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The epithelial-mesenchymal transition (EMT) is characterized by the loss of cell-cell adhesion and cell polarity in epithelial cells and the acquisition of motile and invasive properties. While essential for development, the EMT is one mechanism by which tumors can acquire the capability to undergo tissue invasion and metastasis. It is therefore important to identify novel therapies that can inhibit the EMT, but few assays for EMT inhibitors in high throughput screens (HTS) have developed. A change in fibroblast growth factor receptor 2 (FGFR2) splicing occurs during the EMT and using an innovative luciferase-based splicing reporter assay we previously carried out a genome-wide high throughput cDNA expression screen for regulators of this splicing switch. This screen identified the epithelial cell type specific splicing regulators ESRP1 and ESRP2 demonstrating the feasibility of cell-based splicing assays in high throughput, array-based screens. An extensive set of ESRP-regulated exons switch splicing during the EMT, indicating that global changes in alternative splicing occur during this process. A change in this splicing network is a thus a dynamic feature of the EMT and changes in splicing of ESRP-regulated targets can be used as a biomarker for the EMT. In this application we will develop more robust next generation splicing reporter assays using ESRP- regulated exons that undergo profound "switch-like" changes in splicing and configure them for HTS assays using the Molecular Libraries Production Centers Network (MLPCN). In Aim 1, we will adapt existing minigene reporters containing ESRP regulated exons and flanking intronic regulatory sequences for HTS in the context of our established luciferase-based reporter minigenes. The reporters will include exons whose inclusion is activated as well as those that undergo skipping during the EMT. Additional reporters will also be developed for use in counter-screens to prioritize HTS hits. In Aim 2, these screens will be configured for screening in 384 well format and pilot screens will be carried out using several small compound libraries as well as several previously described compounds that have been shown to function as general modulators of splicing. These compounds will be screened in mesenchymal cells for splicing changes indicative of the reverse process of mesenchymal to epithelial transition (MET) and in epithelial cells for inhibition or reversal of an inducible EMT. Successful completion of this pilot phase of this funding mechanism (PAR-10-182) in year one will enable us to submit these assays for the larger scale screening phase using the MLPCN library of compounds. Such screens hold great promise to yield novel small molecule regulators of splicing, including a subset that broadly promote epithelial-specific splicing pathways to inhibit or reverse the EMT and block cancer metastasis. Such compounds will potentially include those that affect signaling pathways or other upstream events that might potently activate broad transcriptional and post-transcriptional gene expression programs that inhibit the EMT. PUBLIC HEALTH RELEVANCE: The epithelial to mesenchymal transition (EMT) is the process by which cancer cells can escape from the primary site and metastasize to distant sites and is therefore a target for novel cancer therapies. We have identified regulators of alternative splicing that control an epithelial splicing network that is lost during the EMT, suggesting that a mesenchymal splicing program can promote the EMT and that these splicing changes serve as biomarkers for this process. The current application will use innovative splicing assays to carry out screens for novel compounds that inhibit this splicing transition and thereby identify lead compounds for drugs to prevent tumor metastasis.

[unreadable] The specific aim of this application is to help the UPR for repair, renovation, and modernization of its unique animal research resource (ARC) at the CS facility of the CPRC. The CPRC is an unrivaled national and international research resource for comparative studies in the biomedical and behavioral sciences that has received NCRR funding through a P40 award or the equivalent for the past three decades. The proposed renovations will improve the care of research animals and will provide much needed infrastructure support for the ongoing and planned biomedical and behavioral research program at CS. The free-ranging population of rhesus monkeys (Macaca mulatta) on the island of CS provides scientists with an unparalleled opportunity for performing biomedical and behavioral research projects utilizing primates residing in a semi-natural habitat. This population has the most extensive computerized demographic and genetics database available to researchers anywhere in the world. The population management program for CS has been designed to optimize the health and well-being of the monkeys, to enhance the value of the colony for research. In addition, the goal is to provide healthy animals to the scientific community for biomedical research, including AIDS and SlV pathogenesis and vaccine development as well as the support of National defense programs. Many of the existing structures on CS island were constructed in the 1930s and 1940s and are in dire need of renovation. This application seeks funds to renovate the dock to the island and funds to repair the roads, drinking water purification system and the three feeding and trapping corrals on CS. Renovations and modernization of these facilities are essential in order to prevent the continued deterioration of the infrastructure on CS, to maintain and enhance animal health and well being, to retain AAALAC accreditation, to comply with federal regulations and guidelines, and to continue to promote the research program and resources of the UPR. [unreadable] [unreadable]

EXCEED THE SPACE PROVIDED. The proposed research is a continued study of mobile introns in bacteria. The mobile group I and group II introns are both self-splicing, but their RNA-splicing and DNA-mobility pathways are distinct. In each case, the intron typically transfers to an allelic intron-less site, in a homingprocess initiated by intron-encoded endonucleases. For group I intron homing, recombination events are strictly DNA-based whereas for group II intron homing, also termed retrohoming, RNA is involved at levels of both the intron template and the cleavage enzyme for mobility. Transposition to ectopic sites also occurs at low frequency, and is the process responsible for intron dissemination in nature. During the current funding period we showed the following: Homing of the group I td intron is regulated by the very endonuclease that promotes the process, by acting as an autorepressor. Also, retrohoming of group II introns is dependent on host polymerases in a way that suggests that intron movement is related to the cell's DNA damage response. Furthermore, group II intron retrotransposition occurs into single- or double-stranded DNAs in a host-dependent fashion. Additionally, transposed group II introns retain their ability to move, reflecting the high fidelity of the group II intron- encoded reverse transcriptase. Finally, in a study of a small regulatory RNA, DsrA, included in a non-intron- related specific ami, we showed not only that DsrA undergoes conformational changes to effect global regulation, but also that DsrA promotes acid tolerance in pathogenic bacteria, thereby enhancing virulence. In the upcoming funding period we will follow up on these findings using genetic studies in bacteria, as well as biochemical and structural approaches. Further, we will address exciting questions relating to intron evolution and the relationship of group II nitrons to spliceosomeal introns in yeast, all as described in the original application. Our overall goal of studying DNA- and RNA-based rearrangements of model introns remains unchanged. In addition to the innate mechanistic importance of these studies, the invasiveness of group II introns and their similarities to human retrotransposons and spliceosomal introns have great evolutionary, biotechnological and medical significance.

A number of recent studies suggest that cognitive processes rely on spatial and temporal patterns of activity in extended neural networks. Optical imaging techniques can provide information on a microscopic level about the individual and collective behavior of cells involved in these processes. We propose to develop an advanced image probe and digital acquisition system designed for high performance functional neural imaging using intrinsic light scattering signals. The first goal of the project is to improve the image probe design for high sensitivity to small and rapid changes in light scattering. Two methods of reflectance mode illumination will be explored for fluorescence and polarized light measurements. The system will incorporate an electronically tunable filter to illuminate tissue with specific wavelengths for spectroscopic measurements, and an intensified detector for dynamic fluorescence measurements. The second goal is to implement hardware and software improvements to the data acquisition system. Application performance requirements and the need to integrate of a number of data modalities require that we develop custom data acquisition hardware; the proposed system will control CCD camera circuitry, and capture, process and archive 2000 frames per second, with 256 channels of concurrent electrophysiological data. We will integrate this hardware into inexpensive, multiple processor. Pentium computer systems, designed for distributing tasks across multiple processors. The third goal for the project is to apply the device to investigate the nature and origin of light scattering changes associated with neural activation. Our preliminary studies in the hippocampus and medulla have demonstrated several different optical changes associated with neural activation, including fast light scattering changes concurrent with neural swelling and electrical transmission, and slower changes in light absorbance associated with hemodynamic coupling to metabolic demand. We will examine the spectral nature of the optical signals, characterize the timing of at least four components that we have identified, and employ physical and physiological manipulations to identify and characterize underlying mechanisms.

Complex developmental processes that define organ morphology and cellular differentiation appear to be governed by only several signal transduction pathways, suggesting the likelihood of extensive interactions among those pathways. However, the mechanisms that coordinate the activity of different signaling cascades involved in this process remain ill defined. In this proposal, I wish to tackle this important issu by focusing on the interplay between two prominent signals that control development; the transcription coactivator Yes- associated protein (YAP), the prime target of the Hippo kinase cascade, and Sonic hedgehog (Shh) signaling. The main goals of this proposal are to 1) dissect the function of YAP in spinal cord development, and 2) define how the Shh and Hippo signaling pathways act in concert to control development in the vertebrate spinal cord. The overall hypothesis of this proposal is that YAP serves as a novel downstream effector of Shh during the control of NPC proliferation and specification of ventral cell fates in the CNS. Specifically, I postulate that the YAP-dependent signaling communicates with the Shh signaling pathway at multiple levels, providing an effective means of coordinating these two transduction cascades during neural development. I will test this hypothesis using an ensemble of molecular methods, chick embryos and mutant mice. The specific aims of this proposal are as follows: 1) to determine the role of YAP in cell-type specification during neural tube development and 2) to define how the Shh and Hippo signaling pathways act in concert to control development in the vertebrate spinal cord. Besides defining the basic mechanisms by which YAP and Shh signaling coordinate key aspects of normal CNS development, this study should also uncover important principles applicable to other signaling networks that regulate organogenesis.

DESCRIPTION (Directly taken from the application) Polycystic kidney disease (PKD) is characterized by proliferating epithelial cells, alterations in basement membrane composition, and aberrant localization of cell surface proteins. These cellular properties are consistent with a reversion of the renal epithelial cell to a more embryonic phenotype. Thus, genes regulating the differentiation and proliferation of the normal renal epithelium may be aberrantly affected in PKD and may directly contribute to the initiation and progression of the disease. This proposal will address the function of the transcription factor, Pax-2, in the development of normal and cystic renal epithelium. Pax-2 is required and sufficient for the conversion of the kidney mesenchyme to an epithelial phenotype by acting as a differentiation and proliferation stimulus. Pax-2 expression in the developing kidney is repressed as the normal epithelium matures, but is detected at higher levels in the cystic epithelium of experimental animals. The proposed experiments will determine the developmental expression pattern of Pax-2 in the cpk mouse, a well-characterized model for PKD. Furthermore, the cpk mutation will be crossed into the Krd mutant mouse that has one Pax-2 allele deleted. This genetic cross will test for modification of cpk by Pax-2 using gene dosage. If continued expression of Pax-2 in cpk mice is a proliferation stimulus, reducing the Pax-2 gene dosage may result in a slower progressing form of PKD. Additional experiments will address the regulation of the Pax-2 gene by identifying promoter elements required for tissue specific transcription. Finally, preliminary data indicates several cell adhesion molecules of the cadherin gene family may be regulated directly by Pax-2. The cadherin regulatory sequences will be cloned and tested for their ability to bind Pax-2 and mediate trans- activation. These experiments will elucidate basic molecular mechanisms of renal epithelial morphogenesis and may lead to a more complete understanding of the aberrant developmental events underlying PKD. Ultimately, understanding the regulation and function of transcription factors in renal diseases may lead to novel therapeutic interventions based on modulating gene expression.

The object of the proposed investigation is to gain additional insight into the molecular events and regulatory implications in messenger-RNA metabolism in cultured mammalian cells. Human (HeLa S3) cells infected by and rat embryo cells transformed by adenovirus will be employed in these investigations. In the lytic infection the following aspects of viral RNA metabolism will be explored. Transcription of the genome will be examined by electron microscopy following dispersal of nuclear contents. Processing of apparent large molecular weight nuclear-precursor RNA will be examined by analysis of 5'-termini and by analysis of RNA synthesized in the presence of adenosine analogues. Fate of cytoplasmic viral RNA will be determined in enucleated cells (cytoplasts). Much of the proposed study will depend upon limited RNA structure analysis, especially if viral RNA sequences which remain in the nucleus can be isolated. In addition, information bearing on the mechanism of formation of virions and restriction of host-cell RNA synthesis should also be elucidated. The feasibility of extending these investigations to adeno 2-transformed rat embryo cells, which will depend on the incorporation of sufficient radioactivity, will be constantly evaluated. These investigations should not only bear on the cell biological implications of the biogenesis of messenger-RNA but also on the infection and transformation of cultured mammalian cells. BIBLIOGRAPHIC REFERENCES: Nuclear and cytoplasmic adenovirus RNA: Differences between 5'-termini of messenger and non-messenger transcripts. McGuire, P. M., Piatak, M. and L. D. Hodge. J. Mol. Biol. 101 379-396 (1976). Visualization of a unique adenovirus DNA-protein complex. O. L. Miller and L. D. Hodge. J. Cell Biol. 67 284a (1975).

Molecular Core Abstract: Dr. Harris will direct the Molecular Core and a staff of two full-time scientists. This essential Core will service the Program Project entitled "Critical Interactions of APOBECSs: Molecular Approaches to Novel HiV Therapies" by producing plasmid DNA constructs for APOBECS and Vif protein expression in E. coli and mammalian cells. We anticipate completing 10-20 constructs per week and providing a strong foundation for the program investigators.

The consequences of alcohol abuse on the American public are profound, both in terms of individual well-being and impact on the family structure, as well as the enormous cost to society in terms of lost productivity and associated health care expenses. Despite increasing efforts, our understanding of the neurobiological mechanisms that underlie the effects of alcohol and the development of alcohol use disorders (AUD) remains incomplete. Epidemiological research has pointed to adolescence as a critical period in the development of alcohol disorders. The prefrontal cortex (PFC) is a brain region that is not yet mature at the onset of human adolescence and continues to develop during this period, during which some individuals may be highly susceptible to the effects of alcohol. The PFC mediates control over goal-directed behaviors and dysfunction of the PFC is thought to underlie compulsive drug-taking and relapse in substance abusers. Binge drinking is highly prevalent in adolescents, and episodes of high alcohol intake have been associated with decreased PFC activity and function (hypofrontality). Imaging studies suggest that hypofrontality persists in chronic alcohol abusers, and may therefore be a contributory factor in the development of AUDs and behavioral pathologies in adulthood. The underlying mechanisms of this PFC hypoactivity are unknown, and the development of robust animal models would therefore be useful in investigating the underlying changes in neuronal excitability. A better understanding of these changes would enable possible molecular and therapeutic interventions in order to prevent the development of alcoholism. One plausible mechanism for hypofrontality involves the depression of persistent activity, a mode of firing that can be observed in recordings from pyramidal neurons in the PFC of rodents. This type of activity is seen at more depolarized membrane potentials and is associated with performance in working memory tasks, and is dependent on the Ih current, which is mediated by a family of hyperpolarization-activated and cyclic nucleotide modulated (HCN) channels. We propose that chronic changes in persistent firing might result from prolonged alcohol exposure. To date there have been few detailed studies of excitability in the PFC after drinking and none in adolescent rodents. In three separate but integrated aims, we plan to test the overall hypothesis that the HCN1 channel that contributes to the Ih current in PFC PNs is important for the regulation of alcohol drinking, and specifically that (a) binge drinking of alcohol during adolescence inhibits persistent firing and excitability in the PFC via reduction of Ih and (b) reduction in HCN1 channel activity in layer 5 of PFC can mimic the effects of alcohol consumption during adolescence, while (c) activation or over-expression of HCN1 channels can restore persistent activity and normal levels of excitability in PFC of binge drinking adolescent animals.

We have constructed (and continue to develop) a laser-based facility for time-resolved fluorescence spectroscopy of biomolecules. This facility provides rapid collection and analysis of luminescence data related to macromolecular size, flexibility, folding and structural fluctuations. Our time-correlated laser fluorometer was used to study the folding and dynamics of several proteins. We focused most of our effort on DNA- binding proteins that control the transcription of DNA blueprints into the "field copies" (MRNA) used to build proteins in cells. Fluorescence was used to measure distances between proteins and the sections of DNA they control, to look at the wobbling of proteins in the complex, and to reveal internal changes in the bound protein. We studied "oct-pou", a bipartite factor able to bind two different classes of DNA control sites and accelerate transcription 100-fold. It was seen to "read" DNA by changing shape. We also began studies on HIV integrase and Heat Shock Factor. The former incorporates HIV DNA into human DNA, and the latter "turns on" a stress response by self-association. This year we also continued efforts to adapt our laser instruments to the imaging of tissues. In particular, we demonstrated an electronic laser scanning device that uses discoloration to find BB-sized objects almost an inch inside tissue.

G Proteins participate in the transduction of cellular signals by cycling between an active conformation (complexed with GTP) D and an inactive conformation (complexed with GDP). The ras proteins are guanine nucleotide binding proteins believed to participate in the transduction of proliferative signals, although their specific role is still unknown. Using a variety of experimental approaches, we showed that they are not regulatory elements of either of the two main signaling pathways in mammalian cells, namely the adenylate cyclase and phosphoinostide pathways; however, using Xenopus oocytes, we were able to show that they exert a regulatory role (even in the absence of protein synthesis) in cascades of phosphorylation associated with the induction of proliferation and cell division. The HIV nef gene product has also been proposed to act as a regulatory G protein based on reports attributing GTP binding and GTPase activity to these proteins. We expressed different cDNA clones of nef using the same bacterial and mammalian vectors used previously for ras. Unlike ras, nef from the different HIV isolates lacked GTP binding activity but showed autophosphorylation using either GTP or ATP as the phosphate donor. In addition, unlike ras, HIV nef did not exhibit oncogenic potential in focus-forming assays with NIH 3T3 cells, nor did it cause meiotic maturation of Xenopus oocytes. It therefore appears that the biological function of nef does not follow the pattern of G proteins; however, its phosphorylation, either by protein kinase C or through its autokinase activity, may be functionally significant in vivo.

OVERALL DESCRIPTION: This application continues and amplifies the work already undergoing at the Center. The overall goal of the MU proposal is to foster interdisciplinary research that will enable the systematic evaluation of the safety and efficacy of botanicals. The PI's of the current proposal have already established collaborations, which has been shown a productive one. The proposed studies are aimed to understand the molecular mechanisms of phytochemicals and phytonutrients in human disease. The Center grant contains five projects, pilot studies, core units and career development. The concepts directing the research program include 1) the influence of phytonutrients in the progression of prostate carcinoma, this proposal examines the effect of ERKO mice will be studies in the TRAMP mouse model. 2) The effects phytoestrogens in the innate immunity of ER deficient mice, this proposal aims to determine whether phytoestrogens act to innate immunity through either the ER alpha or beta, or both, in SCID mice. 3) This project will determine whether treatment of CF cystic fibrosis with soy derived isoflavones, (primarily Genistein) acts through a non-ER-mediated pathway, on the mutated cystic fibrosis transmembrane conductance regulator (CFTR). 4) To identify and characterize botanicals with primary emphasis on those used in food supplements. 5) To examine alternative molecular mechanisms of phytoestrogens when considered as polyphenols in neurodegenerative disease. The research is facilitated by resources and several well established core facilities. The center Advisory Committee will provide guidance from distinguished representatives that will meet twice a year.

