal to continue an innovative and successful pre-doctoral training program in cancer biostatistics. The objective of this program is to train individuals to be rigorous and independent academic investigators able to use the range of approaches in biostatistics to address questions in cancer research. The program is specifically built upon existing collaborative relationships among biostatistics, statistics, and cancer research faculty in the CCEB and Department of Biostatistics and Epidemiology (DBE), the Abramson Cancer Center (ACC), and Wharton's Department of Statistics (STAT) at Penn. [unreadable] [unreadable] This pre-doctoral training program for PhD students in Statistics and Biostatistics who have advanced to the dissertation stage of their training, provides didactic training in fundamental skills, methodologies, and principles of biostatistics, with emphasis on the areas of most importance to cancer research. Trainees are required to obtain a strong background in substantive areas related to cancer through participation in research seminars and a series of interdisciplinary courses in cancer research and cancer biology that examine methodologic issues, scientific approaches, technologies, concepts, and applications of statistical approaches in cancer research. Specifically, the training program is designed to: 1) provide in-depth knowledge of the biostatistical techniques appropriate to cancer research; 2) provide research experiences with mentors in both biostatistics and cancer research; and 3) provide an inter-disciplinary infrastructure, bringing together faculty and students in the CCEB, DBE, ACC, and STAT, designed to support graduate education in cancer biostatistics. [unreadable] [unreadable] The strengths of the program are its training program in biostatistics, including comprehensive course offerings available to students; the wide-ranging experience of the biostatistics faculty in multiple areas of biostatistics methods and cancer research; the commitment of the faculty to collaborative research and training; the established teaching program in Statistics offered by the Wharton School; the long history of successful clinical research training programs offered by the CCEB; and the existing collaborative links among CCEB and DBE faculty in biostatistics and epidemiology, the ACC, and STAT. The resources available to students include a broad array of ongoing research projects, including clinical trials, observational studies, translational research, and experimental studies. Penn's commitment to collaborative research and training and the broad range of expertise and experiences of faculty participating in this training program provide an ideal environment for this training program. [unreadable] [unreadable] [unreadable] [unreadable]

Research efforts are currently devoted to synthesizing analogs of Coenzyme A which potentially can be used as active-site-directed irreversible enzyme inhibitors. Photoaffinity labels of the nitrene type and thiolesters of arsinous acids, e.g., S-cacodyl-CoA, are under investigation. These agents are to be tested as inhibitors on enzymes of the following classes: Hydrolase (e.g., acyl-CoA hydrolase), Transferase (e.g., 1-acylglycerol 3-P acyltransferase), and Lyase (e.g., citrate synthetase).

Project highlights: assay optimized, miniaturized to 1536-well plate format and screened MIPE and Pilot libraries (25,000 compounds). During this period, the NCGC has fostered and maintained over 180 active collaborations with both NIH and extramural investigators, facilitating drug discovery efforts across the entire spectrum of human disease. These efforts have led to over 100 high-throughput screens and nearly 60 medicinal chemistry campaigns, providing our collaborators and the general research community a wealth of publications and promising small molecule leads. In addition, the NCGC has undertaken a number of informatic challenges to make better use of existing drug and disease target information and provide the general public with easily accessible resources, further catalyzing the development of new therapies for human disease.

Work of others has been repeated and extended that carnitine levels in various rat tissues is significantly increased in choline fed rats consuming a diet low in methionine. Choline is not participating per se in carnitine synthesis in this situation, but rather may be concerned in carnitine transport. Experiments to support this view will be sought and include (a) determining the distribution of long chain fatty acyl carnitive vs free carnitine in tissues from rats on low methyl diets with or without choline supplementation, (b) (3H) carnitine uptake following liver perfusion in such experimental animals, and (c) a consideration of the possibility of a phosphatide exchange between lecithin and carnitine. Biosynthesis and characterization of "lysine-lipid forms" accumulating in the tissues of rats on low lipotropic diets administered radioactive lysine will be undertaken. A series of carnitine binding proteins have been postulated to exist and to function in the transport of carnitine from its site of synthesis to its site of action. The liver carnitine binding protein (LCBP), the red cell carnitine binding protein (RCBP), the cardiac carnitine binding protein (CCBP) and the muscle carnitine binding protein have been identified. The proteins are distinct from other proteins which are known to bind carnitine as a substrate (carnitine acetyltransferase, carnitine palminoyltransferase and carnitine acylcarnitine translocase). Experiments are in progress to purify, characterize and determine the mechanism of action for each of these carnitine binding.

The purpose of this consent is to screen for p24 antigen levels to determine eligibility for antiretroviral drug studies.

Tumors are able to actively create tolerance to their own antigens. This acquired tolerance includes both anergy and potent Treg-mediated immunosuppression, and represents a fundamental barrier to the immunotherapy of cancer. The current proposal focuses on the tolerance-inducing enzyme indoleamine 2,3-dioxygenase (IDO), which functions as a key molecular mechanism linking two potent tolerogenic pathways in tumor-draining lymph nodes: IDO-expressing immunoregulatory DCs, and regulatory T cells (Tregs). The proposal will examine the role of IDO in the critical window of time during recovery from cytotoxic chemotherapy. Chemotherapy releases a wave of tumor antigens and transiently perturbs tolerance. Preliminary data show that IDO is a key molecular mechanism by which the tumor re-establishes tolerance to itself following chemotherapy. IDO acts both by directly anergizing tumor- specific CDS" effector T cells, and by markedly activating the suppressor function of pre-existing Foxp3+ Tregs. In the post-chemotherapy period, IDO-dependent tolerogenic mechanisms are selectively preserved at high levels in tumor-draining lymph nodes. The hypothesis underlying the proposal is thus that IDO represents a key molecular mechanism required to maintain tolerance to tumor antigens following chemotherapy. The overall goal of the proposal is to develop mechanistically-based, clinically applicable strategies to break tolerance to tumor antigens, based on the combination of IDO-inhibitor drugs and chemotherapy. Aim 1 will test the hypothesis that, prior to chemotherapy, the tumor-draining lymph node drives acquired systemic tolerance to tumor antigens via the synergistic combination of IDO+ pDCs and activated Tregs. Aim 2 will test the hypothesis that chemotherapy is able to transiently disrupt tolerance to established tumors, but only if the IDO pathway is blocked during the post-chemotherapy recovery period. Aim 3 will test the hypothesis that the window of disrupted tolerance opened by the combination of IDO-inhibition plus chemotherapy will allow curative anti-tumor T cell responses to be generated by active immunotherapy delivered in this window. The significance of the current proposal is that it aims to develop clinically-applicable treatment regimens for breaking tolerance to tumors, based on a strong and novel mechanistic rationale for synergy between chemotherapy and IDO-inhibitor drugs. Since a number of vaccines and immunomodulators are currently limited by the barrier of tumor-induced tolerance, a tolerance-breaking regimen based on IDO-inhibitors plus chemotherapy has the potential to enhance the efficacy of a variety of existing immunotherapy strategies. [unreadable] [unreadable] [unreadable]

Arterial spin labeling (ASL) has many advantages for the measurement of cerebral blood flow (CBF) and regional brain perfusion. Such measurements are significant for research and diagnosis and treatment of stroke, ischemia, brain tumors, and dementia. ASL is non-invasive, inexpensive, and produces high-resolution images that are easily compared with other anatomical and functional magnetic resonance imaging (MRI) scans acquired as part of the same session. The ASL method involves labeling inflowing arterial spins by inverting them at a plane proximal to the imaging volume. A major factor in the calculation of CBF is the transit time for the labeled protons to pass from the imaging plane and into the labeling plane. Most standard ASL sequences suffer from the limitation that they cannot measure transit times directly. Instead, they rely on a method of factoring out the effect of travel times that is introduces multiple sources of confound and limits signal-to-noise ratio, especially in white matter. Furthermore, standard ASL measurements are unable to determine whether the changes detected are truly reflections of changes in flow or the result of the alterations in transit times known to be involved in neural activation and stroke. In this proposal, we describe a novel method of arterial spin labeling that is able to directly measure transit times. Our method offers the advantages of standard ASL while removing some of its limitations. This is made possible by labeling the arterial spins according to a pseudo random sequence. The mathematical properties of the sequence used for pseudo random amplitude modulation, or PRAM, allow recovery of all the transit times present in the imaged tissue as part of a single integrated acquisition. Implementation of this method should provide novel insights into the details of cerebral blood flow at both the macroscopic and microscopic levels. Detailed specific aims are: Specific Aim 1a: Implement the pseudo random arterial modulation (PRAM) scheme on a Siemens 3T Trio scanner. Specific Aim 2: Validate the PRAM sequence developed in SA 1 using standard spin tagging and ASL techniques in phantoms. Specific Aim 3: Extend the validation studies to human volunteers undergoing visual stimulation by comparing the PRAM results to those acquired by standard PASL sequences.

Objective: Autoimmune retinopathy (AIR) is an ophthalmic disorder in which autoantibodies damage the retina and its components, causing progressive vision loss. AIR has no established treatment, but systemic immunosuppression has shown favorable responses. Rituximab is an immunosuppressive agent which binds specifically to B lymphocytes. The objective of this study is to investigate the safety of rituximab as an effective treatment for AIR. Study Population: Five participants with AIR and visual acuity of 20/200 or better in at least one eye will receive rituximab. AIR must be confirmed by immunohistochemical demonstration of serum anti-retinal antibodies on normal, unfixed, frozen rhesus monkey or human retinas, as well as visual field and electroretinography (ERG) changes. Up to seven participants may be enrolled in order to obtain the five participants to be included in the analysis if participants withdraw prior to receiving rituximab. Design: The study duration is 18 months. Rituximab is administered as a cycle consisting of two separate rituximab infusions of 1,000 mg each, two weeks apart. Participants will receive their first rituximab cycle at baseline and evaluated for a second cycle six months later. Treatment success is defined as experiencing a ≥ 25% improvement in ERG response amplitudes or ≥ 3 decibel (dB) improvement in mean deviation on Humphrey Field Analyzer HFA (30-2) or improvement in threshold values > 0.5 log in the existing scotomas on Goldmann Visual Field (GVF) or ≥ 25% improvement in the area of scotomas on GVF assessment as compared with baseline. As a result, participants could receive a maximum of two cycles in this study. Participants will return to the clinic six weeks and three months after their first infusion of each cycle for a safety visit. Study visits will continue every three months for the study duration. Outcome Measures: The primary outcome is the number of participants who meet the definition of treatment success within six months from baseline. Secondary efficacy outcomes include changes in visual acuity, the number of treatment successes at 9 and 12 months, the number of partial responders at 6, 9 and 12 months, changes in ERG or visual field as demonstrated by the HFA (30-2) or GVF, changes in optical coherence tomography (OCT), changes in fluorescein angiography (FA), changes in serum anti-retinal autoantibody or anti-retinal antibody staining, changes in color vision, positive visual symptoms or nyctalopia and changes in the participants quality-of-life as assessed by the NEI visual function questionnaire. For participants with ≥ 2 ERG measurements available prior to enrollment, an attempt will be made to compare the rate of decline pre-study period to the rate of decline post-enrollment period. Safety outcomes include the number and severity of systemic and ocular toxicities, adverse events, and infections and the proportion of participants with a loss of ≥ 15 ETDRS letter score.

This project seeks to (i) extend what is presently known about the features of environmental mechanical stimuli that find representation in the primary somatosensory receiving areas in somatosensory cerebral cortex of prmates; (ii) obtain quantitative descriptions of the central neural activity evoked by mechanical stimuli that can be dscriminated in psychophysical experiments; and (iii) provide information about the nature of the mechanisms that underlie stimulus coding by cortical somatosensory neurons. The activity of individual cortical neurons shall be recorded extracellularly in the absence of general anesthesia; mechanical stimulation of the skin will be achieved by servo-controlled DC motors that move fine brushes at constant velocity over the skin; the orientation, traverse length, traverse width, direction, and stimulus location are stimulus parameters whose influence on cortical neuron response shall be investigated. Bilateral simultaneous or sequential moving tactile stimuli will be utilized to study the response properties of neurons in area S-II/r. It is anticipated that the data obtained in these studies will enable the distortions of somatic sensory performance which accompany neurological damage or drug administration to be interpreted in terms of altered stimulus coding by individual neurons.

DESCRIPTION: The overall aim of this investigation is to understand how the CNS uses afferent input from the upper GI tract to regulate energy balance and body weight. The working hypothesis is that the normal internal signal for termination of food ingestion involves integration of multiple negative feedback signals produced by the presence of food in the upper GI tract, well before digestive absorption of metabolic fuels. The proposal outlines a series of neurophysiological experiments to elucidate the neuro-humoral basis of energy balance. These experiments will: 1) identify and characterize short term meal-related gut neurophysiological signals, 2) determine their representation and integration at peripheral vagal and central brainstem nervous system sites, and 3) evaluate how they are interpreted in the context of the neuro-humoral signals related to the long-term control and mobilization of stored fuels. Focusing on the sensory vagus nerve and its CNS projections as the main neuroanatomical pathway linking the upper gastrointestinal sites exposed to nutrients during a meal and the CNS sites mediating the control of food intake, experiments will characterize vagal afferent responses arising from nutrient contact with the duodenum. Further experiments will determine the extent to which these responses are secondary to and/or modulated by exogenous administration of gut-brain peptides normally released by the duodenal presence of nutrients. Specific chemical blockade of macro nutrient absorption will evaluate the degree to which vagal afferent transduction of duodenal nutrient chemosensitivity is mediated by the absorptive process. Single unit extracellular neurophysiological recordings will assess the neural brainstem representation of signals arising from individual and combined meal-related stimulation of multiple alimentary tract compartments, including stomach and duodenum. Central administration of putative long-term energy balance peptides modifies meal-related brainstem neurophysiological signals from the gut.

The goal of this project is to develop gene therapy as a potential treatment for HIV-1 infection. In our early work on anti-HIV gene therapy approaches we reported that retroviral vectors designed to express a variety of gene can inhibit HIV-1 in vitro. Based on our successfully in vitro studies we are now collaborating on a clinical trial to evaluate these approaches in HIV-1 discordant identical twins. A phase I/II pilot study was initiated in August 1996 to evaluate the safety, relative survival, and potential efficacy of infusions of activated, genetically engineered, syngeneic CD4+ T lymphocytes obtained from HIV seronegative identical twins. T cells from each seronegative twin were obtained by apheresis, enriched for CD4+ cells, induced to polyclonal proliferation with anti-CD3 and rIL-2 stimulation, transduced with a control (Neo-containing) retroviral vector and up to two additional retroviral vectors containing potentially therapeutic genes (antisense TAR and/or transdominant Rev). The engineered T cell populations were expanded and then infused into the seropositive twins (between 4-20 X 109 CD4+ T-cells were administered). In vitro gene transfer efficiencies ranged from 1.0% (for a low titer PA317 packaged vector) to 40% (for a PG13 packaged vector). 8 patients have been treated to date with a total of 15 separate infusions (each patient will receive 2 infusions of different combinations of vectors). The relative survival of the uniquely engineered T cells is being monitored by vector-specific PCR. Initial results from this study demonstrate good levels of gene transfer and persistence of vector signals for at least 30 weeks post infusion. The amount of engineered CD4+ cells detected in total PBMC correlated with the amount of in vitro gene transfer and varied between 2.0% to 0.1% of total PBMC immediately following infusion. In most treatment cycles, vector signal gradually declined 1 log over the next 4-8 weeks and then stabilized for varying lengths of time. The ratio of therapeutic v ector/control vector for the majority patients studied is slightly increased in vivo in comparison to the in vitro transduced lymphocyte populations. Transient increases in CD4 counts were seen in all patients following one or more cell infusions and persisted from 4 to 28 weeks.

In the last ten years great strides have been made in our knowledge of the nucleus locus coeruleus (LC), its brainstem and spinal cord connections and their effects. For example, in 1978 it was commonly held that the LC consisted of cells which contained norepinephrine (NE) and only NE and that their activity produced inhibition of all the neurons they contacted. Both of these concepts have been shown to be woefully incomplete, since we now know that the postsynaptic effect can be facilitation or inhibition dependi g on the NE receptors involved; the discovery of colocalization of neuropeptides further compounds the postsynaptic possibilities of action. There still remains much that is unknown about the LC, its inputs, outputs and effects. It is the intention of conference organizers to bring togethe an international group of 24 scientists--each of whom is working on the LC and its brainstem or spinal cord effects utilizing a different technique or approach. Seminars, each with approximately four scientists making presentations, will address the following topics: The Physiology of LC cell , Inputs and Transmitters, Anatomy of Coerulospinal Pathways, Brainstem Interactions, and Interneuron and Motoneuron Effects. It is anticipated that through the formal presentations, as well as through informal discussions among the participants during the conference, there wi l emerge a better understanding of the overall functioning of the LC. This, in turn, should stimulate more focused investigations while encouraging a spirit of collaboration among the participants.

Integrins, the principal receptors that mediate cell-ECM interactions, are composed of heterodimeric transmembrane and subunits. The 18 and 8 subunits found in mammals combine in a restricted manner to form specific dimers with different ligand binding properties. 1, the most abundantly expressed integrin subunit in the kidney, binds at least 12 subunits. Its short cytoplasmic tail interacts with multiple intracellular molecules tht promote integrin- mediated adhesion, migration and signaling. The best defined of these proteins are talins and kindlins, which we and others showed are required for normal integrin function. These proteins bind to two canonical NPXY motifs found in all integrin cytoplasmic tails: talins to the membrane proximal and kindlins to the membrane distal motifs. In the last funding period, we utilized integrin beta1flox/flox mice to demonstrate that this integrin subunit s required for normal uretereic bud (UB) development, as deleting it using hoxb7cre mice resulted in severe abnormalities in branching morphogenesis. We further showed that mutating canonical NPXY motifs of the integrin 1 cytoplasmic tail also resulted in abnormal UB development. Kidneys with a Y/A mutation in both motifs (thus affecting binding of talins and kindlins) are severely dysmorphic and dysplastic, but have a significantly less severe branching defect than the UB integrin 1-null mice. Thus a key unanswered question in the field of integrin biology and kidney development is how the NPXY motifs regulate integrin function and whether they have distinct functions from each other. We propose to answer this question by testing the hypothesis that binding of talins and kindlins to specific NPYX motifs of the integrin 1 cytoplasmic tail differentially regulates integrin functions required for normal UB development. This hypothesis will be tested by the following three specific aims. 1) Determine if talin binding to the membrane proximal NPXY motif of the integrin 1 cytoplasmic tail is required for normal UB development. 2) Determine if kindlin binding to the membrane distal NPXY motif of the integrin 1 cytoplasmic tail is required for normal UB development and function. 3) Define the mechanisms whereby integrin 1 NPXY motif binding proteins regulate collecting duct cell branching morphogenesis. Completion of these aims will help define the fundamental mechanisms whereby 1 integrins regulate renal tubule formation in the context of UB development. This has implications for our understanding of both congenital renal hypoplasia/dysplasia syndromes and adult renal diseases as UB branching is a key determinant of nephron number. PUBLIC HEALTH RELEVANCE: We anticipate that this study will generate novel insights into the role of integrins and their binding partners, talins and kindlins in the development of the collecting system of the kidney. This knowledge is fundamental to our understanding of how the renal collecting system functions and could potentially define new etiologies for dysmorphic dysgenic kidneys in infants.

The aim of this research is the total synthesis of the Cephalotaxus group of alkaloids, especially te harringtonines which have shown significant inhibitory action against experimental lymphoid leukemia. The harringtonines (l-4) are esters of the parent alkaloid cephalotaxine (5), which we have already synthesized. Neither cephalotoxine nor the acid moieties show activity alone. Esterification is vital for antitumor activity. In addition we hope to synthesize the structurally related oxycephalotaxines, some of lycorine group of Amaryllidaceae alkaloids, and the newly discovered "homolycorine" alkaloids cocculin and cocculidine. We hope that properly esterified members of these groups or their analogs will show antitumor properties. In addition we hope to synthesize the unusual loline group of Pyrrolizidine alkaloids in order to investigate potential antitumor activity in this series. Finally, we wish to investgate biologically active metabolites produced by the basidiomycete Phlebia strigosozonata.

