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DESCRIPTION: The goal of this research is to gain an integrated view of the mechanisms that underlie development an, maturation of the sensory endorgans of the inner ear. Experiments will focus on the auditory and vestibular hair cells of the amphibian, Xenopus laevis. The proposed research will exploit the advantages that Xenopus offers as a classical system for developmental studies. The potassium channels of the inner ear will be a central subject of these investigations, and will be used to test hypotheses about hair cell development and differentiation. A major objective of this research is to establish the pattern of potassium channel gene expression in the developing inner ear. Electrophysiological methods (patch clamp) will be used to study ion channel function in dissociated hair cells. Molecular techniques will be used to clone ear potassium channels (RT-PCR, RACE, recombinant DNA) and will be combined with anatomical methods (in situ hybridization) to examine potassium channel mRNA expression in the ear. The specific aims of this research are to test the following hypotheses: (1) An outward potassium channel homologous to drkl is expressed in the Xenopus inner ear, (2) Xenopus saccular hair cell bundle morphology can be correlated with saccular hair cell electrical membrane properties, (3) The types of ion channels expressed in Xenopus saccular hair cells change as hair cells develop, (4) The properties of ion charmers differ between hair cells of the Xenopus sacculus, amphibian papilla, and basilar papilla. Experimental results are expected to advance fundamental understanding about the genetic basis of hair cell diversity, and about the genetic control of ear development. The long-term goal of this research is to lay the foundation for future studies that will determine how genes expressed in the ear are regulated during development and regeneration. Results of these investigations will provide essential knowledge that can be used to develop effective treatments for hearing loss such as that caused by trauma or genetic disorders.

The objectives of this Project are two-fold. First, in parallel with Project by Griffin, we will continue to study the effect of survival time and of gene polymorphisms on the manifestation of specific pathological features in traumatic brain injury which mimic the pathology of Alzheimer's disease (AD) in the belief that this will offer valuable insights into the early molecular mechanisms underlying the development of Alzheimer-type pathology. We hypothesis that the genotype of an individual modulates the extent of Abeta deposition and expression of glia-derived cytokines. Seen in the brain post-injury. Having already established that the apolipoprotein E (ApoE) genotype is important in modulating post-traumatic pathology, we will study the molecular basis of this effect in a novel in vivo paradigm of Abeta-induced neurotoxicity. Tissue from a large cohort of head-injury patients with different post-injury survival times and from non-injured control patients will be genotyped with respect to the polymorphisms in genes in the interleukin-1 gene cluster on chromosome 2. Aim 1 will determine quantitatively the changes in expression of the glia-derived inflammatory cytokines IL-1, IL-6, and S100beta throughout the brain after fatal head injury. Aim 2 will assess whether alterations in cytokine immunoreactivity following head injury are spatially and temporally correlated with changes in proteins that are important in the pathogenesis of AD. Aim 3 will determine whether inter-individual variation in the extent of the inflammatory reaction to head injury is associated withy specific polymorphisms in the genes encoding ApoE and IL-1. Aim 4 will investigate the mechanisms by which ApoE4 increases susceptibility to AD using an in vivo model of Abeta-induced neurotoxicity. Results should allow us to determine if genotype affects post-trauma pathology and will provide information on how ApoE4 produces its effects at the molecular level. The effect of genetic makeup on the extent of the inflammatory activity in the brain will be an important factor in predicting long-term sequelae of head injury (i.e., AD) and in targeting any potential therapies for the disease.

Fibroblasts are a major cellular component of the tumor microenvironment and influence cancer cell behavior directly and indirectly through secretion of soluble factors, including growth regulators, angiogenic factors and chemokines. While genetic alterations in breast fibroblasts may exert pro-tumorigenic effects, little is known of the cellular and molecular signals that regulate fibroblast functions in the breast cancer microenvironment. De-regulation of TGF-B signaling significantly contributes to metastatic breast cancer, and may also enhance expression of chemokines in breast fibroblasts, to promote cancer progression. Based on previous studies and preliminary studies, we hypothesize that inflammatory chemokine expression in fibroblasts including CCL2 and CXCL1 mediate epithelial cell and immune cell motility and invasiveness to promote breast cancer progression. The objective of this proposal is determine the functions of chemokines, which are normally suppressed by TGF-B signaling, in fibroblast interactions with tumor cells during breast cancer progression. [unreadable] [unreadable] The following Specific Aims are proposed to address this hypothesis: Specific Aim 1: To determine the mechanisms of CCL2 and CXCL1 inflammatory chemokine expression in mammary fibroblasts as it relates to TGF-B signaling using cell biology, molecular and biochemical approaches. Specific Aim 2: To determine the functional contribution of CCL2 and CXCL1 chemokines expressed by mammary fibroblasts in breast cancer growth, invasion and metastasis, using transplantable and transgenic mouse models of breast cancer, cell biological and biochemical approaches. Specific Aim 3 : To determine the contribution of CCL2 and CXCL1 chemokines expressed by mammary fibroblasts in immune cell trafficking, homing and recruitment in breast cancer progression using transplantable mouse models of cancer, cell biological and biochemical approaches. [unreadable] [unreadable] Specific Aim 1 would be addressed during the mentored phase, while Aims 2 and 3 would be addressed during the mentored and independence phases. This research will address the functions and mechanisms through which inflammatory chemokine expression in fibroblasts contributes to breast cancer. Through these studies, we seek to further understand the functions of stromal cells in the host microenvironment, and how the host microenvironment contributes to tumor progression at the molecular, cellular and in vivo levels. By further understanding the role of the tumor microenvironment in cancer progression, it will be possible to identify new molecular targets for therapy and to develop improved methods for diagnosing and treating metastatic breast cancer. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by investigator): Invasive brain intervention often results in complications and long recovery times. In addition, the delivery of therapeutic agents via the blood supply is often impossible because the Blood-Brain barrier (BBB) protects the brain tissue from foreign molecules. Laboratory experiments have shown that focused ultrasound (FUS) beams can be used for noninvasive interventions. However, the utilization of FUS in the brain has been seriously limited by the difficulty of delivering FUS through the skull bone. The hypothesis of this grant has been that transcranial therapeutic ultrasound exposures can be delivered noninvasively through an intact skull. During our current grant we continued our initial research and developed methods to allow FUS propagation through the skull at large entrance angles thus making whole brain sonications feasible. We have collaborated with industry to develop a prototype device currently in clinical testing. We have further studied the impact of ultrasound exposures on brain tissue and demonstrated chemotherapy delivery across the BBB and an order of magnitude reduction in the required acoustic power for focal tissue destruction when intravascular microbubbles are used. We have also developed computer simulation programs and new array technology and treatment methods that will improve the delivery of FUS energy. Our study plan is to extend our current research and further explore the feasibility of using intravascular microbubbles to enhance the trans-skull sonications. Successful utilization of the bubbles will eliminate skull heating problems and will enable new therapies. Our goals are: First, to modify our prototype phased array ultrasound system to allow acoustic signal feedback, new treatment planning, and advanced sonication methods to be tested for the localization and control of bubble-enhanced treatments. Second, to perform in vivo dual photon microscopy to increase our understanding of ultrasound induced BBB disruption and tissue destruction at the microscopic resolution. Third, to explore sonication methods for localization of bubble-enhanced therapeutic effects. Fourth, to develop and test treatment planning methods for bubble-enhanced therapy that incorporate image-derived information of the blood vessels networks. Finally, to further test the effectiveness of the ultrasound-induced disruption of the BBB for the delivery of chemotherapeutic agents for the treatment of malignant brain tumors. Our vision is that successful trans-skull delivery of FUS could have a major impact on the treatment of many brain disorders. Therefore this research may have a major impact on patient care. PUBLIC HEALTH RELEVANCE: Invasive brain intervention often result in complications and long recovery times. In addition, the delivery of therapeutic agents via the blood supply is often impossible because the Blood-Brain barrier (BBB) protects the brain tissue from foreign molecules. During our current grant we continued our initial research and developed methods to allow focused ultrasound propagation through the skull such that brain sonications are feasible. We have collaborated with industry to develop a prototype device currently in clinical testing. We plan to further develop the ultrasound methods for brain surgery and targeted chemotherapy delivery. Our vision is that successful trans- skull sonications could have a major impact on the treatment of many brain disorders. Therefore this research may have a major impact on patient care. [unreadable] [unreadable]

Research is to be conducted on the visual mechanisms by which the human observer resolves fine details and accurately localizes objects in two and three dimensions. The retinal components of these abilities will be analyzed, using optical and psychophysical techniques, by distinguishing between the limits set by the discrete nature of the mosaic of retinal receptors, by the sensitivity to intensity differences that can be detected in small adjacent retinal locations, and by the interaction between retinal neural entities. Identification of Snellen visual acuity letters and stereoscopic vision require, in addition, the elaboration and relative localization of individual features, and these are functions of the cortex. The interest is in the interaction of separate features and the development of the visual attributes of two-dimensional position and three-dimensional depth. The long-term goal is twofold: (1) to specify rigorously the ultimate performance characteristics of the human visual system and to delimit the range of possible neural circuits that may serve as their substrate, and (2) to gain insight into the optical and neural processes subserving visual acuity, hyperacuity and stereoscopic localization that will aid in the differential diagnosis of patients with reduced acuity and the development of tests of etiological and prognostic value.

PROJECT SUMMARY/ABSTRACT Juvenile myelomonocytic leukemia (JMML) is the most common myeloproliferative neoplasm (MPN) in childhood, and tends to occur in very young children less than 4 years of age. JMML is traditionally characterized as being Ras-driven due to mutations in NF1, CBL, KRAS, NRAS, or PTPN11. Traditional cytotoxic chemotherapeutic agents are ineffective in JMML, and the only curative modality is allogeneic hematopoietic stem cell transplantation. Unlike other MPNs, JMML rarely progresses to blast crisis; rather, mortality is due to extramedullary tumor cell expansion leading to organ failure, respiratory failure, bleeding, or infection. Notably, following allogeneic stem cell transplant, 50% of children succumb to leukemia relapse. This relapse rate in JMML is substantially higher than that of individuals who receive allogeneic stem cell transplant for chronic myelogenous leukemia (CML) in chronic phase (approximately 7% leukemia relapse), implicating a strong hematopoietic stem cell (HSC)-independent component of JMML development and progression. We envision two distinct mechanisms that potentially account for a HSC-independent means of JMML relapse after allogeneic HSC transplant. First, the JMML-initiating malignant cells may emerge during embryonic development prior to and independently from HSCs, and persist postnatally as self-replenishing malignant tissue macrophages. Alternatively, regardless of the origin of the JMML cells, the hyperinflammatory nature of JMML may damage the bone marrow microenvironment, prohibiting the expansion of normal donor cells following transplant, permitting residual leukemia cells to outcompete the normal graft, and leading to leukemia relapse. To address these possibilities, we will use the tamoxifen-inducible Cre recombinase system, which will permit yolk sac-restricted expression of the common JMML mutation, Shp2D61Y, to determine if yolk sac-restricted oncogene expression is sufficient for the post-natal development of MPN. Further, we will examine if inhibition of the pro-inflammatory protein, PI3K p110?, improves homing, engraftment, expansion, and myeloid differentiation of WT donor cells into diseased, Shp2D61Y-expressing recipients.

A novel anti-HIV activity of CCR6 via APOBEC3G: relevance to CNS infection Abstract We have shown that when defensin hBD2 and chemokine MIP-31 (CCL20) bind to their cellular receptor, CCR6, they induce an intracellular antiviral activity against HIV that is absent in cells that do not express CCR6. While direct inactivation of enveloped viruses including HIV has been implicated as one mechanism of defensins, to our knowledge, HIV inhibition mediated through CCR6 is novel. The mechanism occurs post entry but at or before reverse transcription, and appears to be due to increased expression of the intracellular antiviral protein APOBEC3G. Inhibition is abrogated by pertussis toxin, suggesting the involvement of G1i-dependent signal transduction. CCR6 is expressed on cells that are highly relevant to HIV infection; they include memory T cells, dendritic cells, activated macrophages, microglia and Th17 cells. Defensins that bind to CCR6 are primarily expressed by epithelial cells and astrocytes, while MIP-31 is produced by mucosal epithelial cells, activated peripheral blood mononuclear cells and astrocytes. Since both CCR6 and its ligands are expressed in the central nervous system in cells highly relevant to HIV infection, an HIV suppressive mechanism that targets CCR6 may play a critical role in controlling HIV infection and CNS symptoms. Further, elucidation of the intracellular pathways and the step(s) in the HIV life-cycle limited by CCR6 activation could provide insights into virus-host interactions early in infection and identify new targets for antiviral therapy. We propose to identify APOBEC3G as the effector molecule of the antiviral activity mediated by CCR6 in primary cells; to demonstrate that CCR6- induced signal transduction increases APOBEC3G expression and identify what is the critical specific pathway; to demonstrate the efficacy of CCR6-mediated HIV inhibition on a broad spectrum of primary and non-B viral isolates; and to measure the expression of CCR6, CCR6 ligands, and APOBEC3G in clinical specimens. PUBLIC HEALTH RELEVANCE: The studies proposed here will investigate the mechanism of inhibition of HIV by a cellular receptor called CCR6. These studies are highly relevant to prevention and treatment of HIV infection because they will contribute knowledge that can be used to develop novel anti-HIV drugs that will target CCR6.

The promise of cancer immunotherapy is based upon the exquisite specificity of the immune system, through which a potent machinery can eliminate targeted cells. However, despite some notable examples of success, progress in developing this form of cancer therapy has fallen short of expectations. Major insights explaining the limitations of T cell-based cancer immunotherapies have come from the discovery of inhibitory co-receptors or pathways termed immune checkpoints, which restrain T cell functions in normal physiologic settings as well as in the context of neoplastic disease. Recent evidence suggests that tumors may usurp immunological checkpoint mechanisms to create a barrier against antitumor immune responses - including endogenous responses and those induced by immunotherapies such as cancer vaccines. Animal cancer models demonstrate that blocking the interaction of inhibitory molecules on tumor cells with their co-receptors on tumor-specific T cells can release the brakes on antitumor immunity and cause tumor regression. Thus, checkpoint inhibition, applied alone or in combination with vaccines, represents an important new therapeutic approach for enhancing antitumor immunity. One of the most interesting inhibitory co-receptors is PD-1, that is induced on activated T cells and down-modulates critical functions in both CD4+ (helper) and CD8+ (killer) subsets. The major ligand for PD-1 is B7-H1 (PDL1), a B7 family member normally expressed by several leukocyte subsets upon activation, and aberrantly expressed in many human cancers. These findings, highlighting multiple mechanisms by which PD-1/B7-H1 interactions may inhibit antitumor immunity, have provided a rationale for clinical trials in cancer patients using fully human antibodies blocking PD-1 or B7-H1. Notably, objective tumor regressions were observed in the first phase I trial of PD-1 blockade in patients with advanced treatment-refractory metastatic solid tumors. It is now critically important to better understand the regulation and function of PD-1 and B7-H1, and to discern the effects of PD-1/B7-H1 blockade on antitumor immunity. In current proposal, we will test hypothesis that modulation of B7-H1/PD-1 inhibitory pathway could vastly enhance efficacy of cancer immunotherapy by improving tumor microenvironment and protecting ongoing T cell activity. The current proposal integrates basic and clinical science, and will use animal models and human in vitro systems to achieve the following aims: 1) To define mechanisms regulating B7-H1 expression by tumor cells and other cell types in the tumor microenvironment; 2) To characterize factors influencing PD-1 expression by T cells, particularly in the context of vaccine-induced stimulation; and 3) To characterize immunological mechanisms underlying the clinical effects of B7-H1/PD-1 blockade in cancer therapy. Taken together, results from these studies will enable the rational clinical development of PD-1/B7-H1 blockade, alone or in combinatorial regimens, in cancer therapy.

The proposed research outlines a proprietary, potentially patentable fee- for-service method to predict viral leakage, such as HIV, through condoms - - in manufacturers' production lots during or immediately post production. The study would statistically expand results indicating that among 31 brands, some condoms from brands with lowest ASTM/ISO scores leaded HIV; high scorers did not. 100 condoms from each of four lowest scorers will be challenged with high titre bacteriophage phiX-174, (diam.<HCV/HBV). Leakage will be quantitatively assayed with a detection limit of a single phiX-174 particle leaked through the condom. Test condoms will be pre-stressed on a Coital Simulation Instrument to subject them to reproducible conditions akin to those during sexual intercourse. If leakage is adequately confirmed, future work will similarly more fully assess leakage in high and intermediate scorers, correlating all results with individual ASTM/ISO tests values. Goal: a rapid method predicting production lots' leakage potential, through an ASTM/ISO Index of critical score values. The method has potential as a commercially useful step toward lessening the number virally leaky condoms currently on the market in the AIDS/herpes/chlamydia epidemics. Like FDA water leak monitoring, the method would test production lots, not individual condoms.

Critical to cryopreservation is that lethal intracellular freezing (IIF) not occur. Its occurrence depends on two major factors. One is the cooling rate. It has to be low enough so that the cells lose nearly all their water osmotically before cooling to the temperature at which IIF becomes possible. Second is the temperature at which IIF occurs. The higher that temperature, the greater the difficulty in avoiding IIF by slow cooling. Our current grant and this renewal proposal deal primarily with the latter. IIF in mouse and Xenopus oocytes and in Arabidopsis protoplasts was found to require the presence of extracellular ice in close contact with the cell membrane. One strong piece of evidence in the mouse oocyte is that IIF occurs at temperatures where about 95 percent of the external medium has frozen. That temperature varies from -14[unreadable]C to -40[unreadable]C depending on the concentration of cryoprotective compounds in the medium. Another factor affecting the IIF temperature may be cell size, for IIF occurs at much higher temperatures in 1 mm Xenopus eggs than it does in <0.1 mm mouse oocytes. The two tools used above were a cryostage that permits us to observe cells during cooling and warming and manifests IIF as "flashing", and a differential scanning calorimeter (DSC) that detects IIF as an outburst of heat. In this renewal application we propose to introduce two new instruments. One, in collaboration with the Oak Ridge National Laboratory, is a Scanning and Transmission Electron Microscope (STEM) equipped with a newly designed sample chamber that will permit high resolution images of hydrated cells in aqueous solutions. The other instrument is a directional freezing stage. It permits a cooling rate to be resolved into the thermal gradient (G) and the crystal growth velocity (V). Differences in G and V affect the ice crystal morphology which in turn may affect the temperature of IIF. We will add three new cell types to the study: Yeast and two types of hamster tissue culture cells. These cells are 10-times smaller than mouse oocytes thus permitting us to further explore the role of cell size. Second, cells have to be that small to fit in the STEM liquid sample chamber. Third, IIF has never been observed directly in these cells and we intend to use DSC for this purpose. Other major aims in this renewal proposal are (1) to determine whether the high correlation between observed IIF temperature in mouse oocytes and the frozen fraction holds for other cell types. (2) To determine whether there is a relation between pores in the plasma membrane and the IIF temperature. For this, we will study the freezing of mouse morulae, the 8 to 12 cells of which possess gap junctions and aquaporin pores. Second, we will introduce pores in mouse oocytes and plant protoplasts by exposing them to a toxin from Staphylococcus bacteria. The final goal will be to use the mechanistic data on IIF to devise better methods of avoiding it and thus improve success in the cryopreservation of cells that currently can not be well cryopreserved.

The training and research proposed in this application will be sponsored by Dr. Adams and will take place in both the Arteriosclerosis Research Center and the Comparative Medicine Clinical Research Center. Dr. Wagner is seeking SERCA support to further her development as an independent investigator with special emphasis on cardiovascular health of aging females. The studies will utilize a well characterized primate model, the female cynomolgus monkey, which like women in most Western societies, develops less severe atherosclerotic coronary heart disease (CHD) than its male counterpart. Pathogenetic mechanisms of atherogenesis are difficult to study in women, since atherosclerosis develops slowly over a period of many years. Also, while CHD is the leading cause of morbidity and mortality in American women, clinical events occur relatively infrequently. For these reasons we propose to utilize a primate model to study the effect of sex hormone deficiency, sex hormone replacement and oral contraceptive treatment on the pathogenesis of atherosclerosis. Studies of both human and nonhuman primates have found that only a portion of the beneficial effects of estrogens can be attributed to changes in plasma lipoprotein risk variables. This suggests that estrogens may retard atherogenesis by interacting by either directly at the arterial wall or by modifying plasma components in addition to lipoproteins. The studies proposed will explore molecular and cellular mechanisms by which estrogens and progestins may be acting. The effects of sex steroids on the following parameters will be investigated: 1) functional parameters of early atherogenesis eg., endothelial dysfunction as determined by arterial LDL uptake and degradation, monocyte adhesion to the endothelium, and intimal cell proliferation, 2) whole body and hepatic LSL metabolism, 3) plasma HDL composition and its function in reverse cholesterol transport, 4) expression of cytokines and growth factors by arterial cells, and 5) atherosclerosis-related impairment of vascular responses. If, as suspected, beneficial effects are found, this would provide a rational basis for the prophylactic or therapeutic use of these steroids in CHD.

