Datasets:

haritzpuerto

/

the_pile_00_NIH_ExPorter

like 0

African Americans are underrepresented in clinical trials for breast and colon cancers. Public Health Television (PHTV), in partnership with the Case Comprehensive Cancer Center, proposes to increase clinical trial participation among low-income African Americans for these two cancers by developing DVDs to assist patient navigators with guiding low-income African Americans through a process of culturally relevant education about cancer treatment and clinical trials participation. Patient navigation refers to using lay members of the community to guide and support underserved populations through the process of obtaining diagnosis and treatment for cancer. NCI endorses patient navigation as a means to help underserved populations obtain more timely cancer treatment. PHTV will combine the concept of patient navigation with its proven process of culturally specific health communication to increase the rate of low-income African Americans participation in clinical trials for colorectal and breast cancers. Two specific aims will accomplish this objective. First, PHTV plans to produce a DVD series to assist patient navigators to guide low-income African American patients with an orientation to colon cancer treatment and participating in clinical trials. Second, PHTV will develop content for a DVD series to train patient navigators to assist low-income African American women with an orientation to breast cancer treatment and participation in clinical trials PHTV will identify and meet target group information needs with our proven culturally specific production process that uses members of the target population to design messages. Content outlines for DVDs will be developed with a culturally specific production process that uses focus groups and community meetings with low-income African Americans residing in Cleveland's public housing neighborhoods. A Patient Navigator Advisory Panel comprised of patient navigators working in urban sites across Ohio and in Harlem, NY will review content outlines for DVDs and validate feasibility. A Medical Advisory Panel comprised of representatives from cancer centers and the American Cancer Society will help formulate solutions to address barriers identified by target population and validate feasibility for evaluation plans that include a web-based tracking system. These efforts will enable PHTV to successfully launch and test a DVD based clinical trials navigator program in Phase II. [unreadable] [unreadable] [unreadable]

Recent evidence suggests that CD8+ T-lymphocytes of the Vbeta 3 or Vbeta 13.1 subsets are activated and clonally expanded in skin lesions of psoriasis vulgaris. This study is a clinical and pathological examination of potential disease-mediating functions of these T-cell subsets. The study involves administration of a bivalent peptide vaccine containing epitopes from Vbeta 3 and Vbeta 13.1 constant regions to patients with chronic psoriasis vulgaris. Part A of the study is concerned with clinical evaluation of outcomes. Part B of the study is concerned with assessment of T-lymphocyte subsets in psoriatic skin lesions before and during treatment, as assessed in skin biopsies.

Our goal is to achieve a comprehensive understanding of the neural circuitry of the cat retina. This seems realistic now that fundamental neuron "types" have been defined in cat retina, and it is realized that there are only three to five dozen. Seventeen types are already synaptically linked and twelve are associated with a specific transmitter. Our approach is to reconstruct adjacent retinal neurons from electron micrographs of serial sections. In such a series certain neurons are labeled beforehand by the accumulation of a tritiated transmitter, by a monoclonal antibody coupled to HRP, or by a marker of activity, such as the reaction product of cytochrome oxidase. The strength of this approach is in the detail gathered on adjacent neurons for they can be "typed" by multiple criteria and then linked in synaptic circuits. Data can be gathered quantitatively, permitting the development and testing of specific hypotheses regarding function. For example, we have demonstrated within a single series multiple synaptic connections between eight types of neurons. Among these is a Beta/X-on cell with input from two types of cone biolar, one of which may be inhibitory. The receptive field center of the Beta/X-on cell, we hypothesize, may be generated by excitation from a depolarizing cone bipolar and simultaneous withdrawal of inhibition from a hyperpolarizing bipolar. We shall test the qualitative anatomical predictions of this "push-pull" hypothesis using the methods described above. We shall also develop an electrontic model of the Beta/X-on cell in order to test a quantitative version of the hypothesis. We anticipate that additional hypotheses to explain the physiology will develop naturally as we delineate new circuits. When retinal circuits are truly understood, it should be possible tgo mimic them with integrated semiconductor circuits. Such feature-detecting devices may have broad industrial applications and will be medically useful, most obviously for the blind.

This study will test safety & assess dose levels of the mouse monoclonal antibody D612 in patients with metastatic gastrointestinal cancer. Patients will be treated in groups of three with each group receiving a higher dose of antibody than the previous group. Each patient will also receive a radiolabeled antibody dose followed by imaging.

Artificial intelligence and machine vision systems often use shape analysis as a fundamental approach to characterization of complex objects and motion. Such concepts have been used to advantage in industry but have not been extensively exploited for cardiac diagnosis. Aims: (1) To develop a method of quantitative regional function analysis based on analysis of dynamics of ventricular shape that will replace traditional methods used to assess wall motion so that assumptions regarding coordinate, reference and indexing systems, geometry of the ventricle and uniformity of its contraction pattern can be abandoned. (2) To quantify the regional shape of the normal right and left ventricles and establish the patterns of shape abnormality seen in the setting of coronary artery disease. (3) To establish the relationship between regional shape and regional function. (4) To develop a clinically useable, automated method for ventricular feature extraction and determine its usefulness in predicting the presence and location of coronary disease by analysis of ventricular images obtained during an ischemic stress. (5) To assess the prognostic significance of regional shape analysis. (6) To automate the requisite edge detection by using integrative methods of artificial intelligence. The hypothesis is that the normal ventricle has a characteristic shape and rate of shape change throughout the phases of the cardiac cycle and that abnormal ventricles have abnormal shapes and shape changes by virtue of abnormalities of function. Therefore, quantitation of abnormalities of shape parameters will provide an indirect measure of ventricular dysfunction. This will be determined by frame-by-frame curvature analysis of cineventriculograms in patients with normal and stenotic coronary arteries. Animal studies will compare results of regional thickening and thinning determined by implanted sonomicrometers with shape changes assessed from simultaneous contrast ventriculograms and ventricular outlines determined from implanted subendocardial lead pellets. The success of this approach will allow abandonment of numerous controversial assumptions mandated by traditional approaches and will solve a long-standing problem in cardiology. The concepts to be explored will provide new and unique information of potential prognastic importance and it will advance the neglected field of computer vision in the study of non-rigid bodies in motion.

This is an application for funds to continue and expand the Computer Graphics Facility at Columbia University. The material which follows is organized into four major sections as follows. Section I. Gives a brief history of the Facility and describes why we believe that it should be expanded and how we propose to increase the services we provide for users outside New York City. Section II. Gives a detailed justification and explanation of the budget requested in this application. Section III. Gives a summary of the work which has been carried out in the Facility in the general areas of molecular structure and the relationship between molecular structure and biological function. It also describes the present and proposed activities in this area in both core and user research. Section IV. Gives a summary of the work which has been carried out in developing and using CARTOS, the system for Computer Reconstruction by Tracing of Serial Sections. In addition we describe the developments which we propose to continue and extend as part of the core research of the Facility and how these developments should contribute to the scientific activities in neurobiology which the Facility supports.

Arterial pressure (AP)and regional blood flows are finely tuned by the central nervous system CNS) via the synpathetic system. This neural control is essential to match circulation with respiration and behavior. It is also critical to the maintenance of a stable level of AP. Most of the work will be focused on a region of the brain (rostral ventrolateral medulla, RVLM) that plays a critical role in regulating sympathetic tone and AP. RVLM also deserves intensive study because it is the main site of action of numerous drugs that have actions either beneficial or detrimental to circulation. RVLM controls sympathetic tone via a small group of neurons (BS neurons) that project to the spinal cord. A key to understanding how RVLM controls AP is to find how BS neurons are inturn regulated and what type of transmitters they release. A third of the work proposed in this renewal is to analyze with state of the art patch-clamp recording methods the local neuronal cicuitry that regulates the activity of BS neurons. We will first identify the major synaptic inputsof these neurons. We will then examine in detail how catecholamines regulate their excitatory inputs since our preliminarydata suggests that theantihypertensive clonidine and its new imidazoline congeners may work by mimicking the effect of noradrenalirie at this level. Next we will study the presynaptic regulation of the synaptic inputs of BS neurons by other substances of pharmacological or toxicological interest like neuropeptides, nicotine and opiates. We will then determine whether RVLM contains interneurons that regulate the discharges of the BS neurons and we will identify the transmitters used by these interneurons. A second major aspect of the work will be to analyze the structure of various subclasses of BS neurons and to identify the type of transmitters that they release. This will be done with a new method that permits to localize an enzyme or an mRNA within a single neuron physiologically characterized "in vivo". In the last part of the work we will examine the possibility that sympathetic tone could also be regulated by GABAergic inhibitory inputs that originate from the brainstem and,in particular, from the nucleus of the solitary tract. The research will contribute to the basic physiological knowledge needed to understand how syrr pathetic tone is regulated in health and in diseases such as arrhythmias, hypertension and dysautonomias.

The focus of our structural studies is the E. coli Rep helicase. The crystals are tetragonal and diffract to a resolution of 4 E. We hope to extend the resolution of diffraction so that structure determination may be achieved at moderate atomic resolution by multiple isomorphous replacement technique. This study is the first attempt to solve the structure of any helicase using X-ray crystallography. The crystal structure determination of Rep helicase will contribute significantly to our understanding of the mechanism by which DNA is unwound.

Post-translational fatty acylation of internal protein residues is a powerful but poorly understood biological mechanism for altering a protein's behavior. It is important in diverse physiologic strategies, for example, signal transduction The activation of Escherichia coli hemolysin (HlyA) toxin is used in this proposal as a model to study protein internal acylation. Pathogenic E.coli produce nontoxic proHlyA which is made toxic by the post-translational addition of 2 fatty acyl groups to internal residues, causing an extraordinary change in protein behavior. Acyl-acyl carrier protein (ACP) is the obligatory acyl-donor. HlyC is the internal protein acyltransferase, which forms an acyl-HlyC intermediate, evidence suggests an acyl-histidyl HlyC. Using a precise and direct assay, the reaction employing separately subcloned, purified proteins is a unique opportunity to explain the biochemical mechanism of an internal protein acyltransfer (the only one to be so studied) and its role in changing protein behavior. The following specific aims will be pursued with the goals of further understanding the biochemistry of protein internal residue acylation and associated changes in protein behavior and what defines an internal protein acylation site: 1. Site- directed mutation analysis, site-directed fluorescence, and chemical modification will be used to define the roles of several residues shown to be important in HlyC's catalytic function, and the acyl-HlyC intermediate will be characterized. 2. The reaction is likely the sum of 2 partial reactions, a ping pong kinetic mechanism; the reversibility of the first has been shown. Using HlyA labeled with selected radioactive fatty acyl groups, the reversibility of the second partial reaction will be shown. 3. Site-directed mutations of subcloned proHlyA, proHlyA- fragments containing either both or one or the other acylation sites, HlyA, and the respective acylated HlyA fragments will be studied to learn what defines an internal protein acylation site and what changes a protein undergoes upon acylation. Alterations in biological activity and fluorescence characteristics of site-directed fluoroprobes will be observed. E. coli hemolysin typifies one of a family of homologous, similarly activated, infectious protein toxins produced by diverse Gram negative bacteria. The toxins have remarkable organism and cellular specificities. The hemolysin scheme is an important recurring biological motif of rendering a protein toxic and secreting the infectious cellular-specific protein. Insight into the mechanism of this unique acyltransfer is of practical importance in providing a rationale for design of inhibitors of toxin activation as a potential therapeutic approach to severe infections with pathogenic E. coli where antibiotic therapy and subsequent toxin release worsen the clinical outcome.

VA and NHLBI to support the Systolic Blood Pressure Intervention Trial (SPRINT), a multicenter clinical trial to determine whether maintaining blood pressure levels lower than current recommendations further reduces the risk of cardiovascular disease, chronic kidney disease, dementia and age-related cognitive decline.

Neurocysticercosis (NCC) is a parasitic infection of the human central nervous system caused by the helminth Taenia solium. NCC is recognized as a leading cause of seizures worldwide. Seizures in NCC are evoked by localized granulomatous responses to dying parasites in the brain. The mediators of the seizures are unknown, identification of seizure mediator(s) in NCC may result in treatment with specific antagonists. The neuropeptide substance P (SP) stimulates granuloma growth and Th1 cytokine production. Another neuropeptide, somatostatin, stimulates Th2 cytokine production and impairs granuloma formation. SP evokes epileptiform responses in neurons, whereas, somatostatin has anticonvulsant properties. We divided granulomas associated with murine cysticercosis into 4 stages based on the histologic appearance of the degenerating parasite. Early stage granulomas expressed Th1 cytokines and SP, whereas Th2 cytokines and somatostatin were only expressed in later stages. Preliminary results also noted that behavioral seizures and increased hippocampal activity were induced when extracts from early granulomas were injected into brain of rats. Pretreatment with SP receptor antagonist inhibited these effects. Similarly, injection of SP into rat brain also induced seizures and altered hippocampal activity that was completely blocked by pretreatment with SP receptor antagonist or somatostatin. We hypothesize that SP mediates and somatostatin inhibits the granulomatous response and seizures in NCC. Specific aim 1: To test the hypothesis that SP and somatostatin modulate granulomatous responses in cysticercosis. Granuloma size, Th1/Th2 and pro-inflammatory cytokine levels in infected, wildtype mice, SP knockout mice (SP KO), SP receptor KO mice and somatostatin KO mice will be compared. Specific aim 2: To determine if SP and somatostatin are respectively responsible for the mediation and modulation of seizure responses in NCC. SP protein expression will be examined in brain biopsies from NCC patients with seizures. Epileptogenic activity of granuloma extracts from infected, SP KO and somatostatin KO mice will be compared to that from wildtype mice. Specific aim 3: To determine if seizures in NCC are directly due to SP and/or indirectly due to SP induced cytokines. Epileptogenic activity of early granuloma extracts will be tested with or without inhibition or blocking of SP, IL-1beta, TNF-alpha or IL-6. Also epileptogenic activity of early granulomas from infected IL-1beta, TNF-alpha or IL-6 knockouts will be tested. Specific aim 4: To test the hypothesis that somatostatin inhibits seizures in NCC. Epileptogenic activity of early granuloma extracts with or without somatostatin analogues or SOM antagonist will be studied. These studies will determine the importance of SP and SOM in pathogenesis of NCC, and may lead to future use of SP antagonist and SOM analogues as anti-epileptic agents for treatment of seizures in NCC and other seizure related diseases.

The aim of this protocol is to determine whether the increase in leucine oxidation tha tis seen in insulin dependent type I diabetes is attributable to the hyperglucagonemia that occurs during insulin deficiency.

Estrogen hormone action in target tissues of the body involves the interaction of the hormonal substance with a receptor protein. The specificity and responsiveness of tissues to hormonal stimulation are governed in most part by the presence and biochemical action of this receptor protein. Nuclear localization of the receptor protein and its activity was shown to be increased by exposure to growth factors. suggesting a coupling of these two signaling pathways. Gene regulation studies using mutant proteins in transfected cells show that growth factors regulate activity through the N-terminal region of the receptor while estrogen acts through the C-terminal end. Estrogen has an associated effect on bone homeostasis which has now been shown to occur through a receptor mediated mechanism, Prenatal or neonatal estrogen exposure of mice has shown for the first time a highly sensitive and significant influence on adult bone density. Bone cells have extremely low estrogen receptor levels and inconsistent response to estrogen. Estrogen was shown to specifically stimulate exogenous and endogenous genes in the cell lines. Regulation of responsiveness was shown to be influenced by cellular protein kinase C activity by decreasing receptor binding levels with no influence on the receptor gene or protein. This example of signal coupling illustrates an additional level of regulation of hormone response than previously thought. The finding also helps to explain the multi hormonal effects on bone homeostasis. Estrogen receptor is being expressed in yeast for analysis of the gene transcriptional activity of the protein using a variety of hormonally regulated promoter sequences linked to beta-galactosidase reporter genes. Site directed mutagenesis of the ligand binding domain of the receptor protein has identified specific amino acids which influence transactivation activity with no effect on ligand binding. Combined site directed and deletion mutagenesis has shown a clear interaction between the N and C terminal regions of the receptor related to its transcriptional activity. These findings have isolated a peptide region in the N terminus of the receptor which interacts with a specific amino acid in the ligand binding site. Such observations are needed in order to determine the structural conformation of the estrogen receptor related to hormone activation by a variety of estrogenic compounds to explain differential hormonal agonist activity in various estrogen target tissues.

The goal of this project is to investigate whether particular toxicants, including some putative endocrine disrupting chemicals, can increase intracellular estradiol concentrations by inhibiting estradiol metabolism and depuration, and thus increasing estradiol-responsive growth. Further, the role of the Pregnane X-receptor (PXR) and its ligands in regulating estradiol metabolism and elimination will be investigated. Steroid hormone metabolism and depuration is controlled by the same enzymes that metabolize and eliminate toxicants in phase I, II and III reactions. Perturbing these metabolic processes can result in toxicant interactions, as well as, perturb steroid hormone homeostasis. MCF-7 breast cancer cells will be stably transfected with the PXR receptor and putative PXR ligands, such as 4-nonylphenol, DEHP (antagonists) and rifampicin (agonist) will be used to determine whether PXR ligands can significantly alter intracellular estradiol homeostasis and increase estrogen-responsive proliferation. The PXR regulates induction of CYP3A and MRP2 and may be involved in the induction of glucuronosyltransferases, as well. CYP3A is a major steroid hydroxylase, glucuronosyltransferases are important in conjugating steroids and MRP2 is a steroid hormone transporter. Hence, these enzymes are important in metabolizing and eliminating estradiol. Recent evidence in our lab and others suggests that some chemicals may bind the PXR and act as antagonists. Our hypothesis is that these chemicals, including the endocrine disrupters, DEHP and 4-nonylphenol, can down-regulate estradiol metabolism and depuration in this manner, thus increase effective estrogen concentrations and induce proliferation. This alternate mechanism of endocrine disruption could cause developmental abnormalities and/or cause estrogen-responsive cancers, such as breast and ovarian cancer.

Rotator cuff tears (RCTs) are an extremely common cause of shoulder pain and disability. Up to 20% of patients greater than the age of 50 years of age have evidence of a rotator cuff tear. In addition, patients with asymptomatic cuff tears tend to progress to larger, symptomatic tears. The outcomes of surgical repair of small RCT are good, but there has been limited success in the surgical treatment of massive RCTs. Massive RCT have been found to be associated with atrophy of the rotator cuff muscles. In addition, massive RCT are associated with the development of fatty infiltration, a phenomenon that appears to be unique to the rotator cuff in humans. Importantly, patients with large RCT with atrophy and fatty infiltration have poorer clinical outcomes than those that do not have atrophy and fatty infiltration. Thus, it appears that the natural history of large rotator cuff tears is th development of muscle atrophy and fatty infiltration, which leads to poor patient outcomes. The molecular mechanisms that lead to the development of rotator cuff muscle atrophy and fatty infiltration have not been defined. The purpose of this study is to evaluate the role of specific pathways that are critical for the development of atrophy and fatty infiltration in an animal model of rotator cuff tears. We will establish the likely pathways critical to the development of muscle atrophy and fatty infiltration. We will subsequently determine how these pathways are modified by muscle denervation, since this feature is likely a key modifier of the relevant pathways. We will focus on key pathways that have been found in other models to regulate muscle atrophy and expression of fat related genes. Specifically, we will evaluate the Akt/mTOR pathway as it relates to muscle atrophy, and the PPARy-gamma pathways are they relate to fatty infiltration. Understanding these mechanisms may allow for therapeutic modalities that would allow for inhibition or even reversal of the fatty infiltration and atrophic process following repair of massve RCTs. PUBLIC HEALTH RELEVANCE: Rotator cuff tears are one of the most common orthopaedic conditions, affecting up to 20% of patients greater than 50 years of age. Large rotator cuff tears do not heal, result in poor shoulder function, and lead to irreversible muscle changes. The purpose of this study is to understand the expression of specific proteins that lead to the irreversible muscle changes found in the setting of large rotator cuff tears.

