Project Summary Every year, a substantial number of children injure a growth plate, a cartilaginous region found at the end of all long bones in children that provides signals for the bones to lengthen. The growth plate is the most fragile structure in a child?s developing bones, making it prone to injury. Damaged cartilage within the growth plate is often replaced by unwanted bone, forming a ?bony bar?, which can lead to angular deformities or halt bone growth completely. Current surgical methods to correct bone growth defects are invasive, prone to infections and have low success rates. There is no current treatment that leads to complete repair of an injured growth plate. Innovative treatment strategies that prevent bony bar formation, restore functional growth plate cartilage, and permit normal longitudinal bone growth in affected individuals are greatly needed. This proposal seeks to develop an injectable hydrogel biomaterial system that could prevent bony bar formation and promote the formation of cartilage tissue, and which could ultimately be examined for its ability to heal growth plate injuries in children. It has been shown that mesenchymal stem cells (MSCs) infiltrate the injured growth plate and undergo osteogenic differentiation. Here, two important areas for inhibiting the osteogenesis of MSCs via a biomaterial system will be studied: (1) the role of biopolymer hydrogel substrate mechanics in preventing osteogenesis, and (2) the delivery of short interfering RNA (siRNA) from biopolymer hydrogels that can block osteogenic differentiation. We hypothesize that hydrogel systems that are less stiff and that provide sustained exposure of MSCs to p38 MAPK siRNA will inhibit osteogenesis. This proposal seeks to engineer hydrogel systems with these characteristics as a first step towards creating new technologies for helping to heal growth plate injuries in children. This will be accomplished as by two Aims: Aim 1 - To engineer a hydrogel system with mechanical cues that prevent osteogenic differentiation of MSCs. Aim 2 - To design a hydrogel that would provide sustained release of siRNA targeting p38 MAPK to prevent osteogenesis of MSCs. The p38 MAPK pathway has been linked to osteogenesis in various cell types, including MSCs. Local inhibition of this pathway by siRNA could prevent MSC osteogenesis from occurring after growth plate injury. For both Aims, these studies will be conducted in vitro and also in vivo in a rat growth plate injury model. This project is a first step towards successful development of a biomaterial system that can prevent MSC osteogenesis, which could ultimately aid in growth plate tissue repair.


The inhibitors of DNA or differentiation (Id proteins), members of the helix-loop-helix transcription factor family, act in a dominant negative fashion to control cell cycle and differentiation by dimerizing and inhibiting the function of the basic helix-loop helix (bHLH) transcription factors. We hypothesize that removal of 3 out of 4 copies of the Id1 and Id3 genes, and the resultant deficiency of Id, disrupts normal bHLH transcription factor regulation, thus allowing the formation of normally regulated or restricted protein-DNA complexes. The deregulation of bHLH transcription factors may result in the inhibited tumor-associated angiogenesis phenotype previously described in these mice. Therefore, the major goal of this study is to identify and characterize the protein-DNA complexes and the interactions that occur in spontaneous tumors as a result of the deficiency in Id and to determine changes in gene expression. To perform these studies, Id 3 out of 4 copies knocked out mice (Id1 +/ Id3-/-) will be crossbred with the p53-/- murine tumor model. The resulting mice will be a source of tissue that will be examined histologically and on a molecular and biochemical basis to identify novel interactions and gene expression that the absence of Id allows. These studies will help us understand the role of Ids in regulating tumor-associated angiogenesis and may identify new targets for use in blocking tumor vascularization and designing more specific anti-angiogenic drugs.


Hypertensive kidney disease commonly progresses. The primary objective of the AASK (African American Study of Kidney Disease and Hypertension) Cohort Study is to determine prospectively the course of kidney function and risk factors for kidney disease progression in African-Americans with hypertensive kidney disease who receive recommended antihypertensive therapy. A secondary objective is to determine the occurrence of cardiovascular disease and assess its risk factors. The AASK Cohort Study is a prospective, observational study that is an extension of the AASK trial. The AASK trial tested the effects on kidney function of 3 medications used as initial antihypertensive therapy (ramipril, metoprolol and amiodipine) and 2 levels of blood pressure control. Of the 1,094 trial participants, approximately 650 to 700 individuals who have not reached end stage renal disease (ESRD) will likely enroll in the Cohort Study. Risk factors to be studied include environmental, genetic, physiologic, and socio-economic variables. The primary renal outcome is a composite clinical outcome defined by doubling of serum creatinine, ESRD, or death. Medication treatment for hypertension, beginning with the angiotensin converting enzyme inhibitor ramipril, is offered to all participants. In this fashion, the study directly controls two of the major determinants of kidney disease progression (treatment of hypertension and use of reno-protective, antihypertensive medication). The minimum duration of follow-up in the Cohort Study is 5 years (total of 9 to 12 years, including the period of the AASK trial). Ultimately, data from the AASK Cohort Study should enhance our understanding of the determine the progression of kidney disease. Such results might eventually lead to new ESRD.