Our studies focused on the interrelationships of organelles (endoplasmic reticulum, Golgi apparatus, GERL, lysosomes, peroxisomes, microperoxisomes, mitochondria, cytosolic lipid, coated vesicles, plasma membrane, and microfilaments) to gain a better understanding of organelle functions in normal, pathologic, and malignant cells. To search for differences between normal and malignant cells we used microscopy (light and electron) in combination with enzyme cytochemistry, immunocytochemistry, tracers, freeze cleavage, biochemistry, and molecular biology (e.g., in situ rDNA hybridization). Our key accomplishments have included developing immunocytochemical procedures for localizing preneoplastic antigens, epoxide hydrase, and gamma-glutamyltransferase (gamma-GT) in 2-acetylaminofluorene (AAF)-induced hepatomas; describing organelle translocations in hepatocytes during regeneration; studying hormone effects of functioning islet cells on hepatic ultrastructure; studying the uptake and transport of asialoglycoproteins in hepatocytes; localizing apo-protein B and A-I using immunocytochemistry in rat hepatocytes and absorbtive cells of the intestine; isolating fractions enriched in the Golgi apparatus and GERL from rat liver homogenates and characterizing these fractions biochemically; performing an extensive biochemical, ultrastructural, and enzymatic study of 3T3-L1 cells, including the isolation and characterization of microperoxisomes; describing an extensive lysosomal compartment in Kupffer cells and hepatocytes; developing immunocytochemical procedures for localizing lysosomal and glycoprotein processing enzymes; and localizing antimetabolites in neurons. (E)

Mycoplasma pneumoniae is the leading cause of pneumonia in older children and young adults and accounts for 20% of all community-acquired pneumonia. This cell wall-less prokaryote moves by gliding motility, which we contend facilitates colonization of the conducting airways of the respiratory tract. Gliding motility is poorly understood, and no homologs of known motility genes, gliding or otherwise, are found in M. pneumoniae. In the current project period we identified a diverse set of gliding- associated genes, demonstrated conclusively that the terminal organelle alone is the gliding motor, defined the requirement and function of several terminal organelle components in gliding, and generated direct evidence that gliding is required for colonization of mucosal epithelium. The studies proposed here build upon that foundation to define further the role of gliding in pathogenesis and explore the mechanical basis for gliding, encompassing three specific aims. Aim 1 addresses the gliding mechanism, focusing on gliding-associated proteins P65, P41 and P24. The terminal organelle detaches from the cell body with the loss of P41 but retains gliding function. Insertions in the P65 gene result in the dragging of surface adhesin P30 from the terminal organelle to the trailing end, where it detaches to leave a trail behind the gliding cell. In the absence of P41, P24 foci appear to move along the long axis of the cell. In the next project period we will apply biochemical and cell imaging approaches including electron cryotomography to explore further the roles of P24, P41, P65, and other selected proteins in gliding. In Aim 2 we will analyze in detail certain gliding mutants exhibiting a distinctive lawn-like growth as a result of disruption of the genes for the only annotated M. pneumoniae protein phosphatase and cognate ser/thr protein kinase. We will confirm cause and effect for these mutants and explore the impact of loss of protine kinase or phosphatase function on the phosphorylation of terminal organelle proteins HMW1 and HMW2. We will also examine cell behavior of these mutants in detail by microcinematography to establish how lawn-like growth is achieved. In Aim 3 we will use a differentiated normal human bronchial epithelium model and wild-type and gliding-defective mycoplasmas to explore how gliding motility specifically contributes to resistance of mucociliary defenses in the colonization of conducting airways. Mycoplasma pneumoniae is the leading cause of pneumonia in older children and young adults and accounts for 20% of all community-acquired pneumonia. Most infections result in respiratory disease that is chronic and protracted, impacting attendance at school and productivity in the workplace, and permanent lung damage can result. In addition, a growing body of evidence supports a significant, contributing role for M. pneumoniae in onset and recurrence of asthma. The studies proposed here will elucidate the mechanism and role of gliding motility in the colonization of the conducting airways.

PROJECT SUMMARY ABSTRACT Focal Adhesion Kinase (FAK) is a major cancer drug target that is overexpressed in multiple tumor types. FAK is a critical regulator of tumor survival, invasion, proliferation, metastasis, and immune evasion. Current FAK inhibitors that target the ATP-binding pocket of the kinase domain do no effectively inhibit FAK in cancer because FAK also functions as a scaffolding protein. The Focal Adhesion Targeting (FAT) domain of FAK is an interesting alternative drug target due to its requirement for FAK localization, activity, and downstream effects. Disruption and mutation of the FAT domain causes significant effects on tumor cell apoptosis, proliferation, invasion, and metastasis. Specifically, the FAT domain interacts with the alpha helical LD2 and LD4 motifs of Paxillin to promote its biological effects. The structure of the FAT-Paxillin complex has been solved by x-ray crystallography however has been challenging to target with small molecules. In this project, we will use hydrocarbon stapled alpha helical peptides that have the advantage of enhanced proteolytic stability, cell permeability, and potent inhibition of the entire protein interaction interface. We have preliminary data of Stapled Peptide 3 showing low micromolar inhibition of FAK-Paxillin binding and NMR/SPR data validating the binding site of the peptide. In the first specific aim, we will perform structure-activity relationships (SAR) on stapled alpha helical peptides for enhanced binding and competitive inhibition. We will perform SAR on stapled peptides by changing hydrocarbon stapling strategy, modifying N- and C-terminal amino acids, and adding alternative amino acids. In addition, we will utilize molecular modeling to optimize peptide-protein contacts, synthesize stapled peptides of homologous peptide sequences, and characterize biophysical/biochemical properties of stapled peptides. In the second specific aim, we will characterize and optimize lead peptides for cellular effects. We will perform robust assays to measure cell permeability of stapled peptides, characterize peptides for protease resistance and effects on membrane lysis, and test peptides in cellular efficacy assays to assess the effects of stapled peptides on cancer cells. In the third specific aim, we will test lead peptides with in vitro DMPK assays and preliminary in vivo efficacy models. We will characterize peptides using plasma binding, metabolic stability, and CYP inhibition/assays. We will also test lead peptides in mouse xenograft models alone and in combination with chemotherapy. In all, these specific aims will be used to discover peptide inhibitors of FAK non-catalytic function that can be the basis for future clinical development.

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. Peptidoglycan recognition proteins (PGRPs) are pattern recognition molecules of the innate immune system that bind and, in certain cases, hydrolyze peptidoglycans (PGNs) of bacterial cell walls (1). They are highly conserved from insects to mammals. PGRPs bind PGNs with high affinity and are important contributors to host defense against bacterial infections. Insects PGRPs, of which there are at least 19, are involved in activating signaling pathways that induce expression of anti-microbial peptides. By contrast, mammalian PGRPs, of which there are four, do not act through host signaling pathways, but are directly bactericidal against both Gram-positive and -negative bacteria (1). Indeed, these PGRPs are a new class of bactericidal proteins that have a different structure and mechanism of action than currently known mammalian antimicrobial peptides. Mammalian PGRP-L is an N-acetylmuramoyl-L-alanine amidase that hydrolyzes PGN. Very recently, it was found that PGRP-L also has a proinflammatory function, unrelated to its amidase activity (2). We previously published structures of human PGRP-Ialpha and -Ibeta in complex with PGN fragments, and have proposed a mechanism for their bactericidal activity (3,4). We now plan to extend our work to PGRP-L, which contains a large N-terminal domain with no sequence homology to other known proteins, and whose structure remains to be established. The PGRP-L structure will provide insights into how three different types of pattern recognition molecules (PGRPs, Nod2 and TLR4) play interdependent roles in inflammatory responses to bacterial pathogens. Recently, We obtained a few small crystals which are too small (~50um) for data collection with our in-house detector. They diffract to a maximum resolution of 7 [unreadable] with unit cell dimensions of

Multicenter collaboration to reduce the risks inherent in allogeneic hematopoietic cell transplantation (HCT) is essential to dampen the dangers of regimen toxicity, infection, transfusion dependence, graft vs. host disease (GVHD) and malignant relapse. Ongoing efforts to address all these risks have been a focus of Network participation for investigators at the University of Minnesota. Our commitment to the Network has included participation as Steering Committee chair, national PI of four protocols, leading the first four Network publications and committing institutional resources to develop and successfully execute Network trials. Because umbilical cord blood (UCB) transplantation is still limited by delayed or failed engraftment, we initiated studies to augment the homing and hematopoietic recovery of UCB grafts based on preclinical data suggesting that complement fragment C3a can accomplish these goals. An ongoing University of Minnesota phase 1 trial testing C3a priming of one of a pair of UCB units to augment engraftment and enhance predominance of the primed unit provides the clinical background for a proposed multicenter phase 11. We propose a protocol testing whether C3a priming of one UCB unit can increase the likelihood of that unit's predominance as the engrafting hematopoietic source;can augment and accelerate multilineage hematopoietic recovery;and can enhance immune reconstitution thereby lessening risks of infection without excess graft vs. host disease. We offer our continued commitment to the Network's success in developing new protocols, identifying patients suitable for Network trial enrollment and performing protocol specific procedures. We will try to exceed the Network's standards in our commitment to enhance multicenter trial success and advance scientific progress within the Network. Multicenter collaborative trials are essential to formally test new ways to enhance the safety of allotransplantation and improve outcomes for our patients. PUBLIC RELEVANCE: Improvement in the outcomes of allergenic transplantation requires control of early toxicity, risks of infection, transfusion dependence, graft vs. host disease and relapse. Multicenter trials within the BMT CTN have tackled all these areas and at the University of Minnesota we are committed to advancing the development and execution of high-quality Network trials. We propose augmenting the success of umbilical cord blood engraftment by priming one of a pair of UCB units with complement fragment C3a, to extend preliminary University of Minnesota data to a multicenter phase II trial. Improving engraftment and post transplant immune recovery may limit these hazards and augment the success of all transplantation.

Many evidence-based treatments (EBTs) for common children's mental health disorders have been developed, but they are rarely delivered in real world care. EBTs must be complemented by evidence-based implementation strategies if they are to achieve their promise. Accordingly, the NIH has prioritized efforts to identify, develop, refine, and test implementation strategies (PAR-10-038). Yet, this research is not likely to be fruitful if it is conducted without a thorough understanding of the service systems in which these strategies and EBTs will (hopefully) be deployed. This mixed methods multiple case study will involve seven organizations that are participating in the control arm of an NIMH funded RCT, Testing an Organizational Implementation Strategy in Children's Mental Health (Glisson, PI). This affords the opportunity to examine implementation as usual (i.e., implementation processes that occur in usual care settings) in order to ensure that newly developed implementation strategies will be acceptable, feasible, sustainable, and scalable in the real world. Semi-structured interviews (with agency and clinical directors) and document review will be used to identify and characterize the implementation strategies used in routine children's mental health care (Aim 1), and to explore how organizational leaders make decisions about which EBTs to implement and how to implement them (Aim 2). Focus groups and a project specific self-administered online survey will be used to determine organizational leaders' and clinicians' perceptions regarding the relative importance, acceptability, feasibility, and appropriateness of implementation strategies (Aim 3). Finally, data from the first three aims will be linked to their corresponding organization's scores on a standardized measure of organizational social context (culture and climate) in order to examine how context influences implementation processes and perceptions about specific implementation strategies (Aim 4). By shedding light on implementation as usual, this study will inform efforts to develop and tailor strategies, propelling the field toward the ideal of evidence-based implementation. It will also benefit the RCT by illuminating strategy patterns in the control group, which may aid in the interpretation of results. The proposed research and training plan will allow the fellowship applicant to receive training in: 1) implementation and mental health services research, 2) organizational behavior and change processes, 3) mixed methods research, and 4) scholarly writing and research dissemination. This training will facilitate the successful completion of the proposed project, and will prepare the applicant for an independent research career dedicated to improving the quality of mental health services for children, youth, and families through the advancement of implementation science and practice. The proposed project is consistent with priorities set forth by the NIH and the Institute of Medicine, and is the first step in a larger research agenda to improve the quality of mental health services for children, youth, and families by developing, refining, and testing implementation strategies. PUBLIC HEALTH RELEVANCE: Mental health problems affect a staggering 20% of children and youth each year. Yet, this vulnerable population continues to receive substandard mental health care, largely because we do not understand how to effectively implement evidence-based treatments (EBTs). This study will address this gap by examining implementation as usual in children's mental health organizations, which will inform efforts to identify, develop, refine, and test implementation strategies that can effectively integrate EBTs into real world care.

Desialylated (i.e. neuraminidase-treated) lymphocytes (DL) induce significantly more cytotoxicity in isolated rabbit hepatocytes than do intact lymphocytes. This phenomenon can be selectively blocked by a desialylated glycoprotein and is attributed to the receptor for desialy- lated glycoproteins (hepatic binding protein, HPB) on the surface of hepatocytes interacting with its specific ligands on the surface of DL. Purified HBP increases the cy totoxicity of DL to target cells which do not bear HBP on their surface presumably by an analogous mechanism. Infused desialylated periperal blood mononuclear cells (PBMC) and DL are selectively taken up by the liver in rabbits. The phenomenon is unaffected by depletion of complement but can be blocked by preincubation of desialylated PBMC with IgG-Fab fragments. These findings suggest that the selective hepatic uptake of desialylated PBMC or DL is not mediated by HBP. They are compatible with desialylated PBMC or DL becoming coated with naturally occurring antibodies in vivo with the result that subsequent recognition of cell bound antibody by Fc receptors on Kupffer cells mediates their selective hepatic uptake.

Recent advancemens in protein separation, mass spectrometry and bioinformatics have enabled the simultaneous analysis of a large number of proteins in cells and tissues. The interaction of these proteins, especially those that respond to specific challenges, are rapidly identified by ingenious screening techniques such as the phage expression or yeast two hybrid systems. These studies, collectively termed proteomics, have greatly enriched our knowledge of cellular physiology and pathology in normal and diseased tissues. A systematic identification of molecular interfaces between interacting proteins in natural cellular environment represents the next major challenge toward a molecular understanding of the complex and dynamic cellular events. Such a profile of molecular interfaces would also be invaluable as a tool in drug development by targeting crucial molecular interfaces. We are initiating such a proteomics project and focusing on the application of chemical cross-linking approach toward a systematic and global identification of protein-protein interfaces in complex systems such as the contractile machinery of muscle cells, the cytoskeletal filaments and organelles of nonmuscle cells. First, high-resolution gel electrophoresis and fractionation techniques are used to identify and index expressed proteins in skeletal and heart muscle tissues. Second, the neighboring relationship is defined by chemical crosslinking with a wide range of reagents. Third, the molecular interfaces of neighboring proteins are identified by isolating the crosslinked complexes, cleaving the proteins and isolating crosslinked peptides for protein sequencing with conventional chemical methods or by mass spectrometry. Fourth, the peptides at the interfaces will be synthesized and their interaction characterized further by capillary electrophoresis and biosensor techniques. A protein sequence database of molecular interfaces of proteins with known and unknown identities will be established to complement and enhance the utility of genome and proteome databases for basic and clinical research. We are evaluating the feasibility and integration potential of each steps of this multifaceted project. In particular, we have investigated the choice of crosslinking agents and the identification of extent, the stoichiometry of crosslinked complexes to define nearest neighbors. As a first step, contractile machinery, myofibrils from vertebrate skeletal muscle (rabbit psoas) and a novel tubular muscle from Midshipman fish sonic muscle were crosslinked by a zerolength crosslinker. The crosslinked complexes were identified by the gel mobility and Western blot of the complexes. Further efforts are made to purify selected complexes, followed by digestion, purification and sequencing and mass spectrometric analysis. Our work so far has clearly indicated the needs to employee or develop crosslinking agents with spectroscopic and mass tags to facilitate the identification and separation of crosslinked peptides. As a second method, protein interface is identified by screening synthetic peptides. The feasibility is demonstrated by the identification of nebulin/calmodulin interface by capillary electrophoresis of clmodulin with a set of nebulin peptides. The data are being compared with direct crosslinking and mass spectrometry. We conclude that, for our purposes, electrospray mass spectrometry is inferior to laser assisted matrix desorption mass spectrometry. The latter provides simple and interpretable spectra that are sufficiently accurate to calculate the composition of the complexes. - proteomics, protein interface, mass spectrometry, capillary electrophoresis, protein sequencing, crosslinking, genomics

Humans are able to hear across a wide range of sound levels, both in quiet and in the presence of background noise. The auditory system may achieve this wide dynamic range by turning down its response when there is ongoing sound. Though there is physiological evidence for a feedback loop to the cochlea which adjusts gain in a frequency-specific manner. Temporal effects have been measured in simultaneous masking which would be consistent with a decrease in gain with sound stimulation. It is not clear whether these temporal effects are due to the processing of the feedback system. The proposed research examines this important question in light of more recent evidence about the peripheral auditory system. The experiments are designed so that two measures of gain in the cochlea, input-output functions and frequency selectivity, may be estimated with and without preceding acoustic stimulation designed to activate the efferent system. Listeners with normal hearing and listeners with mild cochlear hearing loss will be tested in the experiments. The hearing-impaired listeners provide an interesting control case in which the gain in some frequency channels has been permanently decreased. The data will be analyzed within a well-established physiologically-based model of the peripheral auditory system, to determine whether temporal effects are consistent with a decrease in gain within the auditory channel which is stimulated. This research is important to our understanding of basic auditory processing in normal hearing, and the effects of cochlear hearing impairment. Listeners with normal hearing may have a type of automatic gain control that works independently in each of the thousands of auditory channels in the cochlea. This gain control could help in the perception of speech in background noise, by improving the signal-to-noise ratio within a channel, and by turning down channels containing only noise. The loss of this mechanism could help explain why listeners with cochlear hearing impairment have particular problems in background noise. This research could suggest novel processing strategies that might be used in hearing aids and cochlear implants to improve perception in background noise.The proposed research examines the hypothesis that listeners with normal hearing may have a type of automatic gain control that works independently in each of the thousands of auditory channels in the cochlea. This gain control could help in the perception of speech in background noise, by improving the signal-to-noise ratio within a channel, and by turning down channels containing only noise. The loss of this mechanism could help explain why listeners with cochlear hearing impairment have particular problems in background noise. This research could suggest novel processing strategies that might be used in hearing aids and cochlear implants to improve perception in background noise.

Administration of manufacture of bulk chemicals and bulk pharmaceuticals for preclinical trials.

Throughout the years, several practical problems have discouraged drug testing in children. These include the lack of a suitable infrastructure to conduct pediatric pharmacology research; the unforeseeable nature of some clinical responses in immature individuals; the possibility of unanticipated adverse reactions; the threat of effects on growth, development or health, long after the drug's administration, and difficulties predicting dose or concentration-response relationships by extrapolating data obtained in adults or experimental animals. Researchers and clinicians involved in pediatric drug development face daily challenges that include, but are not limited to the following: the need to obtain parental permission and/or the child's assent; ethical issues related to the conduct of non-therapeutic research in children; the lack of technology applicable to children; and the lack of incentives that would encourage pharmaceutical companies to study drugs in neonates, infants and children. There is a significant unmet need for pediatric therapeutic and diagnostic device development. In spite of these challenges, the NICHD is aware of the critical need to establish and maintain an environment in which safe and effective pediatric clinical and device trial activities can quickly be initiated and managed. The NICHD provides direct support for clinical research and clinical research networks that focus on particular diseases or public health issues. One of the goals of NICHD is to fund research to better understand the effects and effectiveness of pharmaceuticals on maternal and child health. Continued use of this Task Order mechanism will ensure that NICHD is able to contract quickly and efficiently to acquire experts with the ability to support studies relating to pediatric drug and device development.

The Genomics Shared Resource (GSR) is a state-of-the-art genomics facility dedicated to providing the latest genomic research tools at low-cost. The primary goal of the GSR is to facilitate high-impact genomic-based cancer research by Stanford Cancer Center members. This goal is accomplished by providing Cancer Center members with access to cutting-edge technologies and providing the services, expertise, and scientific support necessary for utilizing these genomic tools. Technologies and services currently provided by the GSR include: high-throughput sequencing (Illumina Genome Analyzer II, ABI SOLID, Roche Genome Sequencer FLX, and Helicos HeliScope), microarray services utilizing Affymetrix, Agilent, Illumina, Nimblegen, Stanford, and other platforms, genotyping services utilizing the Affymetrix and Illumina platforms, real-time quantitative PCR. Genomic reagents, such as clones, microarrays, and spike-in controls are also provided. Expert assistance with the use of these technologies is also provided throughout the entire experimental process. Guidance with experimental design, data analysis, data archiving and publication is a major component of the GSR. The resources of the Stanford Functional Genomics Facility (SFGF), Protein and Nucleic Acid Facility (PAN), and Stanford Microarray Database (SMD) have combined to provide the full spectrum of services offered by the Genomics Shared Resource and has a total combined operating budget of $2.8M. John Coiler, PhD (SFGF), is the Director of the shared resource, and Catherine Ball, PhD (SMD) and Michael Eckart, PhD (PAN) co-direct the GSR. Patrick Brown, MD, PhD and Gavin Sherlock, PhD are faculty advisors. There are currently 96 Cancer Center members using the shared resource, representing all program affiliations. Future goals are to increase access to high-throughput sequencing technologies and improve bioinformatics support and infrastructure required to implement the latest generation of genomics tools.

Glutamate excitotoxicity is the presumed mechanism underlying many neurological disorders including acute ischemia and chronic neurodegeneration. Recent work from our laboratories demonstrated that mice lacking the neural isoform of the stress activated protein kinsase SAPK/JNK (JNK3) has remarkable resistance to kainic acid-induced seizures, AP-1 transcriptional activity and apoptosis of hippocampal neurons. These results strongly suggest that the SAPK/JNK signaling pathway is a critical component in the pathogenesis of glutamate excitotoxicity. Based on this hypothesis, the present proposal comprises two sets of closely related in vitro and in vivo experiments. Specific Aim I: Mechanism of JNK -mediated glutamate neurotoxicity ( in vitro studies). Keys issues to be addressed include: (1) the JNK - mediated neurotoxicity through gene regulation and de novo biosynthesis; (2) the regulation of intracellular calcium oscillation through JNK signaling; (3) the interplay between JNK - signaling And oxidative stress, (4) the specificity of activation route of JNK signaling. These potential mechanisms will be tested in primary culture of dissociated neurons using a combination of morphological, physiological and biochemical approaches. Specific Aim II: Neuroprotection of the blockade JNK signaling pathway (in vivo studies) Potential clinical applications of inhibition of the JNK signaling may include: (1) the prevention of ischemic apoptosis; (2) attenuation of chronic glutamate excitotoxicity in amytrophic lateral sclerosis; (3) the prevention of neurodegneration (4) enhanced survival of grafted neurons in transplantation therapy. Preliminary evidence of feasibility has been obtained for each Specific Aim. The potential applications will be tested in experimental models of neuropathology using wildtype and mutant mice lacking the neural-specific JNK3 isoform. In summary, the overall goal of the present proposal is both to understand the mechanism underlying JNK- mediated glutamate neurotoxicity and to test the clinical potential of targeting the JNK signaling pathway for therapeutic intervention.

The proposed research concerns a comparative approach to diel and seasonal activity patterns in feral Dipodomys species both in the field and in laboratory experiments. Some of the specific research proposed is to measure surface activity of Dipodomys deserti automatically throughout hundreds of nights with the intention of evaluating the effects of moonlight, temperature, of competition with sympatric species, the effects upon foraging patterns of seasonal factors and food availability, and the effect upon the activity patterns of males during the reproductive season. Proposed laboratory experiments involve prey visibility in various lighting conditions simulating twilight, a search for a "lunar clock", the effects of food availability upon temporal foraging strategies, and the effects of the presence of a predator upon the timing of activity.

