Current and recent work generally has focused on understanding and improving cognitive and behavioral phenotypes and applying these in analyses of whole genome association data. In Dickinson et al (2011) we extended earlier analyses of cognitive structure in schizophrenia cases and controls (Dickinson et al., 2006; Genderson, Dickinson et al., 2007). To achieve greater cognitive phenotype reliability and avoid redundant statistical comparisons, we used exploratory and confirmatory factor analyses separately in the CBDB samples (schizophrenia n=496, unaffected siblings n= 504, and controls n=823) and identified six positively correlated cognitive factors (for verbal memory, visual memory, n-back working memory, processing speed, card sorting, and span working memory). Data also supported a higher-order factor reflecting general cognitive ability, so-called g. We found that this structure was reasonably consistent across schizophrenia cases and controls, as had been shown previously (Dickinson et al., 2006), and extended this finding to include unaffected siblings. These findings guided construction of cognitive composite scores, which are in wide use in CBDB data analyses. One application of these composite scores has been to explore genome-wide associations in the CBDB samples. One exciting finding has been presented at genetics and psychiatry conferences and will be submitted for publication in the near future. We have identified an exciting and novel genome-wide significant association between our global cognitive composite and a sodium channel gene polymorphism that explains substantial variance in the cognitive impairment in our sample of people with schizophrenia and in their unaffected siblings an association that would not have been detected without the cognitive data aggregation strategy and composite scores developed by the Neuropsychology Lab. To gauge better which cognitive variables are best suited for genetics analysis, separate work has taken different approaches to estimate the heritability of these variables. Recently, a novel technique (GCTA) has been developed that permits estimates of heritability based on genome-wide genotype data from samples of unrelated people. The technique correlates pair-wise distances between genotypes for all possible pairs from a sample, with pair-wise differences in performance on a trait of interest (in our case, cognitive variables). We will present our first results contrasting GCTA methods with more traditional family-based methods at an upcoming conference and are beginning work on an associated manuscript. Wallwork et al. (2011) describes our efforts to determine a more empirically sound dimensional structure for patient data from the Positive and Negative Syndrome Scale (PANSS). The 30-item scale was developed with three syndrome scores, but previous analyses suggest that the scale really captures 5 or more dimensions of schizophrenia-associated symptomatology. We used existing literature to build a consensus five-dimension model of PANSS data then tested variations of this model using confirmatory factor analysis in the CBDB schizophrenia data and in an independent data set from Japan, supporting construction of new PANSS composite scores for positive, negative, depressive, agitated, and concrete/disorganized symptoms. Analogous lines of work are examining the dimensional structure of typical and abnormal personality in the CBDB data. We have elaborated and are refining five-dimension models for the Tri-dimensional Personality Questionnaire (TPQ) and for the SCID-II Personality Questionnaire (SCID-II). The TPQ targets personality more in the non-clinical range. The SCID-II is used to assess maladaptive personality disorder symptomatology. All of this work has been presented at scientific conferences and a paper on the SCID-II analyses is nearing completion. We design cognitive batteries for and collect, score and manage data from pharmacological trials seeking evidence of treatment-related changes in cognitive performance in schizophrenia patients and healthy controls. Recently, we have helped to develop the assessment and analysis strategy for a protocol examining an experimental agent (LX6171) and have worked on data analyses of information from tolcapone and modafinil treatment trials. We have completed an update of earlier meta-analyses of cognitive impairment in schizophrenia (Dickinson et al., 2007), showing that the cognitive impairment seen in people with schizophrenia has been consistent in magnitude and pattern over the past 30 years, and across different geographic regions around the world (i.e., North America, Europe and Asia). An early version of this work was presented at a conference and is accepted for publication as a chapter in an edited collection (Dickinson et al., in press), and a journal manuscript describing the final analysis is in preparation. Illness heterogeneity is a major challenge to the development of improved treatments in schizophrenia. As described in Cole et al. (2012), we have utilized latent class growth analysis (LCGA) in an effort to derive illness subtypes based on pre-morbid academic and social adjustment. We found evidence supporting three developmental trajectory subtypes: good/stable, insidious onset, and poor/deteriorating. These classes differed significantly in terms of age of onset, processing speed, and functioning after onset. The finding illustrates a potentially powerful methodology to attack heterogeneity challenges in schizophrenia research. Higher degrees of intra-individual variability (IIV) across neuropsychological tests have been recently linked to risk for schizophrenia, other clinical disorders, and normal aging. Cole et al (2011) is the first report showing decreasing IIV across cognitive domains in people with schizophrenia, their siblings and controls, respectively. Ongoing work is examining whether IIV might be useful as an intermediate phenotype. We have a large sample of unaffected siblings in the CBDB dataset, who, on average, share half their genetic code with an ill sibling, but do not share confounds, such as medication history and low motivation. In Wisner et al. (2011) we further characterized this sample, showing how sex and psychopathology history associate with sibling cognitive performance.


The mechanism of action and regulation of the alpha-ketoglutarate dioxygenase reaction are being investigated through the study of thymine 7-hydroxylase, pyrimidine deoxyribonucleoside 2'-hydroxylase and prolyl hydroxylase. Streptomyces antibioticus and the simple eukaryotes Neurospora crassa and Rhodotorula glutinis are serving as sources for enzyme purification. The intact cells of the above mentioned organisms, as well as of mammals, are being studied, especially in regard to the factors which regulate the activities of the alpha-ketoglutarate dioxygenases. The objective of this proposal extends to the elucidation of the regulation and physiological functions of the oxidative and reductive pathways of pyrimidine catabolism and to gaining some insight into whether prolyl hydroxylase itself has a regulatory role.


The overall objective of the proposed research is to determine the mechanism for increased ACTH secretion during pregnancy. This increase is necessary for normal maternal and fetal homeostasis, and occurs without symptoms associated with increased steroid action, such as hypertension. We have hypothesized that action of corticosteroids at mineralocorticoid receptors (MR) leads to reduced MR action in pregnancy, and that progesterone mediates this effect as a competitive antagonist at MR. The studies done thus far have supported this hypothesis, showing changes in hippocampal MR consistent with reduced activation and the presence of antagonist activity, during progesterone treatment or pregnancy. The proposed studies will extend these studies to test the following hypotheses: 1) Progesterone treatment in vivo blocks MR effects on 5HT1A expression in hippocampal neurons, resulting in increased plasma ACTH levels. The magnitude of the effects on both 5HT1A expression and ACTH is related to the levels of cortisol relative to progesterone. 2) Progesterone treatment in vivo has selective effects on neurons in hippocampal regions expressing mineralocorticoid receptors (MR) but not progesterone receptors (PR) (including CA1 and dentate gyrus). 3) The increase in ACTH caused by pregnancy or progesterone treatment is caused by an increase in serotonin effects in the brain; 4) The interaction between cortisol and progesterone in hippocampal cells in vitro involves competition at MR, producing opposing effects on 5HT1A mRNA and on the hyperpolarization response to 5HT (serotonin); and 5) Progesterone inhibits activation of MR, resulting in reduced MR binding to specific response elements, including those in the 5HT1A promoter. The first 3 aims will be studied in experiments in sheep. The sheep will be studied either after chronic progesterone treatment, ovariectomy or during pregnancy; MR binding and 5HT1A mRNA, as well as MR, GR and PR mRNA will be measured in hippocampus, hypothalamus and medulla. To test whether changes in 5HT1A occur in the same types of cells in hippocmapus as express MR (but not PR), mRNA will also be determined by in situ hybridization. To test the role of 5HT1A during pregnancy, the ability of a 5HT1A agonist to inhibit the ACTH levels in pregnancy and in progesterone-treated ewes will also be determined. The final 2 aims will be tested in cultures of hippocampal neurons. The effect of progesterone on MR binding, 5HT1A mRNA and electrical responses to a 5HT agonst will be tested; single cell PCR will also be used to determine if cells contain MR, but not PR. The action of progesterone at MR will also be tested by using MR-selective antagonist and agonist, and GR-PR antagonist. Finally, the ability of progesterone to inhibit MR activation will be tested in gel-shift and super-shift assays using specific response elements in the 5HT1A promoter, including a nGRE and the SP-1 site.


Understanding the genetic and environmental mechanisms that underlie the development of reading ability and disability has important implications for literacy education and the prevention of learning disabilities in reading. To that end, the goal of the proposed competing continuation of HD38075 is to conduct a systematic developmental genetic examination of reading comprehension. The Western Reserve Reading Project (HD38075 "Environmental Influences on Reading: A Twin Study") includes a representative sample of 350 same-sex twin pairs born between 1996 and 1998 who, by the proposed start date, will have been assessed longitudinally across four measurement occasions from kindergarten through third grade on a broad battery of measures of reading skills, oral language skills, cognitive skills, mathematics, and family environmental factors. The proposed continuation will extend testing via three additional annual home visits spanning middle childhood to early adolescence, with a focus on emerging reading comprehension skills. We propose to examine the univariate genetic and environmental influences upon reading comprehension, the covariance between reading comprehension and oral language skills, decoding skills, and other related skills, as well as the relationship between proximal and distal measures of the home and school environments that are associated with reading comprehension and related skills. In doing so, the proposed research will offer a unique opportunity to examine the genetic and environmental etiology of the longitudinal development of reading comprehension and related skills during the critical transition from when children are "learning to read" to when they are "reading to learn." Extending the unique resources of the Western Reserve Reading Project is an important opportunity for a highly cost-effective and powerful genetically-sensitive investigation of the etiology of reading ability within its various developmental, cognitive, behavioral, and environmental contexts. Developing a more informative model of how genes and environments work together to affect the development of reading comprehension has important implications fostering reading development as well as to detect, ameliorate and prevent reading problems.