The oncogene c-myc is abnormally expressed in several forms of human cancer, notably Burkitt's lymphoma, colorectal carcinoma and promyelocytic leukemia. It is known that this gene is highly expressed in these cells, due to its amplification or chromosomal translocation, but how this results in abnormal cellular growth has not been clear. Recent work in this laboratory indicated that c- myc protein participates in DNA replication of human cells. Thus, overexpression of this gene product would confer a proliferative advantage to the cell, by making it less dependent on external growth stimuli. It is proposed to exploit this finding to determine more precisely the step or steps in DNA replication which are dependent on the c-myc gene product. The DNA replication complex will be dissociated and reconstituted following an exposure to affinity-purified polyclonal and monoclonal antibodies to c-myc (alpha-myc). Re-addition of individual components of the replication machinery will identify cellular proteins that react with alpha-myc. Studies with replicating molecules of SV 40 virus will provide conditions analogous to an amplified mammalian replication unit, and thus permit a closer analysis of the stepwise maturation of nascent DNA. The role of oncogene c-fos, in cells which express this oncogene, in DNA replication or maintenance of chromatin structure will be addressed by sequential immunocytochemical and in situ hybridization studies, and related to DNA replicative activity. These studies should provide a beginning for the clarification of the physiological functions of oncogenes encoding nuclear phosphoproteins.

The summer course ?Molecular Mycology: Current approaches to Fungal Pathogenesis?[unreadable] has been offered at the Marine Biological Laboratory since 1997 with the purpose to[unreadable] make the area of Medical Mycology accessible to those whose training involved other[unreadable] organisms, and to provide molecular training for classical Medical Mycologists. There is[unreadable] a great need for effective training of researchers who can address the molecular aspects[unreadable] of fungi in an effort to prevent, diagnose and treat infections caused by the fungal[unreadable] pathogens in immuno-compromised individuals. Eighteen students who are outstanding[unreadable] graduate, post-graduate and experienced investigators are immersed in an intensive[unreadable] three-week lecture-laboratory intensive schedule. The aims of the course are to provide[unreadable] the students with: an introduction to the biology and pathogenesis of medically-important[unreadable] fungi; laboratory training in manipulation of these fungi, especially the applications of[unreadable] molecular and classical genetics, and assays of virulence and host-pathogen interaction;[unreadable] and a broad perspective on experimental issues pertinent to these fungi, such as the[unreadable] definition and determination of virulence, the determination of host responses relevant to[unreadable] infection, and the identification of antifungal drug targets. The course provides an[unreadable] introduction to the biology and pathogenesis of medically important fungi, including[unreadable] Candida albicans, Cryptococcus neoformans, and Aspergillus fumigatus. Specific[unreadable] laboratory exercises include biolistic transformation and manipulation of the[unreadable] Cryptococcus genome, tetrad dissection and meiotic analysis in fungal pathogens[unreadable] having sexual cycles, the use of gene disruption in analyses of gene function, gene[unreadable] cloning strategies, in vivo pathogenecity assays, and the identification of antifungal drug[unreadable] targets. During a relatively short period of time, this course has established a strong[unreadable] reputation in the field. Both students and faculty are of the highest quality. Funding is[unreadable] requested for partial support to continue this unique training opportunity in its present[unreadable] form.

Erythropoietin (Ep) is the glycoprotein hormone which is responsible for maintaining erythropoiesis in animals and man. Of many putative growth factors affecting hematopoiesis, Ep is the only one with a defined physiological role. However, understanding of the mechanisms of Ep's action on responsive cells has been limited due to 1) insufficient quantities of pure Ep, 2) lack of appropriate laboratory probes and 3) difficulties in obtaining sufficiently enriched populations of target cells for study. We have purified Ep in microgram amounts and we propose to utilize recent developments in molecular and physiological interactions to study the biochemistry of Ep and its molecular and cellular-biology with responsive cells. We will use monoclonal and polyvalent antibodies to Ep and to defined polypeptide sequences to further purify Ep by immunoaffinity chromatography. As larger quantities of Ep become available, we will determine its biochemical properties and amino acid sequence. We will develop site-specific antibodies to Ep, both polyvalent and monoclonal, to immunologically characterize the molecule. Functional properties will be correlated with antibody-defined structural domains. We will clone the Ep coding sequences from anemic baboon kidney. The cDNA libraries will be screened by immunological methods using anti-Ep antibodies and oligonucleotide probes developed from amino acid sequence data. As the protein sequence is determined, it will be compared to the nucleotide sequences for Ep coding regions. Concurrently, we will attempt to develop labeled, biologically active Ep probes using different iodination schemes, tritium labeling of carbohydrate and amino groups, photoaffinity labeling, and chemical derivatization. Alternatively, we will use labeled or flouresceinated antibody or Fab fragments to identify Ep. With the acquisition of additional purified Ep as well as biologically active probes we will purify populations of Ep-responsive cells and begin studies to identify the Ep receptor and its properties. From the sequence of experiments outlined in this proposal we can expect greater understanding of Ep, its protein structure, its genomic coding sequences, identification of target cells bearing presumed receptors and characterization of Ep interactions with the receptor. These studies may serve as a model for understanding the function of other hematopoietic growth factors and, ultimately, may lead to therapeutic benefit in disorders accompanied by Ep deficiency.

Consumption of n-3 polyunsaturated fatty acid (PUFA) supplements enriched in 20:5 n-3 (EPA) and 22:6 n-3 (DMA) has been shown to reduce coronary heart disease (CHD) and plasma triglyceride (TG) concentrations. Despite the beneficial effects of n-3 PUFAs, consumption in the American population remains low. Vegetable oils containing 18:3 n-3 (alpha-LNA) have only limited impact on tissue concentrations of EPA and DHA, since the conversion of alpha-LNA to EPA is limited by the inefficiency of the delta-6 desaturase-catalyzed step. One strategy to enrich tissues in EPA and DHA is to use botanical oils that are enriched in fatty acids that bypass the delta-6 desaturase step of fatty acid elongation and desaturation. Echium oil, derived from the seeds of Echium Plantagineum, is enriched in stearidonic acid (SDA;18:4 n-3), which is the immediate product of delta-6 desaturation of alpha-LNA. Our preliminary studies in hypertriglyceridemic human subjects have demonstrated that echium oil supplementation resulted in lower plasma TG concentrations. The goals of this project are to determine the mechanisms by which echium oil reduces plasma TG concentrations and whether echium oil supplementation will retard atherosclerosis progression relative to palm oil and fish oil (controls). The moderately hypertriglyceridemic apoB100 only-LDL receptor knockout (B100-LDLrKO) mouse will be used of these studies. In specific aim 1, we will test the hypothesis that echium oil supplementation will reduce plasma TG concentrations to a degree similar to that observed with fish oil supplementation. In specific aim 2, we will test the hypothesis that echium oil supplementation decreases plasma TG concentrations by down regulation of genes involved in hepatic TG synthesis, resulting in less hepatic TG synthesis and reduced VLDL TG secretion. In specific aim 3, we will test the hypothesis that echium oil supplementation will increase the lipolysis and catabolism of plasma TG enriched lipoproteins. In specific aim 4. we will test the hypothesis that echium oil supplementation will reduce aortic atherosclerosis. If echium oil supplementation proves to be atheroprotective, it could be used in conjunction with dietary guidelines to meet the AHA suggested intake of n-3 fatty acids necessary for cardioprotection.

The specific aim of the investigation is to correlate the optimal temperature for growth with the compositions of helical and non-helical segments of 16s and 23s rRNA from selected psychrophilic, mesophilic, thermophilic, and extremely thermophilic prokaryotes. The compositions of the isolated and fractionated RNA's are being determined by ion exchange chromatography and spectrophotometric characterizations. The extents of secondary structure are being assessed by the ability of monoperphthalic acid to react with an RNA component, viz., adenine residues. In helical domains (i.e., hydrogen bonded) the adenine is less accessible than the adenine residues in the non-hydrogen-bonded regions. This condition allows an assessment of the organization of the ribopolymer.

In December, 1988, the National Cancer Institute sponsored a workshop to develop uniform descriptive diagnostic terminology for cervicovaginal cytopathology to replace the Papanicolaou Class designations. The proceedings of that meeting, known as the 1988 Bethesda System, have had a significant impact on the practice of gynecologic cytopathology. A survey conducted in early 1991 by the College of American Pathologists, revealed that 87% of the labs surveyed had already implemented TBS (in whole or in part), or were planning to do so in the near future. Two years after its initial publication, the time had come to critically evaluate the advantages and disadvantages of TBS in actual laboratory practice. A "Second Conference" on The Bethesda System was held April 29 and 30, 1991, at the National Institutes of Health in Bethesda, Maryland with a capacity attendance. The two day meeting organized by the Cytopathology Section, provided open exchange of data, lively debate and a forum for critical analysis of TBS. An Editorial Committee was established to revise TBS terminology based on this input as well as written commentaries, scientific data, and laboratory surveys. The revised TBS has been significantly streamlined and simplified. A Criteria Committee is completing a reference atlas which will include morphologic criteria and accompanying photomicrographs. I have been an active participant in both committees. In addition, I have been involved as a cytopathology resource person in numerous meetings and working groups including: The College of American Pathologist's Cytopathology Committee; Executive Committee of the American Society of Cytology; several CDC workshops on quality assurance in breast and cervical cancer; a CDC expert panel on cervical disease in HIV-infected women; a videodisc collaboration with the National Library of Medicine on cervical cancer; and the development of the National Strategic Plan for Breast and Cervical Cancer.

Administration Core The Administration Core of the MCDTR provides leadership, infrastructure, and resources to: Raise awareness of, and interest in, type 2 translational research in diabetes and create an environment that supports such research. Support new and established investigators engaged in research to translate interventions with proven efficacy into real worid health care settings, communities, and populations at risk. Administer Cores that provide Center members with services critical to type 2 translational research in diabetes. Provide education and training and to foster interdisciplinary collaborations. Administer the Center's P/FS Grants Program. Maintain the Center's website. The Administration Core is responsible for identifying and supporting researchers engaged in type 2 translational research; for developing and implementing Cores to provide services to funded T2 translational researchers locally, regionally, nationally and internationally; for overseeing the allocation of Center resources; for implementing an Enrichment Program; for implementing a process for solicitation, review selection, monitoring, and follow-up of the Center's Pilot and Feasibility Study Grants; and for developing and maintaining a website that integrates seamlessly with the NIDDK Centers program website.

The Genomics Shared Resource (GSR) located in the Eckstein Medical Research Building adjacent to the Holden Cancer Research Laboratories supports research performed by investigators in all HCCC programs. It provides a broad spectrum of services and resources designed to make the state-of-the-art techniques used in DNA sequence and transcript analysis readily available to the UI research and clinical communities. The research-based services and resources include: 1) DNA sequencing and genotyping, 2) custom oligonucleotides, 3) DNA microarrays using both the Affymetrix GeneChip and Illumina BeadArray systems, 4) real-time PCR, 5) molecular biology computing, and 6) genome sequencing using next-generation sequencing (NGS) technologies. The clinically based services include whole exome sequencing (WES). It is the aim of the GSR to provide high quality services to HCCC members with a rapid turnaround, and to support these services with well-trained personnel. GSR also continues to evaluate and update services to remain cutting-edge and provide new services needed, to continue to meet the needs of HCCC investigators. In 2014, the GSR provided services to 124 HCCC members, including members from each of the HCCC programs.

Control of neurotransmitter receptor function may occur by a previously undescribed mechanism: serine protease cleavage. Preliminary data show that thrombin can cleave the NMDA receptor NR1 subunit in transfected cells and brain homogenates. Interestingly, mRNA for the precursor to thrombin is found widely distributed in the developing and adult brain. Pathologies associated with disruptions in the blood-brain barrier cause an elevation in the concentration of thrombin in cerebrospinal fluid. Both the NMDA receptor and thrombin are implicated in brain damage associated with several pathologies, including stroke, head trauma, and Alzheimer's Disease. The interaction of these two proteins may exacerbate disease states. The goal of this project is to define the physiological consequences of thrombin proteolysis of the NR1 subunit of the NMDA receptor. Toward this end, experiments are designed (a) to examine the effects of thrombin proteolysis of the NMDA receptor on receptor function, localization and turnover; (b) to define the thrombin-sensitive site of the NR1 subunit; (c) to find whether thrombin sensitivity is unique to the NR1 subunit in the glutamate receptor family; and (d) to determine if thrombin proteolysis affects NMDA receptor-mediated neurotoxicity. This research project has the potential to uncover a novel mechanism for control of NMDA receptor function during a variety of brain pathologies, and may suggest new strategies for developing better treatments for these diseases.

Several epidemiologic studies have identified infection with oncogenic type human papillomavirus (HPV) as a necessary cause of cervical cancer. More than 99% of cervical cancers contain genes of "high risk" HPVs, of which there are at least 15 different types. Our goal is to eliminate HPV-related cancer through the development of a single vaccine that is protective against all oncogenic HPV types. The two HPV capsid proteins, L1 and L2, are both independent protective antigens. Vaccination with HPV L1 virus-like particles (VLP) induces neutralizing antibodies and protection in patients with strong type restriction. Broad protection against the >15 known oncogenic HPVs is necessary for the eventual cessation of cytologic screening and eradication of cervical cancer. Broad protection may require an expensive highly multivalent L1 VLP vaccine, but current commercial clinical studies utilize VLPs from only two oncogenic types. We propose L2 as a single conserved protective antigen. Protection in animal models is mediated by L2 neutralizing antibodies which also cross-neutralize diverse HPV genotypes. Vaccination of cattle with bovine papillomavirus type 4 (BPV4) L2 11-200 protects against BPV4 challenge on the soft palate. Furthermore, vaccination of rabbits with HPV16 L2 11-200 protects against both cutaneous infection with cottontail rabbit papillomavirus (CRPV) and mucosal challenge with rabbit oral papillomavirus (ROPV). Thus, vaccination with L2 11-200 produced in bacteria protects against both the homologous virus type as well as evolutionarily distant heterologous types supporting the possibility of an L2-based pan-oncogenic HPV vaccine. Unlike currently available L1 VLP vaccines, a single L2-based antigen produced in E. coli is inexpensive to produce. As such, a pan-oncogenic HPV type that is less costly to produce would have its greatest impact in underserved areas in the US and in developing nations. The Rapid Access to Preventive Intervention Development (RAPID, NCI) program is producing GMP-grade HPV 16 L2 11-200 for this proposed clinical trial. HYPOTHESIS 1: Vaccination of patients with HPV16 L2 11-200 polypeptide is safe with or without adjuvant. Specific Aim #1: To evaluate whether vaccination using GMP grade HPV16 L2 11-200 with and without adjuvant is safe in healthy women. HYPOTHESIS 2: The HPV16 L2 11-200 polypeptide is immunogenic in patients and formulation with adjuvant enhances its immunogenicity. Specific Aim #2: To determine which formulation and minimal dose of GMP grade HPV16 11-200 polypeptide with adjuvant induces the maximal titers of neutralizing antibody and also the spectrum of HPV types neutralized by sera of patients vaccinated with the optimal formulation of GMP grade HPV16 11-200 polypeptide. HYPOTHESIS 3: Transudation of L2-specific HPV neutralizing antibody into the genital tract is the relevant correlate of protection. Specific Aim #3: To determine whether passive transfer of mice with L2-specific human IgG or IgM will confer protection from vaginal challenge of mice with HPV pseudovirion and determine the minimal neutralizing antibody titer for protection. PUBLIC HEALTH RELEVANCE: Several clinical and molecular epidemiologic studies have identified infection with oncogenic type human papillomavirus (HPV) as a necessary cause of cervical cancer. Unlike currently available L1 VLP vaccines, vaccination with L2 11-200 produced in bacteria protects against both the homologous virus type as well as evolutionarily distant heterologous types, supporting the possibility of an L2-based pan-oncogenic HPV vaccine. Furthermore, it is known that HPV type distribution is markedly different throughout the world and a pan-oncogenic L2 HPV vaccine, that is substantially cheaper to produce in E. coli, may have a substantial global impact, particularly in developing nations and low resource settings where 80% of cervical cancer occurs.

Juvenile periodontitis is a disease of teenagers and young adults characterized by rapid alveolar bone loss. An accumulation of microbiologic and immunologic evidence strongly suggests an association of the gram-negative rod, Actinobacillus actinomy-cetemcomitans with juvenile periodontitis. The exact role of this bacteria in the disease, if any, is not known. The objective of the work in this proposal is to make and begin to utilize monoclonal antibodies with specificity for Actinobacillus-antigens. Somatic cell hybridization techniques will be used to fuse antigen-sensitized Balb/c mouse splenic cells with the plasmacytoma, Sp2/0. Hybridomas synthesizing monoclonal antibodies directed against Actinobacillus will be detected and cloned. Monoclonal antibodies that identify antigens which are species-specific, strain-specific, and indicative of a group of A. actinomycetemcomitans isolates will be characterized. In addition, monoclonals against antigens on the Actinobacillus-associated leukotoxin will be made and utilized to examine the leukotoxin-host cell interactions. Well characterized monoclonals will be used to investigate the immunobiology of all available A. actinomycetemcomitans isolates and to examine the feasibility of using monoclonal antibodies to identify and localize A. actinomycetemcomitans-associated antigens on cells, in plaque, and in tissue. The information on the antigenic character of A. actinomycetemcomitans isolates and the monoclonal antibodies will prove to be of value in the microbiological and immunological approach to detection, treatment, and prevention of juvenile periodontitis.