In previous studies we have gained considerable insight into the mechanisms by which adenylate cyclase is regulated by cations, hormones, guanine nucleotides, fluoride, and adenosine. We have found that adenosine inhibits adenylate cyclase non-competitively with respect to metal-ATP and in a cation dependent manner. Stimulated form(s) of the enzyme were potently inhibited whereas the basal enzyme was not. We intend to take advantage of these earlier observations: a) To identify sub-cellular fractions in addition to plasma membranes that may be enriched in adenylate cyclase(s) sensitive to stimulation by fluoride, guanine nucleotides, or glucagon, or sensitive to inhibition by adenosine. b) To identify and then purify by affinity chromatography a protein to which adenosine binds to exert its inhibitory effect on adenylate cyclase. c) To ascertain whether the effect of adenosine to lower cellular cAMP levels is due to inhibition of adenylate cyclase or to stimulation of cyclic nucleotide phosphodiesterase. D) To ascertain whether adenosine behaves cooperatively with insulin to enhance insulin's effects in isolated fat cells to decrease cAMP levels, to decrease lipolysis, and to increase glucose metabolism. e) To ascertain whether the effects of adenosine on fat cell metabolism and cAMP levels may be due to a direct action of adenosine or may be mediated by a factor generated in response to adenosine.

PROJECT SUMMARY This is a proposal to take a novel approach to the genetics of bipolar disorder (BP) through sequencing of all known synaptic genes (the synaptome). The project will take advantage of the talents of a next-generation sequencing leader, a BP genetics expert, and a synapse neurobiology specialist. Together we hope to discover rare BP susceptibility variants. BP, the sixth-leading cause of disability worldwide, is highly heritable. Molecular genetics work in BP is currently focused on uncovering common disease variants. The first four genome-wide association (GWA) scans have, however, been disappointing yielding no genome-wide significant signals, although one signal surpassed that threshold in a combined analysis. Interestingly, the two strongest genes in that analysis encode synaptic proteins, and in a pathway analysis of two of these GWA studies, the most significantly enriched gene set was for synaptic transmission. We propose to determine the genetic variation in genes encoding components of the synapse including neurotransmitters and their receptors, adhesion/cytoskeletal proteins and scaffold proteins. Advances in sequencing technology and the ability to target specific genomic areas will allow us, in Aim 1, to resequence exons and promoters of 1,500 synaptome genes in 800 BP probands and 400 controls, and to similarly screen the whole exome in 80 probands from our largest BP families and from 40 controls. In Aim 2 we will bioinformatically assess the likely functional impact of variants, and compare variation in cases to variation in 800 controls (our sequenced controls plus 400 sequenced by the 1000 Genomes Project) to determine whether genes and/or clusters are enriched for rare deleterious variants. We will similarly compare whole-exome variation in 80 cases and 80 controls. In Aim 3 we will genotype extended families of Aim 1 probands carrying likely susceptibility variants to assess for linkage, and genotype 1,600 cases and 1,600 controls to replicate gene and cluster enrichment of functional variants in BP. We will also resequence in a subset of genes to replicate enrichment in BP of functional variants. Our study holds out the possibility of finding, not merely variants in linkage disequilibrium with BP susceptibility variants, but the functional disease variants themselves. Further, it is important to emphasize that the great majority of psychiatric drugs modulate synaptic mechanisms. We therefore consider that discovery of BP genes encoding synaptic proteins has very high translational potential as these potentially represent the most "druggable" targets in BP. PUBLIC HEALTH RELEVANCE: PROJECT NARRATIVE This is a proposal to take a novel approach to the genetics of bipolar disorder through sequencing of all known genes that code for proteins in the brain's synapses. Our study holds out the possibility of finding, not merely gene variants that lie near those that confer susceptibility to bipolar disorder, but the actual disease variants themselves. Further, it is important to emphasize that the great majority of psychiatric drugs modulate brain synapse mechanisms. We therefore consider that discovery of bipolar disorder genes encoding synaptic proteins has very high translational potential as these potentially represent the most "druggable" targets in bipolar disorder. )

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Cellular therapy is a potential technique under study for repair of kidneys damaged by disease. In order to effectively develop such approaches, an understanding of the development of the metanephric kidney is essential, particularly for directing human embryonic stem cells towards renal precursors.

The proposed experiments are intended to provide basic information on the relative frequency and specificity of base substitution and frameshift mutational events and, more importantly, to address the question of the mechanisms through which these events are produced at the molecular level. Focus will be on studies with purified replication proteins including DNA polymerases. These will be used to synthesize defined viral DNA probes in vitro which will permit the selection of various mutational events in in vivo biological assays. The exact nature of the mutational events will be determined by DNA sequence analysis.

The response to inhalation of ambient aerosol particles from indoor and selected outdoor environments will be investigated using high sensitivity elemental analysis by proton induced X-ray emission, PIXE. The response may include both deposition of particles in the respiratory tract and growth in particle size due to exposure to high relative humidity during breathing. Both responses may depend on particle size and on chemical composition of the particles and may be different for different component particles in a mixed aerosol. The different components may be distinguished by their different elemental composition, and the respiratory response of each may be determined in a single experiment. Samples will be taken by 5 stage cascade impactor with fine particle filter and will provide resolution of particles less than 0.25 to greater than 4 mm equivalent aerodynamic diameter. Each size range is analyzed for its elemental consituents by PIXE, including S, Cl, K, Ca, Ti, V, Cr, Mn, Fe, Ni, Cu, Zn, Br, Pb, Zr, Cd, and additional heavy metals if present in the aerosol. The manogram sensitivity of the PIXE technique permits fine particle size resolution at a sampling rate of 1 liter/minute and requires of human subjects only 10 minutes or less of breathing time per sample. Experiments are carried out using ambient aerosol from different actual environments and do not require the use of special tracer materials. A major objective of the research will be to standardize experimental conditions and develop a compact and portable sampling unit which cam be used in more specialized medical tests of inhalation response to pollution aerosol particles. BIBLIOGRAPHIC REFERENCES: Akelsson, K.R., G.G. Desaedeleer, T.B. Johansson, and J.W. Winchester. Particle size distribution and human respiratory deposition of trace metals in indoor work environments. Annals of Occupational Hygiene 19:225, 1976. Desaedeleer, G.G., J.W. Winchester and K.R. Akselsson. Monitoring aerosol elemental composition in particle size fractions for predicting human respiratory uptake. Nucl. Instr. Meth., in press, 1976.

Vascular inflammation is a hallmark of vulnerable atherosclerotic plaques, at high-risk for causing acute clinical events. 18F-labeled fluorodeoxyglucose (18F-FDG) Positron Emission Tomography (PET) imaging in combination with computed tomography (CT) has emerged as an accurate, reliable and reproducible tool to quantify vascular inflammation in carotid, aorta and femoral arteries. Recently, novel PET tracers such as 18F- labeled sodium fluoride (18F-NaF) have been used to target many biological processes in atherosclerosis other than inflammation, including active micro-calcification, which appears to be an important marker of unstable atherosclerotic plaques. Use of PET to quantify inflammation in coronary arteries is challenging due to respiratory and cardiac motions, and the limited spatial resolution of PET. These challenges can be overcome by using motion and partial volume correction strategies that may require long and repeated scans. However the use of CT exposes the subject to X-ray radiation and is unsuitable for such long repeated scans as well as for longitudinal tracking of interventions. Recently, systems that combine PET and Magnetic Resonance Imaging (MRI) have become available that allow for simultaneous, co-registered PET and MRI acquisitions. MRI requires no ionizing radiation and produces high spatial and temporal resolution images with excellent soft tissue contrast. These characteristics are ideally suited to repeated, tomographic imaging for motion correction, repeated scans in longitudinal studies, improving partial volume error (PVE) correction of the PET data, and in providing complementary information about coronary plaque morphology. As part of this grant proposal, we will develop and test methodologies for motion correction using MRI to optimize coronary PET imaging using phantoms and in an in vivo setting (Aim 1). We will also examine strategies for partial volume corrections of PET data to improve coronary PET imaging (Aim 2). As the final Aim (Aim 3) of this proposal, we will use methodologies developed and optimized in Aims 1 and 2 to evaluate in vivo, combined MR/PET imaging of FDG and NaF uptake in the coronary arteries of individuals following an acute myocardial infarction to determine the ability of these techniques to discriminate between the culprit lesions responsible for the clinical event and a non-culprit vessel.

The affinity shown by 67Ga, 111In, and the higher atomic number rare earth radionuclides for nonosseous tumors in man has provided the basis for a diagnostic test (scanning) for the detection of cancer. Our aim in this project is to determine the cellular binding sites for these radionuclides and to identify the nature of the process(es) and the biochemical agent(s) involved. An understanding of the mechanism(s) involved should lead to methods for enhancement of the affinities of these agents for tumor tissue and also to the identification of other tumor-localizing agents of perhaps even greater tumor specificity. Our goals for the coming year are: (1) We will scale-up our procedure for the separation and purification of a 40,000 (40K) dalton 67Ga-binding glycoprotein that appears to be unique to tumor tissue permitting us to obtain mg quantities of the protein for production of antibody to it. We will in turn then be able to separate 40K glycoprotein and antibody directly from tumor tissue extracts and the serum of immunized rabbits using affinity chromatography techniques; (2) We will complete our characterization of the 40K 67Ga-binding protein and the subcellular microvesicles with which it appears to be associated; (3) We will study the subcellular distribution of 67Ga in the thymus, the organ that we now postulate to be the endogenous source of tumor tissue 40K 67Ga-binding glycoprotein. Nude mice implanted with rat hepatomas will be used to test our hypothesis; and (4) The general nature of the effect of the presence of malignancy on the increased body retention of 67Ga in animals following X-irradiation will be assessed, and, if the effect is found to be general, we will initiate an investigation of the sera of tumor-bearing animals for abnormal 67Ga-binding components.

Studies designed to provide insight into the specific substances and regulatory mechanisms that underlie the tumorous state in plants and on the reversal of that state have continued during the past year. A cAMP (3'5')-like compound was isolated and purified from both normal and tumor cells of plants. Although this substance has certain properties in common with authentic cAMP (3'5') and is a nucleotide-size compound, it differs strikingly from that compound in a number of essential respects. In contrast to authentic cAMP (3'5') this compound has been found to be a very potent inhibitor of both beef heart cyclic monophosphate phosphodiesterases and of a cAMP-dependent protein kinase. The role played by this substance in the regulation of cell division in higher plant species was further investigated. A second area of the plant tumor problem that was studied was concerned with the persistent but potentially reversible suppression of the tumorous state in crown gall teratoma tissues of tobacco. The differential expression of the Ti plasmid-specified functions, namely oncogenicity and nopaline synthesis, were investigated under conditions in which the tumorous state was suppressed. It was found that while nopaline is synthesized persistently, a reexpression of oncogenicity depends on the development of new cell divisions. These new cell divisions appear to be required either to reestablish positive feedback control mechanisms upon which the development of autonomy in plant tumor cells appears to depend, or they are required to establish the pattern of metabolism concerned with cell growth and division which is then maintained in the plant tumor cells by positive feedback control mechanisms.

Considerable information is available about the antibody mechanisms that initiate experimental and human renal injury. Despite this knowledge and the use of aggressive therapeutic maneuvers to modulate nephritogenic antibody responses, renal damage often progresses relentlessly to renal failure, necessitating costly and only partially satisfactory measures such as dialysis. Studies herein will examine the humoral and cellular processes leading to progression of immunologically-induced tubulointerstitial nephritis in the rat. This model has an apparent antibody-associated first stage, with sequential stages in which lymphocytes predominate and subsequently monocytes/macrophages, with fibrosis, become prominent. Studies are outlined to determine the major immunopathogenesis of these three stages and to understand how one stage influences the sequential evolution of the next. These include quantitative serum transfer experiments and complement depletion (also polymorphonuclear leukocyte depletion, if needed) to evaluate the first stage. A central question is whether the lymphocytes (predominately T helper cells) in the second stage may be sensitized by the initial injury so that the lesion becomes self-perpetuating. To answer this, selective cell proliferation assays in response to both immunizing and autologous tubular antigens will be done. In addition, subrenal capsule transfer using cells extracted from the active lesions (total cells or populations depleted of T cell subsets or B cells) will be used with or without in vitro antigen-specific propagation and the addition of specific antibody or tubular antigens. These studies coupled with modulation (by specific antisera) of the lymphocyte and macrophage stages will provide an overview of the interrelated facets in the evolution of this model of progressive immune renal injury. The production of the lymphocyte factor, interleukin-2, by the infiltrating cells will be studied since, in association with antigen and accessory cells, it could stimulate local propagation of T cells. Macrophage-produced interleukin-1 will be studied at it may relate to the fibrosis of the later stages of the lesion. An understanding of the elements responsible for the sequential evolution of this lesion and the steps necessary for their interruption will provide a basis for understanding progression and its manipulation in human immune renal injury.

Besides its roles in leukocyte adhesion to endothelial cells during inflammation, E-selectin has recently been shown to be involved in capillary tube formation, which is the critical for solid tumor growth, metastasis, and many angiogenic diseases. Identifying specific E-selectin ligands on endothelial cells is very important to understand this process. In this proposed research project, specific E-selectin glycoprotein ligands will be isolated from bovine capillary endothelial (BCE) cells using E-selectin-IgG protein A Sepharose. The N-terminal and internal peptide sequences will be determined. The cDNA encoding this novel E-selectin ligand will be cloned from a cDNA library prepared from BCE cells induced to form capillary tubes.The cDNA clone will be characterized by database searching, Northern and Southern blotting analysis. A soluble form of the identified E-selectin ligand will be expressed and antibodies specific to this E-selectin ligand will be raised. The soluble form of this novel E-selectin ligand and antibodies specific to this ligand are expected to have high potentials in the development of treatments of solid tumor growth, metastasis, and angiogenic diseases.

Although arsenic is a well-established human carcinogen and induces cancers the skin, liver, bladder, and lung, the underlying carcinogenic mechanism(s) for arsenic is unknown. Although arsenic induces chromosomal aberrations and sister chromatid exchanges in cultured mammalian cells it has not been shown to be active as a gene mutagen. Using the human-hamster hybrid (A/L) cells that are sensitive in detecting multilocus deletions, the applicant has obtained preliminary data suggesting that arsenic is mutagenic in mammalian cells. The first objective of this proposal is to examine the quantitative mutagenic yield and the spectrum of mutations induced by graded doses of the trivalent sodium arsenite and the pentavalent sodium arsenate. Specifically, the quantitative mutagenic data will be used to obtain dose response relationship for the mutagenicity of arsenic. The molecular spectrum will be used to ascertain qualitatively the presence or absence of specific signature changes at the gene level. The second and third objectives are to examine the underlying mechanisms for the observed mutagenic events induced by arsenic compounds, particularly, the roles of reactive oxygen species and cellular glutathione levels. Mutations will be scored at both the S1 and HGPRT loci using the A/L cells. The A/L cells contain only one copy of human chromosome 11 and mutations on marker genes located on this chromosome can be readily scored using an antibody complement lysis technique. Since the A/L cell also contain the HGPRT gene located on the hamster X chromosome, mutations induced by arsenic on an essential (-X) versus a non-essential chromosome (human chromosome 11) will provide useful information on the types and sizes of the induced molecular alterations. By using specific DNA probes of other genes that have been regionally mapped to various sites on chromosome 11, the molecular mechanisms of mutation in A/L cells will also be examined.

The long-term objectives of this proposed research are to determine the genetic and antigenic basis for the diversity of spotted fever group rickettsiae in eastern Asia. This knowledge is the foundation for the development of a rational epidemiologic and immunologic approach to development of effective preventive vaccines and diagnostic tools for these rickettsioses. Important aspects of this proposal are the active participation of leading rickettsiologists of the People's Republic of China as visiting scholars at the University of North Carolina to learn molecular rickettsiologic techniques and the work in the field, hospitals, and laboratories of the People's Republic of China by the American principal investigator. This combination of opportunities offers the prospect of opening the knowledge of this important geographic area to modern scientific access. Isolators of spotted fever group rickettsiae from China including Jing-he rickettsia and Rickettsia sibirica will be compared with R. sibirica from the USSR and other Asian spotted fever group rickettsiae such as R. conorii, JC-880, and TT-118. Methods which will be employed include microimmuno-fluorescent antibody analysis, Western immunoblotting, monoclonal antibodies, and restriction endonuclease analysis of rickettsial DNA. North Asian tick typhus is a widespread spotted fever group rickettsiosis of public health importance in China, USSR, and Mongolia. India tick typhus and Southeast Asian spotted fever group rickettsioses border upon China but are of unknown distribution within the People's Republic of China. A major interest of the principal rickettsiology laboratory in China, located at the National Center for Preventive Medicine in Beijing, is spotted fever group rickettsioses of China. This research proposal offers the fulfillment of long-term cooperative aggreement with that laboratory and the elucidation of the ecology and molecular rickettsiology of spotted fever rickettsiae in China which will be subsequently carried forward by the Chinese scientists.

Despite remarkable advances in our understanding of the biology of malaria over the last 30 years, malaria elimination remains elusive in Malawi, among other sub-Saharan African countries. The applicant has focused her academic career on using genetics and epidemiology to address key public health questions related to treating and preventing malaria, and to training U.S.- and Malawi-based investigators to conduct patient- oriented malaria research to this end. This K24 award builds on her overarching goal of using translational research to improve malaria treatment and prevention now and will prepare her and her trainees to develop strategies for malaria elimination in Malawi and throughout sub-Saharan Africa. The specific research aims of this proposal focus on interventions to prevent malaria in two vulnerable populations: pregnant women and their babies, in whom the malaria risk is well known but for whom preventive strategies are inadequate, and school-age children, a population newly recognized to suffer from high rates of malaria infection. Pregnant women routinely receive intermittent treatment two to three times during pregnancy to prevent malaria, but the efficacy of currently used medication is compromised due to widespread resistance and new strategies are urgently needed. The applicant and her team recently completed a trial comparing intermittent preventive treatment to continuous prophylaxis to prevent malaria during pregnancy in Malawi. In this proposal, clinical, molecular, genomic and immunological data will be analyzed to test the hypothesis that intermittent treatment and continuous prophylaxis to prevent malaria during pregnancy will have different effects on maternal and neonatal outcomes, maternal susceptibility to malaria in subsequent pregnancies and infant immunity to malaria. To further investigate the burden of malaria infection and transmission potential in school-age children, the applicant has added school-based surveys to on-going cross-sectional studies. They will test the hypothesis that children at high risk of carrying malaria infection, including gametocytes, can be reached through school-based interventions. The applicant will take advantage of the career development components of this award to enrich her knowledge base and international partnerships that will allow her to expand the scope of her research and develop new interventions in Malawi. This proposal includes an integrated, comprehensive mentoring plan to train U.S.- and Malawi-based investigators in the technical, scientific and professional skills required to succeed in patient-oriented research. The applicant and her research team strive to design hypothesis-driven studies that lead to evidence-based interventions to improve the health of those who suffer from malaria and contribute scientific insight to help eradicate malaria.