One of the most important mediators of host resistance to a vast number of pathogens is the cytokine Interferon gamma (IFN?). During infection, IFN? is released from specialized immune cells and induces multiple antimicrobial response pathways in most mammalian cell types. An important component of the IFN? response is the induction of cell-autonomous resistance towards bacterial pathogens residing and replicating within vacuoles, including Mycobacterium tuberculosis and Legionella pneumophila. To exert their antimicrobial activities, numerous IFN? -induced host response proteins like Nitric oxide synthase 2 (Nos2), Immunity Related GTPases (Irg) and Guanylate binding proteins (Gbp) translocate to pathogen-containing vacuoles (PCVs). The principles that underlie the ability of the host cell to recognize PCVs and target IFN? -induced host proteins to them are currently not well understood. The goal of this research proposal is to fill this gap in our knowledge by defining the molecular players that direct the localization of IFN? -induced antimicrobial proteins to PCVs. Towards this goal, we are taking two complementary, genomic approaches that aim to identify host factors that either facilitate the translocation of IFN? -induced proteins to a Legionella-containing vacuole (LCV) or translocate to LCVs directly. In Specific Aim 1, we will use gain- and loss-of-function screening approaches to identify host factors required for IFN? -mediated restriction of L. pneumophila replication inside macrophages. Host factors critical for providing resistance to L. pneumophila replication inside IFN? -activated macrophages will be subjected to mechanistic studies to further characterize their role in targeting IFN? - induced proteins to PCVs. In Aim 2, we will use an epitope-tagged kinome expression library to directly identify kinases localizing to PCVs in IFN? -stimulated cells using a high-content imaging approach. This second approach will potentially allow us to identify functionally redundant host factors, as well as capture the dynamic sequence of events involved in the host response. Identification of these targeting pathways is a first and critical step towars understanding how host cells recognize PCVs as 'non-self' and potentially dangerous vesicular structures and, conversely, how host-adapted pathogens can evade recognition. This knowledge will open up avenues for the development of novel therapeutics that aim to boost the inherent ability of the host organism to detect and eliminate intracellular bacterial pathogens. PUBLIC HEALTH RELEVANCE: This proposal aims to deploy new (genomics-based) approaches to identify the key host factors responsible for the delivery of antimicrobial proteins to vacuoles that contain pathogens such as the bacterium Legionella pneumophila. Host factors identified in these studies are likely to play an important role in host resistance to many pathogens, including Mycobacterium tuberculosis and Salmonella enterica, and may serve as drug targets for novel host-based antimicrobial therapies.

ABSTRACT/PROJECT SUMMARY The research theme of the proposed consortium focuses on the early detection of cancer, deciphering cancer pathogenesis, and the identification of potential biomarkers of Kaposi?s Sarcoma (KS) and ocular surface squamous neoplasia (OSSN), two of the most prevalent AIDS-associated cancers in Zambia. The Zambia AIDS Malignancies Diagnosis and Pathogenesis Program (ZAMDAPP)?s goal, which will link key expertise and resources of the Nebraska Center for Virology at the University of Nebraska-Lincoln, University of Zambia University Teaching Hospital, and Zambia?s Cancer Disease Hospital (CDH), is to develop the CDH cancer research infrastructure in three areas critical to the consortium?s overall research theme: cancer diagnosis, viral oncology, and cancer treatment. An Administrative Core will provide the administrative and programmatic leadership and infrastructure necessary to ensure the consortium functions efficiently and effectively. The Administrative Core will pursue four specific aims: 1) provide the administrative expertise and resources necessary to support the consortium?s Career Enhancement Core, Shared Resources Cores, and research projects in the accomplishment of their goals and specific aims; 2) implement a consortium-wide series of operational processes designed to create a collaborative research environment and build capacity for increased self-sufficiency in Zambian health institutions; 3) provide communications infrastructure to engage key stakeholders and promote the consortium locally and globally; and 4) establish and cultivate a strong working relationship with an External Advisory Group composed of key stakeholders and expertise leaders to connect research efforts with Zambian communities and identify research outcomes that will inform healthy policy changes. The organization of the Administrative Core is designed to be responsive, inclusive, and robust, which is essential to successfully address the broad mission and complex relationships within ZAMDAPP, among partnering and collaborating institutions, and with NIH. This core will focus on meeting the needs of the partnership and collaborators in the current climate of rapidly changing scientific and technological advances, which will result in a sustainable consortium with vibrant research programs that work toward the reduction of cancer in Africa.

Support services for the National Kidney Disease Education Program (NKDEP)

Human papillomaviruses (HPVs) represent some of the most common of the sexually transmitted disease. More than 80 HPV types have been identified and several types have been strongly implicated as causative agents in the onset of cervical vaginal, penile, oral and skin cancers. HPV DNA is present in virtually all cases (93 percent) of cervical cancer and its precursor lesions. Current microbicidal compounds with papillomavirus activity have not been described. In addition, there are not published reports of animal models of genital papillomavirus infections for testing intra-vaginal formulations and co-incidental STD infections. Recently, several in vitro and in vivo papillomavirus infectivity models have been documented that make feasible the screening and testing of microbicidal compounds and formulations for potential anti- papillomavirus activity. The purpose of this renewal proposal is to establish the validity of two in vitro and two in vivo papillomavirus infectivity models for the testing of microbicidal papillomavirus compounds. The in vitro models we propose to use are (I) BPV-1-induced focus formation of mouse C127 cells, and (ii) transient in vitro HPV-11 (and HPV-40) infection of human A431 epithelial cells using an ELISA-based RT-PCR detection of HPV E1 E4 spliced mRNA transcripts. We will use the in vitro models to screen for effective anti-papillomavirus compounds to be later tested in two animal model systems. These include a novel human vaginal xenograft animal model system using infectious HPV-11 and HPV-40 virions, and a rabbit genital papillomavirus model recently developed in our laboratory. During the first 3 years of this Program Project, we have identified several potential papillomavirus microbicidal compounds and have begun to characterize in vivo model of genital papillomavirus infections. These microbicidal agents (neutralizing monoclonal antibodies and alkyl sulfates) represent the first compounds with anti-papillomavirus activity. We will continue their characterizing and expand the testing using formulations delivered intra-vaginally (into human vaginal xenografts and into intact rabbit vaginas) for in vivo efficacy.

DESCRIPTION: Recovery from measles is associated with life-long immunity that is reflected by the persistence of antibody. Protection after infection with wt virus or after live vaccine is closely associated with levels of neutralizing and hemagglutination-inhibiting antibody; experiences in immunosuppressed children also suggests that a virus-specific cellular immune response is essential for recovery. The currently licensed live attenuated measles virus vaccine is safe and effective when given at 9-15 months of age, but the vaccine is less effective in infants under the age of 9 months, and urban outbreaks have recently involved many young infants. Although immunization of infants as young as 4-6 months has been successful using a 10-100 fold higher dose of vaccine virus, such immunization has been associated with a poorly understood increase in susceptibility to subsequent infections and has been abandoned as a strategy for improving the vaccine and lowering the age of immunization. An inactivated vaccine was one of the earliest measles vaccines introduced. After 3 doses of alum-precipitated, formalin-inactivated vaccine, neutralizing antibody adequate to protect against wild-type viruses was present for a few months, but protection rapidly waned. In addition, approximately 15% of children immunized with this vaccine who subsequently contracted measles developed a severe form of the disease known as atypical measles. This complication has been ascribed to the low levels of antibody to the fusion protein that were induced by the inactivated vaccine, but this pathogenic mechanism has never been proven. Failure to understand the pathogenesis of atypical measles continues to impede the development of newer vaccines for all paramyxoviruses. The current proposal represents a competitive renewal of an original award granted in 1993 to investigate immune responses to measles and atypical measles in non-human primates. In this proposal the investigators propose follow-up studies and other work to determine the pathogenesis of atypical measles, characterize the immune responses to prototype live and inactivated vaccines, and to continue development of cDNA-based measles vaccines. The specific aims of the proposal are: 1) to characterize the immune responses to formalin-inactivated vaccine and live attenuated vaccines by comparing the specificities and biological properties of anti-measles virus antibodies, T cell effector functions, and the cytokines induced by immunization; 2) to define the nature of the immune responses after challenge with wild-type measles virus in unimmunized monkeys and in monkeys previously immunized with live or formalin-inactivated vaccines; and 3) to determine the nature of the immune response to and the efficacy of H, F an d/or N cDNA vaccines delivered by different routes of inoculation in monkeys.

Research at an early stage of medical education is the best predictor of a future career in research. The College of Physicians & Surgeons of Columbia is especially strong in research focusing on the basic and applied features of diabetes, kidney and endocrinologic diseases. Investigation of the health issues dealing with the mandate of NIDDK requires preparations for the next generation of investigators and this application aims to introduce our medical students to research in these fields mentored by exceptional faculty with proven track record of excellence in research and funding. The curriculum at P &S has changed such that every student is now required to produce a scholarly effort and this proposal will support students performing research either during the summer months between the 1st and 2nd years or later. Students at P& S have traditionally been active in research and this application will provide them with a structured program that includes the research as well as a series of seminars covering topics in modern biomedical research. In addition they will be required to attend a series of lectures in the Responsible Conduct of Research. The Program Director will be assisted in managing the Program by an External and an Internal Advisory Committees. Applying students will be chosen by a Recruitment and Admission Committee and their progress in research will be followed by a Mentoring Committee. Finally, after they complete their research they will be advised by a Career Development committee that will aid them in making important career decisions. Recruitment of under-represented minorities, among mentors as well as students, is strongly supported by an ongoing commitment of the College and by targeted efforts of the Program.

We have recently shown that the specific amino acids in the M1 and M2 proteins of influenza A virus are important genetic determinants of filamentous virus morphology. In addition, we have demonstrated that the host cell cytoskeletal complex is an important cellular determinant of filamentous virus formation. The objectives of this proposal expand upon these recent observations and focus on determining the cell biological dynamics associated with filamentous influenza virus entry, and on the pathogenic potential of filamentous influenza virus as it relates to spread of infection and severity of disease. In specific aim 1, experiments are described to address specific questions relating to the mechanisms of filamentous virus attachment and entry. Employing radiolabeled and fluorochrome-labeled viral filaments, we will quantitatively examine whether viral filament entry occurs by partial or complete endocytosis or possibly by a phagocytosis-like mechanism. Membrane fusion assays will be used to determine pH requirements and extent of cell fusion of viral filaments. Live cell video microscopy, confocal and electron microscopy will be used to follow the fate of internalized viral filaments and examine whether budding viral filaments can mediate cell-to-cell spread of infection. In specific aim 2, we will focus on the pathogenic potential of filamentous influenza virus. Using primary human nasopharyngeal organ and epithelial cultures we will determine whether infection is restricted to a subpopulation of epithelial cells and determine whether filamentous virus strains have enhanced ciliopathic and tissue damaging capabilities. Using a fluorochrome-based binding assay, we will assess whether viral filaments can enhance bacterial binding to the mucosal epithelium. Using the mouse and ferret animal models, we will compare in vivo the pathogenic potential of genetically similar variants of influenza A/Udorn virus, which differ only in morphology. Pathogenic criteria that will be assessed include: i) the 50 percent minimal infectious dose, ii) tissue specific viral loads iii) tissue-specific histopathology, and iv) rates of weight loss recovery. In the animal studies, we will examine whether filamentous strains promote a descending spread of infection, from an initial localized infection in the nasal tract to the lower respiratory tract. These studies will set the foundation for establishing further determinants of influenza virus pathogenesis and severity of disease in humans.

The biologic changes that occur following menopause have heightened interests in the relationship between declining plasma estrogen levels and health in this aging female population. Specifically, in postmenopausal women, the development of atropic oral epithelial cells, which are easily stripped from the underlying connective tissue, hinders proper oral hygiene and impairs the prescribed utilization of dental prostheses. The broad aims of this research are 1) to understand the actions of estrogens on oral tissues and 2) to elucidate the relationship(s) between extracellular matrix proteins secreted by fibroblasts from young versus senescent gingiva, and the effects of such proteins on normal and aberrant epithelial cell growth. As a model system, human fibroblasts derived from the gingiva of premenopausal, postmenopausal and affected postmenopausal females (i.e., those with gingiva exhibiting a hormone-induced desquamative lesion) will be used to define estrogen action in the gingiva. This proposal will examine estrogen effects on proliferation, glycosaminoglycan synthesis, collagen synthesis, noncollagen protein synthesis and collagen expression in fibroblasts from both young and elderly females. Experiments will be performed to determine if maintenance of estrogen sensitivity is a characteristic of the premenopausal fibroblast secretory phenotype and whether it is lost in fibroblasts of elderly individuals with hormone- induced desquamative lesions. Further experiments will analyze cytosol and nuclear estrophiles as well as estrogen metabolism, to determine the mechanism by which estrogen sensitivity is lost. Finally a study of epithelial growth will be examined by in vitro cultivation of epithelial cells on estrogen-stimulated extracellular matrix proteins secreted by gingival fibroblasts, to determine if gingival epithelial cell proliferation, attachment, and keratin proteins are dependent on extracellular matrices secreted by estrogen-stimulated fibroblasts. Knowledge gained from the proposed research on gingival fibroblast and epithelial cell function in young and elderly females, as ell as a new understanding of how these cells respond in the presence of estrogens, should provide the rational for the development of novel therapeutic approaches in the control of hormone-induced oral and other mucosal desquamations.

Neurofilament (NF) proteins, though synthesized in neuronal cell bodies, are normally posttranslationally phosphorylated in axons. In some neuro-degenerative disorders (e.g., ALS), however, they are abnormally hyperphosphorylated in cell bodies. We have been studying the factors regulating these topographic pattern of neurofilament (NF) phosphorylation. One kinase, cdk5, identified in our laboratory, specifically phosphorylates KSPXK motifs which constitutes 20% of the total repeats in high molecular weight NF-subunit, NF-H. The remaining 80% repeats are KSPXXXK motifs kinase(s) phosphorylating these motifs are not known. A synthetic KSPXXXK peptide, KSPAEAKSPAEAKS, which repeats 41 times with minor variations at non-KSP residues was used as a substrate to identify the kinase in rat brain that phospshorylates NF- H. On the basis of biochemical, immunochemical, pharmacological and amino acid sequence analysis, the purified kinase appeared to be MEK- activated MAP kinase. To verify this statement, we demonstrated that bacterially expressed MAP kinase phosphorylated expressed rat NF-H in addition to KSPXXXK, KSPXXK and KSPXK peptides. This study suggests that neuronal MAPK can phosphsorylate all KSP repeats in rat NF-H and may be the principal kinase in vivo that phosphorylates serine residues in KSP repeats in neurofilament tail domains. Since cdk5 specifically phosphorylates KSPXK peptides and not KSPXXXK, we investigated the structural differences of these motifs, by analyzing the conformation of four peptides with either KSPXK or KSPXXXK motifs. The KSPXXXK peptide exhibited a CD spectrum indicative of a helical conformation; under the same conditions, however, the KSPXK peptide showed a mainly extended conformation with beta-turns. These differences revealed a structural specificity for the substrate of this kinase. using two dimensional NMR methods and molecular modeling. We are also studying the mechanism of cdk5 activation. Though, cdk5 is similar to other cdc2 kinases, its regulation is completely different from mitotic cdc2 kinases; e.g., cdk5 is active only in postmitotic neurons, its activity is independent of its phosphorylation, nor is its activity regulated by cyclin binding. Instead association of neuron specific proteins, P35 and P67 are responsible for its activation. These regulator proteins share no homology with cyclins. We have begun to locate the activation domains in P35 using various mutations and truncations of P35 in in vitro assays. These studies suggest the potential residues for kinase activation reside in amino acid residues 137-167 and 291-263 of N- and C-terminal tail domains respectively of P35. Further mutational and truncational studies are in progress.

The long-term goal of this project is to identify novel monoclonal antibodies (mAbs) that broadly recognize the HIV-1 envelope glycoprotein (Env) and block infection in vitro to guide vaccine development. This goal will be pursued in local cohorts of HIV-1 infected individuals who control their infections in the absence of anti-retroviral therapy (JAIDS, 50:403-8, 2009). Approximately 13% of these individuals have circulating broadly neutralizing antibodies providing the study population for our studies. A key element of our approach is the development of a new assay to census Env-specific memory B cell clones (BMem) that allows the rapid and direct cloning of full-length molecular clones of the antibodies expressed by these cells (PNAS, 106:3952-7, 2009). This method permits clone identification using native oligomeric HIV-1 envelope glycoproteins (Env) expressed on cell surfaces and by direct neutralization of pseudoviruses. Env-reactive clones are used to produce mAbs that will be evaluated for epitope specificity and neutralization breadth. This information will be used to test the hypothesis that neutralization breadth is determined by monoclonal or pauciclonal responses comprised of one or a very few neutralizing specificities as opposed to a polyclonal response comprised of a mosaic of neutralizing specificities. There are two specific aims. Aim 1- To develop clonal specificity profiles of Env-specific BMem from NVS who have ongoing broadly neutralizing antibody responses- Clonal specificity profiles of anti-Env responses will be determined by limiting dilution analysis, mAb isolation, and epitope mapping to identify broadly neutralizing mAbs. Aim-2- To compare neutralization breadth between plasma antibodies and mAbs representing a full clonal profile of BMem to determine the number of mAbs that must be pooled to reconstruct the neutralization profile of the circulating antibody pool. Neutralization breadth of mAbs will be determined using standardized pseudovirus assays. This data will be used to determine the clonality of an ongoing broadly neutralizing antibody response. This aim will test the hypothesis that neutralization breadth is determined by monoclonal or pauciclonal responses comprised of one or a very few neutralizing specificities as opposed to a polyclonal response comprised of a mosaic of neutralizing specificities.

This application requests continued support for the long-term objectives of MH 51372, namely to develop and implement high throughput strategies for the detection and fine mapping of mental health-related quantitative trait loci (QTL), for the integration of QTL mapping with functional genomics and for the detection and confirmation of the relevant quantitative trait gene(s) (QTGs). This integrated approach to QTL analysis requires the development and utilization of detailed brain gene expression and sequence datasets. The phenotypes of interest are open-field activity (OFA) and prepulse inhibition (PPI) of the startle response. This renewal application also emphasizes the detection of gene networks associated with OFA and PPI. This competing renewal has 5 specific aims. 1) To fine map in heterogeneous stock (HS) animals (to a resolution of 1 cM or less) high quality QTLs for OFA (2 on Chr 1 at ~ 110 and 175 Mbp) and PPI (Chr 11 at ~ 70 Mbp and Chr 16 at ~ 45 Mbp). 2) To integrate the QTL data with interval relevant gene expression and sequence data. 3) To determine in HS4 animals the gene coexpression networks associated with OFA and PPI. 4) To determine if the modules identified in aim 3 are the same modules that differentiate short-term selective breeding (STSB) lines, selected for High and Low OFA and PPI. 5) To determine if the OFA and PPI modules identified in aims 3 and 4 are also detected in a more genetically diverse mouse population of mice, namely an outbred population derived from the Collaborative Cross. The primary mapping population is a HS formed from the C57BL/6, DBA.2j, BALB/c and LP strains. Mapping in these HS4 will provide QTL resolution of approximately 1-2 cM. As each interval will be completely sequenced, all causative polymorphisms will be detected. In addition to 3' biased oligonucleotide gene expression analysis, we will test the idea that some QTLs may be generated by alternative exon usage. Our ability to sort through the large amount of data generated is made possible by precisely knowing the haplotype of each QTL. The proposed work builds upon data collected during the current grant period and importantly incorporates new genomic technologies which make possible our fully integrated approach. PUBLIC HEALTH RELEVANCE: The proposed work attempts to understand the role(s) of genetic factors in behaviors that are of mental health significance. The eventual goal is to determine which genes and gene networks make some individuals more prone (increased risk) for developing abnormal behaviors such as schizophrenia and anxiety disorders. With this information in hand, it should be possible to develop new therapeutic strategies and interventions. .

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. We will continue investigating the intercellular and intracellular signaling pathways that lead to neuronal death (or survival) following the elimination of excitatory synaptic input to the mammalian cochlear nucleus. A major initiative during the current grant period has been to use modern genetic analysis methods to begin assessing the biological mechanisms underlying differential responses of neonatal and adult animals to afferent deprivation (a critical period) of the mouse cochlear nucleus.

This is an application written in response to RFA PA-00-004. It describes a systematic 5-year training plan to launch an independent research career in the cognitive and emotional neuroscience of human cocaine addiction through the use of functional magnetic resonance imaging (fMRI), a powerful technique that allows simultaneous, non-invasive measurement of brain activation and cognitive performance. This plan will allow the candidate, a Clinical Psychologist with background in Neuropsychology, to initiate technologically sophisticated research of relevance to treating cocaine addiction. This goal will be achieved through a rigorous training plan, integrating didactic and research components tailored to the candidate's goals and experience. Brookhaven National Laboratory (BNL) provides an ideal training environment: 1) BNL is home to state-of-the-art functional neuroirnaging technologies; and 2) The mentors of this project are world renowned for the application of these cutting-edge technologies to study mechanisms of drug addiction. Under their supervision the candidate will conduct fMRI (4T) activation projects designed to evaluate whether adaptations to the frontolimbic circuit underlie deficits in salience attribution and response inhibition in cocaine addicted subjects, and whether these neuroadaptations and cognitive-behavioral impairments can be used to predict and monitor relapse. In Project 1, subjects receive different levels of Monetary Incentive for correct performance on a go/no-go task. In Project 2, subjects perform a Drug Stroop task, naming the color of cocaine-related words, neutral words, or non-words. Project 3 is a longitudinal study, where subjects undergo Projects 1 & 2 at baseline (2-4 weeks after detoxification) and 6 months follow-up. In all projects, we measure the blood oxygenation level dependent (BOLD) responses of the frontolimbic circuit including the orbitofrontal cortex (OFC) and anterior cingulate gyrus (ACG). We hypothesize: 1) Task specificity: the Monetary Incentive task will primarily activate the OFC while the Drug Stroop task will primarily activate the ACG; 2) Group specificity: the activation of the frontolimbic circuit by the two cognitive-behavioral tasks will differ as a function of drug addiction; and 3) Relapse prediction and monitoring: increased frontolimbic activation and impaired cognitive-behavioral performance at baseline will characterize the cocaine addicted subjects and predict relapse at 6-months follow up. For the subjects who will relapse, abnormalities in these brain-behavior measures will remain the same or get worse during the follow-up period while for the subjects who will remain abstinent, normalization of these measures will be documented.