We study transcriptional and translational mechanisms involved in nutrient control of gene expression in the yeast Saccharomyces, focusing on a regulatory system that induces genes encoding amino acid biosynthetic enzymes in response to starvation for amino acids. The transcriptional activator in this pathway, GCN4, is induced at the translational level in starved cells by phosphorylation of initiation factor 2 (eIF2) by the protein kinase (PK) GCN2. Phosphorylation of eIF2 reduces the concentration of the ternary complex (TC) containing eIF2, GTP, and initiator methionyl-tRNA, which transfers tRNAiMet to the 40S ribosome. This impedes general protein synthesis but induces GCN4 translation by a reinitiation mechanism involving small upstream open reading frames (uORFs) in the GCN4 mRNA leader. A reduction in TC levels allows 40S ribosomes scanning the GCN4 mRNA leader after translating uORF1 to bypass uORFs 2-4 and reinitiate at the GCN4 start codon instead. GCN2 is activated in starved cells by binding of uncharged tRNA to a histidyl-tRNA synthetase (HisRS)-like region which functions as a sensor of amino acid availability. Activation of GCN2 also requires the GCN1-GCN20 complex that binds to the N-terminal domain (NTD) of GCN2. GCN1 interacts with ribosomes in cell extracts, but it was unknown whether this activity is crucial for its ability to stimulate GCN2 function in starved cells. We isolated point mutations in two conserved, noncontiguous segments of GCN1 that lead to reduced polyribosome association by GCN1-GCN20 in living cells without reducing GCN1 expression or its interaction with GCN20. Mutating both segments simultaneously produced a greater reduction in polyribosome binding by GCN1-GCN20 and a stronger decrease in eIF2 phosphorylation than did mutating one segment alone. These findings provide strong evidence that ribosome binding by GCN1 is required for its role as a positive regulator of GCN2. A particular mutation in the GCN1 domain, related in sequence to translation elongation factor 3 (eEF3), decreased GCN2 activation much more than it reduced ribosome binding by GCN1. Hence, the eEF3-like domain appears to have an effector function in GCN2 activation. This conclusion supports the model that an eEF3-related activity of GCN1 influences occupancy of the ribosomal decoding site by uncharged tRNA in starved cells. We showed previously that the wild-type GCN2 PK domain is functionally inert when separated from the HisRS domain, but can be activated by replacement of Arg794 with Gly in the PK domain (R794G). We have determined the crystal structures of the PK domain for wild-type and R794G mutant forms both in the apo state and bound to ATP/AMPPNP. These structures suggest that GCN2 autoinhibition results from stabilization of a closed conformation that restricts ATP binding. The R794G mutant shows increased flexibility in the hinge region connecting the N- and C-lobes, resulting from loss of multiple interactions involving R794. This conformational change is associated with intra-domain movement that enhances ATP binding and hydrolysis. We propose that intramolecular interactions following tRNA binding remodel the hinge region in a manner similar to the mechanism of enzyme activation elicited by the R794G mutation. Our GCN2 PK structures lack autophosphorylation of Thr882 in the activation loop, an event critical for PK function, and the configuration of active site residues suggests how autophosphorylation facilitates a second step in kinase activation. The orientation of alpha helix C in the PK domain is incompatible with catalysis, allowing the invariant Glu643 (Glu91 in cAPK) to interact with Arg834 (the R of the HRD motif in the catalytic loop) rather than forming the classical salt bridge with invariant Lys628 (cAPK Lys72) in the ?3 of the N-lobe. This is likely responsible for the failure of Lys628 to properly orient the ?- and ?- phosphate groups of ATP. By analogy with other ?RD? kinases, autophosphorylation of Thr882 would enable this residue to displace Glu643 from Arg834 and allow Glu643 to form the salt bridge with Lys628. Previously, we showed that the eIF3 complex, eIF1 and eIF5 reside in a multifactor complex (MFC) with the TC and mapped the interactions between these factors in the MFC. The N-terminal domain (NTD) of NIP1/eIF3c interacts directly with eIF1 and eIF5 and indirectly through eIF5 with the TC. We investigated the physiological importance of these interactions by mutating 16 segments spanning the NIP1-NTD. Mutations in multiple segments reduced the binding of eIF1 or eIF5 to the NIP1-NTD. Mutating a C-terminal segment of the NIP1-NTD increased utilization of UUG start codons (Sui- phenotype) and was lethal in cells expressing the eIF5-G31R mutant protein that is hyperactive in stimulating GTP hydrolysis by the TC at AUG codons. Both effects of this NIP1 mutation were suppressed by eIF1 overexpression, as was the Sui- phenotype conferred by eIF5-G31R. Mutations in two N-terminal segments of the NIP1-NTD suppressed the Sui- phenotypes produced by the eIF1-D83G and eIF5-G31R mutations. From these and other findings, we propose that the NIP1-NTD coordinates an interaction between eIF1 and eIF5 that inhibits GTP hydrolysis at non-AUG codons. Two NIP1-NTD mutations were found to derepress GCN4 translation in a manner suppressed by overexpressing the TC, indicating that MFC formation stimulates TC recruitment to 40S ribosomes. Thus, the NIP1-NTD is required for efficient assembly of preinitiation complexes and also regulates the selection of AUG start codons in vivo. We have established that optimal transcriptional activation of the amino acid biosynthetic gene ARG1 by GCN4 requires the coactivators SAGA, SWI/SNF, Srb Mediator, and RSC, and that GCN4 recruits these coactivators to target genes in living cells. We recently showed that recruitment of SAGA, SWI/SNF and Mediator to ARG1 is independent of the TATA element and preinitiation complex (PIC) formation, whereas efficient recruitment of the general transcription factors requires the TATA box. This suggests that recruitment of the coactivators precedes PIC assembly. Supporting this prediction, kinetic analysis of coactivator binding at ARG1 reveals that recruitment of SAGA, SWI/SNF and Mediator is nearly simultaneous with binding of GCN4 and is followed 5-10 min later by PIC assembly and elongation by RNA polymerase II (Pol II) through the open reading frame. Despite the simultaneous recruitment of coactivators to ARG1, rapid recruitment of SWI/SNF depends on the histone acetyltransferase (HAT) subunit of SAGA (GCN5), a non-HAT function of SAGA, and also Mediator function. SAGA recruitment in turn is strongly stimulated by Mediator and the RSC complex. These interdependencies in coactivator recruitment were also observed at the GCN4 target genes ARG4 and SNZ1. Recruitment of Mediator, by contrast, occurs independently of the other coactivators at ARG1, but requires SAGA at ARG4 and SNZ1. We found that that Mediator and SAGA not only stimulate recruitment of the TATA binding protein (TBP), but that all four coactivators enhance Pol II recruitment or promoter clearance following TBP binding, and that SWI/SNF and SAGA stimulate transcription elongation downstream from the promoter. Our findings reveal a program of coactivator recruitment and PIC assembly that distinguishes GCN4 from other yeast activators studied thus far.

The long term objective of this proposal is to elucidate the role of norepinephrine (NE) and beta receptors in visual system plasticity. The experiments are based on the finding that depleting NE with 6- hydroxydopamine (6-OHDA) decreases plasticity in the visual cortex. The first experiments attempt to determine whether NE depletion is sufficient for this effect. They make use of our recent finding that very small amounts of 6-OHDA can deplete NE in the visual cortex. We ask if these small doses of 6-OHDA also cause a decrease in plasticity. High performance liquid chromatography (HPLC) is used to measure NE depletion and plasticity is assayed by recording from area 17 in an animal that has had a week of monocular deprivation beginning at 5-6 weeks of age. If small, but depleting doses of 6-OHDA do not alter plasticity, we will conclude that NE depletion is not sufficient for decreases in plasticity. The next experiments ask whether NE depletion or increased beta receptor binding can better account for changes in plasticity. First, we will measure the time course of NE depletion and beta receptor binding capacity in response to 6-OHDA. Receptor binding will be measured with radioactive beta receptor ligands. Next, we will measure the dose dependence of depletion and receptor binding capacity. We ask whether the time and dose dependence of beta receptor binding better parallels changes in plasticity than does the time and dose dependence of NE depletion. The final experiments examine the role of beta receptors in the termination of the critical period. The development of beta receptors will be studied in normal, dark reared, and binocularly sutured animals. Since dark rearing but not binocular suture postpones the termination of the critical period, we hope to find that dark rearing but not binocular suture postpones the development of beta receptors.

The primary objective of this proposal is to establish objective and quantitative morphological parameters for the subdivision of classical varieties of acute leukemia. Computer-oriented image processing techiques, developed at this institution for chromosome analysis and, more recently, for an automatic white blood cell scanner and analyzer will be adapted for the analysis of leukemic cells. Slides from 40 children with acute lymphoblastic leukemia will be scanned, the images of 100 leukemic cells from each recorded, and the digitized images analyzed using cell size, color and texture parameters developed for leukocyte analysis and identification. The resulting quantitative morphological data will be analyzed for significant correlation with clinical data including clinical response to specific therapy, overall survival and cure expectancy. Once developed, these techniques can be extended to the field of acute myeloblastic leukemia where clinical results are relatively poor and where many different combinations of therapy exist. The overall goal is to develop objective criteria which may help to identify those patients who will (or will not) respond to a given mode of therapy.

The Galveston National Laboratory (GNL) will be a new, seven-story building containing 83,433 net usable square feet of state-of-the-art BSL2 through BSL4 laboratory space designed and constructed to support the research of the NIAID Biodefense Network. This proposal describes plans for its operation, and how the GNL will meet critical national needs related to the identification and validation of effective countermeasures for both naturally emerging infectious diseases and the threat of bioterrorism. The GNL will be operated as a consolidated functional entity on the UTMB campus, with multiple linkages to established UTMB campus operations allowing the GNL to take maximum advantage of the depth and breadth of technical and professional expertise and resources residing elsewhere in the University. GNL research and facility operations will report to the Director of the GNL, who will report to the Dean of the School of Medicine and President of UTMB. Biosafety and security functions will report directly to UTMB's Vice President for Business Affairs, who is the federally designated Responsible Official at UTMB. Scientific research, building operations, and business activities will be managed by the GNL's central Administrative Core built around an executive leadership team comprised of the Director, Scientific Director, Associate Director for Research, and Associate Director for Facilities. Through a GNL Operations Council and in consultation with the NIAID Program Office, this leadership team will direct a multidisciplinary research effort in support of both the GNL and NIAID Biodefense Network involving the activities of nine Research Support Cores (Emerging and High Risk Pathogens; BSL3 Research; Advanced Veterinary Services; Insectary Services; Collaborative Research Services; Experimental Pathology Services; Advanced Imaging Services; Advanced Technologies; and Regulatory Services) and four Facilities Support Cores (Facilities and Logistics; Biocontainment Engineering; Health, Safety and Training Services; and Security). Research priorities will be established in consultation with the NIAID Program Office and other investigators of the NIAID Biodefense Network and with the advice of a distinguished external Scientific Advisory Board. A field Investigations team managed by the Emerging & High Risk Pathogens Core will work with laboratory support from the Experimental Pathology Core to assist the GNL in responding to any future national emergency due to a newly emergent infectious disease or bioterrorist attack.

An estimated 25-40% of U.S. pre-adolescents and adolescents are obese. Higher rates have been noted among minority youth. Onset of obesity in late childhood and early adolescence is associated with increased risks of "tracking" of obesity into adulthood, subsequent obesity-related morbidity and mortality, and obesity- related psychosocial morbidity. However, (l) we have limited ability to accurately identify those children who will become obese adults and those who will suffer obesity-related morbidities, (2) existing treatments for child and adolescent obesity have yielded modest, unsustained effects, and (3) single-component prevention interventions have been relatively ineffective. Therefore, we propose an integrated, multiple-component, school- and community-based intervention targeting both primary and secondary prevention of obesity among third- fourth-and fifth-graders ("School- and Family-Based Obesity Prevention for Children"). The proposed intervention model is derived from principles of Bandura's social cognitive theory, and includes activities in THE SCHOOL, THE HOME, and a clinically oriented component for HIGH-RISK CHILDREN. The school component includes: (l) A computer-based classroom curriculum, (2) A physical education curriculum and (3) A school lunch intervention. The home component includes correspondence materials and a videotape for parents. Children identified as "high risk" will be eligible to enroll in an intensive intervention. In addition, several innovative approaches will be included: interventions to influence food preferences and television viewing, interventions promoting health advocacy, and computer-assisted instruction. We propose an "efficacy trial" to evaluate the three-year intervention in a cohort of approximately 1200 3rd graders, in 14 ethnically diverse elementary schools, with follow-up in the 6th grade. 7 schools will be randomly assigned to the comprehensive intervention, and 7 schools will receive an attention-placebo classroom curriculum. Anthropometric measures and assessments of food preferences, cardio-respiratory fitness and self- reported behavior, attitudes and knowledge will occur every 6 months. Parent interviews will occur annually. Although a careful assessment of effects on parents and the schools will be done, the crucial question is whether the overall intervention has an impact on student adiposity and behaviors. The primary objective is to significantly reduce the prevalence of obesity, compared to controls, at the end of the three year intervention. Secondary objectives include maintenance of effects at 6-month follow-up, reducing obesity among high-risk children, improving cardio-respiratory fitness, increasing physical activity, decreasing sedentary activity, reducing the prevalence of unhealthful weight control methods, and improving knowledge, attitudes and perceived self-efficacy regarding the adoption of healthful behaviors. In addition, we will identify personal, behavioral and environmental (including family) factors prospectively associated with development of obesity, maintenance of normal weight, weight reduction among overweight children and obesity-related behaviors. Finally, we will examine longitudinal changes in height, weight, BMI, triceps skin fold thickness, and waist and hip circumferences in girls and boys. 8 - 12 years of age, with resect to stages of pubertal development.

The overall objectives of this proposal are three-fold: 1) to determine the mechanism(s) by which activated macrophages induce an aberrant cycle of reductive division in tumor cells and the relationship of this cycle of reductive division to tumor cell death, 2) to establish several tumor cell lines resistant to macrophage mediated killing as a tool for evaluating the role of macrophage mediated events in tumor growth, 3) to use monoclonal antibodies to macrophages and antisera to Ia antigens to characterize the interrelationships between the biochemical events and the membrane antigens associated with macrophage maturation and/or differentiation. Experiments will involve determining the mechanism(s) by which activated macrophages in vitro induce an amitotic cycle of reductive division in tumor cells. We will determine the relationship of 5-FU mediated reductive division in Ehrlich ascites tumor cells in vivo to macrophage activation to establish whether or not a common mechanism via macrophage mediation exists both in the in vivo 5-FU induced and in vitro macrophage induced reductive division. Ascites and solid tumor models will be used to determine if agents such as C. parvum and pyran, which induce macrophage activation in vivo, can also induce reductive division of tumor cells in vivo. The relationship between activated macrophage tumor cell cytotoxicity and the ploidy (DNA content) of the target cell will be established. Lastly, in this area, attempts will be made to establish in vivo and in vitro the characteristics of these cell lines. Monoclonal hybridoma antibodies to macrophage surface antigens will be used to identify surface antigens associated with the different populations of mouse macrophages. The surface antigens will be characterized by polyacrylamide gel electrophoresis and the relationship of a given surface antigen to macrophage function analyzed. Antisera to different macrophage subpopulations and to Ia antigens will then be used to determine which subpopulation of macrophages are the precursors and ultimate effectors with respect to tumor cell cytotoxicity.

Periodontal disease is one of the most common human diseases afflicting 15% of the population. Recent data has implicated periodontitis as a causative disease for many other systemic complications. Porphoromonas gingivalis is named one of the most virulent periodontopathogens and an etiological agent in the progression of this disease. It also is present in the oral cavity in the absence of the disease. Although the interaction of the bacterium with the host is of major importance for the understanding of the disease mechanisms, both the host as well as the pathogen components involved in the interaction remain poorly understood. In vitro studies have revealed that P. gingivalis can invade a variety of cell types including gingival epithelial cells. It has also been demonstrated to prevent apoptosis in these cells, replicate, and disseminate to surrounding cells. Interestingly, P. gingivalis has been shown to perform these activities in both healthy and susceptible hosts. Studies have also reveled the complex nature of P. gingivalis due to varying results according to, and closely associated with; the host cell type, bacterial strain, initial inoculation, and the use of live (metabolically active) versus dead bacteria, or the presence of specific bacterial components (e.g. cysteine proteinases, LPS). Several studies have focused on the host cell responses mediated by Toll-like receptors challenged with bacterial components to gain insight on the initiation of the innate immune response. Of these bacterial components, little attention has been paid to DNA, especially in oral epithelial cells. To further understand the pathogenesis of P. gingivalis, we will be focusing on one bacterial component, DNA. The specific aims of this study include: (1) The characterization of the response of two oral epithelial cell lines and primary cells harvested from two patients to challenge with P. gingivalis W83 DNA, (2) The role of TLR9 in mediating this response, and (3) other DNA receptors that may play a role in this interaction. Initial in vitro studies revealed that the oral epithelial cell line HN4 responds to challenge with DNA derived from P. gingivalis with gene regulation at the transcriptional level. In addition, we have demonstrated that HN4 cells uptake CpG oligonucleotides. An additional preliminary study has shown that gene regulation does not seem to be significantly impacted in TLR9 knockdown HN4 cell lines that were challenged with P. gingivalis DNA, therefore, we plan to examine the cytosolic DNA receptor DAI, which also may play a role in the pathogenesis of P. gingivalis. Public health Relevance: This study will lead to a greater understanding of the mechanisms of the host response that contribute to the pathogenesis of P. gingivalis W83. These studies may then reveal potential targets for preventing the initiation of periodontal disease.

The metal-ligand stretching frequencies in metalloproteins fall in the far-infrared region. The iron atom in the hemeprotein, myoglobin, binds four nitrogen ligands from the heme group, one nitrogen ligand from a histidine residue in the protein, and the sixth coordination position is the oxygen-binding site. Since other ligands such as carbon monoxide and nitric oxide also competitively bind to the oxygen-binding site, it is important to characterize the structural and electronic intermediates that are involved in the ligand-binding process. Since the iron-ligand bond can be photolyzed at 10 K, we are Hable to use photolyzed/unphotolyzed difference FTIR to probe vibrational modes such as (1) the iron-ligand stretching frequency, (2) the iron-proximal histidine stretching frequency, and (3) heme doming modes, all of which differ in the ligand-bound and photolyzed states. Resonance Raman studies have also been used to identify some of these modes. However, the conditions by which several of them are resonance-enhanced are still unclear, making infrared spectroscopy a necessary alternative. In the past, we have been unsuccessful with myoglobin films in polyvinyl alcohol (PVA) on polyethylene due to (1) the temperature-dependence of the far infrared spectrum of PVA and (2) Hthe optically-opaque nature of polyethylene, making it difficult to Hphotolyze myoglobin at low temperature. Recently, we have shown that Hsolution samples in 75:25 glycerol:water (50 ?m pathlength) between Hsapphire windows are reasonably transparent in the far infrared region Hbelow 100 K and completely transparent in the visible. These findings Hwill greatly improve the ability to (1) photolyze the sample, (2) Hcollect high quality difference spectra, and (3) collect far-infrared Hprotein spectra in solutiona more biologically relevant state of the protein.

The principle objective of this proposal is to develop a high resolution, high detection efficiency positron emission tomography (PET) detector with depth-of-interaction (DOI) positioning that will also support multi-modality imaging (e.g., PET/MR). This detector design can be scaled to support imaging applications from mice to man. The key design features of this PET detector are the use of trapezoidal, salt crystals (TSC) and a maximum likelihood positioning method that we call statistics based positioning (SBP). In addition to the unique crystal geometry, the design will utilize silicon photomultiplier (SiPM) arrays. SiPMs are Geiger-mode avalanche photodiode devices that have very attractive performance characteristics for PET detectors, especially for PET-MR applications. In this R21 proposal, we will investigate a design tailored for small animal PET imaging. The design will support <0.8 mm image resolution and >15% absolute detection efficiency. It is envisioned that the detectors developed under this proposal will eventually be used to fabricate a very compact PET detector ring that will be compatible with operation in high field (e.g., >4.7T) MR systems. For this prototype work the TSC PET detector dimensions will be designed for a system inner ring diameter of ~6.0 cm. This diameter will support dedicated PET and PET/MR whole body mouse and rat brain imaging. The net result of this work is the development and evaluation of a new PET detector design that will provide state of the art imaging performance capabilities and that can be used as part of a compact, stand alone PET system or as part of a multi-modality imaging system (e.g., PET/MR). We believe that this design will provide an outstanding balance between performance and cost and that the design will be translatable to commercial adaptation. This proposal builds upon the previous work of the investigators developing high resolution discrete crystal PET detectors and high resolution monolithic crystal PET detectors. This new design synergistically brings together the inherent strengths of both methods, that is, modularity and excellent spatial resolution control (discrete crystals) and lower fabrication costs and inherent DOI positioning capability using single-sided readout (continuous crystals). The design is scalable to support ultra-high resolution small animal imaging; high resolution organ specific imaging; and whole-body PET imaging. In addition, this project will add to the knowledge based on how to optimize the use of SiPM devices for nuclear medicine imaging detector applications. Two specific areas in which this work will contribute are developing SiPM signal multiplexing techniques to optimize performance versus cost and developing temperature control strategies and temperature/gain dependent calibration procedures for SiPM devices. PUBLIC HEALTH RELEVANCE: The overall goal of this proposal is to develop technology that will be used to advance the biological applications of PET and multi-modality PET imaging. PET is a very important imaging modality for both clinical and research applications (e.g., oncology, cardiology, neurology, drug development, biodistribution studies, cell trafficking, etc.). Further great synergism has been demonstrated when it is combined with a high resolution anatomic imaging modality (e.g., PET/CT or PET/MR). The key component of any PET system is the detector design. In this proposal, we introduce a new trapezoidal, slat crystal (TSC) PET detector that is scalable between small animal imaging and human whole body imaging. We believe that this design will provide state of the art imaging performance and an economical price point such that the design will be translatable to commercial production. Therefore while the focus of this proposal is on a specific PET detector design, the far reaching goal of this work is to develop PET imaging technology that will aid in the battle against human disease.

Urolithiasis can have numerous predisposing etiologic factors. Some of the specific mechanistic steps in the formation of kidney stones have not been determined. Once the concentration of ions and/or macromolecules has reached supersaturation, a state of crystalluria exists within the urinary tract, and the formation of stones can occur through a multifaceted process. These steps include retention of the microcrystallites by the epithelial membranes, and growth by aggregation, matrix adhesion, epitaxial interactions, and/or agglomeration. This research proposal has three major Specific Aims: I) To study the retention of microcrystals by investigating the processes of crystal formation on and crystal binding to rat renal medullary/papillary (RMP) cells in primary culture. II) To study the possible role of epitaxial interactions between crystal structures observed in renal stones. III) To study the role of matrix and the degree of crystallinity on the mechanism of reorganization and maturation of renal stones. In Specific Aim I, we will question if RMP apical cell surfaces can serve as nucleation sites for kidney stone crystal nucleation? Do preformed renal stone crystals bind to the apical surface of RMP cells in primary culture? Are these crystal-membrane interactions influenced by inhibitors, and do the crystals damage RMP cells and their membranes? In Specific Aim II, we will study possible epitaxial interactions between renal stone crystals using our program EPITAX, and will model these interactions in real-time and in three-dimensions using sophisticated computer graphics. In Specific Aim III, we will utilize human kidney stones received by the National VA Crystal Identification Center to study the mechanism of stone maturation. We will determine if stones mature by a two-stage crystallization process where matrix is extruded during recrystallization and maturity. Also, the degree of crystallinity will be correlated with the type of crystal formed and matrix content and composition.