DESCRIPTION (provided by investigator): Fluorogenic minor groove binder (MGB) probes containing an MGB-quencher at the 5'-end and a fluorophore at the 3'-end have been recently reported. These probes fluoresce on hybridization to the complementary targets. The 5'-MGB-quencher group prevents 5' - nuclease digestion by Taq polymerase during homogeneous amplification. The 5'-MGB-quencher-oligonucleotide-fluors can be used as probes in general nucleic acid and SNP detection assays. We propose to use a new software prediction program to develop MGB Eclipse probe assays to detect Clostridium botulinum, Bacillus anthracis and Yersinia pestis. The designed probes and primers will be optimized with use of three modified bases to improve probe performance of AT- and GC-rich sequences. In the Phase 1 effort, we will design and optimize the primers and probes and demonstrate function of the MGB Eclipse probe assays on a set of known target samples. The specificity of the probes will be evaluated against panels of closely related non-pathogenic organisms. In Phase II, we will expand the number of Category A organisms and improve throughput by multiplexing. In addition, commercial prototype MGB Eclipse(TM) probe assays will be developed for each of these organisms.

Lung cancer is the leading cause of cancer related mortality in both men and women and remains a major health issue. More than 162,000 individuals will die from lung cancer in the coming year, more than breast, prostate and colon cancer combined. The majority of lung cancer cases is attributable to tobacco smoking and in some cases other environmental risk factors. Although the relative risk of developing lung cancer declines dramatically in smokers who quit, former smokers remain at risk for the disease. Several recent studies show that greater than 50% of newly diagnosed lung cancers occur in former smokers. It is estimated that there are approximately equal numbers of smokers and former smokers in the United States. Since smoking cessation is a major public health initiative, former smokers will increasingly account for a higher percentage of lung cancer cases. Therefore, two high-risk population groups exist for lung cancer and improved disease management can be beneficial to both current and former smokers. Additionally, the prognosis for lung cancer patients is very poor. Resistance to chemotherapy used in lung cancer treatment remains a major problem and a better understanding of the mechanisms for resistance could lead to more effective therapies. The MAP3K8 gene is a mitogen activated protein (MAP) kinase kinase kinase expressed in a variety of cells and found to be oncogenic and constitutively activated when altered at the 3 end. However, mutation of the gene appears to be a rare event in humans, but altered MAP3K8 expression is associated with multiple tumor types. MAP3K8 possesses the unique characteristic of activating multiple cascades, including both proliferative and apoptotic signal transduction pathways such as the MEK-1 and SEK-1 pathways, respectively. In NIH3T3 transfection assays utilizing lung tumor DNA, our lab identified a 3 alteration of MAP3K8 similar to the previous reports. We first hypothesized that MAP3K8 might be a target for mutation since we were the first group to report an activating mutation in a primary human tumor. However, it has become clear that mutations are not a common event in tumorigenesis for this gene. Subsequently we showed varied levels of expression of the gene in lung tumor cell lines. This led us to investigate other downstream pathways that could explain the tumorigenic potential of MAP3K8. These included transcription factor array analysis and protein kinase array experiments. We were able to confirm other reports in the literature demonstrating upregulation of NF-kappaB and AP-1 as well as identify other important transcription factors not reported in the literature. These and other experiments, as well as published reports lead us to modify our hypothesis that increased expression of MAP3K8 occur in lung cancer and contribute to disease progression. We have recently shown that increased protein expression of MAP3K8 in lung tumor cell lines leads to changes in downstream signaling pathways and ultimately transcription of important genes in cell survival. To test the effects of MAP3K8 over expression on survival in the presence of a commonly used chemotherapeutic, paclitaxel, we stably transfected a normal tracheal epithelial cell line with MAP3K8. These data suggest MAP3K8 expression is altered in lung cancer cells lines and because of its role in the inflammatory response and cell survival, MAP3K8 over expression may be involved in tumor progression. Future experiments will demonstrate the importance of MAP3K8 in resistance to paclitaxel. We are also positioned to test the effect of MAP3K8 on tumorigenesis using the knockout mouse model and the skin two step carcinogenesis model.Lung cancer is the leading cause of cancer related mortality in both men and women and remains a major health issue. More than 162,000 individuals will die from lung cancer in the coming year, more than breast, prostate and colon cancer combined. The majority of lung cancer cases is attributable to tobacco smoking and in some cases other environmental risk factors. Although the relative risk of developing lung cancer declines dramatically in smokers who quit, former smokers remain at risk for the disease. Several recent studies show that greater than 50% of newly diagnosed lung cancers occur in former smokers. It is estimated that there are approximately equal numbers of smokers and former smokers in the United States. Since smoking cessation is a major public health initiative, former smokers will increasingly account for a higher percentage of lung cancer cases. Therefore, two high-risk population groups exist for lung cancer and improved disease management can be beneficial to both current and former smokers. Additionally, the prognosis for lung cancer patients is very poor. Resistance to chemotherapy used in lung cancer treatment remains a major problem and a better understanding of the mechanisms for resistance could lead to more effective therapies. The MAP3K8 gene is a mitogen activated protein (MAP) kinase kinase kinase expressed in a variety of cells and found to be oncogenic and constitutively activated when altered at the 3 end. However, mutation of the gene appears to be a rare event in humans, but altered MAP3K8 expression is associated with multiple tumor types. MAP3K8 possesses the unique characteristic of activating multiple cascades, including both proliferative and apoptotic signal transduction pathways such as the MEK-1 and SEK-1 pathways, respectively. In NIH3T3 transfection assays utilizing lung tumor DNA, our lab identified a 3 alteration of MAP3K8 similar to the previous reports. We first hypothesized that MAP3K8 might be a target for mutation since we were the first group to report an activating mutation in a primary human tumor. However, it has become clear that mutations are not a common event in tumorigenesis for this gene. Subsequently we showed varied levels of expression of the gene in lung tumor cell lines. This led us to investigate other downstream pathways that could explain the tumorigenic potential of MAP3K8. These included transcription factor array analysis and protein kinase array experiments. We were able to confirm other reports in the literature demonstrating upregulation of NF-kappaB and AP-1 as well as identify other important transcription factors not reported in the literature. These and other experiments, as well as published reports lead us to modify our hypothesis that increased expression of MAP3K8 occur in lung cancer and contribute to disease progression. We have recently shown that increased protein expression of MAP3K8 in lung tumor cell lines leads to changes in downstream signaling pathways and ultimately transcription of important genes in cell survival. To test the effects of MAP3K8 over expression on survival in the presence of a commonly used chemotherapeutic, paclitaxel, we stably transfected a normal tracheal epithelial cell line with MAP3K8. These data suggest MAP3K8 expression is altered in lung cancer cells lines and because of its role in the inflammatory response and cell survival, MAP3K8 over expression may be involved in tumor progression. Future experiments will demonstrate the importance of MAP3K8 in resistance to paclitaxel. We ar [summary truncated at 7800 characters]

We have also completed the first characterization of the active site of NiSOD from Streptomyces seoulensis. This study reveals that Ni site is bound to thiolate donors, like other Ni redox enzymes but unlike any previously characterized SOD. Examination of oxidized and reduced samples reveal a coordination number change (5 -> 4) and a shift in the edge energy appropriate for a one-electron process. There is also evidence that the active site consists of two Ni centers, one of which is not redox active in analogy with the CuZnSOD, and therefore must be composed of two subunits in the tetrameric enzyme.

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. Given the ubiquitous nature of the peptide linkage in biological molecules, replacement of the amide bond with isosteres in potential drug candidates has been a continual goal of many laboratories. Successful replacements will provide improved stability, lipophilicity, and absorption. Many surrogates have been introduced already, yet the synthesis of many of these isosteres in a combinatorial way is difficult and requires several steps. Thus, the discovery of new peptide surrogates with easier syntheses is an important achievement that could open new opportunities for the study of amide-containing molecules and the development of inhibitors with novel physicochemical properties.

This research is aimed at the elucidation of the biological function and genetic control of the polydisperse circular DNA complement of Bacillus megaterium. The specific objectives involve: (1) the characterization of the mode of production of these elements; (2) the determination of their physiological function in the cell, and (3) a test of the possibility that these elements reflect a novel regulatory mechanism for DNA replication and/or gene expression which may provide clues to an understanding of certain types of neoplasias in higher organisms. The research involves the use of electron microscopic analysis, including partial denaturation and heteroduplex techniques, and cleavage of specific circular DNA classes with site-specific restriction endonucleases in order to determine the relationships between these molecules. DNA-DNA hybridization will also be employed as will sedimentation velocity procedures for characterization of these elements. Specific inhibitors will be employed to determine the mode of production. Their biological properties will be assessed by attempts to isolate auxotrophic mutants as well as genetic variants defective in megacin production and other properties, and by attempts to transfer these elements to other bacteria by genetic transformation.

In cancer, normal epigenetic silencing and cytosine methylation are disrupted. Dietary intake of methyl donors can directly affect epigenetic mechanisms through cytosine methylation. Our longterm goal is to understand how the risk of human disease, cancer in particular, is affected by epigenetics and diet. We use A10-vy (obese yellow) mice, a model that exhibits a highly variable phenotype of obesity, tumors, and type II diabetes;the expression of the syndrome is under epigenetic control. The epigenetic state of the A-vy allele can be inherited, indicating that the epigenetic marks determining the behavior of the allele are maintained in the germline. Supplementation of the maternal A-vy diet with methyl donors during gestation alters phenotypes in offspring. We hypothesize that continuous supplementation of the maternal diet with methyl donors will produce a cumulative increase in methylation of the A-vy allele, resulting in a multigenerational trend toward suppression of the obese yellow phenotype, and denser methylation of the allele. The changes may persist after supplementation is withdrawn. Our specific aims are: 1. Investigate the effects of continuous methyl donor supplementation on inheritance of the obese yellow phenotype in A-vy mice. Continuous feeding of methyl donors to A-vy mothers may produce changes in phenotype that increase with more generations. 2. Ask if the effects of methyl donor supplementation persist for generations when supplementation is withdrawn. Changes induced by methyl donors may be maintained in the germline, resulting in epigenetic "memory" that persists for one or more generations. 3. Investigate effects of methyl donor supplementation on CpG methylation of the A-vy allele We will use bisulphite allelic sequencing to obtain a detailed picture of the methylation status of the allele in mice bred for Aims 1 and 2.

The airway epithelium represents the first line of defense for the lungs against inhaled-pollutants and infectious agents. Injury to the airway epithelium occurs during mechanical ventilation and in inflammatory diseases such as asthma. Movement of air into and out of the lungs during respiration causes wide variations in the distension of airways, and the epithelial cells lining the airways are thus exposed to both circumferential wall strain depending upon changes in airway diameter and longitudinal elongation or compression depending upon the expansion of the lung volume. Although there has been much interest in the function of epithelium in response to injury and in the pathogenesis of asthma and other chronic obstructive diseases, there has not been an investigation of the role of mechanical strain on the function of airway epithelium. The central hypothesis of this proposal is that physiological levels of mechanical strain regulate the rate of wound closure and the synthesis of eicosanoids in airway epithelial cells by mechanisms involving reactive oxygen species. This hypothesis will be investigated by stretching both primary cultures of cat tracheal epithelial cells and human bronchial epithelial cells as well as lines of human bronchial epithelial cells grown on elastic membranes using a novel biaxial strain device. Preliminary results demonstrate that both cyclic mechanical elongation and compression delay wound closure by inhibiting cell spreading and migration. Furthermore, indomethacin inhibits and prostaglandin E2 (PGE2) enhances wound closure. Cyclic strain inhibits synthesis of PGE2, and other prostanoids by inactivating the enzyme cyclooxygenase (COX). Preliminary evidence also suggests that COX inactivation is oxidant-mediated. In Specific Aim 1, the mechanisms by which cyclic stretch and compression inhibit wound closure in epithelial monolayers will be determined. Measurements of wound width, cell area, and internuclear distances will indicate cell spreading and migration at the wound edge. In Specific Aim 2, the mechanisms by which cyclic strain regulates the metabolism of arachidonic acid to prostanoids will be determined. Western blots of COX-l and COX-2 protein expression and Northern blots of mRNA levels will be used to determine the adaptation response to cyclic strain. In Specific Aim 3, the role of epithelial oxidant/antioxidant balance in the stretch-induced regulation of wound closure and COX inhibition will be examined. Investigation of the mechanisms underlying epithelial repair and injury during cyclic strain may lead to better strategies for the management of patients during mechanical ventilation.

This is a multi-center study to determine if Selegiline and/or Tocopheral will slow the progression of Alzheimer's disease. It is a double blind, multi-center, placebo controlled study.

The long-term goals of this work include developing methods to image the stress response in the brains of experimental animals and humans, and to develop methods of manipulating the stress response in order to protect the brain against ischemia and other injuries. Experiments will determine whether local protein synthesis decreases in non-infarcted regions of cortex following focal ischemia in the rat and following global ischemia in the gerbil; and will determine whether the changes of protein synthesis correlate regionally and temporally with the induction of the HSP70 heat shock protein. It is proposed that the decreases in total protein synthesis could be used to indirectly image the stress response in non-infarcted human tissues. Studies of stress gene induction in ischemic human brain will be performed to show that the patterns of HSP70 and other stress gene induction are similar in human and rodent brain. The induction of the heme oxygenase-1 (HO-1) mRNA and protein will be examined in ischemic brains of normal mice, SOD transgenic and SOD knockout mice. Since HO-1 is induced by oxidative and ischemic stress, it is predicted that HO-1 will be induced to a greater degree in ischemic brains of SOD knockouts compared to SOD transgenics. Suppression of HO-1 induction with HO-1 antisense oligonucleotides is predicted to exacerbate ischemic injury more in SOD knockout compared to SOD transgenic mice. Since heme proteins induce the hemeosygenase HO-1 stress protein, it is proposed that prior treatment of animals with heme proteins will protect the brain against ischemic injury, Blockade of HO-1 induction may block protection produced by heme. Lastly, the cloning of the stress gene, methyl malonyl CoA mutase, will be completed and its induction following focal and global ischemia will be characterized, as well as the factors including lipid peroxidation that regulate its expression, Methods used will include rodent focal and global ischemia models, protein synthesis, cloning, DNA nick end-labeling, in situ hybridization and immunocytochemistry.

The functional nature of the projection from the frontal eye field to the brain stem has been studied in the rhesus monkey. Like the frontotectal projection, the frontopontine projection contains cells which discharge in association with eye movements or visual fixation, but not cells which have exclusive peripheral visual responses. The nature of the visual stimuli evoking smooth pursuit were was studied using open-loop visual methods. Superimposition of open- loop position and velocity errors during pursuit maintenance resulted in the generation of eye velocities that indicated that stimulus position as well as stimulus velocity is an important stimulus for the maintenance of smooth pursuit. The time course and dynamics of uniocular saccadic adaptation were studied in monkeys who were made to adapt to a weakened eye. At first the weakened eye had a hysteresis in orbital position, and an orbital-position-dependent saccadic inaccuracy. Both the hysteresis and the orbital position dependent effects were compensated for in a point by point manner with experience. The results suggest that the oculomotor system has a complicated and sensitive corrective mechanisms for the non-linearity of orbital mechanics. Any physical derangement causes maladjustment of this compensation, which can be adapted in time.

Streptococcus pneumoniae (the pneumococus) ranks among the five leading causes of infectious death worldwide. This single species accounts for a large proportion of respiratory tract (pneumonia, otitis media) and invasive (sepsis, meningitis) bacterial diseases. The development of efficacious conjugate vaccines for children has been based upon protection against the few capsule types that commonly cause disease. However, this strategy is now being eroded by the selection for and replacement by non-vaccine types. The initial step in the interaction of the pneumococcus with its host is colonization of the nasopharynx. Experience with conjugate vaccines has demonstrated that interrupting colonization results in herd immunity that amplifies prevention of disease in the population. To better understand the biology of colonization, during the prior funding period, we utilized a murine model to characterize bacterial and host factors that allow for pneumococcal persistence and its eventual clearance from the mucosal surface. These studies show that carriage, much like infection in normally sterile sites, induces acute inflammation. However, opsonophagocytic killing by this neutrophil-dominated response is not completely effective in clearing colonizing organisms. Complete clearance of carriage requires cellular immunity, and is mediated by the gradual Th17-dependent influx of tissue macrophages into the nasal lumen. The key to the success of the pneumococcus in colonization (and disease), therefore, is its ability to evade the initial inflammatory response it elicits. In specific aim#1, we will identify and characterize the complete set of pneumococcal genes and gene products contributing to evasion of opsonization and phagocytic killing by neutrophils in vitro and in vivo. The key to resolution of pneumococcal carriage appears to be recognition and uptake by macrophages. It remains unclear how the unique population of upper respiratory tract macrophages recognizes colonizing pneumococci. In specific aim#2, we will identify the macrophage receptor(s) (including scavenger receptors and C-type lectins) required for non-opsonic clearance during colonization. Finally, in specific aim#3, we will examine the role of capsule type in colonization and whether type-specific differences are explained by i) evasion of opsonophagocytic clearance by neutrophils and ii) recognition and uptake by upper respiratory tract macrophages. Together these studies will provide mechanistic insight into the three main features of pneumococcal carriage;why it is common, why it is transient, and why it varies greatly among isolates by capsule type. PUBLIC HEALTH RELEVANCE: Streptococcus pneumoniae colonizes the mucosal surface of the human nasopharynx and blocking this initial step in host interaction is the key to prevention of pneumococcal disease in the population. Our prior studies of bacterial and host factors affecting colonization show that pneumococci evade the neutrophil response it elicits, but is eventually cleared by cellular immunity leading to an influx of nasal macrophages. The proposed studies provide a mechanistic understanding of i) its ability to inhibit uptake and killing by neutrophils, ii) its recognition by macrophages, and iii) the role of its capsule type in these interactions with phagocytes.

