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The interaction of anthracycline drugs and selected model compounds with DNA will be investigated by physical techniques including 31P,13C, and 1H NMR, viscosity, u.v.-visible spectroscopy, ultracentrifugation, and equilibrium dialysis. The general goal of these studies is to evaluate structural and thermodynamic aspects of the DNA-anthracycline in solution. Specific experiments with selected DNA samples are designed to answer questions about anthracycline binding specificities with DNA. For example, viscosity measurements with closed circular superhelical DNA will allow calculation of unwinding angles for anthracyclines while equilibrium dialys is measurements with T4 bacteriophage DNA will help in determining major versus minor groove binding of these drugs.

Colony-stimulating factor 1 (CSF1) is the principal colony-stimulating factor produced by osteoblasts constitutively and in response to several hormones and cytokines. It is absolutely required for osteoclastogenesis, but its role in regulating mature osteoclast function remains unclear. In vitro, it is known to prolong mature osteoclast survival and stimulate cytoskeletal remodeling and motility. We have found that CSF1 can regulate electrolyte transporters and gene transcription in mature osteoclasts, pointing to new metabolic pathways regulated by this cytokine. Recent studies have identified CSF1 as a therapeutic target in states of pathologic bone remodeling such as bone loss associated with inflammatory arthritis and bone destruction associated with skeletal metastases. CSF1 also appears to play an important role in mediating estrogen-deficiency bone loss. Therefore, a better understanding of how CSF1 acts to regulate mature osteoclast function is clearly of physiologic and therapeutic importance. The central hypothesis of this application is that CSF1 mediates mature osteoclast function in part by modulating the activity of the small GTPase, Rac1. We postulate that Rac1 lies at the intersection of two important effector pathways, one that targets actin remodeling and a second that targets gene regulation by modulating NF?B signaling. Three Specific Aims are designed to test this hypothesis. Specific Aim 1 explores the role of two Rac isoforms, Rac1 and Rac2, in mediating CSF1's actions in osteoclasts because we have defined CSF1-dependent, non- redundant roles for these isoforms in regulating the osteoclast cytoskeleton. Breast Cancer Associated Protein 3, is a Rac-interacting protein we have recently identified in a yeast two hybrid screen using an osteoclast cDNA library. We have discovered that BCA3 regulates CSF1-induced cytoskeletal remodeling and also augments NF?B p65 signaling. In the second Specific Aim, the role of BCA3 in mediating both CSF1 and RANKL signaling will be explored. Finally, in Specific Aim 3, a genetic approach will be used to further clarify the function CSF1 in mature osteoclasts by deleting its receptor, c-fms, in these cells. In the aggregate, these studies will provide a more detailed metabolic map of CSF1's actions in mature osteoclasts, which is a current gap in the scientific database. This new information will help inform ongoing efforts to develop therapies based on the actions of CSF1 in bone.

This project continues to investigate the immune responses, particularly the thymus-dependent phenomenon of contact hypersensitivity. We will continue to investigate the mechanisms of the development of contact sensitization. We will follow up on our studies of the induction of specific immunologic tolerance and its mechanisms. These include suppressor cells and clone inhibition and the relation between them. We will expand these concepts to include a study of sensitization and tolerance to proteins.

The long term goal of this project is to understand at the cellular level how hormones (steroids and peptides) regulate nervous system development and function. These classes of hormones have profound effects on the nervous system of most animals ranging from lower invertebrates up to humans. The metamorphosis of the nervous system of the moth Manduca sexta is a useful model system in which steroid and peptide hormone action can be studied in relation to identifiable, individual neurons. The action of these hormones can therefore be followed with greater precision in this invertebrate system than in any present vertebrate model of the CNS. Hormones control the proliferation, dendritic growth and programmed death of specific neurons in the insect. One part of the proposal will examine the steroid related death of differentiated neurons in the CNS. Autoradiographic localization of radiolabeled steroid in the CNS will provide information as to the relationship of steroid target cells to the cells that die. Also a study using a series of inhibitors of macromolecular synthesis will provide an initial test of the hypothesis that the onset of neuronal degeneration requires the activation of new genetic information. The steroids also affect other aspects of CNS development. A series of endocrinological studies in conjunction with 3H-thymidine birth-dating of cells will be used to establish the endocrine regulation of postembryonic neuronal proliferation and differentiation. Also, the biochemical basis of the steroid-regulated induction of sensitivity to the peptide eclosion hormone will be examined. The latter studies will involve a biochemical examination of the steroid's effect on molecules in the pathway that mediate eclosion hormone action (i.e. receptors, guanylate cyclase, protein kinase, and cellular substrates). Lastly, the action of eclosion hormone will be studied from an electrophysical point of view in the contex of identified circuits that change their properties in response to the peptide.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The goal of this study is to understand the role of the HIV-1 protein Vpu in HIV pathogenesis. Our previous studies demonstrated that Vpu modulates expression of the adhesion molecule VCAM-1 on endothelial cells (EC) via induction of the cytokine receptor CD40. Modulation of the endothelium in this manner may contribute to the characteristic extranodal presentation of AIDS-associated B cell lymphomas. Ongoing studies have shown that Vpu's effect on CD40 is likely the downstream consequence of Vpu modulation of another protein or pathway. We thus undertook an MS-based proteomics approach, stable isotope labeling with amino acids in cell culture (SILAC), to identify novel cellular proteins affected by Vpu. We compared the protein profiles of Hela-CD4 cells expressing wildtype Vpu to the same cell line expressing a mutant of Vpu that does not interact with beta-TrCP and thus cannot interact with the Ubiquitin-proteosome pathway for stabilization or degradation of Vpu targets. We identified 7 proteins that were up- or downregulated at least 1.5-fold by wildtype Vpu. One of these proteins, BST-2, was recently identified as a host antiviral factor whose function is to restrict the release of HIV virions from infected cells. The protein was thus renamed tetherin. Our novel finding that HIV Vpu could downregulate surface expression of BST-2/tetherin reveals a mechanism for HIV to counteract the host restriction. Based on the potential significance of these findings, we are focusing our current efforts on determining the mechanism and significance of Vpu-mediated BST-2 downregulation.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The long-term goal of this application is to develop therapies for protecting the gonadal function of children with SLE through therapeutic interventions whose beneficial effects have been verified by high-quality clinical evidence. The expected results of the current application are to determine the safety of triptorelin, its dose and the time necessary to achieve complete ovarian suppression (COS) prior to chemotherapy, as well as obtain preliminary evidence of its efficacy to provide ovarian protection for young females with SLE. The central hypothesis of this application is that triptorelin can safely achieve COS and may effectively preserve ovarian function of adolescent females with SLE requiring gonadotoxic chemotherapy with cyclophosphamide. The rationale that underlies the investigation is that identification of more effective interventions to preserve ovarian function and avoid premature menopause will translate into increased fertility, fewer risk factors of cardiovascular disease, osteoporosis and other long-term health complications of menopause and by this lower the overall societal costs associated with SLE.

It is widely recognized that colorectal cancer (CRC) is one of the most preventable cancers. If adenomatous polyps detected by screening were removed before they transformed into cancers, a decrease in new CRCs and in related mortality would follow. Unfortunately, cancer screening rates are low throughout Appalachia, including for Latino residents in PA. Further, Latinos are more likely to be diagnosed at later stage than Whites, have a lower survival rate and less access to quality treatment. Our long-term goal is to increase CRC screening uptake among Latinos in urban and rural central Pennsylvania (PA). Our central hypothesis is that a targeted CRC screening intervention can increase uptake over a non-targeted approach for this population. The specific objectives of the current proposal are to develop, implement and evaluate a targeted CRC screening intervention to increase CRC screening uptake. We will measure actual CRC uptake in both the control and intervention arm as a method to determinate completion rates. To prepare for my K01 application during my post-doc training, I led the design, implementation, and analysis of a CRC-related Latino study that provided preliminary data for theK01 work. In 04/09, utilizing the ACCN coalitions in PA, I conducted eight focus groups (FG) to identify barriers to CRC screenings among Latinos. We identified several substantial barriers to CRC screening for this population, including a significant barrier heretofore unmentioned in literature related to CRC screenings. Spanish-speaking patients want to have family members or close friends in the doctor office and health-related education classes. This barrier was reported across all groups and likely links to the desire for a strong social support that influences their CRC screening behavior. Another finding is that urban residents had some prior knowledge of risk factors associated with CRC screening while rural residents reported no prior knowledge and were unable to list any associated CRC risk factor. A high acceptability of the provider-recommended take-home fecal immunochemical test (FIT) for these Latinos was also reported. We exceeded our study recruitment goal. The same partnered Latino leaders are committed to the K01 proposed work. The K01 study design is a randomized, community-based, participatory design to determine completion of a provider-recommended, take-home FIT kit without (control) and with social support (intervention) among a sample of average-risk, urban and rural Appalachian Latino adults age 50 and older not currently adherent to national CRC screening guidelines (N=264).Each consented participant will attend a one-time, community- based CRC screening educational program offered at one of the eight study sites located in Central PA. The proposal is innovative because it combines provider-recommended FIT screening test with an education program and social support for Latinos in a randomized, community-based participatory research design. Further, it will provide preliminary data for evidence-based methods to increase CRC screening rates among Latinos throughout Appalachia, the focus of a future NIH NC1 R-series grant application. PUBLIC HEALTH RELEVANCE: In an effort to reduce this ethnic disparity among Latinos, screening rates among this population must increase. The specific objectives of the current proposal are to develop, implement and evaluate a multi-component targeted colorectal cancer (CRC) screening intervention to increase CRC screening uptake. The research will provide preliminary data for evidence-based methods to increase CRC screening rates among Latinos;and a future NIH NC1 R-series grant application.

System justification theory proposes that individuals develop belief systems to explain and legitimize existing status differences between groups in societies. Although people differ in the extent to which they endorse system-justifying beliefs, research shows they are widely endorsed and have important implications for mental health. In particular, recent research has found that system justification is related to mental disorders among members of low-status groups. However, the current research is limited by its focus on college samples and use of cross-sectional methods. The long-term objective of this application is to extend the current research on system justification by examining system justification as a risk factor for mental illness among low-income racial/ethnic minorities and recent immigrants, exploring relationships longitudinally and studying associations between maternal system justification and mental health and child behavior problems. The current project, comprised of two studies, relies on data from NYU's Center for Research on Culture, Development and Education (CRCDE), which have a number of advantages. First, the sample is composed of 200 low-income, immigrant and ethnically diverse women who recently gave birth in New York City. Second, mothers and their infants are followed longitudinally over three years and assessed using well- validated epidemiological screening tools for maternal mental disorders and child behavior problems. Third, the CRCDE combines quantitative survey measures with an embedded qualitative study of a random sample of survey members, allowing for a more nuanced understanding of relationships between constructs. The first study, focused on survey data, employs regression-based change score analyses and structural equation modeling techniques to explore longitudinal associations between maternal system justification, maternal mental illness and child behavior problems. I hypothesize that system justification will be positively related to maternal risk for mental illness and child behavior problems. The second study employs a mixed-methods approach, combining survey and qualitative data to explore the types of system- justifying beliefs endorsed by diverse low-income women, and identify factors that mitigate or exacerbate the relationship between system justification, maternal mental disorders and child behavior problems. The proposed project has direct implications for public health. System justification may be an important previously unidentified risk factor placing mothers and their children at risk for mental disorders and behavior problems. Understanding the role of system justification in mental health can inform policy and intervention efforts aimed at improving the mental health of vulnerable groups in the US.

DESCRIPTION: Bacterial resistance to beta-lactam antibiotics continues to become more prevalent and more clinically important. A large part of the resistance can be understood and investigated experimentally in terms of the chemistry of the interactions of beta-lactam antibiotics with the active sites of two groups of bacterial enzymes, the beta-lactamases on one hand, which catalyze the hydrolysis of the antibiotics, and the D-alanyl-D-alanine transpeptidase/carboxypeptidases on the other, which catalyze the synthesis an maintenance of the peptide cross-links of bacterial cell walls, and which are inhibited by beta-lactam antibiotics. There is now good reason to believe that all of these beta-lactam binding sites have much in common. An understanding o the structure and function of these sites and of the relationship between them is fundamental to future antibiotic design--both beta-lactam and otherwise. Th object of the proposed research is to explore further the chemical functionality and the substrate binding properties of a series of these active sites, using a number of modified substrates, novel inhibitors, and potential effecters. A mechanistic study of these sites, designed to determine the role of the functional groups present and the relationship between the proteinases, will be performed. Computational methods will be employed in order to interpre the results in terms of available crystal structures of these enzymes and to thus establish new guidelines to inhibitor design. In order to understand the structural and mechanistic basis of bacterial beta-lactam-resistance through mutation of transpeptidases, one important example of such beta-lactam-resistant enzymes, penicillin binding protein 2a of the methicillin-resistant Staphylococcus aureus (MRSA), will be studied in detail. These studies will lead to a clearer view of the chemistry of beta-lactamase and transpeptidase active sites, and thus to new directions in antibiotic design.

We are investigating a new experimental approach for the treatment of malignant brain tumors which utilizes a new class of potent, targeted anticancer compounds, called immunotoxins. We have initiated a dose escalation trial of regional therapy with the immunotoxin transferrin-CRM107 (Tf-CRM107) for the treatment of recurrent malignant brain tumors. Tf-CRM107 is a conjugate of human transferrin (Tf) and and diphtheria toxin with a point mutation (CRM107). Tf-CRM107 binds to the transferrin receptor, which facilitates iron uptake and is present in higher number on tumor cells than on the normal cells of the brain, and the diphtheria toxin mutant kills these tumor cells to which the Tf-CRM107 binds. The purpose of the study is to evaluate the toxicity of Tf-CRM107 when delivered by intra- and peritumoral slow interstitial infusion in a dosage-escalation schedule, and to assess antitumor activity in these patients. Ten patients with malignant brain tumors (5 glioblastoma, 3 anaplastic astrocytoma, 2 metastatic lung carcinoma) that have failed standard therapy (surgical resection or biopsy, radiation therapy, and chemotherapy in some), with evidence of tumor progression, have been treated. For treatment, single or multiple silastic infusion catheters were stereotactically placed intratumorally and Tf-CRM107 was infused over 2-6 days using an external syringe pump (rates 0.5-6.0 mu l-min). The initial Tf-CRM107 concentration was 7x10-10M which has been increased by 1-2 log increments every 4 patients; the last patient was treated with 7x10/9M Tf-CRM107. Total dose has increased from 0.05 to 27.3 micrograms. Patients were to be treated monthly, and of 19 total treatments, 5 patients have been treated twice and 1 patient has been treated 5 times. Tf-CRM107 infusions were well tolerated with no severe drug-related neurologic or systemic toxicity identified to date. Three patients suffered transient worsening of a neurologic-deficit that resolved with steroids and/or mannitol. Two seizures (1 generalized, 1 focal) occurred during a total of 19 treatments. Two patients required increased steroid dosages after treatment due to prolonged increased peritumoral edema. The only systemic effect of treatment detected has been a mild transient elevation of the liver enzyme SGPT in 6 of 10 patients. Of the 4 patients with MRI follow-up >1 month, all showed areas of decreased gadolinium (Gd) enhancement within their tumors after treatment, consistent with necrosis. In these patients, evaluation of Gd-enhancing volume showed reduction of 1 of 4, no changes in 2 of 4, and progression in the last patient who had a bilateral glioblastoma. In 2 change in 2 of 4, and progression in the last patient who had a bilateral glioblastoma. In 2 of 2 patients with accessible lesions that were resected after a single infusion, histology revealed extensive necrosis in the tumors with residual tumor at the edge of the necrotic areas. We anticipate treating up to 20-30 patients to establish the maximum tolerated dose of Tf-CRM107 when delivered to brain tumors by direct infusion. Evaluation of long-term toxicity, antitumor efficacy, immune response and Tf receptor immunohistochemistry in these patients is ongoing. Also, we plan to study the distribution of 111In-transferrin achieved (when co-infused with Tf-CRM107) with single photon emission computed tomography (SPECT). This data would be used to optimize drug delivery.

Deciphering the extra-telomeric function of Rap1, a metabolic regulator counteracting obesity Agnel Sfeir Project Summary: The telomere-binding protein Rap1 is part of the protective protein complex that binds mammalian telomeres. It was recently found to have additional non-telomeric functions, acting as a transcriptional cofactor for different biological processes. To explore its function more thoroughly, we disrupted mouse Rap1 in vivo and reported its unanticipated role in metabolic regulation and body-weight homeostasis. Rap1 inhibition resulted in dysregulation of hepatic and adipose function, leading to glucose intolerance, insulin resistance, liver steatosis, and excess fat accumulation, resulting in eventual late-onset obesity. At the cellular level, Rap1 appears to play a pivotal role in the transcriptional cascade that controls adipocyte differentiation. Using a separation-of-function allele, we found that the metabolic function of Rap1 is independent of its recruitment to TTAGGG binding elements found at telomeres, we identify a number of possible interactors that might aid Rap1 in its metabolic function. In conclusion, our recent study, together with ongoing experiments, underscores an intriguing function for the most conserved telomere-binding protein, forging an interesting link between telomere biology and metabolic signaling. In this project, we will decipher the underlying mechanism by which Rap1 controls metabolism. Specifically, we will explore the in vivo function and mechanism of Rap1 using a set of molecular and genetic tools. We hypothesize that Rap1 regulates adipose tissue function, mainly by impinging on the transcriptional cascade that controls the remodeling of white-to-beige fat. The impact of Rap1 on metabolic gene expression is most consistent with its propensity to behave as an adaptor protein, acting within the context of a larger transcriptional complex that we plan to characterize. The extra-telomeric function of a bone fide telomere binding protein raises the intriguing possibility of telomeres behaving as a storage site for this transcriptional regulator, thereby regulating Rap1 nucleoplasmic pools available to participate in metabolism. All in all, our study is expected to provide insight into Rap1 function in metabolic control, which is pivotal for understanding dysregulation that arises when this process is mismanaged, for example in age-dependent metabolic disorders. Furthermore, our study might help identify potential therapeutic strategies for regulating excess fat accumulation and protecting against metabolic derangements.

The proposed research will utilize nuclear magnetic resonance relaxation techniques, some of which are being developed in this lab, to elucidate our understanding of the conformational flexibility of proteins and nucleic acids. Although few details are known about the conformational flexibility of biopolymers, it apparently plays an important role in many biological processes. Consequently, we will study the motions in enzymes and the influence of substrates, inhibitors, activators, and unfolding on those motions. The dynamics in nucleic acids and nucleic complexes with proteins will be investigated. Systems to be examined include DNA, tRNA, synthetic RNA and DNA, ribosomes, messenger ribonucleoproteins, and viruses. In certain cases, stable isotopes will be incorporated into the proteins or nucleic acids to provide a nucleus for monitoring the internal motions.

There is a great need to develop new techniques for preparing blood samples. In clinical laboratories, preparing plasma for analysis is one of the most time consuming and dangerous tasks a technician faces. Since centrifugation is a part of the present problem, we propose to devise a system for preparing plasma by filtration. This technology will process blood approximately ten times faster than conventional procedures. Furthermore, the proposed technique can be automated to dispense aliquots of plasma into separate test tubes for further analysis. The proposed device will reduce the dangers associated with processing blood, decrease the time for preparing samples, and eliminate the number of skilled technicians needed to process blood samples.

Essential hypertension is typically thought to attack the brain late in its course, primarily through stroke. Accumulating evidence from both human and animal studies, though, shows that essential hypertension is associated with mild cognitive deficits, altered cerebral blood flow, decreases in grey matter volume, altered sympathetic nervous system function, and signs of premature brain aging. This evidence suggests that the brain is an early target of the disease, and, perhaps, the initial target. We propose to establish this point by examining pre-hypertensive individuals to establish that the brain sequelae of the disease are present prior to diagnostic levels of high blood pressure. We propose a cross sectional study of 250 participants followed by a 2 year longitudinal study of 200 participants with initial blood pressures in the pre-hypertensive range. Presence of brain sequelae in pre-hypertensive individuals will suggest that the currently accepted view is no longer tenable, the view that the brain is only affected late in the disease. Longitudinal results should indicate whether brain alterations predict later blood pressure or both brain and vascular effects of the disease occur in parallel. Risk factors associated with hypertension will be closely examined to see if such factors may implicate co-occurring pathology rather than hypertension in the induction of brain alterations. Successful completion of the project should direct clinical attention toward early sequelae of essential hypertension in both the brain and vasculature. PUBLIC HEALTH RELEVANCE: This project is designed to show that brain structure and function is altered by essential hypertension even prior to the appearance of hypertensive levels of blood pressure. Current medical thought suggests that essential hypertension is a vascular disease that late in its course can interact with atherosclerosis to pose a high risk for stroke. Current evidence and that proposed in this project suggest in contrast that the brain is altered early in the course of the disease. Recognition of this should direct clinical intervention to the reversal of these brain effects in addition to the reduction of peripheral blood pressure. The significance of such a re-orientation is clear given the pandemic proportions of essential hypertension.