Perinatal arterial stroke (PAS) includes infants with neonatal arterial ischemic stroke and presumed perinatal arterial stroke and has an estimated incidence of 1 in 1150. The majority will develop hemiparesis, often resulting in lifelong and debilitating disability, including lowered cognition and quality of life. There are no evidence-based forms of rehabilitation designated as standard-of-care for these infants. We propose a Phase III trial to address this lack of evidence-based treatments. The I-ACQUIRE Trial is a rigorous, adequately powered, multi-site randomized controlled trial comparing 2 dosages of a pediatric rehabilitation - known as Infant ACQUIRE - to Usual and Customary treatment (U&CT). Prior studies of I- ACQUIRE show high safety, patient acceptability, and evidence of significant and enduring benefits. Briefly, I- ACQUIRE will be used to treat 8 ? 24 mth old infants with PAS in either 3 hrs/day (Moderate Dose) or 6 hrs/day (High Dose), for 5 days/wk for 4 wks. Treatment occurs in a home or natural setting where operant conditioning techniques are applied to achieve goals paced for each infant. I-ACQUIRE includes the core features of pediatric Constraint-Induced Movement Therapy (CIMT). Standardized treatment at all sites is monitored weekly via a Fidelity of CIMT Implementation tool. Clinical Trial Design: 240 infants (N=80/group) will be randomly assigned to: 1) Moderate Dose I-ACQUIRE, 2) High Dose I-ACQUIRE, or 3) U&CT. (N=80). Double-blinded assessments occur at baseline, end of 4 wks of treatment, and 6 mos post-treatment. A Parent Council will be active throughout the trial, supporting NIH's goal of increased stakeholder engagement in clinical trials. The specific aims are: 1) Primary: Determine the efficacy of I-ACQUIRE at 2 dosage levels compared to U&CT to increase upper extremity skills on the hemiparetic side (using a minimal clinically meaningful threshold that is achieved at end of treatment and 6 mos later); 2) Secondary: Determine the efficacy of I- ACQUIRE at 2 dosage levels compared to U&CT to improve use of the hemiparetic upper extremity as an ?assisting hand? in bimanual activities; and 3) Exploratory: Explore the association between I-ACQUIRE treatment at Moderate and/or High Doses and the infant's gross motor development and cognition (i.e., potential cross-domain effects of treatment). The Statistical Analysis Plan controls for multiple comparisons; after testing the major study hypotheses, sensitivity analyses will consider factors that may contribute to variation in treatment outcomes. Public Health Impact: Each year, an estimated 3400+ infants in the U.S. have PAS with high likelihood of lifelong impairment in neuromotor and often cognitive functioning resulting in a high cost burden for families, the healthcare system, and society. If I-ACQUIRE proves efficacious, then the field will have the critically needed Phase III confirmatory evidence and the specific clinical protocol needed to transform rehabilitation and improve clinical outcomes and quality of life for infants with PAS.

Development, survival and activation of B cells are regulated by signals derived from the surface expressed B cell antigen receptor (BCR). Src-family protein kinases (SFK) are implicated in both negative and positive regulation of BCR signaling. Changes in the activity and expression of SFK have been linked to immunodeficiency, autoimmunity, and malignancy in mice, suggesting that regulation of SFK activity is essential for normal B cell function. The activity of SFK is regulated by phosphorylation of a conserved tyrosine residue at their C-termini by the ubiquitously expressed C-terminal Src kinase (Csk). Recently, we identified and characterized a novel transmembrane, lipid raft-associated Csk binding phosphoprotein (Cbp) and demonstrated its ability to mediate SFK inhibition by Csk. The overall goal of the experiments described in this application is to reveal the role of Csk and Cbp in regulation of B cell signaling and function. In particular, we will address whether and how Csk controls the threshold for B cell activation in vitro and in vivo. We will also study the role of Csk in autoreactive B cell responses to self-antigens. Our second aim is to investigate the mechanism of BCR mediated regulation of Cbp phosphorylation and its association with Csk in resting and activated B cells. Finally, using mice with conditional Cre mediated inactivation or modification of the cbp gene in B lineage cells, we will investigate the role of Cbp in regulation of lipid raft-associated SFK and B cell activation. Collectively, these studies will shed further light into the regulation of signal transduction in immune cells and provide data that may be useful in the treatment of diseases related to immune dysfunction.

A variety of experimental approaches have established that the vestibular system contributes to making adjustments in the activity of thoracic and abdominal respiratory pump muscles as well as muscles that regulate the resistance of the upper airway during movement and changes in posture. However, these effects are complex, such that the physiological role of the vestibular system in regulating the movement of air in and out of the lungs is not yet clear. Specific Aim 1 of the current proposal will determine this role by examining the consequences of a bilateral labyrinthectomy on the pressure, volume, and flow rate of air exchanged during inspiration and expiration as body orientation with respect to gravity is altered. Prior experiments have also shown that some of the neurons that relay vestibular signals to respiratory motoneurons are located in the medial medullary reticular formation, but that these cells are insufficient to produce vestibulo-respiratory responses. Our anatomical studies and physiological studies by others have revealed that additional premotor respiratory neurons are located in the spinal cord. Specific Aim 2 will test the hypothesis that spinal interneurons are elements in the neuronal circuit that mediates vestibular system influences on the movement of air in and out of the lungs. Patterned contractions of respiratory muscles are additionally responsible for producing vomiting;vestibular signals trigger this motor activity during emesis associated with motion sickness. However, the neural circuit that elicits vomiting has not yet been established, although recent evidence suggests that cells in the medial medullary reticular formation are important components in this pathway. Specific Aim 3 will test the hypothesis that medial medullary reticular formation neurons coordinate the patterned discharges of respiratory muscles that underlie vomiting, including emesis associated with motion sickness. Together, these experiments should establish the particular role that labyrinthine signals serve in adjusting the activity of respiratory muscles to compensate for the effects of gravity on these muscles, the airways, and the lungs. In addition, the location and physiological characteristics of premotor neurons that mediate labyrinthine influences on respiratory motoneuron firing, both during postural alterations and emesis, will be ascertained.

Absence of Oxalobacter formigenes (Oxf) from the GI-tract is a risk factor for development of hyperoxaluria and calcium oxalate kidney stones. Recent data now indicate that patients with recurrent urolithiasis have a lower prevalence rate of Oxf in their stools than non-stone forming individuals. In addition, urinary oxalate levels are higher in non-colonized individuals as compared to colonized individuals. This indicates a clinical need for a test that can identify non-colonized individuals. Phase I of this grant resulted in the development of a reliable and rapid PCR ELISA-based microtiter plate assay for the detection of Oxf in human fecal samples. Pre-clinical performance data for the test (Le. its analytical sensitivity, analytical specificity, precision and reproducibility) were obtained as per the NCCLS guidelines. In Phase II of this project, we propose to complete the development of a production prototype of the XEntrix(TM) O. formigenes Monitor Kit. Analytical and clinical performance of the prototypic test kit will be determined in a multicentre clinical trial. The clinical studies have been designed not only to validate the test kit but also to strengthen the utility of Oxf determination as a diagnostic tool for the assessment of risk for calcium oxalate urolithiasis. By end of the proposed Phase II studies, we will have sufficient analytical and clinical performance data on this diagnostic kit to seek a pre-market approval under section 510K of the Food Drugs and Cosmetic Act, from the Center for Devices and Radiological Health.

The overarching goal of the proposed research is to define the cellular, molecular and metabolic mechanisms determining the response of the heart to myocardial ischemia and LV pressure overload, in order to develop novel therapeutic approaches for patients with cardiovascular disease. Our group discovered that macrophage migration inhibitory factory (MIF) is secreted from the heart during ischemia-reperfusion, activating AMP- activated protein kinase (AMPK) and constituting a protective autocrine-paracrine pathway. The protective action of MIF is mediated by the cell surface CD74 receptor. However, MIF has additional pleiotropic effects to activate the chemokine receptors CXCR2/4, which may be detrimental during pathophysiological stress and render it less than ideal for therapeutic development. We have recently discovered a non-redundant second ligand for CD74 called D-dopachrome tautomerase (DDT), which is more selective than MIF for the CD74 receptor. Our initial results indicate that cardiomyocyte-derived DDT has important actions to prevent both injury during ischemic-reperfusion and heart failure consequent to LV pressure overload. Our Chemical Biology group has also designed small molecule agonists that specifically facilitate activation of CD74. Thus, we will study the molecular signaling and physiological effects of these reagents in the heart and then investigate their therapeutic potential in ischemia-reperfusion. Our specific aims are 1) to determine the signaling mechanisms that mediate DDT action downstream to CD74, 2) to determine the role of endogenous cardiomyocyte-derived DDT in regulating the response of the heart to pathological stress, and 3) to develop and optimize the CD74 pathway as a therapeutic strategy in the heart. Understanding CD74 signaling has broad implications for patients with cardiovascular disease, but might have particular significance in specific individuals who have an increased susceptibility to ischemia in the heart and potentially other solid organs. Such patients include those with a common polymorphism (>5%) in the human MIF promoter, which we have shown leads to impaired MIF secretion and CD74-dependent AMPK activation during hypoxia. The elderly might also benefit, since the MIF- CD74-AMPK axis is also impaired with aging in mice. We have assembled a multi-disciplinary team with expertise in the areas of cellular metabolism and signaling, ischemic heart disease, MIF biology, inflammatory disease, recombinant protein and chemical synthesis. We have generated novel recombinant proteins, small molecule activators, antibodies and genetic mouse models to enable us to test whether the strategy of CD74 receptor activation is protective. Thus, we are poised to define novel biological pathways in ischemic heart disease and develop translational strategies that have potential to result in new treatment strategies for patients with cardiovascular disease.

There are approximately 4 million Americans with severe speech and physical impairments (SSPIs) who must rely on augmentative and alternative communication (AAC) systems to express themselves. People with SSPI are at a significant disadvantage when communicating. AAC systems place heavy cognitive and physical demands on users, resulting in very slow communication rates. The AAC user spends so much time and energy constructing messages, there is often little time for typical turn taking sequences or sustained conversation. Cultural rules for successful interactions are violated. The communication partners are disadvantaged as well, often waiting for minutes with no means for reciprocal engagement before a single message is generated. Currently, the communication partners have no clear mechanism to help the AAC user formulate messages, even if they have shared knowledge about the vocabulary. Is there a way for us to capitalize on the physical skills, language skills and shared knowledge of the communication partners to enhance the user's message production and communication performance? We propose to develop an innovative technology-based system for AAC conversation that is grounded in the success and efficiency of dialogue co-construction and word prediction. We combine dynamic contextual word/phrase prediction from non-disabled communication partners with a sophisticated bigram-based language model residing in the AAC system to increase the speed and informativeness of message production. In Phase 1, we will develop two Android apps (Construct-AAC and Constructor) that communicate via Bluetooth, one for the AAC user and one for the non-disabled partner (called the co-constructor). The AAC user spells out a message with Construct-AAC. The second app, Constructor, displays a copy of the AAC user's message as it is being created and enables the co-constructor to suggest words or phrases in real time by either typing out words, or by choosing words from a prediction list. The word and phrase predictions, along with other lexica stored in the device, are prioritized by the language model within the AAC user's app, and presented in standard word prediction lists to the AAC user. The AAC user maintains independence to choose or ignore the co-construction suggestions during message generation. This written interaction mimics the co-construction behavior observed in speaking conversations and offers the two partners a common written milieu for reciprocal, face-to-face, verbal interaction. Feasibility of this new AAC system will be tested using a single case ABAB withdrawal design with ten dyads of non-disabled participants and five dyads of AAC users and their non-disabled co-construction partners. Participants are shown the Boston Cookie Theft Picture and instructed to produce a language sample by describing the scene. In condition a, participants use only Construct-AAC to describe the picture. In the experimental condition B, the co-construction partner contributes to the user's picture description with the Constructor app. We predict that having access to contextual predictions will improve speed and informativeness of AAC output. This, in turn, will positively influence conversational performance. In Phase 2, we will optimize the system and examine the effect of enhanced AAC output on conversation.

The goal of the proposed research is to develop and apply theoretical simulation techniques to study the nature and energetics of protein- substrate and protein-inhibitor interactions, the mechanism of uncatalyzed solution phase reactions, and enzymatically catalyzed reactions. By increasing our understanding of existing inhibitors as well as our ability to design new inhibitors will have a major impact on improving human health. An increase in our knowledge of protein structure and function will have a major impact on biotechnology, because it will allow for the ab initio design of new proteins with increased or more selective catalytic properties. To address these issues we will subject two classes of zinc metalloenzymes to intense theoretical scrutiny. These are the human carbonic anhydrases (HCAs) and the matrix metalloproteinases (MMPs). In particular, we will look at HCAII and the MMPs stromelysin-1 (S1) and matrilysin, HCAII is of great biomedical importance because of its role in processes involving CO2, and because inhibitors are used in the treatment of glaucoma. S1 and matrilysin are important in structural modification of tissue and inhibitors are potentially useful in the treatment of arthritis. Moreover, related MMPs are involved in diseases ranging from cancer to periodontal disease. Thus, an understanding of S1 and matrilysin will shed light on related MMPs. In order to understand the structure and function of these enzymes we will develop quantum mechanical/molecular mechanical (QM/MM) methods that have improved performance, improved treatment of long-range interactions as well as the ability to determine absolute and relative binding free energies. These new methods offer capabilities beyond traditional methods and, therefore, we will obtain novel molecular-level insights regarding enzymes. These methods will be applied to the study of condensed phase reactions as well as enzyme/substrate interactions, metal ion binding, catalysis and inhibition in S1, matrilysin and HCAII. These studies will provide a molecular-level picture of the factors involved in enzyme inhibition and catalysis. We will also supply our QM/MM programs to the scientific community in order to allow others the ability to probe the structure and function of biomacromolecules using our tools.

The principal objective of this research is to develop convenient and selective chemical liquid chromatographic procedures to separate, characterize and determine various classes of compounds which could serve as potentially useful clinical and biochemical markers in uremia and chronic renal failure, and which may be significant in associated disease states. We have recently developed selective liquid chromatographic procedures to analyze for guanidino compounds and polyamines. Both of these methods utilize ion-pairing chromatographic techniques and selective on-line post-column reaction detector systems. A detailed study of vitamin loss for patients on hemodialysis was also completed recently. During the coming year, it is planned to continue this work by conducting extensive clinical screening programs for guanidine and polyamine levels in uremic hemodialysate, serum and other physiological fluids. Additional studies will be conducted on two other classes of compounds: middle molecular weight molecules (polypeptide fragments) and tryptophan and its metabolites. All of these compounds can be separated by liquid chromatography and can be detected in trace quantities. Peak identification will be accomplished by mass spectrometry.

The overarching goal of this proposed training program is to integrate interdisciplinary and translational aspects of the current, rich training environment in pain research at the University of Pittsburgh. In addition to exposure of trainees to fundamental principles of pain mechanisms and pain presentation, we will establish new program elements (clinical experiences, multi-laboratory projects, courses) that are typically very difficult for individual investigators to support. We are committed to an integrated approach to the study of pain because major breakthroughs in this field can only be achieved through multidisciplinary approaches, and thus will incorporate and facilitate interactions between [1] laboratories (horizontal integration) and [2] researchers and clinicians (vertical integration). Accordingly, horizontal and vertical integration will be essential components of this training program, which will consist of three core elements: 1) Research - Multidisciplinary research projects will not only be encouraged, but expected, as will exposure to clinical management of pain/pain-related problems. These integrations will be achieved through formation of multidisciplinary mentoring committees which will include at least one clinical faculty member among a four-member committee. 2) Theory - Trainees will participate in four required for-credit courses: Mechanisms and Clinical Presentation of Pain, Pain Journal Club, bi-weekly Current Research on Pain presentations, a new course Pain Models - Rationale, Testing and Interpretation as well as the monthly Pain Seminar Series, where trainees interact with prominent pain researchers. 3) Practice - Trainees will be exposed to the assessment, diagnosis and treatment of chronic pain patients through a 2nd new course - Pain Perspectives - that will provide trainees the opportunity to shadow pain physicians as they interview, diagnose and manage chronic pain patients within the Pain Medicine Program at the University of Pittsburgh Medical Center.

Introduction: The relationship between brain activity and reading performance was examined to test the hypothesis that dyslexia involves a deficit in a specific visual pathway, known as the M pathway, from the eye to the brain. Methods: Functional magnetic resonance imaging (fMRI) was used to measure brain activity in dyslexic and control subjects in conditions designed to preferentially stimulate the M pathway. Results: Dyslexics showed reduced activity compared to controls both in primary visual cortex (V1) and in a secondary cortical visual area (MT+) that is believed to receive a strong M pathway input. Most importantly, significant correlations were found between individual differences in reading rate and brain activity. Conclusions: These results support the hypothesis for an M pathway abnormality in dyslexia and imply a strong relationship between the integrity of the M pathway and reading ability.

Recent research indicates that infants can draw on at least two sources of information to individuate objects involved in occlusion events: (a) featural (i.e., the shape, size, color, or pattern of an object) and (b) physical (i.e., knowledge about the lawful ways that objects move and interact). However, whether infants demonstrate this ability depends on the task used. When an event monitoring task is used (i.e., infants must judge whether successive portions of an event are consistent), infants demonstrate the ability to use featural information by 4.5 months of age, and some forms of physical information by 3.5 months of age (Aguiar & Baillargeon, 1997; Wilcox & Baillargeon, 1997). In contrast, when an event mapping task is used (i.e., infants must retrieve a representation of one event and compare it to a subsequent event), older infants often fail (Wilcox & Baillargeon, in press; Xu & Carey, 1996). The goal of the proposed research is to examine more closely the mapping problem and to identify the processes that infants engage in when they attempt to retrieve representations of occlusion events. The proposed research has been organized into two sections. The first section investigates infants ability to map occlusion events that involve two featurally distinct objects. These experiments address the question of how infants organize and map events that require binding features to individuals. The second section investigates infants ability to map occlusion events that involve two identical objects. These experiments address two issues: (a) infants ability to draw on different forms of physical knowledge to individuate objects and (b) event ambiguity and how it contributes to mapping difficulties. Together, the results of the proposed research will provide new insight into the way that infants form representations of occlusion events and how infants use these representations when reasoning about other physical events. The results will also provide direction for future research that will focus on the investigation of event representations in infancy.

Despite extensive research and novel treatments, conditions associated with deranged cardiac metabolism such as heart failure (HF) or ischemia are still associated with a substantial risk of arrhythmic sudden death. Cardiac injury from many causes is associated with altered metabolism and downregulation of the cardiac sodium channel (SCN5A). Recently, data demonstrated that the SCN5A was substantially and immediately modulated by pyridine nucleotides. Physiologically relevant elevations in intracellular NADH resulted in a rapid decrease in INa in both HEK cells and cardiomyocytes that was large enough to be clinically significant. The immediacy of the NADH effect on reducing INa and the lack of change in mRNA abundances under various experimental conditions suggested that the effect of NADH was post-transcriptional. Internally or externally applied NAD+ antagonized the downregulation of current seen with a rise of internal NADH. The finding that the balance of oxidized and reduced pyridine nucleotides regulates the Na+ current suggests that the metabolic state of myocytes may influence INa. The results identify a heretofore unknown regulation of cardiac Na+ channels that may help explain the link between metabolism and arrhythmic risk and may suggest that NAD+ could lessen arrhythmic risk resulting from reduced INa. This application proposes to extend these findings to better understand the mechanism whereby changes in pyridine nucleotides cause SCN5A regulation and to establish the relevance of these changes to arrhythmogenesis in myopathy models. Specific Objectives. Specific aim 1: To determine the mechanism by which NADH acts on the Na+ channel to mediate downregulation of INa. These experiments will differentiate between two leading hypotheses for how NADH mediates its effects on the Na+ channel, either by direct action on the channel complex or by causing channel isolation from the sarcolemma. Insight into Na+ channel regulation may allow mitigation of arrhythmic risk associated with low INa states. Specific aim 2: To determine which proteins are modified in the mitochondrial electron transport chain (ETC) by NADH and NAD+. As we have recently shown, the effects of NADH and NAD+ on mitochondrial ROS production are kinase dependent. Our preliminary data show that NADH activates mitochondrial ROS production via PKC, and NAD+ prevents this via PKA. Using ETC inhibitors and activators, we localized the source of ROS to either complexes I or III.17 We propose to use proteomic techniques to establish if PKA and PKC phosphorylate these complexes and whether that phosphorylation results in alteration of mitochondrial respiration, complex activity, or ROS production to better understand the mechanisms regulating mitochondrial ROS production. Specific aim 3: To determine whether NAD+ can mitigate reduced INa in ischemic and nonischemic cardiomyopathy models. Our preliminary data suggests that NAD+ may serve to mitigate arrhythmic risk in states where INa is decreased. We will test this hypothesis in two cardiomyopathy models as preliminary data for NAD+ use in humans.