Experimental autoimmune thyroiditis in mice is a very useful model of autoimmune disease since it shows the strongest genetic control by gene(s) within the major histocompatibility complex so far described for such a disease. Preliminary experiments suggested that monoclonal anti-Ia antibodies given before immunization of mice with mouse thyroglobulin emulsified in complete Freund's adjuvant, completely prevented the development of thyroiditis. These experiments will be expanded, giving antibodies after the immunization, to observe a suppressive effect on the established disease. The minimum amount of monoclonal antibody needed for suppression and the effect of several antibodies directed against different Ia antigens will be investigated to show the particular Ia antigen(s) on antigen presenting cells involved in presenting thyroglobulin to T lymphocytes. In mice given monoclonal anti-Ia and immunized with thyroglobulin in Freund's adjuvant, several parameters will be monitored namely, the in vitro lymphocyte stimulation by thyroglobulin, the number of T and B lymphocytes (and the number of their subsets) in the spleen, the number of plaque forming cells in the spleen, the skin delayed-type hypersensitivity reaction after challenge with thyroglobulin, the titer of circulating thyroglobulin antibodies, the IgG concentration in serum, the number of cytotoxic T lymphocytes in lymph nodes, and the magnitude of thyroid infiltration with mononuclear cells. Adoptive transfer experiments will be performed to detect the possible induction of suppressor cells after treatment with monoclonal anti-Ia. The expression of Ig antigens in the thyroid of mice (on thyroid cells, antigen presenting cells and endothelial cells) during the development of EAT and the possible modulation of Ia antigens by anti-Ia antibody will be investigated by immunofluorescence and electron microscopy. The effect of various monoclonal anti-Ia antibodies on in vitro lymphocyte stimulation by thyroglobulin will also be investigated in mice immunized with thyroglobulin. The experiments just described will shed light on the role of class II major histocompatibility antigens in the development of autoimmune diseases and will help to better understand the genetic control of these diseases. The experiments will also suggest methods for specific treatment of human autoimmune diseases.

This application for a KO1 Career Development Award seeks five years of support for education and research activities that will prepare Dr. Justeen Hyde to examine the consequences of child abuse and neglect among adolescents in the foster care system in Los Angeles. Previous research has identified children in foster care with histories of abuse and neglect as a population at high risk for a number of developmental and behavioral problems. Researchers, child welfare advocates and journalists have demonstrated that child welfare programs often fail to address the complex needs of maltreated youth. Given the scarcity of published data on adolescents in foster care, the proposed research and education activities are designed to build a foundation of knowledge about the experience of foster care from multiple perspectives. Education and training activities include: coursework in child/adolescent development, public policy making, and quantitative research methods; participation in formal trainings offered to social workers and foster parents/guardians; and one-on-one mentorship provided by an interdiscplinary group of advisors at the University of Southern California, among others. These activities will prepare her to conduct an ethnographic study of young adolescents, ages 12 to 15, entering foster care for the first time. The specific aims of the proposed research are to: 1) document the histories of maltreatment among adolescents entering foster care for the first time, particularly the age at which maltreatment was first experienced and the types and frequency of abuse; 2) characterize maltreated adolescents' experiences entering and adapting to foster care; 3) identify mediating factors among maltreated adolescents that influence variations in behavioral, emotional and psychological adaptations to foster care placements over time; 4) explore, from multiple perspectives, the most pressing needs of adolescents entering foster care for the first time and appropriate strategies for addressing these needs. A total of 30 adolescents will be recruited from two types of placements and interviewed at three points in time over a 6-month period. A subsample will be recruited for in-depth case studies, which include interviews with case managers and guardians. This project will provide depth to current understandings of the consequences of abuse and neglect on adolescents in foster care. Study findings will be widely disseminated to a range of key stakeholders.

To determine whether phlebotomy to an iron depleted state improves response of chronic hepatitis C infection to alpha-interferon therapy in patients who have previously failed such therapy. A secondary objective will be to correlate iron reduction therapy via phlebotomy with hepatic iron concentration. The primary efficacy endpoints are the response rate (based on loss of hepatitis C RNA in serum) at the end of a 6-12 month course of interferon, and the ability to sustain that response for 6 months after the end of therapy.

This application seeks support for Pediatric Oncology Group activities by Southwestern Medical School. Since 1981 our institution has grown to become the Group's third or fourth largest member, among 40 participating institutions, with regard to patients enrolled on therapeutic studies. Moreover, investigators from our center have risen to positions of leadership on major Group committees, including the Executive Committee, New Agents and Pharmacology Committee, and Lymphoid Diseases Committee (Relapsed ALL Subcommittee). Investigators from Southwestern Medical School also have served and are serving as study coordinators of key Pediatric Oncology Group protocols, including the SIMAL-3 study for relapsed patients with ALL, the Group's second largest study in patient accrual. We are now proposing a major commitment of resources from our center to continue Pediatric Oncology Group research studies. This grant proposal describes the personnel and facilities in our center and its affiliate institution, Cook Children's Hospital in Fort Worth, with which we aim to conduct Group activities. The past history of active participation in clinical cancer research within and separate from the Pediatric Oncology Group and our initial contributions to the Group during the past 3 years provide evidence of this commitment. Specifically, we aim to participate in Pediatric Oncology Group research activities by: 1) Actively enrolling as many patients as possible on Pediatric Oncology Group treatment and ancillary protocols, among the 130-140 new children with cancer seen each year at Southwestern Medical School and its affiliate institution; 2) collecting recording, and submitting all necessary data in a timely and organized fashion in order that our protocol entries continue to receive a high rate of evaluability; 3) making scientific contributions to the Pediatric Oncology Group by actively serving on key committees, as protocol coordinators, and as consultants to the Group's leaders in a variety of areas, particularly regarding new therapeutic approaches to ALL and development of Phase I and Phase II drug trials.

Amyloid b-peptide (Ab), the major molecular component of the cerebral amyloid plaques, appears to play a central role in the neuropathology of Alzheimer's disease (AD). Compounds that prevent the formation of Ab aggregates or that selectively destroy these aggregates are attractive candidates for the development of therapeutic reagents for prevention and treatment of Alzheimer's disease. Also, compounds that interfere with the interactions of amyloid precursor protein (APP) with other factors that are involved in directing it into pathological pathway as well as those that are capable to prevent or destroy intraneuronal accumulations of Ab, are of great interest for developing therapeutic molecules for Alzheimer's disease. In this project we are proposing the possible immunological intervention for prevention and treatment of AD applying phage display technology for the construction of the first anti-Ab single chain fragment variable (scFv) antibody library and the selection of individual phage clones expressing Ab-specific scFv antibody, capable of preventing the aggregation of Ab or dissolving the preexisting aggregates, scFv phage display library enriched in anti-Ab antibodies will be constructed using the first strand cDNA synthesized from mRNA of spleen and lymph node cells isolated from mice immunized with Ab. The constructed library as well as a human scFv library will be used in bioselection procedure to isolate Ab-specific scFvs expressed on phage. The amino acid sequences of antibody complementarity-determinig regions (CDR) will be prepared and used as "mini-antibodies". Evaluation of in vitro Ab aggregation and neurotoxicity in the presence of the selected compounds: scFv antibodies expressed on phage or in soluble form and "mini-antibodies" will be performed. The most promising molecules selected in this project will be proposed for further evaluation in animal models to all researchers interested in these studies. If successful, these molecules could be of interest for passive immunization in humans, may be after modification with substances that would increase their blood-brain barrier permeability. [unreadable] [unreadable]

In order to understand how a pathogenic change in a gene causes disease, it is necessary to recognize how pathogenic mutations could affect a protein structure-function, protein-protein interactions in protein networks and how these changes could be associated with clinical parameters describing the disease phenotype. We imply molecular modeling to build protein structure, simulate the effect of pathogenic missense changes, and provide a quantitative analysis of their impact on protein structure and stability. Here we use oculocutaneous albinism, autosomal dominant maculopathy, and X-linked retinoschisis (XLRS) as our disease models. 1. Oculocutaneous albinism (OCA) is a rare genetic disorder of melanin synthesis that results in hypopigmented hair, skin, and eyes. There are currently four types of OCA. For the first time, three full-length protein atomic structures, TYR (OCA1), TYRP1 (OCA3), and SLC45A2 (OCA4), were successfully modeled by homology and in silico analysis of missence changes from the NEI/NHGRI molecular diagnostic study has been performed. TYR is a type I trans-membrane monooxygenase. The 4-helix bundle is structurally conserved in different species to carry CuA and CuB ions essential for the catalytic reaction. The active site is formed by 6 His residues structurally coordinating the copper positions. Several missense changes from the collaborative NEI/NHGRI molecular diagnostic study were analyzed. S50L is found in the Cys-rich motif 1 of tyrosinase, whereas R298W is located within the Cys-rich motif 2. The mutations N364H, P384A, D394N, D437N and R403V disrupt the coordination of the copper ion center. A490D affects conformation of helix located in melanosomal membrane. TYRP1 is a type 1 membrane protein. This protein also has 2 Cys-rich motifs and an active site with 6 His residues coordinating 2 metal ions of unknown nature that could be either copper or zinc. A heterozygous missense mutation, A24T, was found in the border between a signal peptide and Cys-rich motif 1 of TYRP1. The structure of SLC45A2 is predicted as a multi-pass trans-membrane protein. The missense mutation L60R is predicted to be deleterious. To test our predictions on tyrosinase activity we engineered a construct of human C-terminal truncated tyrosinase, hTyrCtr. The expression of hTyrCtr in E. Coli was confirmed by Western blot analysis. Metal affinity chromatography shown poor binding to the column which suggests that C-terminal His-tag peptides have decreased binding capacity to the IMACS resin. In addition, L-Dopa enzymatic assay demonstrated that hTyrCtr is expressed as a non-active enzyme which might be due to either the loss of Cu2+ ions in a catalytic site or protein partial misfolding. In contrast, similar protein construct was implied for the protein expression in larvae. We shown that the hTyrCtr, and 2 mutant variants, R422Q and R422W, are active soluble proteins which catalyzes the rate-limiting conversions of tyrosine to DOPA and DOPA to DOPA-quinone. In perspective, a detailed understanding of protein structure and the mechanisms controlling tyrosine-modified tyrosinase interactions would allow to establish molecular chaperone screening for a future medical treatment of patients with the OCA-1B albinism. 2. Assembly of elastic fibers is critical for structural development as well as proper functioning of the extracellular matrix. Elastin and 10-nm fibrillin containing microfibrils form the major components of elastic fibers, which form integral part of extracellular matrices including Bruchs membrane. One of fibrillins, fibrillin-2 or FBN2, is a 2,912 amino acid polypeptide which consists of one amino-terminal trans-membrane domain, 4 epidermal growth factor-like (EGF) domains, 43 calcium-binding consensus sequences (Ca_EGF domains), and 9 transforming growth factor 1 binding protein-like (TB) domains. FBN-2 has 363 cysteine residues. The amino acid sequence of fibrillin-2 been used to generate a native and mutant variant structures for the Ca-EGF motifs 12-19 by homology modeling. Protein fold of Ca_EGF domain is maintained by 6 conserved cysteines which form 3 SS-bridges. In addition, negatively charged conserved residues are either involved in direct ligation to calcium or involved in stabilizing the calcium-binding site. Calcium ion improves the fold stability, help to fix a relative orientation of two neighbor Ca_EGF domains, and stabilize a spatial orientation of FBN-2. Disease-causing mutation E1144K introduces a positive charge into the negatively charged cavity and decreases the Ca-binding affinity. The interaction of K1144 and E1178 change a relative orientation of a neighbor domain. E1438K mutation is expected to have a similar structural effect. Both mutations are associated with a severe phenotype of disease and could change of microfiber packing and elasticity. The M1247T change is affecting the hydrophobic surface loop. The SS-bridge C1246-C1257 stabilizes the native fold of a protein by lowering entropy of the polypeptide chain and by condensing hydrophobic residues from the surface loop into local hydrophobic core using hydrophobic interactions. Thus, the mutation M1247T might affect the SS-bond stability and/or intermolecular interactions. Other mutations are mild changes. Mutations with severe phenotype are likely to cause a change in the fiber flexibility, packaging, and pathogenicity. This might cause the loss of elastic fibers, thickening and calcification of Bruchs membrane which are associated with dominant maculopathy and AMD pathophysiology. 3. Gene mutations that encode retinoschisin (RS1) cause X-linked retinoschisis (XLRS), a form of juvenile macular and retinal degeneration that affects males. Molecular modeling predicted an association between the type of structural RS1 alterations and the severity of full-field ERG phenotype in all but the oldest group of patients. This is now a second study (Hum Mol Genet 19:1302, 2010) that indicates a genotype-ERGphenotype correlation, and it was done with a totally separate and independent cohort. There was a significant association between the predicted severity of RS1 perturbation and both photopic and scotopic ERG b/a-ratios, but only for one age group (15-30 years). Severe RS1 missense changes were associated with a lower ERG b/a ratio than for mild and moderate missense changes, suggesting a quantitatively distinct ERG phenotype. Age-related differences in dark-adapted ERG parameters are consistent with those reported previously in the RS1 knockout mouse. 4. One of possible clinical implications in a human eye disease is using chaperones for the stabilization of native protein structure in mutant variants affected by genetic mutations. This stabilization could be performed in controllable fashion by using small heat shock proteins (sHSPs) with genetically engineered structure. Our recent study have indicated a role for changing of protein hydrophobicity in the thermal adaptation of alpha-crystallin A and suggested ways to produce sHSP variants with altered chaperone-like activity. In this work we use molecular modeling, computational biology, and side-directed mutagenesis to evaluate the effect of mutations and to establish a link between sHSPs hydrophobicity and physiological temperatures. sHSPs maintain cellular homeostasis by preventing stress and disease-induced protein aggregation. In addition, our work provided an evidence for an evolutionary mechanism that has adapted chaperone activity to different environmental temperatures though the alteration of hydrophobicity at crucial locations in the protein structure. This combination of experimental and computational design potentially could be used to create a new generation of artificial chaperones with a purpose to improve stability of mutant variants in inherited eye disease.

The Illinois Prevention Research Center (IPRC) of the University of Illinois at Chicago has a primary mission to reduce health disparities through behavior change approaches by conducting health promotion and disease prevention research across the lifespan, translating research into practice and measuring the real-world effectiveness, dissemination and translation of health promotion and disease prevention programs and initiatives. The IPRC's activities concentrate in seven areas and aim specifically to: (1) Establish and maintain collaborative relationships with community and academic partners, including a Community Committee and CommunityAdvisory Board, university faculty, students, public health agencies and non-traditional partners to address data driven health disparities; (2) Engage the community and establish a research agenda to conduct participatory research on modification of health behaviors associated with increased risk for major chronic conditions such as obesity and diabetes; (3) Conduct community training, graduate student mentoring and continuing education opportunities in evidence-based public health research and practice; (4) Implement and evaluate innovative programs and interventions and communicate findings to community and professional audiences; (5) Develop a cadre of well trained researchers, practitioners, students and community members to enhance the capacity of communities, public health agencies and organizations, and prevention centers to conduct evidence-based participatory research; (6) Disseminate and translate intervention protocols and research findings to the public health community, local, state, and national policy makers, community-partners, and the research community; and (7) Identify contextual factors that may influence implementation of activities and achievement of outcomes. IPRC activities are guided by the CDC's Logic Model for the Prevention Research Center Program and informed by state-of-the-art conceptual frameworks for community-participatory research, development of intervention strategies at the individual, organizational, and environmental level, and diffusion of innovations. The evaluation of the IPRC focuses on our success in achieving the goals related to each of the seven overall aims.

Mature B cell lymphomas are a diverse set of diseases which share the property of having failed to complete the differentiation process into plasma cells or undergo apoptosis. PRDI-BF1 and its murine homologue Blimp-1 are transcriptional repressors and act as a molecular switch to commit activated B cells to become non-dividing plasma cells or undergo apoptosis. In this role PRDI-BF1 has broad significance for normal humoral immune responses and in malignancies of mature B cells and plasma cells. We hypothesize that failure to induce PRDI-BF1 expression or function contributes to the persistence of lymphomas and as such therapies designed to rescue PRDI-BF1 function may be important in treating lymphoma. Artificial overexpression of PRDI-BF1 leads to apoptosis in multiple lymphoma lines. Furthermore the hypothesis is supported by our finding that treatment of lymphoma cells with chemotherapeutic agents such as proteasome or histone deacetylase inhibitors leads to an early induction of PRDI-BF1 expression. We have made the recent discovery that PRDI-BF1 directly recruits the histone methyltransferase G9a to mediate silencing of interferon-beta. This suggests chromatin remodeling is the means by which PRDI-BF1 drives terminal differentiation of B cells and kills lymphoma cells. The impact of chromatin remodeling driven by PRDI-BF1 in this process will be investigated. PRDI-BF1 is transcriptionally regulated by unknown mechanisms. This will be investigated and will provide the first detailed understanding of transcriptional regulation at this critical developmental time point and may suggest new avenues to induce PRDI-BF1. Finally, PRDI-BF1 function requires a highly conserved SET-like domain but its activity is unknown. We will identify the proteins interacting at this site and determine their role in PRDI-BF1 activity. Thus this proposal will shed new light on the events occurring during differentiation and may indicate new targets to affect the growth and persistence of lymphomas. [unreadable] [unreadable]

Our objectives are to promote excellence in biomedical research through development and maintainance of high quality animal resources. To meet these objectives we propose three major projects within the animal resources program: 1. Renovate animal surgery facilities to provide a suitable environment for recovery surgery and meet DHHS guidelines. 2. Improve satellite animal housing facilities to meet DHHS guidelines. (No funds are requested for this project.) 3. Improve the central animal facilities to provide better utilization of space and meet the needs of the research faculty by upgrading animal housing and procedural areas. These improvements will benefit animal research projects totalling approximately $22 million, and will add to the substantial improvements the College has already made to upgrade animal resources, which have cost more than $5 million over the last two years.