Prior immunity to an unrelated pathogen can lead to beneficial effects such as enhanced viral clearance or detrimental effects such as increased viral load or remarkably increased immunopathology occurring as a consequence of T-cell cross reactivity (XR). Initially described under highly controlled conditions using animal models infecting with human respiratory pathogens such as influenza A (lAV), vaccinia (W), and lymphocytic choriomeningitis (LCMV) viruses, examples of such XR T-cell responses have now been associated with enhanced pathology in humans infected with Epstein-Barr (EBV) (by us), lAV, dengue, and hepatitis C (HCV) viruses. The overall objective of this grant is to exploit our developed mouse models to determine under controlled conditions, not possible in human studies, how T-cell XR impacts T-cell selection and function leading to detrimental effects resulting in exacerbation of disease outcome during respiratory infections. Simultaneously we will use our developed human T-cell expertise during viral infections to translate our findings in mouse models to human disease, specifically examining the association between CDS T-cell XR and the severity of lAV infection. We have identified networks of cross-reactive CDS T-cells in both mice and humans, in human studies, we found that the frequency of lAV-Mlss-specific memory CDS responses XR: with EBV-BMLFI280 in HLA-A2+ patients directly correlated with the severity of acute infectious mononucleosis. We have also observed in 5 EBV-seronegative HLA-A2+ middle-aged adults a unique XR between Mi-specific T-cells and EBV lytic epitopes, BMLF1 and BR.LF1, raising the intriguing possibility that these may be protective. We will focus this proposal first on proof of principle animal models examining the role of t-cell XR in mediating detrimental effects during respiratory infections in lAV-inimune mice subsequently infected with LCMV (lAV+LCMV), category C and A human pathogens, respectively. This model leads to severe lung pathology very similar to that observed in lAV pandemics. Our work will focus on the role of XR memory CD4 and CDS T-cells in mediating detrimental effects, either induction of lung pathology or enhancing viral load (Aim 1&2). At the same time we will determine in translational studies if there is any evidence that this unique XR CDS T-cell network between lAV and EBV is associated with the severity of disease during acute lAV infection in patients when both viruses are present (Aim 3). The overall objective of this proposal is to determine how XR T-cells impact T-cell selection and function and influence disease outcome, for better or worse, as the host is exposed to acute respiratory infections such as lAV.

Osteoporotic fractures are a major source of morbidity and mortality in older women. The incidence of hip fractures in women doubles every five to six years after age 40. Vertebral crush fractures also increase with age and may result in substantial pain and decline in function. The risk of fracture increases with decreasing bone mass. Although women over 70 are at the highest risk for osteoporotic spine and hip fractures, most research has focused on prevention of fractures in younger postmenopausal women. Many women in the older age group, however, present for diagnosis and treatment of low bone mass. A fundamental question is whether treatment at this stage is beneficial. We have shown in a dose-finding study that low dose estrogen (0.25 mg/d) reduces bone resorption in older women with fewer side effects than usual dose estrogen. This proposed study will examine the effect of low dose ERT plus calcium and Vitamin D on hip and bone density compared to calcium and vitamin D alone in a randomized placebo controlled trial. If our hypotheses are correct, then a substantial decrease in fracture risk in older women could be achieved with a safe and well-tolerated regimen. Moreover, if risk factors for cardiovascular disease are also decreased, this regimen may have even greater public health benefits.

DESCRIPTION (Applicant's abstract): Toward the goal of developing an automated, miniaturized device to detect the body's reaction to exposure to environmental toxins, Advanced Sensor Technologies, Inc. proposes to develop a miniature device capable of providing differential blood cholinesterase analysis. The device will provide analysis of cholinesterase levels (acetylcholinesterase and butyrylcholinesterase) using a single drop of blood obtained from finger prick sampling. The system will be automated such that it may be utilized by minimally trained individuals, and will provide results in less than five minutes. The proposed device will provide health clinics with units capable of screening and confirming exposure of lawn care worker, crop dusters, athletes, and first responders (i.e. EMS personnel) to chemical agents, pesticides, or other toxic chemicals. It has been demonstrated that these, and other drugs, especially those that are organophosphorous or carbamate based, inhibit cholinesterase (ChE) activity in human blood. These inhibitory effects often persist for up to 100 days in a healthy individual after exposure. Additionally, the device, designed similarly to home pregnancy tests, can be utilized by parents to test their children for exposure to environmental toxins, including pesticides on insufficiently washed fruits and vegetables. PROPOSED COMMERCIAL APPLICATION: NOT AVAILABLE

Chronic obstructive pulmonary disease (COPD) is a major public health problem and is the fourth leading cause of death in the United States. Despite pharmacologic and surgical therapy, COPD continues to cause considerable morbidity, and patients suffer from exercise intolerance and decreased physical activity, which are independent risk factors for exacerbations, hospitalizations, and mortality.Pulmonary rehabilitation (PR) has been shown to improve symptoms of dyspnea, health-related quality of life, and exercise capacity and national guidelines now recommend that all patients with COPD participate in such programs. Unfortunately, long-term exercise adherence after initial rehabilitation is very poor, and it is clear that benefits gained during PR diminish rapidly if patients do not continue to exercise. There remains a critical need for novel interventions that may sustain the benefits of regular exercise after PR, as well as promote long-term exercise adherence and exercise self-efficacy in the post-rehabilitation period in patients with COPD. Tai chi is a popular mind-body exercise that incorporates low-moderate intensity physical activity, self awareness, relaxation, and meditative slow breathing. It may be particularly suited to deconditioned individuals with COPD who suffer from limited exercise tolerance and dyspnea. Importantly, tai chi exercise comprehensively integrates several key elements of PR (aerobic exercise, upper and lower extremity training, breathing, and stress management) into one relatively low-cost and accessible activity. Studies in varied populations have suggested that tai chi can improve exercise capacity, quality of life, and promote exercise self-efficacy; and that adherence may be favorable when compared to conventional exercise modalities. In this application, we propose a three-arm randomized controlled trial in patients with COPD (N=126) to evaluate the benefits of six months of tai chi exercise, versus group walking, versus general recommendations for self-exercise (standard care) in maintaining clinical improvements after the completion of standard, supervised PR. Our primary aim will be to investigate whether tai chi can maintain gains in exercise capacity three and six months after PR. Secondary aims will examine quality of life, dyspnea, mood, activites of daily living, as well as exercise self-efficacy, intervention adherence and overall activity. Long-term follow-up assessment will be conducted at 1 year. The results of the proposed project have the potential to transform the clinical care of patients with COPD by providing a new strategy that may substantially extend the benefits of PR and promote continued physical activity in this population.

The purpose of the Antioxidant Enzyme Core (AEC) is to provide antioxidant enzyme expertise and analysis for the individual research projects. Discussion between a project investigator and the AEC Director will be held to determine which experiments and/or assays are run. The AEC staff will carry out the proposed work or will advise the investigator on how to perform the experiments. Some of this work, particularly transfections of new cDNAs or in new cell lines as well as the development of antisense reagents, will be developmental in nature. The AEC will aid in enzyme activity assays, western, northern, and Southern analysis. The enzyme assays will be run on the antioxidant enzymes copper-and zinc-containing superoxide dismutase (CuZnSOD), manganese-containing superoxide dismutase (MnSOD), catalase (CAT), and glutathione peroxidase (GPx, both cytosolic and phospholipid). For Project 1, work will also be done on the white cell enzyme myeloperoxidase (MPO). For reagents, we have antibodies and cDNA probes to all primary antioxidant enzymes. In addition, the AEC will aid in transfecting the cDNAs for these enzymes into cell lines of interest to the project investigators. We have experience with several methods of transfection, as well as many different expression vectors for these enzymes. In the course of this work, the AEC will continue to maintain and develop reagents i.e., antibodies, cDNAs, and expression vectors for the antioxidant proteins. The AEC will also have as a goal the development of antisense reagents to inhibit the levels of all of the major antioxidant proteins.

Fast transient A-type K+ currents (IA), such as the subthreshold-activating somato-dendritic A-type K+ currents in neurons (ISA) and the fast transient outward K+ current in cardiac ventricular myocytes (Ito) are essential for the proper functioning of the brain and the heart. During pathological conditions, abnormalities in these currents can contribute to disease conditions, as recently found in a patient with temporal lobe epilepsy. This project addresses the molecular nature of the ion channels responsible for the generation of these currents in mammalian neurons. It seeks to establish the molecular composition of these channels and to elucidate the physiological significance of the identified components. Work supported by this grant led to the discovery of a novel family of proteins that associates with ISA channels known as DPPLs, of which two members are currently known DPPX (or DPP6) and DPP10. It is now believed that ISA channels in neurons are ternary complexes that include principal or pore-forming subunits of the Kv4 family and two types of associated proteins KChIPs and DPPLs. This application is focused in CA1 hippocampal pyramidal cells, neurons that are important in spatial learning and in the pathogenesis of epilepsy, and prominently express one Kv4 protein Kv4.2 and one DPPL, DPPX. The goal of the proposal is to test the hypothesis that DPPX is an important component of Kv4 channels in CA1 neurons, determining the proper distribution, biophysical properties and dynamic modulation of the channels. Aim 1 will utilize highly specific antibodies raised during the last funding period to investigate the localization of DPPX in CA1 neuron dendrites and its relationship to the other Kv4 channel components. Aim 2 will utilize DPPX knockout mice also developed during the last funding period to investigate the effects of DPPX ablation on the distribution and function of Kv4 channels in CA1 neurons. Aim 3 explores aspects of DPPX actions on Kv4 channels that have thus far received little attention: the effects of this auxiliary subunit on channel modulation by protein kinases and in controlling channel trafficking and expression in the plasma membrane, to test the hypothesis that DPPX regulates the stability of Kv4 channels at the plasma membrane. Mutations in the gene encoding DPPX have been associated with autism, underscoring the importance of understanding the function of these proteins.

This is a proposal to identify genetic susceptibility factors for biliary atresia (BA). BA is a devastating pediatric disease associated with medical complications related to a rapidly developing biliary cirrhosis that accounts for 50% of all pediatric liver transplantations. The etiology of BA is unknown, although it is proposed to be multi-factorial, with potential infectious, environmental, inflammatory and genetic causes. In the proposed study, we will test the hypothesis that there is an underlying genetic susceptibility to BA. We will identify susceptibility genes for BA using a large, well-characterized cohort of patients that has been identified through an NIDDK sponsored research consortium. We will conduct a genome-wide association study, testing for association of genetic variation (both single nucleotide polymorphisms and copy number variants) to identify genes or genomic regions associated with BA disease susceptibility. We will perform exome sequencing of a family with two affected and one unaffected sibling, and three additional unrelated BA patients. We will follow up on regions and genes identified, via next generation sequencing to identify causal variants. This proposal will build upon the resources of a large multi-center consortium and our own cutting-edge genomic and bioinformatic facilities. We anticipate that our studies will contribute new knowledge about the biologic pathogenesis of BA and will lead to the development of rationale therapeutics and diagnostics.

Mucosal surfaces are the site of HIV transmission except for direct intravenous injection, and mucosal epithelial is therefore likely to e an important initial site of virus and host interaction. Furthermore, infectious HIV has been shown to be able to transcytose form the apical (luminal) to the basolateral (lamina propria) side of epithelial cells in vitro. Thus, immunity at mucosal surfaces could play a pivotal role in resisting HIV infection. Recent attention has focused on mucosal vaccine approaches for the prevention of HIV transmission. Since the major immune defense mediator at the mucosal surface is IgA, there is an urgent need for improved understanding of the function of IgA antibodies against HIV and other viruses. Secretory IgA at mucosal surfaces has been shown to inhibit viral infection through (s) immune exclusion, its traditional function, and other mechanisms. Particularly, the novel mechanisms recently identified by our group, including (b) intraepithelial cell neutralization of viral replication during IgA transepithelial transcytosis and (c) excretion of antigens from the basolateral (laminal propria) epithelia cell surface to the apical (luminal) surface in the form of IgA immune complexes, merit deeper investigation. Since infectious HIV virions may enter mucosal epithelial cells at the apical surface and be transcytosed to the basolateral surface prior to infecting target cells in the lamina propria, we believe that HIV-specific IgA antibodies may sere yet another function by (d) intercepting virions that are traversing the mucosal epithelium and thereby blocking the spread of virus. Our hypothesis is that HIV-specific IgA antibodies, primarily IgA, can counter HI infection of mucous membranes. The theme of this project interfaces closely with the significance of secretory antibodies in the outcome of gastrointestinal infection (Project 1 and Project 2) and respiratory infection (Project 4). The research also has significance for vaccine strategies against mucosal infection. Development of such vaccines is an integral part of Projects 1 and 2. Project 3 is dependent on Core B for multiple anti-HIV monoclonal antibodies.

It is abundantly evident that cancer is a genetic disease. The integrity, replication, segregation and regulation of chromosomes play critical roles in the formation and progression of all types of cancer. In the past decades a large number of oncogenes and tumor suppressor genes have been identified, expression of which is critical in cancer formation and progression. Altered expression can result from epigenetic changes or alteration of gene or chromosome structure through a variety of mechanisms including DNA mutations, amplifications, rearrangements and loss. Understanding these and other basic mechanisms that affect chromosome metabolism is therefore critical to the study of cancer, and improved knowledge of fundamental mechanisms could improve cancer detection and treatments. Despite this, research on cancer is often focussed on specific types of cancer, specific chromosome aberrations, and specific molecular pathways, while research on fundamental genetic and epigenetic mechanisms frequently proceeds in basic science labs with little or no appreciation of the relevance for cancer cause and treatment. The Chromosome Metabolism and Cancer Training Program (CMCTP) seeks to bridge this gap by bringing together cancer researchers and basic scientists in one of the nation's leading Cancer Research institutions, the Fred Hutchinson Cancer Research Center (FHCRC). The purpose of the CMCTP is to excite young basic scientists who are researching universal mechanisms of chromosome structure and function and to encourage them to apply their results to cancer biology, while at the same time helping trainee cancer researchers learn more about innovative methods and ideas in fundamental science. For the last two project periods, the CMCTP has been funded for two predoctoral and five postdoctoral positions. For the last year, an ARRA Competitive Supplement has allowed us to increase to seven postdoctoral positions. We have had no trouble identifying qualified candidates. Trainees from the past 10 years have been successful in publishing the research supported by the CMCTP, and in building professional careers in cancer research. Former trainees in the CMCTP are now productive independent researchers in academia and biotechnology. Therefore, we are requesting continued support for this successful postdoctoral and graduate training program, and request funding of two predoctoral positions and six postdoctoral positions. PUBLIC HEALTH RELEVANCE: Training of new cancer researchers is important if past successes are to be continued. To this end, research laboratories take new students and post-doctoral scientists into their laboratories to provide them with training for careers in cancer research. This is an application for a research training grant to train pre-doctoral and post-doctoral scientists in the methods and interpretation of results from investigations into the structures of chromosomes and the expression of genes, in normal cells and in cancer cells.

The aim of this proposal is to continue to develop an exemplary education program in pediatric oncology for medical students, pediatric residents, clinical associates, dental and surgical trainees, practicing physicians, nurses, social workers, and educators in the community. This will be accomplished by the use of the established clinical & research programs of CHNMC together with formal lectures, conferences and teaching aids. The program director in collaboration with the multidisciplinary Cancer Education Committee is responsible for the coordination and implementation of this Oncology Education Program. The program will reflect the changing needs of pediatric oncology. There will be particular emphasis on all aspects of supportive care including nutritional support, pain control, home care, reintegration of the child into society, and the epidemiology and genetic transmission of childhood cancer. Teaching aids will be developed including audiovisual tapes for instruction, a newsletter and booklets for parents and patients on solid tumors and leukemia. Frequent and indepth evaluation of the program will be performed.

Not required.

Subanesthetic concentrations of volatile anesthetics potently depress upper airway defense mechanisms. In particular, hypoglossal motor control of the tongue that maintains airway patency during inspiration is severely compromised. The overall goal of this proposal is to characterize the actions of volatile anesthetics on specific synaptic mechanisms and neuronal excitability within spontaneously active mammalian inspiratory hypoglossal motoneurons (IHMNs) in vivo. While in vitro studies have identified potential mechanisms for anesthetic-induced depression, this proposal is unique because we focus on functionally identified IHMNs in an intact neuronal network under in vivo conditions, where neurotransmitters are released at physiologically relevant levels. Our overall hypothesis is 1) that the activity of IHMNs is dependent on synaptic inputs mediated by ligand-gated glutamatergic, GABAergic, and glycinergic and G-protein coupled serotonergic and adrenergic receptors, and 2) the effects of volatile anesthetics on neuronal activity are due to depression of excitatory and enhancement of inhibitory synaptic neurotransmission, rather than non-synaptically mediated intrinsic membrane mechanisms such as changes in K+conductance. Recordings of action potentials from IHMNs in conjunction with localized pressure microejection of neurotransmitter agonists and antagonists will be used to examine the relative importance of anesthetic-induced alterations on excitatory and inhibitory neurotransmission and intrinsic neuronal excitability. Three specific aims have been developed to test our hypothesis in a decerebrate canine model. In specific aim 1, we will identify the key contributing neurotransmitters in IHMNs and determine their physiological receptor activity. In specific aim 2, we will determine the effect of subanesthetic concentrations of volatile anesthetics on overall synaptic transmission of the key neurotransmitter systems in IHMNs. In specific aim 3, we will determine the effects of volatile anesthetics on the postsynaptic receptor responses for the key contributing neurotransmitters in IHMNs. The insights that will be gained from our proposed studies will contribute to our understanding of the effects of volatile anesthetics on upper airway function in the perioperative period and may suggest rational mechanism-based therapeutic interventions to mitigate these effects in the postoperative setting when patients are at high risk of airway- related morbidity.

The organelle that most typifies a synapse is the synaptic vesicle. We and others have shown that synaptic vesicles have unique integral and peripheral membrane proteins. Our data suggest that synaptic vesicles owe their unique composition and size to a neuronal specific modification of an endosomal pathway. We plan to identify the hierarchy of sorting signals that sends an integral membrane protein to the endosome and to the synaptic vesicle. We will use two proteins of contrasting membrane topology, synaptophysin and synaptobrevin. If synaptic vesicles turn out to be related developmentally to the transcytotic vesicles of epithelial cells, we will be able to short-cut the search for sorting information by drawing on the wealth of data available for the transcytotic vesicle. To help understand the association of the tyrosine protein kinase pp60(c-src) with synaptic vesicles, we will identify its synaptic vesicle targeting mechanism. Cytosolic proteins involved in the sorting of synaptic vesicle proteins and in generating their unique size will be analyzed using a recently developed in vitro reconstitution system from the neuroendocrine cell line, PC12. Association of synaptic vesicles with the subcortical cytoskeleton will also be examined in semi-intact PC12 cells. Our data will tell us what regions of synaptic vesicle proteins are required for their targeting, and what cytosolic factors are necessary for assembling these proteins into synaptic vesicles.

We propose to organize a psychotherapy research center dedicated to the development, evaluation and refinement of psychotherapies for drug abuse. The organizing theme is psychotherapy development and empirical testing based on the 'technology model' of psychotherapy research (Waskow, 1984). The Center will catalyze the process of developing promising drug abuse treatments from the point where they are merely 'good ideas' to one where they are capable of being disseminated to the clinical field as empirically validated treatments. This process requires research at sequential, progressively demanding phases. Phase I consists of pilot testing, manual writing and training program development; Phase II consists of controlled efficacy trials and studies on effective components of treatments; Phase III consists of multisite studies to evaluate generalizability of efficacious treatments. Research in the proposed Center will be organized around this sequential program with a CORE facility and 6 individual research components. Core staff will provide scientific oversight, administration and technical assistance to individual Center projects and also provide consultation and technology transfer services to investigators and clinicians at other sites. Three Phase I projects will develop new approaches: Cognitive Therapy for Post Traumatic Stress Disorder in Drug Abusers, Expectancy- based Coping Skills Training for Cocaine Abuse and a Relational Psychotherapy Parenting Group Therapy for Methadone-Maintained Mothers. Three Phase II projects will evaluate efficacy of more established approaches: CRA to improve Naltrexone Treatment, Relapse Prevention and Dynamic interactional group treatments for adolescent drug abusers, and a study of focused Coping Skills Components in cocaine abuse treatment.