Aging is associated with a decline in cellular proliferative capacity. In culture, mammalian fibroblasts undergo a limited number of population doublings (senescence); this number generally decreases with increasing age. The mechanisms responsible for cell senescence are largely unknown. Cell proliferation is governed by environmental factors, of which polypeptide growth factors play a major role. Current evidence suggests that the defective growth shown by senescent cells is the result of an abnormality at a post-growth factor receptor site, and that some growth-related genes may be abnormally expressed in senescent cells. This proposal explores how growth factor- generated signals modulate specific genes in cultured human fibroblasts undergoing active proliferation or senescence. The genes of primary interest include the proto-oncogenes c-fos, c- myc and c-ras, ornithine decarboxylase, and two genes of unknown function (JE and JB) whose expression is growth factor-inducible. These genes probably serve different functions, and are likely regulated at different sites (pre- or post-transcriptional) by growth factor-generated signals. We will define the extent and levels of regulation by growth factor signals, and we will determine one or more post-receptor systems are altered during cellular senescence, resulting in specific changes in the regulation of certain genes. These studies are designed to provide a molecular basis on which to build our understanding of the control of cell proliferation both in early passage human fibroblasts and during cellular senescene.

Ductal branching morphogenesis is one of the many processes associated with the development of the prostate gland. An understanding of this developmental feature is important because it is responsible for the acquisition of adult glandular architecture and because inappropriate ductal branching appears to occur in prostatic disease. The embryonic urogenital sinus mesenchyme is essential for the development, growth and function of the prostatic epithelium. However, the structural organization and characteristics of the specific components of the prostatic stroma remain largely undefined. Since marked heterogeneity of ductal branching patterns occurs within different regions of the prostate, it is possible that variation in temporal and spatial organization of extracellular matrix proteins, and differential production of proteases associated with tissue remodeling, may be regulatory factors. To test this hypothesis, rat prostatic tissue will be examined for ultrastructural localization of specific components of the basal lamina and stroma, using quantitative,indirect, immunogold cytochemistry. This technique will be employed to study the organization of stromal components in the fetal and neonatal development of the gland, using dorsolateral and ventral tissue for comparison. In vitro organ culture will facilitate examination of branching morphogenesis at defined stages of pattern formation. To test whether regional differences in stromal tissue can modify branching patterns, heterotypic recombinants of dorsal epithelium and ventral urogenital mesenchyme will be either grafted into host animals or cultured,then examined by immunocytochemistry. It is anticipated that results from these studies will improve understanding of the normal morphogenesis of the prostate and lend insight into the possible mechanisms of abnormal growth patterns.

Data from these experiments are expected not only to help characterize the visual processing impairments associated with Alzheimer's Disease but also to provide evidence bearing on the adequacy of current information-processing models of human vision.

PROJECT SUMMARY The main purpose of this proposed U19 program project (PPG) is to comprehensively identify male osteoporosis risk genes and their functions. This PPG application will conduct multi-/inter-disciplinary convergence/integration analyses to study and integrate the crosstalk/interaction among multi-level omics obtained from the three individual component projects. The program project involves extensive data management and complex analyses. Such analyses require organic and simultaneous consideration of data from multiple component projects, and demand powerful and innovative integrative analysis methodology. Thus, it is necessary to set up/maintain a Biostatistics and Bioinformatics Core (BBC), focusing on the repurpose of existing, and development and application of novel, integrative analysis approaches. The main Objective of the BBC is to serve as a backbone support resource for experimental design refinement, data quality control, management, integration, analyses and interpretation, and serve as a synergizer to foster data and information exchange and collaboration for individual projects/cores in the PPG. Built upon the Core members? close and long-time collaboration and extensive experience in multi-omics data analyses, this BBC will provide indispensable services through the following Specific Aims: 1) To deliver efficient support and services for data management, including high quality data entry and management database implementation and maintenance, data quality control, safety, monitoring, sharing, etc. 2) To provide strong support for and to conduct extensive biostatistics, bioinformatics, and integrative analyses. Closely working with the PPG project investigators, the BBC will support both single-level omics data analyses and perform integrative analyses of multi-level omics data. Particularly, the BBC will pioneer a sophisticated integrative analysis strategy. This highly innovative strategy will link DNA/miRNA/methylation data through anchoring on gene- based mRNA hubs for constructing unified function multi-omics modules, which will then be used in functional gene unit and disease association analyses. 3) To evaluate, validate, and apply novel, robust and powerful integrative analysis methods for identifying/characterizing (epi-)genes and variants for complex diseases. The novel methods will be developed (mainly in a recently funded R01) under rigorous statistical frameworks, and characterize and incorporate among- omics crosstalk/interactions, along with prior biological information, for studying the causal relationship between multi-omics data and diseases. The innovative methods will be applied to and empirically tested on the multi-omics data obtained from this PPG. The services provided by the Core will be used by all other PPG projects and cores. Overall, the BBC will be efficient, capable and powerful to serve all the PPG projects, and in particular the overall goal of this PPG that intends to integrate the individual project results.

PREVIOUS WORK:[unreadable] The recent development of methods for engineering bacterial artificial chromosomes (BACs), and for the efficient production of BAC transgenic mice, has simplified the design of in vivo approaches for the analysis of gene expression and function in the brain. BAC transgenic mice carrying an exogenous 150 kb DNA sequence containing the Nurr1 promoter directly followed by the Tau green (TG) gene have been generated. The exogenous DNA, was randomly integrated in the genome when injected into oocytes. As the carrying capacity of BACs is several hundred kilobases, we were able to insert a 150 Kb DNA fragment overlapping the Nurr1 gene (as compared to other carrying vectors allowing only 10-15 kb inserts). [unreadable] [unreadable] In these transgenic mice, Tau green expression was expressed only in the olfactory bulb, and absent in other brain areas known to express Nurr1. This data suggests that the BAC (containing the Nurr1 promoter) used in this study is lacking some of the regulatory elements necessary for driving TG expression in brain regions other than the olfactory bulb. We are currently developing additional transgenic mice by homologous recombination, for mapping/visualizing the midbrain dopaminergic system. These mice will be used in our laboratory to conduct several anatomical/functional studies in which mapping/visualization of the Nurr1/midbrain dopaminergic system is necessary (see below)[unreadable] [unreadable] CURRENT UPDATE:[unreadable] Embryonic stem cells are being targeted by homologous recombination with a construct that introduces cDNA encoding for Tau-Cyan (knock-in) expression after the start codon of the tyrosine hydroxylase (TH) gene. In these stem cells, Tau-Cyan expression will be driven by any elements present in the cell that activate the TH promoter. As a result all TH positive cells and their terminals will also be Cyan positive. This unique stem cell line will be used in various studies:[unreadable] [unreadable] 1) TC transgenic mice will be generated. These transgenic mice will be useful for anatomical/functional studies in which mapping/visualization studies of the TH system are desirable. Laser capture microdissection (LCM), fluorescence activated cell sorting (FACS) and patch-clamp studies will be simplified by using these mice, since no immunostaining or labeling will be required to visualize TH-Cyan positive cells. In our laboratory, these mice will be backcrossed with mice containing targeted mutations in dopamine cells of the ventral mesencephalon (see table 1) to further facilitate molecular and electrophysiological studies of the mesolimbic dopaminergic system modified with specific mutations.

The long term objective of this research proposal is to determine the role of the primary cilium in podocytes. In the progression of proteinuric renal diseases, there are alterations in podocyte appearance, e.g. pseudocysts and foot process effacement. Renal collecting duct cells have now been shown to sense fluid flow by the apical plasma membrane's primary cilium in vitro. Since podocytes are anatomically located in an area of high fluid flow, one hypothesis would be that the podocyte's primary cilium senses fluid flow. This major hypothesis is integrated into four specific aims. The first aim will employ standard imaging techniques to determine the podocyte cilia in terms of length, frequency, and microtubular arrangement. The second aim will address the hypothesis that podocytes can sense fluid flow by monitoring changes in intracellular Ca++. Intracellular calcium will be monitored using a standard perfusion apparatus and microscope. The third aim will use biophysical and biochemical manipulation of the primary cilium to determine if the primary cilium is a flow sensing organelle. The fourth aim will identify which intracellular and extracellular calcium channels are involved, e.g. polycystins and transient receptor potential channels (TRPC), in the flow induced response. Ca++.

Two critical hallmarks of chronic Pseudomonas aeruginosa infection in patients with cystic fibrosis (CF) are the appearance of alginate over-producing mucoid colonies and the development of antibiotic resistance. A rapid emergence of antibiotic resistance in P. aeruginosa isolates in CF patients due to in vivo selection of strains that overproduce AmpC [unreadable]-lactamase, an enzyme that hydrolyzes the [unreadable]-lactams, contributes to both morbidity and mortality. Previous studies investigating the regulation of the amp genes in P. aeruginosa using genetic and molecular biological techniques led to the identification of a second [unreadable]-lactamase, PoxB oxacillinase. We showed that the expression of both ampC and poxB are under the control of AmpR, a member of the LysR family of transcriptional regulators. We have demonstrated that AmpR is a global transcriptional regulator which regulates expression of many virulent genes that are important for establishing chronic infections. [unreadable] [unreadable] Our long-term goal is to establish the regulatory circuit that exists between the amp and pox genes and their role in the development of resistance to [unreadable]-lactams and the establishment of chronic infection. To date, we know very little about the molecular mechanisms that govern the expression of pox operon. The experiments proposed in this grant will extend our understanding as they will focus on (1) characterizing the poxAB operon and determining if AmpR regulation is by transcriptional derepression, and (2) determining if expression of the poxAB operon is regulated by a previously uncharacterized two-component system. These analyses will provide invaluable insight into the bacterial adaptation to the onslaught of antibiotics and the immune response. It will likely lead to novel therapeutic approaches in combating the persistent P. aeruginosa infection that ultimately leads to the untimely death of CF patients. [unreadable] [unreadable] [unreadable]

The overall objective of these studies is to examine the alterations in cellular growth control that occur during neoplastic transformation of rat mammary epithelial (RME) cells. The working hypothesis is that normal cells have a finite proliferative lifespan and that an important alteration in cellular growth potential occurs during cancer development which results in the acquisition of an enhanced, perhaps indefinite, proliferative lifespan. Acquisition of this enhanced proliferative potential is necessary but insufficient for complete neoplastic transformation and therefore represents a truly preneoplastic phenotype. The experiments will employ a newly developed culture system in which normal RME cells undergo 15 to 20 population doublings over 4 to 5 in vitro passages before exhibiting signs of senescence. This culture system will be used; a) to examine the hormone and growth factor requirements that induce long term proliferation of normal RME cells in vitro, b) to isolate populations of preneoplastic cells from mammary tissues of carcinogen treated rats and populations of neoplastic cells from mammary carcinomas based on their enhanced proliferative lifespan, 3) to examine the hormone and growth factor requirements of preneoplastic and neoplastic RME cells relative to those of normal RME cells in vitro. These studies address the mechanisms of the alterations in growth control that occur in RME cells during the stepwise acquisition of enhanced proliferative potential and neoplastic potential.

Insulin resistance is a classic feature of non-insulin dependent diabetes mellitus (NIDDM). Compelling evidence exists, from a variety of ethnic populations, to indicate that insulin resistance and hyperinsulinemia are early defects characterizing the prediabetic state. Thus, NIDDM features a primary, probably genetic, component of insulin resistance. However, the magnitude of insulin resistance is much greater in NIDDM than in prediabetic conditions. We hypothesize that this additional insulin resistance in NIDDM results from hyperglycemia through a form of glucotoxicity. Specifically we hypothesize that hyperglycemia causes impairment of insulin receptor tyrosine kinase activity though activation of serine and threonine kinase. This secondary component of insulin resistance is reversible, by correction of the hyperglycemia. A detailed elucidation of the cellular mechanisms underlying this phenomenon will open the possibility of new therapeutic approaches to NIDDM. We propose a combined in vivo and in vitro approach to test this hypothesis. We will induce in vivo hyperglycemia in various groups of human subjects at different insulin levels and conduct a thorough characterization of insulin action in vivo and in vitro before and after the hyperglycemia. We will assess the impact of hyperglycemia on insulin receptor signaling in adipocytes and skeletal muscle, in biopsies taken before and after hyperglycemia. We will test our hypothesis that diminished insulin receptor kinase activity results from increased serine/threonine kinase activity, probably through protein kinase C activation. We will first study lean control subjects but plan to extend the study to obese subjects, subjects with IGT and healthy elderly subjects, any or all of whom may be more susceptible to this secondary form of insulin resistance.

Virtual elimination of vertical transmission of HIV is within reach in South Africa. Despite high antenatal HIV prevalence, implementation of currently available technology and guidelines in the South African Prevention of Mother-to-Child Transmission (PMTCT) program, and particularly the longstanding and comprehensive program in the Western Cape Province, South Africa, has resulted in substantial reductions in vertically transmitted HIV to 3.5% across the country, and 3.3% in the Western Cape. Yet, elimination remains elusive due to persistent coverage gaps and drop-offs at each of the steps required in completion of the PMTCT and infant care continuum, with upwards of 1000 vertically infected infants born in the Western Cape each year. Improving the performance of the current PMTCT and antiretroviral therapy (ART) guidelines at each point in the care continuum through meticulous surveillance of system failures with resultant intervention, are key to further mitigating the effect of HIV on children, irrespective of possible future introduction of different guideline and service design options. Strengthening the health system with this active surveillance approach will provide long term benefits that will both complement and endure beyond future changes in drug protocols or service delivery models. This system will foster a paradigm in which identified cases of infant HIV exposure not covered by PMTCT drugs and HIV infected infants are viewed as a public health emergency requiring urgent intervention to identify reasons for program failure and mitigate its effects in an individual child. This project focused on a primary care obstetric facility, aims to implement and evaluate three linked enhancements to the existing service platform that will iteratively identify and close all PMTCT and early infant diagnosis and ART coverage gaps. First, existing paper registers at antenatal, obstetric and infant clinics will be digitized, internally linked, and merged with laboratory data using context-appropriate technology that has been applied to other priority programs, namely ART and TB monitoring. The combined PMTCT e-register will be linked to a system of urgent reporting of laboratory results of low CD4 counts in pregnant women and positive infant HIV-PCR test results to clinics, with tracing to ensure prompt ART initiation. This will strengthen the health system to close these key PMTCT coverage gaps. Second, a system of routine cord blood testing for HIV, and, if positive, the presence of antiretroviral drugs (ARVs) will identify and link to care HIV-exposed infants with no/suboptimal peripartum ARVs to ensure interventions to prevent postnatal transmission, prompt infant diagnosis and ART if infected. Third, a program of clinical quality assurance, improvement and audit will be established. This will be based on data from both the e-register and cord blood surveillance that will generate an early warning system of coverage gaps. Using these data, PMTCT program failures will be regularly and systematically analyzed together with Provincial and Local Departments of Health service managers and providers, with a view to achieving iterative service improvements.

In response to arterial injury, vascular smooth muscle cells (VSMC) proliferate and migrate from the media to transform the intima from essentially a single layer of endothelial cells to a multilayered neointima that subsequently compromises the vascular lumen. Neointimal proliferation plays a critical role in the pathogenesis of atherosclerosis and restenosis after balloon angioplasty and arterial stenting. The fibrinolytic system is activated by vascular injury and plays a major role in modulating the ensuing vascular response. This application will focus on the interconnected functions of plasminogen activator inhibitor-1 (PAl-l), vitronectin, and fibrin(ogen), key components of the fibrinolytic system that appear to play major roles in regulating VSMC function, but whose in vivo actions during neointima development are poorly defined. The Specific Aims of our studies are as follows: Aim 1: Test the hypothesis that PAl-1 mediates the proliferative response to vascular injury by fibrin(ogen)-dependent and fibrin(ogen)-independent pathways. Aim 2. Test the hypothesis that PAI-1-RR (a mutant that lacks anti-proteolytic activity, binds vitronectin normally) can inhibit the pathologic vascular actions of PAl-1 by a dominant negative mechanism. Aim 3. Test the hypothesis that vitronectin modulates the interactions of VSMC with fibrin matrices. We anticipate that these studies will greatly enhance our understanding of the role of the fibrinolytic system in the pathogenesis of neointima formation, and that they may lead to new strategies for treating or preventing this important clinical problem.

Infants with congenital heart defects (CHD) demonstrate impaired ability to regulate autonomically controlled neurobiologic processes, such as those revealed in feeding and arousal. An impaired ability to regulate neurobiologic processes compromises infants' capacities for physical growth and maintenance and may have long-lasting effects on regulation of social and emotional behavior. The quality of interactions between mothers and infants may affect neurobiologic regulation. This study is designed to describe the relationship between maternal feeding behaviors and physiologic regulation and regulation of arousal in infants with CHD. The long term goal of this research is to identify specific maternal behaviors that contribute to physiologic regulation and regulation of arousal in these infants and to develop interventions directed at supporting mothers in interacting with their infants in ways likely to enhance this critical function. Because each type of congenital heart defect has unique functional and physiologic characteristics, this study will be limited to infants with transposition of the great arteries, one of the most common CHDs. The specific aims are: (1) to describe infant physiologic regulation and arousal during feeding and (2) to examine the effect of mother's feeding behavior on infant physiologic regulation and arousal. Data will be collected during the immediate post-operative period and again two weeks later in order to capture early patterns of maternal behaviors and their association with physiologic regulation and arousal during feedings. Heart rate variability (HRV) will be analyzed and used as an index of physiologic regulation. Infant state during feeding will be used as an index of regulation of arousal. Videotapes of infant feedings will be examined to identify maternal task and social-emotional-behavioral competencies. Multilevel modeling will be used to model patterns of HRV change within feedings and over time, and to examine maternal feeding behavior as a predictor of HRV and arousal. This research will provide important information about how interactions between mothers and infants with congenital heart defects influence the infants' physical, emotional, and social outcomes. This knowledge will be used to develop interventions to assist mothers in establishment of patterns of caregiving supportive of their infant's development. [unreadable] [unreadable] [unreadable]

The marine mollusk Aplysia has a relatively simple nervous system with large nerve cells, many of which can be individually identified and studied from animal to animal using cellular electrophysiological techniques. We have recently found that a group of neurosecretory cells which are known to release a hormone that induces egg laying behavior in this animal appears to alter the physiology of other neurons and synapses for an hour oR more following stimulation of the neurosecretory cells. From previous studies it is known that some of the affected neurons are involved in the neural control of the cardiovascular system. We are now studying these changes in detail to determine if a substance released from the neurosecretory cells acts hormonally to cause the long-lasting changes, to analyze the cellular mechanisms underlying them, and to determine if they serve to alter the neural control of the cardiovascular system during egg laying.