The mechanism of cell division is studied, with special reference to cell cleavage in sea urchin eggs. A variety of experimental approaches are to be applied toward a deeper understanding of events which immediately precede the onset of cleavage furrowing in sea urchin eggs, because during this period the responsible mechanisms are established and begin to be expressed. In particular, the ultrastructure of the surface and of the cytoplasmic cortex will be examined and correlated with mechanical properties of the eggs which are explored by various physical determinations. In addition, living cells at critical times will be manipulated by physically damaging selected cell parts with a simple ultraviolet microbeam device.


The supplementation of vitamin D (vitD) during lactation has been poorly investigated: An expert panel convened by the CDC released a report stating that there are insufficient data to evaluate vitD supplementation requirements during lactation. A CDC study revealed a serious public health matter: 42.4% of African American women in their childbearing years exhibited hypovitaminosis D. Using new setpoints for hypovitaminosis D now renders >90% African American women affected. Nationally, there has been a steady and significant rise in nutritional rickets in breastfed (BF) infants, mainly in the African American population. Health organizations have recommended universal oral vitD supplementation of BF infants to diminish the risk of vitD deficiency in this group. Prolonged supplementation with 25X's the current DRI [10,000 lU/day) for up to 5 mos was shown to be safe in adult men and nonlactating women. Recent data suggests that a minimum of 6,400 lU/day vitDS is essential to prevent or reverse infantile hypovitD, particularly in darker pigmented BF infants. Due to compliance/dosing issues with infants, it is hypothesized that supplementation with higher dose maternal vitD will be safe and as effective as infant oral supplementation in the prevention of vitD deficiency in BF infants and will ameliorate vitD deficiency in lactating women, including women with darker pigmentation. The Aim of this proposal is to determine the effectiveness and safety of maternal and infant vitD supplementation (as a function of ethnicity and latitude) in the prevention of hypovitD in the BF dyad. Mothers from two study sites at different latitudes will be randomized to receive 1 of 3 treatment regimens of vitD3. Mothers, lactating or nonlactating controls will be randomized to either Group A: Standard tx (400 IU D3/d), Group B; 2,400 IU D3/day, or Group C: 6,400 IU D3/day. Infants of mothers randomized to Group A will receive 200 IU D3/d (recommended practice) and infants of mothers assigned to Groups B or C will receive placebo. By measuring an array of indicators, calcium homeostasis and skeletal remodeling in the postpartum mother and the breastfeeding infant will be monitored. Through this study, the prevalence of vitD deficiency in the BF dyad and the utility of maternal therapeutic intervention with vitD3 will be assessed. Lay Description: The Aim of this proposal is to determine the effectiveness and safety of maternal and infant vitamin D supplementation as a function of ethnicity and latitude in the prevention of vitamin D deficiency in the breastfeeding mother-infant pair. The findings of this study will generate important new information for health care professionals and policy makers with regard to vitamin D requirements and the potential benefit to both mother and infant. [unreadable] [unreadable] [unreadable]


Over the past five to ten years, magnetic resonance imaging (MRI) has become an important tool for medical diagnostic applications. Newer techniques are leading to the use of higher static magnetic fields; more rapidly switched gradient fields and higher radiofrequency (RF) magnetic fields. Although use of the increasingly stronger electromagnetic fields is causing concern about patient safety, to date, only simplified homogeneous spherical, cylindrical and disc models have been used to obtain induced current density distributions and rates of energy absorption (specific absorption rates or SARs). Since this knowledge is vitally important, particularly for the critical regions of the body, we will adapt the anatomically based modeling techniques that we have successfully used for electromagnetic dosimetry. We propose to use the previously tested highly efficient impedance method and its new generalization for higher frequencies to calculate internal induced current densities produced by gradient magnetic fields, and SARs for RF magnetic fields, of realistic polarizations and variations typical of present and planned MRI systems. We also plan to use the newly developed modified finite-difference time-domain method, which has previously been used successfully for a wide variety of RF electromagnetic exposure conditions. For the various calculations we will use the new high- resolution model of a human volunteer based on MRI scans (with resolutions of 1.875-3 mm) and its scaled versions where different scaling factors alphax, alphay, and alphaz on the order of 0.6-1.0 will be taken to obtain models of different heights and weights representative of human adults and children. This latter step is needed since induced current densities and SARs are strongly dependent on the size and shape of the body. The current densities and SARs thus calculated will be compared with the safety guidelines suggested by USFDA and NRPB (U.K.).


The career development plan described in this competing renewal application will allow the applicant to continue the development and expansion of his research career into new areas of investigation that complement current areas of expertise. The applicant has extensive training and experience in behavioral neuropharmacology and substance abuse with primary expertise in the neuropharmacology of CMS stimulants. During the previous funding period, the applicant developed in vivo microdialysis techniques in conscious monkeys trained to self-administer cocaine and related stimulants in order to determine neurochemical changes associated with the behavioral and reinforcing properties of abused stimulants. In addition, the development of positron emission tomography (PET) imaging techniques in conscious monkeys expanded further the applicant's ability to characterize and quantify the CMS effects of abused stimulants. More recent efforts have focused on the development of functional magnetic resonance imaging (fMRI) in conscious monkeys to document with high temporal and spatial resolution drug-induced changes in CMS activity. A major focus of the research plan will involve the pharmacological manipulation of monoamine and glutamate systems to assess potential changes in sensitivity to the CNS effects of cocaine. The studies proposed will provide an innovative preclinical model to identify useful pharmacotherapies for stimulant abuse. In addition to research activities, the applicant will commit effort to educational activities related to biomedical research and substance abuse. The host institution, Emory University, has a well-established program related to neuroscience and substance abuse research that is ideally suited to the career development plan. The University will provide the necessary resources and adequate time to ensure the development of the applicant as a productive, independent research scientist. The long-term commitment and support provided by continuation of the Independent Scientist Award will provide the opportunity to develop and integrate new skills and expertise into a unique multidisciplinary research program in substance abuse. [unreadable] [unreadable] [unreadable]


QUALITY OF DYING AND DEATH IN PERSONS WITH AIDS aims are to l) use qualitative interviews and draft questionnaire to develop and select items for an instrument administered to loved ones and health care providers after a patient's death from AIDS that can be used in evaluation of the quality of dying and death; 2) assess the psychometric properties (measurement model, internal consistency, discriminate validity, and convergent validity) and respondent burden of the instrument; 3) compare physician\casework evaluations with that of family and friends. Up to 3 loved ones and 3 health care workers will be interviewed following the deaths of up to 40 persons with AIDS to elicit domains and items that evaluate the quality of dying of death of the person with AIDS and to pilot existing questions. Respondents will be asked to describe last 7 days of person's life in detail. Content of final instrument to be determined by psychometric evaluation of existing items, new domains and items, and comparability among loved ones and health care workers.


This proposal describes the development of a Data Coordinating Center (DCC) at the University of Chicago. Progress in defining the pathogenesis of inflammatory bowel disease (IBD) through genetic approaches will be accelerated greatly through the establishment of an IBD Genetics Consortium. This proposal outlines a data coordinating center (DCC) at the University of Chicago. The overall objective of the IBD Genetics Consortium will be the identification of genes or genomic regions that are associated with increased risk of IBD and with specific phenotypic manifestations. Specific Aim I. To identify and implement IBD genetics projects which can most efficiently be completed through a Consortium structure. The specific objectives of the Consortium will be established through the Steering and Planning Committee and will reflect the nature of the studies proposed through individual Genetic Research Center (GRC) applications. It is expected that the DCC will play a key role in the planning, development and implementation of Consortium objectives. Potential joint issues include: mega-analysis of completed genomewide searches, stratified analysis of genome wide data based on established associations, support of positional gene identification efforts, candidate gene studies, and acquisition of data on clinical covariates, such as tobacco use, age of onset, and disease location. Specific Aim II. To perform the functions of a data coordinating center and genetic analysis center. Functional specifications of the DCC are described. In particular, careful development of a study protocol and Manual of Procedures will be the initial task of the Consortium, to be developed by the DCC with iterative input from the Steering and Planning Committee and individual GRCs. The use of both existing and novel methods of genetic analysis is proposed. Prior experiences in collaborative genomewide screens have been critical in advancing understanding of the challenges associated with pooling genomewide linkage data. These key concepts have been incorporated into the proposed mega-analysis. Methods to reduce genetic heterogeneity are proposed through stratified analyses based on established gene associations, locus-locus and gene-environment interactions. Specific Aim III. To collect common patient specimens and facilitate common genotyping requirements. The creation and maintenance of a DNA and cell line repository at the Coriell Institute containing appropriate patient and control samples is proposed. Provisions for common genotyping are described, if so requested by the Steering Committee or individual GRCs.