Prevalence of HIV-associated neurocognitive disorders (HAND) remains high despite the introduction of combined antiretroviral therapy. HAND is associated with elevation of pro-inflammatory factors in blood and subsets of activated infected monocytes, both shown to cause blood brain barrier (BBB) impairment that contributes to HAND. Therapeutic strategies that disrupt monocyte migration can slow progression of HIV infection and BBB injury, thereby ameliorating HAND. During the previous period of funding, we focused on studies of (1) molecular mechanisms of BBB injury and (2) anti-inflammatory and barrier protective properties of glycogen synthase kinase (GSK) 3? inhibition in neuroinflammation driven by HIV infection. We uncovered molecular mechanisms of BBB dysfunction [tight junction (TJ) phosphorylation, CD40/CD40 ligand interactions at BBB and signaling events behind the direct effects of HIV on brain endothelium]. We demonstrated barrier tightening following GSK3? suppression in brain endothelium due to TJ stabilization. We uncovered potent anti-inflammatory effects of GSK3? inhibition in brain endothelium (suppression of monocyte migration, diminution of inflammatory factor production, and BBB protection) in vitro and in vivo. GSK? inhibition in monocytes attenuated their adhesion/migration across the BBB, down regulated active integrin expression via suppression of the small GTPase, Rac1, and protected the BBB. Yet, BBB shielding properties or inhibition of monocyte migration/adhesion were not fully attained in vitro or in vivo, suggesting that additional pathways complimentary to GSK3? are necessary for the restitution of BBB function. In search of such molecules, we turned our attention to poly(ADP-ribose) polymerase-1 (PARP-1) and its inhibitors, recently recognized as anti-inflammatory/immune modulating agents with significant neuroprotective properties. Based upon preliminary data, we propose that PARP inhibition will attenuate BBB injury caused by HIV-1 via effects on monocytes, brain endothelium, activated microglia and HIV-1 infected macrophages. Indeed, preliminary data indicate that PARP suppression in primary human brain microvascular endothelial cells (BMVEC) improved BBB integrity and augmented expression of TJ proteins. PARP inhibitors prevented barrier disruption caused by inflammatory factors, diminished monocyte adhesion/migration across a BBB model, down regulated adhesion molecules/pro-inflammatory molecules and decreased activity of RhoA/Rac1. In monocytes, PARP inhibitors down regulated the active ?-integrin that paralleled RhoA/Rac1 suppression. PARP inhibitors decreased expression of pro-inflammatory molecules and diminished HIV replication in human macrophages. Although the modulatory effects of PARP inhibitors on immune cells have been studied to some extent, nothing is known about their effects in the setting of HIV CNS infection. PARP inhibitors have now reached the stage of clinical testing for cancer treatment, assuring quick translation to therapy of immune/inflammatory disorders.


This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. This project looks to expand the research that outlines the developmental trajectories of young adult and alcohol use and related outcomes. It also seeks to examine the effects of genetic and non-genetic factors on these trajectories.


Freeze-fracture electron microscopy has revealed organized sites of aggregated intramembranous particles in granular cell luminal membranes from toad urinary bladder after vasopressin (ADH) treatment. This response appears to be a primary hormonal effect which is reversible and quntitatively and specifically related to induced alterations in membrane water permeability. The proposed research is to investigate the aggregation response in terms of onset-offset mechanisms, to determine wether sites of aggregation are actual sites for water passage, and to determine the applicability of this to the mammalian kidney.


PROJECT SUMMARY Although genome-wide association studies (GWAS) have been extremely successful in identifying numerous germline variants associated to risk for schizophrenia and bipolar disorder, at the vast majority of these loci, the causal mechanism between genetic variation and disease risk remains unknown. This limits the development of novel drug targets and/or personalized treatments. Schizophrenia and bipolar disorder share many genetic risk loci thus motivating approaches to gain insights into the shared molecular basis of these two diseases. Post-GWAS studies are experiencing a ?big data? revolution driven by the exponentially decreasing costs of high-throughput genomic assays, including transcriptome levels, epigenetic modifications, and localization of tissue-specific regulatory sites, which are being collected in increasingly large cohorts of individuals. Here we propose a rigorous framework aimed at loci where shared or disease-specific risk for schizophrenia and bipolar disorder is mediated through alteration in gene expression levels, regulated via epigenetic control. To increase power for discovery while also facilitating validation of new findings, we will generate new disease-specific expression and chromatin variation data in subjects with known disease status. We propose to examine risk loci for schizophrenia and bipolar disorder to prioritize causal variants and genes and to validate them in functional assays.


Although posttranslational modifications of lens proteins play a causative role in biogenesis of human senile cataracts, surprisingly little is known concerning the identity of specific amino acids that are modified in these lenses. Nonenzymatic deamidation of asparagine and glutamine residues is probably the most prevalent modification occurring in the human lens, and studies from other tissues have suggested that resistance to deamidation is a general property of stable proteins. The central hypothesis of this proposal is that the aged human lens contains gamma/beta crystallins with very low rates of deamidation, and that if deamidation does occur, it could result in deleterious effects to the lens, such as the formation of high molecular weight aggregates, low molecular weight cleavage products, and eventual cataract. To identify and quantitate deamidation, we have developed a novel approach involving the use of synthetic peptide standards, HPLC, mass spectral analysis and N-terminal sequencing. This methodology will be used to identify and quantitate deamidation of specific residues from the high molecular weight aggregate and low molecular weight cleavage products, as well as total proteins from dissected nuclei of human cataractous and aged-matched normal lenses. Molecular modeling will be used to correlate known structural properties of crystallins, with their observed rates of deamidation. Finally, human gamma/beta crystallins containing specific residues found to be deamidated will be expressed in a recombinant system and characterized by various biochemical and biophysical methods. Together, this project will identify and quantitate deamidation of specific residues of gamma/beta crystallins in the aging normal and cataractous human lens, followed by determination of the consequences of these modifications upon the stability and structural properties of the expressed deamidated proteins.


The goal of this project is to show that sphincter prosthesis placement is an alternative treatment to external sphincterotomy in patients with spinal cord injury (SCI). The sphincter prosthesis consists of a surgical alloy wire mesh prosthesis inserted transurethrally under endoscopic control and is intended to hold the external sphincter open permanently. This project is designed to test the hypotheses that: 1) Sphincter prosthesis placement is as effective as external sphincterotomy for detrusor sphincter dyssynergia (DSD) based on urodynamic parameters of voiding pressure, residual urine volume, and bladder capacity. 2) Sphincter prosthesis placement has less complications than external sphincterotomy of bleeding, erectile dysfunction, autonomic dysreflexia, and reobstruction. 3) Sphincter prosthesis is associated with shorter length of surgery, shorter length of hospitalization, and reduced hospitalization costs than external sphincterotomy. Surgical alternatives to external sphincterotomy and improved treatment of DSD has not been examined in a randomized controlled manner. A total of sixty SCI patients with DSD will be entered into a prospective randomized study between stent placement and external sphincterotomy at three centers: 1) Regional Spinal Cord Injury Center of Delaware Valley (RSCICDV), Jefferson Medical College, Philadelphia, PA, 2) Ranch Los Amigos Medical Center, Downey, CA, and 3) Spinal Cord Injury Service, Department of Veterans Affairs, Palo Alto, CA. Urodynamic studies and testing will be done prior to surgery and at 1, 3, 6 and 12 months post-surgery. The protocol will provide a direct comparison of effectiveness between treatment of DSD patients with a sphincter prosthesis and transurethral sphincterotomy, the currently accepted surgical treatment. The short- term health related benefits of this project are to improve management of the dysfunctional bladder, reduce complications such as bleeding, impotence, urinary infection, multiple operations, and to reduce health care costs. The long-term benefits are to enhance the ability of subject population to more fully participate in regular life activities including school and work environment and to avoid long-term urological complications and death from renal failure and urosepsis.