DESCRIPTION (Adapted from applicant's abstract: Studies of cancer and AIDS typically record clinical events and laboratory measurements for participating subjects at several time points. Frequently, the times to the clinical events and their association are of primary interest. The relationship between the serial laboratory measurements and the times to clinical events is of interest also for the improvement of patient management and for the identification of potential surrogate markers. These data are usually plagued by problems of missingness and censoring due to missed study visits or to the discrete time observation of continuous processes. Interval censored data arise when exact event times are censored within intervals that are unique to each individual, and thus are overlapping. The broad goal of this proposal is the development of methods for analysis of interval and right censored data that addresses medical questions frequently posed by clinicians. The first specific aim of this proposal is the estimation of the distribution function for bivariate and univariate interval censored failure time data. Both nonparamaetric and "loosely parametric" estimators will be derived. The second aim is he analysis of failure time data with accompanying right and interval censored intermediate event times. Smooth estimates of the hazard functions for the terminal event before and after the intermediate event, an estimate of the survivor function adjusted for the intermediate event, and estimates of the latency distribution between the times will be derived. The third aim is the development of methods for testing for independence between bivariate interval censored data, assessing the impact of covariates on multiple interval censored outcomes, and comparing adjusted survivor curves. The fourth aim is the development of methods for analysis of interval censored data that are derived from serial laboratory measurements. These include a new parametric frailty model for interval censored data, adjustment for the measurement error and biologic variation of the underlying processes, and estimation of smooth hazard functions for interval censored data in the presence of a time-varying covariate. To accomplish these aims, local likelihood estimation, multiple imputation, estimating equations, approximations of first passage time distributions for continuous stochastic processes, and convex optimization theory will be used.

The biochemical explanation for the existence of multiple liver microsomal mixed-function oxidase activities has not been obtained. There may be multiple forms of cytochrome P450 or there may be one hemoprotein and several inducable effector proteins which impart substrate specificity and spectral properties to the hemoprotein. The objective of this proposal is to distinguish between these two possibilities. Microsomal proteins have been resolved by SDS-polyacrylamide gel electrophoresis and proteins which are induced by phenobarbital and 3-methylcholanthrene have been identified. A method has been developed to identify protein bands in SDS-polyacrylamide gels. The proposal is to determine if the proteins that have been observed to be induced by phenobarbital and 3-methylcholanthrene are different cytochrome P450 molecules or if there is one cytochrome P450 and several inducable effector proteins. The method is based upon the fact that antibodies to microsomal proteins, purified after either SDS- or protease-solubilization, will precipitate detergent-solubilized enzymes. The immunoprecipitates obtained will be identified spectrally and the electrophoretic mobility of the antigen will be determined. The antibodies will be used to purify the detergent-solubilized proteins for peptide mapping.

Physician smoking cessation advice and assistance in quitting have been successful preventive health services, and are still needed. Approximately 40% of pregnant women are exposed to tobacco smoke during pregnancy through either active or passive smoking. Low birthweight, preterm birth, stillbirth, and sudden infant death syndrome are among the most prominent effects of maternal smoking and secondhand smoke exposure during pregnancy. These consequences are in addition to the additional risks of smoking; active smoking in women alone caused 2.1 million lost years of life during each year in the 1990s. Pregnancy is an opportune time for interventions to help women to quit smoking, because a greater proportion of women quit smoking during pregnancy than at any other point in their lives. A vital component of tobacco education and counseling in the U.S. is the physician, and physician counseling is effective in increasing quit rates and decreasing secondhand smoke exposure. For these reasons, the U.S. Public Health Service (PHS), the U.S. Preventive Services Task Force (USPSTF) and the American College of Obstetrics and Gynecology (ACOG) recommend that physicians counsel all patients about tobacco use. However, many physicians do not assess smoking or counsel pregnant patients about tobacco use or secondhand smoke exposure. We propose to characterize the physicians who do not identify smoking and who do not counsel pregnant patients about tobacco use or secondhand exposure. This will be done in a three-stage process directed at physician behavior and community needs: 1) a local physician survey, 2) a mapping approach based on Geographic Information Systems (GIS), and 3) analysis of a national physician survey dataset. Tobacco use identification and counseling of pregnant smokers and nonsmokers will be the main outcome variables from both the local and national surveys. In the local survey, Los Angeles County obstetricians will be asked about their knowledge, attitudes, and behaviors regarding tobacco use and secondhand exposure in pregnant women. We will explore the relationships between counseling behavior of obstetricians and their knowledge and attitudes about tobacco use and secondhand smoke exposure. We will use GIS to create spatial density maps of maternal smoking in Los Angeles County, and study the relationship between density of maternal smoking and obstetrician counseling behavior. In the national survey, providers were asked about personal and practice characteristics, and patient visit information was collected. Vital statistics information will be used to compute the rate of maternal smoking in the provider's county, and along with provider and patient characteristics, this rate will be studied as a predictor of smoking identification and counseling. With the variety of tools and datasets obtained and compiled for this project, we will gain a better understanding of which physicians follow PHS, USPSTF, and ACOG guidelines, and how to identify those who do not.

Low frequency S-band spectra were obtained on cobalt-dehydrogenate samples to compare signals from nickel and iron-sulfur clusters to determine whether these signals are from monomeric centers with true g-values of clusters for which the energy levels are affected by zero field splitting.

The Nuclear Receptor Program is a network of 20 NIH funded basic scientists focused on understanding the contribution of nuclear receptor transcription factor and chromatic modifying coregulator function to cancer development. Members have a total of $14,612,144 in peer-reviewed research support, $4,167,979 of which is from NCI and the remainder from other NIH institutes, the Department of Defense, and cancer foundation funds. Members of the Program have a strong record of both intraprogramatic collaboration and interprogramatic interactions with both basic and clinical programs throughout the cancer center. During the last three years, members published 202 peer reviewed manuscripts of which 39% were the result of intraprogrammatic interactions and 34% from interrogrammatic publications. A major goal to identify novel therapeutic targets among members of the nuclear receptor superfamily and nuclear receptor interacting coregulator proteins for prevention of and therapeutic intervention in cancer. To achieve this goal, we have adopted an integrative approach with three central components: 1) nuclear receptor and coregulator discovery and analysis of their mechanisms of regulation of cellular homeostasis, 2) preclinical assessment of their roles in cancer development using genetically manipulated mouse model systems, and 3) A translational component involving interaction with clinical programs to rapidly transfer new information into receptor profiling and assessment of therapeutic potential in human cancers. Major accomplishments include elucidation of a breast cancer cell selective posttranslational code that is responsible for overexpression of the pi60 coactivator I breast cancer cells, SRC-3 in breast cancer cells;identification of a critical role of coactivators SRC-1 and SRC-3 in breast and prostate cancer metastases;discovery of the orphan nuclear receptors, NR4A1 and NR4A3 as novel tumor suppressors of acute myeloid leukemia and discovery of their widespread gene silencing in human AML patients regardless of genetic heterogeneity;and discovery of the orphan COUP-TFII as a potent driver of epithelial tumor associated angiogenesis and metastasis.

Most etiologic studies on drug use, specially on injecting drug use, are based on treatment samples. This study aimed to clarify some potential differences between treated and untreated drug users that might be considered in studies that only have drug users recruited from treatment settings as participants. This study further shed light on different suspected determinants of entry into a detoxification treatment only and entry into a methadone maintenance treatment program. Our analysis were based on baseline and six-month follow-up interviews of initially 2,879 injecting drug users (IDUs), who had been recruited through extensive community outreach. We first examined characteristics associated with being in treatment at baseline and up to one year before baseline. A further investigation was focused on active injecting drug users, who had not been in treatment in that time period, and who returned for follow-up assessment within 9 months after baseline. We studied hypothesized determinants of those who entered a detoxification treatment, and those who entered a methadone maintenance program, comparing them to active IDUs who did not enter these treatments. Of 1,039 drug users, who reported injecting drugs at the time of the follow up interview, a total of 144 entered a detoxification program, a total of 64 entered a methadone maintenance treatment program. Multiple regression analysis indicated that enrollment in a detoxification program was associated with a recent episode of drug overdose, recent higher frequency of injecting drugs, and a history of arrest or treatment. Being married or living with a partner, female sex, long duration of drug use (>10 years), and a history of treatment had separate associations with enrollment into a methadone maintenance treatment program.

This is a second revised application for an exploratory/developmental research grant award (R21). Positive social relationships have consistently been associated with better health, although the neurobiological underpinnings of these observed effects are not well understood. Valuable insight may be gained by a life course perspective as it is becoming increasingly apparent that early life social experiences are crucially related to later life functioning and well-being. The overall goal of the proposed work is to explore novel biological pathways that may help to explain how social relationships influence health throughout the life course. Oxytocin is a neurohypophyseal hormone hypothesized to coordinate both the causes and effects of positive social interactions, and may be involved in positive physiological adaptations such as buffering the deleterious effects of stress. The proposed research will examine whether and how oxytocin influences responses to stress in humans and will consider these effects in relation to those of social support. More specifically, experimental research will be used to determine whether exogenously administered oxytocin (intranasal) influences psychological and physiological outcomes under conditions of stress across gender and age in adulthood. Hypotheses to be tested are: 1) Oxytocin ameliorates the deleterious neuroendocrine, cardiovascular, and subjective effects of stress;2) Oxytocin and social support have similar and additive stress-buffering effects;3) Effects of oxytocin are stronger in women versus men;4) Effects of oxytocin are similar across a range of both younger and older adult ages. Hypotheses will be tested with a placebo-controlled double blind study using a sample of healthy men and women recruited from the community. Participants will be randomly assigned to receive either oxytocin or placebo. They will undergo a social stress manipulation with and without social support (randomly assigned), and outcome measures will be obtained at multiple times during the procedure. The proposed research represents a novel area of investigation by a multidisciplinary team of investigators. This work will provide a solid platform from which to launch a larger program of research aimed at identifying how positive social and emotional experiences influence adult health and longevity. Ultimately, a more neurobiological understanding of resilience can inform efforts for prevention and intervention of diseases or problems common in later life. PUBLIC HEALTH RELEVANCE: The proposed work has the potential to provide new insight into the biological mechanisms underlying the protective effects of positive social relationships on health. This experimental study utilizes a multidisciplinary approach focusing on the interplay between molecular, psychological, and social processes as they relate to aging, and will provide a solid platform from which to launch a larger program of research. Ultimately, a neurobiological understanding of resilience may inform efforts for both prevention and intervention of diseases or problems common in later life.

The requirements of drug dose and dosing frequency are unique to pregnancy, in that the renal clearance is altered due to physiologic changes in the maternal renal system, and the addition of the utero-fetal compartment. In addition, the dose frequency required during the third trimester has never been studied. Penicillin is cleared exclusively by the renal system, and during the third trimester of pregnancy, the renal blood flow and renal filtration rates are many times that found outside pregnancy. The current dosing recommendations for GBS prophylaxis by the CDC are not based on physiologic parameters found during pregnancy. Rather, the dosing is based on data from non-pregnant males, from experiments conducted during the 1950's. We therefore propose to determine the proper dose and dosing frequency during the third trimester of pregnancy. We further hypothesize that if the proper dose and frequency of penicillin is used, this will thereby decrease the rate at which resistance to penicillin by Group B Streptococcus will develop over time. With the uncontrolled use of penicillin, an increased frequency of drug resistance for GBS may result. This in turn could lead to higher infant morbidity and mortality from multi-drug resistant GBS.

This research is concerned with the elucidation of the mechanism of energy conversion in photsynthetic systems; algal, bacterial and green plant. In green plant and algal photosynthesis, the reduction of NADP is coupled to the esterification of ADP and Pi. The resultant products, NADPH and ATP, are necessary for the fixation of carbon dioxide into cellular material. We will continue our studies on the identification and nature of the cofactors involved in the pathway of electron flow leading to NADPH formation. In support of the biochemical approach, we have recently initiated a collaborative program (with Dr. R.K. Togasaki of this department) of mutational analysis using the green alga, Chlamydomonas reinhardi. We have available now numerous mutants which we are attempting to categorize with respect to the imposed lesion; photosystem I mutants, photosystem II mutants, carbon metabolism mutants, photophosphorylation mutants, and mutants deficient in the pathway interconnecting the two photosystems.

Extracellular secretion and targeted delivery by the type II secretion (T2S) system is considered a major virulence mechanism in gram negative pathogens, as many of the proteins secreted via the T2S pathway constitute important virulence factors, including toxins and degradative enzymes. The T2S apparatus is comprised of at least 13 different proteins, EpsC-EpsN and PilD, that assemble into a complex that spans the entire cell envelope of Vibrio cholerae. The dynamic and perhaps transient nature of this complex may be a prerequisite for function as its assembly and disassembly may drive extracellular secretion. The energy required for this process is thought to be generated from ATP hydrolysis by EpsE, a cytoplasmic protein that is associated with the cytoplasmic membrane via interaction with the membrane proteins EpsL. EpsM and EpsF. EpsE's interactions with these components modulate its ATPase activity and promote its localization to distinct sites within the V. cholerae cell envelope. The experiments described in this proposal are designed to test the hypothesis that specific protein-protein interactions and acidic phospholipids drive T2S in an ATP-dependent process at discrete sites In the cell envelope of V. cho/erae. Specffically, this proposal will i) determine the mechanism by which the enzymatic activity of EpsE is controlled by components of the cytoplasmic membrane including phospholipids. EpsL and EpsF;ii) investigate the ordered assembly of Eps components and determine the mechanism by which EpsD and EpsC drive focal assembly of the T2S complex;iii) map the cleft that forms when EpsM assembles and identify the cellular factor that binds to the cleft. Resolving the mechanisms of regulated assembly and spatial localization of the T2S system will further our understanding of T2S and may identify ways to manipulate the secretion process for preventative, therapeutic and/or biotechnological use.

The goal of this program is to develop novel inhibitors of human palmitoyl acyltransferases (PATs) that are effective as cancer therapeutic agents. PATs represent new targets for anticancer drug development because of their pivotal roles in regulating the subcellular localization of specific oncoproteins. Most importantly, certain forms of ras-encoded proteins require palmitoylation for their targeting to the plasma membrane and for their ability to transform cells. Since post-translational processing of Ras proteins is critical for their function, the enzymes that catalyze these processing steps have been considered as potential targets for anticancer drugs. Inhibition of farnesylation has been the main focus of this therapeutic effort to date. However, the PATs may be even better targets, since palmitoylation is a dynamic process whereas farnesylation is not. A major reason that inhibition of palmitoylation has not yet been therapeutically exploited is that mammalian PATs have yet to be molecularly and biochemically characterized. Recent studies of palmitoylation in the yeast Saccharomyces cerevisiae have identified two putative PATs. Using the sequences of the yeast enzymes, we performed a search for homologues in vertebrates. Based on sequence and structural homologies, we selected a human protein called HIP3 for further study, i.e. to determine if this protein is an authentic human PAT (hPAT). Using RNA interference, we have shown that HIP3 has PAT activity, and that it affects the subcellular localization of palmitoylated proteins. In this SBIR program, these findings and our previously published work on hPATs will be used to develop small molecule inhibitors of hPATs to be evaluated as anticancer therapeutics. The following specific aims will be addressed in the Phase I project: 1. To identify compounds that inhibit hPAT activity by screening a diverse collection of small molecules in an established ass ay and a high-throughput yeast-based assay. 2. To use computational and medicinal chemistry to optimize these compounds. 3. To determine the in vitro activity, in vivo toxicity, pharmacokinetics and antitumor activity of lead PAT inhibitors.

Dopamine (DA) release in the cortex and basal ganglia is strongly implicated in modulation of CMS function and behavior and is thought to occur through a variety of potential secretory sites. Among these are axonal projections where small clear synaptic vesicles appear clustered in varicosities that resemble presynaptic terminals for typical fast-acting neurotransmitter secretion. Given that DA acts on much longer time scales than fast-acting neurotransmitters, the mechanism involved in controlling the presynaptic machinery may well be different than for those more typical "fast" synapses. Here we propose to examine details of the presynaptic vesicle cycle for these dopaminergic release sites. The long term objective of this proposal is to characterize the mechanism that control the presynaptic vesicle cycle for small clear dopaminergic veshicles. We will make use of technologies previously developed in the lab to examine many aspects of the molecular and biophysical nature of the presynaptic vesicle cycle in cortical and hippocampal cultures. These approaches rely heavily on optical techniques using exogenous organic probes such FM dye family members as well as genetically-encoded tags of presynaptic proteins that allow dynamic and quantitative information about the vesicle cycle to be obtained. These will be adapted to primary dissociated cell cultures of mid-brain neurons from the ventral tegmental area (VTA). We propose 3 specific aims to accomplish this initial characterization of the cell biological, physiological and biophysical aspects of the dopaminergic vesicle cycle. These include characterizing the properties of the vesicle pool in turns of depletion rates, replenishment rates, the sensitivity of pool turnover to stimulation at varied calcium concentrations, as well as the kinetics of endocytosis. Finally we will take advantage of the ability to detect dopamine sectretion directly using carbon-fiber amperometry to examine how details of the vesicle cycle impact neurotransmitter release.

This program will demonstrate the fabrication of high energy density ceramic capacitors, suitable for use in implantable cardiac defibrillators. Proprietary dielectric compositions and innovative processing techniques will be used. In Phase I, small scale capacitors will be produced with full scale capacitors being produced in Phase II. The research effort will investigate a composition found to be favorable in previous work. Processing conditions, capacitor design, and proper termination procedures will be investigated. The successful implementation of this technology will result in significantly smaller capacitors than are currently being used. Since the capacitors occupy a significant amount of the space in an implantable defibrillator, much smaller devices, closer in size to pacemakers, will be able to be constructed. Pectoral area implantation of a small device will lead to more patient acceptance of this life-saving therapy. Other advantages of solid state ceramic capacitors over the electrolytic capacitors, which are the current state-of-the-art, include improved reliability, since there is no corrosive electrolyte with the potential for leakage. Ceramic capacitors can be readily shaped to conform to the case shape of the defibrillator; they have extremely low current leakage and do not require periodic reforming of the dielectric oxide.

Management: This Core coordinates all IDDRC activities and sets policies for Center governance. The Directorate and Steering Committee jointly manage day-to-day operations and work with Core directors to assure efficient administration of each unit. This team meets quarterly to establish policies with respect to the admission of new projects, the provision of core services, staffing issues and general operating procedures. Internal and External Advisory Committees provide an important consultative function. The Administrative Assistant liaises with directors and users. Her/his responsibilities include: (a) Distributing timely announcements of meetings and seminars; (b) Recording minutes of Steering Committee meetings; (c) Soliciting and processing of applications for Center membership; (d) Managing the New Program Development Award: announcing the award, collating applications, responding to applicants and soliciting reviews; (e)' Organizing the monthly seminar series; (f) Maintenance of a database of users and projects; (g) Responding to all questions and problems that may arise.

A central problem in genetics is how substantial shifts in the overall gene expression programs of individual cells are achieved during development and in adult cells when they adapt homeostatically to a disturbance. As a framework against which to investigate this problem, we propose a "two-state" hypothesis, which we will test in relation to the complex homeostatic shifts in gene expression program that accompany the development and regression of left ventricular cardiac hypertrophy, an important cardiovascular risk factor independent of hypertension. The hypothesis states that adult cells (such as cardiac myocytes) when stressed sufficiently can switch without cell division to an alternative state in which the expression of many genes differ co-ordinately. Specific aim i) will test whether individual cardiac myocytes switch expression of representative genes coordinately between two states, and whether the degree of hypertrophy is mediated by the proportion of cells in the two states. Specific aim ii) will increase the stringency of the tests by using a much broader panel of discriminators with several categorically different ways of inducing hypertrophy, and will also test whether the hypertrophic cells replicate the expression pattern of normal cells at an earlier stage of development. Specific aim iii) will test the hypothesis that the expression programs of cardiac myocytes in the hypertrophic state can under appropriate circumstances switch back to the normal state in animals in vivo, or in tissue culture ex vivo. Our proposal is, to our knowledge, the first attempt to formalize how complex shifts in gene expression programs are achieved in cells responding to homeostatic challenges, but the value of the proposed experiments does not depend on the correctness of the two-state hypothesis since each specific aim also addresses a gap in current knowledge related to cardiac hypertrophy.