The proposed study will extend an innovative investigation of biologic markers for autism using archived prenatal and newborn specimens from mother-baby pairs. Funded by the NIMH (R01 MH72565) for an initial three-year period, this project, known as the Early Markers for Autism (EMA) Study, is the first large, population-based case-control study to utilize these very early biologic specimens to elucidate underlying causes of autism. The study includes three groups: children with autism spectrum disorders (ASD), children with mental retardation (MR) but not autism, and children selected at random from the general population (GP). The scientific and conceptual focus of the EMA Study is on immunologic and genetic susceptibility factors, environmental exposures, and the interplay of genes with environment, operating either directly on neurodevelopment, or indirectly via dysregulation of the immune system. Preliminary results from the first funding cycle indicate that the mid-pregnancy immune profile of mothers of children with ASD is dysregulated in comparison to mothers of control children. Levels of select cytokines (IFN-gamma, IL-4, IL-5, IL-6) are elevated, and maternal antibodies to fetal brain proteins are present more often in case than control mothers. In addition, polymorphisms in select immune function genes are more common in mothers (IFN-gamma) and children (IL-6) with autism. This application seeks funding to further these initial findings by conducting a much larger case-control study among a new sample of 1,200 mother-baby pairs (400 ASD, 400 MR, 400 GP controls), with an added family-based component that includes 60 siblings of ASD cases and 60 siblings of MR controls. Funding to assemble this new study cohort has already been received by Autism Speaks and the study cohort will be assembled by September 2009. In this application, maternal mid-pregnancy specimens will be analyzed for polyfluoroalkyl compounds (PFCs), ubiquitous persistent organic compounds that have developmental toxicity and are likely endocrine disruptors. This large sample size will ensure sufficient statistical power for the examination of relatively rare exposures and phenotypic subgroups of ASD. Findings from this study will likely define areas for further investigations of physiologic mechanism and will provide normative data on unaffected controls. In the long-term, a better understanding of the underlying biology may suggest appropriate strategies for early intervention and contribute to the eventual prevention of this often devastating and usually life-long disability. PUBLIC HEALTH RELEVANCE: The primary goal of this study is to identify early biologic markers for ASD. We will use existing blood specimens taken during pregnancy to explore the interplay between markers of endocrine function and environmental exposures during critical periods of fetal brain development and identify factors that contribute to ASD risk. In the long-term, a better understanding of the underlying biology may suggest appropriate strategies for early intervention and contribute to the eventual prevention of this often devastating and usually life-long disability.

Organelle identity and development rely on a complex set of intracellular protein trafficking systems that mediate the specific targeting of nucleus-encoded proteins to their proper subcellular compartment. Not only must these trafficking systems maintain a high degree of specificity, they often must adapt to accommodate dramatic changes in the levels and composition of trafficking substrates imposed by developmental, stress and physiological events. Chloroplasts have evolved from the original endosymbiont to perform specialized functions in different tissues and cell types, giving rise to a diverse group of inter-related organelles called plastids. Plastid function and development rely on the coordinated expression and post-translational import of ~3000 different nucleus-encoded proteins. As such, plastids are an ideal model for studies of preprotein recognition and membrane translocation, and thereby, contribute to our knowledge of the basic processes of intracellular protein targeting and organelle biogenesis. A major aim of this proposal is to define the determinants and molecular interactions that mediate recognition of the targeting signals (transit peptides) of chloroplast preproteins by the TOC GTPase receptors at the chloroplast surface, and understand the process by which transit peptide recognition is coupled to GTP-dependent transfer of the preprotein into the membrane channel at the outer membrane of the organelle. The proposed roles of the TOC GTPases are reminiscent of the roles of the signal recognition particle (SRP/Ffh) and SRP receptor (SR/FtsY) in preprotein targeting to the ER and bacterial cytoplasmic membranes. In this context, the TOC translocon represent a novel, but analogous example of the GTP-regulated switches that regulate numerous intracellular protein targeting pathways. Our studies also suggest that multiple TOC pathways have evolved to balance protein targeting with the changes in gene expression that accompany developmental, physiological and stress events in cells. We will use the chloroplast system to understand the network of processes that 1) control the overall levels of organellar proteins expressed from the nucleus, 2) maintain the stichiometry of multi-protein complexes that contain both organelle and nucleus-encoded subunits, 3) respond to organelle status or dysfunction and, 4) coordinate changes in protein profiles during developmental transitions from one organelle type to another. PUBLIC HEALTH RELEVANCE: The goal of this project is to understand the molecular mechanism by which proteins are targeted to their proper cellular compartment or organelle using chloroplasts as a model system. The facile biochemical and molecular genetic tools available to study protein targeting in Arabidopsis thaliana makes this an ideal system to define general principles of protein targeting that apply to organelle systems in other eukaryotic cells and in bacteria. As such, these studies will contribute to our understanding of a fundamental process that is required for normal cellular function.

The need for a reliable method for estimating dietary consumption in the past has become critical in case referent epidemiological studies of the relationship of diet to cancer. The research objective of this study is to estimate the reliability of estimates of past dietary intake which are derived from present diet histories or from retrospective diet histories. A population on which a current diet history was performed in 1963 is located in Evans County, Georgia. Under this proposal this population would be reinterviewed about their recollections of their diet in 1963, and their current diet in 1977. The reliability of the retrospective diet history will be assessed by comparing it to the diet history obtained in 1963. The reliability of the current diet history is an index of past dietary intake, as well as the degree of dietary change in this population in 14 years, will be assessed by comparing the 1977 current diet history to the 1963 history. Comparisons will be made of average consumption of specific food items. Correlation coefficients of individual consumptions as reported on the respective surveys will also be calculated. Rigorous evaluation of the available methods for assessing past diet is needed. This study will contribute towards a quantitative estimate of their value. Should their reliability prove low, waste of resources on unreliable studies will be avoided. Should their reliability prove high, studies will not fail to be undertaken because of unwarranted negative prejudice against these methods.

Current projections indicate that the number of individuals 65 years and older is expected to double by the year 2030. Because thoracic aortic aneurysm (TAAs) occur most frequently in persons 60-70 years of age, it follows that over the next two decades the frequency of aneurysm disease diagnosis is going to increase dramatically. TAA disease is an insidious process which often causes death by rupture in the absence of antecedent symptomology. There are presently no available indices to determine which patients may be at greater risk for acceleration of TAA development, warranting more immediate treatment. Therefore, further diagnostic and therapeutic advancement is critical, especially for those patients who have not yet reached surgical criteria. TAA development proceeds by a multifactorial process that is regulated by both intracellular and extracellular mechanisms which drive the pathological remodeling of the aortic extracellular matrix (ECM). The matrix metalloproteinases (MMPs) are a family of proteolytic enzymes that play an obligate role in TAA-induced ECM remodeling. In addition to structural remodeling, changes in the cellular constituents have also been demonstrated, and recent data has identified the aortic fibroblast as a critical mediator. When age-dependent changes in aortic structure and composition are overlaid with other risk factors for aneurysm development, the threshold, severity, and prognosis of aneurysm disease is likely accelerated. Therefore, the present application proposes to test the central hypothesis that age-related changes in thoracic aortic aneurysm development are driven by alterations in signaling through the transforming growth factor-beta pathway and result in altered aortic extracellular matrix mediated by phenotypically unique fibroblasts. This hypothesis will be addressed through three specific aims: 1) Demonstrate that differential acceleration of TAA development in young and old mice is a direct result of structural and compositional differences in aortic substrate; 2) Establish that age- dependent changes in TAA progression are a direct result of alterations in aortic fibroblast number, phenotype, and function; and 3) Establish that defects in TGF-b signaling are responsible for age-dependent changes in TAA progression in the old aorta. The outcomes from this project will establish that the structural and compositional changes in the aortic ECM of old mice are mediated by age-dependent defects in collagen and MMP/TIMP gene expression. These age-dependent changes are affected by reduced signaling through the TGF-b pathway and result in a weakened aortic substrate that is hyper-responsive to TAA stimuli. Collectively, these studies will provide significant insight toward understanding the pathophysiology of TAA development, and will form the basis for novel, rational drug design for therapeutic modulation of TAA progression in patients with this devastating disease.

Trichotillomania (TTM) is a chronic impulse-control disorder involving the repetitive pulling and removal of one's hair that results in notable hair loss. A growing body of research has noted the public health significance of the disorder. TTM can result in a variety of debilitating physical, psychosocial, occupational and educational consequences. Empirical support for the efficacy of both pharmacological and nonpharmacological interventions for TTM is extremely limited, and there is growing recognition that traditional behavior therapy alone is likely insufficient. At a scientific meeting jointly sponsored by the National Institutes of Mental Health and the Trichotillomania Learning Center (http://www.nimh.nih.gov/scientificmeetings/trichotillomania.pdf), the paucity of treatment outcome research on TTM was noted and a specific recommendation made for researchers to conduct larger randomized clinical trials to evaluate enhancement to current behavior therapy relative to treatment as usual... To guide the enhancement of behavior therapy procedures, research on the psychopathology of TTM has discovered at least two distinct processes that may underlie the disorder. These include a habitual or automatic process and an emotional/cognitive regulatory or focused process. Preliminary feasibility and pilot studies conducted by the PI suggest that a treatment designed to address both of these processes (Acceptance-Enhanced Behavior Therapy for TTM; AEBT-T) may be an effective intervention for adults with the disorder. The current application proposes to compare a 10 session manualized version of AEBT-T to a manualized psychoeducation and supportive therapy (PST) in adults with TTM. Primary outcome variables will be assessed by blind independent evaluators, using a multi-element assessment battery. The relative efficacy of AEBT-T and PST for alleviating TTM-related distress and impairment will also be assessed, and treatment gains will be monitored over a six month follow-up. The primary aim of this study is to compare the benefits of AEBT-T to PST for reducing TTM severity in 84 children and adults (age 18-65) with TTM over a 12-week (10 session) acute treatment period. A secondary aim of the study is to examine the durability of both treatments in acute-phase treatment responders over a 6-month follow-up interval. In addition, a number of exploratory analyses are proposed to explore potential predictors of treatment response. Trichotillomania (TTM) is a disorder that has negative physical, social, occupational, academic and psychological effects. Despite its profound consequences, there exists no well-supported empirically based intervention to treat those with the disorder. The current study addresses the mission of the NIMH by determining if children and adults with TTM can benefit from behaviorally oriented treatment to decrease pulling severity and related distress.

ABSTRACT Melanoma is a major world health problem with an incidence rate that is rising rapidly. Within the past several years, there has been tremendous progress in novel melanoma therapies ? particularly with regard to immunotherapy as highlighted by the FDA approval of ipilimumab (anti-CTLA-4 antibody) in 2011 and pembrolizumab and nivolumab (anti-PD-1 antibodies) for melanoma in 2014 and 2015. Furthermore, when CTLA-4 and PD-1 blockade are combined, response rates are significantly increased, leading to the FDA approval of this regimen (ipilimumab + nivolumab) in 2015 ? however, toxicity is admittedly high. Despite these advances, there are still a significant proportion of patients who do not achieve clinical response to these agents. Therefore, there is tremendous need to identify biomarkers that may predict response or resistance to immune checkpoint blockade ? either as monotherapy or in combination ? and to identify actionable strategies that will enhance the effectiveness of these potent therapies. Our group has been actively engaged in efforts to better understand responses to immune checkpoint blockade therapies. In these studies, we performed deep immune and molecular profiling in a cohort of patients on immune checkpoint blockade, and recently demonstrated that an immune infiltrate in early on-treatment tumor biopsies is highly predictive of response. However it remains unclear what contributes to enhanced responses, and there is a critical need to identify actionable strategies to improve responses to therapy in all patients. There is a growing appreciation of the role of the gut microbiome in shaping immune responses in health and disease, and pre-clinical evidence that bacteria present within the gut may modulate differential responses to immune checkpoint blockade in melanoma, though this concept has not been studied in patients. This represents a significant knowledge gap, and insights gained could lead to therapeutic strategies to enhance responses to immune checkpoint blockade in melanoma. We have begun to address this knowledge gap in clinical samples from melanoma patients treated with immune checkpoint blockade, and are also studying this in a murine melanoma model. We have preliminary evidence suggesting that differential signatures exist in responders versus non-responders to therapy, and have insight into mechanisms via immune profiling and metabolomic studies. We have also generated data in a murine melanoma model, demonstrating differential tumor growth in C57BL/6 mice with identical genomes but differing gut microbiomes. We will now build on these studies through this proposal to explore the role of the microbiome in shaping anti-tumor immune responses and clinical responses to immune checkpoint blockade in melanoma.

We are currently discussing possible new vaccine candidates for therapy of herpes simplex, and planning new clinical trials.Among these is a live, replication-defective HSV-2 vaccine that we showed this past year to be effective in the immunotherapy of genital herpes in the gunea pig model.

Phase I study to determine the safety, tolerance, and pharmacokinetic profile, biological effects, and maximally tolerated dosage level of escalating of both recombinant human tumor necrosis factor and Cisplatin administration by i.v. route to patients with histologically documented solid tumors resistant but non-amenable to conventional chemotherapy.

This application represents the first competing renewal of our program project, which uses an interdisciplinary team approach to address the unifying hypothesis that most, if not all, aging-related neurodegenerative disorders are caused by the intracellular or extracellular accumulation of specific proteins that have assumed pathogenic conformational states (proteinopathies). The resulting neurodegenerative disorders, which include Alzheimer's disease (AD), Huntington's disease (HD), Parkinson's disease (PD) and other Lewy body diseases (LED), remain largely untreatable and represent a complex biomedical, behavioral and social problem. Medical breakthroughs are urgently needed in this area, and the surest way to such breakthroughs is to determine how exactly these diseases result in the dysfunction and degeneration of nerve cells. Our program addresses this important need by bringing together investigators with diverse areas of expertise, widely overlapping interests in proteinopathies, and an established track record of fruitful collaborative interactions. Our approach takes advantage of a great number of valuable resources and technologies, including robotic microscopy, molecular imaging, genetically engineered mouse models, RNAi mediated gene silencing, and cell type-specific expression of mechanistically informative viral constructs. Using these and other strategies, we will study the processes by which diverse proteins impair neuronal function and survival and compare our results to determine whether there are common mechanisms of neurodegeneration. We will also study the susceptibility of different neuronal populations to common versus disease-specific pathogenic processes to elucidate why these diseases so selectively attack specific neuronal populations. Project 1, Mechanisms of Cell-Specific Huntingtin-lnduced Neurodegeneration aims to elucidate cell autonomous and cell non-autonomous mechanisms that contribute to the susceptibility of striatal neurons to mutant huntingtin. Project 2, Microglial Kynurenine Pathway and Selective Neuronal Vulnerability, will test if genetic or pharmacological inhibition of the microglial kynurenine pathway is protective in mouse models of AD and HD. Project 3, Apolipoprotein E in Alzheimer's Disease: Cellular Mechanisms, will study the regulation of apolipoprotein E expression in neurons and explore Apin dependent roles of different apolipoprotein E isoforms in the pathogenesis of AD. Project 4, Causes and Consequences of a-Synuclein Aggregation, will assess in combined models of AD and PD if interactions between a-synuclein and A lead to neurodegeneration of specific neuronal populations through activation of glutamate receptors and proteases that cleave a-synuclein. Project 5, Mechanisms of Aft-Induced Neuronal Deficits, will test whether the modulation of specific neuronal or glial molecules can block aberrant neuronal overexcitation and ameliorate behavioral abnormalities in mouse models of AD and other proteinopathies. The Cores (A: Administrative, B: Tissue Culture, C: Animal, D: Microscopy/Neuropathology) will provide the common services necessary to accomplish the goals of the program project. Our studies will shed light on diverse neurodegenerative diseases and could provide the knowledge needed to better treat and prevent them.

To determine whether mixed Beta, alpha-adrenergic stimulation of fat from upper body and leg fat in women and me compared with pure Beta-adrenergic stimulation.

Project Summary. Increased lung microvascular permeability, resulting in protein-rich alveolar edema and chronic inflammation, causes ARDS (Acute Respiratory Distress Syndrome), the lethal form of acute lung injury (ALI). During the last funding cycle, we demonstrated that endothelial cell (EC)-specific deletion of focal adhesion kinase (FAK) disrupts adherens junctions, causing chronic pulmonary edema. We also showed that FAK is markedly reduced in lungs of ARDS patients. These findings suggest that FAK expression is critical for endothelial barrier repair and hence lung-fluid homeostasis. Cellular therapy can resolve inflammatory lung vascular injury in animal models, but bone marrow-derived mesenchymal stem cells (MSCs) or MSC-derived exosomes failed to do so in EC-FAK-/- mice. In investigating the role of EC-FAK in regulating the effectiveness of cellular therapy in resolving lung vascular injury, we made the fundamental observation that EC-FAK is required to maintain the expression of sphingosine-1-phosphate receptor1 (S1PR1) in ECs, which is known to strengthen the endothelial barrier and prevent lung injury. Thus, we postulated that impaired SIPR1 synthesis is responsible for defective endothelial barrier repair in EC-FAK-/- mice and for the loss of efficacy of stem cell therapy. Intriguingly, S1PR1 expression and barrier function could be rescued in FAK-depleted ECs following transduction of the transcription factor, Kruppel like factor 2 (KLF2), indicating that FAK functions by upregulating KLF2 activity. Active KLF2 thus overcomes FAK depletion by restoring S1PR1 expression and function. These findings led to our second seminal observation that loss of KLF2 transcriptional activity in EC-FAK-/- mice is due to epigenetic modification of KLF2-DNA caused by activation of DNA methyltransferase 3a (DNMT3a). DNMT3a converts cytosine to 5-methylcytosine (5mc) repressing gene transcription. Hence, DNMT3a methylation of the KLF2 promoter led to impaired S1PR1 synthesis and barrier repair. Based on these findings, the planned research will define the novel role of FAK in inducing EC barrier repair following injury by suppressing epigenetic modification of KLF2, thereby enabling S1PR1 transcription and function. We will use state of the art approaches, including EC specific knockout mice, cellular and nuclear imaging and biophysical approaches such as measurement of cellular tension to establish this concept. Our Specific Aims are: #1: to address the concept that FAK maintenance of S1PR1 transcription in the endothelium is required for intrinsic endothelial barrier repair and thereby for resolving lung vascular injury, and #2: to define the role of FAK suppression of endothelial KLF2-DNA methylation by DNMT3a as a mechanism for S1PR1 synthesis by KLF2 and the role of this pathway in restoring endothelial barrier integrity, resolving lung vascular injury and promoting tolerance to secondary lung injury in EC-FAK-/- mice. We believe our studies will define selective inhibition of DMNT3a as a potential clinical target for preventing the lethality of ARDS.

This project proposes to continue our studies on Carbohydrate Binding Protein 35 (CBP35), a predominantly intracellular lectin that binds to galactose-containing glycoconjugates. The polypeptide (M/T about 35,000) consists of two domains: a proline- and glycine-rich domain at the NH2- terminal half and a carbohydrate recognition domain at the COOH-terminal half, with extensive homology to other S-type lectins. The addition of serum growth factors to quiescent cultures of mouse 3T3 fibroblasts increased the expression of CBP35, in terms of elevated transcription rate of the gene, increased accumulation of mRNA, and increased amount of the protein that is translocated to the nucleus. Analysis of CBP35 in the cytoplasm and nucleoplasm suggests that the lectin is associated with a ribonucleoprotein complex, as indicated by its position of sedimentation on sucrose and cesium sulfate gradients. Using a cell-free assay for the splicing of intervening sequences from pre-mRNA, we found that saccharides and antibodies that bind to CBP35 perturb the splicing reaction, whereas control reagents failed to yield the same effort. On the basis of these results, the specific aim of the proposed research includes: (1) to determine the effect on cell-free splicing activity when a nuclear extract is depleted of CBP35 and to attempt to reconstitute the system by readdition of CBP35; (2) to transfect cells with CBP35 cDNA in the sense and antisense orientations and to monitor the growth and the status of mRNA in these cells; (3) to identify the RNA and protein components of the CBP35- ribonucleoprotein complex; and (4) to search for a homolog of CBP35 in yeasts and to study the in vivo consequences of a gene knockout via integrative disruption.

This study aims to develop a more efficient and timely system for assessing renal function and acute change in renal function. It proposes to determine if any of four commercially available grades of technetium 99mTc-DTPA can be used to provide good estimates of GFR. Clearance results from these four chelates of technetium will be contrasted with a gold standard of inulin clearance to determine the best marker of GFR for clinical use.