The long-term objective of this research is to investigate the function of a newly discovered mitochondrial progesterone receptor, PR-M. We believe that progesterone acts via this receptor to increase cellular ATP production in multiple tissues to meet the increased metabolic demands of pregnancy. Cardiac function dramatically changes with pregnancy with increased heart rate and contractility requiring increased ATP production. Inadequate cardiac adaptation may contribute to placental insufficiency related to preeclampsia and intrauterine growth restriction. Our aim is to investigate the hypothesis that PR-M increases cellular respiration in the heart. To test this hypothesis we will temporally express human PR-M in the mouse heart using a heavy myosin polypeptide 6 promoter in a Tetracycline-On (Tet-On) system. Tissue and temporal expression of the transgene will be determined by realtime RT-PCR and western blot analysis. Cardiac function in transgenic animals will be evaluated during late gestation and in non- pregnant models of cardiac hypertrophy to mimic volume and pressure overload. Assays will include echocardiography, heart morphometry, microscopic analysis of capillary density, fibrosis and apoptosis and determination of tissue ATP levels. Transgenic overexpression is a valuable tool to analyze the tissue function of PR-M. We believe that enhanced ATP production secondary to PR-M expression will diminish cardiac hypertrophy secondary to volume and pressure overload in the study models. These studies will further develop a novel mechanism in which progesterone modulates cellular respiration via a direct affect on the mitochondria. Additionally, the created PR-M transgenic mouse can be cross-bred with other tissue specific Tet-On strains for future studies of PR-M function in the mammary gland, liver, skeletal muscle, adipose and brain. PUBLIC HEALTH STATEMENT Pregnancy is associated with dramatic changes in a woman's body including a great increase in the workload of the heart. This process requires an increase in ATP which serves as the major fuel source for the cell. Our studies are investigating a new receptor for the reproductive hormone, progesterone, located in the mitochondria of the cell where ATP is produced. We believe that this receptor provides a mechanism whereby progesterone increases cellular ATP formation to meet the many metabolic demands of pregnancy. Dysfunction of this system could contribute to pregnancy related diseases.

The overall goal of the project is to validate the feasibility of a microwave tomographic (MWT) imaging technology for assessment of functional and pathological conditions of extremity soft tissue. The successful management of a fractured bone involves an understanding of the two major components (boney and soft tissue elements) of any extremity segment. The diagnosis and evaluation of the boney component is obvious to the treating physician by radiographic studies. The accurate assessment of the soft tissue component of the injured extremity segment remains a major deficiency in management of fractures. Consequently there is an important need to develop an effective and rapid method of non-invasive assessing of extremities soft tissue viability. Previous studies (NHLBI grant No HL65657) demonstrate that microwave tomography is feasible for non-invasive assessment of myocardial viability. Our most recent studies suggests that the technology might be feasible for rapid soft tissue functional imaging of extremities Both the imaging capabilities of this technology for assessment of functional conditions of extremities soft tissues within a very short msec range of acquisition cycle and the use of low, safe levels of non-ionizing radiation with cost efficiency have potentials for significant improvement of public healthcare. In this three year study we plan to (i) develop 2D MWT system; (ii) develop an appropriate image algorithm and (iii) assess feasibility of the technology for extremities soft tissue imaging, including sensitivity and resolution of the technology. Relevance of this project to public health. There is important need to develop an effective and rapid method for assessment of conditions of extremities soft tissue during trauma and recovery. The goal of the project is to validate the feasibility of novel imaging modality (Microwave Tomographic Imaging) for non- invasive (non-destructive) imaging of functional conditions of extremities soft tissue. There is an important clinical need to develop an effective, rapid and cost-effective method of non-invasive assessing or imaging of extremities soft tissue viability. The overall goal of the project is to validate the feasibility of a microwave tomographic (MWT) imaging technology for assessment of functional and pathological conditions of extremity soft tissue. The imaging capabilities of this novel technology and the use of low, safe levels of non-ionizing radiation with cost efficiency have potentials for significant improvement of public healthcare. [unreadable] [unreadable] [unreadable]

Sounds are encoded by the cochlea in the timing of spikes in the auditory nerve. Auditory nerve fibers converge onto bushy cells in the cochlear nucleus, through synapses called "endbulbs of Held". This convergence leads to changes in the timing information passed on by bushy cells to higher auditory centers, which may affect sound localization and processing. Experiments have indicated that the timing of bushy cell spiking is greatly affected by the size of the endbulb synaptic current, which is subject to 2 major influences in vivo. First, endbulb synapses show considerable depression when activated at normal rates. Second, they are subject to a number of neuromodulatory systems. Both these influences can affect the information carried by bushy cells, but neither is well understood. The specific aims of this project are to determine (1) the dynamics and mechanisms of use-dependent changes in the endbulb synaptic current, (2) how the different components of the synaptic current control bushy cell timing, and (3) how neuromodulation changes these relationships. This work will be carried out using patch-clamp recordings of bushy cells in brain slices taken from mice and gerbils. The mechanisms of depression will be considered first, by testing the contributions of presynaptic vesicle depletion and postsynaptic receptor desensitization and saturation. In addition, an unusual form of depression at the endbulb will be examined, which has been proposed to involve reduced presynaptic calcium influx, but has never been directly tested. It will also be determined how depression could be mitigated by both facilitation and the activation of NMDA receptors. In addition, current-and dynamic-clamp studies will test whether delayed release during high levels of activity disrupts the precisely timed responses of bushy cells. Taken together, these studies will provide important information about the mechanisms by which timing information is transformed by convergence of auditory nerve synapses during realistic activity. This work will examine the mechanisms used by cells in the cochlear nucleus to process sound information. It will also provide important insights into the functional role that different receptor types and neuromodulatory systems play in neuronal computation. This work may lead to improvements both in existing cochlear implants and in implants that stimulate the cochlear nucleus directly. [unreadable] [unreadable] [unreadable]

ABSTRACT Neurological and neuropsychiatric illnesses are prevalent and debilitating, affecting more than a quarter of the global population. Learning and memory are implicated in every neurological illness from stroke to Alzheimer?s disease and every neuropsychiatric dysfunction from depression to drug addiction. Understanding the neuroscience of learning and memory, integrating across approaches and methods, is one of the most formidable challenges of the 21st century. While research in these areas has made significant strides in understanding and treating brain disease as well as promoting brain health, progress has been stalled. There are fundamental gaps in our knowledge that stem for lack of integration across approaches and insufficient dialogue among investigators addressing neuroscience questions from different perspectives. Integration across animal and human research as well as basic and clinical research will have substantial impact on facilitating team science, which is essential for successful translation. The 2018 International Conference on Learning and Memory (LEARNMEM2018) covers numerous facets of the neuroscience of learning and memory with an emphasis on integrating across animal and human studies as well as across fundamental and translational science. The 5-day conference that will feature plenary and keynote talks by distinguished neuroscientists, diverse and balanced symposia, short talks, poster sessions, a panel discussion on open science and open publishing, as well as community engagement sessions. Anticipated attendance is ~800 and the conference is open to everyone. AMA PRA Category 1? Continuing Medical Education (CME) credits will be offered to physicians and healthcare professionals. Travel awards for early career scientists will be available, and special emphasis will be placed on multidimensional diversity of contributions across gender, race, seniority, country of origin, and level of analysis. The conference will achieve the following aims (1) Identify knowledge gaps and opportunities for scientists, clinicians, nonprofits, businesses, and community members to share and discuss cutting-edge research in the area of learning and memory; (2) Form collaborative teams among domestic and international investigators, early career and established scientists, animal and human researchers, fundamental and translational scientists, as well as user and developer communities; (3) Disseminate knowledge generated from the meeting through scientific and public venues; and (4) Facilitate engagement between scientists and community members across all levels from schoolchildren to retirees. LEARNMEM2018 will have substantial impact on the field by accelerating the pace of team science and leapfrogging towards a more complete fundamental understanding of learning and memory mechanisms as well as novel therapeutic and preventative approaches to treating brain illness and promoting brain health.

The technique of free tissue transfer, has revolutionized many areas of reconstructive surgery, such as limb salvage and reconstruction of the esophagus, where other options have been unacceptable. As free tissue transfer is now being done routinely at institutions where inexperienced staff are unable to adequately monitor viability, the need for a reliable method to monitor the patency of microvascular anastomoses has become increasingly evident. Accurate and objective assessment of blood flow both at the time of surgery and during the post operative period is essential, because if the tissue viability is lost, the consequences can be staggering. Early recognition of postoperative vessel thrombosis is of utmost importance if reexploration is to prove successful. To this end, numerous monitoring techniques have been investigated. Those classified as direct monitors of perfusion have proven to be of little or no value - particularly as free flap monitors. Probes are bulky, very expensive, and require great care in positioning and removal. Indirect monitors such as those which monitor the tissue concentration of a physiological gas such as oxygen, rate ahead of the direct monitors due to advantages of low cost, simplicity, ease of placement and removal, etc. However, these monitors lack the needed specificity of the direct monitors for measuring tissue perfusion. Adding to this specificity problem is the fact that the sensor cannot be calibrated once it is implanted. Our approach is to preserve the simplicity afforded by indirect monitoring devices, while providing the specificity for tissue perfusion measurement, by utilizing a self-calibrating tissue PO2/PCO2 probe, which manipulates the local tissue gas conditions. Tissue PO2 and PCO2 are monitored under alternating depleting and nondepleting conditions. A physilogically based mathematical relationship relates tissue perfusion to the PO2 and PCO2 measurements. This proposal is directed toward the in vivo validation of the method.

We have developed a method for preparing a covalent conjugate of proteins with purified gangliosides in the micellar form. The ganglioside is oxidized with periodate, and the protein is attached to it by reductive amination with sodium cyanoborohydride. The ganglioside moiety of the product retains the ability to transfer irreversibly from the micellar form to a lipid bilayer, thereby associating the protein moiety with the lipid bilayer of choice, for example, a liposome or viable cell membrane. We have demonstrated that a ganglioside conjugate with rabbit IgG can associate with human or sheep erythrocytes, since the cells may be hemagglutinated with goat anti-rabbit IgG after incubation with the conjugate. We have also demonstrated that a ganglioside conjugate with monoclonal mouse anti-glycophorin can associate with Sendai virus and confer upon the virus the ability to agglutinate and hemolyze desialylated human erythrocytes. The method will be of considerable importance in cell biology and immunology by allowing one to study: 1) antigen-directed fusion; ii) T-cell, T-cell hybridoma production; iii) delivery of genetic material to cells and the insertion of artificial receptors. We wish to improve the method by examining alternative conjugation methods, by examining the potential of different purified gangliosides as a hydrophobic anchor, and by optimizing conditions for insertion of the conjugates into cells. We will develop a method for detection of ganglioside GD3 in leukemia cells and leukemia cell extracts by production of monoclonal anti-GD3 coated erythrocytes. We also wish to explore the potential usefulness of the method by examining the insertion of conjugates into viable cells. We will characterize the effect of insertion of cell growth and viability, and determine whether conjugates can undergo patching, capping and endocytosis or shedding. We will also examine the usefulness of this technique for antigen-directed fusion in the production of T cell, T cell hybrids. Anti T4 or T8 conjugates will be inserted into BUC cells and fused with human T cells. The resultant hybridomas will be scored for T4 and T8 antigens to establish whether the use of antigen-directed fusion can increase the frequency of specific hybrids.

The goals of this Phase II STTR are to evaluate the outcomes of REAL media, an interactive, self- paced, e-learning substance use prevention media literacy curriculum and prepare it for marketing to community organizations, including our partner, 4-H. Substance use increases in frequency and risk through mid-adolescence, yet prevention interventions primarily target early use, are time intensive, and are implemented in a limited number of settings such as schools. Moreover, they often fail to address the media-saturated lives of youth despite research demonstrating the deleterious effects of advertising and entertainment media. This provides a market niche for the proposed project that addresses this curriculum gap through the innovative use of both technology and prevention science. Guided by the new Theory of Active Involvement, REAL media develops critical perspective taking about substance use decisions and confers resistance to pro-drug (e.g., alcohol, cigarettes, e-cigarettes, marijuana, smokeless tobacco) messages through youth analysis of pro-drug media messages combined with interactive media manipulation and active involvement of youth participants in creating their own anti-ATOD prevention messages. These youth-created messages are then entered into an online contest via a social media proliferation strategy (i.e., youth recruit others to view their messages on social media (e.g., Facebook, YouTube) to win the contest) in which messages are diffused to the wider community. The curriculum demonstrated promising results when administered face-to-face during an NIH-funded pilot study, and the Phase I project demonstrated excellent usability and feasibility for online delivery through 4-H clubs. During Phase II, we will finalize the program and conduct a group-randomized clinical trial among 4-H clubs in four states (NJ, OH, MD, and PA). Clubs will be randomly assigned to use the curriculum or continue current practices with the option for delivery at the end of the study. 4-H members (ages 13-15) will complete a pretest, immediate posttest and follow-up posttests at 3 and 9 months to assess effects. Results will guide preparation of REAL media for the market. The flexibility of the brief, online format (four 15-25 minute levels plus a fifth message planning and production level) for youth in individual or group settings makes this ideal for both community and school implementation. Thus, REAL media is well-suited for rapid dissemination through our existing partners, 4-H, D.A.R.E., and Boys and Girls Clubs, as well as other potential community partners (e.g., YM/WCA, Boys and Girl Scouts) and is appropriate for Phase II funding.

To support the development and assessment of two live microbiome-based products.

In an integrated program of laboratory and clinical investigation, we study the molecular biology of the heritable connective tissue disorders osteogenesis imperfecta (OI). Our objective is to elucidate the mechanisms by which the primary gene defect causes skeletal fragility and then apply the knowledge gained from our studies to the treatment of children with these conditions. Structural defects of type I collagen molecule are well known to cause the dominant bone disorder OI. A severe recessive form of OI was first postulated in 1979. We hypothesized that the cause of recessive OI with abnormal collagen biochemistry and normal collagen gene sequence would involve a gene(s) whose products interacted with type I collagen. Ten years ago we identified defects in two components of the collagen prolyl 3-hydroxylation complex, CRTAP and P3H1 (encoded by LEPRE1) as the cause of recessive OI. Our work generated a new paradigm for collagen-related disorders of matrix, in which structural defects in collagen cause dominant OI, while defects in proteins that interact with collagen cause the rare forms of OI. Recessive OI is now a major area of investigation for the BEMB. The phenotypes of types VII and VIII OI are distinct from classical dominant OI, but difficult to distinguish from each other. Biochemically, both groups full overmodification of the helical prolines and lysines by prolyl 4-hydroxylase and lysly hydroxylase, indicating delayed folding of the collagen helix. We showed that mutual stabilization of CRTAP and P3H1 underlies the phenotypic and biochemical similarity of types VII and VIII OI. With collaborators at the Boltzman Osteology Institute, we recently focused on the bone of the non-lethal subset of type VIII OI patients. We demonstrated that there is no redundancy for collagen 3-hydroxylation function, comparing null mutations in bone and skin. Bone histology was similar to type VII OI, although it had the distinctive feature of extremely thin trabeculae and patches of increased osteoid, suggesting mineralization is slower in type VIII than VII OI. BMDD yielded increased mineralization of type VIII bone, as in classical OI and type VIII OI, but the proportion of bone with low mineralization was increased in type VIII bone vs type VII. Type IX OI has a distinctive phenotype without rhizomelia, and distinctive biochemistry. We generated a CyPB KO mouse, which has reduced bone density and strength, but increased brittleness. Only 1-2% 3-hydroxyltion is detected in KO cells, showing the importance of CyPB to complex function. Collagen folds more slowly in the absence of CyPB, but CsA treatment revels the potential existence of another collagen PPIase. CyPB supports LH1 activity and in its absence there is significant reduction of hydroxylation of crosslinking residue K87. The decreased crosslink ratio alters fibril structure and reduces bone strength. With collaborators at the University of North Carolina we showed that CyPB interacted with all LH forms (LH1-3). The effect of CyPB KO in type I collagen of tendon had distinct patterns in the collagen helix versus telopeptide domains. yPB modulates crosslinking by differentially affecting lysine hydroxylation in a site-specific manner. We have extended our work on PPIB function in a collaboration with investigators at Hebrew University in Jerusalem. CyPB was shown to be critical to the folding of presenilin-1, a protein linked to familial Alzheimers Disease. Some substitutions in presenilin-1 make it resistant to folding properly by CyPB. Conversely, the brains of CyPB knock-out mice were shown to have reduced quantities of processed, active presenilin-1. ER-chaperones may thus be targets for the development of counter-neuro degeneration therapies. We delineated a mutation in IFITM5, which encodes the transmembrane protein BRIL, that establishes a connection between types V and VI OI. The BRIL S40L substitution results in minimal expression and secretion of PEDF by mutant FB and osteoblasts. Om contrast to the gain-of-function BRIL mutation that causes type V OI, the BRIL S40 causes decreased mineralization and expression of bone markers. Only type I collagen shows similar expression pattern in both mutations, with decreased expression, secretion and matrix incorporation. Type XIV OI is a moderately severe form of OI which was identified in 2013. It is caused by recessive defects in TMEM38B, which encodes TRIC-B, an ER cation channel. We identified 3 probands with recessive null defects in TRIC-B and studied the function of TRIC-B deficiency in their fibroblasts and osteoblasts. TRIC-B deficiency impaired ER calcium flux, although the calcium channels themselves had normal stability. The impairment in calcium flux resulted in ER-stress along the ATF4 pathway. TRIC-deficiency was demonstrated to be collagen related because it impaired collagen synthesis and assembly at multiple steps. Collagen helical lysine hydroxylation was reduced, although the levels of LH1 protein were increased. Because calcium flux impacts multiple ER chaperones, the collagen was also misfolded and substantially retained in the ER. Further investigations will focus on type XIV osteoblast differentiation. This year we delineated the first X-linked recessive form of OI, made even more exciting by its novel bone mechanism. X-linked OI is moderately severe with pre- and post-natal fractures of ribs and long bones, dysplastic bone with bowing and crumpling. It is caused by missense mutations in MBTPS2, which encodes Site 2 protease (S2P). S2P is a critical component of Regulated Intramembrane Proteolysis (RIP), a process in which S1P and S2P located in the Golgi Membrane sequentially cleave regulatory proteins that are transported from the ER membrane in times of cell stress or sterol metabolite deficiency. The S2P substitutions in X-OI are located in or near the S2P motif critical for metal ion coordination. The levels of S2P transcripts and protein are normal but RIP function on substrates OASIS, ATF6 and SREBP are impaired. At the bone tissue level, hydroxylation of collagen K87 residues in type I collagen is reduced by half, altering collagen crosslinking in bone. The osteoblasts with S2P defects also have a differentiation defect.

Considerable controversy surrounds the question of whether the two different ytpes of asbestos -- amphilboles and chrysotile -- have different carcinogenic potencies. The "amphibole hypothesis" holds that chrysotile is less likely to cause lung cancer and mesothelioma than are the amphiboles. The abiolity to clarify this question is limited by a lack of quantitative data with which to estimate separate dose-response curves for the two fiber types. This proposal seeks funds to study lung cancer and mesothelimoma risks in a Sovenian cement asbestos plant in which excellent historical exposure data are available, distinguishing the two broad classes of asbestos fibers. The small Grant Investigator, an occupatinal physician and epidemiology doctoral student, would conduct a detailed historical exposure reconstruction and case control studies of lung cancer and mesothelimoma. The cohort of workers employed in the cement asbestos manufacturing plant of salonit Anhovo, Slovenia will be constructed from existing detailed wage lists which date from the 1940s. Cases will be all incident cases of primary lung cancer or mesothelioma from 1964 to 1994 in those who were hired after 1959 and who worked at least one month between 1964 and 1994. They will be identified using data from the slovenian National Cancer Registry, one of the oldest national registries in Europe. Exposure measurements are available for most exposed jobs begining in 1961. Three different methods of measurement were used: konimeter measuring particles/cm3, a gravimetric method measuring milligrams/m3, and membrane filter measuring fibers/cm3. Regression analyses will be used to calculate operation-specific conversion factors among these methods. Cumulative lifetime exposure to amphilboles and to chrysotile will be estimated sparately for all cases and controls. Logistic regression models will be used to estimate separate exposure-resonse curves for the two fiber types controlling for smoking (smoking history data available on each worker from medical records).

Binge alcoholism is a growing problem worldwide. Young adults (16-24 years of age) are particularly prone to the adverse effects of alcoholic binging, which is often consumed with secondary drugs of abuse including marijuana. Previous research has suggested that alcohol may share pharmacological effects with cannabinoid agonists such as D9-tetrahydrocannabinol (THC), the primary psychoactive substituent of marijuana. Further, previous work has found that these effects may be modulated by alcohol's effects on the endocannabinoid system. Although much of the preclinical work on both alcohol and cannabinoids has been conducted in male rodents, results of sparse studies in female rodents have suggested sex-selective differences, with females often exhibiting enhanced sensitivity to the pharmacological effects of ethanol as well as THC. In the parent grant, our goals are to characterize some of these differences for THC and to investigate potential mechanisms. In this supplement, we propose to incorporate examination of ethanol/ cannabinoid co-abuse into our overall research strategy. As with the parent grant, integrated in vivo and in vitro measures will be used. To take advantage of local expertise on the hepatic effects of alcohol, we have expanded the dual brain and behavior focus present in the parent grant to incorporate research on the effects of alcohol and THC on the liver. In the proposed project, we will investigate sex differences in the acute in vivo pharmacological effects of alcoho and THC co-administration, as well as their rewarding / aversive effects following repeated intermittent co-administration (Aim 1). These behavioral effects will be correlated with in vitro measures of endocannabinoid system functioning in the brain and the liver and with determination of the extent of liver injury induced by co-abuse of both substances (Aim 2). Understanding the sex-selective effects of co-abuse of alcohol and marijuana is particularly critical at this time since the recent loosening of legal restrictions on marijuana use in the U.S. will likely be associated with increased rates of alcohol-marijuana co-abuse in both sexes.