The present investigation will focus on the effects of alcohol on the liver and employs both in vitro and in vivo model systems. One of the major biologic effects of alcohol is the capacity to inhibit liver repair following cellular injury. Therefore, we will explore the action of ethanol on hormone stimulated hepatocyte DNA synthesis and perform detailed studies on the mechanisms of the ethanol effect using primary hepatocyte cultures. These investigations will examine the effects of ethanol on the binding of epidermal growth factor (EGF) and insulin to liver cell membranes and to observe whether ethanol modifies the action of these hormones by studying receptor binding, polyamine biosynthesis and metabolism, and phosphorylation of tyrosine and serine residues. We also plan to examine the usefulness of nuclear magnetic resonance (NMR) to study liver injury steatosis following in vivo ethanol administration. Such investigations employ NMR spectroscopy and imaging, NMR chemical shift imaging and potential use of paramagnetic probes designed to enhance spin-lattice relaxation times (T1 and T2). For example, we are developing paramagentic agents such as Gd+++ covalently linked to asialoglycoproteins in an attempt to enhance or change NMR contrast within the liver; these studies will hopefully provide in vivo diagnostic information on liver injury and steatosis. Finally, we believe that the capacity of ethanol to inhibit hepatocyte DNA synthesis may have biologic relevance to why patients with severe alcohol induced liver injury fail to recover despite cessation of alcohol intake. The use of emerging NMR technology will further permit us to explore the hepatic changes produced by ethanol and may allow for the early identification of alcohol induced liver injury and steatosis in man.

Project Summary Most kinase inhibitors target the ATP binding site, and frequently inhibit numerous kinases in the human kinome. Due to the nature of pan-kinase inhibitors, severe side effects are frequently observed in pre-clinical and clinical studies. Therefore, more specific therapeutics are desirable to achieve safe and effective treatment of human diseases. Allosteric modulators (agonists and antagonists) have greater potential than ATP- competitive inhibitors to achieve selectivity due to the much less conserved allosteric binding sites. High and consistent potency can also be achieved by the allosteric modulators. Furthermore, allosteric modulators can show different efficacies and pharmacological effects in cell and in vivo, compared to ATP competitive inhibitors. These agents can be applied to investigate specific biological and pathological functions of kinases in human diseases. However, identifying allosteric lead compounds and their binding pockets is a great challenge that retards the development of allosteric modulators of kinases. So, in this Award a novel concept and several approaches are proposed to develop allosteric modulators of Phosphatidylinositol 3-Kinase (PI3K?), which is involved in human cancer, metabolism, innate and adaptive immunity, and Autism spectrum disorder. The peptide fragment trapped at the interface between the kinase domain and the Ras binding domain of PI3K? was employed as an allosteric starting compound to generate allosteric modulators of PI3K?. Our early stage pilot studies supported our hypothesis and produced proper starting agents for the development of allosteric agonists and antagonists of PI3K?. Therefore, we will develop highly potent allosteric modulators of PI3K? by applying structure-guided drug design techniques such as linker search, scaffold hopping, and virtual synthesis and screening (Aim 1). Solid phase peptide synthesis and solution phase organic synthesis will be applied for generating a peptidomimetic library and a series of small molecule analogs, respectively. X-ray co-crystal structures of PI3K? in complex with allosteric modulators will be determined and used for the structure-based design. Once low nanomolar inhibition and activation are achieved in biochemical assays, cellular potency of allosteric modulators will be assessed in a series of cell- based proliferation and functional assays (Aim 2). Furthermore, cellular signaling pathways mediated by PI3K? will be investigated with highly potent allosteric modulators in combination with ATP-competitive inhibitors. In summary, the proposed research will have high impact on biomedical research and drug discovery in terms of new types of chemical entities in kinases and a new concept for the discovery of allosteric modulators. The allosteric modulators developed in this project will facilitate the investigation of new kinase functions mediated by PI3K? to determine its specific roles and requirement in human diseases. Finally, the concepts and techniques established in this project will provide solid strategies for discovery of allosteric modulators of kinases and a large pool of new chemical entities in kinase drug discovery.

Abstract The focus of this BRAIN Initiative funding opportunity is to use ?innovative approaches to understand how circuit activity gives rise to a specific behavior?. Cognitive behaviors arise from collective interactions of multiple brain systems. Yet, for most cognitive processes, we do not yet know which brain areas are involved and how multi-regional interactions mediate specific cognitive processes. This gap in knowledge arises because separate parts of the brain are studied individually, yet, the brain circuits driving behavior vary from one behavior to another. The goal of this proposal is to establish a working example of how brain-wide activity dynamics collectively generate one cognitive behavior. We address this question by studying how a mouse flexibly generate a volitional movement based on short-term memory. Neurons in multiple parts of the brain, including the frontal cortex, thalamus, midbrain, and cerebellum respond robustly during this short-term memory and causally contribute to the behavior. Taking advantage of this opportunity to establish how activity distributed across multiple brain systems orchestrates one coherent behavior, in this proposal, we will use newly developed experimental frameworks to analyze the underlying neural circuitry at brain-wide scale and establish causal relationships between specific activity patterns and behavior. First, we will use brain-wide loss-of-function screen, high-density silicon probe recording, and anatomical techniques to produce multi- modal maps of core neural substrates of the short-term memory. The outcome datasets will be put into standardized brain coordinates, making it possible to link the functional data to existing connectional and gene expression atlases. Next, we will use simultaneous recordings and spatiotemporally-precise perturbations to probe multi-regional interactions underlying the observed activity patterns and relationships to behavior. Finally, we will build multi-regional models that offer interpretable description of the behaviorally-relevant dynamics and relate them to underlying circuit connectivity. The outcome will disambiguate competing models of how information distributed over multiple brain regions is coordinated during cognitive processes, how information is dynamically routed and gated. The experimental, analysis, and modeling approaches will be broadly useful for analyzing distributed circuits driving behavior, as is the focus of multiple collaborative U19 grants. All the data and code will be published in the well document Neurodata Without Border (NWB) format.

In this application, the David Geffen School of Medicine at the University of California Los Angeles (UCLA) is partnering with the University of California San Francisco School of Medicine (UCSF), and the University of Washington (UW) to seek continued support for a formal 8-12 week program of training in aging research for 20 medical students to: 1) Expose them, early in their training, to the excitement of a research career. 2) Support their first steps in developing a research career in areas important to the NIA. 3) Increase the pool of physician scientists engaged in areas of research necessary to continue the mission of the NIA. With UCLA as the core site, our three institutions are particularly well suited to reach these objectives. All three of our programs have: 1) outstanding faculty who conduct NIA-funded research in aging; 2) experience training others to conduct aging research, and 3) additional funding to specifically support geriatrics related projects such as the John A. Hartford Foundation, which supports dementia and palliative care programs; the Eisner Foundation, which supports intergenerational programs; and other innovative collaborations between academic and community nonprofit organizations. To meet the objectives outlined above, we will rely on methods we have been using successfully over the past 25 years to identify research mentors, match medical students with these mentors, monitor students? research progress, facilitate this progress when needed, teach essential general research skills (including the responsible conduct of research), and provide a forum for presentation of students? research and discussion of ideas. In addition, we will offer small group discussion of common topics in clinical geriatrics, career options in aging, and the opportunity to observe and to participate in the clinical care of geriatric patients in a variety of settings. By accomplishing the aims outlined in this application, this program will encourage and support medical students' interest in aging research and geriatrics, and develop a new cadre of physician scientists who will be able to address key issues related to the research priorities of the NIA. This program will also aim to enhance the research programs in aging at the participating institutions and to foster collaboration in medical student research training in aging among UCLA, UCSF and UW.

Significance The visual system of primates contains a large number of separate representations of the visual world, at least 20 at latest count. One of the central problems of visual science is to establish how the numerous areas together form our seemingly unified visual experience. Objectives The multiple areas clearly are connected in a complex network that contains both serial and parallel organization. I have projects exploring both aspects of the organization, employing single-neuron physiological methods to measure information processing in the cortex of rhesus macaques (Macaca mulatta). We employ a combination of physiological and behavioral approaches to explore the relationship of signals in different cortical areas to different aspects of visual perception. Results Cortex, MT and MST. These form a perfect example of serial processing, and we already know a great deal about how they are organized and what kind of information they represent. Both appear to be specialized for the analysis of visual motion, but MST (the later stage of processing) appears to represent much more complex aspects of image motion, such as rotation, expansion, and contraction. We have established that area MST is involved in one complex motion task, recovering the direction of self motion from optic flow (analogous to the "Star Wars" opening graphic), using the method of introcortical microstimulation to minutely perturb the signals carried by neurons there. We have also explored parallel organization by measuring color responses in area MT, long thought to be specialized for motion analysis and not color. We have discovered robust color signals in MT and are carefully characterizing their nature. Future Directions We will continue to explore the mechanisms of perception of self-motion, and will also initiate a similar study on a related complex motion task, in which individual motion cues can be better isolated and controlled. KEY WORDS visual perception, motion processing, neurophysiology, modeling FUNDING NIH Grant EY10562 PUBLICATIONS Britten, K.H. Clustering of response selectivity in the medial superior temporal area of extrastriate cortex in the macaque monkey. Visual Neuroscience 15(3):553-558, 1998. Britten, K.H. and van Wezel, R.J.A. Electrical microstimulation of cortical area MST biases heading perception in monkeys. Nature Neuroscience 1(1):59-63, 1998. Britten, K.H. and Newsome, W.T. Tuning bandwidths for near-threshold stimuli in area MT. Journal of Neurophysiology 80(2):762-770, 1998.

This proposal is part of a long-term effort to study the auditory system at the molecular level. The studies outlined in this application are designed to characterize both normal and pathological auditory nerve proteins with emphasis on rapidly turning-over glycoproteins (RTGP's) and hair cell-associated proteins (HAP's) RTGP's are a group of 5 or 6 rapidly transported proteins with similar molecular weights and isoelectric points that are degraded with half-lives of less than 2.5 h in auditory nerve terminals. HAP's are two proteins (or groups of proteins) rapidly transported in the auditory nerve whose expression appears to be dependent on the state of the hair cell. After damage to the hair cell in the waltzing guinea pig or animals treated with ototoxic drugs, the labeling of the HAP's in the auditory nerve increases more than sixfold. The present studies will include in vivo labeling and characterization to gain insight into the function of RTGP's and HAP's. The effect of hair cell damage by the ototoxic drug, neomycin, on proteins transported in the auditory nerve will be determined. Studies are designed to determine the biochemical properties of the RTGP's and HAP's and priovide data essential for their purification. The similar properties of the several forms of RTGP suggest a functional or biochemical relationship and it will be determined if the several forms are due to modifications of a single protein. To allow further characterization, the RTGP's and HAP's will be purified. Two methods of purification are proposed, one by standard techniques and the other with monoclonal antibodies. The purified proteins will be characterized with respect to gross and subcellular localization. Proteins specific for the auditory nerve will be identified and characterized with monoclonal antibodies. It will be determined if these proteins can be used as markers for Type I and Type II spiral ganglion cells.

(Revised Abstract) DESCRIPTION (provided by applicant): Opioid receptors are over-expressed by a variety of human and animal tumors. Markers for in vivo characterization of tumors expressing opioid receptors may have value as diagnostic aids, and would present opportunities for novel studies of the roles played by opioids in neoplasia. Little attention has focused on this arena despite the fact that opioid receptor antagonists are in clinical trials as antitumor agents, and widely prescribed opioid receptor agonists, such as morphine, may be tumor promoters. Our goal is to develop and validate imaging radioligands and mouse tumor models for in vivo studies of opioid receptors on breast and lung cancers in order to provide a foundation for future imaging studies of opioid receptor involvement in human cancers. Our emphasis will be upon radioligands labeled with 1-123, Tc-99m and In-111 for SPECT / microSPECT scanning. Further, we plan to determine the frequency and extent of expression of opioid receptors in human breast cancers by in vitro screening of biopsy samples and associated normal breast tissues, and to ascertain correlations of opioid receptor expression with a limited panel of tumor markers, including steroid hormone receptors. Specifically, we propose: (1) to design, synthesize and chemically characterize novel opioid receptor ligands labeled with SPECT imaging radionuclides; (2) to determine radioligand affinity, selectivity and specificity for opioid receptor types in vitro; (3) to characterize in vivo radioligand binding to opioid receptors in normal and tumor bearing rodents; (4) to evaluate radioligand pharmacokinetics and pharmacology in mice by microSPECT, and to use microSPECT and microCT imaging to investigate the effects of opioid receptor agonists and antagonists on MCF-7 mammary tumor xenograft growth in nude mice; and (5) to establish the frequency and extent of opioid receptor expression in human breast cancer biopsy specimens.

Whole cell vaccines are often immunogenic without added adjuvants, but production costs and safety issues have prompted the development of protein-based alternatives. However, protein-based vaccine design is challenged by limited immunogenicity and the necessity of adjuvants, few of which are currently available for human use. We have developed a peptide-based platform that self-assembles, and when these peptides are fused to short peptide antigens, they elicit substantial antibody responses in mice to the short peptide antigens without the need for additional adjuvant (1). Antibody responses have been broad, including all IgG subclasses and IgM, and they have been strongly persistent for over 36 weeks in mice. These findings suggest these self-assembling materials as attractive novel candidates for vaccine development. We propose to expand on the capability of these materials to display full-length proteins, and to test them as a protective modality for clinically important community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA). Antibodies (Abs) against the S. aureus alpha-hemolysin (Hla), a pore-forming cytotoxin, have been shown to protect against lethal pneumonia and skin infection in mice (2-6). In the first specific aim, we will assess the ability of the Hla conjugated to the self-assembled peptide platform to elicit anti-Hla Ab production. To this end, we will utilize a novel protein conjugation technique based on a mutant version of the fungal cutinase enzyme to covalently bond cutinase-Hla fusion proteins to "suicide" phosphonate ligands on the self- assembling peptide platform (7). Immunoelectron microscopy and analytical ultracentrifugation will be used to validate fibrillar assembly and epitope availability, and we will assess the ability of the Hla assemblies to stimulate anti-Hla Ab production in mice. These studies are designed to provide proof-of-principle that these self-assembling peptides can display full-length proteins in a highly immunogenic manner. In the second specific aim, we will assess protection afforded by Hla self-assemblies against CA- MRSA infection. We will utilize two CA-MRSA infection models in mice, a pulmonary and a skin infection model. Both models have been developed and utilized extensively by our collaborative team. Measures of protection include survival, S. aureus CFU recovered from the lung and skin, histopathology, and size of dermonecrotic skin lesions. We will further determine the extent to which Hla-specific antibodies elicited by the Hla-peptide vaccination are the basis for protection, by transferring sera from immunized mice into naive mice infected with CA-MRSA. These studies are designed to provide proof-of-principle that these self-assembling peptides presenting Hla can elicit antibody responses that are protective against CA-MRSA. Success in these studies will provide us with insights into the utility of these peptide assemblies as highly engineered vaccines for the prophylactic control of diseases caused by pathogens, and as a modular platform for designing sophisticated immune-modulating reagents. PUBLIC HEALTH RELEVANCE: Vaccination is the most effective means for the prevention of disease from multiple pathogens, but numerous challenges are faced by both whole cell vaccines and protein-based alternatives, which require adjuvants to enhance their efficacy. We have developed a self-assembling peptide system that, when coupled to peptide antigens, raises strong antibody responses to the peptide antigens in mice, without the need for additional adjuvant. In this proposal we seek to expand the capability of these materials to display clinically important full-length proteins and to conduct mechanistic studies to understand the basis of their immunogenicity, with the anticipation that success in these goals will provide us with critical insights into utility of these peptide systems for vaccination against pathogens.

5-HT mechanisms are implicated in common age-associated diseases, including treatment-resistant mood disorders. Our previous work indicates that the neurotrophin BDNF promotes 5-HT axonal growth in the adult brain. However, 5-HT neurons may lose the ability to respond to BDNF in aging. The present proposal examines whether age-associated dysfunction of 5-HT neurons in the rat forebrain may be caused by deficient neurotrophic (e.g., BDNF) support mechanisms. Specific Aim 1 of this proposal examines whether 5-HT neurons lose their structural plasticity with age, as manifested by an impaired regenerative response to injury and a reduced responsiveness to BNDF. Specific Aim 2 examines whether their is an endogenous loss of BDNF availability/signal transduction in 5-HT neurons during aging. Finally, Specific Aim 3 uses mutant BDNF-deficient mice to examine whether endogenous BDNF expression and signal transduction are necessary for the normal integrity and plasticity of 5-HT neurons adulthood 44 and aging. The goal of this research is to gain a better understanding of the molecular interactions between 5-HT neurons and their trophic support mechanisms in aging and whether a deficiency in this interaction contributes to an age-associated pathophysiology of the 5-HT system. It is hoped that this knowledge will facilitate the design of more effective therapeutic approaches to treat age-related psychiatric diseases that may be associated with dysfunction of 5-HT neurons.

To determine whether poisoning with the organophosphate insecticide methamidophos results in a syndrome of persistent subclinical sensory and motor neuropathy, a clinical and epidemiologic study will be undertaken among a population of 50 seriously poisoned agricultural workers. Two comparison groups will also be examined -- one in which a history of methamidophos exposure is common, but with no history of poisoning, and the other with no history of exposure. Specific aims are: 1.To determine whether inhibition of lymphocyte neuropathy target esterase (NTE) measured in peripheral lymphocytes is a sensitive and specific index of peripheral neurotoxicity. 2.To determine whether previous poisoning with methamidophos results in diminished motor and sensory function, as reflected in electrophysiologic studies, pinch strength, and elevated vibrotactile threshold. 3.To determine whether a dose-response relationship exists between lymphocyte NTE inhibition and motor or sensory function. 4.To determine whether there exist threshold levels of methamidophos exposure below which either sensory or motor neuropathy is no longer evident. The public health significance of this study is that methamidophos and other organophosphate neurotoxins are widely used by workers in the United States and throughout the world.

Facility Core Summary The Facility Core for Small Molecule Mass Spectrometry (SMMS) is integral to the Center's research mission by enabling high-resolution detection and isolation of halogenated organic compounds (HQCs). This Core does not replicate any analytical services or facilities currently available at the Scripps Institution of Oceanography (SIO). The SMMS Facility Core will be directed by P3 Leader Aluwihare, and technical oversight for the facility will be provided Co-Director Dr. Yongxuan Su. Daily operation and routine maintenance will be the responsibility of Mr. Matthew Woolery. The facility will include several uniquely configured gas chromatography-mass spectrometry (GC-MS) instruments that are intimately integrated with the Center's research mission to identify the suite of HOCs accumulating in the marine environment, discover new HOCs of concern in wildlife and humans, and evaluate environmental sources of HOCs. The overall goal of PI is to identify organisms and habitats of interest with respect to HOC production in the Southern California Bight. A GC Electron Capture Detector (|JECD)/MS capable of screening these samples for relevant HOC production will be available through the Core. Project 2 aims to identify and characterize the prevalence of polybrominated HOC biosynthetic pathways in the marine environment. As such, P2 will rely on the same instrumentation to screen cultures and insure that organisms under investigation are producing compounds of interest. The overall goalof P3 is to identify the suite of small, natural HOCs available to enter human populations through seafood consumption. This research will be supported by the analytical capabilities of two GCXGC instruments integrated into the proposed Facility. The >200 HOCs that bioaccumulate in apex predators and humans cannot be adequately separated from co-eluting compounds for definitive identification and quantification without comprehensive GCXGC separation. To establish the biogenic origin of HOCs of interest, P3 will also conduct stable isotope studies on pure compounds that are isolated from apex predators, purified and concentrated for isotope measurement. A uniquely configured, preparative GCXGC instrument that is included in the Facility will support this research. Two liquid chromatography-mass spectrometry (LC-MS) instruments will also be housed in the Core to analyze fermentation extracts and synthetic organic preparations. The Internal Advisory Committee will discuss Facility business during monthly meetings, and the External Advisory Committee will evaluate the Facility Core for its performance annually.