Long term objectives: To continue to offer a Master's degree in International Research Bioethics of the highest quality to developing country academics, researchers, health professionals, and members of non government organizations from countries in the Asia Pacific Region with the poorest match of ethics capability and research activity (with a special focus on China, Vietnam and Thailand, where increasing amounts of NIH- supported research are being conducted, and where we have existing institutional linkages). To thus foster the development of national capacity for the ethical design, review and analysis of both clinical and public health research in countries in the Asia Pacific Region, with a special focus on China, Vietnam and Thailand. Specific aims: i) Through the Master of International Research Bioethics, provide a comprehensive program consisting of 12 core units that encompass bioethics, research bioethics in an international setting, research methodology in international health, relevant law and practicums;ii) Continue to offer, evaluate, refine and improve the existing Master of International Research Bioethics;iii) Select a cohort of participants from the above-mentioned priority countries who have demonstrated capacity to benefit from the program and to take up leadership positions in research bioethics, including the provision of education in research ethics, in ethical review and in providing guidance to their institutions, governments and other relevant bodies in their home countries, to undertake the Master of International Research Bioethics;iv) Continue to provide ongoing support to participants subsequent to the program.


The proposed Administrative Core under the direction of Dr. Ellner (PI) and Mr. David Hom (Director) will take overall responsibility of management (fiscal, regulatory, operational), coordinating (operational, logistics), and oversight of all TBRU activities. The Administrative Core will assist proposed sites with study development (biostatistics, protocol, operating procedures), implementation and performance monitoring. The Core will also communicate and work with NIH program staff to monitor study progress and achieve proposed milestones. Lastly, the Administrative Core will work to leverage expertise across other TBRU programs with collaborative projects proposed by TBRU investigators. The Core will also operate a Data Management Center (DMC) to coordinate approved protocol and unit-wide scientific activities in concert with scientific decisions of the TBRU. The DMC will oversee the collection, storage, quality control, and evaluation of all study data. It will provide support and coordination for the statistical and data processing functions for proposed studies at all sites - clinical, animal, and translational. A detailed Management Plan will outline general activities, leadership structure, typical work-flow, chain of responsibility, review/approval processes, review committees and management activities across the TBRU members. Dr. Ellner has overall responsibility for the scientific, administrative, and fiscal activities of the TBRU and will provide vertical integration by directly interacting with leadership at each site. He will be ultimately responsible for scientific conduct and management of the TBRU.


Prostate cancer (PCa) is the second leading cause of cancer deaths in men in the U.S. with yearly treatment costs estimated to be near $8 billion. The increasing amount of research on bioactives and functional foods has fueled the development and marketing of foods and dietary supplements for prostate health, including both soy and tomato phytochemicals, and men may consume combinations of these products. However, it can't be assumed that combinations of supplements and functional foods will be more effective, and it is important to identify interactions that could alter the efficacy or safety of dietary interventions. The long range goal of the proposed research is to quantitatively define and understand the mechanisms by which consumption of soy or tomato products may reduce the risk of PCa. The objective of this application is to investigate the effects of soy germ and/or tomato powder containing diets on the progression of PCa in a transgenic adenocarcinoma of mouse prostate (TRAMP) model. In parallel, we will investigate the bioavailability of tomato carotenoids and soy isoflavones when tomato and soy germ are consumed together. To answer the questions raised in this proposal, diets containing 2% soy germ and/or 10% tomato powder will be fed to TRAMP mice prior to puberty and throughout progression of carcinogenesis. The specific aims for this proposal are 1) To investigate the effects of tomato and/or soy germ containing diets on progression of prostate carcinogenesis and identify their anticarcinogenic mechanisms of action in TRAMP mice 2) To measure bioavailability of tomato carotenoids and soy germ isoflavones in TRAMP mice. It is hypothesized that consumption of soy germ or tomato will reduce the incidence of poorly-differentiated carcinoma, and the combination of the two will not be as effective as when consumed individually. Potential anticarcinogenic mechanisms of the dietary interventions will be investigated through cancer pathway findway PCR arrays and immunohistochemistry. It is hypothesized that an interaction between soy germ and tomato alters bioactive bioavailability. Carotenoid bioaccumulation, serum lycopene metabolites, and urinary isoflavones will be measured to determine if combined consumption alters bioavailability of these bioactives and if the alteration correlates with how soy germ and/or tomato powder modulate prostate carcinogenesis. The proposed research is a unique approach to understand the efficacy of dietary interventions for PCa prevention. We will use whole foods rather than isolated bioactives and investigate a combination of foods to identify potential interactions. The research will benefit both consumers of supplements and the scientific community by providing a better understanding of the safety and efficacy of dietary interventions as alternative strategies for PCa prevention.


The increased availability and reduced cost of small desk-top computer has created increased interest in automating data acquisition and process or experiment control in areas where such things were not feasible before due to cost or complexity. With these changes also came a large increase in the use of the IEEE-488 GPIB by instrument makers. By combining the two, sophisticated instrumentation and data acquisition systems can be assembled quickly and inexpensively. The BEIB is continuing to develop the expertise to provide guidance and assistance to requirements where this approach provides the optimum solution. This capability is further asisted by the BEIB-SERP specifying the IEEE-488 interface on new equiment acquisitions whenever possible.


The affective behaviors symptomatic of the depressed adult are variously expressed in the parent role. How they are expressed by the mother and experienced and responded to by the child are the focus of this project. To be considered, also, is the fact that the child's affective expression, although a response to maternal interaction. The naturalistic laboratory paradigm (MH-02144) is the source of data. Measures of affect include: (a) a minute-by- minute rating of emotions and moods of mother and child, (b) a detailed accounting of physical touch and affection, and (c) an experimental intervention in which depressed affect is elicited from the mother in response to a standard stimulus and the subsequent mother-child interaction is recorded. Analyses are at various stages for these several sets of affective data. Among the findings are: The overall exposure of children to expressed negative mood or emotion of the mother is 17%, 11%, and 6% interaction time with unipolar, bipolar, and normal mothers, respectively. These mean percentages far underestimate the variability related to maternal diagnoses. There are extremes in the depressed groups in which 80 to 90% of the minutes are coded in negative affect. High frequencies on negative affect in the children (the outliers) tend also to be in the offspring of depressed mothers. Children of the unipolar mothers, more than other children, become involved in the mothers's sadness. In such interactions the young child's empathy for the mother and his/her caregiving responses appear to be exploited. Affect and touch were investigated in the interchange of mothers and their 2 to 3 year olds. Touch is initiated more often by the mother (75% of the occurrences) than the child. The major functions of mothers; touch is to monitor and regulate child behavior and to give affection. The young child's initiations of touch are predominantly to be physically close to (in contact with, leaning on) the mother


Human growth hormone has proven to be an extremely important and valuable therapeutic whose full potential has yet to be realized. Recombinant DNA technology has increased the supply of this drug through commercial production in bacteria, but the extremely high cost of this product has put it out of reach from many patients. A cheaper and more abundant source of this therapeutic would be its production in the milk of transgenic animals. In phase I of this proposal we intend to test the feasibility of this approach by producing human growth hormone in milk of transgenic mice.


Physical activity among youth has declined over several decades. Low levels of physical activity have been associated with increased risk of both obesity and certain types of cancers. It is imperative that this trend be halted and/or reversed. Current methods attempting to increase physical activity among youth do not appear to be effective at promoting increased physical activity among youth, suggesting that innovative approaches are needed. Barriers, such as lack of time and interest, are commonly cited reasons youth do not engage in more physical activity. Self efficacy, a critical mediator of behavior change and a common target of interventions, affects the likelihood one will attempt to overcome barriers. Procedures to increase self efficacy for physical activity among youth need to be developed, tested, and theoretically explicated. Information processing techniques have been used to increase cognitive effort, and, thereby, impact information storage and retrieval. Thus, interventions to effect change in physical activity among youth would appear to benefit from including an emphasis on physical activity self efficacy to overcome barriers in a manner likely to enhance information processing. Computerized programs offer interesting possibilities for systematically manipulating characteristics of materials (e.g., visual and verbal information channels) that would affect information processing. Dual Code Theory predicts that information presented in a manner that utilizes visual and verbal processing channels simultaneously will result in more information processing and, therefore, learning. The studies in this proposal examine the effect of presentation format and processing channel on both information processing and physical activity self efficacy. Computerized multi media programs will be developed using Dual Code Theory and administered to 11-15 year old youth. In Study 1, the effect of presentation format on information processing and physical activity self efficacy will be examined. In Study 2, a factorial design will be used to more closely examine the effect of processing channel (i.e., visual vs. verbal) on information processing and physical activity self efficacy in computerized programs. The significance of these studies is that they will contribute to our understanding of the role of presentation format and processing channel in computerized multi media programs promoting increased physical activity self efficacy among youth.


Presently, several clinical Phase III clinical trials are ongoing at the University of Puerto Rico and affiliated Institutions for the treatment of our cancer patients. We are acutely aware of the need for an early clinical trial program at our Institution. The goal of this pilot project is to establish a program of early phase clinical trials at the University of Puerto Rico. To establish this program, we are starting a collaboration with Dr. Daniel Sullivan of the H. Lee Moffitt Cancer Center (HLMCC). This collaboration will emphasize the development of Phase I and Phase III clinical trials, targeting our Puerto Rican population. In addition, we propose to develop basic science infrastructure that will support investigator-initiated studies. Via this collaboration, the HLMCC, will have access to a unique population of patients that have a higher prevalence of specific malignancies in Puerto Rico, such as, esophageal, cervical, penile, anal, breast, and liver cancers. We propose to achieve these goals through the following three specific aims. In specific aim 1, we will evaluate the facilities of UPR and PRCC to conduct early Phase I and Phase II clinical trials. We will identify the availability of the infrastructure to perform pharmacokinetic, pharmacodynamic, and molecular analyses. In specific aim 2; we will delineate a plan to establish an early phase clinical trials program within the PRCC. Clinical and basic investigators from the UPR and HLMCC will join efforts in the development of specific clinical trials. Finally, in specific aim 3; we will initiate clinical trials that are based on basic research data.