Hemoglobin-based blood substitutes catalyze a number of oxidation-reduction reactions which can lead to both the destruction and formation of reactive oxygen species (ROS) and reactive nitrogen species (RNS). In order to assess the contribution of these reactions to the cellular toxicity of blood substitutes, we have developed in vitro models to elucidate the molecular mechanisms by which blood substitutes enhance or inhibit vascular endothelial cell damage by ROS and RNS. We have developed an in vitro model system for assessing hemoglobin-induced damage of bovine aorta endothelial cells (BAEC), the role of reactive oxygen species as mediators of damage, and the relative contribution of necrotic and apoptotic cell death pathways. Using this system we demonstrated that the pseudo-peroxidase activity of hemoglobin protected against hydrogen peroxide induced necrosis. Cross-linked hemoglobin derivatives were shown to undergo oxidative damage and to undergo a transition to a stable, ferryl (Fe+4) state in the presence of hydrogen peroxide. The isolated ferryl-hemoglobin derivatives were toxic to BAECs and induced apoptotic cell death. We also examined the interaction of bacterial lipopolysaccharide (LPS) with hemoglobin and the enhanced toxicity to endothelial cells resulting from the LPS/hemoglobin complex. Hemoglobin had no effect on apoptosis of BAEC or on CD 54 (an endothelial cell adherence protein) expression induced by LPS interaction with the high-affinity LPS receptor. Enhanced toxicity was only observed at relatively high concentrations of LPS in the absence of high-affinity LPS receptor interactions. Anti-oxidants selectively protected against enhanced toxicity of the complex, suggesting that the enhanced toxicity is primarily due to LPS induced changes in the oxidation of hemoglobin. Recent studies have implicated peroxynitrite, a highly reactive species produced by activated inflammatory cells, as a mediator of endothelial cell damage. Preliminary studies suggest that peroxynitrite induced toxicity towards BAEC is partially dependent on the oxidation of serum components. In this system, hemoglobin selectively protected against the formation of toxic serum components upon the addition of peroxynitrite. Future studies will address the serum components undergoing oxidation and the mechanism by which hemoglobin protects.

This study (which is in its fourth of five years, and for which we are requesting four additional years of support) is designed to address and resolve as-yet unanswered questions about the effects and effectiveness of a lower-than-standard range of lithium (0.4-0.6 mEq/1) in the maintenance treatment of patients with bipolar affective illness. We are comparing and contrasting this lower level with a more standard one (0.8-1.0 mEq/1) regarding protection against relapse, the presence of less severe affective symptoms, and association with unwanted and toxic effects. This double-blind study is being run concomitantly at both the Massachusetts General Hospital and the Long Island Jewish-Hillside Medicare Center. Projected intake to completion is 175 subjects at the two sites, with patients randomly assigned to the two different treatment regimens.

Angiogenesis is a rate-limiting step in the progression of human tumors. Key proteins involved in tumor vessel formation are good targets for therapy, since the forming vasculature is composed of genetically stable cells unlike the malignant cells in the tumor proper. The vascular endothelial growth factor (VEGF) receptors are thought to be master regulators of embryonic and tumor angiogenesis; thus, are currently targets for anti-angiogenic therapy. The transparency of the zebrafish embryo makes it an ideal model for angiogenic studies since blood flow can be easily observed in a living animal. In preliminary studies, a human VEGF receptor inhibitor, PTK787/ZK222584, was used to induce potent inhibition of embryonic vessels in the zebrafish. This chemcial genetic approach allowed an examination of the VEGF receptor signaling pathway as an upregulation of a downstream effector, AKT/PKB, can override the receptor block to provide a robust vessel rescue (Chan et al., 2002). In this grant proposal, chemical inhibition is combined with proven forward genetics to identify critical genes in angiogenic signaling as enhancers or suppressors of the anti-angiogenic phenotype. A pilot screen has been conducted using F2 heterozygous embryos under the influence of PTK787. Angiogenic defects have been confirmed genetically in F3 homozygous recessive embryos. Thus, a large scale screen will allow us to identify physiologically relevant players in the VEGF signaling pathway as additional targets for inhibitor therapy in cancer treatments. The application has two specific aims. Aim 1. To perform an enhancer screen for angiogenic mutations under drug-sensitized VEGF receptor function. Aim 2. To perform a suppressor screen for mutations that can override the drug-induced anti-angiogenics effects in zebrafish embryos.

Parasites of the trypanosomatid protozoan genus Leishmania are responsible for a spectrum of tropical diseases that afflict more than 10 million people worldwide, and depending on the specific speciesand mmune status of the infected person, can be severe or fatal. In several countries, leishmaniasis is a common opportunistic infection in AIDS patients, and many USsolidiers stationed in the Middle East have been infected. The goal of our research is to develop and apply new methods for the identification of genes used by this parasite to carry out its infectious cycle. Our premise is that the availability of powerful molecular genetic tools, and identification of genes important to parasite virulence, will radically advance our ability to develop improved control strategies. In previous studies methods were developed for manipulating the parasite genome in a variety ofways by forward or reverse genetics, and to develop functional genomic approaches such as transposon mutagenesis and expression profiling. Success with expression profiling now enables us to incorporate this powerful methodology more comprehensively into our research program, and undertake a broader survey of patterns and mechanisms of gene expression in Leishmania, making use of both genetic and biological methodological advances from the prior grant period. The goal is to identify genes showing patterns of expression suggestive of functional roles in the infectious cycle, for example stage-specific expression or alterations in response to relevant stimuli, whose predictions and implications can then be tested experimentally. One example that will be pursued involves a new perspective on the differentiation of the parasite into the infectious metacyclic form transmitted by sand flies, involving global changes in parasite gene expression probably mediated by changes in chromating structure, specific changes in expression arising from a combination of mechanisms, and biological changes involving massive cellular remodeling and 'shrinkage' possibly mediated by autophagy.

An ALPS-like patient without an identified mutation has been found to have a unique in vitro defect in T cell apoptosis associated with IL-2 withdrawal but normal T cell and B cell apoptosis in response to staurosporine, etoposide, cisplatin and radiation. This suggests the possibility of a distinct defect in the mitochondrial cell death pathway with a number of candidate proteins that could account for this in vitro defect. This lead to protein analysis by Western blot using B cell lines from the patient that revealed normal levels of Bim, Bad, phosphorylated-Bad, Bak, Bax and Bcl-2. In addition sequencing of genomic DNA revealed no evidence of a mutation in the gene encoding Bim and using cDNA, no evidence of a mutation in the gene encoding Bak. In light of the clearly demonstrated defect in apoptosis uniquely associated with cytokine withdrawal and the negative studies of candidate proteins, we have begun evaluation of this patients lymphocytes compared to normals using apoptosis-specific and whole human genome microarrays. At the same time we are further evaluating the nature of cytokine withdrawal induced cell death based on this patients since the in vitro functional findings suggest that this death pathway has distinct features that differ from other mitochondrial death pathways induced by drugs (etoposide, cisplatin) and irradiation. The Bim sequence is normal including over 600 bases upstream from exon 1 that includes the forkhead binding domain as well as sequencing demonstrating that the poly A site is normal. In light of the consistently lower levels of Bim including at baseline studies are currently underway to evaluate the function and sequence of the forkhead O proteins including Fox O1, Fox O3 and Fox O4. The data from these studies is currently being assembled for the first submission.

A. Summary and Objectives The overall aim of the Research Education and Career Development (REC) Core of the Institute for Clinical and Translational Research (ICTR) is to develop a diverse, multidisciplinary, clinical and translational research workforce capable of accelerating the translation of research findings into evidence-based policies and practices that improve the health of individual patients and all populations in the United States. This goal is consonant with the NIH Roadmap and the recent transformation of the UW School of Medicine into an integrated School of Medicine and Public Health. The REC Core will capitalize on the momentum at UWMadison to: 1) advance multidisciplinary collaborations; 2) implement new curricula that complement existing research education programs; 3) establish and evaluate mentor-training programs; 4) design interventions that build on the career development scholarship of our faculty; and 5) transform institutional culture to increase the participation and advancement of women and underrepresented minority researchers in the academic enterprise. To achieve these goals we will pursue the following Objectives: Objective 1: Create a menu of educational and career development opportunities with multiple portals of entry and varying levels of intensity to accommodate the needs of all learners; build activities around six core competencies in clinical investigation and six core competencies in teamwork. Objective 2: Launch a new cross-college MS/PhD in clinical investigation to complete a menu of graduate degree and certificate programs in clinical or translational research. Objective 3: Move conceptually and programmatically from independent training programs (CTSA K12, CTSA T32, individual K-awards) to ICTR Trainees and Scholars, who are recruited and selected through a uniform process; matched with the appropriate funding sources according to their discipline and stage of career development; and provided appropriate educational opportunities, career support, and well-trained mentors to support their success. Objective 4: Increase the gender and ethnic/racial diversity of future academic leaders by: 1) coordinating minority recruitment across all UW Madison T32s; 2) assisting Pis in obtaining minority supplements to their NIH IRQ 1s; 3) emphasizing opportunities for research on health disparities1; 4) establishing a Minority Scholars Program; 5) including diversity training in professional development; and 6) piloting and evaluating programs to assist young investigators in balancing personal and professional worklife. Pursuing these objectives is essential to strengthen the educational and career development programs that are currently offered at UW and to embrace a more comprehensive approach to training clinical and translational investigators. The programs developed to meet these objectives are founded upon the following principles: [unreadable] Build on local strengths and multidisciplinary culture [unreadable] Take an evidence-based approach to education, career development, and institutional transformation by applying research from adult education, social and organizational sciences whenever possible [unreadable] Incorporate gender and ethnic/racial issues as they apply to all programmatic elements, including recruitment, retention, career development and research on health disparities [unreadable] View the ICTR as a "living laboratory" evaluating interventions and re-directing resources accordingly The objectives for the REC Core are so intertwined that they are approached in a comprehensive manner throughout the section, with many of the initiatives serving as action items for more than one objective. Hence, unlike many of the other sections, action items pursuant to a single objective are not described.

Liver is the major site of metabolism, and there are a multitude of known human genetic disorders due to hepatic deficiencies. These include phenylketonuria, glycogen storage diseases, urea cycle enzyme deficiencies, hemophilia, alpha-1-antitrypsin deficiency, etc. In these well characterized monogenic diseases, the metabolic imbalance can be restored by proper expression of the missing functions in the hepatocytes of the patients, thereby effecting a permanent cure of the disorders by hepatic gene therapy. As the human genes responsible for many of these disorders are being isolated and shown to be functional after transfection into mammalian cells, the limiting step is undoubtably the lack of suitable technologies to reintroduce these genes into the patient's liver cells. During the past several years, we and others have reported successful isolation of primary hepatocytes and demonstrated that functional genes can be efficiently transduced into them by retroviral mediated gene therapy. Most recently the feat of reintroducing these cells back into experimental animals and demonstrating long-term survival and functional of the engrafted cells in vivo has been accomplished. Primary mouse hepatocytes were effectively transplanted into recipient mice through direct injection into the portal vein or the spleen, and we demonstrated unambiguously that the engrafted hepatocytes migrate to the liver, incorporate themselves into the normal parenchyma and continue to function indefinitely in vivo. Based on this break through, we propose to 1) attempt to correct certain hepatic deficiencies in two different animal models, 2) investigate the regulation of hepatocyte growth and differentiation, and 3) explore and perfect hepatocyte transplantation technologies for larger animals. This ambitious program pulls together a group of investigators with expertise in molecular biology and genetics, cell biology and differentiation, as well as surgery and pathology, who will function as a unit to expedite the development of necessary technologies and clinical protocols for the correction of hepatic deficiencies by gene therapy.

The goal of this work is to gain a better understanding of the role of central factors in auditory masking in listeners with cochlear hearing loss. The work has theoretical significance for the study and remediation of hearing impairment by aiming to better understand the causes of the communication difficulties experienced by listeners with cochlear hearing loss. The proposal is based on a theory that identifies two types of masking that occur at different physiological levels. Peripheral or "energetic" masking occurs because of overlapping patterns of excitation in the cochlea and has been studied in detail for many years. Central or "informational" masking involves cognitive processes related to the perceptual organization of sound images and the analysis of sound patterns. Informational masking is not well understood, and occurs despite a robust representation of the signal in the auditory periphery. The purpose of this study is to extend the work on informational masking to listeners with hearing loss. This knowledge is critical because of the prevalence of auditory pathologies affecting the sensory mechanism and the extreme difficulty in communication such pathologies often cause, particularly in noisy listening conditions. Although it is clear that sensory pathology affects the spectral and temporal analyses performed at the periphery, there appears to be a significant component to masking that cannot be attributed purely to peripheral deficits. The plan is to test the hypothesis that listeners with sensorineural hearing loss experience abnormally large amounts of informational masking in certain conditions and make poor use of the cues that normally reduce informational masking. This hypothesis will be examined through a series of psychoacoustic experiments employing listeners with cochlear hearing loss. The goal is to relate the amount of informational masking to factors such as etiology and configuration of loss, auditory filter characteristics and processing efficiency, age and speech recognition in noise.

Our aim is to define the details of how the enhancers of the human virus, BKV, regulate the biology of the virus and to determine what role the BKV enhancers play in viral transformation and tumorigenesis. The enhancer element of BKV consists of three 68 bp tandem repeats which stimulate early viral promoter transcription. Recently, we have discovered that the BKV enhancers are necessary for viral replication. Also, reducing the number of BKV enhancers is known to increase the transformation efficiency of the virus. By extrapolation, it is likely that the BKV enhancers affect episomal persistence of the viral genome and tumorigenesis. Our approach will be to generate a variety of different enhancer mutations and assay for altered viral regulation, transformation and tumorigenesis. The types of enhancer element mutations that will be generated are: enhancer elements with one, and two repeats; 5' and 3' enhancer terminal deletions; internal deletions; site-specific mutations of interesting landmark sequences; reverse orientation; and direct substitution of BKV enhancers with those of other viruses. We will test the enhancer mutations both in the context of virus and plasmids, in which specific viral functions can be studied independently of one another. These viruses and plasmids will be tested in distinct assay systems to determine the effect of the mutated enhancers on viral replication, transcription, episomal persistence, transformation and tumorigenesis. We will also use a variety of techniques to explore the binding of specific proteins to the BKV enhancers with the aim of defining how these proteins may regulate the different enhancer functions. In particular, we will investigate the role of the TGGCA nuclear protein which specifically binds to the BKV enhancers. We will use our site- specific enhancer mutants to define the relationship between altered replication and binding of the TGGCA protein. We will also attempt to develop an in vitro replication system to further define the role of the BKV enhancers in replication.

Georgetown University Medical Center is a participating institution of the National CNS Tumor Consortium (NCNSC) whose Central Operations Office/Coordinating Center is at the MD Anderson Cancer Center (W.K.A. Yung, M.D., P.I.). The Georgetown Brain Tumor Center within the Lombardi Cancer Research Center provides: (l) Experienced full-time physician investigators with expertise in Phase I/II studies; (2) A multidisciplinary neuro-oncology team; (3) Resources to comply with data reporting requirements of the NCI; (4) Patient populations to support adequate patient accrual including patients with malignant glioma and patients with multiple nervous system tumors due to genetic disorders; (5) State of the art imaging facilities; (6) Appropriate drug control procedures for NCI-supplied experimental agents; (7) Capability of meeting FDA requirements of Phase I and Phase II trials. The Georgetown Brain Tumor Center has research expertise in defining and using the molecular differences between tumor cells and their normal nervous system counterparts to develop therapeutic protocols. Current research includes: (l) Angiogenesis induced by nervous system tumors: molecular mechanisms and therapeutic blockade; (2) Development of genetically engineered viruses to modulate or kill malignant nervous system tumors; (3) Molecular mechanisms of radiation resistance of malignant gliomas; (4) Regulation of growth and differentiation of malignant pediatric and adult brain tumors by protein kinase C; (5) Peptide growth factors, their receptors and carrier proteins in the invasiveness and motility of human brain tumors. As a part of the National CNS Tumor Consortium, we will begin a study within the first year of the protocol: "Phase II randomized evaluation of recombinant interferon beta as a single agent and cis-retinoic acid (isotretinoin) plus recombinant interferon beta in the treatment of patients with recurrent primary malignant gliomas". We have also performed the basic studies necessary to provide a foundation for a second protocol using the antiangiogenesis agent TNP-470 to treat patients with brain tumors. The Georgetown Brain Tumor Center is an ideal setting for performing multi-center, multidisciplinary therapeutic trials within this CNSC. Our research strengths complement those of the other participating institutions.

Pteronotus parnellii and Rhinolophus ferrumequinum utilize a Doppler shift type of sonar, that is they lower their pulse frequencies so that the echo frequencies remain constant. Studies are being carried out on both species using a pendulum device to monitor echolocative behavior under simulated flight conditions. Both species accurately Doppler shift compensate during acceleration and deceleration phases of the pendulum swing; neither compensate for negative shifts during the backward swing. Further experiments will be carried out in order to directly compare and/or contrast the sonar systems of these two species. In addition, anatomical studies using the LM, TEM, and SEM are continuing on the cochleae of Rhinolophus and Pteronotus in order to compare structures of the inner ear. Results to date indicate the evolution of very different specializations. Work will also continue on quantitative comparisons of nerve fibers in the sparsely and densely innervated regions, and the unusual lining of the scala tympani will be further examined with TEM and X-ray microanalysis. Frequency mapping after acoustic overstimulation will be completed.

One of the most promising approaches in immunotherapy is activating antitumor immunity in patients by 'checkpoint blockade' referring to the use of antibodies that block immuno-inhibitory pathways switched on by cancer cells. A growing number of cancers that respond to these checkpoint blockades have now been described in patients. Antibodies that block CTLA-4 (ipilimumab) and PD-1 (pembrolizumab) have been approved to treat patients with melanoma, renal-cell carcinoma and non-small-cell lung carcinoma with durable clinical responses. However as checkpoint blockade becomes a promising therapeutic avenue for cancer therapy, current preclinical models are not able to recapitulate the antitumor immune responses mediated by checkpoint blockade therapies. This is partly because of the inherent differences between the mouse and the human immune systems. HuMurine Technologies was founded in 2008 as a CRO to standardize and optimize the construction of 'humanized' mice (Hu-M(tm) platform) stably engrafted with a human immune system. The Hu-M(tm) platform has been successfully implemented for evaluating therapies that specifically target the human immune system in a wide range of diseases models. In order to address the high unmet need for a robust preclinical platform for testing the efficacy and safety of novel immunotherapies HuMurine has partnered with Champions' Oncology to develop and validate the PDX/ Hu-M(tm) platform using the approved immune- checkpoint modulators Ipilimumab and Pembrolizumab. This development process will also included consultation with potential end users of the platform including major pharmaceutical companies and biotech's which demonstrates a strong commercialization plan for the platform post-validation. This proposal aims to address a part of NCI's provocative questions initiative about the development of appropriate model systems to understand the human immune responses within the tumor microenvironment. Validated of this novel PDX/Hu-M(tm) platform would significantly reduce the high attrition rates of immunotherapeutics in clinical trials. The Specific Aims of this proposal are: Aim 1: Determine if Ipilimumab treated HLA-A2- matched melanoma PDX/ Hu-M(tm) mice generates a tumor- specific T cell response. Aim 2: Determine antitumor efficacy of combinational checkpoint blockade of CTLA-4 and PD-1 in HLA-A2 matched melanoma and NSCLC PDX/ Hu-M(tm) mice.

Description: (Taken directly from the application) The Nutrition Core will oversee the dietary requirements and standardization of all dietary regimes for all three studies in the program project. In addition, in collaboration with Core B, it will provide the methodology for tracking food intake, carrying out somatometric determinations on the animals, body composition measurements, Bioelectrical Impedance (BIA) and Dual energy X-ray Absorptiometry (DEXA) and blood collections for micronutrient determinations for all three studies. Quality control measures will be incorporated into the protocol and included for all laboratory measurements to allow evaluation of laboratory and procedural variability. Maximum utilization of the data across all three projects will be possible due to the standardization of the study parameters. A manual of operation will be developed with detailed information regarding all procedures and protocols and a training session will be carried out to standardize the procedures across all staff carrying out study tasks. Retraining of personal on study procedures will be required yearly. The Nutrition Core will work with the Statistical Core to develop data forms for their study data and set quality control procedures and limits for all data. The Core Leader has had extensive experience with data entry and analysis on the study methods outlined in the ongoing HIV program project.