To date, there is insufficient empirical evidence exploring the long term health consequences of childhood sexual abuse on adulthood reproductive health. Although negative health behaviors subsequent to the abuse and mental health correlates have been identified, the future health consequences are not as well understood. The purpose of this dissertation is to investigate the long term reproductive health outcomes associated with childhood exposures to physical, sexual, and emotional abuse. In order to examine this relationship, an observational dataset of a large cohort of female nurses, Nurses Health Study II, is being explored to see whether a relationship exists between childhood maltreatment and adult pregnancy outcomes, such as, preterm delivery and spontaneous abortion. Furthermore, the relationship of psychopathology to reproductive outcomes will be explored, by assessing the relationship between post-traumatic stress disorder (PTSD) symptoms with these pregnancy outcomes. Utilizing a life course model of health linking childhood exposures to adult health outcomes, this research will help explore the potential role of childhood abuse as an exposure with repercussions that last far beyond the immediate period of childhood and adolescence. There is a persistent and growing need for research to provide empirical evidence with regards to the possible long term health impacts related to childhood maltreatment. This research can be used to support the promotion of high quality care to meet the needs of children who have suffered maltreatment, as well as increasing awareness of the need for prevention services that will be of benefit not only to children, but may also serve to decrease the burden of disease across the life course. PUBLIC HEALTH RELEVANCE: This research will investigate whether childhood abuse increases risk for poor pregnancy outcomes, and the possible role of mental health and health behaviors in the development of these conditions. This research would be important to increasing awareness among researchers, clinicians, and policy makers as to the long term consequences of childhood abuse, as well as beginning to address how these experiences might lead to poor health.


This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. In addition to the offered training of biomedical research investigators the Resource takes every opportunity to disseminate information regarding its function, capabilities and accomplishments. Major dissemination approaches range from the publication of scientific publications and our Newsletters, active involvement and participation in symposia and workshops, to regular seminars in areas relevant to the Resource. An up-to-date web page additionally has the objective to attract new users as well as inform prior users about new developments at the Resource. The training of medical research investigators in research, technology, use of laser instrumentation and data analysis related to spectroscopic experiments is another important goal of this Resource. By offering an integrated chemical-physics and biomedical environment, visitors, post-doctoral fellows and graduate students gain a wide range of expertise in the application of laser spectroscopy methods to biomedical research questions.


EVALUATION OF ELECTRODE MATERIALS


The dramatic increase in the use of interventional spine procedures and the rising health care costs over the past decade have raised lots of questions about the utilization and outcomes of treatments for back and neck pain. Several articles have addressed some concerning scenarios related to spinal surgeries and catastrophic adverse events after epidural steroid injections have been reported, related to steroid use. The purpose of this study is to describe and characterize the treatment of back and neck pain, while examining temporal and geographical trends, physician specialty, and setting variations. We will characterize treatment trajectories in newly diagnosed back and neck pain patients, and identify efficient treatment paths and potential gateways to high cost and utilization. The characterization of patients treatment paths will include tracking of manual and percutaneous treatments, use of opioids, imaging utilization, surgical procedures, and safety events. We will examine the outcomes and adverse events following interventional spinal procedures by tracking patients for at least one year after their procedures. Identifying efficient and safe treatment paths for back pain will lead to better guidelines for back pain management. A characterization of the outcomes and side effects of interventional spinal procedures will help patients and physicians make more informed decisions about treatment options. Data This project currently focuses on Medicare administrative health records for patients diagnosed with back pain between 2000-2011. A contract to access this data has been established between NIH and CMS. Since the previous annual report, we have achieved the following: Renewed our seat for access to a CMS virtual research data center (VRDC) that allows the analysis of CMS data in SAS, on CMS servers. Produced preliminary results based on a subset of the data of interest and evaluated the feasibility of future work. Presented preliminary results at academic conferences and meetings. Submitted a data addendum request to CMS, who approved the proposal. Obtained access to CMS Carrier, Inpatient, Outpatient, and Part D files for the years 2000-2011 for almost 47 million patients with diagnoses of back pain. Started collaborations with academic partners. Identified codes of diagnoses, procedures, drugs, complications, and outcomes relevant to our projects. Additional data runs have been conducted on the updated dataset and cohort that focus on patient demographics and numbers of injections over the period under study. Differential complication rates by steroid type and injection approach are being examined. Preliminary results have been presented at Grand Rounds style conferences.


Abstract: Unexplained medical symptoms in functional medical illnesses present a challenging diagnostic dilemma for physicians. These conditions usually involve pain or pressure together with other symptoms which suggest a combination of biological and unexplained medical factors. Interstitial cystitis (1C), more recently labeled Painful Bladder Syndrome (PBS), is a prime example of this type of disorder. Recently, intense interest has focused on the psychological/psychiatric components of these disorders. In particular, our team has demonstrated that somatization disorder, when present, may influence the course and management of these functional medical syndromes. Patients with somatization disorder disproportionately consume clinicians'time and medical resources. Thus, understanding the role of somatization disorder in IC/PBS has the potential to yield clinical and economic benefits. In the present study, 120 patients with IC/PBS will be assessed for physical and psychological symptoms, medically unexplained symptoms, and somatization disorder, and they will also monitored over 12 months for temporal symptom patterns and outcomes. The study will demonstrate the proportion of patients with IC/PBS who also have somatization disorder and the degree to which these patients represent a significantly distinct IC/PBS subgroup with different illness presentation, progression, and outcome. Previous research by this team from similar studies of Irritable Bowel Syndrome (IBS) has generated highly useful information and will serve as a template for this research. The comparability of the findings of this study to previous work on IBS will help guide recommendations for modification of pharmacological and psychological interventions for this patient population. Recognition and appropriate management of somatization disorder in patients with IC/PBS has the potential to benefit a substantial proportion of patients with IC/PBS and inform future research. This proposed study truly investigates the interface of medicine and psychiatry in its approach to these disorders.