This application for a Midcareer Investigator Award in Patient Oriented Research (K24) seeks support for a physician nutrition specialist and established investigator in clinical and translational nutrition science. The P.I. is committed to performing patient-oriented research (POR), focused on nutrition, and in mentoring clinical residents, fellows and junior faculty in clinical nutrition research to develop a new cadre of investigators in Clinical Nutrition. This proposal will provide protected time to enable the P.I. to advance his research and mentoring efforts in nutrition-oriented research, and provide additional training and resources that will in turn enhance his ability to mentor physicians and other clinical investigators in nutrition-related studies. The applicant has a unique training background in nutrition and medicine and an excellent record of federal, foundation and industry funding for his investigator-initiated POR and translational research. He has a strong publication record and a history of leadership roles in national nutrition-related societies and service on editorial boards of front-line nutrition journals. At Emory, the P.I. serves in the leadership of the NIH-funded GCRC, K12 Emory Mentored Clinical Research Scholars (EMCRS) and K30 Masters of Science in Clinical Research (MSCR) programs, the Center for Clinical and Molecular Nutrition and the graduate program in Nutrition and Health Science - all of which provide opportunities for mentoring in POR. The P.I. has mentored more than 18 trainees, including a current cadre of 5 physician junior faculty members in Medicine, Surgery, Neurology and Pediatrics, 2 Ph.D. junior faculty clinical investigators, and 3 Ph.D. students who conduct both clinical and translational nutrition research. The candidate's research program includes GCRC-based studies on the efficacy and mechanisms of glutamine in catabolic states, including critical care and sickle cell anemia, on metabolic adaptation in short bowel syndrome and after medical/surgical weight loss, and studies on diet-related redox control. The specific aims of this K24 are: 1) to provide protected time enabling the P.I. to increase his mentoring activities among current and new trainees in POR; 2) to facilitate the P.I.'s development of new research skills in nutritional metabolomics and new redox-related measures within the context of his current clinical research, enabling him to train mentees in these new measures; and 3) to faciliate the career development of the applicant. [unreadable] [unreadable] [unreadable]

Project Summary The goal of this project is to focus on the relationship between dental use, overall health, comorbidity, and medical services use. The central hypothesis guiding this study is that dental care use is highly correlated with better-than-average general health and lower levels of comorbidity and utilization of medical services. We will test the hypothesis and conduct this study using secondary data available from the Medicare Beneficiary Survey (MCBS) and Chronic Conditions Warehouse (CCW), Area Health Resources File (AHRF) and the University of Michigan Institute for Social Research (ISR) Health and Retirement Study (HSR). We will also take advantage of a HRS dental specific module co-developed by members of this research team and the survey staff at the ISR. This proposed project builds upon and is an extension of several collaborative studies previously conducted by this research team. Results obtained from this study will have a high impact on the health and welfare of the Medicare eligible community population by describing which co-morbid states are associated with the presence of oral diseases, whether or not preventive treatments are associated with lower levels of those co-morbid states, and whether or not the use of corrective treatments are associated with amelioration of those co-morbid states in comparison to the frequency and severity of those co-morbid states among individuals whose oral diseases are not treated. Specifically we will: Aim 1. Estimate the relationship between utilization of dental care services by elderly persons and co- morbidities over time. The working hypothesis is that elderly persons not using dental care service are more likely to experience specific co-morbidities compared to those using dental services on a regular basis. Aim 2. Estimate the relationship between preventive dental care service use, oral health problems, and co- morbidities over time by elderly persons. The working hypothesis is that elderly persons with regular use of preventive dental care or without oral health care problems are less likely to experience specific co-morbidities over time than elderly persons not regularly using preventive dental care or with oral health care issues. Aim 3. Estimate the relationship between oral disease and co-morbidities over time for elderly persons. The working hypothesis is that elderly persons with oral disease are more likely over time to experience specific co- morbidities compared to elderly persons without oral disease.

The p53 tumor suppressor and master regulator is central to human DNA repair, damage checkpoints and many aspects of human biology. It controls cell fate in response to many types of internal and environmental stress. Importantly, most cancers are altered for p53 function. We have discovered a greatly expanded universe of p53 targets and human diversity as well as variations in responses to specific stresses. The DNA binding and transactivation of p53 is critical for tumor suppression. There is considerable variation in p53 dependent expression across 100s of targeted genes leading to differences in p53-mediated biological consequences. Much of that is due to target RE sequence. As described below the universe of genes subject to direct p53 control is much larger than originally anticipated based simply on previously established response element (RE) consensus target sequence, consisting of 2 copies of the sequenceRRRCA/TT/AGYYY (R, purine;Y, pyrimidine) separated by a spacer of up to 13 bases. There is considerable flexibility in what constitutes a functional RE. We focus on RE functionality, i.e., the ability of REs to support transactivation by p53. To directly assess transactivation responsiveness of human REs, we developed promoter systems in budding yeast for variable p53 expression (i.e., rheostatable). This has been used to establish the functional evolution of REs across species (discussed below) and has been summarized in our piano model that describes functional variability within a transcriptional network. We have translated many of the findings in yeast to p53 in human cells in culture and ex vivo. Recently, we established functionality of and sites. Also, increases in spacer length of only a few bases in a target RE greatly reduces functionality in terms of p53 transactivation. With Gilbert Schoenfelder (Berlin), we identified a novel mechanism of p53 transcriptional control of genes in studies of a SNP located in the promoter of the Flt1 gene. This C to T SNP results in generation of a perfect p53 -site RE and is present in 5% of the population. Our findings provided the first report of a functional p53 -site. Endogenous p53-dependent induction of FLT-1 mRNA was only observed in cell lines containing the FLT1-T allele in response to several DNA damaging agents. These results established that p53 can differentially stimulate transcription at a polymorphic variant of the FLT1 promoter and placed the VEGF system directly in the p53 stress-response transcriptional network via FLT1. We recently examined transactivation capacity towards a panel of 15 p53 -sites in p53-null human osterosarcoma cells (SaOS2) transiently co-transfected with a WT p53 vector. Some 1/2 site REs supported p53 transactivation to levels similar to a weakly responding full-site RE. This finding greatly expands the potential p53 master regulatory network. We are currently using p53 ChIP-seq approaches to identify targeted genes and networks, investigate crosstalk between regulatory networks especially other stress and DNA response networks. INTERACTION OF p53 AND ER REGULATORY NETWORKS. We identified a -site estrogen receptor RE (ERE) that greatly increased p53 induced transactivation at the FLT1 -site p53 RE, thereby establishing a new dimension to the p53 master regulatory network. Recently, we addressed the generality of synergistic transactivation by p53 and ER acting in cis. The 1/2 site in the FLT1 promoter was replaced with various 1/2 sites as well as canonical weak and strong human p53 REs. p53 transactivation was greatly enhanced by ligand-activated ER acting in cis. Enhanced transactivation extends to several cancer-associated p53 mutants with altered function, suggesting ER-dependent mutant p53 activity for at least some REs and possibilities for reactivation of cancer mutants. We propose a general synergistic relationship between the p53 family and ER master regulators in transactivation of p53 target canonical and noncanonical REs which might be poorly responsive to p53 on their own. We are developing a functional matrix as a tool for genome-wide searches for putative p53 target genes via noncanonical sites and augmenting transcriptional factors. In a limited search to identify motifs containing both strong p53 noncanonical RE sequences (including decamers with a CATG core) and associated ER responsive sequences (FLT1-like motifs), we identified the human RAP80 gene and Toll-like receptors (TLRs) that determine innate immunity. The identification of p53 target REs associated with the TLR genes has led to exciting new findings. Among the 10 human TLRs, nine had canonical and noncanonical p53 REs that may be functional. Using primary human cells obtained in a collaboration with the Clinical Research Unit we examined expression of the entire TLR gene family following exposure to anti-cancer p53 inducing agents. Expression of all TLR genes, TLR1-10, in blood lymphocytes and alveolar macrophages from healthy volunteers is inducible by DNA metabolic stressors. However, there is considerable inter-individual variability. Similarly p53 dependent TLR expression is detected in human cancer cell lines. For some TLRs the p53 control seems to enhance the inflammatory responses, mediated by activation of TLRs in the presence of natural ligands. Furthermore, a polymorphism in the TLR8 promoter provides the first human example of a p53 target RE sequence specifically responsible for endogenous gene induction. CANCER-ASSOCIATED P53 MUTANTS. Nearly all cancers have mutant or reduced expression of the p53 tumor suppressor gene. Using yeast-based and human cell systems, we show that functional p53 mutations can lead to considerable diversity in the spectrum of responses from REs including 1) decrease/loss-of-function;2) subtle changes;3) altered specificity;and 4) super-transactivation all of which lead to variation in biological responses. Most of the functional mutants (8/9) were able to function at 1/2 sites. We extended our analysis of p53 mutants in yeast-based systems to a clinical study of breast cancer patients undergoing neoadjuvant treatment. Several transcriptionally active p53 mutants identified in breast had subtle defects in transcription at REs that can only be revealed at low expression levels achievable in the yeast-based system. It appears that nonfunctional as compared with functional missense mutants are more likely to exhibit stage III progression at diagnosis, high grade cancers and to relapse with cancers at distant sites. P53 NETWORK EVOLUTION. We are investigating evolution of REs in terms of responsiveness to p53. Individual REs exhibited marked differences in potential transactivation as well as widespread turnover of functional REs during p53 network evolution. Only 1/3 of the REs found in humans are predicted to be functionally conserved in rodents. Surprisingly, the p53 responsiveness of the DNA repair/metabolism set of 15 p53 targeted genes in humans has evolved separately from mice suggesting differences in responsiveness to cancer-inducing agents between mice and humans. Importantly, we found functional conservation of weakly responding REs including 1/2 sites. Among validated p53 REs conserved between rodents and humans, one third were comprised of 1/2- or -sites, each with a perfect consensus -site suggesting a selective advantage in retaining weak p53 REs. Importantly, the integration of the TLR gene family into the p53 network also appears unique to primates.

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. The goal of this research continues to refine and improve the methodology of diffusion tensor imaging (DTI) at very high fields (3T and 7T) for the investigators and collaborators on the P41 RR09784 "Center for Advanced MR Technology at Stanford" effort. Preliminary studies from a variety of studies suggest that DTI may predict the cognitive and motor performances of a patient from scans requiring several minutes. Diffusion is measured along at least six non-collinear directions. For each gradient direction, typically four images were acquired and averaged. Two images with no diffusion weighting (b = 0s/mm2) are acquired and a set of Inversion Recovery (IR) images for CSF nulling are acquired with b = 0s/mm2;these images were used to unwarp the diffusion weighted images, which resulted in a more robust unwarping than using the non-IR b=0 images. The development of SENSE and more robust parallel imaging sequences and post-processing methods has accelerated the use of high fields for diffusion studies. Issues regarding the opimal protocol and set of sequence parameters needed for various diffusion studies are under investigation, which includes more rapid protocols for the assessment of stroke and more high resolution protocols for diffusion tensor imaging of white matter tracts. The effort is being translated from 3T to newer field field studies at 7T.

OBJECTIVE: To determine whether behavioral and hormonal responses to stress in marmosets are altered by social and reproductive status. RESULTS In human neuropsychiatric disorders, chronic alterations in baseline levels of the stress hormone cortisol are often associated with altered hormonal responses to stress. I have been investigating whether chronic suppression of baseline cortisol levels in socially subordinate, reproductively suppressed female marmosets are similarly associated with altered hormonal or behavioral responses to psychological stressors. Results indicate that psychological stressors, including restraint and exposure to novelty, cause dramatic elevations in circulating levels of both cortisol and the pituitary hormone adrenocorticotrophic hormone (ACTH), as expected, and that the magnitude of the endocrine stress response increases with increasing stressor intensity. Preliminary analyses, however, do not support the hypothesis that individual differences in baseline hormone levels, associated with differences in reproductive and social status, are accompanied by differences in the hormonal response to stress. If confirmed, these results woud indicate that the neuroendocrine mechanisms mediating the endocrine stress-response, unlike those controlling baseline cortisol levels, are not highly responsive to social and reproductive factors. FUTURE DIRECTIONS In the coming months, I will complete the analysis of the hormonal and behavioral stress esponses to either confirm or disprove the hypothesis that suppression of baseline plasma cortisol in socially subordinate female marmosets is associated with altered endocrine and behavioral responses to stressors. KEY WORDS dominance, subordination, ovarian cycle, adrenal cortex, adrenocorticotrophic hormone, glucocorticoids. stress

That early postnatal experience may have long-term repercussions has been demonstrated for visual, auditory, tactile and chemical sensory systems. Multiple studies employing abnormal stimulation of these systems shortly after birth revealed significant alterations in their functionality in the adult individuals. These studies also pointed to three types of most-frequently observed early experience-induced alterations in the sensory circuitry which are likely to underlie the aberrations in the functionality of sensory systems in animals subjected to abnormal early experience; (i) alterations in connectivity of the neuronal processes, detectable as changes in the density and distribution of projecting sensory fibers; (ii) alterations in neurochemical organization of the circuitry, detectable as changes in the density and distribution of specific neurotransmitter and receptor-expressing cells as well as modifications in the levels of expression of neurotransmitters and receptors in the relevant centers of there central nervous system; and (iii) alterations in receptive field size and/or stimulus-response properties of neurons within the sensory circuit, detectable as changes in cells electrophysiological responses to appropriate sensory stimulation. Nociceptive circuitry undergoes significant postnatal maturation, and thus should also be vulnerable to modulations by early pain-associated experience. Indeed, recent studies demonstrated that exposure to pain-associated events early in life could induce long-term alterations in responses to pain by the affected individuals. Furthermore, experiments conducted in newborn rats have demonstrated that even a single local inflammation of a handpaw lasting for approximately 24 hours can result in significant changes in future withdrawal responses to noxious stimulation in the affected animals. However, the specific parameters of the "window(s) of vulnerability" of the developing nociceptive circuitry to such short- lasting inflammation, as well as long-term changes in nociceptive circuitry underlying the altered nociceptive behaviors, have not been well investigated. It is unknown how global are the long term-effects produced by local inflammatory insult. Therefore, in this project: I. We will characterize the early postnatal sensitive period(s) of nociceptive circuitry to modulation by short-lasting inflammation; II. We will test the hypothesis that short-lasting early local inflammation is capable of inducing long-term alterations in morphological, biochemical and electrophysiological properties of the peripheral and spinal nociceptive circuitry serving the affected peripheral region (this specific aim will be performed in conjunction with Project 3, which focuses on the ability of early local inflammation to affect ion channels and electrophysiological properties in cells of the dorsal root ganglia); and III. Together with Projects 1 and 4, we will test the proposition that early local inflammatory insults may have a global long-term effect on nocifensive responses of the organism and that this global effect may be associated with alterations in the descending modulatory circuitry.

The goal of this proposal is the characterization of a locus that confers resistance to mammary and intestinal tumor development. These studies will provide insight into factors that control the complex process of tumor development. The identification of genes that affect tumor development will aid in our ability to design prevention or treatment strategies. Mouse models provide an excellent system with which to identify genes that affect cancer development. ApcMin/+ mice are predisposed to develop intestinal and mammary tumors. Gtrosa26 mice carry a retroviral gene-trap insertion of LacZ-neoR on chromosome 6. These mice express the fusion protein ubiquitously and are thus a useful tool in chimeric analyses. We have found that when ApcMin/+ mice also carry the Gtrosa26 insertion, they are resistant to mammary tumor development and the intestinal tumors are reduced in size, but not number. The first aim of this proposal is to test two hypotheses for the molecular mechanism of resistance to tumor development in mice carrying the Gtrosa26 insertion. One is that the resistance is a function of the high levels of expression of the beta-galactosidase-neomycin resistance fusion protein that is encoded by the insertion. The second hypothesis is that the disruption of the expression of two non-coding transcripts at the insertion site is the cause of the resistance. In the second aim, we propose experiments that explore the mode of action of the insertion. Specifically we will test for an effect of the insertion in other mammary tumor models. This will test whether the effect is a general effect on tumor development or is specific to the Apc pathway of tumor development. Second, we will test whether the Gtrosa26 insertion acts in a tissue autonomous manner. This information will be vital in the understanding of how the insertion exerts its effects.

The overall objective of this research effort is to identify mechanisms which may be responsible for or contribute to differences in disease severity among patients with bronchial asthma. The research effort considers both immunologic mechanisms. A lymphocyte beta-adrenergic receptor defect has been demonstrated to be present among some patients with bronchial asthma. The magnitude of the receptor abnormality, as determined by specific dihydroalprenolol binding assays, appears related to disease severity and degree of airway obstruction. Drug consumption and documentation was noted on all patients studied. A significant relationship was shown between quantitation of airway reactivity by methacholine challenge and disease severity score. Higher disease severity scores also correlated with urinary norephinephrine and dopamine excretion. The results suggest that a major determinant of disease severity among patients with bronchial asthma is differences in nonspecific airway hyperreactivity among individuals. Abnormalities of beta-adrenergic receptors in conjunction with alpha-adrenergic hyperresponsiveness contribute to disease severity. The importance of adrenergic factors in disease severity may have important clinical and therapeutic implications.

The primate visual cortex uniquely possesses a regular array of metabolically active, cytochrome oxidase (C.O.)-rich zones (blots or puffs) in the supragranular layers with distinct physiological properties, particularly those related to color processing. Our previous studies indicate that unilateral retinal impulse blockage in the adult severely affects the most metabolically active neurons and induces synaptic reorganization within the puffs. These findings suggest that the mature visual cortex is not static but, rather, responds dynamically to altered functional demands. Besides changes in puffs, our preliminary light microscopic analysis of surrounding C.O.-poor interpuff regions indicate compensatory increases in C.O. levels within zones related to the non-treated eye. Such dynamic changes in the adult are of obvious clinical and functional importance, consequently our initial ultrastructural and quantitative analyses of the puffs will be extended to the interpuff regions. Selective vulnerability of the most metabolically active neurons deserves further investigation, since it appears to implicate a specific neurotransmitter type, GABA. Whether all puff neurons with intense C.O. activity are GABAergic, or whether GABAergic neurons encompass a wide range of oxidative metabolic capacities, will be examined by means of combined C.O. histo- or immunohisto-chemistry and GABA-immunohistochemistry on the same histological section. To directly address the relationship of C.O. levels to physiological activity and visual processing, single neurons will be recorded extracellularly from C.O.-rich and poor zones in normal cortex and during periods of physiological modification: 1) following monocular retinal impulse blockage in the adult and 2) during the critical period of postnatal development, when the innately determined cortical organization undergoes further physiological maturation. To understand the anatomical basis of these changes during development and specifically to explore the dynamics of maturational plasticity, we will examine structural reorganization in developing visual cortical neurons following retinal blockade. In summary, our approach is to combine histochemical, immunohistochemical, ultrastructural, and physiological observations to yield an integrated understanding of metabolic adjustments to altered functional demands within developing and mature neurons of the primary visual cortex.

Chromatin structure is critical to the regulation of eukaryotic gene expression. The basic unit of chromatin, the nucleosome, regulates transcription by limiting the accessibility of transcriptional factors to specific DNA sequences within promoters. A better biochemical understanding of how eukaryotic genes are regulated will require reconstituted transcription in the context of genomic DNA packaged with properly organized nucleosomes. Our lab recently used the latest in DNA sequencing technology to show that the in vivo pattern of nucleosome positioning can be recapitulated in vitro across the S. cerevisiae genome by chromatin remodelers. Here we propose to develop a genome- wide immobilized template assay that will allow us to ask fundamental questions regarding nucleosome dynamics, transcription factor occupancy, and RNA transcription on a genome-wide scale in the context of proper chromatin structure. With this assay aspects of gene expression regulation will be investigated in regards to transcription factor binding and the transcriptional requirements of histone modifications. These studies are intended to shed light on two fundamental questions in gene regulation: Can transcription factors intrinsically access their binding sites in native chromatin, or is the cooperation of remodelers needed? Does the physiological pattern of active histone modifications arise before or after transcription occurs? The development of this assay will lead to a technique in which this in vitro system will be programmed to contain specific aberrant chromatin states as are seen in disease states in order to determine how these changes in chromatin structure lead to the development of cancer, along with cardiovascular, neurodegenerative, and autoimmune diseases. PUBLIC HEALTH RELEVANCE: Chromatin structure is critical to the regulation of eukaryotic gene expression, and alterations of chromatin structure are known to cause aberrant regulation of gene expression that can lead to cancer. Here we are proposing to develop a biochemical assay in yeast that will later be used to study the aberrant regulation of gene expression caused by specific alterations of chromatin structure to understand how it leads to the development of cancer.

This Unit investigates the mechanism of action of excitatory amino acids as synaptic transmitters and neuromodulators in the vertebrate CNS, utilizing cell culture and electrophysiological techniques. Substantial progress has been made in developing a fast perfusion system for applying drugs and ions to nerve cells, and this is now used routinely. The divalent cations, zinc and cadmium, have two major effects on hippocampal neurons: (1) block of excitatory responses to NMDA receptors (Zinc Kd = 13 micromolars); (2) an increase in due to block of postsynaptic inhibitory GABA receptors (Zinc Kd = 11 micromolars), and zinc suppression of a transient potassium current, which normally slows repetitive firing. Zinc block of NMDA responses is reduced on raising the extracellular calcium concentration suggesting competition between zinc and calcium or screening of the zinc binding site by calcium. Low concentrations of zinc (50 micromolars) also potentiate responses to kainate, quisqualate, and response to glutamate at non-NMDA receptors. Fast application of excitatory amino acids produces three patterns of response: fast (tau = 20 ms) desensitization of quisqualate receptors: slow (tau = 200 ms) desensitization of NMDA receptors, sustained activation of kainate receptors. Concanavalin-A, which binds to glycoproteins, reduces desensitization at quisqualate but not NMDA receptors, and does not alter responses to kainate. Low concentrations of L- glutamate @ 1 micromolar) were found to depress excitatory synaptic transmission via activation of a novel presynaptic receptor: L-AP4 mimics this effect. Neither agonist produces a substantial postsynaptic response in glycine free medium. Culture medium is conditioned by substances secreted into the extracellular space, including L-glutamate, which tonically inhibits synaptic transmission; on the other hand neuronal survival in F-12 medium reflects activity of a glial sink for neurotoxic amino acids, which rapidly reduces the L-glutamate concentration from 100 less than 10 micromolars.