This proposal aims at the design, preparation, and testing of microfluidic devices containing porous polymer monoliths that will provide unmatched performance in "sample preparation" and the identification of proteins in complex mixtures and enable full exploitation of the rich potential of proteomic research. It will provide access to an enhanced array of tools for handling biological samples.The devices made of glass or plastic will contain functional monoliths prepared in situ from liquid precursors via a simple free radical polymerization process. This will afford a generic macroporous monolithic polymer with porous properties optimized for the desired application that can subsequently be functionalized by UV intiated photografting. The photografting enables the placement of sevral different functionalities (such as hydrophobic, hydrophilic, ionizable, or affinity) in precise locations placed in series or as layered or gradient chemistries. This proposal also aims at the development of new approaches to the covalent immobilization of enzymes and ligands that will enable independent immobilization of multiple biological molecules in the same channel. Using the benefits of sequential photografting, monolithic devices integrating sample collection, enzymatic digestion of proteins, and separation of peptides will be prepared. Difficult protein mixtures will be separated into less complex samples using separation modules with carefully designed selectivities. Optimization of structural properties of the monoliths is expected to afford recoveries close to 100% for all of the proteins present in the sample. Specifics attention will also will be paid to designing and controling all preparation processes to ensure high batch-to-batch reproducibility of both building blocks and composite devices. Direct coupling of these devices to a mass spectrometer will simplify and accelerate proteomic research. We expect that this project will result in the practical implementation of a totally new array of versatile components and devices for sample preparation and the mass spectroscopic study of proteomes or their components extending microanalytical capabilities well beyond the current state of the art.

The overarching goal of this research is to integrate family systems and developmental psychopathology theory and research to understand the processes underlying the association between destructive marital conflict and children's mental health with a specific emphasis on the role that sibling relationships play for understanding patterns of child adjustment and maladjustment. Destructive marital conflict poses significant risk to children's mental health and adjustment (for a review see Davies &Cummings, 1994) with significant implications for society. Moreover, sibling relations can either exacerbate child difficulties, or alternatively, promote positive child adjustment (for a review see Brody, 1998). Thus, high social importance is attached to understanding the role of sibling relations in the association between marital conflict and child outcomes. Toward this endeavor, there are four main aims of the proposed research. First, this research addresses theoretical and methodological gaps in the field. The sibling relationship literature has been heavily influenced by social learning theory (see Dunn &Davies, 2001;Jenkins, 1992;Stacker &Youngblade, 1999). As a result, the survey measures in the area commonly focus on warmth and conflict measures without consideration of family systems constructs. To address this gap, the first aim of the proposal is to develop and test the validity (i.e., predictive, discriminate, convergent) of a semi-structured interview assessment of sibling relationships based on family systems theory. Next, the processes by which marital conflict is associated with child psychopathology will be explored. Integrating family systems within sibling relationships and child psychopathology, the quality of children's sibling relationships will be examined in three models: additive (Aim 2), mediational (Aim 3), and moderator (Aim 4) to illuminate the link between marital conflict and child maladjustment. To test the proposed pathways, a multi-method, longitudinal study will be completed with children, their primary caregivers, and their teachers. This research complements and advances existing knowledge by (a) elucidating the impact of inter-parental discord on children's mental health;(b) examining children's sibling relations as potential processes that highlight the link between marital conflict and child maladjustment and;(c) incorporating an innovative assessment of children's sibling relationships designed to identify undetected patterns of sibling relations. PUBLIC HEALTH RELEVANCE: The multiple-method, multi- informant, and longitudinal design will yield a rigorous assessment of the study's main constructs. Results will be distributed and discussed as they pertain to future research and prevention/intervention efforts to enhance children's mental health.

Prescription drugs have become an increasingly important component of mental health treatment and the costs of psychotropic drugs have increased rapidly in recent years. However, there are major gaps in our knowledge about the economics of psychotropic drug treatment. This Mentored Research Scientist Development Award would allow Dr. Haiden Huskamp, a health economist with expertise in mental health policy and economic institutions, to supplement her economic tools with the knowledge and skills needed to conduct clinically-relevant and policy-significant research on the economics of prescription drugs used in the treatment of mental illnesses. The specific aims of this career development proposal are to: 1) develop a greater understanding of clinical decision-making related to the use of psychotropic drugs; 2) acquire basic knowledge of psychopharmacology; and 3) expand knowledge of the important economic institutions influencing the prescription drug market. In this undertaking, Dr. Huskamp will be guided by her sponsor, Richard Frank, PhD, and co-sponsors, Andrew Nierenberg, MD, and Ernst Berndt, PhD. Her career development plan includes guided study with Dr. Nierenberg on clinical issues related to treatment decision-making and Drs. Berndt and Frank on economic institutions of the pharmaceutical market, as well as coursework and participation in psychopharmacological "Grand Rounds," relevant seminar series, and professional meetings. Dr. Huskamp will use the knowledge and skills developed through these career development activities to conduct three research projects. The first project examines the effect of generic entry in the class of selective serotonin reuptake inhibitors (SSRls) on utilization patterns, costs, and market share among antidepressants as well as the competitive response of brand antidepressant manufacturers with respect to drug prices and promotional spending. The second project assesses the economic incentives created by three-tier drug formularies and how these arrangements affect costs, utilization patterns, and adherence to treatment guidelines in a non-elderly population. This project includes an economic welfare analysis of the tradeoffs associated with restrictive formularies. The third project examines the effect of a three-tier formulary on psychotropic drug costs and utilization patterns in a retiree population and explores the impact of formularies on the mental health costs of adding a prescription drug benefit to Medicare and on access to appropriate psychotropic drug treatment under such a benefit. The proposed plan of career development will provide Dr. Huskamp the training, mentoring, time and resources to develop the skills that will put her in a position to lead independent research on the economics of pharmaceutical treatment for mental illnesses.

Thyroid hormones stimulate growth of normal and malignant cells, and influence metabolism and development. They are, therefore, excellent signals to study as a model for understanding general features of regulation of these processes in mammalian systems. In the proposed studies, we plan to investigate the molecular mechanism of thyroid hormone action. To do so we hope to purify chromatin "receptor" proteins which appear to be mediators of the thyroid hormone response. We plan to study the properties of these receptors in terms of potential biological function and determine the way in which they interact with chromatin and DNA. We also plan to investigate using thyroid hormone-responsive cultured cells whether there are direct hormonal influences on RNA synthesis. We hope to establish cell-free conditions to investigate actions of thyroid hormone and of purified receptors bound by the hormone on RNA synthesis by chromatin. We hope to determine the interrelation of different thyroid-induced responses and gain information as to whether diverse responses such as those on RNA synthesis and transport all occur through nuclear events. We plan to determine the extent to which thyroid hormones stimulate growth or normal and malignant cells and to study the mechanisms of this regulation. Finally, we plan to select cells which vary in their responsiveness to thyroid hormone and to use these to facilitate understanding of thyroid hormone action and genetic regulation.

This research proposes to construct and analyze a longitudinal model of the relationship between family composition and the labor force participation of married women. The model will be estimated for two separate cohorts of women drawn from the National Longitudinal Surveys. Family composition is conceptualized as a dynamic set of variables including the number, ages, and spacing of children. The dimensions of labor force participation to be included in the model are labor force experience, the intensity of labor force participation, and the nature of the occupational task. The second stage of the analysis will address the manner in which the parameters of the model are influenced by a set of exogenous variables. Once the model has been estimated for the two cohorts the parameters will be utilized as dependent variables hypothesized to be influenced by the exogenous variables. The respondent's education, the availability of childcare, the family's economic well-being, and the familial context are all thought to affect the nature of the basic relationship between family composition and labor force participation as defined in the model. The successful completion of this research will generate one model of the reciprocal causal relationship between family composition and labor force participation from a national sample of women who have completed their families and another model from a sample of women who are in the process of family building. The inclusion of the exogenous variables will provide additional information on the specific conditions under which the relationship takes various forms.

Morphometry and Physiology Core. The purposes of Core C are to evaluate the morphometric changes that occur during graft arteriosclerosis (GA) that culminate in constrictive remodeling and to assess the vasomotor function during the progression of GA in vitro. The combination of histological assessment and morphometric evaluation in conjunction with vessel physiology will allow us to dissect when vasomotor function contributes to GA remodeling. Core C will interface with Projects 1, 2 and 3 to assess the degree of remodeling and function in vessels undergoing GA in vitro and vessels modified in organ culture.

The Resource catalogs gene expression data in the endometrium. The EDR has established a List of genes whose expression has been associated with uterine function. Currently, this list includes 1262 genes. This list is divided by species. The genetic information has been accumulated for human, mouse, monkey, rat, rabbit, guinea pig, hamster, pig, sheep, goat, bovine, and horse. The data for each gene is listed in a table and gives basic genetic information, expression data, phenotypic analysis and links to other informatics resources. The basic genetic information includes chromosomal location and gene ontology. The expression data includes a description of cellular expression, timing of expression, and endocrine or paracrine regulation. The phenotypic analysis includes gene ablation studies, as well as, any clinical reports of gene function. Finally, all genes are linked to OMIM, MGI, Unigene, and Genbank, as well as, to the homologous gene in other species within the EDR. Over the last 12 months, we have established this format and first began filling the database with genes and are now annotating these genes for the above information. In the accumulation of genes for the EDR, we have exploited publications using high density DNA microarray as a tool to identify expression differences of the genes in the uterus. In order to accomplish this, we have listed all uterine microarray publications in the EDR and have imported all genes that have been identified as differentially expressed in the uterus in these assays into the EDR. These genes are inserted into the EDR and identified as being expressed in the uterus as determined by microarray experiments. The EDR also has a separate site that lists all genes identified by microarray and lists common genes between different microarray experiments. This approach allows the identification of genes that are in common among similar microarray experiments, as well as, genes that are identified as differentially expressed in the uterus under different endocrine or physiological conditions. This approach will allow investigators to determine if other researchers have identified the gene they are interested in as being subject to differential regulation. 2. The Resource catalogs transgenic and knockout mouse models for the regulation of endometrium development and function. The EDR has a separate page that lists all knockout mouse models that have been associated with a uterine and/or reproductive phenotype. This will allow investigators to identify which genes are pivotal in regulating uterine function. As new models develop, they will be added to the EDR. Currently, there are 32 genes listed. 3. The Resource will compile protocols for the investigation of endometrium function in various animal models. The EDR has a section dedicated to invite investigators to write short essays on research topics, clinical topics, model systems, and experimental protocols. As the EDR develops, investigators will be recruited to submit such topics. Dr. DeMayo will request reviewers for such essays and once they pass an initial review they will be posted in the EDR. 4. The Resource established a forum where investigators can post commentaries and reviews on specific topics related to endometrium function and disease. The EDR has established a forum section for investigators to discuss current breaking results in endometrial research. This section has just been established and will encourage investigators through the SCCPRR Endometrial Focus Group to utilize this resource. Although the majority of genetic information in the EDR is deposited by the curator of the EDR, this site offers investigators the ability to enter information. An investigator who wants to enter information into the EDR can register and enter information into the EDR data page regarding any specific gene. Dr. DeMayo reviews the information prior to being entered into the EDR main database and the investigator is given credit for the entry. In summary, the EDR offers a unique bioinformatics resource for endometrial researchers. Over the last 12 months the EDR has established a template to accumulate this information. Over the next 12 months, we will annotate the genes in the EDR and recruit outside investigators to participate in the EDR by posting essays, protocols and discussions.

This is a revised competing renewal application for an environmental toxicology training program for 6 pre-doctoral and 3 postdoctoral fellows. Since this program started in 1990, training has been provided to 9 postdoctoral fellows and 29 pre-doctoral fellows. The 14 trainees who have completed their formal education have professional positions where they are utilizing their toxicology knowledge for basic research, biotechnology, or practical problems of environmental risk assessment. Three have academic faculty positions. Long-term goals of those who are pursuing further training in fellowships (6) or in law school (3) are also to address health problems of environmental agents. 100% of the 9 who sought an individual postdoctoral fellowship were successful, including 5 F32 awards from the NIEHS, which the investigators consider a positive reflection of their requirement that every trainee submit a competitive research proposal. The investigators propose research training in three areas where they have a critical mass of experienced faculty and superb physical resources for state of the art research. These three areas of pathophysiology, DNA damage and repair, and biotransformation and transport share a unifying theme of oxidant injury. Training in the broad area of pathophysiology will focus on toxic agent injury to the GI tract, CNS (i.e., brain and ocular tissue), and lung; this latter site meshes with new participation by clinician-scientist faculty in their recently approved Asthma and Allergic Disease Center. An additional, unique feature of the proposed training is that the research by several faculty in each area includes assessments of the influences of aging and infection. A totally updated curriculum builds upon the reorganization of the graduate school towards an interdisciplinary common first year curriculum. Core courses in mechanistic and advanced toxicology culminate in student preparation and peer review of research proposals. Novel aspects of the toxicology program curriculum for pre- and postdoctoral fellows: are 1) a distance education toxicology course that is conducted by trainees with faculty supervision, and 2) mini-courses on specialized topics that is taught by visiting experts. Hallmarks of institutional commitment include full funding of stipends for the first 4 terms of graduate school and remarkable support for new and bridging faculty grants from endowments. Key elements in the investigators? outstanding climate for training are a multiplicity of environmental-health relevant research centers supported by the NIEHS and a local foundation, notably a new (March 2000) Sealy Center in Environmental Health and Medicine.

Although lithium displays remarkable mood-stabilizing properties and has served as one of the most effective therapies for bipolar disorder (BPD), the mechanisms underlying its actions on the nervous system remain unclear. The long-term goal of this project is to understand-at the molecular and cellular levels-how lithium affects nervous system function. The objectives of this proposal are to identify genes that are involved in the lithium-responsive neurological process and to determine their roles in lithium's therapeutic action. To accomplish these objectives, we will utilize genetic tools that are uniquely available in the fruit fly Drosophila melanogaster. These include a neurological mutant Shudderer (Shu), whose phenotypes are largely rescued by lithium administration at therapeutic concentrations. Our central hypothesis, which is based on strong preliminary data, is that lithium reduces the severity of neurological defects caused by up-regulation of the Ca2????dependent phosphatase calcineurin, and that it does so by suppressing the innate immune response. The rationale for the proposed research is that, once the genes involved in lithium-responsive neurological processes in Drosophila have been identified and their roles have been revealed, this information should be readily translated into the vertebrate system, which is known to use signaling mechanisms that overlap extensively with those in Drosophila. Thus, our study is expected to provide novel and important insights into lithium's therapeutic action-with respect not only to BPD, but also other neurological disorders. Our central hypothesis will be tested by pursuing three specific aims: 1) Determine how up-regulation of calcineurin causes the lithium-responsive neurological defects; 2) Delineate the mechanisms responsible for lithium's therapeutic action on the Shu phenotype; and 3) Identify novel genes whose up- or down-regulation mimics lithium's actions in the nervous system. For the first aim, particular cell types and developmental timing of the calcineurin overexpression leading to the Shu phenotype will be defined by genetically manipulating calcineurin activity in a spatially and temporally specific manner. For the second aim, various genetic variants will be utilized to determine the extent to which the innate immune system is involved in manifestation of lithium-responsive neurological defects. For the third aim, molecularly defined mutants and RNAi will be employed to confirm the involvement of novel genes (from candidates that have been identified in genome-wide screens) in the lithium-responsive neurological processes. The proposed research is innovative in that it capitalizes on the power of Drosophila genetics to identify and characterize genes that are involved in lithium's actions in the nervous system. The proposed research is significant because it is expected to lead to the recognition of molecules and molecular interactions that are responsible for lithium's actions in the vertebrate nervous system. This would open up new avenues toward a better understanding of the etiology and pathophysiology of BPD, and result in improved therapies for BPD and other disorders of the nervous system.

The overall objective of this competing R01 renewal is the continued development of the Elastin Fusion Protein technology as a genetically encodable, bioinspired analog of "smart" -stimulus responsive- protein-polymer conjugates. In this renewal application, we seek to greatly expand upon our ability to modulate the solution behavior of ELP fusion proteins by introducing a greater level of sophistication beyond simple physical triggers of the ELP phase transition and uncontrolled aggregation as the final state, by developing new strategies for the: (1) stimulus responsive self-assembly of ELP fusion proteins into micelles or other self-assembled nano-mesoscale structures;and (2) coupling molecular recognition to the phase transition of ELPs, so that ELP fusion proteins can be programmed to exhibit an isothermal phase transition in response to ligand-binding. These "second-generation" ELP fusion proteins will, we believe, provide a new set of applications in Biotechnology and Medicine by virtue of the increased sophistication of the functionality embedded in these ELPs. For example, the ability to trigger an LCST transition by biochemical ligand-binding to an ELP fusion protein will provide a simple, convenient and modular approach to design biopolymer actuators that exhibit ligand-binding triggered phase transition behavior by splicing together ligand binding domains with ELPs. Similarly, developing a generic methodology to convert a peptide or protein into a nanoscale construct such as a spherical micelle has implications for the delivery of protein therapeutics because: (1) it will increase the apparent size of the biomolecule and will thereby increase its in vivo half-life;and (2) will provide a rational strategy to enhance the affinity and specificity of a protein or peptide drug for its target receptor via multivalency. PUBLIC HEALTH RELEVANCE: This project will develop bioinspired "smart" -stimulus responsive- Elastin fusion proteins. In this renewal application, we seek to greatly expand upon our ability to modulate the solution behavior of ELP fusion proteins by developing a new generation of ELP fusion proteins that will self-assemble into nanoparticles of defined size and shape or that will exhibit soluble-insoluble phase transition behavior upon binding to another molecule. These ELP fusion proteins will provide new molecular building blocks that will enable new approaches for targeted drug delivery, regenerative medicine and bioanalytical assays.

Stroke is the 3rd leading cause of death and one of the leading causes of adult long-term disability in the US, with high impact for African Americans. Since 90% of stroke survivors are functionally impaired, improving stroke outcomes is a major public health issue. Agencies such as CMS and the Joint Commission have implemented performance programs including quality indicators (QIs) which would ideally be selected based on impact on stroke-related outcomes, but such evidence is currently scant. The Institute of Medicine has stated that unbiased, reliable information about what works in health care is essential to reducing geographic variation in the use of health care services, and improving quality. We propose to systematically study Qls related to acute ischemic stroke (AIS) care and their associations with the outcomes of cardiovascular disease [CVD] mortality, CVD events, and functional independence. The overall goal is to develop a set of optimal AIS care Qls that maximizes long term outcomes both for patients overall and for specific groups of patients, such as African Americans, and AIS patients with chronic kidney disease (CKD) or diabetes, both more common among African Americans. We aim to 1) examine variations in guideline-concordant process of AIS management, hypothesizing that guideline concordant care will vary by patient, hospital and regional factors, and that adherence to CMS Qls but not other potential Qls will improve over 2003-12; 2) examine associations between processes of AIS management and outcomes, including incremental and relative effectiveness and cost-effectiveness in the >70% with CMS data. hypothesizing that guideline concordant care is associated with lower in-hospital complications and mortality, and that one year later, guideline-concordant inpatient care during the initial AIS hospitalization is associated with lower recurrent stroke, myocardial infarction (Ml), CVD mortality and higher functional independence; 3) examine the association between processes of AIS management and outcomes in AIS patients with CKD or diabetes, hypothesizing that patients with CKD or diabetes will receive less guideline-concordant care than others, and that CKD and diabetes patients receiving guideline-concordant care will have better outcomes than others, but the set of Qls that optimizes quality of life for CKD and diabetes patients will differ compared with those for AIS patients overall. The study will consist of formal chart abstraction of processes of AIS care among 1200 REGARDS subjects, and will use publically available data on hospital characteristics, as well as CMS data.

The overall goal of this project is to characterize and stimulate the control of phonation in vocal tremor. The working hypothesis around which the project is organized proposes the vocal tremor results from modulation of vocal fold vibration due to an interaction between central and peripheral oscillators that control (or undermine control of) the voice and speech mechanism. This project focuses on one aspect of the system, vocal fold vibration and the control of voice production at a peripheral (laryngeal) level. The specific aims to be accomplished during this period are as follows: 1) Determine the physiological basis for laryngeal biomechanics in production of vocal tremor through measurement of laryngeal muscle activity, subglottal pressure, and respiratory movement patterns. 2) Investigate the role of peripheral reflex feedback loops in vocal tremor modulation through system perturbation via electrical stimulation of laryngeal muscles. 3) Simulate vocal tremor with computer models of phonation, using a) hypothetical input parameters for laryngeal stiffness and subglottal pressure, and b) the same physiological input parameters from patient recordings incorporated into the models, and comparison of simulations with patient data. This training grant focuses on acquisition of skills and experience in laryngeal modeling and phonation simulation. Training will be gained in electromyographic signal analysis, multi-channel recording and instrumentation for speech research, and statistics and research design. The project will also be heavily computer-oriented and investigate an area of laryngeal modeling that is relatively unexplored, phonatory control under pathological conditions. Theoretical studies will examine modulations in laryngeal stiffness, subglottal pressure, phase, and muscle activation, their effects on modulation (amplitude and frequency) of vocal fold oscillation, and develop feedback loops to simulate hypothesized peripheral oscillators. The experimental phase consists of studies of neuromuscular and respiratory input to the larynx. This information about vocal tremor will be applied to model simulations with the use of physiological signals as input model parameters.