This is a General Clinical Research Center grant supporting eight beds for inpatient research, without laboratory facilities. The studies currently being carried out are designed by investigators of the Departments of Medicine, Dermatology, Psychiatry, Gynecology and Neurosurgery of this hospital. The unit provides facilities for clinical research, particularly research requiring careful control of diet, complete collection of urine, timed collections of blood, and innovative and complex diagnostic, therapeutic and physiological techniques. Facilities are not available for the management of acutely ill or post-surgical patients, or other patients requiring extensive nursing care.

The purpose is to study the interaction of calcitonin with its specific receptor target organs. The current investigations should provide further insight into the structure function relationship in calcitonin. Calcitonin is a small polypeptide hormone and therefore lends itself well to studies using synthetic peptide fragments. The system is also useful for characterizing hormone receptors in kidney, bone and other tissues. Studies are in progress to characterize further the interaction of calcitonin with tissue receptors. It also will be of interest to solubilize the receptors and characterize them chemically.

(1) The reactivity of functional groups attached to cross-linked polymer beads will be studied as a function of the network structure, to provide background information for the "solid-state synthesis" of polypeptides and proteins. (2) The attack of enzymes on side chains of synthetic polymers will be studied to provide background information for a possible method of sustained drug release. (3) The interaction of reactive groups attached to two polyanions will be studied to aid the interpretation of data on DNA renaturation. (4) The adsorption of flexible macromolecules onto monodisperse latex particles will be studied in connection with the use of such latex as a tool in medical diagnosis.

Research has been initiated to determine the adenovirus gene products required for cell damage and to establish the molecular mechanisms effecting the pathogenesis of adenovirus diseases. The cotton rat is an ideal animal model for these studies because the pathology of the pneumonia and upper respiratory disease produced by type 5 adenovirus in this animal is similar to that observed in humans. Basic experiments have been carried out to establish the parameters of the infection: kinetics of viral replication and clearance of virus from the lung; the temporal relationship of viral replication to the development of pneumonia; the cellular sites of viral replication in the lung; the immune response to infection; and the establishment of viral latency. Two species of cotton rats, Sigmadon hispidus and Sigmadon fulviventir, were compared in these studies; the S. hispidus proved to be the better species for investigating pathogenesis of pneumonia, but the S. fulviventir, appears to have distinct advantages for studies of the latent state. Experiments have been initiated using a variety of deletion, insertion, and conditionally lethal mutants in an effort to determine the gene product or gene products responsible for cell damage and subsequent development of pneumonia. An adenovirus expression vector, using the vaccine strain of type 4 adenovirus, is being constructed to develop an alternate viral vaccine containing the rotavirus gene encoding the major antigen that induces neutralizing Abs. This vaccine would have a number of advantage for prevention of rotavirus infections throughout the world.

[unreadable] The proposed construction is a renovation of 26,918 net square feet of an existing laboratory building at the Southwest Foundation for Biomedical Research that will provide offices and laboratories for 8 faculty and 2 senior staff scientists in the Department of Genetics. This proposed construction is an integral part of a multiphase master plan to modernize the laboratory facilities at SFBR, with the intent of providing facilities that adhere to current safety standards for biomedical research laboratories. Phase I was started in 1997, Phase II started in 2000, and the proposed construction represent a portion of the Phase III of the plan. The specific aims of this proposal are to provide "wet" and "dry" laboratory facilities for investigators with research programs in molecular and statistical genetics that will promote the efficient use of laboratory space by increasing the amount of core space shared between research programs, that will place these programs in biosafety level 2 conditions that do not exist in their current laboratories, and that will foster scientific interactions between the molecular geneticists and the molecular and statistical geneticists by locating laboratories in adjacent space and locating all offices in adjacent space. Thus, the design of the proposed laboratory facility will provide efficient space currently needed by these programs while also improving the collaborative research environment for faculty and staff. [unreadable] [unreadable]

PROJECT SUMMARY/ABSTRACT Although there is some consensus that brief interventions (BI) in general medical settings can reduce self-reported alcohol consumption among patients who drink risky amounts, effect sizes vary widely, most patients continue to drink risky amounts, and there are many null studies. Despite the lack of understanding of why BI effects can vary so much across settings, screening and brief intervention are still widely recommended for implementation in general health settings. Knowing what works and what does not, how much is necessary to achieve desired effects, and in what settings and circumstances BIs work more or less, has become critical. The proposed study will therefore use meta-analytic techniques to analyze variability in the effects of BIs for drugs and alcohol delivered in general healthcare settings. The specific aims of the study are to 1) assess the overall effects and variability in effects of BIs for drugs and alcohol delivered in general healthcare settings, 2) examine variability in effects of BIs according to active intervention components, participant characteristics, provider and setting characteristics, and study methodology, and 3) create predicted profiles of the contexts and settings in which BIs may be most or least effective in reducing drug and alcohol use. The proposed study will compile a meta-analytic database from the corpus of experimental studies examining the effects of BIs in general healthcare settings. Studies eligible for inclusion will be those involving a BI delivered in a general healthcare setting, intended to reduce drug or alcohol use among patients (adults and youth) screened for heavy substance use. Randomized controlled trials conducted in any country and reported in any language will be eligible for inclusion. Detailed information and aggregate data will be extracted from study reports. Individual participant data will also be collected for all eligible studies, to permit more in- depth exploration of variability in intervention effects across different types of patients. Random-effects meta- analysis and mixed-effects meta-regression models with robust variance estimates will be used to synthesize findings across studies and create the predicted profiles identifying the contexts in which BIs are most or least effective. An expert panel of advisors will be used to guide the study, providing feedback on data collection and data analysis strategies, facilitating collection of individual participant data, and ensuring the study addresses clinically meaningful patient profiles for BI research. The results from the proposed study will significantly advance the field of BI research by documenting and exploring the variability in BI effects. Inconsistent findings across primary trials currently present a challenge for researchers and practitioners interested in implementing BIs in healthcare settings. This study will identify the intervention contexts and patient profiles for which BIs may be most or least effective, providing timely and valuable guidance for future research on and implementation of this form of drug prevention program.

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. 1. Determine the effect of FSH-stimulated granulosa cells on androgen responses to hCG in PCOS women. 2. Determine the contribution of adrenal androgen production to total androgen output in women with PCOS. 3. Determine the role of insulin on basal and stimulated androgen production in women with PCOS.

The mechanisms of cardiac defibrillation are subject of major interest in the treatment of human heart disorders. Significant progress in the understanding of defibrillatory action has been made recently through advanced high-resolution mapping studies. The research focus s has now advanced from the traditional empirical approach to the mechanistic interactions of shocks and wavefront propagation. Recent progress in optical imaging techniques has enabled such studies to detailed spatial information of the transmembrane potential (Vm) distribution around the entire heart throughout the defibrillation episode, providing unobstructed panoramic views of the epicardial wavefronts. Similarly, experimental results using optical imaging have promoted the concept of cardiac bidomain from theoretical considerations to a valid representation of myocardium, thus generating new hypotheses in defibrillation mechanism and prompting the necessity to re-evaluate traditional ones. The major goal of this proposal is to study basic defibrillation mechanisms for the perspective of cardiac bidomain. Furthermore, this project is the first attempt to use dynamic high resolution Vm images interactively in defibrillation by including the imaging result as a feedback component in a real-time, intelligent multi-electrode shock delivery system. The interactive shock system allows the test of important hypotheses pertaining to the mechanism of defibrillation, which historically have been examined empirically without such capability. We will investigate 91) the interaction of direct activation sites with the tissue status during the shock; (2) the optimal timing for the shock delivery during different stages of the formation on f a reentry pattern; and (3) the optimal timing and sites to deliver the shock during the interaction of multiple spiral recenter patterns. Bu7 combining various predictions and hypotheses related to the concept of bidomain continuum for sycytial cardiac tissue, it is expected that the results from this proposal will offer new insights of the fibrillation mechanisms based on spatia-temporal wavefront interactions as predicted by the cardiac bidomain, and could provide practical guidelines for the future, improvement of arrhythmia intervention.

We wish to define the pathogenesis of Crohn's disease (CD). Using patients' tissues and athymic (nu/nu) mice, we plan to identify isolate and characterize putative antigen(s) related to CD. In our initial studies, lymph node and intestinal filtrates of CD tissue produced lymphoma or hyperplastic lymph nodes in 16% of nu/nu mice. Both lymphomas and hyperplastic lymph nodes immunologically react with 50-80% of sera from patients with symptomatic CD and not with control sera. We further demonstrated that immunoreactive B cells occur in normal nu/nu lymph nodes. Using hybridoma technology, we will "immortalize" antigen(s) present in these cells. We will examine the effects of chemical and physical treatments of the tissue filtrate on appearance of antigen in lymph nodes. Since the "antigen" detected by CD sera in nu/nu B cells may be an "anti-idiotypic antibody", we will purify IgG from CD tissue filtrates and inject it into nu/nu. Secretory proteins from nu/nu Beta cells-myeloma hybrids and nu/nu serum IgG will be examined for anti-idiotypic antibodies. Using a fluorescence-activated cell sorter, we will separate immunoreactive cells. Using immunohistochemical techniques, EM and immunoelectron microscopy, we will localize the antigen and search for viral particles in these cells. If viral particles are seen, detailed virologic studies will be performed. Using gel filtration techniques, CD and control intestinal tissue homogenates, we identified several CD tissue-specific proteins of 160 kd, 120 kd and 110 kd mol. wts. Specific recognition of the proteins by CD serum IgG was demonstrated by transblot. Using HPLC, we purified the 160 kd protein which reacted with CD sera and not with control sera by dot blot assay. We plan to purify the protein(s), determine their molecular structure and raise monoclonal antibodies. These will enable us to examine their origin (from host tissue or an extrinisic agent(s)), determine whether they are derived from a single source, and whether they are associated with lymphoid hyperplasia and the appearance of immunoreactive cells in nu/nu, and perform molecular studies for cloning their genes. These proteins may provide important probes for studies of the etiology of CD and lead to a better undertanding of its pathogenesis.

DESCRIPTION. Statetheapplication'sbroad.long-fflWbbjectivesandspecificalms, makingreferencetothehealtmBitednessof theproject.Describeconcisely the researchdesign and methods for achievingthesegoals. Avoidsummaries of past accomplishments andthe useofthe first person.This description is meant to serve as a succinct and accurate description of the proposed work when separated fromthe application. If the application is funded, this description, as is, will become public information. Therefore, do not include proprietary/confidential information. DO NOT EXCEED THE SPACE PROVIDED. The Molecular and Cell Biology (MCB) Training Program is an interdepartmental program devoted to the training of individuals for careers as independent research scientists in universitiesand health related research laboratories. The trainers are tenure track faculty members drawn primarily from the Department of Biochemistry and Molecular Biology (BMB) and the Department of Moelcular Genetics and Cell Biology (MGCB) plus selected faculty from the Departments of Chemistry, HumanGenetics, Medicine, Neurobiology,Pharmacology and Physiology, Organismal Biology and Anatomy. Pathology, Pediatrics, Radiation and CellularOncology, as well as the Ben May Institute. The program leads to the Ph.D. degree which is awarded by the Departments or degree-granting Committees. The students take similar fundamental courses in molecular biology, cell biology, and genetics during the first year. They meet uniform standards in degree requirements. The training grant is administered by the Director and the Steering Committee of trainers in the major participatingDepartments and degree-granting Committees. Trainees are selected from students admitted through the various Departments and Committees on the basis of previous academic record and specific interest in cell and molecular biology. Progress is monitored on a regular basis by DepartmentaiyCommittee thesis advisory committees and the MCB Steering Committee. Common features of the program are coursework, laboratory rotations, teaching and assisting in courses, seminar programs, journal clubs,joint research meetings, a student seminar program, an interdepartmental retreat, and minisymposia focusing on current interest in molecular and cell biology. A total of 31 trainees in the first year, with a yearly increment of one to a total of 35 trainees in year five is proposed for a primary training faculty of 63 individuals. The research laboratory space of the trainers exceeds 90,000 sq.ft. Special facilities includeanimal care, transgenic mouse/embryonic stem cell, biostatistics, computing, DNA sequencing, oligo-nucleotide and peptide synthesis, electron microscopy, monoclonal antibody, flow cytometry, NMR spectroscopy, image analysis, confocal microscopy, and DNA microarray facilities, a greenhouse and fly media rooms. PERFORMANCE SITE(S) (organization, city, state) The University of Chicago, Chicago, IL KEY PERSONNEL, See instructions on Page 11. Use continuation pages as needed'to provide the required information In the format shownbelow. Name Organization Role on Project Ursula B. Storb Yair Argon Gayle Lamppa Susan Lindquist Bernard Roizman Andrea Sant Tobin R. Sosnick Edwin Taylor The Universityof Chicago The University of Chicago The Universityof Chicago The University of Chicago The University of Chicago The University of Chicago The Universityof Chicago The University of Chicago Principal Investigator Steering Committee Steering Committee Steering Committee Steering Committee Steering Committee Steering Committee Steering Committee PHS 398 (Rev.4/98) Page 2 QQ Number pages consecutively at the bottom throughout the application. Do not use suffixes such as3a, 3b. NN Program Director (Last, first, middle): Storb, Ursula B. Type the name of the programdirector at the top' printed page and each continuation page. (For typS|[unreadable]cifications, see instructions on page 6.) INSTITUTIONAL NATIONAL RESEARCHSERVICEAWARD (Substitute Page)

This application seeks funds to enable participation in the Eighteenth Pacific Science Congress, which will be convened the week of June 5-12, 1995 in Beijing, China. This year's theme is "Population, Resources and Environment: Prospectives and Initiatives", issues which relate not only to the Pan-Pacific region, but to the scientific community at large. The Chair for the Botany Committee has requested the presentation of a session discussing plant exploration and its relation to drug discovery in the Pacific region, with particular attention to Southeast Asia. As the National Cancer institute, through its support of Dr. Soejarto's project, has been involved with a successful ongoing program of plant exploration and collection, it is believed that participation in this Congress will promote an exchange of information, enabling a more global level of cooperation and communication in the sciences. Further, the presentation of sessions discussing NCI's support with regard to plant exploration will provide the opportunity to engage in scientific dialog with other Pacific Asian entities, establishing friendly relations and ensuring continued plant exploration/drug development programs.

The overall objective of this project is to define the role of chemerin receptors CMKLR1 and CCRL2 in leukocyte trafficking, function, and the pathophysiology of experimental autoimmune encephalomyelitis (EAE), a mouse model of multiple sclerosis. Preliminary results suggest that CMKLR1 is an integrin-triggering chemoattractant receptor expressed by macrophages, NK cells, and upregulated by activated dendritic cells; CCRL2 is a non-signaling chemerin delivery receptor expressed by mast cells, activated macrophages and endothelial cells that binds chemerin and serves to regulate the bioavailability of the attractant; and that the chemerin receptors play critical roles in modulating inflammatory responses in vivo. Studies under Aim 1 will determine if chemerin can induce CMKLR1-mediated integrin triggering and rapid adhesion in macrophages and NK cells. These studies will have important implications for understanding how CMKLR1 contributes to macrophages and NK trafficking in vivo where chemerin is activated by inflammation or coagulation-associated proteases. Studies under Aim 2 test the hypothesis that CCRL2 is a non-signaling chemerin delivery receptor that serves to concentrate and present active chemoattractant. These studies will elucidate the functional significance of CCRL2 in regulating chemerin activity and its signaling via CMKLR1 in vivo. Aim 3 will define the roles of CMKLR1 and CCRL2 in the pathophysiology of experimental autoimmune encephalomyelitis (EAE). Mice genetically deficient in CMKLR1 or CCRL2, as well as anti-mCMKLR1 blocking mAbs, will be used to investigate the contribution of these receptors to disease progression in animals. Local leukocyte infiltration, function, and cytokine production will be examined to define the mechanisms by which the receptors contribute to or modulate inflammation and immune responses in vivo. Together, the studies proposed promise to define a key regulatory mechanism in macrophage, mast cell, and NK cell biology and function. They may lead to novel targets or approaches for the prevention or treatment of autoimmune disease. PUBLIC HEALTH RELEVANCE: This research project has the potential to identify novel targets for the prevention and/or treatment of autoimmune diseases, such multiple sclerosis. This project will improve our understanding of white blood cell trafficking, and may offer new methods for therapeutically altering the accumulation and/or function of critical white blood cell populations at sites of tissue damage and inflammation.

Detailed chemical knowlege of the mechanism of interconversion of various nucleotides has progressed to a point at which questions concerning the energetics of the reactions may be answered. This study is designed to explore the energy changes occurring during thymidylate synthesis form the precursor, uridylate. The enzymes involved in this conversion are one of the few successful targets for tumor chemo therapy. Methotrexate, a folate analog competes with dihydrofolate thus preventing cell replication. The purpose of this study is to provide detailed thermodynamic information, about this enzyme system, which may contribute to the developmental compounds of increased efficacy in chemo therapy. BIBLIOGRAPHIC REFERENCES: I.M. Klotz, D.E. Darnall, N.R. Langerman, "Quaternary Structure of Proteins", The Proteins, 3rd Ed. (1975) 293. Mangold, A. and N. Langerman, "The Enthalpy of Oxidation of Flavin Mononucleotide. II. Temperature Dependence of in vitro Bacterial Luciferase Bioluminescence", Arch. Biochem. Biophys. 169:126 (1975).

PROJECT SUMMARY Adolescent cocaine abuse increases the risk and severity of lifelong addiction and decreases the likelihood that cocaine-abusing individuals will seek treatment. Developing and understanding therapeutic approaches that mitigate maladaptive decision-making and cocaine-seeking behaviors in organisms with a history of cocaine exposure during adolescence could reduce the high societal cost of cocaine addiction. During adolescence, dendritic spines in the prefrontal cortex (PFC), a brain region critical for complex decision-making, proliferate markedly and then prune, refine, and mature. This process is believed to optimize cellular connectivity and set the neural stage for adult functioning, but such dramatic cellular reorganization within a narrow developmental period may also open a window of vulnerability to insults. We have shown that in adolescent mice, experimenter-administered cocaine derails neural development in the orbital PFC (oPFC), eliminating dendrites and dendritic spines. It also impairs complex decision-making, accelerating the development of stimulus-response habits in adulthood. We have also shown that adolescent mice develop marked individual differences in their cocaine self-administration patterns, and that mice self- administering escalating amounts of cocaine most readily develop stimulus-response habits in adulthood. In Aim I, we will test the hypothesis that individual differences in cocaine self-administration in adolescence determine effects on oPFC dendritic spines, such that mice that escalate are more susceptible to spine deficiencies in adulthood. We will then assess whether ifenprodil, an NR2B-selective NMDA receptor antagonist that blocks the reinstatement of heroin-, nicotine-, and alcohol-seeking behaviors in rodent models will also have therapeutic-like effects after adolescent cocaine exposure, occluding cocaine-induced habits. In humans, adolescent cocaine exposure increases the risk of substance use, dependence, and relapse in adulthood. In Aim II, we will examine whether individual differences in cocaine self-administration in adolescence are associated with individual differences in cocaine self-administration and the reinstatement of cocaine seeking in adulthood. We expect that mice with a history of escalating cocaine exposure will respond more for cocaine as adults and be more likely to reinstate responding after extinction conditioning. In these experiments, ifenprodil will be paired with extinction training in an attempt to mitigate the reinstatement of cocaine seeking. This approach models the use of ifenprodil as a therapeutic adjunct to behavioral therapy in humans and is strongly supported by our preliminary findings. Finally, we will test the utility of ifenprodil as an adolescent-targeted intervention strategy. Cortical development is characterized by a reduction in NMDA NR2B:NR2A subunit ratios, which facilitates spine and synapse stabilization. We hypothesize that application of the NR2B-selective antagonist ifenprodil during critical adolescent developmental periods will have corrective behavioral benefits that persist into adulthood.