In the current project, genetic determinants of type 2 diabetes mellitus and obesity are being sought using techniques of genetic linkage and association analysis. Lymphoblast cell lines have been established from informative pedigrees. DNA is available from other families in nuclear pellets extracted from blood specimens obtained in the epidemiologic studies and is amplified by whole genome amplification when needed. An autosomal genome-wide linkage study identified strong evidence for a locus influencing diabetes and diabetes/obesity on chromosomes 1q and 11q. Efforts to identify the causative polymorphism or polymorphisms in both of these regions are currently underway using both a systematic analysis of linkage disequilibrium and analyses of candidate genes. Genome-wide association mapping methods are also being used and exhaustive association analyses are being conducted of regions identified by the genome-wide association studies and of other candidate genes. Whole genome sequencing studies are also being pursued. Genetic variants in the trehalase gene, which is in a region linked to diabeteson chromosome 11q, are strongly associated with plasma trehalase activity and one of these variants is also consistently associated with diabetes. Several candidate genes that have been associated with type 2 diabetes in other populations have been evaluated. The majority of genes seen in other populations appear to have consistent effects in Pimas, though only a few show statistical significance. Some variants identified in other populations (e.g. TCF7L2) appear to have little effect. Variants in established type 2 diabetes genes, MOB2, KLF14 and KCNQ1, are subject to parent-of-origin effects and these parent-of-origin effects replicate in Pimas. The effect of the KCNQ1 variants is particularly strong; with consideration of the parent-of-origin effect these variants account for 4% of liability in susceptibility to diabetes. Recently genome-wide association studies initially with 100,000 markers and later with 1,000,000 markers have identified several additional potential susceptibility genes for young-onset diabetes and for obesity. These include potential diabetes-susceptibility variants in a number of genes and potential obesity-susceptiblity variants in SIM1 and MAP2K3. Replication studies in larger sample sizes are planned, as are functional studies. Variants in candidate genes, such as LEPR, LPGAT1 and SIRT1 show nominally significant association with diabetes, obesity and related traits in Pimas; rare variants in MCR4 are also associated with obesity particularly in childhood. Currently fine-mapping studies with additional variants are being conducted to extract more of the genetic information in regions identified as potentially involved in diabetes susceptibility. Through collaborations, studies are being conducted to determine if any of the signals identified in the present mapping studies replicate in other populations. Variants reproducibly associated with type 2 diabetes from other populations are also being typed to determine their role susceptibilty to diabetes and obesity in the Pimas. Whole genome sequencing is also being conducted in a small number of participants. The data from these studies will initially be used to impute untyped variants from the genome-wide association data; data from these studies suggest that existing reference panels are not accurate for imputation in American Indians. Additional American Indian participants are being recruited for replication studies.


The detailed mechanisms underlying the fundamental membrane events involved in the visual process will be investigated at the sub-molecular level by x-ray and neutron diffraction and nuclear magnetic resonance and optical spectroscopy. These studies center initially on a determination of the dynamical structure at the sub-molecular level of the rhodopsin-containing retinal receptor outer segment disk membrane and the nerve axonal membrane. The dynamical structure of the natural and structurally modified membranes together with their various structurally modified functionally relevant model membranes will be studied "at rest" and during the time course of their natural or artificially perturbed visual function. "Functionally relevant" here refers to model membranes which exhibit selective light-modulated, chemical-modulated and electric field-modulated ion binding or permeability. The detailed mechanisms will primarily be determined through a correlation of systematic time-independent and time-dependent dynamical-structural variations of the membranes with their resulting modified functionality.


Since our neurodegerative disease research group received COBRE grant support we have administered 5 separate research pilot grant programs and provided financial support to investigators totaling in excess of $1,000,000. The purpose of the pilot grant program has been to increase the scope of research conducted at UND related to the theme of the COBRE grant and to increase the competitiveness of UND investigators for funding from extramural organizations. Resulting from this investment in research pilot grant support, the recipients brought into the institution over $5.2M. Thus, pilot grants have been an excellent investment in the on going success of our COBRE grant supported neurodegenerative disease research group. The following is a brief description of the research pilot grant program that we are proposing for the COBRE Phase III funding period. First, we will solicit proposals for our research pilot grant program. Second, the applications will be submitted electronically to the PI/PD. Third, the grants will be distributed to the grant review committee consisting of Dr. Geiger (PI/PD), Drs. Vaughan and Brown-Borg of our Internal Advisory Committee, and Dr. Fyffe of our External Advisory Board. Fourth, the reviewers will meet with the applicants to discuss the hypothesis proposed, the methodological and scientific approaches to be taken, the respective involvement of named investigators, and their timelines and benchmarks of success. We have found that this; site visit approach provides to the applicants a very high quality review and an outstanding opportunity for mentorship. Fifth, midway through the pilot grant year investigators will submit a progress report and then again meet with the grant review committee to evaluate progress, receive advice, and review plans for the remaining one-half of the pilot grant year. At the end of the pilot grant period, the investigators will submit to the review committee a final report in which they describe their original or modified specific aims, their research findings, and most importantly detailed information about abstracts submitted and presented, manuscripts submitted and published, and grants submitted and funded.


Training and Career Development The effective replication and spread of evidence-based practices in cancer communication across and within healthcare systems will be facilitated if healthcare providers and researchers understand not just the practices of better patient-provider communication but also underlying ideas of translational knowledge-toaction processes and cycles.193 In the organizational studies literature, such objectives have been termed double-loop learning as opposed to just single-loop learning; that is, understanding the reasons why change efforts can be effective and not only that certain efforts are effective or not. Of course, teaching principles in addition to how-to knowledge is the basis of pedagogy in the service of enlightened life-long learning. In the Training and Career Development purpose of the CRN Clinical Communication Research Center, we will help targeted audiences with learning of both types: How-to knowledge and principles knowledge. We identify the following targeted audiences for proactive dissemination of research results and practical tools: > Second-year doctoral students from communication and public health programs; > Post-doctoral cancer research scholars; > Healthcare practitioners and researchers in Cancer Research Network institutions; > Healthcare practitioners nationally through three professional societies. In addition, we will also disseminate research results and practical tools through the traditional channels of conference presentations, peer-reviewed publications, and hosting of a special issue of a peer-reviewed journal.