Studies performed in our laboratory implicate the Porphyromonas gingivalis HtpG stress protein, the prokaryotic homologue of Hsp90, in the etiology of periodontal disease. We have reported that elevated levels of anti-Hsp90 antibodies, concomitant with P. gingivalis colonization, are associated with periodontal health. Transcription of HtpG message was also found to be upregulated 7-10-fold in P. gingivalis obtained from diseased subgingival plaque. There is a precedence for Hsp90 homologues contributing to pathogenicity of other microorganisms. Immunity to a single Hsp90 epitope of Candida albicans has been demonstrated to confer protection against systemic candidiasis. Studies performed by our laboratory have revealed that P. gingivalis HtpG has a significant degree of homology with human Hsp90, but remains clearly distinct from other HtpG proteins due to its unique C-terminal region. We have found that HtpG is localized to P. gingivalis membranes and extracellular vesicles, and that it cross-reacts with other prokaryotic and eukaryotic Hsp90 homologues. Our findings suggest that HtpG is readily accessible to participate in host cellular invasion processes, as well as to interfere with normal host cell functions one P. gingivalis enters the host cytoplasmic compartment. Transfection of KB cells with the P. gingivalis htpG gene stimulates IL-8 production by these cells. This application proposes to extend our investigations into the role that molecular mimicry by HtpG plays in the pathogenicity of P. gingivalis. Previous studies of other pathogenic microorganisms which appear to use the Hsp90 homologue as a virulence factor have been purely descriptive. Our application is unique in that while will propose to evaluate the role of HtpG in adherence and invasion mechanisms, we also propose to elucidate novel pathogenic mechanism(s) by which microorganisms such as P. gingivalis utilize molecular mimicry to disrupt normal eukaryotic cell function(s). Since the most clearly defined eukaryotic Hsp90-mediated mechanisms involved signal transduction pathways, these will be the primary foci of our investigations. The hypothesis to be tested in this study is: 1) HtpG plays a role in adherence and invasion of host cells; and 2) once internalized, signal transduction mechanisms mediated by Hsp90/TRAP1 within eukaryotic cells are disrupted by the HtpG of P. gingivalis through molecular mimicry. This leads to disruption of normal inflammatory cytokine responses to microbial invasion by P. gingivalis and other oral microorganisms.


The initial aim of this project was to examine the relations between two assessment methodologies. The CBCL (Child Behavior Checklist) is a quantitative tool which scores behavior along a continuum within each of eight behavioral syndromes: Attention Problems, Aggressive Behavior, Delinquent Behavior, Conduct Problems, Oppositional Behavior, Withdrawn Behavior, Somatic Complaints, and Thought Problems. The DSM- IV ( Diagnostic and Statistical Manual) is a categorical system of conceptualizing emotional and behavioral problems as discrete disorders. For example, this phase of the study included the prediction that a deviant score on Attention Problems of the CBCL ( T-score > 67) would also indicate the presence of an Attention Deficit Hyperactivity Disorder according to the DSM. In fact, the data showed this particular relationship to be true 100% of the time.Currently, the data collection phase of the Vermont Family Genetic Study is underway. The primary hypothesis predicts siblings of children who are deviant on CBCL syndromes / meet a DSM diagnosis are at increased risk for the same emotional / behavioral problems than siblings of a control group.Potential participants are selected from a pool of patients seen either at the Center for Children Youth and Families or the Pediatric Clinic at the University Health Center, University of Vermont. Eligibility is determined by the availability of at least one biological parent, at least one full sibling between the ages of 6-18, age of the patient must be between 6-18, and deviance on the Attention Problems, Aggressive Behavior, both, or neither of these syndromes on the CBCL. Parents are contacted by telephone and invited to participate. The study consists of two parts: completing several questionnaires, and a two hour visit during which parents are interviewed in person about their children's behavior, and children are administered a brief battery of cognitive tests in order to rule out learning disabilities or developmental delay. Parents are given the option of coming in to the Clinical Research Center at Fletcher Allen Hospital or be provided with a home visit. Saliva for genetic testing is obtained at the time of the visit.Data has been collected in this manner since 07/96. To date 75 families have participated. The goal is to schedule and interview 4 families a week for at least another year.The main implication if this study is to develop more accurate and efficient diagnostic methodology to ensure early intervention of common childhood emotional and behavioral problems.


Histologic and ultrastructal studies of heart, lung, liver and kidney were made of mice treated with interleukin-2 singly and in combination with interleukin-1 and or interferon-alpha. The vascular leak syndrome induced by interleukin-2 was ameliorated by concomitent treatment with interleukin-1. This reduction in toxicity may be useful in the treatment of neoplasms with interleukin-2.


Calpain 3 (C3) is the muscle specific member of the calcium dependent protease family, mutations in which result in a form of muscular dystrophy called limb girdle muscular dystrophy type 2A (LGMD2A). In the previous funding period for this grant, we made good progress in understanding the biological function of C3. We showed that C3 is important for proper sarcomere structure, in vitro and in vivo. Based on our findings, we hypothesize that during normal development and muscle adaptation, C3, in conjunction with the ubiquitin/proteasome-system, displaces and removes myofibrillar proteins, and allows for the subsequent replacement by new proteins. Once sarcomeres are fully assembled they form such a dense compact entity that their subunits are protected from proteolytic action. C3's placement in the sarcomere, due to its interaction with titin, allows it accessibility to myofibrillar proteins that are to be turned over following damage or remodeling. We further showed that pathogenic LGMD2A mutations that do not affect C3s proteolytic capabilities can affect its ability to bind titin, and that titin serves as an important anchor for C3. Finally, we have shown that there is a generalized loss of protein turnover in C3KO muscles, leading to protein aggregates and a cellular stress response. The above observations have elucidated the biological function of C3 but have left unanswered questions. How does loss of C3's normal activity result in muscle disease? How does loss of anchorage to titin affect C3's normal cellular role? In our preliminary data, we show that C3 has cellular substrates that fall into two major categories that include myofibrillar proteins and mitochondrial proteins. This finding suggests that in addition to its role in myofibrillar protein turnover, C3 might be important in mitochondrial protein turnover. This hypothesis is supported by our preliminary data showing abnormal mitochondrial morphology and oxidative stress in C3 knock out mice. In this funding period, we will investigate the following aims: Aim 1: To characterize the biochemical consequences of disrupted calpain 3-titin interactions on calpain 3 activity and substrate accessibility. Aim 2: To characterize the biological consequences of disrupted calpain 3-titin interactions on muscle morphology and function. Aim 3: To characterize the mitochondrial defect and features of oxidative stress in calpain 3 KO muscles. [unreadable] [unreadable] [unreadable]


Core A takes care of the tissue processing needs for all projects. It is directly supervised by Dr. Pelayo Correa and his assistant Dr. M. Blanca Piazuelo. In Nashville our personnel receive all biopsies from Colombia. Biopsies for histopathology evaluation are fixed immediately in buffered zinc-formalin, embedded in paraffin, sectioned and stained with hematoxylin eosin and modified Warthin- Starry silver stain. Biopsies for frozen sections are received in OCT and frozen immediately. The laboratory performs all immunohistochemical stains, including Ki67, TUNEL assays, inducible nitric oxide synthase, nitrotyrosine and a variety of white blood cell markers. Fully trained and experienced personnel are available for all procedures.


Racial and ethnic health disparities abound, with decreased quality of health care in these underserved communities. The reasons for these health inequities are complex and multi-layered. As noted by the Institute of Medicine, there are historic and contemporary social inequities that have been embodied in these populations and in the health systems that care for them. In addition, racial biases of health care providers also may play a role in the unequal treatment of minority populations. Racial stereotyping and bias have been identified as factors in the delivery of cardiovascular disease care, pain management, and mental health care. Given the well-documented disparity in the receipt of total knee replacements between African-American and white patients with severe osteoarthritis, research is sorely needed to better understand what role racial stereotyping and bias plays in creating these inequities in the provision of this treatment. In the proposed research project, we will objectively measure physicians'racial stereotyping and bias and evaluate its association with those physicians'recommendations of patients for TKR. We also propose an innovative educational intervention aimed at reducing the effect of racial bias on clinical decision-making and will assess its efficacy. Findings from this exploratory study will be used to develop a larger, more long-term study of racial stereotyping and bias in clinical decision-making and methods for intervening to mitigate their effects. Specific Aim 1 will test whether physicians express explicit racial bias, using a Web-based survey instrument. Specific Aim 2 will test whether physicians show implicit racial bias, using Web-based Implicit Association Tests (IATs). Specific Aim 3 will evaluate whether the magnitude of implicit bias predicts physician recommendation of TKR for African-American and white patients with severe osteoarthritis. Specific Aim 4 will assess whether use of the Web-based IAT as an educational intervention decreases the effect of implicit racial bias on physician recommendation of TKR. Public Health Relevance: The proposed research project tests an intervention to help physicians manage and, perhaps, eliminate their racial bias. If this intervention proves effective, we will have a powerful tool to help reduce racial health disparities.