In the United States (US), stroke affects more than 700,000 people annually in the U.S. and is the third leading cause of death. Stroke patients may present to the emergency room with a wide variety of symptoms that can be caused by other conditions not related to stroke, collectively called stroke mimic. Even at the most advanced stroke centers, the diagnosis of ischemic stroke is made by clinical examination after ruling out hemorrhagic lesions by imaging. These imaging tests can be used to diagnose hemorrhagic stroke but is relatively ineffective in detecting most ischemic stroke. A reliable and rapid method to distinguish stroke and stroke mimic is a necessary aid to current examination and imaging-based stroke diagnosis and will become more crucial with the implementation of new therapies for the time-critical treatment of ischemic stroke. In contrast to cardiovascular disease and oncology, no approved immunoassay or molecular test is available to aid diagnosis. Analysis of a previous dataset demonstrated that our advanced GeneRx algorithm technologies were able to develop an accurate diagnostic model where other multi-marker panels failed. In this phase I proposal we will perform a validation study, collecting blood samples from 200 admitted with stroke-like symptoms and diagnosed as stroke or stroke mimic. We will measure stroke biomarker protein levels markers present in the top combinations and algorithms from our previous study, and utilize our proprietary GeneRx technology to develop a multiple marker predictive model for accurate differentiation of stroke from stroke mimic. Project Narrative: Currently no blood test is available to help accurately diagnose stroke patients. This study develops a diagnostic to help differentiate stroke patients from the many non-stroke patients that arrive at the ER with stroke-like symptoms. Successful development of this diagnostic will increase the effectiveness and speed of stroke treatment to increase a patient's chances of improvement and safety of treatment. [unreadable] [unreadable] [unreadable]

This study was initiated in October of 1996 and therefore there are no major findings or accomplishments to date. Four patients have been accrued, no toxicity has been seen.

Regular physical activity is associated with a favorable cardiovascular risk factor profile, a lower prevalence of morbidities and reduced premature death rates. However, individual differences are observed in the magnitude of benefits derived from a physically active lifestyle. This phenomenon was investigated in the previous phases of the HERITAGE Family Study in which 742 Blacks and Whites from 214 nuclear families, all adults, completed a standardized and fully monitored 20-week exercise training program. There were large inter-individual differences in responsiveness but this heterogeneity was not randomly distributed, as there was significant familial resemblance in the magnitude of the risk factor responses to the exercise program. These differences in response have been associatedwith a number of candidate genes. Moreover, extensive analyses of the data have made it possible to identify several quantitative trait loci (QTLs) for the responses in important risk factors. In this renewal period (Phase 4;2005 to 2010), our main goal is to conclude the positional cloning efforts of four QTLs for the response of cardiorespiratory fitness and hemodynamic phenotypes to regular exercise, to resolve them in terms of candidate genes and allelic variants, and to functionally confirm them. Investigators from the Pennington Biomedical Research Center and from Washington University are submitting a single revised application to continue the close collaboration established over the last 12 years in pursuing the proposed positional cloning goals. The hypothesis to be tested in Phase 4 of the HERITAGE Family Study is that human cardiorespiratory fitness and hemodvnamic changes in response to regular exercise are regulated by a minimum of four QTLs. Two of these QTLs have yielded strong candidate genes: titin (TTN;QTL1), and kinesin 5B (KIF5B;QTL2). We propose to finalize the positional cloning efforts of two other QTLs for cardiorespiratory fitness as well as exercise heart rate phenotypes, i.e., QTLS and QTL4 (Specific Aim1). Furthermore, we will continue the ongoing in vitro studies to characterize the functional properties of the alleles of the SNPs that have been associated with the response to regular exercise (Specific Aim 2). This research will generate unique data concerning the biology of adaptation and the molecular basis of human heterogeneity in the responsiveness to regular exercise. Studies designed to understand why some people are fundamentally more likely to benefit from a physically active lifestyle than others are very important as such a lifestyle is recommended by all national and international public health authorities.

The Tufts IRACDA Program will serve the national need for university and college faculty trained in bio-medical research and optimally prepared for the multiple challenges faced by professors in their first independent position. To be successful, these individuals must set up and manage a productive research lab, obtain grant funding, and develop and deliver innovative science courses. They must understand the value of diversity in the scientific workforce and be able to inspire the next generations of scientists through the teaching and research opportunities they offer. Tufts IRACDA will address these challenges by providing exceptional postdoctoral scholars with the research portfolio and career skills needed to succeed in an academic research environment that includes mentoring of future biomedical researchers. To achieve our goals, we will partner with three local institutions: the University of Massachusetts, Boston, Pine Manor College and Bunker Hill Community College, all of which are committed to educating students from groups underrepresented in the biomedical research workforce. Our Tufts IRACDA program builds upon our successful TEACRS postdoctoral training program. With TEACRS, we developed valuable teaching and research collaborations with our partner faculty and students. Tufts IRACDA will utilize new initiatives to enhance the research and scholarship of our postdoctoral scholars and build upon our existing strengths in teaching and career development. These initiatives will broadly prepare our scholars for academic positions at the nation's top universities and colleges and increase the impact of Tufts connections at our partner schools. To do this, we will: ? Recruit and prepare the nation's best postdoctoral scholars for successful careers in tenure-track, academic positions in the biomedical sciences by providing strong research training. ? Prepare our scholars to create and deliver impactful science curricula to diverse student populations. ? Foster research and mentoring partnerships that encourage undergraduates at our partner institutions to engage in independent research and pursue biomedical science careers. Tufts provides outstanding opportunities for research training in the traditional biomedical disciplines as well as areas such as biomedical engineering, nutrition, and behavioral sciences. Scholars will spend on average 75% of their time conducting research and 25% of their time in career development and teaching activities, including teaching. Similar training, along with comprehensive career mentoring, enabled us through TEACRS to place 88% (36/41) of our alumni in academic faculty positions well-suited to their career goals, a percentage markedly above the national IRACDA average of 73%. In the new Tufts IRACDA program, we will strengthen the training in research and research-related career skills such as grant and manuscript writing, so that we place scholars in a more balanced mix of research-intensive and primarily undergraduate institutions. We plan to support a total of nine scholars each year from IRACDA funds and three from institutional support.

Senescence, a permanent form of growth arrest following oncogene activation or telomere attrition, is generally considered a tumor-suppressive mechanism active in vitro and in vivo. However, senescent fibroblasts that are near epithelial cancer cells may promote tumor formation, although in vivo evidence for the existence of such cells and how they act to promote tumor formation remains elusive. Also unknown is how cancer cells communicate with tumor-promoting fibroblasts, if such fibroblasts do exist in vivo. We may have found a novel means for this communication: in the course of profiling RAS-transformed ovarian surface epithelial cells and their isogenic immortalized counterparts, we identified a chemokine, Gro-1, that is up-regulated in RAS- transformed ovarian cancer cell lines and is critical for transformation of ovarian epithelial cells. Unexpectedly, we found that Gro-1 induced senescence in ovarian stromal fibroblasts. Knockdown of the receptor for Gro-1, CXCR2, abrogates the senescence and leads to uncontrolled proliferation of the fibroblasts. We further demonstrated that Gro-1-induced senescent fibroblasts have an increased proangiogenic factor vascular endothelial growth factor (VEGF-A) and decreased antiangiogenic factor thrombospondin-1 (TSP-1). The ratio of VEGF-A:TSP-1 in senescent fibroblasts is 80 fold higher than that in control fibroblasts, suggesting that senescent fibroblasts provide critically needed factors to enhance tumor angiogenesis. We also observed, in the human ovarian cancer specimens, that the stromal fibroblasts near epithelial ovarian cancer cells are senescent. Because Gro-1 is a secreted molecule activated by RAS and can diffuse from epithelial cancer cells to neighboring fibroblasts, Gro-1 may be a signaling molecule by which cancer cells use to accelerate the senescence of neighboring fibroblasts. Our central hypothesis is that RAS activates Gro-1 expression in ovarian tumor cells. Gro-1 signaling through its receptor CXCR2 activates multiple downstream effectors to create a senescent phenotype. Senescent fibroblasts upregulate their VEGF-A:TSP-1 ratio to act on endothelial cells to induce an angiogenic switch, which in turn leads to tumor promotion. We propose the following two specific aims: Specific Aim 1. Define the mechanisms by which Gro-1 induces senescence and creates an angiogenic phenotype of senescent fibroblasts. Specific Aim 2. Determine the mechanisms by which senescent fibroblasts promote the tumor growth. PUBLIC HEALTH RELEVANCE: Development of cancer requires not only genetic alterations in epithelial cells but also changes in the stroma, a heterogeneous group of cells interacting with cancer cells. The predominant component of the stroma is fibroblasts. We have found that the RAS oncogene can send a signaling molecule, Gro-1, a small secreted protein, to fibroblasts to accelerate their aging process (senescence) and that this process promotes tumor formation. This project aims to define the molecular mechanisms by which aged fibroblasts initiate ovarian tumor growth and the signaling involved in the aging process. The improved understanding of the molecular mechanisms will be required for an eventual exploration of the therapeutic relevance of this observation. [unreadable] [unreadable] [unreadable]

Project Summary The purpose of this individual National Research Service Award (NRSA) is to provide postdoctoral research training for a neuroscience nurse to become an independent investigator focusing on the behavioral and biological mechanisms that contribute to neuropsychiatric syndromes such as apathy in patients with neurodegenerative disease (ND). In addition to coursework and the acquisition of new research skills, I propose to focus this NRSA on impairments of goal-directed behavior (GDB) that are very common but understudied in ND. The syndrome of apathy, defined as a reduction in self-generated or voluntary behavior,1 has profound consequences for morbidity and mortality in the patient and for family caregiver burden. Treatments for apathy are hindered because of our poor understanding of the mechanisms underlying this behavior. In my cross-sectional dissertation data (funded by NINR F31NR013306), I identified three subtypes of apathy in patients with behavioral variant Frontotemporal Degeneration (bvFTD): difficulty with initiation, planning or motivation. Each subtype is related to disease in distinct frontal regions. Based on preliminary longitudinal data, I will collect additional data to test the hypothesis that subtypes of apathy will worsen over time, and that decline will be restricted to the subtype of initial impairment. I will also extend my research program to investigate how biologic and environmental factors contribute to worsening in apathy. I have had more than 8 years of experience as an Advanced Practice Nurse and Research Nurse Coordinator in a nationally ranked laboratory that studies the neural basis for behavior in ND. I have direct access to research participants as well as a large database of genetic and neuroimaging data that I will incorporate into my analyses. My research training will be guided by two internationally known scientists, Dr. Ann Kolanowski, a well-known gerontological nurse expert and leader, and Dr. Murray Grossman, a highly acclaimed cognitive neurologist and experienced scientist. The interdisciplinary training during the performance of this work will serve as an essential component in attaining my career goal to become an independent neuroscience nurse researcher. The long-term impact of this research training will be profound for patients with ND, their caregivers, and families. Knowledge of the natural history of apathy is essential in the development of interventions for subtypes of apathy. Consistent with the National Institute of Nursing Research (NINR) priorities, this innovative work will lead to a better understanding of these brain behavior relationships and with this new knowledge, tailored interventions will be designed to reduce apathy and the poor health outcomes that are associated with this devastating condition.

Several, but not all, enzymic reactions that involve carbanionic intermediates have associated oxygen-consuming side reactions that do not appear to require oxygen activation. With the exception of the oxygenase reaction of ribulosebisphosphate carboxylase, these reactions have only recently been discovered and a rigorous determination of the products and mechanisms of these reactions has not been completed. Expression of an oxygenase side reaction clearly involves more than accessibility of a carbanionic reaction intermediate to molecular oxygen. A determination of all products of various oxygenase reactions, evaluation of the mechanism of these reactions, and the variability in levels of activity for the same enzyme from various sources will contribute to an understanding of these reactions. Oxygen toxicity is generally not thought to be associated with triplet oxygen per se, but requires conversion of O2 to its singlet state, superoxide, hydrogen peroxide, hydroxyl radical, or the formation of other radicals. The oxygenase side reactions of acetolactate synthase (Salmonella typhimurium) and glutamate decarboxylase (Escherichia coli) give peracetate and hydrogen peroxide as products, respectively, while that of fructosebisphosphate aldolase (Staphylococcus aureus) gives a product capable of reacting with proteins, hydroxypyruvaldehyde phosphate. The relevance of these reactions to the acute or chronic toxicity (aging- related loss of function) of oxygen in mammals is unclear, especially since the potential oxygenase activities of relevant enzymes have not been examined. To this end the mammalian glutamate decarbexylase will be examined for an oxygenase reaction similar to that recently discovered for the enzyme from the bacterium Escherichia coli. A combination of enzymic and chemical methods will be used to determine hydrogen peroxide and superoxide production by these enzymes. An infrared- sensitive chemiluminometer will be used to detect singlet oxygen. Organic and inorganic products of the oxygenase reactions; oxidative products of the enzymes and cofactors will be assessed by mass spectroscopy. Losses of enzymic activity by these enzymes will be assessed in the presence and absence of oxygen and substrate; in the presence and absence of protectants. Acute oxygen toxicity associated with exposure to hyperbaric oxygen is thought to be a consequence of inactivation of glutamate decarboxylase in the brain. A knowledge of the molecular mechanism by which oxygen inactivates and/or inhibits this enzyme could lead to the development of therapeutic agents to prevent or to treat acute oxygen toxicity. These results could also be relevant to chronic effects of oxygen on neural function (e.g. aging). Similarly, the oxygenase activities of other enzymes may contribute to aging in nonneural mammalian tissues.

Early initiation of intensive intervention may lead to improved outcomes for children with autism spectrum disorders (ASD);yet there are currently no reliable techniques for diagnosing ASD before the age of two years. This research is designed to identify variations in frequencies of occurrence, delayed onset, and/or atypicalities in vocal, motor, and communicative skills that may index later ASD diagnosis. The approach taken involves describing the development of these skills in infant siblings of older children who have been diagnosed with Autism. Infant Siblings are known to be at elevated risk for ASD and language delay. The goal will be to distinguish prospectively between those Infant Siblings eventually diagnosed with ASD, those eventually diagnosed with Language Delay but not ASD, and those with no apparent atypicalities. One hundred fifty Infant Siblings will be observed longitudinally from ages 5 to 14 months, with follow-up at 18, 24, and 36 months. Audio- and videotaped observations designed to sample behavior in contexts that are consistent across participants, standardized instruments, and parental reports will be employed to study vocalizations, rhythmic limb movements, vocal-motor coordinations, postural and locomotor abilities, gross, fine, and oral-motor skills, words, and gestures. Diagnostic outcomes will be assessed at 36 months with the Autism Diagnostic Observation Schedule, Autism Diagnostic Inventory-Revised, and clinical judgment using DSM-IV criteria. Data from all three subgroups of Infant Siblings will also be compared to parallel data from infants with no known risk for ASD currently being followed as part of separate NAAR and NIH-funded studies. Analyses will focus on identifying behavioral markers in infancy that predict a later diagnosis of ASD, assessing their positive predictive value, sensitivity, and specificity, and on evaluating a set of hypotheses concerning variation in patterns of production and developmental trajectories of infant vocal, motor, and communicative behaviors that may differentiate groups and predict an eventual ASD or Language Delay diagnosis. We currently lack reliable methods for diagnosing autism spectrum disorders (ASD) in children younger than 2 years of age. To identify possible predictors of later ASD diagnosis, this study will focus on the development of vocal, motor, and communicative skills in infants at particular risk for ASD and relate atypicalities in infant development to diagnostic status at 3 years of age.

The Clinical Research Unit is a facility where patients may be admitted for special study. The studies range from the evaluation of new surgical techniques to studies of endocrinologic mechanisms, evaluating new medical procedures, drug evaluations and the study of disease mechanisms. All of the studies are aimed at evaluating or evolving new treatment methods or gaining better insights into metabolic processes and disease mechanisms so that better therapeutic approaches can be devised. The facility provides a meeting place for investigators whose different ideas and approaches can be joined on specific problems. The studies involve investigators from all clinical departments and most basic science departments. The Unit is housed in an area specifically designed for its use. It is hoped that such an area designed for clinical research will foster higher quality clinical research and research not previously feasible on the usual service unit.

Cerebrospinal fluid concentrations of homovanillic acid, 5-hydroxyindoleacetic acid, norepinephrine and 3-methoxy 4-hydroxyphenylethylene glycol did not differ significantly between patients with Alzheimer's disease and age matched controls, and were not correlated with age in healthy men. Spinal fluid concentrations of choline increased with age in healthy men, and were higher in young adults with Down syndrome than in age matched controls. CSF biopterin, a coenzyme for tyrosine and tryptophan hydroxylase, was lower in patients with Alzheimer's disease that in controls, and was correlated with concentrations of 5-hydroxy- indoleacetic acid and homovanillic acid. Corticotropin releasing factor, a neuropeptide, was significantly reduced in the cerebrospinal fluid of patients with Alzheimer's disease as compared to controls, as was peptidyl-alpha=amidation activity, suggesting a loss of neurons which produce amidated neuropeptides. Rations of albumin and immunoglobulin between cerebrospinal fluid and plasma were normal in Alzheimer's patients, suggesting that the blood brain barrier is intact.

Isolate by ultracentrifugation and characterize by electrophoresis the aqueous humor lipoproteins. Lipids will be characterized by chromatographic and chemical methods. Human aqueous humor cholesterol levels will be assayed by gas-liquid chromatography. Utilization of aqueous humor lipoproteins by rabbit and bovine lens as an exogenous source of lipid for fiber cell membrane synthesis will be investigated using in vitro incubation and labeled lipoprotein components. The balance between exogenous and de novo synthesis of both protein and lipid components of cell membranes will be investigated using appropriate radioactive precursors. The intraocular site of aqueous humor lipoprotein synthesis will be investigated by incubating selected, isolated intraocular tissues with radioactive precursors and isolating lipoprotein from the incubation medium. Lens membrane protein isolation and synthesis will be investigated by SDS-gel chromatography and radioactive precursors. Finally, the mechanism of triparanol cataractogenesis will be investigated with desmosterol-lipoprotein or triparanol in vitro incubation. The effect of desmosterol on de novo cholesterol synthesis will be investigated. In addition, the amount of desmosterol in the aqueous humor of triparanol-fed rats will be assayed.

Alcohol abuse represents a significant problem in our society. Chronic intermittent ethanol (CIE) treatment of rats (60 doses of intermittent intoxication and withdrawal), encompasses all of the major characteristics of human alcoholism, including anxiety, lowered seizure thresholds, and enhanced alcohol preference after withdrawal. At least some of these symptoms may be explained by the measured reduction in the function of the 3-aminobutyric acid (A) receptor (GABAAR) and altered sensitivity to its allosteric modulators. GABAARs mediating synaptic (phasic) and extrasynaptic (tonic) inhibition appear to be altered differently. Thus, tolerance develops to acute ethanol (EtOH) potentiation of hippocampal extrasynaptic GABAARs, while synaptic GABAARs develop high sensitivity to EtOH. Such paradoxical changes in EtOH sensitivity are proposed to underlie both the development and persistence of alcoholism. Preliminary studies suggest that altered subunit composition and localization of GABAARs may account for the observed alterations in GABAAR function within the hippocampus. However, it is unknown whether other key brain areas implicated in symptoms of alcohol withdrawal and dependence experience similar neuroadaptations. It is also unknown whether EtOH-induced neurodegeneration may account for these neuroadaptations. Underscoring the persistence of CIE-induced changes is a new observation of fundamental importance: a single intoxicating dose of EtOH results in GABAAR changes similar to those seen after CIE treatment, but recovery is seen by 1-2 weeks after this single dose. The specific aims of this proposal were designed to address key hypotheses regarding GABAAR involvement in mechanisms of alcohol withdrawal and dependence by: 1) determining the dose-, duration-, and frequency-dependence of CIE treatment to produce long-lasting symptoms of EtOH dependence;2) studying changes in GABAAR subunit composition and function within nucleus accumbens and basolateral nucleus of the amygdala (brain areas known to be of major importance for the mechanisms of reward and dependence) and relating them to the behavioral measures of withdrawal from single or multiple EtOH treatments;and 3) determining whether neurodegeneration plays a role in altered GABAergic inhibition after EtOH intoxication through the use of histochemical and stereological techniques. The knowledge acquired from the proposed experiments will increase our understanding of the alcohol-induced alterations in GABAAR function, which has profound effects on various emotional and intellectual aspects of brain activity. This knowledge will also be useful to the development of therapeutics targeting the GABAergic system for the treatment of alcoholism.