Modified Project Summary/Abstract Section PROJECT SUMMARY FAM57B is an autism risk gene, and part of the 16p11.2 disease locus. 16pdel syndrome is a severe and prevalent (1:2000 people) haploinsufficient disorder, caused by deletion of ~600 kb of chromosome 16. This syndrome is tightly associated with autism, language and intellectual disability, seizures, ADHD, hypotonia and obesity, however genetic contributions to each symptom are unclear. Our proposal addresses the novel hypothesis that alteration of FAM57B function is associated with autism and 16pdel syndrome, by changing cellular lipids of the ceramide pathway. Lipid cohort alteration may change neuronal plasma membrane composition and associated proteins, so altering synaptic activity and contributing to 16pdel symptoms. We defined a 16p11.2 functional interactome, and identified FAM57B as a pivotal ?hub? gene (McCammon et al. 2017). FAM57B has been additionally identified as an autism risk gene (Satterstrom et al. 2019). FAM57B contains a TLC domain found in ceramide synthase enzymes (CerS), but residues required for CerS activity are absent. Loss of function of fam57b in the zebrafish model led to significant changes in brain lipid species. Strikingly, we also observed altered plasma membrane architecture, mis-localization of synaptic proteins, depressed movement and decreased brain activity. The Aim will determine the role of FAM57B in ceramide synthesis and its impact on neuronal and brain function. We hypothesize that FAM57B regulates ceramide levels by interacting with CerS. We further hypothesize that FAM57B maintains the synaptic lipid and protein cohort contributing to neurotransmitter exocytosis. We will determine activity of FAM57B human and zebrafish homologues; delineate molecular changes at the neuronal synapse after loss of FAM57B and determine activity of fam567b in the zebrafish brain. This Aim will solve the function of FAM57B, giving insight into mechanisms underlying autism and 16pdel syndrome phenotypes.


Through the proposed training in his K01, I will pursue the additional mentorship that I need and the additional research time to enhance my passion for intestinal epithelial biology. During this award period I will have the opportunity to acquire and refine fundamental skills of becoming a developed scientist. Career development will be fostered through twice-weekly meetings with Dr. Rustgi, convening an expert interdisciplinary advisory committee twice annually, and taking advantage of opportunities in career and professional development through UPenn, the NIH and the AGA. These skills include (but are not limited to) manuscript and grant preparation, lab meeting presentations, participation in the seminars and courses described herein, and presentations at international research conferences (DDW, Keystone). Training will also entail course-work in biostatistics (two semesters), short courses in RNA biology and basic bioinformatics, ongoing training in bioethics, weekly participation in the seminar series (where I will meet visiting professors), journal clubs (presentations semi-annually), and twice yearly floo lab meetings through the NIDDK P30 Center for Molecular Studies in Digestive and Liver Diseases and Division of Gastroenterology. The overarching hypothesis of this project is the following: LIN28B plays a critical role in regulating growth and proliferation in the intestinal epithelium via cooperation with c-MYC, with key roles for intestine stem cell (ISC) maintenance. Two potential interrelated mechanisms will be explored: 1) LIN28B binds to target mRNAs associated with cellular growth and cooperates with the transcription factor c-MYC, which is a master regulator of cell growth. and 2) LIN28B affects the maintenance of the ISC by promoting stem cell identity and growth. LIN28B is normally expressed in the intestinal epithelial crypt where cell division occurs. LIN28B represses the maturation of miRNAs such as Let-7, which is restricted to villus epithelium, where post- mitotic differentiated cells are located. The spatial restriction of LIN28B and Let-7 expression in the intestinal epithelium is likely crucial for maintaining sufficient but limited compartments of differentiation and proliferation. C-MYC is also restricted to the crypt epithelium and is required for epithelial proliferation and growth. W have evidence to support a Let-7-independent function of LIN28B entailing intimate cooperation with c-MYC. The Specific Aims of this proposal to test the hypothesis are the followin: 1) We will explore the functional relationship between LIN28B and c-MYC, in vitro. 2) We will assess how LIN28B cooperates with c-MYC through in vivo studies of Lin28b in the intestinal epithelium. 3) We will examine the potential of LIN28B to modulate the intestinal stem cell compartment. LIN28B function may be relevant to ISC homeostasis and epithelial proliferation. Determining how LIN28B functions in the intestinal crypt will likely provide an important link among the cellular mechanisms governing epithelial proliferation, differentiation, and/or mucosal regeneration. PUBLIC HEALTH RELEVANCE: This project will explore the regulation of cell growth in the lining of the intestine in stem cells and other dividing cells. Stem cells are required to constanly replenish cells of the intestinal lining throughout the entire life of an individual. This study wll provide key insights into how intestinal cell growth is maintained, controlled, and perturbed in disease states.


The primary means by which nerve cells communicate with each other is through the release of neurotransmitter at chemical synapses. The ability of the brain to process information depends on synaptic connections forming precisely and reliably between many different types of neurons. This proposal is directed towards developing a molecular understanding of the signaling between synaptic partners that regulate synaptogenesis. It is well established that even in simple metazoans like the worm C. elegans changes in synaptic activity induce compensatory changes in synaptic strength and structure. We propose to use a combination of genetics, cell biology, molecular biology and live imaging to identify and characterize the role of molecular components of the signaling pathways that coordinate synaptic development at nerve-nerve synapses. First, we aim to describe the order of cellular events in nascent synapse formation by visualizing the recruitment of fluorescent-tagged components to newly forming synapses. We will define the order in which mitochondria, synaptic vesicles, active zone components and adhesion molecules appear at synaptic sites. We will also define the cellular mechanisms that mediate subsequent growth of the presynaptic specializations. Second, we will define the role of novel molecular components that were identified as mutants that fail to form synapses. Using a variety of molecular, genetic and protein interaction studies we will position the genes within the current molecular models of synapse assembly. Third, we will use genetic approaches to isolate and characterize genes which disrupt signaling between mechanosensory neurons and their synaptic partners in C. elegans using a novel synaptic tag which can be easily detected in live animals under a fluorescent dissecting scope. Together these approaches will help define mechanisms that cells use to identify and communicate with one another during the process of synapse formation and synaptic maintenance. While synaptogenesis is undoubtedly less complex in C. elegans than in vertebrates, it is already clear that similar pathways operate in both systems. Thus, analysis of the molecules participating in the process in C. elegans should help define a set of general and likely conserved principles that are common to synaptogenesis mechanisms in general. PUBLIC HEALTH RELEVANCE: Synaptic connections are the primary neuronal communication structures in the brain. In Alzheimer's disease, it is now well established that changes in synaptic density (i.e. loss of synaptic connections) correlate better with cognitive impairment that the hallmark plaque and tangle lesions that are also associated with the disease. Our work is focused on understanding how synaptic connections are formed. Such basic scientific understanding of brain development and function will aid in developing therapies that intervene early in disease hence slowing or arresting synaptic loss.