This application targets the millions of patients suffering from bladder disease/dysfunction related to congenital anomalies, cancer, infection, inflammation or lesions of the central or peripheral nervous system. Regardless of etiology, in all cases these conditions eventually result in largely irreparable damage to bladder tissue structure or function, ultimately affecting the ability of the bladder to fill, store or empty. The ensuing bladder dysfunction significantly reduces the patient's quality of life, and can also lead to significant morbidity due to upstream effects on the kidneys. Tissue engineering and regenerative medicine technologies represent a potential solution to the deficit of viable bladder tissue associated with these conditions. While the first clinical success of these techniques have recently been reported in young patients with congenital spinal cord lesions (i.e., myelomeningocele), there is no doubt that there is room for therapeutic improvement if one wishes to extend these technologies to the unmet medical needs of the much larger potential patient population. Because bladder regeneration in rodents and man shares several common features, we propose novel studies of bladder regeneration following subtotal cystectomy in a rodent model. The rationale is that improved mechanistic understanding of the normal bladder regeneration process in vivo would be an absolute prerequisite to further clinical progress. To this end we shall: Specific Aim #1: Assess progression of host tissue regeneration in vivo using micro CT and MRI imaging techniques and conscious cystometry measures. Specific Aim #2: Assess the progression of the host tissue regeneration response in vitro using pharmacological and physiological assays. Specific Aim #3: Characterize the cellular basis and histological features of the regenerative response of the urinary bladder. The combination of high resolution imaging modalities with direct measures of bladder function in vivo and tissue function and histology in vitro will establish the baseline characteristics of bladder regeneration as well as noninvasive markers for physiological milestones associated with normal bladder regeneration. The power of this approach stems from comparison of the regenerated bladder with the native bladder from the same animal. The over-ridding goal is to leverage the novel insights obtained to maximize the body's potential for achieving enhanced/maximal tissue formation in vivo. PUBLIC HEALTH RELEVANCE Cancer, congenital deformities, infection, inflammation, traumatic injury and nerve lesions produce irreversible damage to bladder tissue resulting in a permanent impairment in bladder function. Such debilitating impairments affect millions of Americans and lead to a poor quality of life. We propose to study the process of bladder regeneration in a clinically relevant rodent model in order to better apply tissue engineering and regenerative medicine technologies to the creation of new functional tissue derived from the patients own cells.

This project is concerned with an analysis of the activity of nerve cells in that system within the primate brain which is necessary and responsible for the process we refer to as attention. Monkeys trained to perform visualy-guided go-no go discrimination tasks are tested whilst extra cellular recordings are made from brain regions thought to be part of an attentional system. The most recent study in this series examined structures in the forebrain. We have found attention-related units in the anterior portion of the upper bank of the cingulate sulcus, in the rostral neostriatum, and in the periarcuate area of the dorsal frontal cortex. These cells responded to the manipulation of attention, e.g., manipulation of the pre-stimulus waiting period or changing the behavioral significance of the task-stimuli. These attention-related units have properties similar to those found in the brainstem reticular formation in previous studies by Bakay Pragay, Mirsky, Ray, and colleagues.

Hypertension (HTN) is the most prevalent modifiable risk for cardiovascular disease (CVD) and disorders directly influencing CVD (i.e. diabetes, chronic kidney disease, obstructive sleep apnea, etc.). Despite lifestyle changes and advances in drug therapy, ~20% of all HTN patients are resistant to (or require ?3) antihypertensive drugs Resistant HTN (R-HTN) is generally neurogenic in origin and associated with a dysfunctional autonomic nervous system. Few treatment options remain available following the recent failure of percutaneous renal artery sympathetic denervation (SYMPLICITY HTN-3, PRAUGE-15). Thus, a mechanism- based breakthrough is imperative to develop novel strategies to control and potentially cure R-HTN. We believe that our evidence of gut dysbiosis and dysfunctional brain-gut-bone marrow (BM) interaction in R-HTN represents this breakthrough. We propose a brain-gut-BM dysfunctional interaction hypothesis: HTN risk factors increase sympathetic drive by influencing autonomic brain regions, setting in motion a sequence of critical signaling events key to establishing neurogenic R-HTN. This includes increased gut stiffness, permeability and inflammation leading to gut microbial dysbiosis. Dysbiosis-associated changes increase BM production of myeloid progenitors and other proinflammatory cells. This contributes to increased peripheral inflammation, and neuroinflammation, as some BM-derived myeloid progenitors migrate to the paraventricular nucleus (PVN), and differentiate into microglia. Therefore, we hypothesize that establishment of R-HTN is caused by an increased SNA-mediated gut dysbiosis, activity of BM proinflammatory cells, and neuroinflammation. Three specific aims are proposed to support/refute this dysfunctional brain-gut-BM linked neuroinflammation hypothesis in R-HTN: Aim 1 will investigate the hypothesis that increased gut SNA is critical in enhanced intestinal permeability, proinflammatory conditions, and microbial dysbiosis in HTN. Aim 2 will define how gut dysbiosis increases production of proinflammatory progenitors and neuroinflammation in HTN. Aim 3 will evaluate the hypothesis that human R-HTN is linked to profound gut microbial dysbiosis and that treatment with minocycline will reverse the dysbiosis and lower BP. These studies will utilize state-of-the-art integrative physiological genomic techniques and will be conducted by an exceptional team of investigators. Thus, the outcome of this mechanism-based translational study spanning from mice to humans will provide the basis for development of paradigm-changing therapeutic approaches for R-HTN without involving more anti-hypertensive drugs.

The adipocyte hormone leptin regulates energy balance, substrate metabolism, immunity, bone formation and reproduction. Our recent work demonstrates that leptin production is regulated at the translational level, and that elements within its 5' UTR stimulate and its 3' UTRs inhibit translation of a reporter gene. The major goal of this application is to identify the cis elements and trans acting factors that regulate leptin translation in response to variations in nutritional state. The objectives of this application are: 1) To identify RNA binding proteins (RBP) that interact with the leptin 5' and 3'UTRs, and define the cis elements involved; 2) To determine the effect of starvation/feeding in vivo and acute treatment with insulin and beta-adrenergic agonists in vitro on expression of RBPs and their interaction with leptin mRNA; 3) To assess the functional roles of specific RBPs in leptin mRNA translation using knockdown and overexpression studies; and 4) To delineate the signaling mechanisms that cause high basal and insulin-resistant leptin translation in obesity. The leptin 3'UTR includes AU-rich sequences and motifs for binding of RBPs that are known to stimulate (HuR) or inhibit (TIA-1, TIAR) the translation of specific mRNAs. We will therefore determine if leptin mRNA 'in vivo' associates with RBPs 'in vivo' by immunoprecipitating ribonucleoprotein complexes from cytosolic extracts of 3T3-L1 adipocytes and rat adipocytes. Pull-down assays using biotinylated leptin UTR probes will verify the interactions of RBPs of interest and point to the motifs involved. We will test the hypothesis that insulin in vitro or nutritional status in vivo (starvation, feeding) affects the binding of specific RBPs to leptin mRNA, and involves an alteration in their expression and/or nucleocytoplasmic shuttling. Finally, we will test the hypothesis that the chronic hyperinsulinemia associated with obesity increases leptin production at the translational level through the activation of stress / energy sensing pathways (e.g. mTOR, AMPK, and MAPK) and/or by altering the expression or binding of specific RBPs to leptin mRNA. Overall, the proposed studies will enhance understanding of leptin biology and broaden knowledge of how the adipocyte functions as an endocrine cell that orchestrates the production of numerous hormones and cytokines in response to nutritional cues. [unreadable] [unreadable] [unreadable]

Many oncoproteins and tumor suppressive proteins shuttle between the cytosol and nucleus and thus interact with changing sets of protein partners in different sub-cellular compartments. Furthermore, increasing evidence indicates that oncogenic mutations of these proteins can result in alteration of cellular localization and rewirin of oncoprotein interaction networks. Characterization of the dynamic interaction network of oncoproteins and tumor suppressive proteins is therefore crucial to understand their roles in tumorigenesis. Robust technologies for mapping dynamic protein interaction networks under physiological conditions within the cell are not yet available. We propose to develop a novel approach for mapping dynamic oncoprotein interaction networks using endogenously epitope-tagged proteins for affinity- purification and quantitative proteomics for accurate network identification. This application builds upon our successful development of an innovative method to introduce epitope tag- encoding DNA into endogenous loci by homologous recombination-mediated knock-in in human cancer cells and our intensive experience in quantitative proteomics analysis. The endogenously tagged proteins are expressed at physiological levels and provide physiologically-relevant environments and compartments for protein complex identification and are therefore ideal for mapping dynamic protein networks. The goals of this application are twofold. First, we propose a detailed quantitative comparison of our technique in terms of distinguishing mutant and wild-type oncoprotein complexes against two conventional approaches for studying protein complexes. Second we test the potential of our new approach for studying the dynamic interactions that occur in response to cell signaling. Successful development of this technology will provide a platform for understanding the reconfigurations to protein interaction networks that result from oncogenic related translocation or mutation. As large-scale projects such as The Cancer Genome Atlas (TCGA) proceed, many more novel oncogenes and tumor suppressor genes will be discovered; our technology provides an important pipeline for understanding the function of these genes at the interaction network level by mapping their dynamic interaction networks.

An estimated 5.3 million Americans are currently suffering from Alzheimer's disease (AD), and AD cases are expected to rise at an alarming rate (AD;Alzheimer's Association, 2010). Therefore, market potential exists for products, approaches, and/or training that can help treat and/or circumvent the challenging behaviors associated with the condition. One approach that has been shown to be effective involves the use of Montessori Activity Programming (MAP;Camp, 1999a;Camp, in press). MAP consists of a set of principles for developing and facilitating activities for engaging persons with dementia. In this study, we will develop a new Montessori-based activity: Hearthside Book ClubTM stories, which will be the first-ever dementia-level appropriate reading materials. Twenty topics will be chosen which are familiar and interesting to many older adults. For each topic, three different versions of the story/essay will be created. Developed for persons in the early stages of dementia, "A-Grade Level Reading Materials" will be 30 pages in length and be written at an 9th grade level;"AA-Grade Level Reading Materials", developed for persons in the Middle Stages of dementia, will be 20 pages in length and will be written at the 6th grade level;finally, "AAA-Grade Level Stories", created for persons in the Late Stages of Dementia, will be 10 pages in length, and will be written at the 3rd grade level. A total of 60 stories will be created for this study (20 topics at three reading levels). Half of the Hearthside Book ClubTM booklets (split equally among all reading levels) will contain pictures. Forty-five older adults with dementia will take part in the study. They will be assigned to one of three groups: the Early Stage Group (Mini-Mental Status Exam (MMSE) score of 18+), the Middle Stage Group (MMSE 11- 17), or the Late Stage Group (MMSE 5-10). The engagement/affect of persons taking part in Hearthside Book ClubTM sessions will be compared against the engagement/affect of participants in baseline activities (e.g., bingo, current events, exercise, etc.) Hearthstone Book ClubTM sessions will also be compared against the engagement/affect of participants in small-group sessions using Reading Roundtable(R) booklets, which are designed for persons with dementia, but they are not adjusted for varying levels of dementia. Proximal outcome measures will involve engagement and affect, communications, and behaviors observed during small group reading activities. Distal outcome measures will assess agitation, depression, and quality of life. Measures will be taken at baseline, after two weeks of taking part in Reading Roundtable(R) sessions, and after taking part in level-matched reading materials sessions. The Specific Aims of this project are as follows: (1) to develop reading materials (with and without pictures) tailored specifically to persons with varying levels of dementia;(2) to determine if matching text materials to levels of dementia is related to proximal and distal outcomes across levels of dementia;(3) to determine if the inclusion of pictures significantly relates to proximal and distal outcomes across levels of dementia. PUBLIC HEALTH RELEVANCE: An estimated 5.3 million Americans are currently suffering from Alzheimer's disease (AD), and AD cases are expected to rise at an alarming rate. Besides affecting the person with the condition, AD also presents a public health concern, due to the significant cost required to care for persons with dementia and due to the mental and physical stress placed upon caregivers of persons with dementia. So, there is an urgent need for products and programs that will engage and stimulate persons with dementia, while at the same time reducing the stress on caregivers. Through the development of special reading materials for persons of varying levels of dementia, the proposed project will help meet this need.

The purpose of this project is to delineate the mechanisms involved in regulating immune responses in filarial and nonfilarial disease states. Immunoregulatory studies have examined the phenomenon of antigen-specific anergy in microfilaremic patients by showing this anergy to be a result of the production of the antiproliferative cytokine, IL-10. Filter immunoplaque assays for the major cytokines have been developed and used to demonstrate that in helminth infections of humans there is an expansion of Th2 CD4+ cells. The phenotypic characterization of these Th2 CD4+ cells has shown them to be CD45RO+ HLA-DR+ CD27-; a novel method for intracellular staining for cytokines has been used to assess further the frequency of these cells. Similarly, new ways of assessing eosinophil activation have also been developed.

Gastric adenocarcinoma is the second leading cause of cancer-related death in the world. H. pylori is the strongest known risk factor for this malignancy, yet only a fraction of infected persons ever develop cancer. One H. pylori determinant that augments cancer risk is the cag pathogenicity island, and several cag genes encode components of a type IV bacterial secretion system which functions to export proteins (e.g., CagA) nto host epithelial cells. A host effector that may influence carcinogenesis is li-catenin, a downstream component of the Wnt pathway. When Wnt signaling is inactive, (l-catenin is constitutively phosphorylated and degraded; binding of Wnt to its receptor inhibits G-catenin phosphorylation, leading to its nuclear accumulation and the transcriptional activation of genes that influence carcinogenesis. Nuclear accumulation of fi-catenin is increased in gastric adenoma and dysplasia specimens, histologic stages that precede gastric adenocarcinoma. Our preliminary studies now demonstrate that a rodent-adapted H. pylori ag+ strain (7.13) rapidly induces gastric cancer in hypergastrinemic (INS-GAS) mice by 24 weeks and in Vlongolian gerbils by 4 weeks and that strain 7.13 induces nuclear translocation of G-catenin and activates a B-catenin-responsive reporter in vitro, indicating that li-catenin is functionally responsive to this prototype strain. IS-catenin activation by H. pylori is dependent upon translocation of CagA into epithelial cells, and nuclear accumulation of U-catenin is increased in gastric epithelium harvested from cag+-infected persons, compared to subjects carrying cag strains or uninfected persons. Our hypothesis is that H. pylori cag* strains selectively activate host signaling pathways, such as those mediated by R-catenin, thereby regulating cellular responses that contribute to the augmentation in carcinogenic risk associated with these strains. Thus, our specific aims are: 1. To define the effects of H. pylori constituents on activation of S-catenin in vitro and in vivo. 2. To determine the eukaryotic signaling pathways that regulate H. py/or/-induced fi-catenin activation. 3. To define differences in epithelial molecular responses to carcinogenic H. pylori versus mutant strains using a transgenic murine model of gastric cancer.

Ubiquitous and secure access by physicians to a patient's medical records regardless of geographic location has tremendous potential to improve the delivery of medical care along multiple dimensions, yet the lack of a unique patient identifier between institutions remains a critical obstacle. While a national patient identifier was to have been established by the federal HIPAA legislation, opposition from privacy advocacy groups led to the suspension of a notice of proposed rulemaking. Probabilistic record linkage, however, represents a cross-disciplinary set of techniques that should permit reliable identification of patients across institutions in the absence of a unique identifier. Here we propose to study the feasibility of these techniques and quantify their reliability in the context of radiologic records, the only digital medical data for which there is a standard for electronic data interchange. In collaboration with the Hospital of the University of Pennsylvania (HUP), we propose to create a virtual patient "film jacket" across two unconnected picture archiving and communication systems (PACS). Adapting tools initially developed at the US Census Bureau, we will use probabilistic techniques to map unique patients between the two PACS based on the standard demographic data available in the DICOM headers of their imaging studies excluding the medical record number (MRN). We propose to compare this statistical approach to a benchmark of "true matches" represented by HUP's own MRN and use frequency analysis to determine appropriate cutoff values for probabilistic matches and evaluate the discriminatory power of the different DICOM attributes. We will also extend this analysis to additional demographic data stored in HUP's radiology information system (RIS) to evaluate the potential benefit of interfacing with both PACS and RIS and explore system performance as the number of records scales upwards. Phase II of the project, intended as a proof-of-concept to set the stage for commercialization, will involve applying our approach to a far more realistic test case - the creation of a virtual patient film jacket across two unaffiliated medical institutions. Commercialization of this technology will take the form of a radiology-specific global master patient index platform that will enable the creation of seamless, patient-centric virtual file jackets across arbitrary sets of institutions and PACWRIS installations. Such a product based upon non-proprietary, open standards would enable true and practical integration of care delivery between hospitals within an affiliated integrated delivery network or even between unaffiliated medical centers.

Abstract Lateral ankle inversion sprains occur frequently in sports and recreation activities. Although many patients relatively recover after their first acute injury, chronic ankle instability is often reported after ankle sprains despite treatment. Chronic ankle instability includes mechanical instability and functional instability. Surgical procedure is effective in restraining the ankle joint to reduce mechanical instability. Functional instability (FAI), however, poses a more difficult issue with little improvement following either conservative treatment or surgical intervention. FAI is primarily identified by a condition of recurrent ankle sprain and/or an incidence of ankle giving way. There have been inconsistent results in the literature about the role of suggested FAI factors including ankle join laxity, proprioceptive deficiencies, peroneal muscle weakness, and elongated muscle response time. None of them provides a reasonable explanation for ankle giving way phenomenon. We have recently suggested an etiological FAI factor, i.e. hyper-reactivity to unloading reaction. We demonstrated in our previous study a hyper-reactivity to unloading reaction in FAI ankles compared to normal ankles in healthy subjects. A major limitation of our previous study was static stretch applied to the tested ankle. To further link the hyper-reactivity theory to FAI, we propose to test FAI ankles using a combination of dynamic stretch and nociceptive stimulation that mimic the condition of an ankle giving way incidence. In our recent pilot study we observed in the majority of the subjects a shifting in reactive movement pattern that is closely mimic the ankle giving way phenomenon. In the proposed study, we will test 25 FAI subjects and 25 healthy control subjects to demonstrate a shift in reactive movement pattern in the majority of the FAI subjects in terms of quantitative changes in kinematic and kinetic variables compared to ankles of healthy controls. The result of the proposed study may fill the last, but most important gap between our hyper-reactivity theory and FAI.

The small intestine contains a significant number of intraepithelial T cells (IELs) that express CD8?? homodimer and ??TCRs. Currenlty these cells function and antigenic specificities remain enigmatic. Our long term goal is to understand how these cells contribute to homeostatic balance in the intestine, and how their TCR repertoires respond to microbial flora. Our central hypothesis is that clonal expansions and selective trophism of these IELs depends on microbial antigens that these cells recognize in vivo. These interactions may also contribute to sustain intestinal equilibrium. To test our hypothesis we propose two specific aims. First we will show how these IELs ??TCR repertoire changes in response to particular microbial species. We will examine this issue in a mouse model mice where T cells express semi diverse repertoire of TCRs and in vivo activated IELs are labeled with fluorescent protein (GFP). Limited diversity of TCRs allows to track individual IEL clones in response to different microbial species and organs using TCR CDR3 regions as clone-specific tags, whereas GFP allows to isolate IELs activated in their native environment. We expect to show that prime repertoire of IELs is broad, but become more oligoclonal following contacts with specific commensal antigens. In our Specific Aim 2 we propose to refine our new method to retrieve original pairs of ??TCRs sequences expressed by individual IELs for high-throughput sequencing (HTS), and use this approach to determine natural diversity of ??TCRs on IELs from wild type mice. This protocol is universal and in the future can be used to analyze native ??TCRs from humans.