In the course of studying inflammatory muscle diseases (polymyositis, dermatomyositis, and related diseases), we have encountered patients with many other muscle diseases. We have studied patients with two genetic metabolic myopathies in detail: phosphofructokinase (PFK) deficiency, and acid maltase (acid alpha-glucosidase) deficiency (also known as Glycogen Deficiency Type II or GSD II or Pompe Syndrome). For the last several years, we have focused particular attention on GSD II because of its close resemblance to myositis. It is a recessively inherited lysosomal storage disease in which glycogen accumulates in the lysosomes, particularly those in skeletal muscle. When the enzyme, known also as GAA, is completely absent, affected infants are usually sick at birth and die in infancy of heart failure, rarely living longer than a year. Apparently the enzyme is needed in the heart only in infancy since affected individuals with even a small amount of effective enzyme survive without cardiac involvement. Survivors generally develop a progressive proximal myopathy with pulmonary failure secondary to diaphragmatic involvement in later years. The long-term aim of our studies is to prevent and to treat this devastating disease, particularly the adult variety, in which the level of enzyme is only slightly (less than two-fold) below the minimum necessary for a normal life. From quite early in the project, it was driven by the belief that GSDII, being a lysosomal storage disease, should be amenable to enzyme replacement and possibly gene therapy, and since there are well over 30 diseases of abnormal lysosomal storage, our results might have wider application. There are estimated to be several thousand cases of GSD II worldwide, and hundreds in the United States, of which many are fatal in infancy (Pompe Syndrome). Our guiding plan has been to do research directed towards therapy, but without trying to move into areas likely to be covered by pharmaceutical companies. We aimed to develop tools that would advance the development of therapy while at the same time learning new biology or developing techniques that might be applicable to other lysosomal or other enzyme deficiency diseases, especially those in muscle. We have followed lines of research designed to provide answers necessary for the development of optimal therapy for GSD II. Starting with the knockout mice, a human GAA expression vector was put in transgenically under the control of either a skeletal muscle or a liver specific promoter controllable by doxicycline [164]. These mice were designed to determine whether the liver, a much easier organ to target, could serve as a suitable depot organ for the synthesis and secretion of GAA that could be taken up by the heart and by skeletal muscle. We discovered that the liver was a superior site to the skeletal muscle for the repair of both heart and skeletal muscle. [164] The achievement of therapeutic levels with skeletal muscle transduction required the entire muscle mass to produce high levels of enzyme of which little found its way to the plasma, whereas liver, comprising <5% of body weight, secreted 100-fold more enzyme, all of which was in the active 110 kDa precursor form. In the past year, we have continued studies on the therapy of the mouse disease. A major step forward was the development of a mouse model that could be used for pre-clinical study of the effects of long term therapy of the disease by repeated injection of the recombinant human enzyme. The barrier to such study has been the immunological response of the mice to the foreign human protein. After a few doses, the animals die of what appears to be anaphylactic shock. In the experiments described above, we developed a strain of mice that produces a minute amount of GAA in the liver ? an amount below the level of biochemical detection, but the mRNA is detectable by PCR. The liver glycogen is very slightly reduced, but the glycogen in other tissues is unaffected so that the mice are phenotypically unchanged. These mice develop no detectable immune response and can receive weekly injections of rhGAA for many months at low or high dose and have no adverse immunological effects. These mice, immunologically speaking, resemble those human infants who have tiny levels of the enzyme and are thereby tolerant to injection of the recombinant protein. The work on this strain has been published, and the mice have been distributed to many investigators and are now used for pre-clinical studies [173]. Our own studies with endogenous expression of the GAA gene in the skeletal muscle {172] and with the intravenous injection of high doses of rhGAA over long periods have shown the remarkable and discouraging finding that unless the enzyme is turned on (controllable transgene) early or the injections are begun early, the stored glycogen in skeletal muscle is only incompletely removed, and clinical recovery of muscle strength is marginal. By contrast, the glycogen stored in cardiac muscle is cleared well. This finding accords with the small clinical experience so far in human infants, in which heart failure is reversed, but skeletal muscle strength has responded little or not at all. This failure of response has now become a major focus of our work. We have discovered that clearance tends to be very good in Type I skeletal fibers and very poor in Type II fibers. We are exploiting the different proportions of these two types of muscle in different fibers to study the possible reasons for this imbalance. The amount of the mannose-6-phosphate receptor is clearly lower in Type II fibers than in Type I fibers or heart muscle, and so are the levels of several other proteins in the lysosomal pathway. We are, with the collaboration of the NIAMS Light Imaging Unit, developing a technique to measure the pH of lysosomes in living, unfixed single muscle fibers in order to determine whether the pH in lysosomes with a large quantity of stored glycogen in Type II fibers is optimal for GAA. We are exploring the difference in gene expression between the two types of muscle fiber in Pompe mice of various ages to look for pathogenetic clues as well as to look for therapeutic avenues. In other studies, we have been working for the past year with the Rapoport lab in NIA to attempt to deliver therapeutic levels of GAA across the blood brain barrier to clear CNS neurons of the large quantities of stored glycogen seen in Pompe mice, and which it is presumed will accumulate in the brains of Pompe infants who survive infancy with the help of exogenous enzyme replacement.

Currently, the standard of care for cancer therapy has limited specificity to only tumor tissue, and can cause devastating side effects. Cancer vaccines utilize the immune system to generate a sustainable and specific anti-tumor response, providing a promising means to improve cancer therapy. However, delivery of vaccine antigen/peptide to dendritic cells (DCs) remains a limiting factor for persistent anti-tumor immune response. Thus, there is a profound need for designing delivery vehicles for vaccine peptides. Current nanocarriers mainly consist of liposomes and polymers, which are larger in size (>100nm in diameter) and tend to stay at injection sites while smaller nanoparticles drain into lymph nodes with subcutaneous injections. They also have known toxicities post degradation. Gold nanoparticles (AuNPs), on the other hand, have three advantages: potential lymph drainage due to their small size, simplistic synthesis/conjugation process and passively collection in antigen presenting cells. Although peptide conjugations on to AuNPs are not new, this proposal aim to be the first to utilize layering techniques onto AuNPs to deliver large does of vaccine peptides for enhanced anti-tumor effect. Thus, the goal of this proposal is to design an effective, simple, versatile gold-based nanovaccine (AuNV) platform for improved anti-tumor immune response. Three specific aims will be carried out to achieve this goal. In aim 1, class I peptides, which stimulate tumor-killing cytotoxic T cells, are conjugated onto polyethylene glycol (PEG) coated AuNPs. Peptides used in this proposal are from a model antigen (ovalbumin) and common melanoma antigens (gp100 and Trp-2). From preliminary characterizations of AuNVs, conjugation yield is very high (~90%) and the overall particle size remained sub-100nm. This will allow potential lymphatic drainage post subcutaneous injection. In vitro Immune response from AuNVs will be measured by interferon-? release, a marker for vaccine anti-tumor efficacy. In vivo efficacy of AuNVs will be assessed by splenocyte sensitization, tumor rejection and treatment assays. As part of aim 1, biodistribution of AuNPs from subcutaneous injections will be explored and be the first to look at distribution at the cellular level. In aim 2, class II peptdes, which stimulate helper T cells, will be incorporated in the AuNV design. In vivo enhancement of antitumor efficacies will be assessed with known class II peptides. However for unknown class II peptides situations, peptide pools will be used to incorporate both class I and class II epitopes. In aim 3, CpG, a known inflammatory stimulant used for cancer immunotherapy, on AuNPs will be combined with AuNVs for a complete vaccine regimen. Future applications of AuNVs are substantial. This design would dramatically lower the cost of immunotherapy compared cellular vaccines. They are easily synthesized, versatile and can potentially target later stage cancers by using inducing anti-tumor immunity over the whole body. PUBLIC HEALTH RELEVANCE: The American Cancer Society estimated over 1.5 million new cancer cases and over half a million deaths in 2010 with national cancer care expenditures estimated to be 104.1 billion dollars since 2006. Cancer vaccines are effective but are limited by vaccine delivery methods and enormous cost for cellular based methods. Gold-based nanovaccines (AuNV) designs are cost-effective, versatile, and can improve the delivery and efficacy of cutting edge cancer immunotherapies and hopefully improve the survival and quality of life for cancer patients.

An understanding of the relationship between malarial parasites and their hosts will ultimately aid in developing vaccine strategies. Malarial parasites release heat-stable soluble antigens into the sera of human infected with Plasmodium falciparum. The immunologic role of these antigens is unclear. We recently described a heat-stable antigen (Py117) that circulates in the sera of mice during Plasmodium yoelii infection and an immunologically-related, crossreactive protein (Pf93) that circulates in the sera of humans with P. falciparum malaria. Preliminary data suggest that soluble Py117 and an Ag of merozoites share common epitopes. This has led us to hypothesize that Py117 may provide a protective mechanism for the parasite by "tying-up antibodies before they reach important receptor molecules on the surface of merozoites. We also hypothesize that the secreted antigen is poorly immunogenic thereby increasing the probability of high levels of circulating antigen. These hypotheses will be tested using the P. yoelii-mouse model system. Experiments proposed include evaluating the antigenic relationship between the secreted and merozoite- associated forms of the antigen, determining if enhancing the immune response to Py117 by immunization or removal of immunoresponsiveness to Py117 by neonatal tolerance will alter the course of malaria infection, determining if the antibody response to Py117 is H-2 restricted and comparing the kinetics of antigenemia, anti-Py117 Ab production and possible formation of immune complexes in strains of mice that differ in susceptibility to malaria. Basic information gained from the mouse model should aid in our understanding of the human situation.

Band 3, the major intrinsic glycoprotein constituent of the erythrocyte membrane is responsible for anion transport. Selective in situ proteolysis of this protein has been used to define domains of the protein, certain of which are glycosylated. It is the objective of this research to determine the sequence, linkage and anomeric configuration of the carbohydrate chains of band 3 and to assign them to regions of the peptide. Isolation of micromolar quantities of band 3 and its proteolytic fragments from single units of blood will be coupled wth carbohydrate structural techniques operating on the micromolar level. Since detergent solubilization of the extremely hydrophobic peptides of band 3 limits the effectiveness of exhaustive proteolysis, two new chemical methods will be used to depolymerize the peptide chain for oligosaccharide isolation. High pressure liquid chromatography will be used to fractionate long oligosaccharides while oligomers up to six residues will be gas chromatographed. The carbohydrate structures of oligosaccharides will be determined by methylation analysis, mass spectrometry of the oligosaccharides, high field proton nuclear magnetic resonance and by circular dichroism.

This program continues to study the behavioral effects of brain lesions in children and adults. The aim of our experiments is directed toward five topics: 1. Language and related nonverbal capacities considered as significant indices of left-hemisphere function. 2. Certain perceptual achievements, particularly the recognition of faces and objects in atypical views, as tentative indicators of right-hemisphere processes. 3. Personal and exptrapersonal orientation, as measured in several tasks, to be varied in such a way as to permit a better distinction of learned from relatively unlearned components in performance, and of predominantly parietal from predominantly frontal lesion effects in either hemisphere. 4. Memory, as assessed in tests involving relatively short-term as contrasted with longer-term memory, and varying conditions of retrieval. 5. Mood: Specifically, we shall continue our assessment of therapeutic outcome, neurologic status, and behavioral test performance after surgical interruption of the cingulate gyrus and bundle in selected cased of persistent pain or severe affective disorders. All except the last two of these questions will be investigated, in a manner as nearly comparable as we can accomplish, in children and adults with brain pathology. We realize that there are severe difficulties, both in establishing comparability of lesions across these age groups, and in achieving any certainty of the comparability of tasks; and it will not be possible to do equal justice to all the questions in the next few years, since so much depends on the rates at which appropriate clinical cases will appear. Nevertheless, by monitoring a properly prepared set of behavioral tasks and by keeping the five topics as signposts, we are more likely to make maximal use of the patients who might come our way.

Breast cancer among Filipina American women represents a major but largely neglected cancer disparity; -first, though not as highly visible as other Asian subgroups, the Filipino population in the US is large, second among Asians only to the Chinese. Second, Filipinas have higher rates of breast cancer incidence than most other Asian subgroups. Third, resources for and data regarding Filipina women with breast cancer are almost non-existent. In 2004, West Bay Filipino Multi-Service Center (West Bay) joined forces with the UCSF: comprehensive Cancer Center (UCSF-CCC) and the San Francisco General (SFGH) Breast Care Program to start the first Filipina breast cancer support group (Sinag Tala) in San Francisco. However, group attendance is uneven and the little research available on Filipino Americans suggests that a "one size fits all" approach to outreach would not be effective in this highly relational (collectivist-oriented) culture. The proposed study will elucidate the meanings of survivorship and breast cancer support in this community, and inform how to design more culturally appropriate outreach building upon existing community resources social networks) for the women who need them most. We will utilize multiple qualitative methods because each taps different types of data (participant observation, individual ethnographic interviews, and small group interviews) to achieve our specific aims to: 1) identify beliefs and values associated with breast cancer, survivorship, and support; and 2) create and pre-test culturally resonant outreach themes and channels based on core cultural values and existing community resources. The conceptual framework is drawn from theories of quality of life, social capital, and relational culture. The product of this study will be new, culturally appropriate outreach themes and outreach communication channels designed to encourage Filipina breast cancer patients and survivors to take part in support groups. Just as access to and participation in adequate and meaningful social support has been shown to improve the quality of life of White breast cancer patients, developing meaningful outreach themes and channels to link Filipina breast cancer survivors to support services is likely to improve the quality of life of Filipina breast cancer survivors and to address survivorship disparities in this community. Formative research into culturally relevant outreach for support services has implications across the stages of survivorship. [unreadable] [unreadable] [unreadable] [unreadable]

Prostanoids exert profound physiological effects on the urinary tract including nociception, induction of smooth muscle contractility, enhancement of cell proliferation, and blood flow regulation. The rate-limiting step in prostanoid synthesis is catalyzed by cyclooxygenase (COX). This enzyme exists in two isoforms: COX-1 (usually constitutive) and COX-2 (highly inducible). COX-2 is substantially induced with distension and stretch of urinary tissues. A new area of interest involving COX activity in urinary tract has been identified: urothelial cell stretch-induced COX-2 expression. In cell culture, urothelial stretch produces a 10-fold induction in COX-2 levels within 6 hours of applying stretch. Since COX-2 upregulation would lead to an increase in soluble prostanoids, this process represents a critical link between urothelial stretch and afferent pain perception, muscle contractility, and cell proliferation. While the specific model of this proposal involves urothelial cells cultured from the ureter, this novel concept has broad implications in the pathophysiology of the urinary tract. The goal of this project is to identify cellular pathways of stretch-induced COX-2 expression and thereby provide several potential targets for drug therapy. Based on titerature reports in other cells, and preliminary data, the hypothesis of this proposal is that stretch activation of PKC and p38 MAP kinase signaling cascades induces COX-2 in urothelial cells. The Specific Aims are: I. To characterize COX-2 induction in human urothelial cells subjected to mechanical stretch. This will be accomplished by determining the optimal duration and degree of stretch for COX-2 induction in human urothelial cells and determining if stretch-induced COX-2 expression is regulated transcriptionally and/or post-transcriptionally in human urothelial cells. II. To determine if stretch induction of COX-2 is mediated by increased intracellular calcium concentrations [Ca++] i and subsequent PKC and p38 MAP kinase activation. This will be accomplished by determining if stretch of urothelial cells increases [Ca++] i, and activates PKC and p38 MAP kinase, if inhibition of calcium, PKC, or p38 MAP kinase attenuate stretch-induction of COX-2, and if p38-dependent transcriptional factors are involved in stretch-induced COX-2 expression. The findings of this exploratory proposal have great potential to expand into several dynamic lines of research and may identify novel approaches to treating urinary tract disorders such as bladder outlet obstruction, ureteropelvic junction obstruction, and interstitial cystitis.

The isolation of alpha-aminolevulinate synthetase (ALA-S) from livers of induced chick embryos is being continued; with purified enzyme, an antibody will be developed to study the induction mechanism and the transport of the enzyme from the microsome into the mitochondria where it is active. Chlorinated hydrocarbons both induce ALA-S synthesis and block the biosynthetic chain so that uroporphyrin accumulates. The mechanism of the block will be sought. Iron and metabolites of the chlorinated hydrocarborns may be involved. These studies may lead to our understanding of the environmental toxic effects of the chlorinated hydrocarbons including tetrachlorodibenzodioxin. In Friend leukemia cells dimethyl sulfoxide caused their differentiation into erythropoetic cells. The steps in the development of the enzymes of the heme biosynthetic chain will be examined to note whether there is a sequence in time of the developing enzymes that follows the sequence of enzymes of the heme biosynthetic chain. In animals, the synthesis of the first committed product of the heme biosynthetic chain is ALA. It is well establised that ALA is formed by an enzyme that uses glycine plus succinyl CoA as its substrates. In plants there is evidence that ALA may be formed from other substrates; the mechanism of ALA formation will be studied with appropriate labelled substrates and with metabolic inhibitors.

To meet the dual threats of emerging infectious diseases and engineered biowarfare/bioterror agents, there is a pressing need for more efficient systems for vaccine development. TRIAD, or the Translational Immunology Research and Accelerated [Vaccine] Development program, based in the Biotechnology Program at the University of Rhode Island, has pioneered the development and application of an integrated "gene to vaccine" in silico, in vitro and in vivo vaccine design program to address this need. TRIAD has selected Category A pathogens F. tularensis, Category B agents Burkholderia pseudomallei and Burkholderia mallei, and emerging infectious diseases (HCV, H. pylori, tick borne diseases) as the focal point of this proposal. Using the TRIAD immunoinfomatics Toolkit, TRIAD investigators will pursue the development of second generation epitopebased immunome-derived vaccines for these pathogens, while addressing the failings of prior generations of epitope based vaccines. We will maximize payload quantity using validated immunoinformatics tools that permit selection of optimal T cell epitopes that are highly conserved and immunogenic. We will ensure payload quality by choosing epitopes that demonstrate antigenicity in human PBMC as well as protection in established murine models of disease/infection. We will select a combination of promiscuous Class II epitopes and Class I supertype epitopes will provide >99% coverage of human populations. We will avoid cross-reactive epitopes and explore the role of regulatory T cells in the context of improving vaccine design. Where appropriate, we will combine our epitope-driven vaccines with broad-spectrum anti-LPS vaccines. We will optimize payload, delivery, formulation, and adjuvanting by exploring a range of delivery options [Dendritic cells, DEC205, DNA, electroporation, mucosal delivery). The TRIAD project aims to develop vaccines demonstrating broad spectrum activity include crossprotective and multiple component vaccines, and delivery technologies that have the potential to be effective against multiple emerging and re-emerging infectious diseases. Our efforst to merge rational design with rcentadvances in vaccine deliverywill manifest in a coordinated toolkit and a cadre of informed users, who will be ready and able to apply the tools to discover new treatments for emerging infectious disease and biodefense.