Methamphetamine is one of the most growing drugs of abuse, causing neurological impairments, and associated with behaviors that favor exposure to HIV, a virus that penetrates the CNS leading to neuroAIDS. Meth users infected with HIV usually present a more severe form of neuroAIDS, due to effects on neurons. IT is known that free radicals participate in the enhancement of brain pathology, but the cell types as well as pathways affected by Meth that lead to free-radical mediated stress. The hypothesis behind this proposal is that Meth interacts with cell types other than neurons will be explored, particularly regarding its potential to induce and/or modulate reactive oxygen and nitrogen intermediates. We will investigate the action of methamphetamine (Meth) on oxidative metabolism, directly on macrophages, and on other cell types in vitro, as well as in vivo, in conditions of co-morbid factor interaction. For that we will examine the action of Meth on cell lines, by investigating how the drug affects the intensity and quality of the oxidative burst, and searching for genes within ROI and RNI pathways that exhibit transcriptional pattern changes upon Meth exposure. Such transcriptional changes will be validated by looking for similar changes in cells obtained from Meth-treated animals infected with SIV, which have been previously characterized for CNS disease parameters. The results from this project will offer important clues on the participation of Meth in pathology mediated by oxidative stress by acting on ROI and RNI induction pathways, not only in the Central Nervous System, but also in peripheral organs. PUBLIC HEALTH RELEVANCE: We will investigate the action of methamphetamine (Meth) on oxidative metabolism, directly on macrophages, and on other cell types in vitro, as well as in vivo in conditions of co-morbid factor interaction. The hypothesis behind this proposal is that Meth affects the intensity and quality of the oxidative burst by acting directly on macrophages and/or glial cells, in addition to its action on neurons, contributing to the development of pathology mediated by oxidative stress, not only in the Central Nervous System, but also in peripheral organs. Initially, we will focus on the effect of meth on various human and mouse macrophage (THP1, RAW264.7), microglia (MG5, EOC 2), astrocyte (C8-D1A, CCF-STTG1), and neuronal (SH-SY5Y, SK- N-MC) cell lines, regarding the production of O2_, H2O2, and other reactive oxygen intermediates (ROI), as well as Reactive Nitrogen Intermediates (RNI) using various chromogenic methods. Mitochondria activity upon interaction with meth will be also addressed on cells stimulated with Meth. Following a characterization of the different cell lines regarding production of free radicals and mitochondrial changes in reaction to Meth, we will choose good responders to investigate transcriptional changes on molecules within ROI and RNI pathways, caused by action of Meth. For that we will use multiplex technology, as well as enzymatic checkpoint inhibition strategies. These approaches will help dissect the molecular basis of respiratory burst induction by Meth, and will distinguish whether Meth interferes over mitochodria, peroxisome, or both. Following the in vitro approach using cell lines, we intend to validate findings in the context of pathology. We will use cryopreserved cells and tissues from monkeys infected with SIV (as a model for HIV infection, a common co-morbid condition) subjected to a Meth treatment schedule. We will identify transcriptionally altered oxidative pathways on cryopreserved brain and peripheral tissues derived from SIV-infected Meth-treated monkeys by qRT-PCR. This brings relevance to pathology in humans. The present proposal will identify the basis of oxidative stress in the brain and other organs in Meth users. Meth is one of the most growing street drugs of abuse, exposing users to HIV infection, and potentially facilitating the penetration of virus across the blood brain barrier (BBB) into the brain. In this proposal we will dissect mechanisms by which Meth can induce and modulate free radical production, causing an aggravation on the severity of damage associated with virus presence in the brain tissue, and consequently aggravate AIDS-associated CNS dysfunctions. The results obtained in this proposal will allow the generation of data on direct effects of the drug on cells, relative susceptibility of different cell types to direct actions, and on pathways, aiming a future R01 application on modulation of oxidative stress in Meth abuse in vivo. These will lead to the development of important therapeutic tools for rehabilitating individuals, especially patients presenting co-morbid factors, such as HIV infection.

The overall goal of this project is to understand the systems-level neural mechanisms by which general anesthetics suppress consciousness and allow its return during emergence in the human brain. Our fundamental hypothesis is that consciousness emerges from brain function as a network phenomenon, and that general anesthetics suppress consciousness by disrupting the communication across large-scale neuronal networks that support information integration in the brain. Our previous findings suggest that anesthetics alter the functional connectivity of thalamocortical systems, particularly in the nonspecific thalamocortical division, as well as in other intrinsic cortical cognitive networks. Here, we build on our decade-long developments in blood-oxygen level-dependent (BOLD) functional MRI (fMRI) and resting-state functional connectivity MRI (R- fcMRI) methods with high spatial and temporal resolution applied to test specific hypotheses about the anesthetic modulation of cognitive functioning, network organization, reorganization, and information integration during wakefulness and at graded levels of suppressed consciousness. In Specific Aim 1, we will employ concurrent behavioral and BOLD fMRI assessment of semantic discrimination to test the hypotheses that deepening anesthesia with propofol will suppress conscious awareness by diminishing integrative functional networks of the brain in a graded, top-down manner, suppressing the most complex systems first and the simplest systems last and that these changes can be characterized by the anesthetics' effect on the behavioral response and neural activity to a series of tasks that depend on different levels of information integration. In Specific Aim 2, we will test the hypotheses that propofol confers differential changes in resting- state (baseline) functional connectivity, modularity and network integration in thalamocortical and cortical intrinsic networks, particularly those involved with attention, executive control, and salience, vs. others, such as the default mode and sensory networks. Finally, in Specific Aim 3, we will test the hypotheses that anesthetic- induced loss and subsequent return of consciousness are mediated in part by different neural mechanisms that show prior state dependency, as reflected by various brain network interaction measures and that the restoration of consciousness from anesthesia requires additional neural resources over those required for the maintenance of consciousness, reflecting the reconfiguration capability of the brain as a self-organizing system for resource management and functional resilience. The proposed work should advance our understanding of the neural mechanisms of anesthesia with respect to its effect on human consciousness at an integrative level. The work should reveal the order in which cognitive functions are lost during sedation and anesthesia and the degree of residual cognitive functions based on direct, noninvasive detection of neural events. It should illuminate how anesthetics alter resting-state intrinsic brain networks and how the latter may reconfigure to cope with anesthetic challenge. Novel quantitative approaches to assess residual cognitive functions by fMRI network analysis may eventually be extendable to neurological patients with disordered consciousness. In a wider context, the findings should facilitate our understanding of the scientific basis of human consciousness, including its aspects for sensory awareness and voluntary action.

We propose to continue the study of glutathione synthesis and metabolism in the lens in which we have already made considerable progress. The maintenance of a high level of reduced glutathione is recognized as essential in the prevention of certain types of cataract formation and progression. Utilizing procedures already established in this laboratory for organ lens culture, enzyme purification, assay, and kinetic studies, we plan 1) to investigate certain key enzymes in glutathione synthesis and metabolism, especially in relation to aging and safe use of pharmaceuticals. These enzymes include coenzyme A-acetyl transferase, glutathione S-transferase and the mercapturic acid pathway, and the first three enzymes in the transformation of methionine to cysteine. 2) To determine the amount of free and bound glutathione in situ in both aging and cataractous lenses by means of NMR spectroscopy; and the concentrations of ADP and substrates of glutathione synthesis in the aging human lens by HPLC or the amino acid analyzer. 3) To establish the roles of glutathione peroxidase and catalase in the prevention of oxidative damage in the organ cultured lens. 4) To study the effect of both reduced and oxidized glutathione on Na-K-ATPase. 5) To examine the possible role of a glutathione analogue, arising from homocysteine, in patients suffering from homocystinuria. These studies will further expand our knowledge of glutathione and its role in the maintenance of a clear lens.

This research project consists of experiments on the induction of rhabdomyosarcomas in rats. The two stage model of tumor induction is used. The initiating agent is a subcarcinogenic dose (0.4 mg) of nickel subsulfide (Ni3S2) injected into the rectus abdominis muscle; the promoting treatment consists of three slight compressions made at weekly intervals, of the area infiltrated with the carcinogen. It is known that a single muscle compression induces promptly proliferation of mononucleated cells located at a short distance from the site of compression. Cell division is considered to be vital importance for neoplastic transformation. The use of the two stage method makes it possible to schedule tissue removal in concomitance with cellular mitotic activity. Study with the electron microscope will lead to establish whether the cells in divisions are cells that have cleaved from injured multinucleated muscle fibers or are satellite cells. Information will also be gained on the role of the dividing cells in tumor formation.

This application is being submitted to continue the University of Pennsylvania's role as a main institution in the Eastern Cooperative Oncology Group beyond its 37 years of continuous particpation. The overarching objectives are to continue to serve as a scientific and administrative resource for the Group, to enter patients onto ECOG protocols, and to coordinate and support a large affiliate network. In the latter role, this application will support the diffusion of knowledge of clinical trials, assist in the proper conduct of clinical trials, and make these trials accessible to the broadest possible population of patients with cancer. Although the University of Pennsylvania has had an outstanding track record in all aspects of cooperative group activities, including accrual, administrative and scientific input and data quality, the specific aims of the proposal for the next six years are to: 1. continue to enhance accrual in all disease site and modalities, beyong our current particpation, particularly in breast and lung cancers. 2. increase physician, nurse, and data management participation in Group activities, including protocol development. 3. attract new affilaites through the Abramson Cancer Center network, allowing for enhanced availability for clinical trials participation in the larger community The relevance of this project to the ultimate cure of cancer is self-evident. Cooperative groups are the main force outside of the pharmaceutical industry dedicated to the definitve tesing of new treatments, studying not just new drug development but also non-drug treatment, such as radiation therapy and surgery, for which there would be nooter funding. These cooperative group activities, and the specific involvement of the University of Pennsylvania, represent the chief way in which the ultimate model of multimodal, collaborative patient care can be performed. The main ECOG grant describes in detail the very specific way in which clinical trials have led to RELEVANCE: Malignancies remain an enormous burden for individuals in the United States and for society as a whole. Without public support of clinical trials, both by financial support as well as participation in clinical trials, progress will not be made. If left in private hands, large areas of cancer treatment will go unexplored, denying patients the possibility of progress.

Abstract Endosomes, the membrane compartments inside living cells, are increasingly recognized as discrete ?hubs? that regulate the network of cell signaling circuits in space and time. Physical phenomena like the clustering of proteins on endosome membranes and the active transport of endosomes are hypothesized to play a key role in these regulatory mechanisms. Unfortunately however, the direct evidence needed to support this level of mechanistic understanding of endosome signaling is lacking. Over the last four years, the research program of my lab has focused on exploring the wealth of physical phenomena involved in the endocytic process. We have uncovered new physical mechanisms of endocytosis in immune cells, but the questions we pose are of general relevance to many kinds of cells. We aim to test the general hypothesis that endosomes are a specialized platform for the spatiotemporal regulation of cellular signal transduction. This proposal highlights two of our developing project areas that are designed to test this hypothesis by identifying the biophysical mechanisms of endosome signaling regulation. Both are enabled by our established biophysical tools that allow us to manipulate and analyze the signaling activities and dynamics of endosomes in living cells. One research direction focuses on the mechanisms of signaling crosstalk on endosome membranes. Our ultimate goal for this research direction is to identify mechanisms by which physical interactions between endosomal receptors lead to their signaling crosstalk. By developing a novel approach that physically manipulates interactions between receptors on endosome membranes, we will establish the quantitative relationship between receptor clusters on endosomes, their signaling crosstalk, and the end-point cell response. The other research project addresses the functional roles of endosome trafficking in signaling. Our ultimate goal for this second research direction is to determine mechanisms under which the transport and subcellular location of endosomes regulate their signaling functions. By developing a particle reporter system that will allow us to magnetically control trafficking of single endosomes and simultaneously detect their signaling activities, we will reveal direct connection between the dynamical, mechanical and biochemical activities of individual endosomes. The proposed research directions are enabled by the novel integration of nanomaterial engineering, quantitative physical measurements, and advanced optical techniques, with live cell experiments. In the long term, we will expand our research scope from endosome signaling in immune cells to that in other cell types. Our ongoing and future research directions share the overarching goal of establishing a quantitative understanding of endosome signaling in living cells.

This training grant proposal is entitled The cellular and molecular foundations of biomedical sciences. It is a renewal of a successful program started six years ago and seeks to train graduate students in the broad fundamentals of biomedical sciences to provide them with the tools to understand important problems in modern biology and health. This is accomplished initially through a two semester Core course covering Genetics, Genomics and Bioinformatics, Eukaryotic molecular biology, Molecular biophysics, Protein structure and function, and Cell biology. Students follow this with electives in specific fields of their interest. This interdepartmental program is based in the Department of Biological Sciences, but has been expanded to include established faculty from Columbia's College of Physicians and Surgeons. Students undertake rotations in up to three laboratories and can do their thesis work in any of the 47 faculty in the program, roughly half of which are on the Medical School campus. This has provided expanded opportunities for training of the students in outstanding laboratories and has fostered collaboration between faculty and students at Columbia's Medical School and main campuses. The faculty members work in a broad range of fields including structural biology, molecular biology, genetics, bioinformatics and systems biology, developmental biology and neurobiology. Experimental systems are also broad including bacteria, yeast, drosophila, C. elegans, frogs and mice. Human tissue culture systems are also utilized and most systems consider problems relevant to human health and physiology. Twelve graduate student traineeships are requested per year to support six students for two years each, usually in their third and fourth years. This will fund 19% of our current training grant eligible student pool of 63. The best students in our program will be chosen to be funded by this grant. This will be determined by their performance in the Core and other courses, recommendations from their rotation and thesis sponsors, and a short research proposal reviewed by the grant's steering committee. The qualifications of the faculty and the research facilities at Columbia are outstanding and provides a superb training environment for the students. RELEVANCE: The training of the next generation of biomedical scientists is essential to continued excellence of research in the U.S. and advances in understanding, controlling and curing human disease. Laboratories in the program address specific basic problems in biomedical sciences, many with direct applications to human disease.

Flow Cytometry Project Summary/Abstract The primary purpose of the Flow Cytometry Module is to provide Principal Investigators with a centralized service for cell phenotyping, sorting (FACS) to obtain viable populations of cells according to specific cellular markers, and biomarker detection. Such a service makes expensive equipment for fluorescent activated cell sorting and analysis available to researchers. In addition, the module helps develop experimental procedures and assists with analysis as well as the interpretation of results. Finally, the module provides full time technical support for the maintenance and calibration of the equipment, a centralized internet access to data and a database for techniques.

Reduction of racial and ethnic disparities in cancer incidence, stage of presentation and outcome requires resilient strategies that link medically underserved communities into the continuum of care. Community- based participatory research has the potential to generate such strategies, through processes of collaborative planning, experimentation and discovery. The proposed Queens Library Health Link program is designed to test the efficacy of a comprehensive participatory research approach to reduce disparities by improving use of cancer prevention, screening and treatments interventions. In this project, public library branches provide a base for neighborhood organizing, education, data gathering, planning and implementation of local health promotion experiments and dissemination of results. We will work with 42 of the 63 branch libraries in Queens, each serving its own large, diverse segment of the Queens community. Although we will follow the same process consultation protocol, every community will undertake unique health promotion activities most suited to local priorities, cultures, preferences and opportunities. This project is designed to address the following aims: (1) To determine whether community-based participatory action research (PAR) leads to more effective cancer prevention and control in underserved communities compared to standard, "top-down" programs; (2) To examine how communities build upon and sustain locally-developed initiatives for cancer prevention and control; (3) To compare whether cancer prevention and control strategies developed through PAR are more widely accepted and more efficacious than standard strategies when they are transferred to new communities. This project will be carried out in two phases. In phase 1, we compare health behavioral outcomes of community-tailored strategies to address cancer needs with top-down alternatives. In phase 2, we examine the sustainability and dissemination of tailored strategies. Queens neighborhoods will be randomized to serve as settings for program development or dissemination. Data will be gathered from multiple sources to examine the participatory processes as well as behavioral outcomes at the individual, library and community levels. Core organizations involved in this project comprise a four-way partnership including the Queens Borough Public Library System, the Queens Cancer Center, the American Cancer Society of Queens, and Memorial Sloan-Kettering Cancer Center.

The goal of this research is to define the relative roles of intra-and extra-mitochondrial genetic systems in determining cellular and mitochondrial phenotype. Major emphasis will be placed on analyzing the regulation of mitochondrial biogenesis. We propose to apply both genetic and biochemical techniques to this end. In this project attention will be focussed on the biosynthesis of heme and its relationships to the formation of cytochromes. In addition, a comprehensive study will be made of the role of copper in regulation of heme formation and cytochrome aa3 biogenesis. Three projects are proposed. In the first, the mechanism by which delta-aminolevulinate (Alv) synthase is transported into mitochondria will be studied. Evidence for specific membrane receptors and pilot sequences on cytoplasmic Alv synthase will be sought employing specific mutants. In the second, a mutant which over-produces cytochromes will be characterized to determine whether the primary defect involves alteration in heme production or in a general regulator of apocytochrome formation. The third project will examine the mutual interactions between copper metabolism, heme biosynthesis and cytochrome formation. This study will include a characterization of copper transport, copper proteins and the regulation of copper metabolism as it pertains to mitochondrial biogenesis.

Development of an effective therapy against post-exposure Anthrax. Anthrax is an infectious disease caused by the bacterium Bacillus anthracis. This rod shaped bacterium infects humans through the respiratory system, skin, or digestive tract. Dependent upon the entry route into the human body, Anthrax can be highly lethal. Although cutaneous Anthrax is rarely lethal, inhalation Anthrax is dangerous and usually fatal. Upon inhalation, the Anthrax spores adhere to the alveolar macrophages and germinate. Bacteria migrate to the lymph node, in which they rapidly multiply and excrete a tripartite exotoxin comprised of protective antigen (PA), lethal factor (LF, a Zn2+ dependant metalloproteinase) and calmodulin-activated edema factor adenylate cyclase (EF). The combined actions of these proteins constitute the Anthrax toxin which induces cell death particularly in macrophages by the action of LF. Once in the cytosol, LF is able to cleave several members of the MAPKK family near their N-terminus preventing interaction with, and phosphorylation of, downstream MAPKs, thereby inhibiting one or more signaling pathways. High liter of the bacteria and concomitant high level of LF in the blood stream will lead to the death of the host organism. Therefore, the developments of treatments that include a combination of antibiotics with orally active, potent, safe and selective LF inhibitors should provide an efficacious countermeasure to post-exposure Anthrax. Our three specific aims are: (I) To increase, through iterative optimizations selectivity, potency and drug-likeness of selected LF inhibitors. (II) To perform in vitro and in cell toxicity studies to refine hits and identify drug-like inhibitor(s) of LF for subsequent in vivo studies. (Ill) To perform in vivo efficacy evaluations and necessary formulation, PK and toxicity studies. Relevance Inhalation Anthrax is the most deadly form of this disease. After infection, Anthrax spores are engulfed by alveolar macrophages where germinate to generate the bacteria, which spread through the lymph nodes to the bloodstream, eventually leading to systemic fatal shock presumably due to the action of the secreted Anthrax toxin. LF (lethal factor) is a key component of the secreted toxin and plays a major role in inducing apoptosis (cell-death) of macrophages by cleaving the specific human cell signaling proteins. Our plan is to develop potent, safe, selective and orally active compounds that inhibit LF, which should prevent macrophage cell-death and promote termination of the disease. Such inhibitors will be tested as single agents and in combination with antibiotics, in order to develop the most efficacious post-exposure therapy that should facilitate and assure the recovery of patients who harbor the disease.

Summary: Despite their high prevalence, the molecular basis for common genitourinary (GU) congenital defects is poorly understood. We have identified and validated gene lesions at the KCTD13/16p11.2 locus as responsible for lower GU tract abnormalities, particularly cryptorchidism and hypospadias. KCTD13 encodes a substrate-specific adapter of a BCR (BTB-CUL3-RBX1) E3 ubiquitin-protein ligase complex, which regulates the actin cytoskeleton and cell migration via ubiquitination and degradation of RHOA. Notably, RHOA has been implicated in the maintenance of Sertoli-germ cell junctions to promote gametogenesis and gonads development. E3 ubiquitin ligases also regulate the androgen receptor (AR), which is key to male genitalia development and differentiation. We found that KCTD13 is robustly expressed in the GU tract and have recently shown that in comparison to normal controls, gene copy number variants are remarkably common in patients with GU birth defects. Moreover, we obtained in vitro and in vivo evidence that KCTD13 loss affects AR levels in testis and penis. We also observed that haploinsufficient and Kctd13 deficient mice had significantly higher incidence of cryptorchidism and decreased size of testes, seminal vesicles and penis size in conjunction with spermatogenic defects, causing severe subfertility. Further, Kctd13 null mice revealed a significant decrease in masculinization factor SOX9 levels and concomitant upregulation of the feminization factor RHOA. While our collective data suggest a key role of KCTD13 in male GU development, the mechanisms of how this molecule impinges upon the AR and/or SOX9 axes remain a major gap of knowledge. We hypothesize that gene dosage changes in KCTD13 alter the signaling of the masculinization axes, leading to abnormal GU tract development, defective gonad formation, undervirilization, and subfertility. In line with our reasoning, we will test two plausible independent mechanisms by which KCTD13 mediates lower GU tract development and differentiation. First, we hypothesize and test that gene dosage changes in KCTD13 affect GU tract development by affecting AR degradation, subcellular localization and downstream gene targets (AIM 1). Second, we hypothesize and test that KCTD13 affects the expression of masculinization factor SOX9 directly by modulating SOX9 ubiquitination or indirectly by regulating RHOA degradation, which in turn leads to defects in testis and penile development and differentiation (AIM 2). Finally, we identify and characterize the mutations in KCTD13- CUL3 pathway that are critical in GU development such that they could be used to generate a diagnostic kit for patients with disorders of sexual development (AIM3). Completion of the studies in this proposal will advance our understanding of the molecular mechanisms that underlie common GU birth defects.

Approximately 34 million people are infected with human immunodeficiency type-1 (HIV-1) world-wide. More than two million new infections occur annually (1,2). These statistics could be reduced if at-risk individuals were provided antiretrovirl drugs for HIV pre-exposure prophylaxis (PrEP). We propose to develop a combination nano-microbicide containing cellulose acetate phthalate (CAP) as a HIV-1 entry inhibitor with dolutegravir (DTG), a HIV-1 integrase strand transfer inhibitor, for highly effective PrEP. Our preliminary data show that novel CAP-DTG combination nano-microbicide reduce cytotoxicity of DTG to cells. In vitro HIV prophylaxis using HIV indicator TZM-bl cells show that CAP-DTG combination nano-microbicide offers significantly higher protection from HIV-1 infection as compared to DTG solution and CAP nanoparticles indicating the importance of combination nano-microbicide. This proposal will extend our preliminary experiments and evaluate CAP-DTG nano-microbicide in thermosentive gel and DTG solution based conventional gel for cytotoxicity, intra-cellular delivery of DTG, in vitro HIV PrEP, and vaginal pharmacokinetics. In order to reach this goal, Specific Aim 1 focuses on development of DTG solution based conventional gel (DTG-gel), optimization and characterization of CAP-DTG combination nanomicrobicide for DTG loading, and incorporation of CAP-DTG combination nanomicrobicide into a thermosensitive gel (CAP-DTG-NPs-Gel) for vaginal delivery. Chemical stability of DTG in conventional gel and CAP-DTG-NPs-Gel will also be assessed. Specific Aim 2 employs various cell lines and primary human cells to evaluate DTG-gel and CAP- NPs-DTG-Gel for cytotoxicity and intra-cellular delivery of DTG. The ability of DTG-gel and CAP-NPs-DTG-Gel to offer protection from HIV-1 infection will be tested in TZM-bl cells, PBMCs, and MDMs. We will evaluate vaginal pharmacokinetics of DTG-gel and CAP-DTG-NPs-Gel using female NOD SCID gamma (NSG) mice for up to 7 days. Drug dissemination will be determined using HPLC analysis to measure DTG levels in dissected tissue. We will evaluate tolerability of DTG-gel and CAP-DTG-NPs-Gel after repeated administration for 7 days by analyzing inflammatory cytokines in cervicovaginal fluid and immunohistochemical analysis of NSG tissue. As a laboratory within an undergraduate biology department with collaborators in attached professional schools, we are uniquely situated to expose undergraduates to state-of-the-art nanoparticle research. This project will incorporate education of undergraduate students in our investigation of the utility of combination CAP and DTG nano-microbicide in a thermosensitive gel delivery system as cost-effect, highly efficacious modality for HIV pre-exposure prophylaxis.