Aggregatibacter actinomycetemcomitans is a gram negative pathogen that is the etiologic agent of localized aggressive periodontitis (LAP) and other systemic infections, including infective endocarditis. LAP, a disease which affects medically-underserved children in both the United States and throughout the world, is characterized by a breakdown of the periodontal ligament and alveolar bone structure that holds the teeth in place. Without intervention, loss of teeth occurs, causing both a cosmetic deformity and a functional defect. The manner by which A. actinomycetemcomitans causes disease has not been determined; however, it is known that the bacterium produces several putative virulence factors, including a leukotoxin (LtxA) that is a member of the repeats-in-toxin (RTX) family. This toxin kills human white blood cells and likely plays a key role in the ability of the organism to evade the immune response during its establishment of an ecological niche in the host. Thus, preventing or interfering with LtxA activity may be one option for successful treatment of the disease. The mechanism by which LtxA exerts its lethal effect on the host cell involves the formation of a physical disruption, such as a pore. Specifically, the interaction of the toxin and membrane results in a bending of the bilayer that leads to the formation of a toroidal, or lipid-lined, pore. A logical extension of this argument would suggest that LtxA will therefore interact most strongly with lipids that are able to bend or curve more easily. The proposed study is designed to explore the ability of LtxA to damage cells by altering the structure of their membranes. In the first aim, the effect of lipid curvature on LtxA-induced membrane changes, such as phase behavior and pore formation, will be characterized. In the second aim, the effect of lipid curvature on LtxA structure will be determined and the relation between those LtxA structural changes and the membrane changes observed in Aim 1 will be examined. The final aim will elucidate the biophysical properties of LtxA- induced pores. This study will allow determination of the role of the physical properties of the membrane, specifically curvature, on LtxA-induced membrane disruption, and will establish the mechanism of this disruption.

The broad goal of this research is an increased understanding of the relationship between personality factors and coronary heart and coronary artery disease. Subjects will consist of cardiac patients with angiographically documented coronary disease and a matched group of apparently healthy men, some of whom are at risk of heart disease by virtue of their personality type. Using a standardized clinical interview, we will be measuring a so-called Type A personality pattern, which has been associated with increased prevalence and incidence of coronary disease. The proposed research will then focus on several related issues. First, we will examine patterns of responding on speed and intellectual tasks as a function of both magnitude of coronary disease documented by angiography, and presence of pre-disposing Type A personality. Previous research indicates that both patients with clinical coronary disease and symptom-free Type A subjects exhibit impaired performance. Therefore, we will conduct a series to examine attitudinal and personality mechanisms which may be involved in impaired performance. We will also examine the effects of participation in an exercise and dietary therapy program on personality and behavior. Subsequently, data obtained from the Type A standard clinical interviews will be analyzed item-by-item in order to determine the particular aspects of personality pattern which most closely relate to magnitude of angiographically documented coronary disease. Implications of the research for developing attitudinal, personality and response measures of CAD presence and CAD risk, as well as for understanding characteristics of coronary-prone individuals are presented.

Epilepsy is a disease affecting 1-2% of the population. Electrical recordings from chronic animal models and human neocortical epileptic foci indicate that the population of neurons underlying each interictal epileptiform discharge varies over time. The spatial relationship between interictal events and the ictal onset zone, thought to be the critical area of epileptogenesis, is not well understood and critical to the surgical treatment of epilepsy. Electrophysiological recording methods, although currently the "gold standard", are inadequate to address these questions based on restrictions due to volume conduction or sampling limitations, many of which can be overcome with optical recording techniques. The PI is a fellowship trained epilepsy surgeon at UMDNJ with extensive laboratory experience in optical recording of neuronal activity, both in vitro and in vivo. In a second post-doctoral fellowship, the PI demonstrated that in vivo optical recording of intrinsic signals can be used to generate high-resolution, real-time maps of the population of neurons participating in an epileptiform event. The goal of the current study is to examine the shifting spatio-temporal dynamics of the epileptogenic aggregate in both acute and chronic experimental models of in vivo rodent epilepsy. In the laboratory of mentor Gyorgy Buzsaki, a world-renowned expert in electrophysiological mapping of rodent epilepsy at Rutgers and part of the joint UMDNJ-Rutgers Graduate Center m Newark-Program in Neurosciences, we will first determine the precise relationship between the optical signal and the interictal and ictal epileptiform events using well-established acute and chronic in vivo rodent models. Optical epilepsy maps will be correlated with maps derived from electrophysiological recordings from a grid of surface electrodes, multicontact silicon probes, as well as c fos hybridization. Additional technical support in optical recording and data analysis will be provided by collaborator Ralph Siegel, also a member of the UMDNJ- Rutgers Graduate Center in Newark-Program in Neurosciences. As a related goal, optically-guided surgical resections of epileptogenic cortex will ascertain the required volume of epileptogenic tissue which must be removed to eliminate seizures. The results of these investigations will not only be important in understanding the pathophysiology of neocortical epilepsy but also critical in optimizing surgical treatment of human clinical epilepsy. Following the period of mentorship, the PI will be able to combine independent basic science research in a separate laboratory at UMDNJ with clinical optical recordings in the operating room during the neurosurgical treatment of epilepsy.

Neutral endopeptidase 24.11 (NEP) is a cell-surface peptidase expressed by prostatic epithelial cells which cleaves and inactivates neuropeptides implicated in the growth of androgen-independent prostate cancer (PC). We show that NEP expression and catalytic activity are lost in vitro in androgen-independent but not androgen-dependent PC cell lines. In vivo, NEP protein expression is commonly decreased in cancer cells of metastatic PC specimens from patients with androgen-independent but not androgen-dependent PC. Overexpression of NEP in androgen- independent PC cells or incubation with recombinant NEP inhibits PC cell growth. Furthermore, in androgen-dependent PC cells, expression of NEP is transcriptionally regulated by androgen and decreases with androgen- withdrawal. Consequently, PC cells which survive androgen-withdrawal can emerge with reduced NEP. These data suggest that decreased NEP expression, common in androgen-independent PCS, is facilitated by the elimination of androgens, and that NEP loss plays an important role in the development of androgen-independent PC by allowing PC cells to use mitogenic neuropeptides as an alternate source to androgen to stimulate cell proliferation. To thoroughly define the involvement of NEP on androgen-independent PC cells, our specific aims are (1) to explore the mechanism by which NEP inhibits cell growth; (2) to establish that the androgen response element (ARE) in the 3' end of the NEP gene is a functional ARE which enhances transcription of the NEP gene; and (3) to assess the antitumor effects of NEP in an animal model of prostate cancer by establishing that recombinant NEP can inhibit the tumorigenicity of androgen-independent PC cells in an orthotopic model of PC, and to establish that overexpression of NEP in androgen- independent PC cells inhibits the tumorigenicity of androgen-independent PC cells. These studies leading to a better understanding of the involvement of NEP in the development and progression of androgen- independent PC may ultimately provide support for novel approaches for the treatment of advanced PC.

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. Following knee and hip replacement, ambulation is often limited by joint pain, greatly decreasing functional mobility, rehabilitation quality, and possibly the ultimate functional outcome. Perineural infusion is a relatively new analgesic technique that involves the percutaneous insertion of a catheter directly adjacent to the peripheral nerves that supply the affected joint. Local anesthetic is then infused via the catheter to provide potent, site-specific analgesia free of significant side effects. The ultimate objective of the proposed research is to determine if perineural local anesthetic infusion in the immediate postoperative period has both short- and long-term benefits following knee replacement surgery for osteoarthritis. The research plan consists of a multicenter, randomized, triple-masked patients, investigators, statisticians, placebo-controlled, parallel-arm clinical investigation. Patients will randomly receive either perineural local anesthetic or normal saline for 4 days following surgery. The primary outcome is the time until three specific, predefined readiness-for-discharge criteria are met following surgery. Secondary outcomes include the relationship between analgesic technique administered in the immediate postoperative period and subsequent pain, stiffness and functional disability.

This study uses a prospective, longitudinal design to determine the impact of grade retention in the early elementary grades on children's academic, behavioral, emotional, and social adjustment during the transition from elementary school to middle and high school. Participants will be recruited from the 679 children [35% White, 22% Black, 39% Hispanic (35% of whom were Spanish language dominant in 1st grade), and 4% Asian] who entered first grade for the first time hi Fall of 2001 or 2002 in one of three school districts, who scored below their school district's median on a test of literacy, and who are currently participating in the study. Of these, 141 (20.8%) repeated first grade and 198 have repeated grade 1, 2, or 3. To provide a comprehensive view of the development of these children, all study children have participated in annual assessments of child variables (e.g., language proficiency, academic achievement and motivation, and behavioral, social, and emotional functioning), peer relatedness variables (e.g., classmates'liking and evaluation of the child's social and academic competencies), family/parenting variables (e.g., family adversity, acculturation, and parent support for schooling), and classroom and school variables (e.g., % free lunch students and participation in remedial and enrichment educational services). As these children approach adolescence, measures of risk behaviors and leisure activities will be added. Based on social- ecological and transactional theories of development (Bronfrenbrenner, 1979;Sameroff, 1979, 1989), processes that mediate the effects of elementary grade retention on achievement and behavior in secondary grades will be investigated. Retained children are expected to experience a decrease in school belongingness and academic motivation and to be more likely to develop networks outside of school that include a high proportion of deviant peers. Second, the study aims to identify characteristics that may affect the academic, behavioral, emotional, and social trajectories of children who enter school with below average literally skill. Of particular interest is the role of social relatedness at school to children's motivation, self-views, and achievement. Third, the study investigates the role of ethnicity, language, and culture as they relate to the first two specific aims in a sub sample of Hispanic students. Study aims will be achieved with a multi-informant prospective design and the use of latent growth curve modeling and longitudinal SEM. The study will identify children for whom grade retention is especially disruptive and (b) processes that buffer or aggravate such disruption.

Information about fast axonal transport in normal and/or diseased mammalian axons (including human) and the regulation of this process is limited. Until this basic information is established, the physiology and pathophysiology of this phenomenon cannot be fully elucidated. We have an opportunity to obtain new basic information on the nature of fast axonal transport in mammalian axons by interfacing sophisticated microscopy, video, and computer technology with ultra-structural analysis and electron probe techniques. We aim to: 1) Pursue our findings of an abnormality in the fast anterograde axonal transport in human amyotrophic lateral sclerosis (ALS) axons by testing the hypothesis that axonal sprouting (as seen in ALS and experimentally reproduced in partially denervated muscle in mice) leads to increased peripheral demand on neuronal somata resulting in augmented organelle delivery rates. Concurrently, we will evaluate the retrograde intra-axonal organelle traffic in the partially denervated mouse model. 2) Test the hypothesis that peptides known to augment intra-axonal organelle traffic speeds may produce neurogenic dysfunction by overloading the neuronal metabolic machinery and/or depleting axonal reserves of critical components when administered chronically. We have shown that both in vitro and in vivo parathyroid hormone treatment will increase fast axonal transport. Hyperparathyroidism in humans can produce an ALS-like syndrome with weakness, muscle atrophy, fasciculation, and neurogenic changes on muscle biopsy. The hypothesis will be tested by sequential subcutaneous implantation (over months) of PTH-loaded Alzet osmotic mimipumps in rats. 3). Test the hypothesis that peptide agonists, specifically PTH, and arginine vasopressin (AVP) increase mean fast organelle traffic speed through the regulation of intracellular calcium. This effort will further probe the thus far unexplored question of regulation of fast axonal transport. 4). Develop further a data base on fundamental aspects of fast organelle transport in mammalian axons in asymmetric segments of neurons and in functionally different classes of neurons. We will evaluate fast axonal transport in a) central vs. peripheral branches of rat dorsal root ganglia, b) sensory vs. motor spinal nerve roots, c) central nervous system vs. peripheral nerve axons and d) myelinated vs. unmyelinated axons. These functionally different axon segment and axon types have not been systematically analyzed for potential differences in fast organelle traffic, differences which might provide clues to the selective vulnerability of different axons in different disease states.

Project Summary Vector-borne Disease Research Training Program in China This research training application seeks funding to promote career development and capacity building in vector biology research in China. Vector-borne diseases are a major public health problem in China and vector control represents the most effective method for dengue and malaria prevention. However, it is remarkable that given these circumstances vector biology research capacity in China is limited and underfunded. Among major gaps in medical entomological research in China are descriptive, non- mechanistic studies of vector ecology, student knowledge deficiency in mosquito taxonomy, molecular biology and genomics techniques, and disconnection between scientific findings from medical entomologists and vector control policy makers. This training program is designed to fill these major gaps in medical entomology research and vector control in China. The scientific objectives of this training application focus on vector population and community ecology, insecticide resistance genetics, and translational research in malaria and dengue vector control. We propose two tracks of training: long-term training of six Ph.D. students and four postdoctoral fellows from China, and short-term training of ten junior scientists and professional vector control staff, including policy makers in charge of vector-borne disease control. In addition to obtaining research experience in both laboratory-based molecular biology and field- based ecological research, each year we will organize a core training curriculum that focuses on biostatistics and data management, ecology, vector systematics, scientific writing and responsible conduct of research. The core training curriculum will be open to a large audience. The superb infrastructure and capacity in the international training sites (Southern Medical University in China and China CDC) and in the U.S. (University of California at Irvine) are particularly suitable for the proposed training. This training program will contribute significantly to the career development of the Chinese scientists by bridging laboratory and field research experience in vector biology, by equipping them with new technologies, and by enabling them to develop independent or collaborative research projects. The main scientific issues addressed in this application are common to dengue and malaria vectors in the world, thus the findings from our program are anticipated to have general significance in vector-borne disease control in other regions.

It is proposed to develop and test the xenon atom as a probe of biological microstructure using nuclear magnetic resonance (NMR) spectroscopy. Systems to be investigated might include simple aqueous systems, micellar solutions, vesicles, multilamellar liposomes, and proteins. The techniques to be used for this investigation include xenon solubility, xenon NMR chemical shift measurements, temperature studies, solvent and paramagnetic ion effects on the Xe chemical shift, 129Xe and 131Xe relaxation time studies, nuclear Overhauser effect measurements, and chemical shift measurements made at high (ca.100 atm) Xe and hydrostatic pressures. The expectation is to show that this small, inert atom is a unique probe capable of reporting structural information on biological microstructures difficult to obtain by other means. In this study information pertinent to the mechanism by which xenon functions as a general anesthetic may be found.

A continuing focus of the project this year was the investigation of ways to improve the retrieval accuracy of DELTA-BLAST through the use of model surgery and of asymmetric but uniform gap costs. Traditionally, PSI-BLAST constructs its position specific score matrix (PSSM) using the query sequence as a template, with each amino acid serving as a place holder for a column of the matrix. However, if the query sequence includes an atypical insertion or deletion, the resulting PSSM will be handicapped in having to imply a corresponding deletion or insertion when aligning to most related sequences. The recently developed DELTA-BLAST first aligns a query sequence to a database of PSSMs, and this opens the possibility of allowing the constucted PSSM to take its length from any aligned PSSMs rather than from the query. Furthermore, it is possible to treat insertions and deletions with respect to PSSMs constructed using such model surgery asymmetrically, for example penalizing insertions less than deletions. We achieved statistically significant improvements using this approach. An updated article on the BLAST algorithm and programs was written for the Encyclopedia of Life Sciences.

The major goal of this project is to determine whether PET scans ( 18F) fluorodeoxyglucose (PET-FDG) to measure metabolic rate for glucose and (11C)-PK 11195 to assess peripheral benzodiazepine binding sites can predict the outcome of newly diagnosed patients with gliomas. The predicative power of PET will be compared with three novel methods of assessing the growth kinetics of gliomas, including the bromodeoxyuridine labeling index, Ki-67 antibody binding, and c-myb proto-oncogene expression. In addition, we will assess the ability of gadolinium MRI to predict outcome. The results of PET imaging, growth kinetic indices, and MRI will be correlated with neuropathological grade and with two measures of outcome, time to progression and total survival. The results of this study may support the routine clinical application of one or more of these new techniques for assessment of patients with gliomas, permitting more rational choice of treatment. This might be especially important in patients with low-grade gliomas in whom initial treatment is controversial. Secondary goals include determining whether PET-FDG scans can improve the diagnostic accuracy of stereotactic biopsies and whether high metabolic rate corresponds to specific pathological characteristics. The results could lead to increased accuracy of pathological diagnosis of gliomas.

We have recently identified and partially characterized a peptidase from human postmortem cerebral cortex that is highly active toward luteinizing hormone-releasing hormone (LH-RH) and probably other neuropeptides including angiotensin II, bradykinin, and neurotensin, but not somatostatin. We propose in this application to: 1) Purify to homogeneity this neuropeptide-degrading enzyme from postmortem human cerebral cortex. 2) Characterize this enzyme in terms of molecular weight, subunit structure, pH optimum, Michaelis-Menton kinetics, behavior in the presence of enzyme inhibitors or activators, and bond and substrate specificities. 3) Raise and characterize an antiserum against the purified enzyme as an initial step toward further investigating the biological role of the enzyme. Based upon preliminary data, the purification procedures will include anion-exchange and gel-filtration chromatography as well as affinity chromatography on organomercurial-Sepharose. Other aspects of the study will employ SDS-polyacrylamide gel electrophoresis, radioimmunoassay for LH-RH, and high performance liquid chromatography. Our long-term objectives are to elucidate the biological role of this enzyme and similar peptidases in normal brain function and to determine the involvement of these enzymes in neuropsychiatric disorders.

The use of neoadjuvant chemotherapy prior to resection or radiation therapy has contributed to dramatic improvement in survival of patients with pediatric malignancies. Precise clinical and radiologic methods of evaluating the primary lesion at diagnosis and assessing response to chemotherapy are necessary to select appropriate sites for biopsy, plan surgical procedures and estimate prognosis. Standard definitions of response rely on changes in uni- or bidimensional measurements and do not take into account the relative amounts of residual viable malignant disease and necrosis within the tumor mass. The development of more accurate definitions of response based on imaging technologies which are capable of distinguishing active disease from non-malignant tissue is therefore an important management goal. This research will be performed within the framework of existing institutional protocols for treatment of Ewing sarcoma, rhabdomyosarcoma, and related malignancies which have a total annual accrual of approximately 25 patients. We will perform routine CT, MR and nuclear imaging of the primary tumor and will investigate volume measurements, dynamic contrast-enhanced MR imaging (DEMRI) and Thallium scintigraphy in assessing response to neoadjuvant chemotherapy prior to radiation or surgery. Our investigations will include: A. use of interactive displays for calculation of tumor volumes from three dimensional magnetic resonance (MR) imaging data after transfer to a Sun Unix workstation. B. Use of dynamic gadolinium-enhanced MR imaging (DEMRI) for measuring the rate of contrast-enhancement in multiple regions of a representative tumor section, displayed in a pixel-by-pixel manner on the Sun Unix workstation. Data will be used to assess response to chemotherapy. C. Use of thallium-201 nuclear imaging as a method of assessing tumor viability by quantitating lesional uptake before and after neoadjuvant chemotherapy. Changes in volume and, results of DEMRI and thallium-201 scans will be compared to standard response criteria based on clinical findings, routine CT/MR imaging, and pathologic evidence of tumor activity on biopsy or histologic maps of tumor activity in resected specimens.

DESCRIPTION (Adapted from applicant's abstract and specific aims): The results from a large number of different experiments with SP-A and SP-D (lung collectins) in vitro suggest these two related proteins participate in at least two major physiologic processes: first, the regulation of lung surfactant homeostasis; and second, the non-specific innate immune response of the lung. The general objective of this project is to establish the physiological consequences of a deficiency in SP-A, SP-D or both proteins in mice to further our understanding of collectin function in the intact animal in health and disease. The specific aims are to: 1) generate mouse models of pulmonary collectin (SP-A and SP-D) deficiency by consecutive gene disruptions of the collectin locus; 2) characterize pulmonary mechanics and surfactant structure, function and extracellular metabolism in collectin deficient mice a) under normal, non-stressed conditions, b) during exercise, c) with protein-rich pulmonary edema and d) after reversing the phosphatidylglycerol to phosphatidylinositol (PG/PI) ratio in the surfactant phospholipids; and 3) determine if pulmonary collectin deficiency a) alters the susceptibility to spontaneous respiratory infection and b) increases the susceptibility to influenza A viral infection.