[unreadable] [unreadable] PROJECT SUMMARY [unreadable] Over 30 million men suffer from erectile dysfunction in the U.S. Constriction and dilation of the cavernosal vasculature determines penile erection. In the absence of arousal stimuli, activation of heterotrimeric G proteins following ligand binding to membrane receptors maintains constriction of the cavernosal arterioles and sinuses, keeping the penis non-erect. Subsequent to heterotrimeric G protein activation, two signaling pathways are brought into play to cause constriction: the well-characterized Ca2+-dependent pathway (phospholipase C) and the recently identified RhoA/Rho-kinase pathway known as Ca2+ sensitization. The coupling of G protein receptors to phospholipase C activation is well characterized, but much less is known about how RhoA/Rho-kinase is coupled to G protein activation. Recent evidence indicates that the RhoA activation is positively regulated by Rho guanine nucleotide exchange factors (RhoGEFs). The regulation of RhoGEF activity is also unclear. Interestingly, one particular RhoGEF, PDZ-RhoGEF is regulated by a proline- rich tyrosine kinase 2 (PYK2), a Ca2+-dependent enzyme. We have obtained preliminary data to support a role for PYK2 in vascular responses to angiotensin II. We hypothesize that angiotensin II activation of PYK2 and PDZ-RhoGEF leads to increased RhoA/Rho-kinase to maintain the non-erect state. Further, -term over- expression of PYK2 and PDZ-RhoGEF leads to erectile dysfunction. These hypotheses will be tested by 3 specific aims: 1A) to demonstrate that activation of PYK2/PDZ-RhoGEF leads to vasoconstriction and penile flaccidity; 1B) to determine if PYK2 expression in the penis can be modulated to alter erectile function and if siRNA for PDZ-RhoGEF will inhibit components of the RhoA/Rho-kinase signaling pathway; 2) to determine if increased PYK2/PDZ-RhoGEF activity contributes to erectile dysfunction in angiotensin II-induced hypertension; and 3) to determine if increased PYK2/PDZ-RhoGEF activity contributes to erectile dysfunction in diabetes. The approach will utilize rat and mouse models of erection. The experiments will determine the biochemical, pharmacological and physiological measures of PYK2/RhoGEF activity with respect to stimulation of the RhoA/Rho-kinase pathway in the intact penis and in isolated cavernosal strips. Gene transfer to over- express PYK2 should lead to a new model of erectile dysfunction whereas; siRNA for PDZ-RhoGEF will reduce signaling via the RhoA/Rho-kinase pathway. Contractile force measurements in isolated cavernosal strips (intact and permeabilized) will provide evidence for Ca2+ sensitization and its regulation by PYK2/PDZ- RhoGEF. Erectile dysfunction induced by diabetes and hypertension will be used to evaluate a possible clinical significance of this signaling pathway. PYK2 (-/-) mice will provide an important approach to studying the PYK2/PDZ-RhoGEF signaling pathway. If supported these studies would provide an explanation for the molecular basis for smooth muscle contraction in the normal state and how long-term changes in the penis contribute to erectile dysfunction. The work will provide insight into novel therapeutic approaches. [unreadable] [unreadable] PROJECT NARRATIVE [unreadable] Over 30 million men suffer from erectile dysfunction in the U.S. Whereas, penile erection is determined by cell signaling mechanisms promoting both constriction and dilation of the cavernosal vasculature, current therapeutic approaches focus on stimulating the dilatory response. Our research has identified novel cellular events regulating constrictor responses, and an understanding of how these events can be inhibited will provide an alternative therapeutic approach to treating erectile dysfunction. [unreadable] [unreadable] [unreadable]

This application proposes a program of training in development and psychopathology that integrates diverse approaches and methodologies used in developmental and child clinical psychology. Research training will take place in the University of Oregon Psychology and Counseling Psychology programs, Oregon Social Learning Center (OSLC) and Oregon Research Institute (ORI). The program is organized around an ecological framework for the study of developmental psychopathology that integrates neuroscience, cognitive psychology, personality, social development, and intervention science. For trainees, the key features of the research training program are: (1) An initial reading and conference with two or more research faculty that provides focus and direction to the student's program of research, (2) a 2-year association with one research laboratory to ensure depth of training, (3) a year-long sequence in developmental and psychopathology, (4) involvement in workshops on specialized topics that bring together research faculty and international scholars, and (5) weekly research meetings. Support is sought initially for three predoctoral students and two postdoctoral trainees for 2-year appointments. Additional slots will be added in Years 2, 3, and 4. Predoctoral students will be selected from psychology and counseling psychology students. Postdoctoral students will be selected from psychology and counseling psychology students. Postdoctoral trainees will be selected after national advertisement. The University of Oregon, OSLC, and ORI include state-of-the-art research projects and equipment for the study of development and psychopathology. Intervention training is accomplished in a yearly child and family practicum at the University of Oregon, involving clinical and counseling psychology students, and at the two research institutes.

: The goal of this proposal is to determine the normal function of synelfin, a neuronal protein abundant in the adult vertebrate telencephalon. Synelfin has been independently isolated from several species, including humans, where it has been referred to as NACP (the Non-Amyloid beta-Component Precursor) to indicate its relationship to a novel peptide recently purified from amyloid plaques in Alzheimer Disease (AD). Studies in songbirds suggest that this highly-conserved protein has a specific but yet-undefined role in the normal regulation of synaptic plasticity. To gain insight into the normal function of synelfin, three research aims are proposed: 1) conduct biochemical studies to test the hypothesis that synelfin has a functional relationship to apolipoproteins, as suggested by observation of conserved structural features. Normal and mutated forms of synelfin protein will be expressed in E.coli and analyzed for their interactions with phospholipids, using the well-characterized behavior of apoliopoproteins as a point of comparison. 2) determine the structural elements in the protein responsible for its accumulation at presynaptic terminals. Mutated DNA constructs will be introduced into primary cultured hippocampal neurons, and the encoded proteins will be localized by immunofluorescence. These studies may give insight into the apparent modular structure of the protein, and help in the design of experiments to identify other proteins with which synelfin interacts. 3) overexpress the synelfin protein in transgenic mice and assay the effect on neuroanatomy and neurological function. This will test the hypothesis whether a constitutive increase in synelfin will promote the development of Alzheimer-like neuropathology, or alter developmental or behavioral processes that depend upon neural plasticity.

Despite acceptance that alveolar basement membranes are critical to normal alveolar structure and function, there is limited information about the cellular sources and regulation of alveolar basement membrane components or about alveolar cell responses to alveolar basement membrane damage. Laminins are major components of basement membranes. Each laminin is a heterotrimer composed of an alpha, beta, and gamma chain. We will focus on recently-identified laminin alpha chains, alpha3A, alpha3B, alpha4, and alpha5, which are more prominent in adult mouse lung parenchyma than alpha1, the alpha chain in the much studied laminin-1. We will define the cellular expression of laminin alpha chains in lung through mouse development. We will determine the distribution of laminin alpha chains in alveolar walls using immunoelectron microscopy. Using alpha5 "knockout" mice, which we have made, we will determine the effect of laminin alpha5 on lung development. We will compare laminin alpha5-deficiency with laminin beta2-deficiency using laminin beta2 "knockout" mice, which we also have. In preliminary observations, we have found that TGFbeta1 and KGF up-regulate laminin alpha5 expression by alveolar epithelial cells. We will extend these observations to other laminin alpha chains and other lung cell types. We will use co-cultures to determine the effects of epithelial-mesenchymal interactions on laminin alpha chain expression. Because we hypothesize that the regulation of laminin alpha5 expression by TGFbeta1 and KGF is transcriptional, we will characterize the 5' end of the mouse laminin alpha 5 gene, including the promoter elements. We will determine the expression of laminin alpha chains in experimental alveolar damage and whether KGF affects expression of basement membrane components in models of alveolar damage. We will assess expression of basement membrane components in human alveolar damage using reagents we are developing specifically for human tissue. Preliminary studies show up- regulation of laminin alpha5 in human pulmonary fibrosis. We believe that better understanding of alveolar basement membrane biology will lead to improved capacity to monitor and influence lung damage and repair in conditions such as ARDS and idiopathic pulmonary fibrosis.

Swarthmore has a sponsored research program that can be considered robust for a small liberal arts college. This project is designed to bring the College's practices up to the level of our R1 partners. This will enable Swarthmore to collaborate more effectively with R1-level institutions, either as a subcontractor or prime grantee. This capacity-building will, in turn, expand our nation's pool of undergraduate researchers - an important pipeline, as Swarthmore is fourth in the country in per capita production of students who go on to achieve a PhD in the natural sciences, engineering, or the social sciences. Furthermore, the sponsored research enhancement proposed here is designed to be replicable at peer institutions; Swarthmore will share information about best practices for (and pitfalls to avoid) in growing from a small- to medium-sized sponsored research program. The project is timely: In December 2011, Swarthmore completed its latest strategic plan, which foregrounds faculty and student research, high-impact learning experiences, community-based learning and research, and creating a more diverse, inclusive campus culture. In the coming years, we expect to hire approximately 25-30 new faculty, as we move from a 5- to 4-course load, with the concurrent expectation that faculty spend increased time on involving students in their own research and mentorship of students' research. Also, over the past decade Swarthmore has pioneered some programs to promote underrepresented minority (URM) participation in STEM research; now, with student enrollment increases planned for the coming decade, the College seeks to redouble those efforts. We also see additional as-yet-untapped potential for sponsored research growth in certain fields (e.g., our emerging neuroscience curriculum; behavioral health research led by faculty in the Economics Department) and will develop explicit sponsored research connections in those areas. Qualified student demand for research opportunities is outstripping the College's ability to provide them: not only laboratory research, but social science research that includes international and local community projects, and Swarthmore faculty (and students) are engaging in increasingly complex and challenging behavioral and social science research. This project seeks to ensure Swarthmore's growth in all these areas is supported by a proactive Sponsored Research Office strengthened by efficient pre-/post-award administration and compliance strategies and tools and backed by clear institutional policies in a campus culture that recognizes the infrastructural needs that make research excellence possible.

Regulation of the expression of the rat tyrosine hydroxylase gene by nicotine Increased risk of cardiovascular disease in tobacco users is in part due to chronic nicotine exposure. Nicotine in the peripheral blood stream is capable of stimulating the adrenal medulla to synthesize and release the catecholamine epinephrine, a potent neurohormone that elevates blood pressure and stimulates cardiac output. Prolonged elevated levels of epinephrine in the blood stream due to chronic nicotine exposure is associated with cardiovascular disease. The long-term objective of this research is to understand how the extracellular stimulus provided by nicotine mediates increased synthesis of the catecholamines in the adrenal medulla. Previous work has demonstrated that rats chronically exposed to nicotine have an increased activity and expression of the enzyme tyrosine hydroxylase (TH) in the adrenal medulla. TH catalyzes the rate limiting step in the biosynthetic pathway of the catecholamines. Understanding the mechanisms by which nicotine regulates the expression of the TH gene in the adrenal medulla may provide insight into the cardiac pathogenicity of cigarette smoking. Since adrenal chromaffin cells demonstrate increased levels of cellular cAMP in response to nicotine exposure, the specific aim of this project is to study the regulation of the rat TH gene promoter in response to cAMP. Using gel retardation assays and DNase I footprint assays potential regulatory elements of the rat TH gene promoter have been identified. By sequence comparison a number of these promoter elements contain sequences that share homology with known cAMP response elements (CREs). A reporter gene assay is presently being used to investigate the functional significance of these potential TH gene CREs. Mutant TH gene promoter constructs will be cloned into a vector upstream to the bacterial gene coding for chloramphenicol acetyl transferase (CAT). The mutated and wild type TH gene promoter CAT constructs will then be used for liposome mediated transient transfections in rat adrenal chromaffin tumor cell lines. The transfected cells in culture will be treated with cAMP analogs to examine the ability of response element mutations to block induction of CAT expression. Once functionally active cAMP inducible elements of the TH gene promoter have been identified the trans factor proteins interacting with these regions will be characterized. A modified southwestern blot technique will be used to determine the molecular weight of the DNA binding proteins. Ultimately, this same blotting technique will be used to clone the trans factor(s) binding to TH gene CREs by screening a rat adrenal expression library. These studies will provide significant insight into the biochemical events involved in chronic nicotine exposure and the signal transduction mechanisms that lead to increased expression of the TH gene. KEY WORDS: tyrosine hydroxylase, nicotine, tobacco, mutations,

Panic disorder (PD) is a severe anxiety disorder characterized by recurrent panic attacks affecting about 2- 5% of the population and resulting in severe disability in about a third of those subjects. During the previous funding period, using our well established animal model we identified that orexin (hypocretin) neurons within the perifornical-dorsomedial hypothalamic region (PeF/DMH) are one of the key regulators of a coordinated panic response and that patients with panic but not depression symptoms did indeed have high levels of orexin in their cerebrospinal fluid. Little is known about how this disrupted panic regulation leads to many of its debilitating consequences such as chronic anticipatory anxiety and agoraphobia. Therefore, the goal of this competitive renewal (MH 52619-14 to -19) is to elucidate the neural mechanisms involved in how a panic- vulnerable state also leads to increased chronic anxiety and enhanced vulnerability to conditioned fears, avoidance and phobias. The project will have significant impact as we hope that the results of these studies will identify novel therapeutic targets for not only panic attacks, but also their many disabling consequences such as agoraphobia. Specifically, we predict that chronic disinhibition of orexinergic neurons in the PeF/DMH results in disruption of neurotransmission of not only orexin, but also its co-transmitters glutamate and dynorphin, resulting in disrupted network functions in 1) the bed nucleus of the stria terminalis (BNST); 2) the central nucleus of the amygdala (CeA); and 3) the infralimbic prefrontal cortex (IL) to delay the extinction of conditioned fear. We will elucidate these mechanisms with behavioral, molecular and electrophysiological endpoints, utilizing pharmacological, gene silencing, optogenetic experiments in both whole animal and slice preparations. Specific Aim 1 will focus on chronic disinhibition of ORX neurons in the DMH/PeF inducing molecular and network changes in the BNST to decrease GABAergic and enhance glutamatergic neurotransmission to induce a chronic anxiety-like phenotype. Specific Aim 2 will study the mechanisms within the CeA that result in enhanced acquisition and expression of conditioned fears seen in panic-prone rats, testing if chronic disinhibition of ORX neurons in the DMH/PeF enhances excitation and reduces inhibition, thus facilitating neuronal plasticity in the CeA. In Specific Aim 3, we propose to stud the mechanisms within the IL which could result in delayed extinction of conditioned fear responses in panic-prone rats. This project is innovative at several level providing a mechanistic model to understand the pathophysiology of PD and its disabling consequences combining the study of whole animal behavior, physiology, and systems level pharmacological and optogenetic manipulations with functional network electrophysiology as well as basic cellular and molecular changes. Finally, at the translational level, the work has already had significant impact in the development of novel therapies for acute panic attacks, but hopefully will provide novel insights into the persistent and hard to extinguish nature of agoraphobia and anticipatory anxiety in patients with panic disorder. PUBLIC HEALTH RELEVANCE: Panic disorder (PD) is a severe anxiety disorder characterized by recurrent panic attacks affecting about 2- 5% of the population and resulting in severe disability in about a third of those patients. Using our well established animal model, we identified that a specific set of brain cells called orexin (hypocretin) neurons are a key regulatos of panic attacks. Therefore, we will study how this chemical pathway also leads to increased chronic anxiety, fears, avoidance and phobias.

Iso-1-cytochrome c and iso-2-cytochrome c from the yeast Saccharomyces cerevisiae are two of the few proteins of known primary structure from a microorganism which is particularly suitable for experimental genetic studies. The isolation of appropriate mutants have been facilitated by enrichment procedures for both forward and reverse mutations. A series of deletions are available, making it possible to conveniently map point mutants and to estimate their positions relative to the iso-1-cytochrome c sequence. The large number of mutants that have been characterized and the selection procedures permit an unprecedented degree of genetic manipulation of nucleotide sequences by recombination. The sequencing of a portion of the gene from frameshift mutations has made it feasible to directly analyze the mRNA of iso-1-cytochrome c with a synthetic oligonucleotide. This synthetic oligonucleotide has been used as a probe for identifying bacteriophage lambda and E. coli plasmids containing segments of yeast DNA corresponding to the cycl gene. Thus the body of information concerning the iso-1-cytochrome c gene, the large number of defined mutants and the available genetic and biochemical techniques are without parallel for any other eukaryotic gene. This iso-cytochromes c system is being employed for investigating numerous problems in molecular biology and genetics, including: the isolation and characterization of structural and regulatory mutants for both iso-1 and iso-2-cytochrome c; the identification of amino acids inserted by various nonsense suppressors and other translational suppressors and the determination of the efficiencies of suppression; the determination of the specific action of numerous mutagens; gene conversion studies and the examination of the relationship of recombination frequencies and nucleotide alterations; the characterization of DNAs and mRNAs specifying the two iso-cytochromes c and their measurements in normal and mutant strains; the determination of DNA sequences of mutations that are in the untranslated regions of the CYC1 and CYC7 gene.

The proposed work consists of the following four projects: 1. Quantitative Characterization of the In Vitro Neoplastic Transformation of Syrian Hamster Embryo Cells (SHE cells, 13-days gestation) - The Development of Carcinogenic Bioassay. 2. Somatic Genetics on the Progression in Neoplastic Transformation: Studies of the Spontaneous Initiation and Progression Toward Neoplastic Transformation of Syrian Hamster Cells. 3. Changes in Gene Expression Accompanying Neoplastic Transformation. 4. Transformation of Syrian Hamster Embryo Cells by HSV-2 DNA. Considerable progress has been made as in the sections reported below: 1. Perturbation of Mammalian Cells by Macromolecules (Nucleic Acids and Enzymes) via Liposomes as Carriers - Neoplastic Transformation by DNase I Entrapped in Liposomes. 2. Detection and Characterization of Subpopulations of Contact-Insensitive and Anchorage-Independent Cells in Syrian Hamster Embryo Cell Cultures. 3. Senescence and Neoplastic Transformation of Syrian Hamster Adult Fibroblast. 4. Effects of Ploidy (Gene Dosage) on Somatic Mutation, In Vitro Senescence, and Neoplastic Transformation. 5. Analysis of Specific Cell Surface Antigens (Proteins) from Tumorigenic Cells. 6. Induction of Heteroploidy in Diploid Human Fibroblasts Antimitotic Agents. 7. Role of HSV-2 Gene Expression in Transformation. 8. Identification of HSV-2 mRNA in transformed Cells by In Situ Hybridization. 9. Identification and Localization of the Oncogenic Fragment of HSV-2 DNA. 10. Neoplastic Transforming Activity of Bg1 II N Fragment. 11. Phenotypic and Antigenic Properties of Cells Transformed by Restriction Enzyme Fragments of HSV-2 DNA.