This study will compare the efficacy and safety of two doses of a new inhaled corticosteroid, fluticasone proprionate (FP) usinf one of two delivery systems. Asthma is a potentially debilitating disease in children, likely accounting.

Neuroendocrine (NE) tumors such as carcinoid, islet cell tumors, and medullary thyroid cancer are the second most common cause of isolated hepatic metastases. These tumors often cause debilitating symptoms due to excessive hormonal secretion. Besides surgery, there are limited curative and palliative treatments available to patients with metastatic NE tumors, emphasizing the need for development of other forms of therapy. In this proposal, we will present data illustrating that GSK3B inhibition may be a viable strategy for the treatment of NE tumors. Furthermore, we will show that the potent GSK3B inhibitor, lithium chloride, suppresses NE tumor proliferation and hormone production. Thus, we propose to conduct a Phase II clinical trial to determine if lithium is a potential therapy for patients with NE tumors. The primary objective in this aim is to evaluate the response rate for patients with NE tumors treated with Lithium. The secondary endpoints are: 1) the progression-free and overall survival of patients with NE tumors treated with Lithium; 2) the effect of Lithium on tumor markers specific for NE tumors; 3) the toxicity and tolerability of Lithium in this patient population; 4) the quality of life of patients with NE tumors treated with Lithium and 5) the expression of GSK3B signaling pathway members in the NE tumor samples from patients enrolled in this clinical study before and during lithium therapy. In summary, these studies are part of our long-term goal of designing and developing novel methods to treat and palliate patients with unresectable, metastatic NE tumors. Neuroendocrine (NE) tumors such as carcinoid, islet cell tumors, and medullary thyroid cancer are the second most common cause of isolated hepatic metastases. These tumors often cause debilitating symptoms due to excessive hormonal secretion. Besides surgery, there are limited curative and palliative treatments available to patients with metastatic NE tumors, emphasizing the need for development of other forms of therapy. [unreadable] [unreadable] [unreadable]

TheVARR&DCenterforNeurorestorationandNeurotechnology(CfNN)attheProvidenceVAMedical Center(PVAMC)unifiesadistinguishedteamofscientists,engineersandcliniciansfromtheVA,Brown University,andaffiliatedhospitalstodevelopandtranslateneurotechnologiesanddevice-basedtherapies thatrestorefunctionforVeteranswithdisordersaffectingthenervoussystem.CfNN?sthreeFocusAreasare eachdedicatedtoperformingtherigorous,peer-reviewedsciencethatleadstonewdiagnosticand therapeuticstrategiesforVeteranswithspinalcordinjury,stroke,amyotrophiclateralsclerosis(ALS), epilepsy,limbloss,post-traumaticstressdisorder(PTSD),depression,chronicpain,andothernervous systemdiseasesorinjuries.CfNN?sthreeFocusAreasare:(1)RestoringCommunicationandMobility(2) RestoringAffectiveandCognitiveHealth,and(3)RestoringLimbFunction.TheFocusAreas?interdisciplinary researcheffortsaresupportedbythreeCoresprovidingessentialinfrastructuresupportandspecialized services:(A)NeuromodulationandNeuroimaging(B)Recording,Decoding,andComputational Neuroscience,and(C)Assessment,OutcomeMeasurement,andImplementation.CfNNresearchisfurther facilitatedbyadedicatedadministrativestaffwhosupportscientificproductivity,coordinatethesynergistic relationshipsbetweenCfNNanditsacademicandclinicalaffiliates,maintainclosecontactwithlocaland nationalVAofficials,andoptimizeourservicetoVeterans.CfNN?sresearchstrategymaximizesinteractions viacomplementaryexpertiseandresourcesamongitsFocusAreasandCores.Thisinturnsupportsahigh- impactresearchcommunityfocusedonapplicationofmedicaldevicestoneurologichealth,bothwithinthe VAandinthewiderregionalandnationalbrainsciencecommunity.Throughthisrenewal,overthenextfive yearsCfNNwillleverageandexpanditscollaborativeresearch,bothwithintheProvidenceVAMCand togetherwithotherVACenters,towardrehabilitationgoalsimportanttothelargenumberofaffected Veteransandtheirfamilies.Theseinclude:restoringarmandlegmovementforVeteranswithstroke,spinal cordinjury,oramputation;?improvingcommunicationforVeteranswithALS;?improvingtheunderstanding, earlydetection,andmanagementofseizures,and;?moresuccessfullytreatingVeteranswithPTSD, depression,obsessive-compulsivedisorder,orotherseverecognitiveoraffectivedisorders.CfNNalsoaims todevelopandvalidatetheoutcomesmeasuresneededforeffectiveclinicaltrialsofnewrestorative neurotechnologiesfortheseconditions.Inadditiontotheresearchofitscoreinvestigators,CfNNisdesigned tofacilitatethetrainingandresearchcareersofjuniorresearchersandtorecruitestablishedcliniciansand scientiststofocustheirresearcheffortsonimprovingrehabilitationforVeteranswithnervoussystemdisease orinjury.CfNNiscommittedtoprovidingseedfundingforpilotstudiestoenableaffiliatedinvestigatorsto launchpromisingresearchandcollectpreliminarydatatoinformandstrengthenlargerresearchprojects. ThroughitscloseassociationwithBrownUniversity,CfNNprovideseducationalopportunitiesto undergraduates,graduatestudents,neuropsychologyinterns,andpostdoctoralresearcherstolearnmore abouttheVA,inspiringthemtoshapetheircareerstowardnotonlygainingabetterunderstandingof fundamentalhumanneuroscience,buttowardusingthatknowledgetohelpdesign,test,anddeploynovel device-basedneurotherapeuticstosupportVeteransHealth.

The specific goals of the research study proposed for this fellowship are to examine interpersonal obsessive-compulsive disorder (OCD) and to assess how these processes can be modified in order to improve patients' treatment outcomes, while simultaneously providing invaluable training in the administration of a family treatment. The investigator will design and implement a brief family intervention for families with the aim of reducing family members' accommodation of patients' OCD symptoms. The intervention will take place during two 90-minute sessions and will encompass psychoeducation regarding OCD and anxiety, as well as modeling and role-play of appropriate responses to requests for accommodation. Half of the family members in the study will be randomized to receive the intervention, and half will be randomized to the no-intervention condition. Measures of family accommodation will be taken at 5 timepoints throughout the study, including a 1-month follow-up after patients have completed treatment. The investigator will also assess levels of depression and anxiety in family members, as well as family members' critical or rejecting attitudes toward the patient. The study will examine: 1) whether participation in the intervention is associated with greater change in family accommodation compared to a no-intervention condition, 2) whether family members' participation in the intervention is associated with greater symptom reductions in patients, 3) whether patients' treatment outcomes are mediated by changes in the level family accommodation, 4) the nature of the relationship between family functioning and accommodation and the effect of the intervention on measures of family functioning, and 5) whether improvements in accommodation and patients' symptoms are maintained at follow-up assessment. The broader aim of this study is to begin determining specific parameters for an effective family intervention, including targets for treatment and appropriate treatment length. In light of the growing acknowledgement of important underlying similarities across anxiety and mood disorders (Barlow, 2002; Brown, 2007; Brown & Barlow, 2009), this deeper understanding of the effective components of family interventions is likely to have significant implications across a range of different diagnoses. The lifetime prevalence of anxiety and mood disorders in America are estimated to be 29% and 21% of the population, respectively (Kessler et al., 2005), and empirically-supported interventions which address patients' broader interpersonal contexts have the potential to bolster the success rates of our current treatments, reducing dropout and relapse rates.

The overall goals of the currently funded competing renewal grant P50 DC000242-21 are the following: 1) To determine the boundaries of selection criteria for the acoustic and electrical speech processing implant. 2) Refine and expand how electrophysiologic measures of auditory function can be used to understand how the auditory system responds to electrical stimulation. 3) Investigate factors that influence music perception and enjoyment of Cl recipients. 4) Describe and explain individual differences in perceptual, linguistic, and social outcomes, of individuals receiving cochlear implants in childhood. The purpose of this supplement is to complement the above goals of the currently funded P50 grant by studying ways to: improve and determine benefits of binaural cochlear implants using more diverse and realistic soundfield hearing tests. Specifically, our goals are to improve: (1) bilateral signal processing; (2) bilateral fitting; and (3) the evaluation of bilateral and unilateral devices. Several experiments are planned and will determine if: (1) moderately directional microphones are superior to highly directional microphones (the latter might exclude important binaural cues); (2) fixed directional microphones are superior to adaptive directional microphones; (3) linear amplitude coding is superior to amplitude compression; (4) synchronous amplitude compression across devices can preserve binaural cues; (5) the use of a "conditioning" stimulus improves binaural hearing; (6) electrophysiological measures that match electrodes across ears result in improved bilateral performance; and (7) bilateral fittings are superior to unilateral fittings in patients who are experienced unilateral listeners. We use several new tests, including ones of distance perception, movement direction, speech recognition with localization cues, and speech recognition while performing a visual thinking task. We believe our proposal is innovative in the comprehensive set of new tests we have designed, in our exploration of new bilateral fitting and signal processing approaches, and our comprehensive evaluation of bilateral and experienced unilateral implant users. The impact of these experiments is very high, in that we hope to be able to improve hearing in challenging everyday conditions and will have direct relevance for the larger population of hearing-aid users. [unreadable] [unreadable] [unreadable]

The long-term goal of these studies is definition of the role of cyclic nucleotides as possible regulators of mammalian gastric secretion, with particular emphasis on the source and activity of gastric cyclic nucleotides and the "second messenger system" of cyclic nucleotides as mediators of gastric acid secretion in response to gastrointestinal hormones, other hormones, cholinergic release, adrenergic blockade, fasting, refeeding, H2 receptor antagonism, hyperchlorhydria and achlorhydria. We plan to determine whether cyclic nucleotide responses in the intact canine stomach are representative of events occurring exclusively in parietal cells by measuring intra- and extracellular cyclic nucleotide concentrations after gastric secretory stimulation by histamine or pentagastrin. In order to resolve the problem of measurement from parietal cells exclusively, we will attempt to study responses in dogs using the antral (acid-secreting) and fundic (acid-secreting) mucosa to secretagogue stimulation. We plan also to measure the responses to a variety of stimulatory and inhibitory agents with measurements in vitro of the key enzymes involved, including cyclase, phosphodiesterase and protein kinase in addition to gastric cyclic nucleotide activities. We also plan to further develop an isolated mammalian parietal cell preparation which would permit direct metabolic study of the parietal cells and resolve the problem of structural localization of presumed key enzymes inth cyclic nucleotide system in the analytical approach of acid generation mechanisms. We plan to further define the localization of cyclic nucleotide activity by developing immunofluorescence techniques which will selectively localize cyclic AMP and cyclic GMP to the parietal cells and indicate their response to secretagogue stimulation. We plan to determine the source of cyclic nucleotide activity in gastric juice by utilizing double isotopic techniques and inert markers to calculate both plasma and gastric cyclic nucleotide content in order to determine whether simple plasma clearance or active secretion from the mucosa accounts for the presence of cyclic nucleotides in canine gastric secretions. BIBLIOGRAPHIC REFERENCE: Schwartzel, E.H., Jr., Bachman, S., and Levine, R.A.: Cyclic nucleotide activity in gastrointestinal tissues and fluids. Fed. Proc. 35:583, 1976 (abstract).

ATAD2 is an important co-activator of the estrogen and androgen receptors. ATAD2 is known to be up- regulated in multiple different types of cancer including breast, lung, gastric, endometrial, renal, and prostate. Up-regulation of ATAD2 is often correlated with poor patient outcomes, and can be used as prognostic marker. Furthermore, silencing the expression of ATAD2 through RNA interference inhibits cell proliferation and promotes apoptosis in ovarian carcinoma, and inhibits migration and invasion of hepatocellular carcinoma and colorectal cancer cells. ATAD2B, is a poorly studied paralog of the ATAD2 gene, and although ATAD2 and ATAD2B are highly conserved, there is little known about the function of ATAD2B or its role in oncogenesis. Both the ATAD2/B proteins contain two conserved domains: an AAA ATPase domain and a bromodomain. The overall objective of the proposed research is to determine how di-acetyllysine recognition by the ATAD2/B bromodomains regulates the cellular function of these proteins. This proposal aims to: (1) characterize how cross-talk between histone modifications modulate acetyllysine recognition by the ATAD2/B bromodomains; (2) outline the molecular mechanism(s) of di-acetylated histone recognition by the ATAD2/B bromodomains; (3) determine the functional significance of di-acetyllysine recognition by the ATAD2/B bromodomains. A unique combination of in vitro biochemical, biophysical, and structural biology studies on the ATAD2/B bromodomains will be coupled with in vivo functional genomic investigations using a breast cancer progression model to characterize the biological roles of the ATAD2/B bromodomains. We will evaluate the impact of neighboring histone modifications on histone H4 tail recognition using peptide array assays in combination with isothermal titration calorimetry (ITC) and nuclear magnetic resonance (NMR) chemical shift perturbation techniques. We will determine the structural features of ATAD2/B bromodomains required for recognition of di-acetylated histone tail ligands using NMR and/or X-ray crystallography. To characterize the binding mode of the ATAD2/B bromodomains with their histone ligands we will carry out analytical ultracentrifugation, size-exclusion chromatography, ITC, and NMR T1/T2 relaxation experiments. Site-directed mutagenesis coupled with NMR and ITC will be used to measure the effects on ligand binding, and identify differences in the binding pockets of the ATAD2/B bromodomains. We will compare the genome-wide associations of ATAD2/B with histone H4 acetylation patterns in a breast cancer progression model to determine the impact of ATAD2/B on breast cancer cell phenotypes using ChIP-seq and RNA-seq, followed by cellular migration and invasion assays. Our multi-faceted approach will correlate specific histone modifications with ATAD2/B binding and action, which will allow us to connect histone H4 acetylation marks to bromodomain function in cancer cell proliferation. Overall, our integrated biochemical, biophysical, structural biology and functional genomics approach will reveal the biological roles of ATAD2/B and facilitate the discovery of novel drug targets to help overcome cancer.

Sustaining adherence to antiretroviral therapy (ART) is central to HIV management. For children in resource- limited settings, no well-validated measures of adherence exist. A reliable, valid measurement tool would provide more efficient, effective HIV care for this vulnerable population. My long-term goal is to work as a clinician-scientist who develops instruments to improve pediatric HIV care systems In resource-limited settings. In the near term, I want to focus on a reliable, valid, cost-effective way to measure children's ART adherence because this is central to the challenge of providing long-term HIV management. The objective of this application is to develop and test a reliable, valid instrument to measure pediatric ART adherence for children ages 0 to 14 years in western Kenya. The rationale for developing a validated adherence measure is to gain the ability to measure adherence accurately, test interventions to improve adherence, and assess if therapies optimize clinical outcomes. This work will be done within a long-standing US-Kenya partnership, the Academic Model for Providing Access to Healthcare (AMPATH). AMPATH cares for over 80,000 adult and pediatric HIV-infected patients in western Kenya, including over 2,800 children on ART. I plan to accomplish the research objective of this application by pursuing the following three specific aims: Aim 1: Develop a reliable, valid comprehensive pediatric ART adherence measurement questionnaire (CAMP - Comprehensive ART Measure for Pediatrics);Aim 2: Develop a reliable, valid, short-form version of the pediatric ART adherence measurement tool (SF-CAMP) for use as an adherence screening measure in busy clinical care environments;Aim S: Evaluate the field-readiness, implementation feasibility, and clinical utility of CAMP and SF-CAMP within the AMPATH HIV clinical care system in western Kenya. I also propose a course of training, study, and mentorship in the design and testing of health behavior measurement tools for International settings, the use of psychometric analyses to develop and validate clinical tools, and evaluation of pediatric HIV care. The combination of research work and my career development plan will enable me to become an independent clinician-scientist who can improve pediatric HIV care provision. PUBLIC HEALTH RELEVANCE: The contribution of this study is expected to be a reliable, valid instrument to measure pediatric ART adherence within a resource-limited setting. The proposed research is significant because we must measure adherence accurately to provide long-term clinical management for HIV-infected children and to find effective interventions to prevent viral resistance and poor outcomes.

Purpose of this grant application is to perform studies aimed at understanding the structure, structure-function relationships, and molecular pathology of the platelet membrane receptor, glycoprotein (GP) Ib-IX complex, GP Ib-IX is essential for normal platelet adhesion to and activation on thrombogenic surfaces. It exerts its function by interacting with the adhesive molecule, von Willebrand factor (vWF). and with the platelet agonist, thrombin. Deficiency or defective function of GP Ib-IX complex leads to the congenital bleeding disorder known as Bernard-Soulier syndrome. The interaction of platelets with vWF through the GP Ib-IX receptor has also been shown to be relevant for the development of pathological platelet thrombi responsible for vascular occlusion. Therefore, studies on the structure and function of GP Ib-IX are relevant for understanding the pathogenesis of the thrombotic complications of atherosclerosis, as well as for unraveling the physiological mechanisms of platelet function. We propose to use an integrated approach based on the use of proteolytic fragments, synthetic peptides, monoclonal antibodies, and expressed mutant molecules to identify the amino acid residues responsible for vWF and thrombin binding to GP Ib-IX. The amino terminal domain of the alpha-chain of GP Ib (the 45 kDa domain),previously shown to contain the vWF- and thrombin-binding sites, will be subjected to limited proteolysis to generate smaller fragments retaining binding function. Synthetic peptides will be constructed to represent the sequence of functionally relevant epitopes of GP Ib-Alpha. The identification of amino acid residues related to the binding function of GP Ib-IX will represent the basis for selective mutagenesis experiments that will be used to obtain final confirmation of the chemical structure of functional domains. Moreover, the molecular pathology of different forms of Bernard-Soulier syndrome, including a newly described variant, will be explored to establish the bases of the genetic abnormalities responsible for this diseases, and also to obtain information on the functionally relevant structures of the GP Ib-IX complex from the study of naturally occurring mutants with defective binding activity. These studies will provide concepts and reagents to probe the functional relevance of the GP Ib-IX complex in normal hemostasis and pathological thrombosis.

A unique method for measuring filtration in single isolated glomeruli has been developed. This technique permits study of the ultrafiltration characteristics of glomeruli from any species. Values from the ultrafiltration coefficient of normal glomeruli of rats, rabbits and dogs have been established. An increase in glomerular size and ultrafiltration in the developing rabbit has been documented. Glomerular function in several forms of acute renal failure has been studied. Further studies will be directed toward developing a more precise description of the changes in capillary volume which occur during filtration in vitro and further defining the relation between glomerular size and cell morphology and filtration. In addition, glomerular function in several physiologic states, eg. intravascular loading and depletion will be studied, and the in vitro effects of several vasoactive substances will be investigated. These studies will enhance our understanding of normal glomerular function and of the mechanisms which regulate glomerular function in normal and disease states.

Aging is the single most important risk factor in human disease in developed countries, and it is thought that an understanding of aging mechanisms would prompt design of rational therapies against age-related diseases. A corollary is that a pharmacological agent that slows aging itself is likely to be effective against a wide spectrum of diseases. To discover what classes of chemical compounds slow aging, we will screen a large and diverse chemical library for extension in the lifespan of the nematode C. elegans. We have developed a number of automated screening methods that exploit C. elegans handling technology. We will also screen a library of compounds generally recognized as safe (GRAS) for extended lifespan to bias our search towards compounds that will prove useful in mammals. We will establish whether the compounds extend lifespan by acting on known pathways and determine their impact on general metabolism and stress resistance. We expect some compounds will prolong lifespan by novel mechanisms in which case we will identify the target proteins. Overall, we aim to identify a range of compounds that slow aging;these compounds will be leads for the development of therapeutics against a range of age-related diseases.

The central objective of this proposal is to determine the regulatory mechanisms controlling the accumulation of specific proteins during mouse central nervous system development. 1) The accumulation of the tissue specific enzyme glutamic acid decarboxylase will be examined to determine if the increase in the quantity of the protein is due to a change in the rate of synthesis, the rate of degradation, or a modulation of both parameters. 2) The tissue specific S-100 protein is actually a class of related proteins; we will complete experiments to determine whether each of the mouse S-100 proteins is a direct product of mRNA translation or whether they result from an interconversion process. 3) Mouse CNS mutants showing the appearance of abnormalities at 10 to 25 days postnatally will be surveyed for quantitative and qualitative alterations in soluble proteins. The mutant spastic which does have alterations in brain amino acid pool levels will be examined in detail to determine if this is a result of a change in the activity of an enzyme. If this is found, we will determine whether this change is due to the synthesis of an altered protein or the production of an abnormal amount of the enzyme.

EXCEED THE SPACE PROVIDED. Scientific research conducted in a Culture of Mentoring is fortified vastly with respect to both qualityand productivity. This culture, which is being actively cultivated at the Oklahoma Medical Research Foundation, has played a vital role in the generation of numerous innovative and fundamentallyimportant biomedical research breakthroughs credited to this Institution. Recognizing the important role of this culture in strengthening the research infrastructureat the OMRF and connected research environments, we describe in this proposal a mechanism by which we seek to formalizeand stratify the mentoring process at OMRF, with the goal of creating a self-sustaining system designed to maximize both individual and collective research potential. Within the context of this mission we seek to: 1) integrate and diversify individual and Program research efforts with the aim of achieving specific research objectives in the areas of immunology and neurobiology, and 2) strengthen individual and collective research efforts by reinforcing selected Core Research Facilities. The cumulative impact of these aims is the cultivation of a strong, supportive, and thoroughly progressive research environment at OMRF, which functionsboth directly and indirectly to reinforce and energize biomedical research here and throughout the state of Oklahoma.