The purpose was to determine if whole body and skeletal muscle glutamine and leucine metabolism are altered in HIV-infected subjects. Six HIV-infected men with chronic stable opportunistic infections and 10% weight loss, 8 HIV-infected men and 1 woman without wasting and 6 HIV- negative age-and weight-matched men were studied. Constant intravenous infusions of stable isotopically labeled leucine and glutamine were used to assess plasma GLN and LEU rates of appearance by mass spectrometric measurements of stable isotope content. Fasting whole body protein breakdown and synthesis rates were increased above control in the asymptomatic HIV-infected subjects and further increased in symptomatic HIV-infected subjects. These findings suggest that the rate of muscle protein breakdown was increased while the rate of muscle protein synthesis was unchanged in symptomatic HIV-infected subjects. Since lymphocytes require GLN release may be increased to provide energy for proliferati ng lymphocytes.

The goal of the proposed research is to understand the mechanisms of how genes are regulated in different tissues. This will be accomplished through the study of the human gene encoding debranching enzyme and molecular analysis of Type III glycogen storage disease. In this disease there are patients who lack debranching enzyme activity in both liver and muscle, and patients with enzyme deficiency confined to liver; yet the enzyme is a monomeric protein and appears to be identical in all tissues. The proposed research hypothesizes that debranching enzyme in liver and muscle is encoded by a single gene; its expression, however, is under separate genetic control. Patients with various clinical and enzymatic subgroups of Type III glycogen storage disease are being followed at Duke Medical Center; fibroblasts from these patients have been collected and stored. The debranching enzyme has been purified from porcine muscle and the antibody characterized. The cDNA coding for debranching enzyme from both human liver and muscle will be isolated by screening lambda gtll human cDNA libraries with antibody against porcine debranching enzyme. The debranching enzyme mRNA in these two tissues will be compared by Northern blot analysis, cDNA sequencing and/or sequencing of mRNA by primer extension. The chromosomal gene(s) coding for debrancher enzyme will be isolated, and the molecular structure and number of copies of the genes will be determined. Information about the number of debrancher genes and the sequences of the mRNA from liver and muscle will be used to design strategies to investigate the mechanism responsible for control of tissue-specific debrancher gene expression. This may involve sequencing of the 5' flanking region for two possible transcription initiation sites, or in the case of two different mRNA sequences encoded by a single gene, sequencing the regions of the gene that specify the mRNA unique to liver or muscle. The defective chromosomal debrancher genes from patients with disease confined to liver will also be cloned and the mutations characterized. Molecular classification of Type III glycogen storage disease will be performed at the protein, mRNA, and gene levels. These molecular findings will be correlated to the type and severity of clinical phenotypic expression. Information gained on molecular structure of glycogen debrancher gene and human mutants lacking debrancher activity may lead to identifying DNA regions important in the control of tissue-specific gene expression, and may provide a model for mechanisms responsible for clinical variability in other genetic disorders.

The effect of experimental conditions related to the induction of atherosclerosis will be studied with an emphasis on changes in elastin fiber formation. The conditions which will be studied include hypertension, hyperlipidemia, and hyperinsulinemia. Hypertension will be induced by galactose intoxication, which in avians produces marked plasma hyperosmolarity. Hyperinsulinemia will be studied using both in vitro and in vivo systems. In addition, properties of proforms to tropoelastin will be studied after isolation and characterization in an attempt to provide information on the metabolism of elastin. One of the key features of the work will be the assessment and identification of events which lead to increases or decreases in net elastin synthesis. One aspect of the work will be the investigation of changes in elastin synthesis in older animals or through the lifecycle of animals exposed to conditions which are known to alter the metabolism of the vascular wall.

We plan to isolate and analyze zebra fish mutants with altered visual systems. Of particular interest initially will be mutants with altered organizations of retinal cell types, and specific enrichment procedures will be utilized to aid in the isolation of such mutants. The mutants will be analyzed by behavioral, anatomical, physiological and genetic methods. In order to increase the frequency of mutants in the populations subjected selection, we will expose fish to mutagens at appropriate stages in their life cycle. Since most mutations are recessive, we plan to generate homozygous fish from mutagenized eggs in order to enable us to recognize mutant phenotypes. Procedures for generating homozygous fish have already been developed in our laboratory.

The importance in the understanding of the basic mechanism of human plasminogen activation is underscored by the wide use of fibrinolysis in the therapy of some blood clotting and cardiovescular disorders. Staphylokinase is a plasminogen activator protein secreted by strains of Staphylococcus aureas. Limited data from ours and other laboratories suggest that this protein activates plasminogen by strong binding of the two proteins, similar to the mechanism of streptokinase. The lack of information on staphylokinase has been due to the difficulty to obtain sufficient amounts of pure protein. This project proposes (1) to obtain pure staphylokinase form cloned gene in E. coli, (2) to examine themechanism of human plasminogen activated by kinetic and binding studies, (3) to determine the functional groups involved in plasminogen binding and activation by differential chemical methods, and (4) to change the functional groups of staphylokinase using site-directed mutagenesis of cloned gene. These changes involve lysines, tyrosines, tryptophan, N-terminal regiona and C-terminal region. The changes on the N- and C-terminal regions will test the functional roles of these residues. The changes in Tyr and Trp will be done in conjunction with 19F-nuclear magnetic resonance studies of the role of these residues. The studies described above will permit a greater understanding of mode of plasminogen activation by staphylokinase in specific and of contact activation of serine protease zymogens in general.

Heart disease is the leading cause of morbidity and mortality in the Western World. As this is mostly related to disorders of the blood supply to the heart, it is very important to be able to assess the perfusion of the heart wall. Conventional methods for assessing myocardial perfusion have many limitations, including relatively low resolution and poor ability to provide quantitative data. Magnetic resonance imaging (MRI) of the "first-pass" kinetics of contrast enhancement by a bolus injection of contrast agent is a very promising method for noninvasive assessment of myocardial perfusion, with higher resolution than conventional radionuclide imaging methods. However, current approaches to MRI perfusion studies have only been "semiquantitative", due to several technical difficulties. We have been achieving very encouraging initial results in making absolute measurements of regional myocardial perfusion, using novel MRI methods that we have designed to overcome the limitations of conventional MRI approaches. In the proposed research, we will further develop our MRI methods, and the associated image analysis methods, for quantitatively measuring myocardial perfusion. We will perform a series of studies on normal subjects to establish the expected range of variability of the results. We will also perform a series of studies of patients with coronary artery disease, comparing the results of our MRI methods with those of conventional methods used to assess the blood supply to the heart, including radionuclide imaging, CT angiography and conventional cardiac catheterization (including invasive measures of flow reserve). The significance of the proposed work is its potential to achieve more accurate and higher resolution assessments of blood flow to the heart wall than current conventional methods can provide. This would be very useful for aiding treatment decisions and for following the results of therapy in patients with ischemic heart disease, a common and serious clinical condition. The quantitative MRI methods we propose to develop for the assessment of cardiac vascular disease may also be applicable to assessment of the perfusion of other important organs, such as the brain, which is subject to stroke, and in other important disease processes, such as cancer, where the disease evolution may be related to blood flow.

The work being carried on is quite varied in method and approach, but has one consistent aspect: the application of conditioning and training theories and methods to the empirical analysis of concepts important to theories of social behavior, motivation, psychopathology, and personality development. The major focus has been aversive control but now the emphasis is shifting to the aversive, motivational after-effects of terminating positively-reinforcing stimuli rather than the aversive, motivational effects of the onset of aversive stimuli. Specific research areas: (1) the interaction of Pavlovian conditioning and instrumental learning; how appetitive and aversive Pavlovian conditioning modify instrumental responses of either appetitive or aversive origin; (2) tests of an opponent-process theory of affect and motivation.

It is known that individuals with peripheral arterial disease are at increased risk for cardiovascular morbidity and mortality. In order to facilitate design of a trial to evaluate the effects of intervention on risk factors on morbidity and mortality in such patients a feasibility study (ADMIT) has been designed to evaluate the ability to recruit, treat and retain volunteers in anticipation of a large scale multi-center study focusing on risk factor reduction.

Importance: Intimate partner violence (IPV) is a serious national problem noted in Healthy People 2010 and the CDC Injury Research Agenda. The Deaf community is an understudied linguistic, cultural, and disability minority group whose IPV risks and experiences are virtually unknown. Preliminary studies, most conducted by this research team, suggest that the deaf population experiences higher levels of IPV than the general population and more frequent and serious IPV injury consequences such as emergency room visits and suicidality. To be maximally effective, IPV intervention and prevention strategies must be responsive to the language, culture, and other characteristics of the target population. Moreover, if, as our clinical and research experience suggests, IPV perpetrator characteristics and manifestations of IPV affecting the Deaf community differ from general population norms in significant ways, then etiologic research identifying those differences must precede and inform the development of IPV prevention and intervention strategies targeting the deaf population, as well as guide further research on this important topic. Objectives: (1) To investigate and describe risk and protective factors associated with characteristics of IPV perpetrators who are involved in violent relationships where one or both partners are deaf and communicate via American Sign Language (ASL). (2) To investigate and describe risk and protective factors regarding the ways IPV behaviors manifest in relationships involving one or more deaf individuals who use ASL, especially behaviors associated with greater injury and mortality risk. (3) To compare and contrast these study findings with current knowledge regarding risk and protective factors associated with IPV perpetrators and perpetration behaviors in the general population. (4) To utilize study findings to make recommendations for future research, intervention, and prevention efforts aimed at preventing and/or reducing the injury consequences of IPV in the Deaf community. Study Design: This etiologic, cross-cultural study will be conducted by a multidisciplinary team of deaf and hearing researchers. A period of study team cross-training precedes the data-collection activities. The majority of study data accrues via 90 semi-structured interviews with three distinct participant groups (see below). We employ a mixed-methods study design based on a social ecological model of IPV origin, continuation, and severity. Rigorous qualitative and quantitative methods will be employed, the latter focused on three well-regarded IPV measures used in this study on an exploratory basis. The study goal is to discover Deaf community-specific IPV risk and protective factors by triangulating data from our three participant groups, and further assess these findings via presentation to three respondent verification focus groups consisting of Deaf community IPV perpetrators (2 groups), and victims/service providers (1 group). Setting: The 90 semi-structured interviews forming the core source of study data will involve geographically diverse/balanced samples from our three respondent categories (see below). Most of these interviews will involve deaf people communicating in ASL and will take place via videophone technology which allows point- to-point, distant audio-visual communication and, therefore, communication in ASL. Participants who are hearing or hard-of-hearing can choose a telephone interview. Two of the three, face-to-face respondent verification focus groups will take place in southern California, where collaborators run the country's only two programs serving deaf IPV perpetrators. The third will take place in Rochester, New York. Participants: Three respondent samples will participate in our semi-structured interviews: (1) individuals (professional and lay) who provide services to deaf IPV perpetrators or victims (N=30), (2) deaf IPV victims (N=20), and (3) individuals (regardless of hearing status) who have perpetrated violence in relationships where one or both partners is a deaf ASL-user (N=40). An additional 15 individuals will participate in the respondent verification focus groups that comprise the final phase of the study - 10 IPV perpetrators (2 groups of 5), as defined above, and one group of 5 deaf IPV victims and service providers. Interventions: This study does not involve interventions. Outcome Measures: We will employ a mixed-method data analysis approach, involving rigorous qualitative analysis of interview and focus group data and quantitative analysis of these data in relation to data accrued through the exploratory use of three well-regarded measures of IPV during the semi-structured interviews with deaf IPV victims and perpetrators (N=60 total). Also, an Evaluation Task Group will investigate and gauge the ongoing and final success of the project through a quarterly series of formative and summative evaluations. PUBLIC HEALTH RELEVANCE: RELEVANCE TO PUBLIC HEALTH STATEMENT Intimate partner violence (IPV) is a serious national problem identified by federal authorities and many professional societies. When IPV occurs in unique subpopulations, such as minority groups, etiologic research must guide the design of prevention and intervention strategies if they are to be maximally effective. The American Deaf population, which is a severely understudied language and disability minority group, experiences high rates of IPV and deserves quality etiologic research aimed at reducing this high-priority public health burden.

With the increased use of prenatal screening by ultrasound, more fetuses are being diagnosed as potentially having congenital renal obstruction (2-5% of all pregnancies in the US). To date, the most common cause of renal failure in children is from renal damaged caused by urinary obstruction. Many controversies exist regarding detection, prognosis, and proper management of children with this condition. We hypothesize that congenital renal obstruction results in the release of specific proteins into the urine that reflect changes in protein secretion and shedding from renal tubular cells and other sources. We predict that these changes will vary with onset, duration and severity of the obstruction. In our previous work, we have demonstrated our ability to use state-of-the-art mass spectrometric and proteomic approaches to identify proteins in the urinary proteome in an animal model of postnatal maturation. With this experience, my objective is to identify new markers of obstructive renal damage that could be potential diagnostic or prognostic clinical tools. Our approach will be to use an animal model of neonatal renal obstruction and follow urine composition over time after initiation of the injury. We will study the urinary proteome during obstruction using advanced qualitative and quantitative proteomic methodologies in order to prioritize and identify candidate clinical markers. The discriminatory power of the candidate markers, and their potential for clinical translation, will be determined using directed quantitative proteomics in select human infant cohorts with and without severe renal obstruction. Overall, my long-term career objective is to become an independent pediatric urologic clinician-scientist with a strong commitment to translational research that focuses on biomarker discovery for renal injury. My immediate goals are to acquire a strong background and research experience in mass spectrometry, proteomics, and biomarker validation. During the early portion of my career, I plan to add to my foundation as a clinician investigator through lectures, strong mentorship by Dr. Michael Freeman and others, scientific collaborations, hands-on experience, and didactic coursework that focuses on proteomics, clinical trials, biomarkers, epidemiology, and bioinformatics. My research will be conducted at Children's Hospital Boston in the Urological Diseases Research Center and in the Children's Hospital Boston Proteomics Center. In summary, this project will provide the necessary foundation for a successful career as an independent investigator and will lead to the identification of novel biomarkers that may be used to inform clinical decisions in children affected with congenital renal obstruction.

The Pathology Core goal will continue to serve as a resource for the investigators interested in neurogenetics research. The resources include a tissue bank and providing neuropathological expertise in the analysis of murine and human normal and lesional tissue associated with Neurofibromatosis 2 (NF2), Schwannomatosis and Tuberous Sclerosis (TSC). To accomplish this goal the core will: 1) Coordinate the collection of frozen lesional tissues and cell culture from patients with inherited nervous system tumor syndromes and from corresponding solitary, sporadic tumors from the general population, as well as collect corresponding fresh and paraffin-embedded fixed tissues from normal controls (autopsy tissues). 2) Maintain a separate database of tissues, cell lines and, DNA for these projects, which can be linked to other databases (clinical database, mutation analysis database, RNA database). 3) Provide immunohistochemical expertise in the study of cellular and subcellular localization of merlin, tuberin, hamartin and their interacting proteins and analyze the expression of novel proteins (identified by CGH and microRNA studies), phosphorylated proteins and neurotransmitter receptors in murine models, in the normal human brain and in TSC or NF-associated lesions 4) Provide histological and immunohistochemical expertise in the classification and analysis of lesions associated with NF2, Schwannomatosis and TSC and their sporadic counterparts. 5) Provide histological and immunohistochemical expertise in the classification and analysis of newly generated lesions in Nf2 and Tsc murine models and evaluate the delivery and effects of experimental gene therapy in these lesions. The core will ensure optimal and uniform processing of all tissues and cell cultures to be used by the investigators of the various projects. In addition, the histological review of all tissues (murine and human) will ensure uniform and standardized classification, which are essential for meaningful comparison and interpretation of the data. The core will continue its work, in collaboration with the various projects, of elucidating the pathogenesis and altered protein expression in TSC-associated lesions in humans and in the Tsc murine models and in defining the molecular, clinical and histological characteristics of NF-associated lesions in humans and murine models. Finally, the delivery and effect of gene therapy on lesions in Tsc and Nf2 mouse models will be assessed.

The pol gene of the human immunodeficiency virus (HIV) encodes three viral enzymes, reverse transcriptase, protease and integrase, which are critical for the replication of HIV. This project focused on the discovery of inhibitors of one of these viral enzymes, HIV reverse transcriptase (HIV RT). The broad objectives are to contribute to the chemistry and biochemistry of conceptually new nucleosides and nucleotides with useful therapeutic potential against the infectivity of HIV. These novel compounds, with stereochemically defined surrogate carbohydrate components, that are regioisomeric with the natural nucleosides and nucleotides, are referred to as isonucleosides and isonucleotides. A compelling rationale for this investigation comes from the discovery in this project of a potently anti-HIV active compound, (S,S)-isodideoxyadenosine [(S,S)-isoddA}. IsoddA 5'-triphosphate is a powerful inhibitor of HIV RT (KI 16 nM). This renewal proposal seeks to expand successful work of the current project to include cyclic monophosphate (MP) pro-drugs (cyclo Sal MPs) and hydrolytically stable phosphonyl derivatives in order to deliver the MPs or their isosteres directly inside the cell. In addition, new classes of isonucleosides with multiple supporting rationaleS for anti-HIV activity are proposed. The synthetic work will involve the development of approaches to the novel, chiral isodideoxynucleosides and their purification and complete characterization by magnetic resonance, mass spectral, and X-ray methods. Collaborative antiviral studies on the target compounds and pro-drug forms will be carried out against HIV-1 and HIV-2, including drug-resistant HIV isolates. Data on inhibition of the cytopathic effect of HIV, on inhibition of HIV RT, on inhibition of proviral DNA synthesis, on inhibition of the expression of HIV-1 p-24 Gag protein, on host cell cytotoxicicty including inhibition of cellular DNA polymerases alphaepsilon beta and gamma, and on therapeutic indexes will be determined and analyzed. Cellular combination drug studies, particularly those having the potential for synergistic inhibition of HIV infectivity, are also planned.

Background and Objective: [unreadable] Previously, all experiments examining protein protein interactions in living cells by FRET have been limited to the use of two fluorescent proteins.[unreadable] Here, we report the development of a novel FRET technology for studying complexes involving three proteins.[unreadable] The system utilizes CFP, YFP and mRFP (monomer red fluorescent protein). CFP->YFP FRET1, YFP->mRFP FRET2 and CFP->YFP->mRFP linked FRET signals are monitored separately by flow cytometry. The technology has been validated by constructing plasmids encoding several FRET-positive and -negative controls, including CFP-YFP-mRFP, CFP-T2TD-YFP-mRFP and CFP-YFP-T2TD-mRFP, whereas T2TD (TRAF2 TRAF domain) acts as FRET insulator because of its structure with a distance of 9 nm from the head to the end.[unreadable] Furthermore, this technology has been used to examine the trimer formation of TRAF2 in living cells.[unreadable] [unreadable] Results: [unreadable] 1. The validity of CFP->YFP->mRFP 3-way FRET: For the first time, we validated the practicality of using flow cytometry to determine CFP->YFP->mRFP linked FRET by directly visualizing 2-step-FRET signals and the quenching of CFP->YFP FRET1 signals.[unreadable] 2. The determination of three protein complexes: with the system, we examined the trimer formation of tumor-necrosis-factor-associated factor 2 in living Hela cells. TRAF2 was tagged separately by CFP, YFP and mRFP. Co-transfection of all three plasmids displayed the 2-step-chained FRET signals, but not in cells co-transfected with any two of them and blank fluorescent protein. [unreadable] [unreadable] [unreadable] Conclusions: we have developed a method to determine interactions among three proteins in living cells using flow cytometry and aCFP-YFP-mRFP FRET system.