The long-term goal of these investigations is to understand how genes cause congenital heart disease (CHD). The first set of objectives involved devising methods of analysis by unifying two approaches: the focus of the current application, involves obtaining clinical data and extending the theory to the analysis of these data. These sets of objectives are clearly synergistic, and examination of empiric data will continue to stimulate fresh ideas for theory, just as exploration of models has pointed to potentially revealing avenues of clinical study. Considerable progress in extending and developing the methods of genetic analysis for CHD has been achieved. Topics sufficiently explored to warrant extension to empiric data include: epistasis; selection; multicompartmental models; bingo models; threshold and tetrachoric models; circular distributions; and, angular homeostasis. The following disorders will be studied. (1) Cardiovascular malrotation in its several forms will illustrate aspects of angular homeostasis; theory predicts how extracardiac manifestations might be associated with some, but not all, types of maldevelopment of malrotation. (2) Papillary muscle position on the ventricular endocardium can be studied by directional statistics applied to echocardiographic and magnetic resonance images. Malposition has been related to flow lesions such as aortic stenosis. Papillary muscle position will be determined by echocardiography, and genetic factors studied in normal families, and in families ascertained through occurrence of CHD flow lesions, especially aortic stenosis. (3) The mild end of the flow lesion spectrum includes bicuspid aortic valve and aortic coarctation; often associated with these lesions is dilatation of the proximal aorta. The possibility that predisposition to these lesions is an autosomal dominant trait will be studied by searching, by magnetic resonance imaging and echocardiography, for subclinical anatomic alterations in relatives of probands. Development of extensive pedigrees will enable investigation of epistasis. (4) The occurrence and progression of aortic root dilatation in the Marfan syndrome, hereditary annuloaortic ectasia, and congenital bicuspid aortic valve will be studied as multiple-hit systems by means of bingo-gamma models. The genetic and evolutionary fitness of the Marfan phenotype can be explored by the method of branching processes. (5) Mitral valve prolapse is thought to be an autosomal dominant trait in some families. Probands will be categorized as to the presence of redundancy of valve apparatus, and relatives studied by echocardiography, with the dual aims of examining mendelian models and exploring ascertainment bias in an age-dependent trait. (6) The conjunction of digital abnormalities and CHD in certain mendelian conditions, notably the Ellis-van Creveld and the Holt-Oram syndromes, provides a way to test pathogenetic models based on quadrangular correction functions.

Recent advances in human islet transplantation have shown great promise as an approach to treat insulin-dependent diabetes. However, the clinical potential of beta-cell replacement therapies will not be realized until appropriate strategies have been developed for the expansion of pancreatic islets ex vivo or for stimulating new islet growth within residual pancreatic tissue of diabetic patients. It is known that certain nutrients and growth factors can induce pancreatic beta-cell growth. The ErbB receptor family plays a fundamental role in the development, differentiation, proliferation and survival of many tissues including the pancreas. ErbB receptor gene ablation studies have suggested an important proliferative role for ErbB signaling in beta-cell biology but these studies have been hindered by the severity of defects found in these receptor-null mice. Members of the EGF-related peptide growth factor family bind to and activate ErbB receptors. Each ErbB ligand has distinct ErbB receptor binding specificities and can activate unique downstream signaling pathways to generate diverse biological responses. For example, exogenous, recombinant betacellulin (BTC) can uniquely induce beta-cell neogenesis, proliferation and differentiation in both in vitro and in vivo models. However, it is important to note that endogenous ErbB ligand precursors have distinct signaling functions and that sequential processing to release soluble ligand is a fundamental regulatory event in ErbB receptor signaling; a regulatory step which can not be duplicated by addition of exogenous recombinant growth factors. The generation and viability of mice deficient in certain ErbB ligands including BTC provides important genetic tools to investigate the specific and/or complementary roles of individual endogenous ErbB ligands in beta-cell biology. The long-term objectives of my research are to determine the unique cellular and biochemical functions of different endogenous ErbB ligands in beta-cell biology. The specific goals of this grant proposal are to investigate the roles of endogenous BTC signaling in beta-cell proliferation, differentiation, neogenesis and survival. Specific aim 1 of this application will characterize the regulated and sequential processing of BTC precursor in beta cells. The second Specific aim will examine the signaling potential of different BTC cleavage products in beta-cells proliferation and differentiation in vitro. In Specific aim 3, the role of BTC in beta-cell development and neogenesis as well as maintenance of beta-cell mass in vivo will be examined utilizing Btc-/-deficient mice alone or under EGFR wa2/wa2 and/or combinatorial ErbB ligand null genetic backgrounds. The results of these studies should enhance our understanding of the role of BTC signaling in beta-cell biology and its application to human beta-cell replacement therapies.

In our recent studies, a novel intrakine approach was developed to effectively inactivate the chemokine coreceptor CCR-5 for the macrophage-tropic HIV-1, and SIV, with a therapeutic application for HIV-1 infection. In this study we are proposing to use a SIV model to evaluate the therapeutic potential of the novel genetic intrakine approach. The hypothesis of this proposal is that the lymphocytes from macaque monkeys can be transduced with the intrakine to inactivate CCR-5, and the transduced lymphocytes would be resistant or less susceptible to SIV infection and may have an advantage in survival and functions in SIV-infected macaques. The goals of this study are two- fold: to use the SIV model to evaluate the therapeutic potential of this genetic intrakine approach for HIV-1 therapy, and to assess virus usage of chemokine coreceptors in vivo under the selection pressure by intrakine. Specifically, we will determine the effects of intrakine expression on SIV infection and cell function in primary macaque lymphocytes in vitro. We will then evaluate the survival of the intrakine-transduced or control PBLs in SIV-infected macaques to determine whether the intrakine expression confers a survival advantage of the transduced lymphocytes in vivo. Whether the autologous administration of the intrakine-transduced lymphocytes has therapeutic benefits, such as, increasing CD4+ cell counts, decreasing virus loads will be evaluated by various assays. Growing numbers of SIV or HIV-1 co-receptors being identified imply that targeting CCR-5 might result in gaining the use of alternative co-receptors, and mutated viruses might emerge and come to predominate. Accordingly, we will serially assess the intrakine-PBL macaques or intrakine-PBL macaques at different time points postinfection. We will use the envelope complementation assay to determine the coreceptor usage of SIV in the macaques with or without the intrakine selective pressure. These experiments should be important for understanding the HIV-1 pathogenesis, and has added significance for therapy since SIV is placed under the intrakine selection pressure. In summary, the SIV model study will not only address the critical issues of the safety and therapeutic potential of the novel intrakine approach for HIV-1 gene therapy, but also may shed light on the selective forces driving the usage of the chemokine coreceptors.

Actin, villin, and calcium play key roles in regulating the finger-like shape of intestinal microvillar membranes. In physiologic calcium concentrations (less than MuM) villin crosslinks actin filaments into a bundle which stabilizes the microvillus membrane. However, when the calcium concentration increases greater than MuM, villin fragments the filaments into short pieces and the unsupported membrane vesiculates. Villin is unusual in that it possesses both actin filament bundling and fragmenting activities. In other cells these activities are properties of separate proteins. To understand how actin binding proteins regulate cell structure and motility we will identify the calcium-regulated changes in villin-actin binding domains using biochemical and structural methods. The goals of this proposal are to describe the molecular mechanisms of villin-mediated actin filament bundle formation and actin filament fragmentation. The specific aims are: 1. to map the positions of cysteine, methionine, and tryptophan residues in villin and actin with an antibody end-labeling method. 2. to identify and characterize actin-binding domains of villin using site specific chemical and proteolytic cleavage, binding assays and gel overlay methods. 3. to map the positions of villin-actin binding domains by chemical crosslinking and antibody end-labeling methods. 4. to identify and characterize the calcium binding sites on villin. 5. to start high resolution structural studies on villin by crystallizing villin fragments or villin fragments bound to actin. The molecular details of calcium-villin-actin interactions have important health-related significance because membrane vesiculation is an early event in the pathogenesis of intestinal malabsorption disorders such as coeliac syndrome and enterotoxin-induced enteropathy.

This investigation is concerned with three aspects of early embryonic development in R. pipiens, and with certain aspects of the cell life cycle in general. 1. The mechanism of meiotic maturation and ovulation is being studied in terms of the interrelationships between: a) protein phosphate turnover and exchange, b) protein kinase- adenyl cyclase activity, c) cation binding to membrane phospholipids and phosphoproteins, and d) electrical and ion selectivity changes at the plasma and nuclear membranes. 2. Changes in macromolecular syntheses are being studied using cell cultures of dissociated blastula and gastrula stage embryos. Turnover, acetylation, and phosphorylation of histones are being correlated with electrical and ionic changes during the induction of tissue specific enzymes. 3. Macromolecular (RNA, DNA, and protein) synthetic events in synchronized dissociated blastula cells and human cancer cells are being correlated with changes in membrane potential and intracellular cation concentrations during the cell life cycle.

The management of children with pediatric mastocytosis poses a challenge. There is a limited information as to the application of clinical and laboratory findings and bone marrow evaluation as they relate to medical intervention and prognosis. The diagnostic criteria for pediatric mastocytosis are largely based on adult studies. There is very little data about bone marrow pathology in children with mastocytosis. We evaluated the use of the World Health Organization (WHO) criteria for the diagnosis of systemic disease in pediatric patients. Bone marrow biopsies were analyzed by histopathology and immunohistochemistry, flow cytometry and KIT mutational analysis. Complete bone marrow evaluations were performed in 50 cases. Seven children had repeat procedures. We identified unique clinico-histopathological features within the biopsies. Bone marrow biopsies displayed mildly atypical hematopoietic maturation, increased hematogones and hypocellularity in a sub-set of patients with urticaria pigmentosa, diffuse cutaneous mastocytosis and indolent systemic mastocytosis. Hypocellularity was most pronounced in those with urticaria pigmentosa. Hematogone increases were highest, on average, in patients with diffuse cutaneous mastocytosis or mastocytomas. There was no evidence of peripheral blood cytopenias, myelodysplastic syndrome, myeloproliferative neoplasm or leukaemia within this cohort. Long-term follow-up of patients within this study (median 6.9 years; range 1-25 years) showed that all patients remained clinically stable without progression to a more aggressive variant. We conclude that the WHO criteria are applicable for the diagnosis of systemic mastocytosis in pediatrics.

DESCRIPTION: Cytokines produced by CD4+ T lymphocytes play essential roles in host adaptive immune responses and are involved in the development of allergic diseases including asthma. Our understanding of the pathways regulating cytokine production in CD4+ T lymphocytes remains incomplete. We have identified a novel pathway regulating cytokine production in CD4+ T cells using molecules that are thought to be primarily involved in neuronal cell function and pathogenesis of Alzheimer's disease. We show that CD4+ T cells lacking the extracellular matrix (ECM) protein F-spondin or its receptor apoE receptor 2 (ApoER2) exhibit defective cytokine production, and asthma development is attenuated in mice lacking F-spondin. Our overall hypothesis is that F-spondin and its receptors ApoER2 and amyloid-2 precursor protein (APP) constitute a novel ligand-receptor pairs whose interaction on the CD4+ T cell surface provides a critical signal to initiate cytokine production. To test this hypothesis, we propose two specific aims: 1. To establish the mechanisms by which F-spondin regulates CD4+ T lymphocyte cytokine production. 2. To examine the signaling pathway mediated by ApoER2 and APP in CD4+ T lymphocyte cytokine production. Results from these studies will not only provide important mechanistic insights into the regulation of CD4+ T lymphocyte cytokine production and asthma development, but also may lead to discoveries of novel drug targets for asthma treatment. Given the current effort by industry and academia to develop Alzheimer's disease treatments, our proposed research may be able to redirect the drugs developed for Alzheimer's disease into asthma treatment. Project Narrative Cytokines produced by CD4+ T lymphocytes are involved in the development of allergic diseases including asthma. Our understanding of the pathways regulating cytokine production and asthma development remains incomplete. Investigation of the pathways regulating cytokine production and asthma development will lead to new discoveries that will help drug designs for asthma treatment. Our recent work has identified a novel pathway regulating cytokine production in CD4+ T cells and asthma development in mice by molecules that are thought to be primarily involved in neuronal cell function and pathogenesis of Alzheimer's disease. Given the current effort in the development of Alzheimer's disease treatment by industry and academia, our proposed research may be able to redirect the drugs developed for Alzheimer's disease into asthma treatment.

Aim 1 Dozens of proteins control the docking and fusion of exocytic vesicles in neurons and endocrine cells. The identity and roles of many of these proteins have been assigned through a combination of genetics, biochemistry, and electrophysiology. However, the spatial organization, heterogeneity, and dynamics of these proteins have yet to be determined. Finding this organization is key to understanding how proteins regulate these systems in healthy cells and might malfunction in disease. Thus, we aim to map key proteins proposed to act during exocytosis in endocrine cells. To accomplish this, we developed a combination of high-throughput live cell imaging, super-resolution fluorescence imaging, and electron microscopy. Through this multi-modal imaging approach, the location, dynamics, and occupancy of individual proteins were mapped at specific populations of vesicles in cells and compared to the underlying cellular architecture that organizes the plasma membrane. This allowed us to determine the fundamental organization of the exocytic membrane system and how specific molecular components responsible for vesicle trafficking and fusion assemble together and function. Specifically, we have been using two-color TIRF microscopy and high-throughput image analysis to detect and characterize over 80 different proteins that have potential roles at exocytic sites. Through this work we developed a universal map of the proteins that control exocytosis and provide a new global network level analysis of vesicle fusion. We were able to identify unique classes of key regulatory molecules that strongly associate with the vast majority of docked exocytic vesicles in endocrine chromaffin and beta cells. Key proteins we identified were Rabs and Rab effectors, SNARE proteins, and several SNARE modulators. The above mapping work was done on single images. Thus, to determine the cellular dynamics of these components we imaged the live-cell local changes of proteins at the exact moment of fusion. In these microscopy studies we discovered an unexpected rapid recruitment of several important endocytic proteins and lipids to sites of exocytosis. These molecules include the regulatory lipid PIP2, and the proteins dynamin, amphiphysin, syndapin, and endophilin. We further discovered that mutations to several of these proteins altered the kinetics of cargo release from single insulin-containing dense-core vesicles in cultured beta cells. Our hypothesis is that these proteins (dynamin, syndapin, amphiphysin, and endophilin) regulate the dilation of the fusion pore to control the amount of cargo released during single exocytic fusion events in cells of the pancreatic islets. Along with these studies we have developed a new super-resolution correlative light and electron microscopy method (CLEM). This CLEM method allows us to image the location of identified proteins in the nano-scale structural context of the cellular environment. Specifically, we have succeeded in developing a robust pipeline for imaging the plasma membrane of human cells with both fluorescence microscopy (TIRF) and transmission electron microscopy (TEM) of platinum replicas. These studies are allowing us to build structural models for how single organelles are organized and how these complexes regulate exocytosis, a central process for commination among cells and tissues in the body. We have expanded our research in this aim to the study of the large terminals of primary bipolar neurons from the retina. These terminals can be over 10 microns in diameter which make them ideal for studying the structure of the synaptic space. In these studies we discovered a new synaptic cytoskeletal structure. We found a thick marginal band of microtubules that extends down from the axon and loops throughout the synaptic terminal. This microtubule loop is structurally similar to those found in red blood cells. Using 3D confocal fluorescence, block-face serial scanning electron microscopy, and transmission electron microscopy we fully characterized this structure. We found that this microtubule structure associates with a substantial population of mitochondria in the terminals. Drugs that inhibit microtubule-based kinesin motors stalled mitochondria in the axon. From this work we conclude that this prominent microtubule band is important for the transport and localization of critical mitochondria into the presynaptic space to provide the sustained energy necessary for continuous transmitter release in these giant synaptic terminals. Aim 2 We discovered that in endocrine cells vesicle material is captured on a dense network of pre-formed clathrin-coated structures following exocytosis. Despite the identification of many molecular components of clathrin-mediated endocytosis, a structural understanding of how these molecules come together to build and retrieve material from the plasma membrane is incomplete. In this aim we sought to directly determine the structure of clathrin-coated vesicles responsible for endocytosis. By understanding how proteins that have been functionally implicated in endocytosis assemble together at the nanoscale we place decades of biochemistry, cell biology, and genetics into a physical model of membrane retrieval. First, using our high-throughput TIRF imaging pipeline discussed above we mapped how over 80 proteins associated with endocytic clathrin-coated structures in PC12 cells. This provided us with the fundamental protein signature of clathrin structure across the plasma membrane. From this analysis we determined that a core group of 30 protein associate with clathrin-coated sites in these cells. This information provided us with a molecular framework to understand the regulation of endocytosis in these cells. Next, we developed a new super-resolution correlative light and electron microscopy imaging method. This development allows us to image the nanometer-scale location of proteins in the context of their local cellular environment. Specifically, we succeeded in developing a robust pipeline for imaging the plasma membrane of cells with both 3D iPALM (interferometric photoactivation localization microcopy) and 3D transmission electron microscopy (TEM) of platinum replicas. In these studies we have imaged the 3D position of the endocytic protein Epsin at single clathrin-coated structures in PC12 cells. From this work we discovered that Epsin sits at the equator of endocytic vesicles where it likely aids in the retrieval of clathrin-coated pits. We have continued to map dozens of other critical endocytic components at the nanoscale with this method to develop a global map of the proteins involved in endocytosis. These studies are allowing us to build structural models for how proteins are organized at single organelles to regulate endocytosis, a central process for all living cells. Aim 3 My lab's major focus is to understand how cellular process work at the molecular scale. To accomplish this, current tools were inadequate. Thus, we focused on the development and application of new imaging methods to uncover the structure of molecules and organelles inside cells. In my group we continue to pioneer four novel imaging technologies. These include, 1) high-throughput microscopy and image processing to map protein distributions across populations of cells, 2) 3D super-resolution fluorescence imaging methods, 3) super-resolution correlative light and electron microscopy to map proteins at the nanoscale, and 4) high-throughput metal ion FRET methods to map distances within individual proteins at the atomic scale. The combination of these methods provides our group with the unique tools to gain a multi-scale understanding of the structure, dynamics, and functions of proteins and protein complexes inside cells.

In this proposed research, benzo[a]pyrene (B[a]P) and it's metabolites, will be used as model compounds to determine the major metabolic pathways of PAHs in lung carcinogenesis. It is generally accepted that cytochrome P4501A1, CYP1A1, is primarily responsible for the metabolism of B[a]P to 7,8-dihydroxy-9,10- epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (B[a]PDE), the ultimate DNA-adduct forming, carcinogenic reactive metabolite;thus connecting CYP1A1 to the toxicological effects of this environmental carcinogen. DNA-adduct formation has been detected at CpG islands located in the promoter region of the tumor suppressor p53. Inactivation of p53 by point mutations is seen in over 40% of all cancers. However, in preliminary research using H358, human bronchoalveolar lung cells, it was found that DNA-adduct levels were higher in non-treated CYP1A1/1B1 null cells compared to H358 cells in which CYP1A1 expression was induced with 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD). This data correlates with literature results of CYP1A1 KO mice stuides in which it was determined that CYP1A1 conferred a protective rather than a toxic effect. This is indicative of the metabolic activity of another CYP in the metabolism of B[a]P. Several other CYP enzymes are capable of catalyzing this metabolism. Preliminary data has also indicated that TCDD may have an effect on the transport of B[a]P-reactive metabolites. It is known that TCDD is able to induce several of the ABCC drug transporter family members;therefore, explaining why less DNA-adduct formation is observed in lung cells pretreated with TCDD . This proposal, which has two aims, will identify the predominate CYP responsible for B[a]P metabolism in the lung and determine the role of B[a]PDE transporters in DNA-adduct formation in lung cells. A stable isotope dilution LC-MRM/MS method, previously developed, will be used for DNA-adduct quantification. DNA-adduct levels will be correlated to mRNA and protein expression determined by RT-PCR and a novel quantitative proteomics method, SI LAC. CYPs including 1A1, 1B1, 1A2, 2B6, 2E1, 2J2, 3A4, and 3A5 will be investigated. DNA-adduct formation will also be monitored in lung cells in which drug transporter expression has been inhibited by siRNA. Continuing research in this area will provide exciting new therapeutic strategies (drug targets) that can be employed to prevent PAH-mediated DNA damage. Relevance to public health: Cigarette smoke is known to cause 90% of lung cancer, but only 10% of smokers develop lung cancer. Examination of alternative CYP metabolism and B[a]PDE transport in lung cells will provide an experimental model to explore whether inter-individual differences in CYP metabolism and transport contribute to these phenomenon.