Interparental conflict is a common early environmental stressor with potential long-term consequences for emotional development and risk for psychopathology. Current research findings suggest that interparental conflict is associated with behavioral and physiological indices of emotion regulation during infancy (Crockenberg, Leerkes, & Lekka, 2007; Moore, 2010). Further, differences in biological systems associated with conflict during infancy have been identified as moderators of risk for school-aged children exposed to conflict (El-Sheikh et al., 2009). However, researchers have not yet explicated how exposure to conflict during infancy may get under the skin to affect development of key neural systems. In the proposed study, we will utilize a neuroimaging technique, resting state functional connectivity magnetic resonance imaging, to examine differences in functional neural networks between infants from high and low conflict families. The cortical and limbic (cortico-limbic) level of the biological stress response system and a set of functionally connected regions known to be more activated during rest states, the default network (Raichle et al., 2001), will constitute the focus of this study due to evidence linking the development of these networks both to early adversity and to risk for psychopathology. This work will build on the applicant's ongoing study of two groups of 6- to 12-month- olds screened for high and low interparental conflict. Within this study, fMRI scans are conducted with infants during natural sleep. The purpose of the proposed investigation is to examine whether, among infants from high conflict families, regions of the cortico-limbic network involved in emotional reactivity are less functionally connected to regions important for regulatory processes. We will also investigate whether functional connectivity between regions of the default network previously shown to be less established in preterm infants (Smyser et al., 2010) will be less strong in infants from high conflict families. The current study represents a step towards examining common early environmental stressors in terms of potential consequences for neural networks that have been implicated in mental health disorders. Eventually, such work may facilitate the identification of targets for prevention and intervention and provide a basis for program evaluation in terms of reducing risk at a biological level. The proposed fellowship training covers stress neurobiology in the context of environmental risk, fMRI methods with challenging populations, and rs-fcMRI analyses. Training also focuses on the collaborative process that plays a central role in translational research.

The gram negative bacterium Neisseria gonorrhoea is the causative agent of gonorrhea in man. Many basic questions concerning gonococcal pathogenicity remain unanswered due to the lack of an adequate animal model for this disease. The research outlined is designed to examine genetic mechanisms by which the gonococcus regulates the expression of a major surface structure, the pilus. Gonococcal pilin is a member of a family of pilins that share amino acid sequence homology. The pilin genes from N. gonorrhoea, Pseudomonas aeruginosa, Bacteroides nodous, and Moraxella bovis all show significant DNA sequence homology within their structural genes, and also show DNA sequence homology to the sigma factor binding sequence present in the 5' untranscribed regions of nitrogen fixation and nitrogen utilization genes. In E. coli, expression of a pilin beta-galactosidase fusion is stimulated 40- fold by expression of a cloned Klebsiella pneumonia nifA gene. Pilin expression has been found to be regulated by two gene products from pilA and pilB. The relationship between pilA and pilB regulation and the sigma factor binding site will be investigated utilizing gene fusions and site directed mutagenesis. The gene encoding the sigma factor will be isolated and the effect of mutations in this activity assayed. The possibility of a regulatory system related to nifA, but distinct from pilA and pilB will also be investigated.

Several investigators have demonstrated cytoplasmic particles from human leukemia cells with characteristics of oncornavirus. We have successfully induced the production of oncornavirus-like particles from cultures of human leukemia cells. The presence of similar particles has been demonstrated in patients suspected to have "premalignant" conditions, two of which subsequently developed leukemia and lymphoma respectively. Based on this work, the broad objectives of this proposal are (1) to determine the biological significance of oncornavirus in human leukemia and preleukemia, and (2) to determine the nature and activity of particles from human leukemia cells. Bone marrow cells from preleukemic, immune deficient, and renal transplant patients will be studied following in vitro culture for the presence of oncornavirus-like particles. Human leukemia cells will be grown in vitro. Proteins from these particles will be studied and compared to known oncogenic agents. Human leukemia cells will be cocultivated with human and possibly non-human cell lines in an attempt to induce a chronically infected cell line. These studies will provide data relevant to the occurrence of oncornavirus-like particles in various disease states. The establishment of long term productive cultures of human oncornavirus-like particles and/or the demonstration of related proteins in easily accessible mammalian oncogenic agents will provide material for further biological survey by biochemical and immunological techniques.

This is a report on progress achieved during the third year of the program. The aims have been to establish a program of social science research and training in the study of drug use. Research has focused particularly on drug behavior among youth, cross-cultural studies of drug use within the U.S. and in other countries, the evaluation of drug treatment programs, and the methodologies of drug research. During this period, drawing on computerized sets of data from drug studies at Columbia, a number of cross-study analyses--as well as new research projects--have been undertaken in these areas of interest. Other activities during this period include the expansion of drug research training facilities at Columbia and the continuation of a Columbia University Seminar on Drugs and Society. Goals for the coming year include conducting additional cross-study analyses of data already collected as well as new research in the U.S. and abroad in the areas of interest. Also planned for the coming year is a conference on the contributions of social science research to policy-making in the drug field.

This project has been expanded to encompass two related research studies. The first study has analyzed data derived from a randomized clinical trial of diagnostic ultrasound use during pregnancy conducted by the team of Norwegian investigators in Trondheim, Norway. The purpose of the analysis is to examine fetal growth patterns using longitudinal measurements throughout pregnancy of: (1) symphyseal-fundal heights; (2) weight gain at each prenatal visit; (3) serial biparietal and abdominal diameter measurements from ultrasound; and (4) maternal hemoglobin level. Regression models have been fit to the serial measurements for each mother. The coefficients of the regressions have been analyzed in relation to various indicators of birth size such as weight, crown-heel length, ponderal index, and birth weight-for-gestational age percentile. Using an analysis of covariance procedure, additional factors (e.g., cigarette smoking, alcohol intake, low maternal prepregnancy weight, etc.) will be tested for significance in modifying intrauterine growth patterns. In addition to the study described above, a prospective study to determine risk factors for intrauterine growth retardation, or small-for-gestational age birth, was begun in 1984 through the research contract mechanism with both the University of Alabama in Birmingham and University of Trondheim, Norway (in collaboration with the Universities of Bergen and Uppsala). The study protocol includes recruitment of pregnant women before 17 weeks gestation. Those enrolled in the study will be carefully monitored throughout the remainder of their pregnancy. Symmetric and asymmetric forms of intrauterine growth retardation will be assessed prenatally and at delivery. Infants born to the study mothers will have follow-up exams during the first year of life to assess catch-up growth and attainment of early developmental milestones.

The bi-directional translocation of lipids from one side of a biological membrane to the other is termed flip-flop. Lipid flip-flop across the endoplasmic reticulum (ER) membrane is required for protein N-glycosylation and GPI-anchoring. These protein modifications are essential in eukaryotes; for example, their genetic abrogation causes embryonic lethality in mammals and renders yeast unviable. Lipid flip-flop across the ER is also required for membrane biogenesis: phospholipids that are synthesized on the cytoplasmic face of the ER must be translocated to the opposite face to enable the membrane bilayer to grow uniformly. The demand for lipid flip-flop at the ER is likely to be exceptionally high when the ER membrane expands and glycoprotein secretion increases; this occurs, for example, during the differentiation of B-lymphocytes to antibody-secreting plasma B cells. Unassisted flip-flop is extremely slow because of the energy barrier to taking the polar lipid head group through the hydrophobic interior of the membrane, yet lipids flip-flop rapidly across the ER membrane on a time-scale of seconds. This is because the ER possesses specific transport proteins (flippases) that accelerate lipid flipping to a physiologically sufficient rate. Lipid flipping in the ER occurs by an ATP-independent mechanism in which the flippases facilitate 'downhill' transport of lipids; this distinguishes ER flippases from other translocators, typically found in the eukaryotic plasma membrane, that couple ATP hydrolysis to concentrative 'uphill' transport of lipids. We estimate that there are as many as six different ER lipid flippases but none of these have been identified at the molecular level. We developed biochemical reconstitution systems that recapitulate the activity of three of the flippases required for ER membrane bilayer expansion and protein glycosylation. These flippases specifically translocate glycerophospholipids, oligosaccharide diphosphate dolichols and mannose-phosphate dolichol. Our aim is to identify these physiologically important translocators with the long-term goal of understanding their mechanism of action. We propose to do this via a two-pronged approach involving protein purification and mass spectrometry on the one hand, and screening of systematic collections of yeast ER membrane proteins on the other. Our purification efforts will be aided by the use of novel affinity matrices. We will also use partially purified flippase preparations to continue our efforts to define the specificity of these proteins. Our published work and preliminary data put us in an excellent position to accomplish these aims. PUBLIC HEALTH RELEVANCE: Flipping of lipids from one side of a biological membrane to the other is necessary for membrane expansion during cell growth, as well as for the biosynthesis of molecules that play critical roles in human and microbial physiology. These molecules include glycoproteins such as the neural cell adhesion molecule, GPI-anchored proteins such as acetylcholinesterase, glycolipids such as the receptor for cholera toxin, components of the cell walls of bacteria and yeast, and the O-antigen of E. coli lipopolysaccharide. We are interested in identifying the transport proteins that catalyze lipid flipping in yeast and mammals and understanding how they work.

PROJECT SUMMARY Microbial transcriptional dynamics have been known to be highly dynamic and heterogeneous between cells, seen from microscopy based and targeted sequencing approaches. Such heterogeneity can be an asset or a liability; from a fitness perspective, transcriptional heterogeneity is a prerequisite for survival under changing environments; however, the modern scourge of antibiotic resistance may be ascribed to such heterogeneities. In either case, there is an enormous need to characterize the transcriptional dynamics at the resolution of individual microbial cell. However traditional approaches relying on one or a few selected reporter genes are inadequate for this challenge. Recent technical advances now allow us to use RNA-Seq to profile single mammalian cells. Massive and early barcoding followed by pooling or manipulation by microfluidics have increased the scale to tens of thousands of cells. However, these technologies have thus far failed to translate to single microbial cells due to (1) difficulty in single microbial cell lysis, especially those with thick cell wall; (2) difficulty to capture and barcode relatively sparse microbial mRNAs, especially when lacking polyA tails (in bacteria); and (3) large population size and complexity of microbial population that require orders of magnitude more cells be sampled in an experiment. We will leverage droplet microfluidics, develop physical, chemical and enzymatic lysis methods, and investigate novel molecular biology and sequencing techniques to develop a single-cell microbial genomics pipeline to (1) Isolate and (2) lyse single microbial cells; (3) capture and barcode the mRNA of single microbial cells; and (4) process 104-105 cells per sample with hundreds of distinct transcripts per cell. Barcoded RNA will then be pooled and sequenced at high depth. These tools will be modular and have broad applicability beyond RNA-Seq, including single cell epigenomics and proteomics. New physical lysis modes investigated will include MEMS, laser ablation, acoustic waves, and plasmon resonance. The proposed project will significantly advance current technologies which are limited in throughput or the number of RNA molecules measured. Currently, there is no successful strategy for single microbial cell RNA- Seq at scale. Our strategy will enable cost-effective and generalized single-cell RNA-Seq in microbes at massive throughput. Novel, hybrid microfluidic devices containing silicon, nanomaterials and elastomeric components will be developed for single microbial cell lysis, barcoding and library prep.

Six studies of the adult mentally retarded living and working in community facilities will test the influence of opportunity to interact; similarity choice for cognitive level and individual differences in sociability on enduring friendship choice.

Lyme disease (LD) is a tick-borne, multi-system, infectious disorder caused by the extracellular spirochetal bacterium Borrelia burgdorferi (Bb). Since Bb lacks exotoxins or known bacterial secretory system, it is widely believed that the inflammatory manifestations of the disease result from the host's innate and co-evolving adaptive immune responses to the bacterium. Monocytes and macrophages are considered to be critical cellular elements of the innate immune response to the spirochete. For more than a decade, Bb-mediated cell activation was thought to occur chiefly as a result of the interactions of the spirochete's abundant outer membrane-associated lipoproteins with CD14 and Toll-like receptors (TLR) 1/2 on the surface of these cells. We now have extensive evidence that phagocytosis of live spirochetes by human monocytes and murine macrophages generates a more intense and far broader inflammatory response than can be attributed to lipoprotein-mediated, cell surface TLR1/2 activation. Phagocytosis of intact Bb also induced transcription of interferon-2 (IFN-2) and type I interferon-stimulated genes (ISGs), independently of TLR2. Recent evidence from the investigator's laboratory provides substantial evidence the TLR2-independent signaling events elicited by Bb in human monocytes are MyD88-dependent and occur via TLR8. On the basis of our collective findings we now propose a new model of Bb-induced monocyte activation, which emphasizes the importance of phagocytosis and the cooperative role of TLR2 and TLR8 signaling. In this model, binding of Bb to the monocyte/macrophage cell surface, through a yet to be characterized phagocytic receptor, is followed by a broad sequence of immune signaling events which mechanistically can only be integrated following internalization of the bacterium and formation of the phagolysosome. The validation of the phagosomal signaling model, the centerpiece of our research strategy, will significantly enhance our understanding for how the bacterium triggers the inflammatory processes that under actual disease conditions cause tissue damage and/or that promote bacterial clearance. To accomplish our goals and examine mechanistic aspects of the proposed model, we have formulated the following Specific aims. In Aim 1 we will use a ex vivo stimulation technique to characterize in detail the mechanisms by which phagocytosis of Bb elicits activation and TLR1/2 and TLR8 dependent inflammatory signals in human monocytes. In Aim 2, we will examine key elements of the phagosomal signaling model in Bb-infected human and murine macrophages. The use a large repertoire of available knockout mice will allow us to define mechanistically how Bb is sensed and triggers immune responses in these cells. In Aim 3 we will characterize MyD88 dependent and independent responses to Borrelia burgdorferi both in vitro and in vivo using human monocytes and macrophages obtained from children and adults with known congenital deficiencies in components of the MyD88 signaling pathway (IRAK-4 and MyD88 deficient). PUBLIC HEALTH RELEVANCE: Lyme disease (LD) is a tick-borne infectious disorder caused by the spirochetal bacteria Borrelia burgdorferi (Bb), which has continued to increase in endemic areas and has spread geographically, paralleling the distribution of its primary vector, Ixodes ricinus complex, and the explosive growth in the white-tailed deer population. To study the inflammatory responses to the LD spirochete, the investigator will use a powerful stimulation model that allows a very complete characterization for how the bacterium activates human immune cells and in parallel experiments takes advantage of the large repertoire of available knockout mice to study these responses. Using this combined translational-experimental approach, in this application the investigator will validate key elements of a proposed new model of spirochetal recognition, where the phagosome is a central platform for recognition of diverse borrelial ligands and which involves a cooperative interaction between TLR2 and TLR8 in pro- and anti-inflammatory cytokine responses, and TLR8 in IRF-7 mediated induction of IFN-2.

The L-type Ca2+ channel provides the initial triggering influx of Ca2+ (I/Ca) for excitation-contraction coupling in cardiac muscle. This channel is an extensively regulated protein complex composed of three subunits: alpha1, beta and alpha2-delta. Stimulation of neurohormonal receptors such as endothelin (ET), angiotensin II, and alpha1-adrenergic receptors result in the production of the second messenger diacylglycerol (AG), which can activate protein kinase C (PKC). The effects of activation of PKC on the contractility of the heart and on the L-type Ca2+ channel are controversial. The objectives of this proposal are to clarify the effect of neurohormonal activation of PKC on native cardiac L-type Ca2+ channel are controversial. The objectives of this proposal are to clarify the effect of neurohormonal activation of PKC on native cardiac L-type Ca2+ channels and determine the molecular mechanisms underlying this regulation. Preliminary data using the perforated whole-cell patch clamp technique demonstrate a potent increase in Ica in response to ET and photorelease of a DAG analogue. The hypothesis that PKCepsilon directly stimulates cardiac L-type Ca2+ channel activity by phosphorylation of the alpha1C subunit will be tested. Experiments will utilize isolated rat ventricular myocytes and cultured embryonic stem cell- derived cardiomyocytes, which will be studied using a series of complementary cellular electrophysiology, biochemical, and molecular biology approaches. We propose to address the cardiac myocytes in response to ET and photorelease of caged diC8; 2) Determine the PKC isoform specificity by modulation of cardiac L-type Ca2+ channels; 3) Characterize the phosphorylation of the alpha1C subunit by PKC and determine the functional consequences. Overall, these results will provide a new molecular framework to understand the regulation of L-type Ca2+ channel by the CAG/PKC system. Activation of PKC has been implicated in myocardial hypertrophy and heart failure as well as in ischemic heart disease. Understanding the detailed regulation of the L-type Ca2+ channel by PKC may provide insights and potential novel therapeutic strategies for these disease processes.

The eventual long term scientific goal of this research proposal is to achieve an understanding of the molecular mechanisms which are important for vesicle transport and digestive enzyme secretion leading to invasive activities of prostatic tumor cells. We are searching for ways to destroy tumors more effectively by identifying critical cellular targets (ATPases) and more specifically studying drugs which inhibit the ATPases critical for cell motility events related to tumor invasion. Estramustine, a synthetic product of nor nitrogen mustard and estradiol represents a class of anti-tumor drugs we have worked with whose effectiveness in inhibiting a specific class of ATPases, vesicle transport, enzyme secretion and invasive activities of prostatic tumor cells will be assessed. We have found that a novel microtubule associated cytoplasmic ATPase, termed kinesin powers secretory vesicle streaming along microtubules isolated from DU 145 cells. The motion is inhibited in vivo and in vitro with micromolar levels of estramustine (Piazza and Stearns, Nature, in preparation), and agents (vanadate) which are known to inhibit dynein and kinesin ATPase activity. Because of these preliminary findings, in depth biochemical studies of estramustine and kinesin are planned. Specifically, these include, (a) purification and characterization of prostatic cell kinesin from a variety of tumor cell lines; (b) immunofluorescent and immunogold localization studies of kinesin(s) and assessment of the relevance of such localizations to secretory vesicle motility along microtubules, (c) drug combination studies with estramustine and several analogues in an attempt to establish if ATPase activity and vesicle motility are inhibited and more importantly, to determine if drug combination studies inhibit collagenase secretion by invasive DU 145 cells or primary tumor cell lines established from human prostate biopsies. The results from these studies should establish if different tumor cell lines contain kinesin or novel kinesin(s) important for critical cellular functions during tumor invasion. The effect of estramustine dosages to cell performance will be determined with respect to its affinity for kinesin and inhibitory effects on kinesin ATPase activity and related motility functions in tumor invasion including vesicle transport and secretion.

We have established the University of Massachusetts Center for Clinical and Translational Science (UMCCTS) which includes the five UMass campuses. Operationally, the Center is located at the University's Worcester campus and managed by the School of Medicine. The Center serves as the "academic home" for clinical and translational scientists across all UMass campuses. The CTSA program will build clinical and translational research upon a foundation of world class basic science as evidenced by the award of the Nobel Prize in Physiology or Medicine to Craig Mello in 2006 and the Albert Lasker Award to Victor Ambros in 2008. The UMCCTS aims are to: 1) Accelerate early phase translational (Tl) studies by recruiting outstanding T1 research leaders;establishing innovative research core facilities;and developing new therapies, devices, and interventions based upon UMass discoveries;2) Integrate unique networks of clinical research and healthcare delivery in Central New England and Massachusetts at-large to build and expand T2 (clinical effectiveness research) and T3 (implementation and dissemination research) capacity by recruiting outstanding T2 and T3 research leaders, and by developing innovative T2/T3 core facilities;and enhancing patient and community outreach and participation in clinical/population-based research;3) The UMCCTS will work collaboratively with all campuses and schools ofthe UMass system in developing programs, curricula, and faculty support systems that promote careers in clinical and translational research. Governor Deval Patrick and the Massachusetts Legislature recently enacted legislation committing $1 billion in state investment to be directed to the Life Sciences in the Commonwealth. With $90 million in seed funding, the University has established the UMass Advanced Therapeutics Cluster (UMATC) which includes the RNA Therapeutics Institute, the Center for Stem Cell Biology and Regenerative Medicine, and the Gene Therapy Center. The UMATC joins the Massachusetts Biologic Laboratories, Meyers Primary Care Institute, and Commonwealth Medicine, four innovative components that are rarely found in academic centers, to speed bench to bedside to community discoveries in clinical and translational research. RELEVANCE (See instructions): The University of Massachusetts Center for Clinical and Translational Science (UMCCTS) will transform the conduct of clinical and translational research by providing an "academic home" for all university clinical investigators. The UMCCTS will provide robust pilot grant programs, comprehensive education and career development programs, and innovative core facilities to support and nurture clinical investigators.

Genomic imprinting is a mammalian-specific phenomenon whereby the expression of a subset of genes is dependent on parental origin of the allele. Monoallelic expression is generally correlated with differential methylation of the parental alleles. Differences in methylation can be inherited as a primary imprinting mark from the gametes or acquired secondarily as a post-fertilization event. The significance of establishing the appropriate methylation pattern and achieving imprinted expression is illustrated by the fact that in the absence of parent of origin-specific expression, developmental defects ensue. Human diseases such as Prader-Willi, Angelman and Beckwith-Wiedemann syndromes result from the inappropriate expression of imprinted genes. The objective of this research is to enhance the understanding of how imprinted expression is achieved via an analysis of methylation acquisition at imprinted loci. The first goal is to conduct a temporal analysis of the acquisition of methylation during male gametogenesis at the paternally-methylated Gtl2 and Rasgrf1 loci. Second, the role of DNA methyltransferase 3L in establishing methylation at imprinted loci during male gametogenesis will be examined. Finally, the post-fertilization establishment of methylation at the Gtl2 promoter and its relationship to monoallelic expression will be investigated. Bisulfite mutagenesis and DNA sequencing will be the primary methodology utilized to examine methylation in these analyses. This research will advance the field of imprinting by determining whether the establishment of imprinting marks is globally or independently controlled. Furthermore, it will test the hypothesis that DNA methyltransferase 3L is the enzyme responsible for methylation establishment in the male germline. In addition, this project will enhance undergraduate education by providing students the opportunity to conduct independent research in molecular genetics.

Current therapy for Rheumatiod arthritis comprises non-steroidal anti-inflammatory drugs (NSAIDs) in early stages of the disease, ultimately given way to oral steroids and the disease-modifying anti-rheumatic drugs (DMARDs) as the disease progressively worsens. Methotrexate (MTX) has become the DMARD of choice of many rheumatologists because of its faster mode of action and better record of prolonged use. However, despite the use of high doses of MTX, many patients only experience partial relief of symptoms and still have features of active disease. Experimental evidence implicates TNF as an important mediator in the pathobiology of RA and underscores TNF as an appropriate therapeutic target. Chimeric A2 (cA2) is a monoclonal antibody that binds with high affinity and specificity to human TNF and neutralizes its biologic activity. The current trial is designed to show in a larger patient population (n=300) that cA2 given for up to one year will provide a rapid and sustained reduciton of clinical signs and symptoms of RA in patients who have active RA despite MTX therapy. Four cA2 treatment groups will be evaluated and compared to a placebo group. The primary endpoint in this study will be a reduction in the clinical signs and symptoms of RA at 30 weeks following the onset of treatment. Continuing reduction in signs and symptoms, as well as disability, joint damage, disease remission and quality of life will be evaluated at 1 year.