The overall goal of Project 4 is to detemine how diabetes affects cardiac progenitor cell (CPC)-mediated myocardial repair after infarction and how CPC therapy could be optimized for the diabetic heart. Although stem cell therapy with CPCs offers new hope for the treatment of heart failure, the efficacy of stem cell therapy in diabetes remains uncertain. Our preliminary observations show that diabetes impairs CPC growth and differentiation, and in contrast to non-diabetic hearts, diabetic hearts do not benefit from CPC therapy. We propose that diabetes decreases CPC competence by inducing insulin resistance. Excessive nutrients in diabetes increase the production of reactive oxygen species (ROS) that triger inflammation and establish insulin resistance in CPCs. This in turn impairs their capacity to promote myocardial repair. To test this hypothesis, three specific aims are proposed. (1) To examine glucose transport and glucose and fattty acid metabolism in CPCs, we will measure the rates of glycolysis and fatty acid metabolism in lin-/kit+ CPCs isolated from the mouse heart, under basal condition and in the presence of high glucose and palmitic acid and determine how nutrient excess affects the fundamental parameters of CPC competence. To determine the effect of diabetes, we will isolate CPCs from db/db and high-fat fed mice and compare their competence with CPCs from non- diabetic mice. (2) To elucidate the mechanisms by which nutrient excess affects CPCs, we will examine the role of inflammation and oxidative stress in inducing insulin resistance and test the hypothesis that in conditions of nutrient excess or diabetes, hyperactivity of the mitochondrial electron transport chain leads to an increase in ROS production which triggers pro-inflammatory responses resulting in insulin resistance. (3) To determine the impact of diabetes on CPC-mediated myocardial repair, we will transplant diabetic CPCs in non-diabetic hearts and non-diabetic CPCs in diabetic hearts after myocardial infarction and measure changes in CPC-mediated changes in myocardial function and test whether anti-inflammatory or anti-oxidant interventions that resolve insulin resistance promote CPC mediated myocardial repair in the diabetic and non-diabetic hearts. RELEVANCE (See instructions): A majority of patients with heart failure have diabetes, yet the effects of diabetes on stem cell therapy are unknown. By providing new understanding of the underlying mechanisms by which diabetes affects stem cell therapy in heart failure and how stem cell therapy could be optimized for the diabetic heart, this project could lead to the development of new therapies for the treatment of heart failure in diabetic and non-diabetic patients..

The goals of the research project are to devise a stereo- and regiospecific synthesis of chemically stable, cyclobutanone analogs of penicillins for testing as inhibitors of penicillinase as antibiotics, as inducers of penicillinase as antigens toward heterogeneous penicillin induced antibodies and as probes (i.e. iodo analogs) for crystallographic characterization of the penicillinase and transpeptictase receptors.

Mycobacterim tuberculosis infects approximately2 billion people, of whom greater than 2 million will die each year of tuberculosis.With the emergence of multidrug resistant strains and the growingHIV epidemic, the global problemof tuberculosisis worsening.In the last several years the role of the innate arm of the immune response in the control of M. tuberculosis infection has become apparent. Although this response is often adequatefor long term containmentof the bacilli in healthy individuals, it is insufficient for clearance of the infection. Our recent efforts, indicatethat M. tuberculosis possesses specific mechanismsfor the subversionof immune effector populations.Through understanding these immune evasionmechanisms we will be able to design and develop more effective tuberculosisvaccines. To do so, we will utilizecutting edge high-throughput technology to develop a full array M. tuberculosis deletion library. This invaluable tool will enable the elucidationof the molecular basis for this immune evasion, part of which we have found is mediated by a large gene cluster conserved within pathogenic Mycobacterial species.

There is little research on predictors of patient satisfaction with health care for the 19 million Americans who are diagnosed with major depression and no research on whether these predictors differ by gender for this group. Patient satisfaction is thought to be a key quality of care indicators and is a central health plan performance measurement for the Health Plan Employer Data and Information Set (HEDIS) and the Foundation for Accountability (FACCT). It is conceptualized as combining patient assessments of technical difficulties in distinguishing the two when assessing satisfaction with health care. The main objective of this study is to test whether patient satisfaction is determined by both types of quality of care for people who are diagnosed with major depression and if there are any gender differences in this relationship. In turn, it is also a main objective to understand if provider switching is a consequence of patient dissatisfaction with care. The long-term objective is to help health plans, understand what predicts satisfaction with care so they can target and restructured care, know if there are gender differences in satisfaction, and whether provider switching is a consequence of patient dissatisfaction. The data for this research are from the Quality Improvement for Depression (QID) study, a large national study of 1,481 patients diagnosed with clinical depression in a variety of managed care settings that provides information on patient sociodemographics, health status, visits, attitudes, behavior and satisfaction with health care every six months for a period of 2 years. Data from baseline and six months are available for the following set of specific aims that are not covered for funding under the original QID grant. Specific Aim 1: To determine the effect of quality of care on overall patient satisfaction and patient satisfaction with mental health care. Specific Aim 2: To determine the bi-directional effect of provider switching on patient satisfaction. Specific Aim 3: To develop a comprehensive predictive model of patient satisfaction and determine if predictors differ by gender. These aims lead to a set of hypotheses that are based on the Donabedian Model of Structure-Process-Outcomes and the Andersen Behavioral Model of Health Services Use. The hypotheses of these aims will be tested using multiple logistic regression.

The Cell, Molecular, and Genetics (CMG) Training Program at the University of California, San Diego is currently in its 29th year. This program is a cornerstone of the Ph.D. training effort in the biological sciences at UCSD, as it supports the very best graduate students from the Biology-Salk and Chemistry-Biochemistry Ph.D. programs. The mission of the CMG Training Program is to provide rigorous basic research training in cell biology, molecular biology, and genetics to outstanding young biomedical researchers in the earlier years of their doctoral studies. Specific emphasis is placed upon the creativity, quality, and impact of the research, the ethical conduct of research, the achievement of racial diversity among biomedical researchers, the ability of the trainees to communicate their results effectively, and the promotion of cooperation and collaboration among scientists. The CMG Program Director is Dr. Randolph Hampton. He has been a member of the UCSD Division of Biological Sciences since 1995, and is currently Associate Professor and holder of the Paul Saltman Chair for Scientific Education. Dr. Hampton works with an Advisory Committee for the CMG Training Program that consists of Drs. Lorraine Pillus, Tracy Johnson, Bart Sefton, Elizabeth Komives, and Bill McGinnis. There are currently 110 training faculty from the UCSD Division of Biology, Salk Institute, UCSD Department of Chemistry and Biochemistry, and Department of Cellular and Molecular Medicine at the UCSD Medical School. The CMG Training Program provides support for 39 outstanding Ph.D. students that are selected from a current total group of 264. The trainees fulfill the general requirements of their respective graduate programs and additionally participate in CMG-specific training activities, which include, training in responsible conduct in research, Fall Reception and Poster Session, biannual CMG Training Program Symposia, annual one-on-one Conferences with the Program Director, CMG Sponsored Workshops, annual Idea and Input Lunch, CMG Hosted Biology Divisional Speaker, CMG-Hosted Informal Events, and participation in Trainee Committees to steer and organize these activities. Over the past 10 years, 126 CMG trainees have successfully completed their Ph.D. theses. Past and present CMG trainees have contributed an enormous wealth of fascinating and important knowledge to the biological sciences in over 400 publications (not including abstracts). With the projected growth of biology at UCSD, we envision the implementation of an even stronger CMG Training Program over the next five years.

The preovulatory LH surge stimulates an increase in ovarian matrix metalloproteinases (MMPs) and their associated inhibitors, the TIMPs, (collectively referred to as the MMP system) prior to follicular rupture in many species. The paramount role of the MMP system in ovulation is forthcoming from numerous experiments where oocyte release is blocked by MMP inhibitors. Yet other than our preliminary data, nothing is known as to progression and temporal patterns of the MMPs and TIMPs involved in the ovulatory process in the human. This question will be addressed in the first Specific Aim by using a unique model where the granulosa, theca, and stroma from human periovulatory follicles will be collected prior to and at three designated times after hCG (early, late and postovulatory). The expression patterns of key members of the MMP and TIMP family associated with follicular rupture in the human will be illuminated. Specific Aim #2 will build upon these observational findings and explore the regulation of these MMPs and TIMPs by known LH stimulated signaling pathways using well characterized models in the rodent and human. However, one of the key questions remains as to what are the MMPs and TIMPs actually doing during the process of follicular rupture? We propose that the ovarian MMP system directs the requisite proteolytic or degradomic changes necessary for ovulation. Thus, Specific Aim #3 will elucidate specific protein targets of MMP action by proteomic profiling allowing us to characterize the function of the MMPs and TIMPs in the ovulatory process. A major strength of this proposal is our extensive expertise with the MMP system that allows an integrated approach to understand the cellular expression, regulation, and impact of the MMP system on follicular rupture. One novel aspect of this study is the use of well characterized human preovulatory follicles as a foundation to explore the role of the MMP system in the process of human ovulation, which has never been accomplished. As such the proposed studies are extremely timely to elucidate the role that this exquisite proteolytic system plays in the coordinated processes of follicular rupture and oocyte release which are key aspects of normal human ovarian physiology. PUBLIC HEALTH RELEVANCE: The proposed studies will investigate the underlying mechanism involved in human ovulation, which is a process where the egg is released from the follicle in the ovary. These studies will focus on a family of proteolytic enzymes, known as metalloproteinases, that we believe breakdown the wall of the ovarian follicle to allow the egg to be released. Only by fully understanding such basic tenets of ovarian function will we be able to promote or inhibit the events associated with metalloproteinase action during the ovulatory process thereby facilitating and increasing fertility or acting in a contraceptive manner to decrease fertility.

PROJECT SUMMARY/ABSTRACT: High-need, high-cost patients (HNHC) often experience poor coordination of care, a key driver of increased cost and utilization. Healthcare organizations and policymakers are investing in care coordination mechanisms that combine health information exchange with individualized care plans to fix the fragmented care HNHC patients receive, but evidence for these approaches is limited. In 2012, the Washington State Health Care Authority (HCA), which oversees the state Medicaid program, implemented a health information exchange (HIE) across all hospitals referred to as the Emergency Department Information Exchange. As part of HIE implementation, HCA provides financial incentives to hospitals to develop individualized care plans to improve care coordination for Emergency Department (ED) high utilizers (? 5 ED visits per year). The current project seeks to apply advanced quantitative methods in causal inference, comparative effectiveness, and qualitative methods to understand gaps in our knowledge in the use of HIE-enabled care plans to improve care coordination for HNHC patients. To accomplish this research, I will link an existing Washington State Medicaid dataset to patients who received a care plan in EDIE. Working under the mentorship of experts in health service research, I will: (1) measure the impact of HIE-supported care plans on healthcare utilization among HNHC patients (2) identify subgroups of HNHC patients with lower ED healthcare utilization after receipt of a care plan (3) describe the implementation of HIE-care plans and content of care plans in HNHC patients. These studies will inform healthcare systems on ways to improve care coordination for complex patient populations. This work will also help me establish a set of skills to become an independent investigator addressing the gaps in care coordination for HNHC populations.

Cytomegalovirus (CMV) infections following renal transplantation are extremely common, occurring in 60-86% of patients studied. This proposal seeks: (1) to establish more firmly the relationship between CMV and hepatic function abnormalities occuring in the post-transplant period and to determine the spectrum and natural history of hepatitis associated with CMV seroconversion, (2) to explore the possibility that CMV infection may be an important cause of graft rejection, (3) to initiate in vitro experiments designed to gain information on how certain epidemiologically significant variables (hydrocortisone, azathioprine, uremia, and antigenic stimulation) might influence CMV replication and the immune response to this infection, (4) to determine whether the "immunological enhancement of viral replication" extends to CMV, and (5) to ascertain the relative contribution of viral multiplication versus the immune response in the causation of cellular injury and death. To achieve these objectives we will study prospectively 50 patients undergoing renal transplantation and set up experiments utilizing human fibroblasts and mononuclear cells, CMV and relevant variables, measuring the results in terms of viable cells remaining at the end of the experiment and virus yield.

The attainment of adequate self-regulation represents a critical developmental milestone that has significant implications for children's social-emotional wellbeing. Prior work has noted the importance of parenting for children's early developing self-regulatory skills, particularly at times in life, such as infancy and early toddlerhood, when children rely heavily on parental support in the regulation of emotion and behavior. Although preliminary evidence suggests that parental self-regulation is important for parenting, most studies have not focused on parenting in early childhood and have not linked parental self-regulation to children's self-regulation through parenting. The aim of the current proposal is to evaluate the effects of multiple aspects of maternal self-regulation (e.g., emotion regulation, effortful control, executive functions, and physiological regulation [heart rate variability]) on parenting of infants and on the emergence of infant self-regulation directly and through parenting. Using a diverse sample (n = 200) of typically developing infants and maternal caregivers recruited from one urban and one rural site, the current proposal evaluates parenting under two conditions. First, the implications of maternal self-regulation for parenting infants in the context of high infant distress is examined using a novel parenting simulation task wherein maternal caregivers interact with a highly distressed, life-like infant simulator. Next, the mother's interactions with her own infant will be evaluated in the context of free play and after a mild infant stressor. Finally, infant self-regulation of emotion during a frustrating task will be examined. It is anticipated that maternal caregivers who have better self-regulation will engage in more adaptive, sensitive parenting behavior during the simulation task as well as with their own infant during play and after a mild stressor. Furthermore, it is expected that parenting will mediate the effects of maternal self-regulation on infant self-regulation. The current study has implications for early developing self-regulatory skills, developmental psychopathology, models of parenting, as well as for parent-infant intervention programs.

PROJECT SUMMARY The objective of the Stanford Research in Anesthesia Training Program (ReAP) is to train leaders in academic anesthesia. We recognize that in order to accomplish this goal, substantial training beyond an MD or PhD is required. ReAP provides the guidance, training, and mentoring critical for the successful initiation of an independent research career and becoming a leader in the broad field of Anesthesiology. Trainees must learn to pose important and well thought out questions, to think critically, and to use cutting edge interdisciplinary tools to answer these questions. Success also requires the development of skills in presentation of results in oral and written format, in preparation of competitive grant proposals, and in the ability to engage in collaboration when this will more effectively advance the research. The training program starts by recruiting the most talented trainees from MD/PhD, MD and, occasionally, PhD applicants interested in pursuing a career in anesthesia research and academic anesthesia. This recruitment is facilitated by our department's research training continuum featuring both a formal residency research track and, later, comprehensive support in transitioning to a junior faculty position. Once appointed, ReAP trainees select a primary research mentor and a secondary mentor to monitor and facilitate their progress. Close interaction with mentors and other accomplished faculty is essential to master critical skills that form the core of our training program. This is supplemented by didactic material, and, in the case of clinical research, may be augmented further by a master's degree in epidemiology or health science research. Administratively the program consists of a Program Director, Steering Committee, External Advisory Committee and a group of 28 highly skilled and successful training faculty from the anesthesia department and 9 other departments within the medical school. There are already established interactions among many of the faculty members. The diverse faculty is divided into three overarching areas: 1) Neuroscience, Pain and Analgesia, 2) Injury, Inflammation and Immunity, and 3) Outcomes Research, Economics and Bioinformatics. These divisions encompass research areas at the forefront of our field. Our institutionally well-supported program and pipeline of highly qualified candidates will easily support a total of four trainees with two appointed per year anticipating two-year training experiences for most candidates.

Simvastatin, like lovastatin is converted to active metabolites by cytochrome P450 3A4 (CYP3A4). We have shown that lovastatin is susceptible to interactions with a commonly used calcium antagonist, diltiazem, that is known to have the capacity to block CYP3A4. Verapamil can similarly block this enzyme. The current study is to quantify the interaction of diltiazem and of verapamil with simvastatin since combinations of either of these calcium antagonists with simvastatin are likely to occur frequently.

The goal of the project is to study the electronic spectroscopy of porphyrins and related molecules such as phthalocyanines using a supersonic expansion to internally cool the molecules and to, thus, greatly simplify the spectra. We are interested in studying both the electronic and vibrational structure of these molecules by laser-induced fluorescence excitation spectroscopy, and also the intramolecular energy transfer properties of the molecules by observation of their dispersed fluorescence spectra.

Project Summary Malaria that results from Plasmodium falciparum is among the most globally devastating human diseases. The principle vector of malaria, mosquitoes of the Anopheles gambiae species complex, are thus central targets for controlling the human health burden of Plasmodium. For nearly two decades, there have been large-scale, coordinated efforts to diminish mosquito populations, generally through spraying and insecticide treated bed nets. Indeed such control efforts have now led to a nearly 50% decrease in the rates of malaria infection in many parts of sub-Saharan Africa. At present, however, control efforts of A. gambiae are being threatened by evolutionary responses within mosquitos: A. gambiae populations have shown increases in insecticide resistance as well as behavioral adaptations that allow mosquitos to avoid spraying all together. Thus adaptation of mosquitos to the control efforts themselves is currently a risk to maintain the gains made in the fight against malaria. In this proposal we lay out an integrated population genomic approach for systematically identifying regions of the A. gambiae genome that are evolving adaptively in response to ongoing control efforts. Our approach centers upon state-of-the-art supervised machine learning techniques that we have recently introduced for finding the signatures of selective sweeps in genomes (Schrider and Kern, 2016), coupled with the large-scale population genomic datasets currently in production by the Ag1000G consortium.

Cognitive control and executive function are similar terms used to describe our ability to direct thought and action based on our goals and intentions, rather than being driven automatically by the world around us. Current theories of cognitive control propose that the prefrontal cortex (PFC) is a brain region that is critical for tis ability. The PFC has extensive, reciprocal projections to both cortical and subcortical regions and therefore is in a privileged position to be a source of top-down signals that could sculpt behavior. Building on the progress we have made in the previous funding period, the overall aim of the current proposal is to further advance and refine our understanding of the functional organization of PFC and the neural mechanisms by which the PFC can provide top-down signals that modulate incoming sensory information. We propose that the PFC stores the highest level of representations such as rules and goals, and it is the active maintenance of these representations that bias information processing elsewhere in the brain influencing how we ultimately make decisions and act. In humans, frontal lobe function has been extensively researched both through the careful study of neurological patients with focal lesions (usually due to stroke and traumatic brain injury) and using functional MRI (fMRI) with healthy young subjects. However, there has been surprisingly little work combining these two approaches. Lesion and fMRI methodologies can complement each other in significant ways, and so when combined can be a powerful approach for studying brain-behavior relationships. In this proposal, such a convergent approach will be implemented. Basic knowledge about PFC function and cognitive control can provide substantial insights into the nature of a large number of psychiatric and neurological disorders affecting PFC function such as schizophrenia, dementia, stroke and traumatic brain injury; as well as many other conditions such as attention-deficit disorder, substance addiction and normal aging, that are proposed to involve selective dysfunction of frontal brain systems. Moreover, cognitive and behavioral deficits from PFC damage are particularly challenging to treat. A greater understanding of frontal lobe function is necessary for developing effective therapeutic interventions.

In many cases naturally occurring animal models of human genetic disorders, especially those affecting the nervous system, have not been described. In those instances where the gene defect has been identified, the introduction of mutant DNA into the germ line of mice can provide a unique opportunity to generate mice having appropriate phenotypes where disease pathogenesis can be studied. Transgenic mice are generated by microinjection of DNA into fertilized eggs, by receptor complex targeting of DNA, and by retroviral gene transfer. Introduction of murine embryonic stem cells into blastocyst stage mouse embryos that have been genetically modified by gene targeting also permits the establishment of mouse strains carrying appropriate mutations. Murine models of Gaucher disease and other inherited disorders are being produced using these techniques. Such transgenic mouse models of human disorders will provide valuable tools for the evaluation of new treatments, including protein/enzyme replacement, cellular transplantation, and gene transfer therapies. Development and tissue specific expression of genes can also be investigated in these transgenic mice.

This project is a collaboration between Massachusetts General Hospital-Harvard Medical School and the International Centre for Diarrheal Disease Research in Bangladesh (ICDDR,B), focused on defining protective immunity to V. cholerae infection and developing an improved cholera vaccine. The central hypothesis of this project is that V. cholerae expresses specific proteins during early infection, which generate immune responses that are protective on subsequent exposure; an ancillary hypothesis is that these proteins are not adequately expressed during colonization with available vaccine strains, and that these differences may explain the lessened efficacy of current vaccines. There are five specific aims in this project. In Specific Aim 1, the investigators will determine the genes and proteins expressed during human infection with V. cholerae in Bangladesh, utilizing gene expression profiling by microarray in samples of stool and vomit, as well as proteomic analysis of these samples. In Specific Aim 2, the investigators will identify proteins that are immunogenic following human infection with V. cholerae, as well as following vaccination with cholera vaccine strain Peru-15, in order to determine whether differences in gene expression between wild-type V. cholerae and Peru-15 may be correlated with differences in specific immune responses. In Specific Aim 3, the investigators will assess the duration of immunity to key cholera antigens following both cholera and Peru-15 vaccination over a 1-year followup period. The duration of immunity will be evaluated in serum, feces, and duodenal biopsies, as well as with a newly described methodology for measuring circulating, antigen-specific memory B cells. In Specific Aim 4, the investigators will examine which of the immune responses to in vivo-expressed proteins are protective following exposure to V. cholerae in household contacts utilizing: baseline antibody liters to key antigens in serum and feces; the increase in baseline to day 3 immunologic responses (as a marker of an anamnestic response), and the number of circulating, antigen-specific memory B cells at baseline. In Specific Aim 5, the investigators will evaluate selected host factors for correlation with development of immune responses following cholera or Peru-15 vaccination, as well as with susceptibility to symptomatic cholera following exposure in household contacts. The long term goals of this project are to develop the theoretical underpinnings for an improved vaccine for prevention of cholera, and to accomplish substantial technology transfer and capacity building at the ICDDR,B. [unreadable] [unreadable]

During this fiscal year, Dr. Bailey-Wilson has been collaborating with Drs. Trent and Carpten of the Cancer Research Branch and Drs. Cristina Leske, Barbara Nemesure and Anselm Hennis of State University of New York at Stony Brook on the planning phases of a proposed study of the genetic epidemiology of prostate cancer and breast cancer in Barbados. These cancers occur at very high rates in the Barbadian population. Dr. Hennis' joint appointments in New York and Barbados have expedited this proposed study. Large case-control and family studies are planned. Questionnaires, study protocols and consent forms have been developed in this year and pilot funding is expected within the next fiscal year.