A new method was developed, based on measurements of the kinetics of cell activation, for determining whether or not the descending limb of biphasic dose response curve falls because of insufficient cross-linking.


The Department of Urology at the University of Oklahoma has a 7-year history of collaborative multicenter research on the correlation of biochemical and clinical features of interstitial cystitis and the testing of new drug therapies. Within the Department are strong programs in all areas of clinical urology, including oncology, andrology, female urology and urodynamics, the basic sciences, emphasizing immunology, molecular oncology and biology, and cellular disease markers. Strong links with epidemiology and pathology have allowed us to be a leader in several areas of research. We maintain an active specimen collection and storage system for urines and other specimens collected from interstitial cystitis patients using standardized, quality-controlled procedures and a patient database and have a followup mechanism for tracking patients. The Department has a comprehensive residency training program. An informal regional network of community and academic urologists has also been developed to help meet patient needs for improved diagnostic and clinical care and for support as well as to recruit patients for specialized studies and experimental treatment protocols. Routine care is provided by community urologists or other physicians while Dr. Roy provides certain specialized services and experimental treatment protocols. The Department has drawn its patient base predominately from Oklahoma and neighboring states, a population base of 32.2 million, containing an estimated 5-6,000 undiagnosed interstitial cystitis patients with an estimated yearly incidence of roughly 200 patients. The largest out-of-state component being from Texas. Patients have come from as far as Michigan and Montana. The University of Oklahoma Department of Urology clearly meets the criteria for a clinical center. In order to meet the overall programmatic objectives, our specific objectives for the overall collaborative program: (1). Expand and further formalize the current informal network of community and academic urologists in the region to ensure standardized collection of data and specimens as well as long-term followup and to aid collaborating urologists who are responsible for routine care of patients to provide the best care based upon the latest findings of the clinical centers. (2) Expand current database and sample collection and storage programs in conjunction with other centers. (3) Expand clinical services offered to interstitial cystitis patients to include bladder retraining and reference to a university pain clinic, both to provide better services to patients while facilitating patient participation in research studies. (4) Recruit 50 patients the first year and 75 per year thereafter. (5) Present an annual seminal for physicians and workshop for patients as a means of educating physicians and the public about interstitial cystitis and reaching patients.


Youth with a history of maltreatment and foster care placement are at risk for a host of mental health, behavioral, and social problems, resulting in adverse life-course outcomes of great public health significance. This R01 application seeks support for a 5-year efficacy trial of the Fostering Healthy Futures Program (FHF), a preventive intervention designed to promote prosocial development, and to reduce problem behaviors for youth in foster care. FHF is an innovative, culturally-sensitive and multi-component intervention for 9-11-year-old children who have been maltreated and placed in foster care. Through a 9- month intervention that includes screening assessments, one-on-one mentoring, and skills groups, FHF targets cognitive, social and behavioral domains in order to build competencies, improve mental health functioning and quality of life, and reduce problem behaviors and adverse life outcomes (e.g. arrests, school dropout, restrictive placements). Assessments with youth, caregivers and teachers will be conducted at baseline (pre-randomization), post-intervention, and 1-year follow-up. Data will also include child welfare, educational, and juvenile justice records. A randomized controlled pilot trial of FHF in one county has produced program manuals for the mentoring and skills group components, and evidence of program feasibility, with high recruitment and retention rates. The FHF pilot study has demonstrated positive preliminary effects on putative mediators, including youths'social functioning, attitudes, coping skills, behavioral regulation, and extracurricular involvement (cfe from .23-72). It has also demonstrated positive effects on distal outcomes, including problem behaviors, competencies, number of restrictive placements, and life satisfaction (cfe from .2S-.75). Finally, the pilot study has generated preliminary effect sizes that have enabled us to specify the sample size necessary for a full-scale efficacy trial. A multi-county randomized controlled trial of the FHF program with 256 youth will enable us to: 1) examine intervention effects on both proximal and distal outcomes, 2) examine potential moderators of the intervention, 3) conduct mediational analyses to identify the mechanisms by which the program may enhance outcomes, and 4) conduct within-group analyses. The goal is to design more efficacious interventions, thereby reducing disability, morbidity, and mortality, not only for youth in foster care, but for all high-risk youth.


The long term objective of this application is for the candidate to become an independent investigator studying promising new neuroprotective botanical treatments for multiple sclerosis (MS) in human subjects using advanced MRI imaging measures. Axonal loss is thought to be the cause of irreversible progression of disability in MS. None of the available therapies for MS directly protect axons. Epigallocatechin gallate (EGCG) is a potent antioxidant and is the main component contained in polyphenon E, an extract from the leaves of the green tea plant (Camellia sinensis). Animal studies done by our group indicate that EGCG protects axons in the spinal cord from injury in an animal model of MS, experimental autoimmune encephalitis. The overall objective of this application is to determine if polyphenon E is a safe neuroprotective therapy that warrants further study as a treatment to prevent the progression of disability in [unreadable] MS. To achieve this objective, a double-blind, controlled, two-arm parallel group clinical trial will be [unreadable] conducted; 46 subjects with MS will be treated for two years with either polyphenon E 400 mg twice a day or placebo. The first specific objective is to determine if there is evidence of a neuroprotective effect. To accomplish this objective the difference between the two groups on the rate of change in n-acetyl aspartate (NAA) will be determined. NAA was selected as the primary outcome because it is a selective marker for neurons and axons that reflects their number and metabolic activity and thus is optimal to show a neuroprotective effect. NAA levels will be measured using magnetic resonance spectroscopy at baseline, six months, a year and two years. Changes in magnetization transfer ratio and brain atrophy will be secondary outcomes. The second specific objective is to determine the safety of polyphenon E in subjects with MS. The subjects will be monitored for adverse events with laboratory exams and physical exams. The third specific objective is to determine if plasma levels of EGCG predict changes in the imaging markers or the occurrence of adverse events. Conducting this study will allow the candidate to obtain extensive training advanced magnetic resonance techniques and pharmacokinetics. Relevance to public health: MS is the second most common cause of neurological disability among young people. This study is relevant to public health because it is expected to result in a new treatment for MS that can prevent the progression of disability. [unreadable] [unreadable] [unreadable]


Background: Estrogen antagonists are widely employed in the treatment of breast cancer. They also comprise the most promising drugs for breast cancer chemo-prevention. Despite these facts, the molecular mechanisms by which these compounds inhibiting cellular proliferation are not fully defined. One of such compound, tamoxifen, leads to highly-significant decreases in the rates of both disease recurrence and death, but tamoxifen is limited by a less that 50 percent response rate in breast cancer prevention and the inevitable development of cellular resistance in breast cancer therapy. Our preliminary studies have demonstrated that depletion of prohibitin protein or introducing dominant negative forms of co-repressors of prohibitin overcomes 4-hydroxytamoxifen-induced growth suppression in breast cancer cells. A potential role for prohibitin gene in breast cancer has also been suggested in earlier studies, in which prohibitin was found to be mutated in some sporadic breast cancers. Objective/Hypothesis: I hypothesize that prohibitin and its co-repressors Brg-1/Brm play a role in estrogen antagonists-induced growth suppression of breast cancer, and that estrogen antagonists target the prohibitin/E2F pathway to affect breast cancer cell proliferation. The studies proposed will test and prove a critical and necessary role for prohibitin/Brm-Brg/E2F axis in the response of breast cancer cells to estrogen antagonists. Specific Aims: I. Determine the mechanism of estrogen antagonist-induced growth repression of breast cancer cells II. Determine the molecular level at which estrogen antagonists regulate prohibitin activity. Study Design: Northern blot or RT-PCR and immunoblot analysis will be carried out to assess the regulation of endogenous E2F responsive promoters by estrogen antagonists. The requirement for prohibitin/Brg-1/Brm in estrogen antagonist-mediated transcriptional repression of natural E2F-responsive promoters will be tested, using anti-sense and dominant-negative strategies. Chromatin immuno-precipitation assays (CHIP) will be employed to study the effect of estrogen antagonists on the interactions of prohibitin, Brg-1, and Brm with the natural E2F-responsive promoters. Relevance: A major problem facing breast cancer chemo-prevention is the low response rate. Estrogen antagonists treatment is further limited by the inevitable development of resistance. The lack of information about the molecular mechanisms of estrogen antagonists-mediated growth suppression prevents the development of strategies to overcome the problems. Our demonstration of the involvement of prohibitin in 4-HT induced growth arrest suggests that the prohibitin/E2F pathway is the/a cellular target for estrogen antagonists, and thereby also implicates prohibitin as a potentially important target for breast cancer therapy.


This project will investigate the ability of two or more potential metabolites of chloramphenicol to induce aplastic anemia in mice. The colony-forming cells (CFC) assay will be used to detect potential toxicity of these metabolites to the bone marrow. The carcinogenicity of the two compounds will also be investigated, as this action may relate to aplastic anemia. The hydroxylamine and hydroxamic acid metabolites will be synthesized by proven methods, then subjected to biological testing. A study of the in vivo metabolism of the compounds will also be performed. All chemical investigations will be conducted at Midwest Research Institute; the biological investigations will be performed at the University of Kansas School of Medicine.