The purpose of the Scientific Core is to make a data set rich in information about substance use, risk and protective factors, and other variables readily available to the five individual research projects in the proposed Center, so that important questions about drug use can be addressed by means of state-of-the-art methodological procedures. From 1986 to 1992 the NIAAA-funded Adolescent Alcohol Prevention Trial (AAPT) study collected data on substance use, risk factors, and numerous other variables on four cohorts of Southern California schoolchildren (approximately 12,000). Data were collected annually during the junior high and high school years. The AAPT participants are now entering young adulthood. The result will be a cohort-sequential data set on individuals ranging in age from ten to approximately 19. This data set will provide an exciting opportunity for the five individual research projects to examine risk for substance abuse across adolescence.


Experiments are proposed to investigate the role of abnormal early experience in producing abnormal neural pathways linking the left and right hemispheres of the brain. The interhemispheric pathway matures quite late during development, and its pattern of development is particularly interesting. There is an initial over-production of connections (mistakes?) followed by a gradual narrowing of the zone containing neurons which send axons to the opposite hemisphere. This narrowing process can be altered when there is unusual experience, resulting in persistence of developmental mistakes, which could provide a substrate for abnormal behavior. We will use anatomical techniques involving retrograde transport of horseradish peroxidase and anterograde transport of isotope to address a number of questions related to the role of abnormal experience in producing abnormal interhemispheric pathways. We will use a model system chosen to allow complete control of early experience. In addition, the anatomy and physiology of the interhemispheric pathway are well understood in this system. The goal of the experiments is to increase our understanding of the link between abnormal early experience and abonormal behavior. It has been known for many years that abnormal experience in childhood can result in severe mental health problems in adulthood. Since abnormal experience can result in retention of developmental mistakes in brain connectivity, this link may form a structural basis of many psychiatric disorders. Interhemispheric pathways may be particularly vulnerable to abnormal early experience. Indeed, recent computed tomography studies of schizophrenic patients have shown that they have a high incidence of abnormal hemispheric specialization. The proposed experiments will explore the circumstances and mechanism for retention of developmental mistakes in interhemispheric connectivity resulting from abnormal early experience.


Two approaches were taken for cloning the gene for type IV collagenase from a tumor cell cDNA library. A human melanoma (A2058) gamma gtll library (custom made for Dr. Liotta by Clontech Co.) was first screened with a polyclonal antibody against type IV collagenase. The collagenase was purified from culture supernatant of the A2058 cell line which was used to make the cDNA library. The second approach was to screen the melanoma library with a mammalian collagenase cDNA clone (supplied by C. Brinkerhoff). After the first screening with the antibody, 18 clones were isolated, of which four were plaque purified after 4-5 rounds of screening. All four clones produced clear plaques, which indicates an insert-bearing phage, but failed to demonstrate an insert by restriction analysis; the restriction pattern was identical to that of a wild type gamma gtll phage DNA. Likewise, negative results were obtained when the same melanoma cDNA library was screened with the mammalian collagenase clone. Six clones which were strongly positive through four rounds of screening turned out to produce no-recombinant blue plaques without an insert as judged by restriction analysis of phage DNA. Since there may be problems with the melanoma library, we will start the cloning process all over again using another tumor cell cDNA library. Presently, we are beginning to focus our attention on the role of endothelial cells in metastasis. Therefore, we are in the process of screening a human endothelial cDNA library, purchased from the Clontech Co. Using the mammalian collagenase cDNA clone under low stringent conditions, we expect to pull out mammalian collagenase and other related genes, expressed by endothelial cells.


This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. The ARF-p53 tumor suppressor pathway is one of the cell's major defenses against the stimulation of uncontrolled cell division induced by activated cellular and viral oncogene. ARF and/or p53 are mutated in over 70% of human cancers. The inappropriate activation of growth promoting cellular signaling pathways by oncogenes can result in the induction of ARF. The expression of ARF can activate p53 leading to apoptotic cell death or cell cycle arrest. The mechanisms by which the ARF-p53 pathway is regulated remains to be precisely elucidated. The expression of ARF can activate p53 leading to apoptotic cell death or cell cycle arrest. We have shown that the polyoma virus oncogene, PYMT, activates an ARF-induced p53 mediated block. We find that the polyoma virus small T-antigen, PYST, via its ability to bind to cellular protein phosphatase 2A (PP2A), can negate the ARF-induced block to cell division induced by PYMT. We intend to use the PY induction and inhibition of ARF signaling to p53 to better define this important tumor suppressor pathway. Our hypothesis is that the polyoma virus proteins are revealing an important new aspect of the ARF-p53 tumor suppressor signaling circuit, and we plan to use these viral proteins as tools to study its molecular basis. To better define the role of PYMT in activating ARF and as an oncogene, and to define the role of PYST in blocking ARF signaling to p53 we plan to characterize the proteins complexed to PYMT, PYST and members of the ARF-p53 signaling pathway. In the first instance proteins bound to TAP fusion constructs would be identified by Mass Spectrometry.


The Pilot Core (PC) will work with the Management and Administrative Core (MAC) to support small-scale and innovative pilot projects that are consistent with the overall theme of the Northwest Roybal Center (NRC). Specifically, NRC pilot studies will focus on innovative approaches to improve the health, wellbeing, quality of life and productivity of older adults with cognitive impairment, midlife and older adults at risk for cognitive impairment, and their caregivers through the translation of basic behavioral and social sciences research. Furthermore, the PC will work with the MAC to ensure that each Pilot Study: (1) addresses one or more of the thematic areas of focus identified in the current FOA, (2) moves basic behavioral and biobehavioral research toward the ultimate goal of translation, and (3) leads to additional, larger research efforts to further implement translation activities into a program or product. Finally, the PC will work with the MAC to involve both junior and established researchers from a variety of disciplines in the work of the NRC, and will foster interaction and collaboration among the pilot investigators and outside agencies and corporate partners.