Cell cycle controls are organized into a loop, so that cycles may occur over and over again. These controls are of several kinds: transcriptional controls, proteolytic controls, protein kinase controls, etc. The development of microarray technology has now made it far easier and more efficient to investigate all kinds of transcriptional controls. Indeed, microarray studies on the transcriptional regulation of the cell cycle have provided a wealth of data. In this proposal, microarray technology will be used to continue the study of the transcriptional control of the yeast cell cycle. In particular, an overview of the circular nature of cell cycle controls will be sought. In Aim 1, we will complete the task of assigning cell cycle regulated genes to their regulating transcription factors. In Aim 2, we will find out how the time at which cell cycle genes are expressed is determined, and we will define the extent and mechanisms of combinatorial control in the cycle. In Aim 3, we will test the hypothesis that an oscillation in the state of histone acetylation is a major mechanism of cell cycle transcriptional control. [unreadable] [unreadable]

Project Summary/Abstract for Phases I and II The purpose of this fast-track SBIR project entitled Paragraph and Essay Writing Instruction for Struggling Secondary Writers is to develop and evaluate the effectiveness of a total of four interactive multimedia (IM) software programs to be used to teach complex writing skills to struggling writers in secondary schools. The foci of the four programs will be (1) basic paragraph-writing skills, (2) advanced paragraph-writing skills, (3) basic essay-writing skills, and (4) advanced essay-writing skills. The lessons within each program will include an introduction to the program and the targeted writing skills, a description of how to use a targeted writing strategy, demonstrations, practice activities, and feedback. The Phase I project will focus on the feasibility of this medium for teaching complex writing skills to struggling writers. Measures will focus on (a) student use of the program, (b) student knowledge of writing skills, (c) student writing skills related to writing topic, detail, and concluding sentences within a paragraph, and (d) student satisfaction. During Phase I, an informal and a formal pilot test will be conducted to determine the feasibility of the prototype program. During each of the three years of Phase II, at least one informal pilot test and one field test of one new program will be conducted. A pretest-posttest control-group design with random assignment of students to groups will be utilized in each field test to determine the effects of each instructional program on student knowledge, performance, and generalization of the writing skills to complete writing tasks. The programs resulting from this project, because they will be designed to include instructional methods that have been found to be successful with at-risk students, should enable these students to respond successfully to the writing demands of their courses and minimum competency tests.

Biomedical scientists require ever improving tools to face the complex challenge of understanding the intricacies of protein regulation and its relationship to disease states. Recent research in the field of protein biochemistry has revealed that in many cases oxidative chemical modifications to protein molecules actually serve as a means of regulating protein activity helping to absorb, informationally register (for the purpose of activating biological feedback mechanisms) and/or alter protein activity, and deflect what otherwise might have been injurious insults. Cysteine sulfenic acid formation stands as one such particularly difficult-to-track modification (due to its inherent instability outside of its native cocoon-like protein environment) that appears to play a key role in the biochemical regulation of many proteins. The analytically elusive nature of this protein modification has made understanding its biological role and thus the full biological role of any affected protein an oft-ignored priority. The driving hypothesis of this application is that oxidative [redox] regulation of proteins via cysteine sulfenic acid formation critically affects the activity of proteins containing one or more unoxidized cysteine residues [i.e., cysteine residues with free thiol functional groups]. The long term goal of this research is, thus, to develop methodology that facilitates the amino acid residue-specific identification and quantitation of cysteine sulfenic acid modifications of specific target proteins directly out of their biological environment such as might be appropriate for making clinical diagnoses. This goal will be realized through completion of three specific aims: 1) Synthesize a custom-designed trifunctional molecular tag that can be used to specifically label site(s) of cysteine sulfenic acid within proteins and simultaneously provide a handle for affinity purification and a signature pattern in preliminary analyses [within single stage mass spectra] that indicates the presence and molecular mass of labeled peptides. 2) Optimize the preparation of blood plasma samples for qualitative identification and relative quantitation of cysteine sulfenation events within specifically targeted proteins. And 3) validate preparatory and analytical procedures according to the Association of Analytical Chemists-recommended method validation criteria and begin to qualitatively and quantitatively test biologically extracted, genuinely unknown, normatively unoxidized [free thiol-containing] proteins for cysteine sulfenic acid modifications. [unreadable] [unreadable] Recent evidence shows that within proteins, a uniquely modified (oxidized) form of the amino acid cysteine known as cysteine sulfenic acid can play important regulatory roles in both day-to-day biological function and in disease states. Because of their unstable nature, however, the biologically important details of such modifications go all but unnoticed under traditional techniques used to directly analyze proteins. The project under proposal here aims to remedy this analytical deficit by providing a robust means to study the detailed qualitative and quantitative nature of cysteine sulfenic acid modifications in proteins extracted directly from clinically relevant biological samples. [unreadable] [unreadable] [unreadable]

Despite the use of effective antibiotics in combination with cardiopulmonary support, the mortality rate from sepsis and septic shock for the last three decades has remained high (29%). Furthermore, the incidence of sepsis appears to be increasing. Lung infection or pneumonia is the most common cause of sepsis in patients hospitalized in MICUs. New therapeutic approaches that augment effective conventional treatments are necessary to lower the high mortality rate of this syndrome.[unreadable] [unreadable] Excessive release of host inflammatory mediators contributes directly to the pathogenesis of sepsis and septic shock. Mitogen-activated protein kinases (MAPKs) are serine/threonine intracellular signaling proteins that have a central role in this inflammatory response. These MAPKs are activated by phosphorylation and mediate gene expression and other cellular functions in response to extracellular signals. Three major MAPKs in mammalian cells are p38, extracellular signal-regulated kinase (ERK), and c-jun N-terminal kinase (JNK). Each of these MAPKs regulates inflammatory mediator production in response to lipopolysaccharide (LPS), a bacterial toxin closely associated with the pathogenesis of gram-negative bacterial pneumonia and sepsis. These mediators include cytokines, chemokines, nitric oxide, reactive oxygen species, and prostaglandin metabolites. Based on substantial data supporting a pivotal role for MAPKs in inflammation, there is growing interest in the therapeutic application of selective inhibitors of these proteins. However, the application of such agents in sepsis has been primarily tested in in vivo models employing LPS challenge only. Since many of the mediators MAPKs regulate participate in protective innate immune or host defense responses as well as inflammatory injury, their inhibition could be harmful during bacterial infection. Data from a study we conducted with Bacillus anthracis lethal toxin (LeTx) supports the possibility that there may be risks as well as benefits with MAPK inhibition. The lethal factor (LF) component of LeTx is a zinc protease that cleaves and inactivates several different members of the mitogen-activated protein kinase (MAPKKs) family and their downstream targets including p38. We showed that when LeTx was administered in sublethal doses to animals challenged with lethal LPS, inflammatory cytokine and nitric oxide release were reduced and hemodynamic function and survival improved. However, similar pretreatment in animals challenged with intratracheal E. coli, while also reducing cytokine and nitric oxide release, decreased survival. These findings suggested that nonselective inhibition of these MAPKs may be harmful with live bacteria. Selective inhibition of these MAPKs may still be beneficial however.[unreadable] [unreadable] SB203580 [4-(4-flurophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)imidazole] is a pyridinyl imidazole that has been employed extensively as a selective inhibitor of p38, in both in vitro and in vivo models of inflammation. This agent competes for the ATP binding site on p38 and inhibits its phosphorylation and activation. In in vivo models, administration of this agent via several different routes, as well as both before and after inflammatory stimulus, has been shown to inhibit cytokine and nitric oxide release, leukocyte trafficking, and organ injury. This agent and a related one have also been shown to improve survival in LPS challenged models. Although the results of such studies have provided a major rational for the use of p38 inhibitors during states of inflammation such as sepsis, an extensive literature search has revealed only one published report (12) assessing the effects of this or related agents in an animal model of sepsis employing live bacterial challenge. This was a very limited study in mice undergoing cecal ligation and puncture (CLP) and did not incorporate antibiotic support or microbiological assessment. The results of this study did suggest however that the effectiveness of SB203580 was increased when its administration was delayed for 12 h after the onset of sepsis. [unreadable] [unreadable] We previously developed a mouse model of E. coli pneumonia in which antibiotics and fluids were shown to synergistically improve survival as they are believed to do clinically. This model is now being used to test the effects of SB203580. In initial experiments we have found that doses of this agent previously reported to be beneficial in very limited studies, actually appears harmful when administered either 1 h before or 1 h after bacterial challenge in our mouse model. Delaying treatment for 12 h is does not result in a harmful effects but is also not beneficial. Reducing the dose of SB203580 3 logs does result in beneficial effects with the agent, but only if it is given prophylactically. Later treatment at these lower doses has no benefit. These results are being submitted in abstract form shortly. Other studies are ongoing to better define the mechanisms underlying the harmful and beneficial effects of the agent as well as deternmining whether there are administration regimes that will produce benefit with later treatment. At present however these studies suggest that targeting p38 may be difficult in the treatment of sepsis and septic shock.

The challenge of eliminating racial and ethnic health disparities requires expanding research beyond the biomedical model since many of the determinants of racial/ethnic health disparities differ from the factors that lead to an overall population decline in disease rates. We think that many of the determinants of racial/ethnic disparities in health outcomes are social, behavioral, cultural, economic, historical, and political in nature. Furthermore, we propose that the solutions for eliminating disparities require community participatory approaches. This proposal is being submitted by Morehouse College on behalf of a consortia of four historically Black undergraduate colleges in Atlanta, Georgia: Morehouse College, Spelman College, Morris Brown College, and Clark-Atlanta University, collectively referred to as the Atlanta University Center (AUC), to develop an Excellence in Partnerships for Community Outreach, Research on Disparities in Health and Training (EXPORT) project. The specific aims of the EXPORT project are: 1) To develop a team of researchers (faculty, staff, students) that engage in conducting interdisciplinary studies of the social determinants of racial/ethnic health disparities; 2) To develop research questions, pilot studies, or feasibility studies that explore behavioral, cultural, economic, or political determinants of disparity; 3) To provide junior level and mid-career AUC faculty with mentoring opportunities to conduct social determinants of health disparities research at the University of Michigan and the Centers for Disease Control and Prevention; 4) To develop community-based participatory research methods for studying intervention approaches to eliminating health disparities; 5) To work directly with grassroots community organizations throughout the southeast to develop projects related to community public health and environmental health issues; 6) To provide African American high school and undergraduate students with educational and research opportunities in science; and 7) to integrate health disparities into the curricula of each of the undergraduate college's research courses and seminars. After 3 years of the EXPORT project, the AUC will have an interdisciplinary team of researchers dedicated to creating an innovative, multidisciplinary framework for studying racial and ethnic health disparities and to developing research techniques that integrate conceptual and analytic social science constructs with behavioral constructs and population-based research. [unreadable] [unreadable] [unreadable]

(1) Clinical applications arising from stem cell and regeneration research will require an understanding not only of de novo tissue synthesis but also of the patterning mechanisms regulating blastema morphology. This project seeks to elucidate the regulatory networks controlled by biophysical epigenetic changes which orient blastema polarity relative to existing tissues. Our hypothesis is that ion transport by the H,K ATPase transporter and gap junctional communication (GJC) together regulate anterior-posterior polarity during planarian regeneration upstream of traditional signaling. Aim 1 is to identify and characterize transcriptional targets downstream of ion transport, through microarray analyses of regenerating worms inhibited for H,K ATPase (completed) and GJC activity. Candidates will be selected using specific criteria, validated by expression analyses, and functionally characterized following RNAi knockdown using in situ hybridization and molecular marker analyses of phenotypes. Aim 2 is to build and test models which outline the mechanisms ion transporters use to control axial identity in regenerating tissue. A preliminary transcriptional network downstream of ion transport will be generated by identifying network nodes, using quantitative PCR to test how RNAi knockdown of a single candidate affects the expression of all other candidates. Hypotheses predicted by this network will then be generated and tested, to elucidate the transcriptional and signaling response to the activity of the implicated ion transporters during the establishment of regenerative polarity. The long-term goal is to compile and validate a systems-level regeneration network model integrating biophysical and biochemical signaling. This has the potential for use as a predictive model of morphogenesis to aid in the translation of basic regeneration research into practical biomedical therapies. (2) The promise of stem cell and regeneration research is ultimately to replace lost or damaged tissues, organs and limbs in patients suffering from injury, aging or cancer. Although the regeneration of new tissue is critical, for that tissue to be functional, it must be properly patterned in relationship to the existing tissues: This research aims to discover how new and old tissues communicate to regulate this pattering, by learning how regenerating flatworms determine whether to form a new head and/or tail following amputation.

The long-term goal of this research is to understand the roles of of this research is to understand the roles of neuroendocrine homeostasis. CRF is the major integrator of these processes. Deviation of major integrator of these processes. Deviation of its regulation is its biological activity by binding two classes of proteins: CRF receptor activity by binding two classes of proteins: CRF receptor and CRF binding modulator. The objective of this proposal is to clarify the role of each objective of this proposal is to clarify the role of each CRF component mutations in each component of the CRF system. This involves: in each component of the CRF system. This involves: 1. Cloning of cDNAs encoding mouse CRF, CRF receptor A (CRF-RA), and CRF- 1. Cloning of cDNAs encoding mouse CRF, CRF receptor A (CRF-RA), and CRF- genomic clones containing these genes for targeted disruption of the CRF-RA of genomic clones containing these genes for targeted disruption of the studied. will be studied. 2. Investigation of effects of targeted disruption of CRF-RA and CRF-BP on 2. Investigation of effects of targeted disruption of CRF-RA and CRF-BP and stressful conditions including neuroendocrine, immune, and reproductive basal and stressful conditions including neuroendocrine immune and developing a strategy to generate mice with the components of CRF system developing a strategy to generate mice with the components of CRF system in the CRF system by intercrossing mice carrying mutations in different in the CRF system by intercrossing mice carrying mutations in different components of CRF system.

Mitochondrial dysfunction has been identified as one of the key players in Parkinson's disease (PD) pathogenesis. While the etiology is unknown in most cases, the development of progressive parkinsonian symptoms has been shown in patients following exposure to various environmental and occupational toxins. Numerous studies demonstrate that these neurotoxins specifically inhibit the mitochondrial respiratory chain complexes of dopaminergic neurons, which initiates a cascade of events ultimately leading to cell death. Furthermore, the susceptibility to environmental neurotoxins is increased in the aged nervous system. Thus for the long-term treatment of PD and parkinsonian symptoms, therapeutic strategies are needed that not only restore dopaminergic neuron function, but also provide mitochondrial protection and restoration from various stresses, including environmental toxin exposure. In the past, neurotrophic growth factors have received considerable attention as potential therapeutic agents for neurological disorders. However, the clinical application of these native molecules has not advanced primarily due to pharmacological disadvantages and challenges associated with directly delivering large protein molecules to the brain. The emergence of physiologically functional propeptides from the neurotrophic factor family provides a wealth of novel, smaller sequences for biotherapeutic exploration and evaluation. Examination of the glial cell line-derived neurotrophic factor (GDNF) prosequence predicts internal dibasic endopeptidase sites that would yield a smaller, amidated eleven amino acid residue peptide named dopamine neuron stimulating peptide-11 (DNSP-11). Recent evaluation of DNSP-11 has shown that it exhibits similar GDNF-like neurotrophic responses in normal and parkinsonian rat models. However, cell culture and proteomic pull-down data suggest that DNSP-11 functions differently than mature GDNF;leading to our hypothesis that DNSP-11's neurobiological actions are through the mitochondria. The research outlined in the current proposal will measure DNSP-11's bioenergetic and protective effects, in the MN9D dopaminergic cell line, from toxins that specifically target the mitochondrial respiratory complexes. The information obtained in this study will further our understanding of this propeptide's neurobiological activity and provide the basis for future evaluation and biotherapeutic development of DNSP-11. PUBLIC HEALTH RELEVANCE: Parkinson's disease (PD), a chronic neurological disorder that affects over 1 million Americans, is characterized by stooped posture, balance impairments, rigidity, resting hand tremors, and bradykinesia. While the cause of PD is largely unknown, parkinsonian symptoms have been shown to develop following long-term exposure to common environmental toxins that specifically target the mitochondria of dopamine neurons. The goal of this pilot project is to investigate the neuroprotective properties of a novel neurotrophic propeptide, DNSP-11, from mitochondrial-specific toxins to pave the way for its downstream evaluation as a potential PD therapeutic.

Gulf War Veterans Illness (GWVI) is a constellation of symptoms reported by Gulf War Veterans shortly after their return from deployment in 1991. Clinical diagnostic criteria for GWVI are based on chronic multisystem illness (CMI) criteria, which are based on statistical symptom cluster analysis resulting in three categories: fatigue, mood/ cognition, and musculoskeletal symptoms. Currently, approximately 40% of Gulf War Veterans (over 1/4 million Veterans) have GWVI by these criteria. The pathophysiological mechanisms underlying GWVI are not understood, and insights into the mechanisms of GWVI could lead to novel concepts, methodologies for study, and treatment interventions. Our Pilot study will assess three facets of vascular and skeletal muscle function in 25 cases with GWVI and 25 Veteran controls without GWVI. The purpose of the proposed three-year pilot study is to determine whether lower extremity (leg) endothelial function, exercise functions, and skeletal muscle mitochondrial gene regulation are different among Veterans with GWVI compared to Veterans' without this illness. Endothelial dysfunction, skeletal muscle functional impairment, and dysregulation of mitochondrial genes are plausible mechanisms for GWVI as exposure to anticholinesterase inhibitors during the Gulf War may affect these functions to cause symptoms of fatigue and other musculoskeletal symptoms. We will recruit subjects from a well characterized cohort of Gulf War Veterans (the Fort Devens Cohort). Approximately 60% of Gulf War Veterans in this cohort meet clear CMI criteria for GWVI. Symptoms and exposure to pesticides, pyridostigmine bromide, and low level sarin exposure are well documented in these cohorts. Cases and controls will have femoral artery microvascular endothelial function assessed invasively in the cardiac catheterization laboratory using Doppler flow wire and intravascular ultrasound. We will use this technique to measure flow and artery responses to intra-arterial infusions of the endothelium-dependent vasodilator acetylcholine 10-6M, the microvascular endothelium-independent dilator adenosine, and the large artery vasodilator nitroglycerin. The PI has a long track record in this type of research. After 1 week, subjects will have an extensive skeletal muscle functional assessment using cardiopulmonary exercise testing to measure anaerobic threshold, objective strength and fatigue assessments, and the 6-minute walk test. One week after this study, subjects will have a skeletal muscle biopsy from the thigh. This will be analyzed for muscle fiber type, and RT-PCR to assess nuclear and mitochondrial genes responsible for regulating mitochondrial respiratory function. The overall aim is to assess differences in pathophysiological and muscle mechanisms, which will allow us to design a more definitive MERIT Review proposal to assess differences in these functions and test potential treatments targeting these mechanisms. This Pilot study may also provide insights into other chronic illness characterized by fatigue and other musculoskeletal symptoms, such as peripheral vascular disease and congestive heart failure.