Changes in gene expression patterns are a hallmark of the aging process. Important insight into the mechanisms controlling such gene expression programs has come from the study of replicative senescence of cultured cells (eg, human diploid fibroblasts), which recapitulates many features of cells from aging individuals. This Project has traditionally studied changes in RBP expression and function during replicative senescence. It has also examined the influence of RBPs in replicative senescence by interventions to elevate or reduce RBP levels, followed by the analysis of changes in senescence-associated mRNA expression patterns. We have studied if a given RBP binds a senescence-associated mRNA using a variety of in vitro binding assays (biotin pulldown, RNA EMSA, etc) and assays to measure binding of endogenous molecules ribonucleoprotein immunoprecipitation (RIP) or crosslinking IP (CLIP). To investigate RBP function during senescence, we employ approaches such as RBP silencing, RBP overexpression, mutant RBP analysis, and RBP-associated mRNA identification (using microarrays, RNAseq, and RT-qPCR). To investigate whether RBPs affect the stability of target mRNAs during senescence, we measure the steady-state levels and half-lives of the mRNAs of interest as a function of RBP abundance. We investigate whether RBPs affect the translation of target mRNAs by studying the relative association of the mRNA with translating polysomes and by quantifying the nascent translation rates of the encoded proteins. We also employ reporter constructs to gain additional insight into the processes modulated by RBPs and use various senescence-associated markers to examine changes in the senescence phenotype. Over the past 12 months, this Project has examined the changes in gene expression that occur in human tissues as part of physiologic aging. Much of our effort in this Project has been directed at understanding how RBPs and noncoding RNAs affect the process of cellular senescence, which is increasingly recognized as underlying age-related changes in tissue physiology and pathology. The studies in this Project examine the RBPs and ncRNAs that modulate cellular senescence and the consequences of their influence on the senescent phenotype. Among the cell systems used for these studies, human diploid fibroblasts have been particularly informative. Senescence-associated RBPs. Following a long-established line of research in our group, we have continued the characterization of several RBPs implicated in aspects of cellular senescence, including the loss of proliferation, the impaired ability to respond to stress, and the implementation of a senescence-associated secretory phenotype (SASP). We discovered that the RBP NF90, which lowers the stability and translation of several mRNAs Tominaga-Yamanaka et al., Impact Aging 2012, associated with many mRNAs encoding SASP factors such as IL-6, IL-8, osteoprotegerin, GM-CSF, MCP-1, HCC4, Gro1, and Gro2. In young, proliferating fibroblasts, NF90 was abundant and inhibited the expression of mRNAs encoding SASP factors; by contrast, NF90 was low in senescent cells, allowing the levels of SASP factors MCP-1, GROa, IL-6, and IL-8 to rise. In collaborative studies, we have continued to investigate the roles of RBPs HuR and AUF1 in senescence, finding that the decreased nuclear export of HuR mRNA by HuR was linked to the loss of HuR in replicative senescence, that the interaction of HuR and AUF1 with the 3UTR (untranslated region) of p16 mRNA was inhibited when NSun2 methylated the p16 3UTR, leading to a rise in p16 levels Zhang et al., Nat Commun. 2012, and that the senescence-associated decline in CARM1, which methylates and hence activates HuR, was associated with the loss of HuR function in senescent cells Pang et al., BMC Mol Biol 2013. Senescence-associated ncRNAs. During the past twelve months, we have also continued to investigate the influence of ncRNAs in senescence. We have reported a number of specific microRNAs differentially expressed in senescent cells. During this evaluation period, we have also contributed several reviews to this emerging field, covering the topics of microRNAs that influence senescence and tumor suppression, the regulation of microRNA biogenesis in senescence, and the global influence of microRNAs on gene expression patterns in senescence Abdelmohsen et al., Ageing Res Reviews 2012. We have also initiated efforts to investigate the role of long ncRNAs (lncRNA) in senescence. A recent high-throughput search using RNA-Seq in collaboration with Dr. Beckers core facility, revealed numerous lncRNAs that are differentially expressed during cellular senescence. Among these senescence-associated lncRNAs, termed SAL-RNAs Abdelmohsen et al., Impact Aging 2013, we identified several transcripts that played a direct role in cellular senescence. SAL-RNAs are the subject of many follow-up studies in our laboratory that will be reported in the near future. In addition, we collaborated with the Prasanth laboratory in reporting that the lncRNA MALAT1 promoted cell proliferation and that silencing MALAT1 triggered cellular senescence Tripathi et al., PLoS Genetics, 2013. Tissue aging. Although our understanding of the post-transcriptional factors that influence senescence is advancing quickly, we still know relatively little about the RBPs and ncRNAs that affect the aging process itself. We recently reported that the levels of several RBPs that regulate translation and stability (HuR, AUF1, TIA-1, and TTP) in human tissues revealed that these RBPs are abundantly expressed in most tissues in all age groups. Expression was widespread in all normal tissues, including nondividing, terminally differentiated tissues, suggesting that these RBPs play life-long physiologic roles in tissue homeostasis.


Marked improvement on the growth of mouse megakaryocytes was observed in cultures maintained in a liquid medium containing 20% horse serum in medium NCTC 135. Degeneration of megakaryocytes appeared less than before. The complete sequential events of maturation of megakaryocytes through the stages of megakaryoblast, promegakaryocyte, granular megakaryocyte, and demarcation membrane formation was observed regularly in all the eight experiments performed up to the present time. This is quite different from previous results in that good granular and demarcation membrane forming megakaryocytes were observed only occasionaly in some experiments. The number of megakaryocytes increased 3- to 5-fold in a period of 3-4 days. A few megakaryoblasts were observed in cultures as late as 15 days after cultivation.


This application, which deals with the effects of alcohol on the liver, requests the conversion of an individual research grant, which has been funded for 25 years to a Program Project. The four individual projects are concerned with (1) the effects of ethanol on signal transduction in intact hepatocytes, (2) mechanistic studies of the effects of ethanol on signal transduction in membrane systems, namely turkey erythrocytes and isolated liver plasma membranes, (3) the effects of ethanol on the interaction of liver cell membranes and extrinsic membrane-associated proteins, involved in signal transduction, and (4) the molecular characteristics of ethanol-hepatocyte membrane interactions. In these projects, we will study the effects of ethanol in vitro and of chronic ethanol feeding on membrane-associated parameters in the hepatocyte, including (but not restricted to) ligand-receptor coupled CAMP and phosphoinositide signal transduction systems, homologous and heterologous desensitization produced by ethanol, alterations in the interactions of phospholipase A2 and protein kinase C with liver cell membranes, and differential binding of ethanol in hepatocyte membranes in naive and chronically intoxicated animals, to distinguish specific from bulk effects. Techniques include ESR, NMR, time-resolved fluorimetry, digital imaging fluorescence microscopy, laser confocal microscopy, and preparative and analytical HPLC. The overall goal of the integrated biochemical and biophysical studies in this program project is a deeper insight into the mechanisms of ethanol-induced liver injury.


Trypanosoma brucei is a eukaryotic parasite that evades mammalian host immune response by antigenic variation: changing its Variant Surface Glycoprotein (vsG) ?coat? by alternatively expressing one among hundreds of highly diverged VSG genes in its genome Most VSG switches occur by the little-studied process of gene conversion. Here, a new strategy is proposed, replacing VSG ORFs with selectable markers, to investigate parameters affecting gene conversion of VSG cassettes. Drug selection will detect recombination of promoter-less markers with the active VSG, allowing isolation and study of rare gene conversion events. The hypothesis that recombination rates differ for internal versus telomeric VSGS will be evaluated by measuring gene conversion from markers placed at each type of site. Creating lines with latent promoters upstream of silent telomeric VSGs will detect otherwise ?silent? switches, which might shuffle and increase the availability of VSGS at ?preferred? chromosornal locations. The reporter constructs can be altered to test the role of conserved sequence motifs of VSG gene conversion cassettes for effects on the rates and order of VSG switching. The influence of chromosomal context and local sequence elements on recombination will be teased apart. A thorough understanding of VSG gene conversion will allow development of more accurate models to explain and predict patterns of antigenic variation. This work is highly relevant to host-pathogen interactions, especially in trypanosomes and malaria parasites.


The goal is to understand the essential hydrophobic and subunit interactions with the mitochondrial electron transport system that enable it to couple electron transport to ATP synthesis. Four specific projects will probe functionally important interactions within cytochrome c oxidase, which is the terminal electron transport complexes in the inner mitochondiral membrane. The first objective will be to determine the phospholipid specificity and the functionally important high affinity lipid binding sites on cytochrome c oxidase. The approaches to be used include measurement of functional binding of synthetic derivatives of cardiolipin that contain defined abnormal fatty acid tails, and photoaffinity labeling the cardiolipin binding sites with cardiolipin analogs. The second objective will be to determine the involvement of the tightly bound cardiolipin in dimerization of cytochrome c oxidase. The techniques to be used will utilize high speed sedimentation velocity to measure the aggregation state of the protein as a function of bound cardiolipin. The third goal is to structurally locate the redox centers within the protein relative to the membrane interior and one of the protein subunits, subunit III. The approach will be measurement of intrinsic protein fluorescence. The last goal will be a direct investigation of the structure of one of the subunits of cytochrome c oxidase, subunit III. The approaches in this project utilize detergent binding methods to measure surface hydrophobicity and hydrodynamic measurements to determine overall size and shape of the purified subunit. Together, these four approaches will probe the importance of specific lipids and subunits in the structure and function of this inner mitochondiral membrane protein.