An experimental peer leadership curriculum in which older adolescents counsel younger peers in how to resist social pressures toward smoking has been developed for experimental study. In a California site near Stanford University one cohort of 7th grade students (n equals 300 plus) in the 1977-78 school year was selected to receive the program, while two matched cohorts (n equals 300 plus) were selected as control units. Baseline and follow-up surveys of smoking and other pertinent variables are being conducted periodically in all three cohorts. Results of the first year of follow-up reveal a significantly lower rate of onset of smoking among students receiving the program, with some evidence of generalization to other substance use and support for the hypothetical model on which the intervention is based. Replications of that work are underway in diverse populations in two sites near Boston.

The NIDA Drug Dependence Epidemiology Training Program is designed to increase the number and quality of expert scientists who identify drug dependence epidemiology as their major lifelong career, with a special focus on the application of advanced statistical methods to the design and analysis of epidemiologic study data and all forms of research dissemination. Now in its fourteenth year of competitively renewed support by NIDA, this proposal is for an additional five years of support. Pre-doctoral trainees earn the Ph.D. degree by completing required course work, research apprenticeships, field research, and examinations followed by the completion of thesis research and defense. Post-doctoral trainees complete an individualized program of research training, typically lasting two years, focusing on research collaboration with two-or-more program faculty to assemble a portfolio of first-authored, published scientific articles and career development plans. The research training plan retains educational and research-related elements from previously approved training plans, with refinements that reflect accumulating experience during the research training process. Recent developments include: (a) expansion of NIDA-funded core faculty specializing in drug dependence epidemiology;(b) a resulting increase in courses and research apprenticeship opportunities that focus on drug dependence epidemiology;(c) the addition of core faculty utilizing bench science methodologies;and (d) the implementation of new training initiatives to facilitate fellows'establishing a record of first-author publication during their training experience. We have also revitalized our commitment to recruit underrepresented minorities into the Training Program facilitated by faculty nationwide. Some challenges currently facing the drug dependence field are unique to our era such that advances in bench sciences are now providing the basis to integrate genetic laboratory methodologies within epidemiologic field studies. Other challenges are long-standing with a sustained need for well-designed studies to help identify the biological and environmental factors that predispose to drug dependence and to develop empirically supported approaches to drug prevention. The Training Program at Johns Hopkins University is well positioned to meet these challenges by fostering the development of expert scientists capable of applying advanced epidemiologic and biostatistical methodologies to the study of drug dependence.

The trichothecene mycotoxins are a group of sesquiterpenoid fungal metabolites, that include some of the most potent protein synthesis inhibitors known. Since trichothecenes are frequently found in cereal grains and are recalcitrant to inactivation during milling and processing, they enter the food chain. Of these vomitoxin is encountered most often in the U.S. food supply. Ingestion of vomitoxin by mice results in marked increases in serum IgA. It is hypothesized that dietary exposure to trichothecene mycotoxins impairs normal regulation of IgA production and that this results in accumulation of serum IgA. The overall goals of this proposal are to understand the cellular basis by which trichothecenes modulate IgA production within the mucosal and systemic compartments and relate this effect to potential health hazards associated with ingestion of these naturally-occurring mycotoxins. Using the mouse model, four (4) specific objectives are proposed. The first objective will be to determine the effects of in vitro exposure to vomitoxin on macrophage and T cell regulation of IgA production in spleen and Peyer's patch (PP) cultures. Second, the effects of in vivo vomitoxin exposure on regulation of IgA production will be assessed by quantitating in vitro IgA secretion in spleen and PP- derived lymphocyte cultures, measuring IgA-secreting B cell and T regulatory cell frequency in spleen and PP, and monitoring mucosal and systemic IgA response to oral antigen challenge. Third, IgA distribution following in vivo vomitoxin exposure will be characterized by monitoring changes in total serum IgA, secretory IgA, and ratio of serum monomeric:polymeric IgA; and by determining potential for IgA deposition in kidney mesangium. Finally trichothecene structure will be related to modulation of IgA production in in vitro cultures.

This proposal addresses the general method of preparation of linear and nonlinear triquinane serquiterpanes, in particular several biologically active coriolins and hirsuitic acids. Our proposed syntheses represent newly redesigned versions of the original research propositions funded in 1981. The new approach is perhaps more flexible in the ultimate obtention of the natural products for medicinal screening. The continuous supply of especially the antitumor and antibiotic coriolins from natural sources seems uncertain and, therefore, it is essential that an efficient and a general preparation of these compounds be available. Our synthetic plan thus focuses on the rapid production of a flexible tricyclic intermediate which is equipped with properly differentiated functionalities to permit its conversion into various coriolin natural products. This intermediate is accessible through the application of intramolecular cyclopentene annulation methodology which has been developed in our laboratory and pursued in conjunction with terpene synethesis. Following our recent work on the synthesis if triquinanes we propose to concentrate our efforts on the specific aims summarized below: a. Synthesis of five coriolins and three hirsutic acids by a redesigned strategy. b. Synthesis of other linear and nonlinear triquinanes of medicinal interest by an extension of our methodology. c. Investigation of the ulity of the intramolecular Simmons-Smith reaction and sequential radical alkylation of dienic dihalides as alternatives to methods utilizing diazoketones for cyclopropanations and d. Explorations of some newly discovered aspects of the Reformatsky reaction of halocrotonates in organic synthesis. The overall significance of the proposed research lies not only in the provision of the biologically important target compounds but also in the development of what appears to be a quite general system-oriented design of cyclopentanoid terpenes. The pursuit of the novel application of the two established reactions will enrich the compendium of the methods available for the carbon-carrbon bond formation. The budgetary period of this applications has been expanded to five years in order to further utilize the Cyclopentene Annualtion Methodology in the synthesis of biologically active cyclopentanoid terpenes and to begin detailed investigations of several new and potentially general synthetic methods.

This proposal describes a comprehensive research project designed to prospectively pilot test the efficacy of prolonged exposure (PE) therapy at reducing symptoms of HIV-related posttraumatic stress disorder (PTSD) and increasing adherence to antiretroviral medication regimens in people living with HIV (PLWH). Recent studies examining the efficacy of cognitive behavioral interventions at increasing adherence to medications in PLWH have produced, at best, small effect sizes. Failure to address mental health issues in PLWH has been suggested as one explanation for the relatively small impact of these interventions. Previous research has found that symptoms of PTSD (particularly avoidance symptoms stemming from diagnosis/living with HIV) are positively related to non-adherence. PE has been found to be an effective treatment for PTSD; however, its efficacy at reducing PTSD symptoms in patients meeting criteria due to diagnosis with a life-threatening disease is unknown. Further, the extent to which decreasing symptoms of PTSD results in consequent reductions in non-adherence has not been examined. The proposed research will examine the efficacy of PE therapy at reducing HIV-related PTSD and non-adherence in 70 PLWH (20% female, 50% African-American, 50% Caucasian) with PTSD. Participants will be randomly assigned to either the 6-week, 12-session intervention or a weekly monitoring/wait list control group. Follow-up time points at post treatment and three and six months will consist of interviews and questionnaires designed to measure symptoms of HIV-related PTSD and co-morbid disorders. Further, medication adherence (via self-report and electronic monitoring) will also be measured. The proposed research will directly test the efficacy of PE at reducing symptoms of HIV-related PTSD and co-morbid disorders, and non-adherence, and will provide pilot data and effect sizes for a large-scale randomized, multi-site trial.

Orthograde degeneration studies have outlined major ascending and descending projections of forebrain structures in reptiles. However, very little is known about the internal organization of the reptilian forebrain. The proposed research will determine the normal ultrastructure of three structures in the forebrain of snakes and turtles and use experimental electron microscopic techniques to establish the synaptic pattern of major afferents within each structure. Nucleus sphericus is an amygdaloid structure associated with the vomeronasal organs. The distribution of degenerating axon terminals in nucleus sphericus will be determined following lesions of the accessory olfactory bulb, contralateral nucleus sphericus, and of focal lesions of ipsilateral nucleus sphericus. The distribution of degenerating axon terminals in each area and layer of cerebral cortex will be determined following lesions of the thalamus and of various cortical areas. The anterior dorsal ventricular ridge is a subcortical structure which receives afferents from thalamic auditory and visual nuclei. The distribution of degenerating axon terminals in anterior dorsal ventricular ridge will be determined following thalamic and cortical lesions. BIBLIOGRAPHIC REFERENCES: Ulinski, P. S. 1977. Tectal efferents in the banded water snake, Natrix sipedon, Journal of Comparative Neurology, in press. Ulinski, P. S. 1977. Intrinsic organization of snake medial cortex: An electron microscopic and Golgi study, Journal of Morphology, in press.

This grant proposal in a continuation of our previous work dealing with the psychopharmacology of the lithium ion. The scope has been considerably expanded; we now plan on concentrating our research efforts in using lithium as a tool to explore the biochemical framework underlying the affective disease entities. In this regard a number of studies are proposed exploring the metabolism of biogenic amines: determination of serum DBH activity; examination of MHPG, HVA, and 5HIAA in CSF; turnover rates add relative specific activities of NE3H2 as well as its major metabolites and biogenic amine transport in platelets. RBC COMT and the RBC/plasma lithium relationship is also proposed in distinguishing different clinical pictures. The effect of lithium on a variety of endocrines and the effects of this ion on the hypothalamus- pituitary axis will be explored; this includes thyroid and adrenocortical studies as well as further examination of carbohydrate metabolism. The lithium-aldosterone relationship will be examined with particular interest. Clinical studies will now concentrate on exploring lithium's potential therapeutic effects in other related disorders. These include aggressive/sociopathic behavior (prisoners), hyperactivity in adolescents and cyclic mood disturbances related to the menstrual cycle (pre-menstrual tension). Continuing to define a role for lithium in acute depressive recurrences is considered of central importance in considering lithium as an agent capable of affecting a disease entity rather than merely the symptoms of the related illnesses (i.e., the manic-depressive disorders).

Previous investigations have shown that recurrent exposure to dietary lead or cadmium results in increased volitional intake of ethanol. The present proposal will help define a suitable rationale for interpreting metal/ethanol interactions. Specifically, it is proposed that metal-treated animals may consume more ethanol because the pharmacologic and behavioral effects associated with the drug are attenuated by toxic exposure. In this context, increased consumption by treated animals would be viewed as compensatory. To test this idea, the impact of lead or cadmium toxicity on ethanol-induced changes in motor performance, operant responding, and thermoregulation will be studied. Experiment 1 will expose adult rats to a diet containing 500 ppm lead (as lead acetate) for 60 days prior to testing for ethanol-induced impairment of locomotor activity, roto-rod performance, and the aerial righting reflex. Experiment 2 will 'involve the same behavioral tests, but animals will be exposed to food containing 100 ppm cadmium (as cadmium chloride). Experiments 3 and 4 will examine the effects of metal exposure on the rate-depressant action of ethanol in an operant context (FR 32 water-reinforced responding). Experiments 5 and 6 will test for possible attenuation of ethanol-produced poikilothermia in lead-exposed and cadmium-exposed rats. Experiment 7 will examine the effect of lead and cadmium exposure on ethanol metabolism. In all experiments, a full range of ethanol doses will be used for testing.

Although P. carinii is a common opportunistic pathogen causing pneumonia in hundreds of thousands of immunocompromised patients, many basic questions about the organism and the disease it causes remain unanswered. Do different strains of P. carinii infect humans? Are patients with recurrent P. carinii infections suffering relapse or reinfection? Do strains resistant to antimicrobials exist and if so, what mechanisms of resistance occur? Answers to these questions may greatly influence clinical practice. Using modern tools of molecular biology and immunology and the greatly expanded patient pool resulting from the AIDS epidemic, these questions can now be addressed. The first aim of this proposal is to determine the degree of variation in the genome of P. carinii from patient specimens acquired in ACTUs at Indiana University and other ACTU sites. Sequences obtained from PCR (polymerase chain reaction) of up to 4 regions of the genome from 30 selected P. carinii isolates will be tested for differences related to geographical origin of patients. P. carinii in the 30 selected BAL (bronchoalveolar lavage) specimens will also be tested with monoclonal and polyclonal antibodies that discriminate among human isolates. The second aim is to compare isolates from patients with recurrent infection to isolates from their initial infection to determine if the recurrence is caused by relapse due to the same strain or reinfection with a different strain. Up to two hundred BAL specimens taken from patients suffering initial infections will be saved each year under conditions suitable to allow later use for PCR; these specimens serve as controls against which to compare isolates taken from later infections. Under the third aim P. carinii from specimens acquired in ACTUs at Indiana University and elsewhere will be compared for molecular differences that may alter susceptibility to trimethoprim/sulfamethoxazole. Portions of rat P. carinii dihydrofolate reductase gene will be used as a probe for PCR, to augment and isolate the dihydrofolate reductase gene from P. carinii in human BAL samples. Using RFLP (restriction fragment length polymorphism) and partial sequencing, homology of isolates from untreated patients and from patients failing therapy will be assessed. Any dihydrofolate reductase genes from human P. carinii showing different sequences will be expressed in dhfr E. coli and the functional consequences of these differences will be determined. The results of the experiments described should allow determination of 1) the inherent degree of genetic diversity in human P. carinii isolates from several geographical areas, 2) the possibility of reinfection versus relapse, 3) the molecular differences in P. carinii from patients failing therapy with trimethoprim/sulfamethoxazole.

DESCRIPTION: (Verbatim from the application) One of the most serious problems of mobility impairment is the tendency of old adults to fall. Falls account for almost 90 percent of all fractures in the elderly. The limitations in performing activities of daily living and falls are undoubtedly associated with alterations on postural muscles. Postural muscles are concerned with steadying a segment of the body on another in standing. This muscle subgroup exhibits a high content of slow muscle fibers. In the soleus muscle of many adult mammals the proportion of this type of fiber approaches 100 percent (more than 80 percent in humans). Although the crucial role in posture, the mechanical properties and the molecular mechanisms involved in single intact slow fibers contraction in the elderly or animal models of aging have not been explored. Unfortunately the information about age-related changes in fast-twitch fibers cannot be extrapolated to slow fibers due to substantial physiological, structural and biochemical differences between the two fiber subtypes. The hypothesis of this proposal is that the decrease in dihydropyridine receptor (DHPR) gene expression in type-1 fibers results in a decline in skeletal muscle force with aging. A lower number of DHPR renders a significant number of RyR1 unlinked and unable to be activated by sarcolemmal depolarization, leading to a substantial decline in sarcopasmic reticulum Ca2+influx and contractile force in aging slow-twitch muscles. This hypothesis will be assessed using the following specific aims: (1) To determine whether the decline in single skeletal muscle fiber contractility is associated with alterations in peak intracellular calcium concentration in single slow-twitch (type 1) muscle fibers from young (7months), middle-age (14), and old (28) C57BL/6 mice. (2) To establish whether the age-related impairment in type-1 fiber contractility results from a reduction in sarcoplasmic reticulum Ca2+ release. (3) To determine whether the age-related decline in functionally monitored DHPR is associated with a decrease in DHPR gene expression measured in the same type-1 single muscle fiber or whole soleus muscle, and (4) To define whether alterations in DHPR and/or RyR1 expression in slow muscle from aging mice results from a decline in nuclear transcriptional activity of DHPR alpha1, alpha2, beta, gamma and delta subunits and/orRyR1 DNA.

Proper connectivity of the central nervous system allows for communication between hemispheres necessary for such diverse functions as locomotion and linguistic processing. A clear understanding of how an individual axon growth cone makes a guidance decision during development is necessary to learn how reproducible connectivity patterns are accomplished in normal development. Knowledge as to how axon guidance receptors convert extracellular cues into a guidance choice will be essential to serve the long-term goal of guiding rewiring in developmental disease, neurodegenerative disease or injury states. This proposal is designed to provide mechanistic insight into the how the regulation of signaling of one axon guidance receptor, Roundabout (Robo) affects its repulsive guidance instruction. Determining whether and how the spatiotemporal profile of activated Robo affects growth cone guidance is a largely unexplored topic and may provide insight into general mechanisms of guidance receptor regulation. The proposed experiments use Drosophila genetics, biochemistry, and cell culture approaches to test the hypothesis that ligand-dependent internalization of Robo, by proteolysis and endocytosis, regulates the spatiotemporal dynamics of signaling within a growth cone during repulsive guidance. Knowledge of the mechanism by which an activated receptor informs wiring decisions will provide insight into optimal points of therapeutic intervention in developmental diseases of miswiring and repair following injury or neurodegenerative disorder.

Tremendous strides in the understanding of DNA structures responsible for expression of antibody molecules have been made through use of DNA cloning and sequencing techniques. In this research, we extended the recombinant DNA approach to the study of human immunoglobulin genes in normal and leukemic cells. We are characterizing the heavy chain genes for human IgM and IgD by isolating and sequencing genomic and cDNA clones of this region and have found that the human delta gene has three constant region domains and an extended hinge, while the mouse delta gene has only two constant region domains and a hinge. We studied the immunoglobulin heavy chain variable region (VH) genes in patients with familial chronic lymphocytic leukemia (CLL). We cloned the expressed partially sequenced VH genes from two brothers with familial CLL and showed that they express extremely similar or identical VH genes. This supports the concept that some aspect of the antigen receptor, such as a particular idiotype or affinity for a particular antigen or T-cell factor, is involved in development of this cancer. (AB)

Our long-term objective has been to develop noninvasive techniques for studying the human retina for both basic science and clinical purposes. By applying current theories of phototransduction to the full-field electroretinogram (ERG), we successfully developed widely used techniques for studying the global activity of the rod and cone receptors, as well as the rod bipolar cells. These indices of objective retinal function are appropriate outcome measures for treatment trials with systemically administered agents. However, many ongoing and anticipated clinical trials involving diseases of the photoreceptors require localized measures of retinal function, such as the multifocal electroretinogram (mfERG), static automated perimetry (SAP), and rod and cone-mediated fundus tracking perimetry, for evaluating treatment efficacy. With recent developments in retinal imaging, we can relate localized measures of visual function to the underlying structure. We have focused on frequency domain optical coherence tomography (fdOCT) and fundus autofluorescence (FAF). This work, supported by the current grant, has benefitted from our novel quantitative approaches for relating the thickness of retinal layers seen on fdOCT to other structural and functional measures. The upcoming focus will be on patients having Inherited Retinal Degenerations (IRDs), including STGD1, with identified genetic mutations. A primary goal is to provide insights into the pathophysiology and rates of progression in molecularly characterized patients with our novel imaging and functional measures. Present outcome measures in RP (primarily SAP and ERGs), and STGD1 (primarily visual acuity and FAF) are incapable of assessing change with modest sample sizes over a relatively short time interval, resulting in expensive and lengthy clinical trials. Our recent work with en face slab OCT represents a radical change in translational research in RP and STGD1. Our work in RP is virtually unique in the steps we are taking to establish quantitative OCT as a viable clinical trial outcome measure. As evidenced at the NEI-FDA endpoints workshop (NIH, Nov 9, 2016), there is considerable enthusiasm for an ?anatomical? endpoint for IRDs. Nevertheless, there is still much to be done to expand the measures to EZ area and to establish the relationship between outer retinal structural alterations (i.e. EZ area) and functional loss. Planned experiments will determine the extent, nature and progression of receptor loss by developing and evaluating novel en face slab methods for quantifying the receptor regions on wide-field OCT scans, relating OCT parameters to rod and cone SAP, developing models relating rod and cone sensitivity to quantitative measures of inner and outer segment length, outer nuclear layer thickness, and inner nuclear layer thickness, and using multimodal imaging methods including OCT, infrared (IR) reflectance and FAF to test hypotheses about disease mechanisms and models of disease progression. Note that these experiments apply equally to RP and to STGD1. Taken together, these studies continue to support novel and more efficient outcome measures for clinical trials.