The free Ca2 ion concentration (Ca2 ion) of the cytosol, an important parameter in the regulation of carbohydrate metabolism, is itself regulated through the combined actions of the Ca2 ion transport systems of cellular membranes. Since these systems have been studied under unphysiological conditions, little is known of the details of cytosolic (Ca2 ion) regulation in an intracellular environment. This laboratory's approach has therefore been to measure directly the functional result of subcellular Ca2 ion transport under simulated in vivo conditions. An ion-selective electrode continuously monitors ambient (Ca2 ion) in a cytosol-like medium containing either isolated fractions of mitochondria and endoplasmic reticulum (microsomes) from rat liver or isolated rat liver cells pre-treated to make only their plasma membranes permeable to solutes. Having already documented joint steady-state regulation of ambient (Ca2 ion) by mitochondria plus microsomes at values approximating those of liver cell cytosol, this project will investigate 1) inter-organellar effects on Ca2 ion homeostasis through reciprocal alteration of internal Ca2 ion content and 2) modulatory effects on Ca2 ion homeostasis of a) phosphate, adenosine triphosphate and oxidized/reduced pyridine nucleotide, which reflect the status of carbohydrate metabolism, and b) glucagon, epinephrine and norepinephrine, which alter the rate of carbohydrate metabolism.

The study of tolerance to self-antigen in B-cells is of the utmost importance to understand how the immune system is regulated in health and disease. Unfortunately, the study of human B-cell tolerance has been hampered by the inability of investigators to identify an abundant population of autoreactive B-cells with homogenous antigenic reactivity and surface markers. We have identified such a population (termed VH4- 34 cells) and over the last three years we have validated the use of these cells for the study of human B-cell tolerance. [unreadable] [unreadable] VH4-34 cells recognize glycolipid autoantigens widely expressed in the surface of multiple cell types as well as microbial glycolipid antigens and probably play a homeostatic role in host defense by connecting the innate and adaptive immune systems. In healthy individuals, VH4-34 cells are prevented from inducing autoimmunity by positive selection into the marginal zone of the spleen and negative selection form the germinal centers. The latter mechanism prevents VH4-34 cells from expanding into the long-lived postgerminal center memory and plasma cell compartments thereby inhibiting the production of significant amounts of pathogenic autoantibodies. [unreadable] [unreadable] In this application we propose to study the phenotypic and functional properties of VH4-34 cells in healthy individuals and to compare them to VH4-34 cells obtained with patients with Systemic Lupus Erythematosus (SLE) in whom B-cell tolerance is defective. This will be accomplished by using multiparameter flow cytometry and immunocytochemistry. Furthermore, we will analyze the signaling processes responsible for the biological responses observed with VH4.34 cells and again determine how signaling differs in patients with SLE. Finally, we will ascertain what type of T-cells regulate the behavior of VH4-34 cells both in healthy subjects and SLE patients. [unreadable] [unreadable] The studies proposed herein should yield important insights into the regulation of autoimmunity and the pathogenesis of autoimmune diseases such as SLE. Furthermore, we believe that our results will improve our ability to treat patients with SLE and similar diseases. [unreadable] [unreadable]

While most women are overweight or obese when they reach menopause, we know little about how obesity affects this transition and its impact on metabolic health. We have hypothesized that the enhanced production of estrogens in peripheral tissues of obese women protects adipose tissue, skeletal muscle, and bone from the menopause-related decline in function. We have merged well-characterized preclinical models of obesity (obesity-resistant/prone rats) and menopause surgical ovariectomy (OVX) to study how pre-existing obesity affects this transition. In the first aim, we examine obesity's effects on the loss of ovarian estrogen production and its impact on energy balance, fuel utilization, and metabolic health. The role of extragonadal estrogen production in obesity's effects will be dissected with the use of aromatase inhibitors. Diet-induced weight loss is common in women with weight control problems, but the body compensates with homeostatic adaptations that drive weight regain. The loss of ovarian function in weight reduced women may exacerbate this metabolic drive to gain weight and create a potentiated vulnerability to metabolic disease. Regular exercise and estrogen have many overlapping effects on energy balance and fuel utilization that may counter these consequences to energy homeostasis and disease. In the second aim, we examine how energy restricted weight loss from obesity affects the drive to gain weight and the decline in metabolic health after the loss of ovarian function. The impact of regular exercise, with actions that reflect an estrogen mimetic, will be examined in weight reduced subjects. These studies will provide insight into how one of the most common preconditions of menopause, obesity, affects the loss of ovarian function and its consequences to energy homeostasis and metabolic disease. Observations from these studies may provide evidence that certain populations of obese woman (treated with aromatase therapy, energy restricted) who may be particularly vulnerable to menopausal weight gain and metabolic disease unless they maintain a program of regular physical activity.

West Nile Virus (WNV) is one of the many neurotropic flaviviruses that is widely spread throughout the world, and continues to cause significant morbidity and mortality. In the US alone there were 24,000 cases of human WNV infection and 1000 fatalities since 2006. Both innate and adaptive immune components, including CDS, CD4 T cells and B cells contribute to WNV clearance and prevent infection of neurons. The adaptive immune components also form memory, which is the hall-mark for vaccination strategies. The generation of adaptive immunity is linked to innate activation, primarily via viral sensing by host pattern recognition receptors (PRRs), which in turn lead to ARC activation and generation of inflammatory mediators. One class of these early inflammatory mediators, type-l interferons (IFN-I) induce an anti-viral state in infected and neighboring cells. Many viruses, including WNV, developed potent IFN-I evasive strategies. Recent studies from our and other labs show that IFN-I can also exert profound influence on T cells responses, and generation of immune memory;but very little is known about the roles of specific pattern recognition receptors and the IFN-signaling in generating flavivirus-specific adaptive response. An understanding of this is critical for generating improved vaccines. We hypothesize that IFN-I signaling plays a critical role in generating WNV-specific adaptive immunity and that strategies to interfere with viral IFN-I evasive mechanisms should yield better vaccines. In Aim 1 we will define the role of dendritic cell signaling via RNA helicases and TLRs for generating WNV-specific CDS T cell responses. In Aim 2 we will examine the role of IFN-I signaling in the generation of WNV-specific CDS T cell responses. In Aim 3 we will assess the importance of the timing of IFN-I signals in programming T cell responses. Using the knowledge gained from these and the studies proposed in the other four projects of this U19, in Aim 4 we will modulate IFN-I signaling as means to enhance vaccination. Thus, this proposal will contribute to attainment of multi-project objectives of this U19.

[unreadable] This is a 5-year training program renewal to support 5 postdoctoral trainees (Ph.D. and/or M.D.), 4 predoctoral students (Ph.D. or M.D./Ph.D) and 3 short-term minority (BS or in MS program) students per year for years 21-25 with an emphasis on a multidisciplinary approach to understanding hematopoietic cell regulation and disease mechanisms. The average training period for postdoctoral and predoctoral students will be 3 and 4 years, respectively. Twenty-three productive preceptors will provide extensive training in highly active and interactive research laboratories pursuing state of the art research on fundamental aspects of hematological, cellular, physiological, molecular, and biochemical aspects of cell growth and disease-related abnormalities. Preceptors are in 6 departments and 4 research centers housed in 4 buildings in close-proximity to each other. Strong track-records of peer-reviewed publications and extramural funding of preceptors are a major strength of the proposed training program, as are the training records of the preceptors. Opportunities for pursing multi-disciplinary research exist due to active, ongoing scientific collaborations among preceptors. A significant emphasis is to promote and foster the development of the next generation of biomedical researchers committed to scientific careers in academic medicine to become independent investigators. The approach for comprehensive training encompasses close and direct contact with seminars, laboratory meetings, and participation in national and international scientific meetings is already in place. The decision of a student to associate with a laboratory will be by mutual consent of trainee and preceptor. The scientiifc development of the trainee will be facilitiated by a research committee composed of preceptors with multi-disciplinary research interests. Major resources that are available include internationally recognized clinical and basic science research programs, an NCI-designated cancer center, an NIH-designated National Gene Vector Laboratory, an NIDDK Center of Excellence in Molecular Hematology, and state-of-the-art infrastructure, and frequent contacts with active investigators at IU and throughout the world [unreadable] [unreadable]

Coronary heart disease is the leading cause of death in the United States. The elderly, those older than 65 years of age, are more likely to have coronary disease and this problem will become even more pervasive as the population ages. Stenting of coronary blockages has become a conventional technique in both the emergency setting of a heart attack as well as more routine therapy to reduce chest pain. In general, there are two types of coronary stents: drug-coated (or drug-eluting stents, DES) and non-drug- coated (or bare metal stents, BMS). Placement of either type of stent requires treatment with clot- preventing medications such as aspirin and clopidogrel or prasugrel to keep them open. The latter two are newer types of medications that work in conjunction with aspirin to reduce the function of cells that help form blood clots in the body (platelets), thereby preventing clot formation on stents (stent thrombosis). Currently, the American College of Cardiology / American Heart Association recommend continuation of these medications for at least one year following DES and for at least one month following BMS placement. Initial studies comparing DES to BMS showed that by reducing scar formation around stents, DES are less likely to close up and require repeat procedures. Recruitment of elderly in these studies was limited and subjects were generally healthier when compared to the community-dwelling elderly. Despite lack of such information, the elderly routinely receive DES during cardiac procedures, requiring the obligatory one-year of anti-platelet drugs. On average, the elderly are more likely to be taking multiple medications, have a lower body weight, and have worse kidney function than the relatively healthier population in randomized trials, thereby putting them at higher risk of drug-drug interactions and unwanted bleeding. Bleeding, which was originally considered just a nuisance in the early days of stenting, has more recently been recognized to increase mortality in patients with coronary heart disease. The balance of possible increased risk of repeat procedures with the use of BMS and potentially increased risk of bleeding with long-term use of dual antiplatelet therapy needs further investigation. The Dual Antiplatelet (DAPT) Study will randomize >20,000 patients to either 12 versus 30 months of dual antiplatelet drugs. This trial is unique in that it will study all-comers, there is no upper age limit, and will collect data on bleeding, interruptions of medications, and adverse cardiac events. We intend to study the DAPT Study elderly subset and compare differences in effectiveness and risk of dual antiplatelet therapy and stent types. Data on the rate of heart attacks, stroke, repeat procedures, and bleeding from a state-mandated registry which collects information on all stenting procedures in Massachusetts will also be analyzed for 3 and 4 year follow-up. Information obtained from this project will provide guidance to physicians caring for elderly patients toward stenting and medical treatment of coronary heart disease, particularly in regards to type and duration of antiplatelet drugs. PUBLIC HEALTH RELEVANCE: An important aspect of the management of coronary heart disease entails placement of drug coated and uncoated stents in the heart arteries to open up blockages. Medications required to prevent the stents from clotting can also increase the risk of bleeding and safety of these stents and medication in the elderly is not entirely clear based on current available data. The goal of our project is to study the safety and efficacy of these medications and stents in the elderly as part of a 20,000 patient randomized trial of these clot prevention medications and a statewide registry that manages information on all coronary stenting procedures done in Massachusetts.

The early development of nerve-muscle synapses is characterized by three major events: (a) the accumulation of junctional acetylcholine receptors (AChRs), (b) the localization of synaptic acetylcholinesterase (AChE), and (c) the elimination of extrajunctional AChRs. This last event is mechanistically distinct from the accumulation of junctional AChRs and seems to result from the suppression of AChR synthesis in extrajunctional regions of muscle cells caused by muscle contraction. The final result is a muscle cell with a highly elaborated apparatus specialized for efficient synaptic transmission. Experiments in this proposal are designed to understand at the molecular level the way in which neurons and muscle cells communicate to establish this apparatus. The proposal is divided into three major sections. The first will make use of immunocytochemistry and antibody microinjection to demonstrate a functional association between the presence of a newly discovered muscle component (a 37 kilodalton nonmyofibrillar tropomyosin) and the ability to cluster AChRs. This molecule was first identified by its absence from vitally transformed muscle cells which are unable to cluster AChRs at all. The second section of this proposal describes similar techniques to probe other cytoskeletal elements involved in clustering and subsequent structural changes in the muscle cell. These studies are based on the observation from this laboratory that clustering causes a subset of organelles, including myonuclei and the Golgi apparatus, to assume a constant sub-cluster localization. The final section is a study of changes in the levels of AChRs and AChE caused by the increase in muscle cell Ca2+ which occurs during contraction. Also, experiments are designed to test the hypothesis that regional differences in the amount of Ca2+ released during contraction or in the levels of particular Ca2+-binding proteins underly the ability of these cells to specify where particular macromolecules are synthesized. Ca2+ concentration will be measured using the Ca2+-sensitive fluorescent dye fura-2 and optical image processing. Ca2+-binding proteins will be investigated using biochemical and immunological techniques. These experiments should add considerably to our knowledge of how neurons influence properties of their target cells and may contribute to an understanding of a variety of developmental and neurological disorders. In addition, certain results may provide information useful in comprehending cell transformation.

The objectives of this protocol are to compare the combination of 10-Ethyl-10- Deaza-Aminopterin (10-EdAM), mitomycin and vinblastin (EMV) with the combination of mitomycin and vinblastine (MV) in the treatment of patients with advanced Non-Small Cell Lung Cancer and to demonstrate that the two regimens are acceptable in terms of toxicity and to compare their relative toxicity. This is an open-label, multicenter, randomized trial.

The E4 allele of the Apolipoprotein E (ApoE) gene is the strongest genetic risk factor for the onset of sporadic Alzheimer's disease (AD) identified to date. The roles by which ApoE influences amyloid-beta (A2) metabolism and non-A2-mediated mechanisms in AD pathogenesis, however, remain to be fully clarified. The literature, and our preliminary data, suggest that early alterations in peripheral lipids (sphingolipids, fatty acids, cholesterol and cholesterol esters) reflect brain functioning and pathology, and may interact with ApoE genotype in the development of AD pathogenesis, warranting human studies. Clinical and epidemiological studies of short duration, while important, will not contribute to our understanding of the earliest phases of AD pathogenesis because Alzheimer's pathology (A2 plaques and neurofibrillary tangles) begins decades before the emergence of symptoms and substantial neurodegeneration. Identifying factors years before the onset of AD that may modify the effects of ApoE4 in initiating and promoting AD pathology and the subsequent emergence of symptoms will uniquely contribute to the development of prevention strategies. Longitudinal studies of cognitively normal individuals with serial measures of Alzheimer's pathology in the living brain, as proposed here in the unique cohort of the Baltimore Longitudinal Study of Aging (BLSA), initiated in 1958, are necessary to understand the relationship between ApoE4 genotype, perturbations in peripheral lipids, their interaction, and later development of AD clinical symptoms and brain alterations. The BLSA is one of few human studies that could provide the unprecedented opportunity to systematically examine this relationship over a long follow-up. BLSA participants, cognitively normal at their first visit in the study (mean age: 63.4), have a mean follow-up of 14.3 years (SD = 6.5) and a maximum follow-up of 38.9 years. In the proposed study we will measure plasma lipid levels (sphingolipids, fatty acids, cholesterol and cholesterol esters) at three early visit in the BLSA study, roughly 5 years apart, for those aged 55 and over (n=1095), and at the last visit, as well as during the neuroimaging sub-study. The specific aims include examining the proposed peripheral lipids, changes in these lipids over a long follow-up, and their interaction with ApoE to predict: 1) decline in tests of memory; 2) incident MCI, all-cause dementia, and AD; 3) change in serial MRI measures of brain atrophy and white matter lesion burden over 10 years; and 4) amyloid-beta deposition on 11C-PIB PET scans. The lipids will be assayed using an already-developed targeted and quantitative lipidomic approach. PUBLIC HEALTH RELEVANCE: Many potent risk factors for Alzheimer's disease (AD), including hypertension and high cholesterol, are most detrimental in mid-life, presumably at the emergence of AD pathology, but have less of an effect on AD risk in late-life. Identifying factors in mid-life that may modify the effects of APOE E4 in initiating AD pathology, and the subsequent emergence of symptoms, will uniquely contribute to the development of prevention strategies. The overall aim of the proposed study is to examine, in the 50-year Baltimore Longitudinal Study of Aging, whether plasma lipids measured in mid-life (sphingolipids, gangliosides, fatty acids, cholesterol and cholesterol esters) modify the association between APOE and cognitive decline, clinical onset of mild cognitive impairment or AD, and neuroimaging measures of brain atrophy and brain pathology.