There are two long-term objectives of the proposal. First, to understand in neuronal detail how simpler nervous systems are able to generate behaviors and how these nervous systems are able to accomplish simple forms of learning. The second is to understand what happens in cortex during epileptic seizures. In order to better accomplish these scientific goals, we also have the methodological goal of improving optical techniques for monitoring neuron activity. We have been using optical methods to monitor neuron activity in the Aplysia abdominal ganglion during the gill-withdrawal reflex. Our preliminary results suggest that between 250 and 420 neurons in the ganglion are activated by a mechanical stimulus to the siphon. However this estimate comes from a recording that is perhaps 35% complete. We propose experiments to improve the completeness of the recording, experiments to allow preliminary identification of many of these neurons, and analyses to allow us to follow the activity of individual neurons during a series of trials involving habituation and sensitization. We also plan experiments to try to see if the same behavioral plasticity can be achieved with fewer neurons. If this is successful we propose to make a model of the neuron interactions that generate the behavior. We have been able to monitor activity in bicuculline induced inter-ictal epileptiform discharges in rat somatosensory cortex. Our results were surprising in two regards. First, they indicated that the optical measurements and the ball electrode measurements could give very disparate results. Second, they indicated that there were qualitative differences between discharges induced by sensory stimulation and spontaneous discharges. Our first aim is to confirm these result. Then we plan a local application of bicuculline to study the spread (or absence of spread) of the epileptic activity away from the treated region. Finally we plan a simultaneous measurement from both the bicuculline treated and the untreated contralateral somatosensory cortex. These experiments are meant to lead toward measurements in more sophisticated models (primates) of the human disease. These experiment may also lead to the use of optical monitoring techniques for determining the location of epileptic foci in surgical patients.


The long term goal of this proposal is to identify mechanisms responsible for the aging associated rise in coronary smooth muscle excitability, with emphasis on the role of K channels. The main hypothesis postulates that KCa channels, as key players of coronary arterial tone, are regulated by potent vasoactive substances and that their expression and/or modulation may decrease with aging. Thus, the capacity of KCa channels to maintain an optimal arterial tone is diminished in the elderly. This hypothesis will be tested by studying in coronary arteries of young-adult, mature and senescent subjects the abundance of KCa channels, their molecular constituents (alpha +/- beta subunits), response to vasoactive substances and mechanism(s) of regulation. KCa channels are abundant in vascular smooth muscle of various species. We and others have shown that KCa channels set the level of smooth muscle contractility in cerebral arteries and myometrium. Our preliminary data indicate that: 1) KCa channels are abundant in human and rat coronary arteries and are positively regulated by a beta subunit; 2) they are modulated by potent vasoactive metabolites like thromboxane A2 (TXA2) and nitric oxide (NO); 3) aging of coronary smooth muscle is associated with a downregulation of KCa channel expression and function; 4) they play a critical role in the control of coronary arterial tone. The questions that we want to answer are: 1) is regulation of coronary arterial tone by KCa channels affected by aging? 2) does the functional expression and/or characteristics of KCa channels change in the aging process? 3) are the alpha and beta subunits of KCa channels equally expressed or distributed at different ages? 4) is the modulation of KCa channels by potent vasoactive substances (e.g. TXA2 or NO) affected by aging? We will use a combined electrophysiological, biochemical, immunochemical, and mechanical approach. The specific aims are to compare in young, mature and senescent subjects (rats and humans): 1) the contractile responses to KCa channel blockers and agonists; 2) the channel functional expression, biophysical properties, and pharmacology; 3) the relative abundance of the pore-forming alpha and regulatory beta subunits; and 4) the channel response to vasoactive substances (e.g. TXA2, NO) and their mechanism(s) of modulation. This study will aid our understanding of age-related changes of coronary vasoreactivity, and possibly in the design of clinical treatments that reduce coronary spasm and heart dysfunction.


Project Summary/Abstract Age-onset diseases including cancer, neurodegenerative disease, diabetes, cardiovascular disease, stroke, and osteoporosis are generating a public health burden that is quickly becoming insurmountable. Exacerbating this problem, co-morbidities are common among the elderly. The ideal therapeutic strategy to confront this crisis is to target a unifying risk factor, but patient age is the only risk factor common to all these diseases. Fortunately, it is increasingly clear that the biological processes of aging are malleable, thus the rate and quality of aging may be improved. Genetic and nutritional interventions causing real or perceived energy- depletion are robust, conserved mechanisms to promote healthier aging. Unfortunately these interventions, e.g. activation of the molecular low-energy sensor AMPK, also carry clinically unacceptable side effects, such as suppressed immunity and fertility. Translating these findings into therapeutics thus requires identification of downstream mechanisms that are sufficient for healthier aging. Through a genetic model of longevity in C. elegans via activation of AMPK, we demonstrated that the negative side-effects of energy-depletion can be uncoupled from the positive effects on healthy aging. Using unbiased, systems-level approaches, we found that the longevity-specific mechanism involves downstream regulation of mitochondrial dynamics and metabolic functions, and we now demonstrate that regulation of mitochondrial dynamics is causal to AMPK longevity. New data indicate that perturbing the unfolded protein response (UPR), which mediates homeostasis of the endoplasmic reticulum (ER), interacts with the AMPK pathway to extend lifespan through a mechanism that also requires mitochondrial remodeling. Given recent studies showing that the ER physically interacts with mitochondria to regulate organelle morphology and metabolic signaling, these data suggest a new paradigm in aging: ER/mitochondrial regulation of longevity occurs through an integrated metabolic mechanism. Physical changes in mitochondrial networks are a hallmark of aging, but how organelle dynamics are mechanistically involved in longevity is unknown. By genetically inactivating mediators of mitochondrial remodeling (fission/fusion), Aim 1 will define how mitochondrial dynamics drive the changes in mitochondrial metabolism associated with low-energy longevity. Through novel transgenics and training in high-resolution microscopy in Aim 2, I will test the hypothesis that UPRER perturbations promote altered mitochondrial morphology and signaling between organelles. Finally in the R00 phase, Aim 3 will build on the tools and insights developed in Aims 1 and 2 to identify genetic mechanisms in C. elegans by which ER-mitochondrial inter-organelle communication can be directly targeted to extend healthy lifespan and protect metabolic homeostasis. Taken together, the goal of this proposal is to identify how evolutionarily conserved energy-sensing pathways coordinately modulate inter-organelle signaling and metabolic function to promote longevity.


Puerto Rican children in the United States experience the highest asthma prevalence and morbidity rates of all racial/ethnic groups and these rates are increasing. Factors such as biological differences in severity and sensitivity, environmental conditions, access to care, and socioeconomic status contribute to, but do not fully explain, these prevalence and morbidity rates. Inadequate self-management behaviors, which are potentially modifiable, also contribute to these asthma morbidity outcomes. This exploratory R21 application will develop and test an intervention to improve asthma self-management for this high-risk population. The intervention is unique in that the education and support provided to all participants from the standardized Core Curriculum can be modified using a Toolkit based on individual needs and strengths. The education incorporates specific skills training in environmental modification, problem solving, self-monitoring, and enlisting social support. Specific Aim 1 is to explore influences of asthma self-management behaviors (including asthma belief models, the use of folk remedies, social support, stress, access to care, and socio-economic status) for Puerto Rican parents of children with asthma. Specific Aim 2 will incorporate these influences into a community health worker asthma intervention and use a small behavioral randomized controlled trial to test the ability of this intervention to improve asthma self-management behaviors for parents of Puerto Rican children with asthma. The long-term goal is to apply these pilot data in the design and implementation of a larger behavioral randomized controlled trial that will test a similar asthma intervention aimed at improving asthma control. The investigators in this application have significant experience with community agencies serving the target population and have expanded these relationships to prepare for this application using the principles of community-based participatory research. A prominent community service agency and a local community college contributed to the design of this application and have committed their services in the study implementation and results dissemination. The data generated from this research will improve our understanding of the specific influences of asthma self-management for Puerto Rican parents of children with asthma and the study will provide important pilot data on an intervention that may ultimately improve asthma control for this high-risk population that suffers disproportionately from asthma. The participatory design serves to build community capacity for research and improve research quality and effectiveness. PUBLIC HEALTH RELEVANCE: The data generated from this research will improve our understanding of the specific influences of asthma self-management for Puerto Rican parents of children with asthma and the study will provide important pilot data on an intervention that may ultimately improve asthma control for this high-risk population that suffers disproportionately from asthma. The participatory design serves to build community capacity for research and improve research quality and effectiveness.