Although we probably know more about the genetics of Drosophila then any other multicellular animal, we do not understand how genes control the development of any organ. The logic behind gene deployment will become more evident once we start classifying genes into those with controlling roles and those carrying "executive" functions. Homoeotic mutants are the prime candidates for controlling elements in Drosophila. These mutants transform one body part into a different body part and represent single loci which determine entire developmental pathways taken by groups of cells. Recently it has been discovered that specific groups of cells are set aside at early stages of development, and their descendants form very specific regions referred to as "compartments". Evidence suggests that these compartments are under the control of homoeotic genes. Until recently most evidence for compartments was derived by examination of adult cuticle. We propose (1) to expand our mapping of developmental compartments within the imaginal discs by using mitotic recombination (Minute technique); (2) to map by histochemical techniques the distribution and Compartment affiliation of isocitrate dehydrogenase, glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase in normal and homoeotically transformed imaginal disc tissue; (3) to adapt recently developed procedures for use in establishing enzyme, protein and/or polypeptide profiles for transplanted differentiating normal and homoeotically transformed imaginal tissue; and (4) to study the transdetermination characteristics of homoeotically affected imaginal disc tissue and to record the biochemical changes occurring at each step of the transdetermination process.


The HIV-envelope protein gp120 plays a critical role in triggering the apoptosis of T-lymphocytes, which is a central pathogenic feature of HIV-mediated immune dysfunction. T-cell apoptosis is also a physiological process that regulates antigen receptor repertoire selection during homeostasis and the maturation of T- cells. We hypothesize that gp120-induced cross-talk between T-cell receptor (TCR) and chemokine receptor signaling pathways leads to apoptosis. Our preliminary data on the role of the TCR signaling components CD45 and SLP-76 in HIV-gpl20-induced apoptosis support this hypothesis. In addition, we have recently shown that cross-talk between TCR and chemokine receptor CXCR4 regulates apoptosis via a novel mechanism that is mediated by AKT/Protein Kinase B (PKB), heat shock protein-70 (HSP-70), caspase-1 and RIP2 (caspase recruitment domain-containing serine/threonine kinase). To analyze mechanistically how the TCR components and chemokine receptors CXCR4/CCR5 regulate gp120-induced apoptosis, we will pursue the following specific aims: Aim 1) we will assess the interaction between CD45 and CXCR4/CCR5 signaling molecules by defining the domain of CD45 and characterizing the CD45 signaling responsible for apoptosis. Aim 2) we will perform structural and functional analyses of the TCR-mediated downstream effector SLP-76 that mediates Ca2+dependent apoptotic pathways. Aim 3) we will analyze the role of the AKT/HSP-70 apoptotic pathway that may lead to the induction of forkhead transcription factors, and will characterize the activation of caspase-1 and its regulation by RIP2. We are also exploring innovative strategies to inhibit gp120-induced apoptosis. In this regard, we have shown that a novel protein, Slit, which binds to the Robo receptor and modulates CXCR4 function can inhibit gp120-induced apoptosis. Aim 4) we will identify which domains in the Slit/Robo complex regulate the observed anti-apoptotic effects. These studies are designed to identify apoptotic signaling molecules and effector pathways involved in T-cell loss, and thus to provide novel therapeutic targets to combat immune deficiency in AIDS. [unreadable] [unreadable] [unreadable]


This study is being done in order to determine safety and potential beneficial effects of a new investigational drug, CP-424,391, in patients with congestive heart failure. CP-424,391 may have value for the treatment of congestive heart failure by causing increased release of a natural hormone in the body, growth hormone. Increasing growth hormone levels could improve the pumping of the heart and thus help the condition. This study will be the first use of CP-424,391 in patients with congestive heart failure. Because this is a research study, CP-424,391 will be available to the patient only during their participation in this study and not after.


New gene expressed in prostate (NGEP) is a prostate-specific gene encoding either a small cytoplasmic protein (NGEP-S) or a larger polytopic membrane protein (NGEP-L). NGEP-L expression is detectable only in prostate cancer, benign prostatic hyperplasia and normal prostate. We have identified an HLA-A2 binding NGEP epitope (designated P703) which was used to generate T cell lines from several patients with localized and metastatic prostate cancer. These T cell lines were able to specifically lyse HLA-A2 and GEP-expressing human tumor cells. NGEPP703 tetramer binding assays demonstrated that metastatic prostate cancer patients had a higher frequency of NGEP-specific T cells when compared with healthy donors. Moreover, an increased frequency of NGEP-specific T cells was detected in the peripheral blood mononuclear cells of prostate cancer patients post-vaccination with a PSA-based vaccine, further indicating the immunogenicity of NGEP. These studies thus identify NGEP as a potential target for T cell-mediated immunotherapy of prostate cancer. Tumor-associated antigens are weakly immunogenic. Human carcinoembryonic antigen (CEA) is overexpressed on a wide range of human carcinomas and represents an attractive target for cancer immunotherapy. A concurrent multicenter, randomized Phase II trial employing a recombinant poxviral vaccine provided evidence of enhanced median overall survival (OS) (p = 0.0061) in patients with metastatic castrate-resistant prostate cancer (mCRPC). The study reported here employed the identical vaccine in mCRPC to investigate the influence of GM-CSF with vaccine, and the influence of immunologic and prognostic factors on median OS. Thirty-two patients were vaccinated once with recombinant vaccinia containing the transgenes for prostate-specific antigen (PSA) and three costimulatory molecules. Patients received boosters with recombinant fowlpox containing the same four transgenes. Twelve of 32 patients showed declines in serum PSA post-vaccination and 2/12 showed decreases in index lesions. Median OS was 26.6 months (predicted median OS by the Halabi nomogram was 17.4 months). Patients with greater PSA-specific T-cell responses showed a trend (p = 0.055) toward enhanced survival. There was no difference in T-cell responses or survival in cohorts of patients receiving GM-CSF versus no GM-CSF. Patients with a Halabi predicted survival of less than 18 months (median predicted 12.3 months) had an actual median OS of 14.6 months, while those with a Halabi predicted survival of greater than or equal to 18 months (median predicted survival 20.9 months) will meet or exceed 37.3 months, with 12/15 patients living longer than predicted (p = 0.035). Treg suppressive function was shown to decrease following vaccine in patients surviving longer than predicted, and increase in patients surviving less than predicted. This hypothesis-generating study provides evidence that patients with more indolent mCRPC (Halabi predicted survival greater than or equal to 18 months) may best benefit from vaccine therapy. Adenoviral transduction with CD40L and poxviral transduction with B7-1, ICAM-1, and LFA-3 (TRICOM) have been used to enhance the antigen-presenting capacity of chronic lymphocytic leukemia (CLL) cells. This study compares the same vector (modified vaccinia virus strain Ankara (MVA)) encoding CD40L or TRICOM for its ability to enhance the immunogenicity of CLL cells. CLL cells from some patients showed differential responses to each vector in terms of induction of autologous T-cell responses. This study supports the rationale for the use of CLL cells modified ex vivo with pre-specified recombinant MVA vectors as a whole tumor-cell vaccine for immunotherapy in CLL patients. In chronic lymphocytic leukemia (CLL), malignant B cells and nonmalignant T cells exhibit dysfunction. We previously demonstrated that infection of CLL cells with modified vaccinia Ankara (MVA) expressing the costimulatory molecules B7-1, ICAM-1, and LFA-3 (designated TRICOM) increased expression of these costimulatory molecules on the surface of CLL cells and thus augmented their antigen-presenting capability. Here, we evaluate the effect of MVA-TRICOM-modified CLL cells on T cells. Following incubation with irradiated MVA-TRICOM-modified CLL cells, allogeneic and autologous CD4(+) and CD8(+) T cells expressed significantly higher levels of B7-1, ICAM-1, and LFA-3. We show that this increase was the result of physical acquisition from the antigen-presenting cells (APCs), and that purified T cells that acquired costimulatory molecules from MVA-TRICOM-modified CLL cells were able to stimulate the proliferation of untreated T cells. These results demonstrate for the first time that T cells from CLL patients can acquire multiple costimulatory molecules from autologous CLL cells and can then act as APCs themselves. Given the immunodeficiencies characteristic of CLL, enhancing the antigen-presenting function of CLL cells and T cells simultaneously could be a distinct advantage in the effort to elicit antitumor immune responses.