Duchenne muscular dystrophy (DMD) is a severe muscle wasting disease and the most prevalent inherited muscle disorder worldwide. It is caused by a mutation in the dystrophin gene, which normally encodes an important structural and signaling protein located at the muscle membrane. In DMD, the dystrophin protein is absent or present at low levels within the body, rendering the muscle susceptible to damage. DMD patients lose motor function over time and die prematurely from respiratory or cardiac failure. Current treatments are merely palliative and do not target the underlying cause. Gene therapy strategies aimed at replacing or correcting mutated genes have shown promise for DMD. Recombinant adeno-associated viruses (AAVs) are popular gene delivery vehicles due to their non-pathogenic nature and ability to establish long-term and efficient gene transfer. Still, restoring dystrophin fails to completely alleviate motor deficits and prevent fatigue after mild activity. While DMD is caused by a mutation in a single gene, many secondary disease mechanisms are involved, such as ischemia, fibrosis and abnormal regeneration. A strategy that addresses multiple pathological mechanisms may provide greater benefit. MicroRNAs (miRNAs or miRs) are small, regulatory RNA molecules that inhibit their target genes. A skeletal muscle-restricted miRNA, miR-206, is highly up-regulated in dystrophic muscle. While its function after disease onset is not well-understood, multiple miR-206 targets have shown therapeutic benefit for DMD, such as vascular endothelial growth factor A (VEGF-A) and utrophin. Down-regulation of miR-206 and its detrimental effect on corrective mechanisms thus presents a novel strategy for treating DMD. This proposal aims to characterize the therapeutic efficacy of a recombinant AAV vector carrying an antisense sequence against miR-206 (AAV-anti-miR-206), exploring its impact on two secondary pathological mechanisms. Functional ischemia is a major contributor to the dystrophic phenotype and exacerbates muscle damage. To better understand the role of AAV-anti-miR-206 on muscle vascularization, the first Aim will investigate its ability to increase the expression of proven therapeutic target VEGF-A. These experiments will also determine if AAV-anti-miR-206 can recapitulate blood vessel growth, function and integrity associated with VEGF-A treatment. Increasing membrane stability using utrophin, a dystrophin paralog, can delay DMD progression and improve motor function. To determine if AAV-anti-miR-206 influences muscle structural integrity and pathology, the second Aim will explore its effect on utrophin expression. The proposed experiments will determine if this treatment improves overall muscle pathology and prevents further damage. Together these Aims will provide a better understanding of miR-206 function and explore a novel, multifunctional therapeutic strategy for DMD.

This application, entitled ?Student Transformative Experiences to Progress Underrepresented Professionals? (STEP-UP) for cancer prevention, is a multidisciplinary initiative led by the Cancer Prevention and Control and Health Disparities Programs' faculty at the University of Arizona Comprehensive Cancer Center. It is uniquely designed to provide 55 undergraduate and 54 Masters' degree seeking students with an intensive, 12-week summer research experience in cancer prevention and control. Educational pedagogy will inform on our methodology in order to assure students receive the quality research experience that not only will increase understanding, but also will motivate students toward a continued educational commitment to advance in cancer prevention and control sciences. The emphasis in training will be largely within the clinical trials to community-based participatory research end of translational research, yet offering students 2 week-long immersion opportunities to experience cancer prevention and control science across the entire continuum of basic to applied and even dissemination research. Over 35 faculty members from diverse disciplines who are funded in cancer prevention and control research are committed to this program. The program fills a gap in our web of training opportunities for underrepresented students and yet builds on a substantial foundation of purpose-driven efforts at the UA and the Arizona Cancer Center to attract and retain a diverse student body in cancer prevention and control research. Importantly, this effort leverages our international reputation in cancer prevention research. Our specific aims include: 1. Recruit undergraduate and Master's level graduate students from URM into the STEP-UP in cancer prevention 12-week summer experiential cancer prevention and control research training; 2. Provide an integrated, innovative, multidisciplinary educational experience in cancer prevention and control research that strengthens self-efficacy and intrinsic motivation to become a cancer prevention and control scientist; 3. Support mentoring and professional relationships with scientists, research programs and community partners; 4. Conduct formative and summative evaluations to improve the program over time. The training program is centralized in Southern Arizona and offers experiential learning within our unique catchment area which is rich in diversity relative to ethnicity (30.7% Hispanic), race (5.8% Native Americans) and age (16.4% over age 65 years). This distinctive location, strong community ties, university facilities and resources for research and education, as well as committed and experienced faculty mentors and program leaders, assures a quality program that will impact the diversity, competence and commitment of the cancer prevention and control workforce of the future.

Project Summary Progress in elucidating the role of the environment in child development and disability has been slow and incremental. Nearly all studies have examined relatively small populations of children; considered only one parameter at a time; had little power to examine interactions among chemical, social, and behavioral factors; had limited ability to examine gene?environment interactions; and suffered from brief duration of follow-up. NYU School of Medicine and Erasmus University Medical Center, in partnerships with multiple other institutions, respond to RFA-16-OD-004, presenting four cohorts for inclusion in the Environmental Influences on Child Health Outcomes Program (ECHO). These cohorts are the NYU Children's Environmental Health Study (n~1000, 2016?2018 births, NYU CHES); the Rotterdam-based Generation R Second Cohort (n~1000, 2016?2018 births, GR2); the First Generation R Study (n=1431, 2004?2006 births, GR1); and the Infant Development and Environment Study II (n=717, 2010?2012 births, TIDES). Together, they would comprise ~9% of all human subjects within ECHO. All four cohorts are well suited to examination of perinatal outcomes; upper and lower airway; and neurodevelopment, and offer substantial flexibility in prospective collection and the use of existing biospecimens. Though our proposed aims focus on chemical exposures, our cohorts also take a broad approach to biological, psychosocial, and physical exposures, which are equally influential on health outcomes. GR1 is widely known as a model for ECHO, given its track record of successful implementation, much like Project Viva. Moreover, GR1 measures could be used to evaluate the promise and feasibility of common ECHO exposures and outcome measures for use in more recently established cohorts. First-trimester recruitment and collection of urine samples in each trimester are common to all four cohorts. A unique benefit to inclusion of GR2 is its nesting within Rotterdam's municipal preconception care program, permitting examination of preconceptional exposures (generally unavailable in US cohorts). GR1's unique abdominal MRI, pulse wave velocity, and echocardiographic measures permit studies of end-organ effects. ECHO funding would extend NYU CHES and GR2 through age 2 years, GR1 through age 13 years, and TIDES through age 9 years, examining prenatal exposures in relation to early life trajectories of body mass. The proposed NYU-Erasmus ECHO Pediatric Obesity, Metabolism and Kidney Cohort Center pairs an internationally known leader in children's environmental health (Trasande) with a leader in the Developmental Origins of Health and Disease (Jaddoe) as multiple PIs and leverages extensive expertise in fetal growth, epidemiology, biostatistics, metabolomics, epigenetics among other disciplines that contribute to high-quality execution of synthetic cohort studies, and guide prevention.

Chronic inflammatory reactions in the brain, has been implicated to play a role in age-related neurodegenerative disorder's, such as Alzheimer's disease (AD). In AD, glial cells in the brain are alerted by the innate immune receptors on the glial cell surface following A[unreadable] deposition, resulting in activation, phenotypic transformation and release of soluble inflammatory mediators by the glial cells. Some of these inflammatory mediators can be directly toxic to neurons and therefore they have been accused in driving neurodegeneration. Furthermore, in the case of AD, it is hypothesized that these inflammatory cytokines can potentially alter APP processing and A[unreadable] clearance mechanisms, creating "feedback loops", that promote further A[unreadable] accumulation. However, we have now generated a wealth of data suggesting that over-expression of inflammatory cytokines (e.g., IL-6, IFN3 and TNF1) does not significantly alter APP levels, APP processing or steady state A[unreadable] generation in vivo. Rather, all three inflammatory cytokines have a beneficial effect, i.e., they significantly "attenuate" amyloid deposition when expressed early in the disease process and have no effect (neither increase nor decrease) on amyloid deposition when expressed in older mice with significant pre-existing amyloid burdens. In contrast, AAV mediated over-expression of anti-inflammatory cytokine IL-10 led to significantly increased amyloid deposition and exacerbated cognitive deficits in APP transgenic mice (details in Specific Aim 2). Thus, depending on the timing and context, the actions of individual cytokines may produce divergent and unexpected effects during the disease process. Growing evidence now suggests that neural- immune interactions in the brain are intricately connected, complex and engage in significant crosstalk to maintain brain homeostasis and protect the brain. In AD, the role of these complex neural-glial interactions and immune responses in the brain are largely unresolved. In this proposal, we will take advantage of our AAV system to explore the effects of individual cytokines and chemokines particularly as it relates to AD-associated pathologies. The studies outlined in this proposal will provide insight into how specific neuro-inflammatory and neuro-modulatory reactions affect amyloid deposition and microtubule -associated protein tau pathology, the two hallmark pathological features of AD. PUBLIC HEALTH RELEVANCE: This study will use mouse models of Alzheimer's disease (AD) to systematically evaluate the role of the neural- immune responses in AD brain. These studies will provide insight into the role of inflammation on AD pathologies, and potentially lead to the development of novel therapeutic approaches for treating AD.

Project Summary/Abstract?Resource Component The objective of the Caenorhabditis Genetics Center (CGC) Resource Component is to promote research on the small metazoan Caenorhabditis elegans by curating important, genetically characterized nematode stocks and distributing them upon request to researchers and science educators. The CGC is the sole general stock center for the curation and distribution of C. elegans. The CGC currently houses over 19,000 different strains, and they are immensely popular: ~30,000 strains are distributed each year and the majority goes to 2,622 user groups in the United States, with users in all 50 states, the District of Columbia, and Puerto Rico. Researchers in all locales supply us with important strains that they have generated. If the CGC were not performing this curation and distribution service, strain sharing would be extremely inefficient and costly; the burden of filling requests would be placed upon individual labs, likely leading to delays in the exchange of research materials. Moreover, the conservation of many published strains might be in jeopardy. This would be a great loss, because research in this model organism has led to fundamental insights into basic biological mechanisms, including programmed cell death, axonal guidance, the discovery of microRNAs, and the mechanism of RNA interference in animals. C. elegans has also provided insights to mechanisms of cancer progression and other diseases including Alzheimer's and Parkinson's. C. elegans also serves as a key model for illuminating the biology of parasitic nematodes, a major health concern. In addition to enhancing research progress, the CGC Resource Component offers major cost-savings to the NIH as a whole, to individual research labs funded by NIH, and to all other labs in the community, through decreasing redundant labor through making new mutations and mutant combinations readily available, decreasing labor and other costs by providing an economy-of-scale approach rather than having individual labs responsible for disseminating useful strains, safeguarding strains made with NIH grant support through curation in redundant sites, and facilitating the use of this relatively low-cost model by investigators working primarily in high-cost models such as mice.

The objective of the Clinical Research Skills Development Core is to develop skilled, productive, and independent translational and clinical investigators in vascular disease. Our specific aim is to train young investigators in designing, performing, analyzing and interpreting translational and clinical research to prevent, detect, characterize, and treat/manage vascular disease. Training for each participant will include a) an advanced course on Vascular Biology taught by prominent researchers;b) biweekly SCCOR-TAAD seminars led by eminent faculty;c) a Clinical Research Curriculum described on the "pink sheets" for the competitive K30 renewal as "an extremely sophisticated and successful program." Basic courses & workshops are given Wednesdays 5-6:30 PM &tailored to help each participant develop &refine translational or clinical study protocols, grant proposals, and manuscripts. Advanced courses may be taken at UT Houston Graduate School &Schools of Medicine, Health Informatics, &Public Health;Baylor College of Medicine &Graduate School;&Rice University, all within or adjacent to our Medical Center. Participants with adequate protected time may pursue an MS in Clinical Research or other MS or PhD degree if important to their career development;d) a focused program of research with intensive mentorship provided jointly from a primary mentor &a Core mentor providing methodologic expertise in translational or clinical research. Young investigators will be fully involved in a SCCOR-TAAD project, initiate their own research, &within 2 years prepare a major research proposal (e.g. a R03, K08, or K23). Intensive mentorship will continue through completion of both this research and the SCCOR-TAAD. The Core Director is Jon Tyson, MD, MPH, Bain Professor of Pediatrics &Internal Medicine, Director of the Center for Clinical Research and Evidence-Based Medicine, and the 2004 Recipient of the Distinguished Educator Award from the National Association of Clinical Training Program Directors. The Core Co-Director is Dianna Milewicz, MD, PhD, Professor of Internal Medicine, Director of Medical Genetics and of the MD/PhD program, &recipient of a Doris Duke Distinguished Clinical Scientist Award. They and other senior Core mentors and investigators in the SCCOR-TAAD provide the complementary background and talents to train skilled, productive, and independent translational and clinical investigators in vascular disease. In this way, our Clinical Research Skills Development Core will help to train the next generation of physician scientists investigating vascular disease and to translate the advances in basic research into improved methods to prevent, diagnose, and treat a common cause of death and disability.

There is a widely held view that with advancing age efferent sympathetic nervous system (SNS) activity becomes elevated at rest and that the magnitude of the SNS response is increased during acute "stress". However, the experimental basis for this concept is rather weak because the data in both humans and animals have been derived primarily from peripheral measurements of plasma norepinephrine (NE), an imprecise and potentially misleading index of SNS activity. The purpose of the proposed studies will be to investigate SNS regulation during acute physical and environmental stressors in mature (i.e., adult) and senescent (i.e., older) Fischer 344 rats. These animals have several important advantages over the human for studying the true biological effects of aging on SNS activity: l) The Fischer 344 rat can be subjected to invasive surgical procedures and a variety of stressors that cannot be safely undertaken in humans; 2) With advancing age, Fischer 344 rats do not develop chronic cardiovascular diseases or increased body weight; 3) Because these animals are cage- confined, marked, age-associated differences in physical activity levels do not occur; and 4) By comparing animals within different age groups that are of the same strain and sex, it will be possible to isolate the effects of age per se on SNS function. To test the hypothesis that a "hypersympathetic state" develops with aging, a comprehensive experimental approach will be employed in these animals. Efferent SNS activity will initially be estimated from measurements of NE depletion (i.e., "turnover") rates in selected sympathetically-innervated organs and regional blood flows. Subsequent studies will assess SNS activity directly by measuring sympathetic neural outflow. Regional SNS diversity will be determined from rates if NE depletion in the heart, kidney, adrenal gland, liver and selected skeletal muscles and from direct neural recordings from the splanchnic, renal, and adrenal nerves. Because age-related changes in SNS behavior may be stressor-specific, SNS activity will be determined both at rest and in response to different types of acute stress including exercise, nonexertional heating, and hypoxia (i.e., physical stressors) and noise and air jet stimulation (i.e., environmental stressors). Since age-related differences in the SNS responses to these stressors may be dependent on the intensity of the stimulus, several levels of a particular stress will be applied. In addition to the physiological questions being posed, these studies should have important clinical relevance. For example, the development of heart and kidney disease and hypertension are thought to be linked to elevated SNS activity at rest and exaggerated SNS responsiveness to acute stressors. In addition, the prevalence of these pathological conditions increases with advancing age. The information gained from these proposed studies in rats may not be directly applicable to human aging; however, the design of these studies and the ability to eliminate many of the confounding variables prevalent in human investigations provides another mechanism to evaluate the effects of age on SNS function, in an animal that is susceptible to aging, but not susceptible to a variety of diseases associated with aging.

Drosophila is one of a handful of model organisms used extensively in biomedical research. Importantly 60% of human disease genes are conserved in the fly. Genetic analysis in Drosophila has focused primarily on a whole organism approach but cell lines have also been at the forefront of research because of their utility in biochemical analysis. Moreover, the recent development of cell-based RNAi screens, which have the potential to assay the function of all genes, has further highlighted the importance of cell lines. With these developments has come the realization that the small number of poorly characterized cell lines, with ill-defined cell origins, will limit the full potential of these approaches in Drosophila. Correspondingly, there is a broad consensus in the field that efforts should be made to develop cell-type specific Drosophila cell lines. The goal of this proposal is to address these needs by 1) improving the utility of existing cell lines through genome-wide expression analysis and 2) to develop a genetic method to immortalize/transform Drosophila primary cells. This method will be targeted to specific tissues to derive cell-type specific cell lines. Preliminary results showing that expression of a highly conserved oncogene can induce cell proliferation in primary cells support this goal. The completion of this work is expected to significantly increase understanding of the mechanisms of cell immortalization and transformation in Drosophila and to generate several cell-type specific cell lines. These cell lines will be distributed to the community and the information about them disseminated on a web site. Subsequent use of the cell lines by individual investigators to study diverse biological problems is expected to impact our understanding of human disease. [unreadable] [unreadable]

The cause(s) of neuropsychiatric systemic lupus erythematosus (NPSLE) remains an enigma. Although numerous reports of associations between specific autoantibodies and CNS manifestations have been published, serum levels of none of these autoantibodies has consistently shown a high degree of sensitivity and specificity for NPSLE. Recently it has been demonstrated that immune complexes containing nucleoproteins become internalized and stimulate Toll Like Receptors (TLRs) to produce type 1 interferon (IFN). IFN has previously been implicated in NPSLE and high concentrations of IFN are known to cause neuropsychiatric symptoms similar to NPSLE. We therefore hypothesize that cell death coupled with the release of nucleoproteins results in the formation of immune complexes that stimulate TLRs to produce high local concentrations of type 1 IFN (and perhaps other cytokines) in the central nervous system (CNS) thereby causing NPSLE. The specific aims of the proposal are: To compare the frequency and activity of interferogenic (IFG) complexes in the serum (and CSF) of patients with and without NPSLE. The ability of serum from NPSLE and non NPSLE, matched for activity and other parameters to stimulate IFN and other inflammatory cytokines from mixed peripheral blood mononuclear cells as well as from cultured human microglia will be compared. A requirement for apoptotic or necrotic cells as a source of antigens will be tested and the ability of NPSLE serum to induce both cell death of neuronal targets followed by IFG immune complex formation will be examined. To determine the autoantibody specificities, nature of the antigens and TLR pathway responsible for the IFG activity. The autoantigens, their nucleic acid composition will be defined by immunochemical techniques and mass spectrometry as required. The cellular origin of cytokines will be determined by positive and negative selection. Identification of specific TLRs will be achieved by selective inhibition of endosomal TLR using oligonucleotides or siRNA. These studies are expected to lead to a new understanding of the pathogenesis of NPSLE that accounts for both autoantibodies with 'cytotoxic' function as well as autoantibodies that form nucleoprotein complexes that are now known to stimulate IFN and other pro-inflammatory and neuromodulatory cytokines. The cause of most cases of lupus affecting the brain is unknown, but there is good evidence to suggest that certain types of antibodies may play a role. Here, we test the idea that the sequential action of different antibodies that cause tissue injury followed by antibodies that capture antigens and enter the cell, causes the release of proteins called cytokines that alter brain function. [unreadable] [unreadable] [unreadable]

The maintenance of genomic integrity following DNA damage depends on the coordination of the DNA repair system and cell cycle checkpoint controls. Increasing evidence suggests that several tumor suppressors, including p53 and BRCA1, participate in these processes. Chk2, a DNA damage-activated protein kinase, regulates p53 and BRCA1 activities following gamma radiation. In addition, heterozygous germ-line mutations in Chk2 have been identified in Li-Fraumeni syndrome (LFS), a highly penetrant familial cancer phenotype usually associated with mutations in the p53 gene. The observation that Chk2 is mutated in a sub-group of Li-Fraumeni patients that lack mutations in p53 gene strongly suggests that, like p53, Chk2 is a tumor suppressor gene. We hypothesize that Chk2 participates in a DNA damage-responsive pathway that is critical for the prevention of cancer development. In this proposal, we will explore the molecular mechanisms by which Chk2 functions as a tumor suppressor. 1: We speculate that Chk2 exerts its tumor suppressor function mainly by regulating p53. Our preliminary studies have shown that Chk2 phosphorylates the C-terminus of p53. Here, we will explore the contribution of these phosphorylation events to the regulation of p53 following DNA damage. 2: We will study the roles of forkhead homology-associated (FHA) domain in the regulation of Chk2 functions. Two tumor-associated mutations within Chk2 FHA domain have been identified, suggesting that the FHA domain of Chk2 is important for its tumor suppression function. Studies of its homologue in yeast also suggest that the FHA domain may play essential roles in regulating Chk2 function. Based on the recently solved structure of the yeast Chk2 FHA domain, we will perform structural and functional analyses of the Chk2 FHA domain and assess the roles of the FHA domain in Chk2 activation and p53 stabilization following gamma radiation. 3: Chk2 is a component of a large protein complex with an apparent molecular weight of approximately 200 kDa. Using mass spectrometry and microsequencing analyses, we have demonstrated that one of the Chk2-associated proteins is a novel protein with unknown function. In this proposal, we will clone the cDNA encoding this polypeptide and explore whether its association with Chk2 contributes to Chk2 function. In addition, we have observed that, following gamma radiation, the Chk2-containing protein complex increases in size. We speculate that there are additional proteins associating with Chk2 following DNA damage. We will identify these damage-regulated, Chk2-associated proteins, perform initial characterization of their interactions with Chk2 and begin to explore the biological significance of these damage- regulated interactions. Collectively, these studies will elucidate the Chk2-dependent DNA damage-signaling pathway and the molecular mechanisms by which Chk2 functions as a tumor suppressor.