Senior citizens typically fail to follow their prescribed medication regimens. Separate studies report that adherence to long-term therapy for chronic illness is less than 50 percent. Failure to take medicines as prescribed significantly increases the likelihood of negative health outcomes. For example, studies suggest that 11 percent of elderly hospital admissions and 24 percent of nursing home admissions are due to medication non-adherence. Our application is to develop an image enabled intervention system whereby a remote informal caregiver (e.g., a family member) can collaborate with a senior citizen living at home to improve medication adherence. A significant innovation is changing the self-medication process model into a real time collaborative one. Today's solutions require physical presence for objective verification and do not encourage dialog related to the root causes of non adherence. Starting with the information on the labels of the patient's Rx bottles, the proposed approach allows a remote participant to literally see that the prescribed medications are properly organized and that they have been removed from the organizer according to the desired schedule. There are a plethora of pill organizers and reminder systems on the market today. The proposed Collaborative Medication Adherence System builds upon perhaps their simplest form (i.e. the $5.00, 7 day compartmented pill box) and adds the dimension of collaboration. A key component of the system are "MediCam" devices that apply existing low cost video conferencing technology in a novel and highly constrained manner to address privacy and ease- of-use concerns. The associated software captures an image history and presents reports that encourage the collaborators to discuss how to improve adherence. T I Works Inc. will collaborate with Oregon Health and Science University (OHSU)'s Oregon Roybal Center for Aging and Technology (ORACTECH) to accelerate the work. This will build upon their expertise, processes, Health Coaching Platform infrastructure, and patient population. To verify feasibility, experiments will be conducted with a small number of home- resident senior citizens that are already participants in ORCATECH's health coaching project. This new dimension of intervention is likely to have both adherence and socialization benefits. Further, the approach has the potential to delay the senior from being institutionalized, saving up to $70,000 annually;because there is high confidence in their medication adherence. These benefits would be compelling motivators for commercial products and services. PUBLIC HEALTH RELEVANCE: Poor adherence to medication regimens by senior citizens is wide spread, costly, and increases the likelihood of negative health outcomes ( e.g. worsening of condition, relapse etc.). The research proposed is relevant to public health because it has the potential to improve the wellness and delay institutionalization of senior citizens. The approach can be implemented at low cost because it uses existing technology and engages a previously untapped resource, namely remote relatives and friends. The vision is that "aging in place" elders can extend their ability to live independently and safely at home, because there is high confidence in their medication adherence.


We plan to study the environment of divalent metal ions responsible for activating enzymes. We will attempt to identify the nature of the metal's ligands, the liganding geometry and the effects of substrates; inhibitors and allosteric cofactors on the nature of the metal-protein interaction. We will then try to interpret these features in terms of the metal's catalytic role. A systemic study of enzymatically active metalloproteins substituted with a number of transition metal ions will be made. We will take advantage of the fact that contributions of metal's d electrons to absorption, circular dichroism, and magnetic resonance spectra are very sensitive to the nature of the metal-enzyme interaction. We will also make kinetic studies, to determine the catalytic properties of the substituted metalloenzymes we will be using.


Synthetic methods for indole derivatives are being investigated. Of particular interest are methods for obtaining indoles with medium sized (7-10 atoms) C-rings which also contain nitrogen and other functional groups. The eventual goal is to couple such compounds with the alkaloid vindoline to provide semi-synthetic analogs of the anti-neoplastic alkaloids vincristine and vinblastine.


Inhibition of the enzyme NQO1 in pancreatic tumor cells using the non-specific inhibitor dicoumarol resulted in increased intracellular superoxide, inhibition of cell growth and inhibition of the in-vitro malignant phenotype of cells. The hypothesis proposed was that inhibition of NQO1 led to increased superoxide levels which inhibited pancreatic cancer cell growth and the in-vitro malignant phenotype. We have recently shown that NQO1 can directly scavenge superoxide providing both a potential approach and a mechanism of action for the use of NQO1 inhibitors in the therapy of pancreatic cancer. In this proposal, we will test the hypothesis that specific mechanism-based inhibitors of NQO1 are effective compounds for the therapy of pancreatic tumors both in-vitro and in-vivo. One of these inhibitors, the indolequinone ES936, is a potent inhibitor of pancreatic cancer cell growth and the in-vitro malignant phenotype. We will therefore test the hypothesis that ES936 can be employed as an effective therapeutic agent in pancreatic cancer and extend in-vitro data to in-vivo xenograft and orthotopic pancreatic tumor models. We will define the mechanism of action of ES936 and will attempt to dissociate NQO1 inhibition from effects on pancreatic cancer cell growth and the in-vitro malignant phenotype using both chemical/pharmacological and genetic approaches. Whether the effect of ES936 is due to increased levels of superoxide as a result of NQO1 inhibition will be characterized and downstream effects of ES936 on modulation of the cell cycle and apoptosis will be determined both in-vitro and in-vivo. This approach represents a novel therapeutic strategy for the treatment of pancreatic cancer, a disease where therapeutic options are very limited and where chemotherapy has made minimal impact.


The interplay of circadian timing and metabolic physiology represents a new frontier in biomedical research. Research on weight gain in humans has focused primarily on macronutrient intake and energy expenditure, the latter predominantly through physical activity, to understand and treat overweight and obesity. Emerging evidence from animal models indicates that circadian physiology impacts weight gain, including the observation of obesity in clock gene mutants and most recently the finding that food intake restricted to the habitual sleep time of mice leads to weight gain as compared to the same amount of food intake during the normal wake episode. We propose to determine if findings from these mouse models translate to humans. To do so, we have designed an exploratory Clinical and Translational Research Center (CTRC) study that uses whole room calorimetry to determine how restricting the majority of food intake to the biological night impacts energy metabolism in humans. Eating at night is common in work schedules with long work hours and with work operations during the nighttime hours (e.g., health care, emergency response, security personnel) and in circadian sleep disorders including, but not limited to, shift work and jet lag disorder. Night eating disorders are also newly recognized conditions that may contribute to obesity. The project addresses several themes outlined in NIH PA NUMBER: 09-124 Exploratory/Developmental Clinical Research Grants in Obesity by testing the following specific hypothesis: i) circadian misalignment will lead to reduced fat oxidation-a physiological state that could contribute to weight gain. This research effort is responsive to PA-09-124 as it will assess how a behavioral phenotype of eating at night increases risk for obesity and will contribute to identification of this behavior as a modifiable determinant of overweight and obesity.


We propose to extend the recently developed Medical Imaging Informatics Bench to Bedside (mi2b2) Workbench software to make medical images collected during routine clinical care available to clinical translational investigators. Today, this gold mine of patient information is not easily available. Well characterized medical images from clinical repositories would be an extremely valuable resource for clinical translational investigators who have specific ideas that require testing in large sets of images from many patients with different diseases. Our hospitals are international leaders in the development and deployment of advanced biomedical imaging technologies (MRI, high speed CT, ultrasound, PET and others) for clinical practice. The Picture Archive and Communication Systems (PACS) within each of the Departments of Radiology in our participating hospitals (Massachusetts General Hospital, Brigham and Women's Hospital, and Children's Hospital Boston) contain a wealth of medical images that often equal the quality of available clinical research imaging data, and greatly exceed its volume in terms of the number of patients and disease types. Though immediately extensible to other research applications and diagnostic scenarios, we apply our approach first to the difficult task of interpreting pediatric brain magnetc resonance images (MRI). The proposed Pediatric MRI module and Harvard Catalyst Radiological Decision Support toolkit that host it are targeted to address the immediate clinical challenge of understanding normal because normal is a constantly moving target, changing rapidly with brain development. These free and open source software tools are natural extensions of currently funded government projects. This includes software from two National Centers for Biomedical Computing; Informatics for Integrating Biology & the Bedside (i2b2) which provides tools that extract and integrate data in a secure, HIPAA compliant manner from electronic medical records, laboratory data, billing information systems and the National Alliance for Medical Image Computing (NAMIC), which provides tools for medical image analysis including registration, visualization and computation of individual subject and large cohort data sets and will form the foundation for the Harvard Catalyst Radiological Decision Support Toolkit.


Specification of the most effective evaluations and exclusion of specific disorders of intermediary metabolism are being sought by testing analyses and performing metabolic challenges. (This is an ongoing study.)


Description (taken from application): The purpose of this program is to facilitate the exchange of information between investigators who have research interests in the areas of diabetes and hormone action. The Enrichment Core has utilized six approaches to achieve this goal: 1) guest speakers program, 2) yearly symposia, 3) workshops, 4) individual research data clubs, 5) insulin-IGF research group meetings and 6) signal transduction research group meetings. This format has proven to be highly successful as it involves all members of the DERC at several levels of information interchange.


Rotaviruses have been studied extensively in many parts of the world predominantly by cross-sectional approaches. Such studies have yielded essentially "numerator" data which indicated that rotaviruses are a major cause of diarrheal illness in infants and young children. There has been a paucity of longitudinal viral gastroenteritis studies that yield not only important "denominator" data but also valuable insights into the natural history of a pathogen or illness, with special emphasis on epidemiologic, immunologic and laboratory parameters. We, therefore, initiated an intensive examination of anal swab and serum specimens obtained during a previous LID long-term longitudinal study (1955-1969) at Junior Village, a welfare institution for homeless, but otherwise normal children. Our aim is to investigate the natural history of rotavirus infections in such a longitudinal setting employing recently developed techniques such as serotyping of rotaviruses with VP7 specific monoclonal antibodies, determining the epitope-specific serologic responses in sequential sera to determine the scope of homotypic and heterotypic responses in order to gain insight into the parameters influencing susceptibility or resistance to rotavirus disease. In addition with the availability of rotavirus strains obtained over 20 years ago, it is planned to compare such strains with current isolates at the genetic level to determine whether significant differences had occurred and the effect of such differences, if found, on resistance to illness.