ABSTRACT Quantifying aging is a major goal in Geroscience research as the availability of a reliable marker of aging can facilitate understanding of the fundamental biology of aging, enable tracking of the aging process in different tissues and cell systems, and support identification and validation of interventions that extend lifespan and healthspan. Traditionally, aging has been monitored by following chronological age, mortality, age-related changes in gene expression, and/or other molecular features, however, there is currently no consensus on the best practices for quantitatively tracking progression through aging. The recent advent of biomarkers based on advanced omics approaches, such as DNA methylation, have provided some hope to support development of precise estimates of age, both in humans and mice. Nevertheless, the majority of such measures are trained as chronological age predictors, with little to no integration of biological, functional, or phenotypic data. Further, the modifiability of aging measures based on DNA methylation in response to lifespan and healthspan extending interventions is almost entirely unknown. We propose to address these challenges by developing a series of novel DNA methylation clocks by integrating information on phenotypic and functional aging, investigating links between DNA methylation and aging hallmarks, and evaluating DNA methylation responses to longevity interventions. We suggest that these clocks will offer a much-needed resource for the Geroscience community. We will develop these clocks using three general approaches. First, we will use cultured cells (MEFs) to induce or establish models of three well-known hallmarks of aging?cellular senescence, DNA damage, and mitochondrial dysregulation. We will then train epigenetic predictors of these hallmarks and validate them in vivo. We will also establish epigenetic alterations in response to novel and established longevity interventions. In doing so, we will develop biomarkers of intervention response that can be used to test mimetics, and/or optimize aging biomarkers. Finally, building on the highly characterized SLAM colony of C57Bl/6 and UM-HET3 animals, we will produce longitudinal methylation data across the lifespan that can be used to develop an epigenetic clock that can serve as a robust predictor of healthspan. We hypothesize that these new clocks will better capture biological age than chronological age trained clocks. Given that they were developed to capture different facets associated with the aging process, they can be combined to create a single aging measure that is more biologically informed and characterized compared to existing epigenetic clocks.

as fresh samples for isolation of normal organoids or breast stem cells. The pathology core will provide normal breast tissue for stem cell studies (Polyak) and for 3-D organoid cultures (Brugge). Freshtumor samples will be provided to establish primary tumor xenograftsin mice Weinberg). In addition, Dr. Richardson will participate in the pathologic comparisons, both molecular and histology, between the parent human tumors and the resultant xenografts. Gene expression array data obtained from human tumors will be used in system interaction analyses of BRCA1 relatedpathways (Livingston). Dr. Richardson will provide both tissue and scientific expertise to analyzefrozen tumor specimens and paraffin tissue microarrays for the expression of BRCA1gene products, including IRIS (Livingston). RELEVANCE (See instructions): Research using actual human breast tissue and breast tumor material will hopefully provide the most relevant answers to pathogenesis and tumor behavior of breast cancer in women. PROJECT/

The objectives of this study are 1) to test a group treatment program for women patients with borderline personality disorder (BPD) designed to decrease borderline symptoms and improve social functioning, and 2) to add to the understanding of the psychopathology of BPD by exploring a "level of emotional awareness" model to conceptualize its learning deficits. The specific aims of the proposed study are: 1) to test the efficacy of an 8-month, psychoeducation treatment program adjunctive to individual psychotherapy in reducing BPD symptoms, 2) to identify the characteristics of patients able to benefit from such treatments, 3) to explore treatment's mechanism of action, 4) to produce a treatment manual for the program, and 5) to provide training in the treatment of BPD. Forty-eight subjects who meet criteria for a BPD diagnosis using DSM-III-R, the Diagnostic Interview for Borderlines (DIB) and the Borderline Syndrome Index (BSI) will be randomly assigned to the treatment and waiting list control groups. The program's content is based on the deficits in emotional awareness, regulation, and expression and the consequent social, communication, and problem-solving skills like increased awareness of emotions, distress reduction, anger management, and need identification. It then moves on to interpersonal skills like emotional expression, communication, and conflict management. Treatment outcome measures include self-report (Symptom Checklist-90 SCL-90), Social Adjustment Scale-Revised), and structured interview measures DBI, Alexithymia Provoked Response Questionnaire). These measures will be given pre-treatment, post-treatment, and at a 6- month followup. A pilot study with nine subjects randomly assigned to treatment group and control provides strong support for the efficacy of this treatment program and the merit of a larger trial. Treatment group members improved significantly on all outcome measures (SCL-90, BSI, and Global Assessment of Functioning (GAF), while control subjects did not change significantly. This study will contribute to the much needed empirical evaluation of time-limited psychosocial group treatment for this difficult population.

The specific aims of this Administrative Core include: 1) to provide the administrative structure to permit the effective day to day operation of the MCRC, 2) to provide the scientific expertise and leadership to identify and focus projects on clinically relevant issues and to insure that important unmet needs of underserved and minority patient groups are addressed, 3) to maintain and enhance internal communication and cooperation among MCRC members and to promote novel approaches to address clinically relevant issues, and 4) to expand the influence of the MCRC within the institution and to identify and secure institutional support to promote and enhance the activities of the Center. The Administrative Core will be housed in the Division of Rheumatology in the Department of Medicine. This Unit will be responsible for the overall direction, organization, operation and integration of the MCRC. The MCRC director, Dr. Richard Pope, will be assisted on a routine basis by the associate director, Dr. Rosalind Ramsey-Goldman, and the Methodology/Data Management Core director, Dr. Rowland Chang. The MCRC administrator, Mr. Christopher Loertscher, will assist in supervising and monitoring all budgetary issues and the scheduling of meetings by the executive committee, as well as MCRC sponsored research meetings and seminars, assisting in the preparation of reports and in dealing with regulatory matters. The scientific expertise and leadership within the Northwestern MCRC occurs at the level of the executive committee, that is chaired by Dr. Richard Pope and includes Dr. Roz Ramsey-Goldman, RowlandChang, Alan Dyer, Thomas Schnitzer, W. Zev Rymer, Lauren Pachman, and Rex Chisholm. A strength of this group is that it is composed of individuals with a variety of interests in clinical research on diseases relevant to the mission of the MCRC. The background that each member brings to the committee is quite different, which allows a truly multidisciplinary view of each issue. The committee meets bimonthly and considers all issues relevant to the scientific direction of the MCRC. The committee is responsible for identifying and pursuing new opportunities for interaction and development. These opportunities are particularly enhanced because of the roles of members of the executive committee both within the university community and nationally. It is through these interactions that the members of the committee are able to identify and promote important issues of national importance.

We propose to study the development of drug resistance in the context of HIV protease inhibition to develop and test structural and synthetic strategies in response to this mechanism. The overall goal of this Program Project is to understand the mechanisms of viral resistance, enabling modeling and design of more sustainable anti-viral therapeutic strategies. The Program consist of four highly integrated Projects and three supporting Core facilities: Project 1, will enhance and extend a computational co-evolution approach to drug resistance by developing and applying detailed atomic models of drug/target interactions, modeling viral population dynamics and patient response under drug selection pressure, exploiting automated learning and hidden Markov modeling approaches to inform and refine these models; Project 2 will exploit the capabilities of high throughput crystallography to find and characterize novel binding sites on the protein target using fragment libraries to help construct new inhibitor leads to maintain efficacy against multi-site PR mutants, linking with optimization and synthetic efforts in Projects 1 and 3 respectively; Project 3 will utilize their "Click Chemistry" synthetic approaches for rapid development and evolution of novel fragment-based inhibitors in conjunction with Projects 1 and 2, and develop resistance probes with Project 4; Project 4 will experimentally characterize the evolution of HIV resistance in response to protease inhibition both within PR and in the rest of Gag-Pol, by exploiting tissue-culture time-course evaluation passaged virus in the presence of identified inhibitors, as well as from deep genetic analysis of selected patient samples; Core A will provide mutant and wildtype proteases, functional assays and chemical probes, and inhibitor analyses for the Program; Core B will provide the necessary x-ray structural data and computational analysis to integrate new information on protease mutants, and protease-inhibitor interactions; and Core C will assemble and make available to projects 1 and 4 time-course anti-retroviral treatment data on HIV infected patients as well as blood samples from highly resistant individuals for in-depth bioinformatic and viral genomic analyses. AIDS remains the major pandemic of our time. While patients infected with HIV can now be treated with drugs that enable them to live productive lives, the virus can subvert this treatment by developing resistance to these drugs. This study is aimed at a detailed understanding of HIV drug resistance, with the goal of developing new therapeutic strategies for more sustainable treatments to prevent AIDS. [unreadable]

Fear of intracranial hemorrhage (ICH) is the primary reason that the only proven therapy for acute ischemic stroke is not provided to otherwise eligible patients. ICH also deters the development of more effective stroke therapies. New reperfusion strategies, using an intra-arterial (IA)approach, which may restore blood flow more effectively by providing therapy directly at the site of an occluded artery, are accompanied by higher rates of the mildest forms of ICH than proven intravenous thrombolytic therapy. While ICHs of all types have been considered adverse events in every acute stroke trial to date, the mechanistic significance and clinical impact of mild ICH subtypes is not known. The primary hypothesis is that acute ischemic stroke patients treated with IA therapies who have the mildest radiological subtype of ICH will be more likely to have early reperfusion and good clinical outcome than those without any ICH. Using the largest available trials with revascularization status, the PI will pursue the following specific aims: (1) determine the relationship between radiological subtypes of ICH and timing of successful revascularization and (2) determine the relationship of ICH subtypes to clinical outcome. The proposed work would: (1) guide the clinician's perceptions of the interplay between risk and benefit in the setting of reperfusion, (2) impact the interpretation of future Phase I and II reperfusion trial safety data, and (3) inform the design of future reperfusion strategies using hemorrhage prediction and hemorrhage prevention strategies. This research project will position the PI to independently design and implement an acute stroke trial with the goal of minimizing clinically significant hemorrhage. With this career development award, the PI will gain practical experience with a large-scale Phase III acute stroke clinical trial, obtain exposure to innovative reperfusion strategies, and pursue formal training in biostatistics and clinical trial methodology through introductory and advanced coursework. The PI will become an expert on acute stroke reperfusion therapies, and particularly hemorrhagic transformation, with the skills to implement an acute stroke clinical trial independently. Relevance to Public Health: Despite the availability of an effective treatment for strokes due to blocked arteries (88% of all strokes) since 1996, stroke remains the third leading cause of death and the leading cause of disability in the United States. Therapies that restore blood flow to more patients, and do so more effectively and safely, are greatly needed to improve outcomes after stroke.

A group of seven qualifying Primary Users have detailed a broad range of experimental plans that specifically employ BIAcore analysis as summarized herein: (i) Characterization of association and dissociation rate constants for the interaction of immunoglobulin E (IgE) and the high affinity IgE receptor (FcepsilonRI) is a major goal of the PI. The effect of point mutations on alterations of k/on and/or k/off values will be determined by measurement of phage-displayed peptide binding to sensor chips C1 and/or F1 covalently derivatized with either recombinant IgE receptor ectodomain or IgE; (ii) Determination of the kinetics of the interaction between the T cell receptor (TCR) and its ligands, specifically MHC class I-peptide complexes. To extend our recently published study of peptide/Class I-TCR binding kinetics, which showed a correlation between the affinity of TCR for particular ligands and the biological outcome, we plan to determine TCR-ligand recognition in lipid monolayers using the HPA sensor chip to model cell-cell TCR-MHC/peptide mediated interactions; (iii) The superoxide generating system of neutrophils is a membrane-associated enzyme complex, NADPH oxidase, which consists of an integral membrane protein, flavocytochrome b245 and cytosolic proteins p47phox, p67phox and Rac. BIAcore analysis will be used to study the molecular interactions of the cytosolic and membrane factors, individually and in combination, and to determine how molecular interactions of the cytosolic and membrane factors, individually and in combination, and to determine how molecular interactions of the cytosolic and membrane factors, individually and in combination, and to determine how molecular interactions of the cytosolic and membrane factors, individually and in combination, and to determine how their affinity is affected by anionic lipids, ionic strength and GTP; (iv) The precise role of Rac in the foregoing system is still unknown, but one current hypothesis is that the association of GTP-bound Rac with p67phox increases the affinity of this protein for the flavocytochrome. This will be tested by determining the kinetics and affinity of binding of p67phox to immobilized, purified, relipidated flavocytochrome in the presence and absence of Rac-GTP in the analyze solution; (v) The energetic processes that drive the binding reactions for DNA and RNA will be determined using the zinc finger protein TFIIA, and truncated or mutated derivatives, in solution with either 5S DNA or 5S RNA immobilized on a BIAcore chip; (vi) Surface plasmon resonance technology will be utilized to study fibroblast growth factor (FGF) receptor- mediated signal transduction. Several proteins, identified using the two-hybrid system, show affinity for the cytoplasmic domain of FGFR-1. The binding components from the 2 hybrid analysis will now be analyzed using the BIAcore system to determine the kinetic binding parameters. (vii) Autoantibody binding to wildtype and mutant fibrillarin, in the presence and absence of mercury, using the BIAcore machine. Quantitation of Hg-dependent changes of antibody k/on and dissociation (k/off) from fibrillarin immobilized on the biosensor chip will then be assessed.

The Center engages in collaborative research that leverages the Center's molecular modeling software, expertise and facilities to bridge experiment and computation. While experimental techniques unveil critical aspects of molecular and cellular architecture, computational molecular modeling describes biomolecular systems in their native states at the atomic level. During the last funding period, the Center completed 35 Collaboration and Service projects with 41 joint publications on subjects such as the assembly mechanism and drug resistance of the ribosome, neurons' synaptic receptors and solid-state DNA sequencing nanopores. Currently, the Center is engaged in 42 ongoing collaboration projects with 56 joint publications so far. To make the Center's technological advances driven by the collaboration projects available to the biomedical community, the Center distributes its software for all major platforms, from laptops to supercomputers and cloud computing facilities. The Center ensures software usability by providing user support, maintaining up-to-date documentation, tutorials, and online discussion groups. Over the last funding period, the Center-developed molecular modeling and simulation packages VMD, NAMD, and Lattice Microbes served a community of over 100,000 users. Technical support for the community was provided mainly through the online discussion groups ?NAMD-L?, ?VMD-L? and ?LatticeMicrobes-Users?, where more than 17,000 software-related messages were submitted over the last funding periode. The Center also o?ers individual consultation for special cases requiring direct personal interaction, operating directly in its laboratory area a well-equipped Visitor Center to host visiting scholars and collaborators. 50 scholars/collaborators visited the Center for up to six months seeking technical and scienti?c expertise, and 107 leading scientists presented their work on the Center Seminar program at the host institution, the UIUC Beckman Institute. All the activities described above were possible only through the Center's Petascale Biology Gateway Facility, which provides the high-end computing and graphics resources required by the demanding preparation, visualization, and analysis tasks of advanced molecular modeling. This gateway also hosts the Center's website and all associated content, from scienti?c research-related material to software documentation and tutorials, as well as the online discussion platforms.

PROJECT 1 (MONTINE): Abstract Genetic risk for PD-related cognitive impairment and its disease mechanisms The inaugural PANUC award was funded to pursue the association of three candidate genes with cognitive impairment and dementia in PD: APOE ?4 allele and variants in SNCA and MAPT; to these we added GBA and LRRK2 mutations. With our collaborators, we demonstrated that cognitive impairment and dementia are significantly more common among PD patients who carry APOE ?4 or GBA variants, less common in patients with LRRK2 mutations, and not associated with variants in MAPT or SNCA. In this project our approach will be to bring insight to disease mechanisms by determining the corresponding molecular pathology. During the current PANUC award, we and others used standard histopathologic criteria for Lewy bodies (LB) or Alzheimer's disease (AD). Unfortunately, this approach has led to unexpected and conflicting results for GBA variants and APOE ?4 in PDD in part because some studies were limited by low number of cases or inconsistent attention to genetic risk. Specific Aim 1 proposes to fill these gaps in knowledge by regional quantification of neurotoxic proteins and synaptic degeneration using a novel technique developed by our laboratory coupled with a large, multisite autopsy cohort assembled by PANUC. Specific Aim 2 reflects that although successful with our candidate gene approach during the inaugural PANUC award, we propose to expand gene discovery for cognitive impairment and dementia in PD by evaluating additional candidate genomic regions recently identified by the PDCGC, and by determining genetic influences on longitudinal change in cognition measured prospectively using consensus assessments.

This is a response to Notice Number NOT-OD-09-058, NIH Announces the Availability of Recovery Act Funds for Competitive Revision Applications. The goal of this revision supplement is to extend our studies of nicotine vaccine efficacy in rats by introducing methods for the delivery of nicotine through inhalation of cigarette smoke. Smoking cessation medications have added substantially to our ability to treat tobacco addiction, but their efficacy is limited and new types of medications are needed. Nicotine vaccines elicit nicotine-specific antibodies which bind nicotine and alter its access to brain. Three nicotine vaccines have shown preliminary efficacy in Phase I-II clinical trials, but efficacy is closely correlated with the serum antibody titer and current vaccines do not reliably produce sufficiently high titers in all individuals. The parent grant DA10714 is using rat models of nicotine addiction to study novel means of enhancing vaccine efficacy. Like essentially all current animal studies of nicotine addiction, DA10714 models tobacco addiction using parenteral (i.v. or s.c.) administration of pure nicotine. In contrast, smokers take in nicotine by inhalation and as one of over 4,000 chemicals present in cigarette smoke. The adequacy of using such artificial dosing paradigms to model cigarette smoking is unknown and largely untested. We have adapted and characterized methods for inhalation exposure of rats to smoke simulating the smoking of 1 cigarette or periods of heavier smoking. In this revision supplement we propose to use these methods to study the effects of nicotine vaccines on the absorption and distribution of nicotine inhaled in cigarette smoke. The purposes of doing so are to 1) expand the range of preclinical models available to study nicotine vaccines, 2) assess whether inhalation models provide novel information for vaccine evaluation, 3) examine the specific role of route-specific factors such as pulmonary antibody in mediating nicotine vaccine efficacy, and 3) develop quantitative models which can be more generally used to study the contributions of the inhaled route and other smoke constituents to tobacco addiction and treatment medications development. Nicotine vaccines are an attractive initial candidate for such study because vaccination is a pharmacokinetic intervention, and accurate pharmacokinetic modeling of nicotine intake may be important in understanding and exploiting its efficacy. Aim 1 will test the hypothesis that vaccination is effective in reducing nicotine distribution to brain over a range of clinically relevant dosing conditions. Aim 2 will test the hypotheses that vaccination is more effective in reducing the distribution to brain of inhaled compared to i.v. nicotine, and that such differences are in part mediated by the presence of pulmonary mucosal or tissue antibody. Because heroin and cocaine are also often smoked, the results of this study may inform ongoing efforts to develop vaccines for these addictions as well. PUBLIC HEALTH RELEVANCE: Cigarette smoking kills 5 million people worldwide yearly. Current medications are helpful for smoking cessation but are incompletely effective. We are studying the use of a nicotine vaccine to help smokers quit, which acts by binding nicotine in blood and reducing its access to brain. Rat models of tobacco addiction use nicotine delivered intravenously, which is informative but does not accurately model the human route of intake of nicotine by inhalation from cigarette smoke. This proposal will develop and study the utility of delivering nicotine to rats via exposure to cigarette smoke, and assess whether it allows better evaluation of nicotine vaccine efficacy and can expedite its further development.

This project is designed to investigate long-term regulation in brain of the enzyme choline acetyltransferase (CAT), catalyzing the biosynthesis of the neurotransmitter, acetylcholine. We shall study the biochemical basis for the observed multiplicity of molecular species of CAT and the mechanisms governing the regulation of its activity. To accomplish this, we shall (a) prepare and purify antibodies to the different forms of CAT found in rat brain; (b) analyze regionally in the brain the molecular nature of the different forms; (c) characterize the biochemical properties of each form; (d) determine their turnover rates; and (e) establish whether the mechanism of altered CAT activity affected by psychoactive drugs, hormones or brain lesions, as well as during growth and development, is one of induction (increase in enzyme protein) or activation (increased catalytic activity/molecule).

The 5-nitro drug, metronidazole (Mz), has been a mainstay of antimicrobial therapy for decades. Several of its simple derivatives such as tinidazole combine similar activity profiles with improved pharmacokinetic properties, but resistance to existing nitro drugs is increasing. Although commercial development of this drug class largely ceased decades ago, work by us and others over the last several years has shown that extensive modifications of the basic 5-nitroheterocyclic ring can lead to marked enhancement in activity against different microbes compared to existing drugs. These data suggest that Mz and other approved nitro drugs do not possess optimal activity in this drug class, yet important questions about the potential utility of novel nitro compounds must be addressed to advance their development as next-generation nitro drugs for clinical use: Is it possible to develop improved nitro drugs with broad-spectrum activity, or do enhanced activities exist only in microbe-specific fashion? Do new nitro drugs have different targets that can be exploited for overcoming resistance to existing drugs? What are the optimal pharmacokinetic properties of novel nitro drugs for maximal efficacy and potency against infections with different target microbes? Can new nitro drugs be developed with improved dosing regimens compared to existing drugs? Answers to these questions are not only critical for assessing the therapeutic potential of new nitro drugs, but are also key for identifying new leads for specific indications. The project will address these questions with a focus on two important protozoan pathogens, Trichomonas vaginalis and Giardia lamblia. We will evaluate a newly synthesized library of ~1,200 nitro drugs for activity against a broad range of drug-sensitive and drug-resistant strains of the target protozoa to identify library compounds more potent than Mz. Electrochemical approaches will be employed for determining the redox properties of the most potent nitro compounds to gain new fundamental clues about their mechanisms of action and potential toxicity. Subsequently, we will introduce new structural modifications into the top leads and evaluate them for bioactivity, cytotoxicity, electrochemical characteristics, and propensity to develop new drug resistance. Finally, we will evaluate the most promising nitro compounds for efficacy, potency, and pharmacokinetics in different murine models of protozoal infections. Upon completion of the proposed research, we expect to have elucidated broadly applicable principles that govern optimal efficacy of next-generation nitro-heterocyclic agents in the treatment of the clinically important parasitic diseases trichomoniasis and giardiasis. The comprehensive data sets to be generated will also be instrumental in selecting the most promising candidates as novel leads for the improved treatment of these infections, and potentially infections with other important pathogens, including Entamoeba histolytica, Trypanosoma cruzi, Helicobacter pylori, and Clostridium difficile, which can be treated with nitro antimicrobials.

Antisense phosphodiester oligonucleotides, conjugated with acridine, and antisense methylphosphonate oligonucleotides, killed cultured Trypanosome brucei in the Phase I program. In the Phase II program, antisense olignucleotides will be edified at the base, sugar, and phosphodiester functional groups to increase their therapeutic potency. These modifications will be designed to increase oligonucleotide cell penetration, stability, and functional inhibition of target RNA or DNA. The nucleic acid target will be expanded beyond the 39 nucleotide spliced leader sequence of trypanosome mRNAs to include the small nuclear RNAs, the coding and non-coding of several mRNA sequences, and the actin gene promoter region. Modified oligonucleotides then will be tested against the trypanosome procyclic form in culture, and those with enhanced therapeutic activity against the bloodstream form. Active oligonucleotides will be tested for therapeutic efficacy using trypanosome infected mice. Positive results will be of interest to the pharmaceutical industry for the development of new antisense oligonucleotide chemotherapeutic agents for trypanosomes and other parasites.

The WHO Collaborating Center for Design, Methodology and Analysis of Epidemiological and Clinical Investigations in Diabetes was designated in 1986. The purposes of the Center are to collaborate with the World Health Organization in the implementation of the WHO/IDF action program to provide advice, consultation and collaboration with other investigators in the design, methodology and analysis of epidemiology and clinical investigations relating to the etiology and pathogenesis of non- insulin dependent diabetes (NIDDM) and its complications. The center will assist in the development and application of standardized methods for epidemiological and clinical investigations, and data analysis relating to diabetes and collaborate with those interested in applying such techniques elsewhere. The Center will advise and help in the design of new studies, including onsite assistance when necessary. The center serves as a central laboratory for the WHO Multicenter Study of Vascular Disease in Diabetes, as well as being a participating study center for this study which is examining the mortality and incidence of vascular complications of diabetes among different ethnic groups in different countries. In addition the center has initiated a collaborative study of impaired glucose tolerance in China, is collaborating in the preparation of a survey manual for diabetes mellitus on behalf of WHO Center personnel are participating in teaching a WHO sponsored course on Clinical Epidemiology and Public Health Aspects of Diabetes.

As implemented, outside of a handful of reference institutions, electronic health record (EHR) systems have not been conclusively shown to improve health outcomes. The New York City Primary Care Information Project (PCIP) is the nation's largest community EHR extension project, with 148 primary care practices and over 1,000 providers in some of the city's poorest neighborhoods. The PCIP's goal is to maximize improvements in the quality of care through this investment of public funds. This public health perspective has led to development and implementation of clinical decision support and integrated registry tools, quality benchmarking, onsite technical assistance in quality improvement and practice redesign, and a novel reward and recognition program. In essence, the PCIP aims to provide even small independent practices with the health system advantages enjoyed by integrated health care organizations. The PCIP is evaluating trends in priority quality measures (e.g., blood pressure and lipid control) among 90 small practices randomized to receive clinical decision support tools and/or financial rewards. This proposed study of 60 comparison practices that have not adopted EHRs will provide a unique opportunity to demonstrate the potential of appropriately implemented health information technology for improving quality of care and reducing health disparities in a large community EHR program.

(Revised Abstract) Description: The rapid spread of HIV in many Eastern European countries has been linked to an increase in injecting drug use among young people. Non-injecting drug use has also increased among young people in this region. Non-injecting drug users are both at risk of initiating injecting drug use and of acquiring through sexual transmission not only HIV but also other sexually transmitted infections. Research is needed into the factors that increase the risk of HIV transmission among young drug users in this region. Such research is needed not only in countries where there is considerable HIV infection among IDUs, but also in countries, such as Hungary, which may be in a pre-epidemic phase, i.e. where HIV prevalence is currently low, but where behavioral and network risk is common among young drug users. In the proposed research, we plan to conduct a pilot study of HIV risk among young drug users between the ages of 16 and 30 in Budapest, the largest city in and the capital of Hungary. The specific aims of the study are to: 1. use ethnographic methods to explore (i) the drug use and sexual risk behaviors and practices of young drug users, (ii) their risk and social networks, especially injecting and sexual mixing patterns with people from high HIV prevalence groups or countries; (iii) the knowledge, attitudes, beliefs and peer norms of young drug users about HIV risk and prevention and, (iv) among young drug injectors, the behaviors, network characteristics, personal biographies and circumstances that may be linked to their initiation of injecting drug use; 2. develop and administer a structured questionnaire to a small sample of young drug users that will utilize questions that are based on the findings from the ethnographic study and questionnaires used in our previous studies; 3. develop and test methods for sampling and recruiting "hidden populations" of young drug users. The ethnographic part of the study will utilize focus groups, in-depth ethnographic interviews, and observational techniques. In addition, existing secondary data will be collected and synthesized. A structured pilot questionnaire and methodology for the sampling and recruiting of study participants will be developed. Procedures for HIV, HBV and HCV testing and counseling will also be developed and assessed. All data collection instruments, protocols, reports, presentations and papers will be both in Hungarian and English. HIV prevention in Eastern Europe needs to target populations that are the most vulnerable for becoming infected, particularly young injecting and non-injecting drug users. This study will provide new knowledge on young drug users in Hungary and will also contribute to advancing research methods and capability in other Eastern European countries.

Examine differences between pre-term and full-term human milk. This study is also designed to identify material factorswhich may influence the concentrations and determine changes in concentrations which may result from storage of breast milk.

The Children's Cancer Study Group is a multi-disciplinary organization formed to cooperate in research into malignant diseases in children: specifically, to study the factors which may cause or influence these diseases, and to develop, evaluate and improve methods of treatment. The primary objective of the cooperative group has been the evaluation of new biological and chemotherapeutic agents in the treatment of acute leukemia and solid tumors in childhood, and finding new and better ways of using the already known agents. This evaluation is done by pediatric oncologists, surgeons, radiotherapists, pathologists and immunologists within the cooperating institutions which make up the group. It is also our objective to provide supportive care for these children with leukemia and solid tumors which will enable all children who desire to do so to participate in our protocol studies. This supportive care includes financial supportive care (domiciliary care, travel assistance, outpatient costs, etc.), social service assistance, as well as medical supportive care.

Studies on long-term effects of two peroxisome proliferators inducing high and low carcinogenic activities respectively, indicated that factors other than oxidative injury may be important in the carcinogeneity of such chemicals. Speculation on the existence of specific receptors for mediating the action of peroxisome proliferators has recently been supported by the isolation of new members of the steroid hormone receptor superfamily which are activated by peroxisome proliferators: the mouse peroxisome proliferator-activated receptor (PPAR), the Xenopus laevis PPAR and the rat PPAR. Moreover, it has been shown that PPARs are capable of activating the promoter of the rat acyl coenzyme A oxidase gene, the key enzyme of peroxisomal fatty acid beta-oxidation, and mediates the induction of rabbit CYP4A6, a cytochrome P450 fatty acid omega- hydroxylase, by clofibric acid. This activation is carried out through specific response elements that have recently been identified. Our approach has been to examine whether differences between human and mouse PPAR could account for the marked differences in responsiveness to peroxisome proliferators on human hepatocarcinogenesis. To achieve this goal, we isolated the human PPAR from a human liver cDNA library and compared its structure and ability to activate the ACO response element with that of mouse PPAR. B-lymphoblastoid cells were used to construct a system which can be easily used to determine whether a chemical will be a peroxisome proliferator. The system uses an episomally-replicating plasmid containing the human PPAR cDNA under control of the thymidine kinase gene promoter, and a reporter gene having a PPAR response element driving expression of the CYP2A6 cDNA. A variety of constructs and promoters were tested to achieve maximal differences between untreated and peroxisome proliferator-treated cells.

PROJECT SUMMARY Breathing is a remarkable behavior fundamental to life that mediates gas exchange to support metabolism and regulate pH. A reliable, non-stop, robust rhythmic pattern of respiratory muscle activity is essential for breathing in mammals. Failure to maintain a normal breathing pattern in humans suffering from sleep apnea, apnea of prematurity, congenital central hypoventilation syndrome, hyperventilation syndrome, Rett syndrome, and perhaps Sudden Infant Death Syndrome, leads to serious adverse health consequences, even death. Various neurodegenerative diseases, such as Parkinson's disease, multiple systems atrophy, and amyotrophic lateral sclerosis, are associated with sleep disordered breathing that we hypothesize results from the loss of neurons in brain areas controlling respiration. If breathing is to be understood in normal and in pathological conditions, the mechanisms for respiratory central pattern generation must be revealed. We focus on two brain sites essential for generation of the normal breathing pattern, the preBtzinger Complex and the retrotrapezoid nucleus/parafacial respiratory group. We propose a broad series of experiments both in vivo and in vitro in rodents using advanced techniques including: viral delivery to express genetically encoded opsins or DREADDs in key subpopulations of neurons in these regions; advanced optical techniques to determine the contributions of the preBtzinger Complex microcircuit to rhythm generation; state-of-the-art neuroanatomical techniques to establish, in appropriate and necessary detail, the interconnectivity of the brainstem respiratory pattern generator. The data from these experiments will provide an extraordinary window into the mechanisms underlying respiratory rhythm and pattern generation.

We are entering an exciting era in pediatric rheumatology. New treatment approaches are improving the lives of children with juvenile idiopathic arthritis (JIA) to such a degree that it's now rare to see wheelchairs or crutches in our waiting rooms. Even splints, commonly used in the past to treat joint contractures, are seldom seen on our patients. Despite our progress, remission in JIA is rare. Recent work by our collaborator, Dr. Carol Wallace, has shown that only 5% of children with the polyarticular JIA (the most severe form of this disease) are in remission 5 years after diagnosis. Part of our problem in achieving remission is that, at the biological level, we don't really understand what "remission" is. It's a classic case of the difficulty of getting somewhere when you don't really know where you're trying to get. This grant application is about learning where we are trying to get. In this application, we aim to achieve a better understanding of what "remission" is using microarray-based biomarkers. Research from the Cobb (acute inflammation) and Jarvis (chronic inflammation) laboratories has demonstrated the feasibility of using genome-wide expression profiling can be used to define disease "states" (e.g., infected vs. not infected;in remission or not in remission). Furthermore, the Jarvis laboratory has demonstrated the promise of using these same technologies to predict clinical outcomes. For each group, these promising preliminary studies must be validated using larger patient populations and prospective study designs. In this application, we propose to validate peripheral blood biomarkers that already suggest that: (1) remission in juvenile arthritis can be identified at the molecular level through distinct gene expression signatures;(2) those signatures include the balance of both pro- and anti- inflammatory gene networks;and (3) the clinical course of children who reach an inactive disease state can be predicted based on molecular signatures that emerge in the peripheral blood mononuclear cells. Furthermore, we will take another step toward clinical application of this work by developing PCR-based whole blood assay to identify the most robust indicators of remission or clinical outcome. This project brings together two experienced investigators from two very different disciplines: pediatric rheumatology (Dr. Jarvis) and surgical intensive care (Dr. Cobb). Furthermore, the project brings together two computational biology groups spanning multiple disciplines, as well as other experienced pediatric rheumatology investigators. Thus, the project is highly responsive to the goals of the most recent NIH roadmap.

DESCRIPTION: (Adapted from the Applicant's Abstract.) This project is designed to describe some salient characteristics of mammalian cochlear hair cells. There are two types of such cells in our ears, inner hair cells and outer hair cells. The former are thought to be the true sensory receptors of the ear; they convey auditory information to the central nervous system. Outer hair cells, in contrast, may have primarily a mechanical effector (feedback) role in that they modify the mechanical input to the inner hair cells. To be studied in this project is the stimulus-response relations (electrical response versus hair deflection) for the two hair cell types in isolated organ of Corti segments. Also proposed is to study the mechanical motile response of outer hair cells to electrical stimulation and to hair deflection. These studies are conducted on single isolated outer hair cells for which a real life-like electrical-chemical-mechanical environment is provided. Hair cells are the key elements in the hearing process and their defects are the cause for the vast majority of hearing loss and deafness. Understanding their properties is a prerequisite for the eventual remediation of most hearing disorders.

[unreadable] The long-term goal of the proposed research is to develop an implantable polymeric device, which delivers gonadotropin releasing hormone(GnRH, aka leuteinizing hormone release hormone, or LHRH) in periodic pulses, mimicking the normal, ultradian endogenous rhythm. Rhythmic pulsed administration of GnRH has been used successfully in the induction of ovulation in amenorrhic women, and in the induction of puberty and maintenance of sexual maturity in both males and females with hypogonadotropic hypogonadism (HH), while continuous administration of GnRH fails. Present pulsatile treatment is administered using wearable pumps, with cutaneous breach by a catheter. For patients who must receive pulsatile GnRH for many years, this mode of delivery leads to significant inconvenience and danger of infection. The proposed device could be implanted subcutaneously or intraperitoneally, and may function for months or years. It is hoped that implantation will ameliorate the disadvantages of pump/catheter systems. The device concept features a hydrogel membrane, which undergoes periodic swelling-deswelling cycles, due to a feedback interaction between the membrane with the enzyme glucose oxidase. Release occurs during the swollen phase of the cycle. Oscillations are "fueled" by endogenous glucose at the site of implantation, whose level fluctuates only moderately in nondiabetics. (Rhythmic behavior of this device is distinguished from the glucose-responsive behavior of closed-loop insulin delivery devices.) While the feasibility of this concept has been confirmed in a model system, considerable work remains in converting the concept to a workable device that can be tested in patients. The specific goal of this proposal is to produce a prototype system whose properties can be tested in an in vitro environment that emulates in vivo conditions. In pursuing this goal, effects of the hydrogel membrane composition and thickness, the enzyme reaction, the device geometry, and the molecular properties of GnRH on the pulsatile release pattern will be studied. We will investigate device behavior in buffers and simulated plasma, when challenged with constant levels of glucose and programs of glucose fluctuations that are observed in nondiabetic individuals. [unreadable] [unreadable]

Mouse leukemia cells bearing TL surface antigens escape from destruction in mice immunized against those antigens, but the same cells are effectively killed when other antigens (e.g., H-2) serve as targets. Correlating with escape is the tendency for TL plus tumor cells to acquire resistance to TL antibody-\and guinea pig complement-mediated lysis following exposure to TL alloantiserum (antigen modulation or evasion). Evasion also affects some mouse lymphomas expressing murine leukemia virus major envelope glycoprotein, MuLV gp70. Evasion-positive tumors grow in all syngeneic mice immunized against gp70 by passive immunization with xenoantisera, whereas evasion-negative tumors are rejected by some immunized mice and growth is markedly suppressed in others. Evasion-positive and -negative variants of FLC745 and RBL-5 lymphomas are being studied to establish the relationship between antigen evasion and tumor escape. Xenoantisera, mouse antisera, and monoclonal antibodies to gp70 and other MuLV components expressed on the cell surface will be utilized, and evasion of cytolysis will be measured in a radiochromium release assay. Antigen lateral mobility, antibody-induced antigen aggregation, and steric hindrance of complement binding (guinea pig Clq) are apparently responsible for evasion. Factors not directly related to antigen evasion include: (1)\quantitative representation of target antigens before and during antibody sensitization and quantitative aspects of antibody binding; (2)\manner of antigen presentation at the cell surface, molecular associations, and molecular and ultrastructural organization; and (3)\differences in detected antigen specificities. Defense mechanisms involved in rejection of evasion-negative tumors are being examined, and a fully homologous (mouse antisera and complement) antigen evasion assay is being developed. Short-term gp70 antigen evasion appears to result from lateral mobility/aggregation of gp70 molecules influenced by surface membrane dynamics, motile cell activity, and cytoskeletal elements as well as antibody binding. There is no apparent involvement of complement (C3), differences in gp70 antigenicity, surface representation or configuration, differential cell sensitivity to complement lysis, or different cell growth characteristics. (IS)