These experiments are designed to determine whether adolescents at risk for substance abuse (SA) exhibit impaired cognitive performance on a task that reveals a deficit in performance by adults with a history of SA. Abnormal cognitive performance in adults after long-term exposure to drugs of abuse can reflect either vulnerability to SA (primary effect) or drug effects on the brain (secondary effect). This study tests the hypothesis that a predisposing cognitive deficit in adolescents contributes to risk for substance abuse. To date, 23 adolescents completed the initial phase of the study. Adolescents at risk for SA showed worse performance on a Gambling task (GT), which targets impulsivity and judgment and depends on orbitofrontal cortical function, and on the Wisconsin Card Sorting Task (WCST), which targets executive function and activates the dorsolateral prefrontal cortex in healthy control subjects. Task performance on the WCST was correlated predominantly with global functioning, a measure of psychiatric symptom severity, and performance on the GT correlated with antisocial and aggressive behaviors. Although both tasks differentiated adolescents at risk from controls, the WCST may be less specific to a neurobiological vulnerability for substance abuse because it relates more to psychiatric status. In contrast, the GT task may be more relevant for substance abuse problems as it is influenced by aggressive and antisocial behaviors, which are known to predict later substance abuse. These results are preliminary and await confirmation by studying larger samples, and assessing directly the significance of the findings in follow-up data. - adolescents, cognitive performance, gambling task, wisconsin card sorting task, substance abuse - Human Subjects: Interview, Questionaires, or Surveys Only


We shall continue to analyze the ontogeny of thymocyte and T cell responsiveness in both neonatal mice and in radiation chimeras. The loss of thymocyte function with age will also be rigorously examined. Particular emphasis will be placed on determining whether or not suppressor T cells are playing a regulatory role in the thymus of both chimeric and normal, aging mice. Preliminary experiments with cyclophosphamide indicate that this will be a useful approach. The responder profiles of T cells and thymocytes from neonatally tolerized (to H-2 angigens) baby mice will be explored in detail. All experiments will utilize the vaccinia virus and influenza virus cytotoxic T lymphocyte systems.


It is well known that increasing an individual's exercise endurance can ameliorate multiple disease processes. We have identified possible quantitative trait loci (QTL) on Chromosomes 2, 8,13, and X that appear to be involved in the genetic regulation of maximal exercise endurance. Therefore, the overall objective of this proposal is to determine the specific genes involved in the control of inherent exercise endurance. This objective will be fulfilled through the following Specific Aims: 1) To complete a genetic linkage analysis and generate high-resolution linkage maps of the high exercise endurance (HEE = Balb/cJ) and low exercise endurance (LEE = DBA/2J) phenotypes; 2) To examine the mechanisms that confer differential maximal exercise endurance phenotypes, we will develop congenic strains of mice that contain the candidate regions from Specific Aim 1; 3) To characterize the maximal exercise endurance, central physiological response, and peripheral physiological response in congenic, HEE, and LEE parental mouse strains; and 4) To characterize and compare the gene expression of the congenic, HEE, and LEE parental mouse strains. The Specific Aims, when fulfilled, will be significant given the health risk of hypokinetic diseases. Understanding the genetic factors that predispose an individual to a certain fitness level could lead to better methods of improving the physical health and well-being of individuals. Unlike other past attempts, this project is unique in that we are proposing to use common, but powerful, genetic research techniques to investigate this area. This will be the first project investigating the genetic basis of exercise endurance to use these techniques that have already proven successful in other fields.


Abstract Simultaneous PET/MR can be considered as an integrated imaging modality only if the information of both modalities is integrated together. In current routine PET/MR applications, the PET and MR scans are performed separately, and the images are reconstructed separately as well. The information is integrated only at the application level. Here we propose unified methodologies of joint PET/MR image reconstruction, a paradigm shifting new way to integrate information of PET and MR to significantly maximize the outcome of PET/MR. The PET and MR scanners indeed measure different physical or physiological signals, but there are still redundant information (e.g. tumor boundary and mutual information) between the images obtained with the two modalities that can be utilized to build connection between PET and MR images in a potential joint reconstruction. In addition, if the compartmental model is taken into account, the physiological parameters estimated from PET and MR can have overlaps, and therefore the parametric image (voxel-wise kinetic parameters) estimated from one modality could be directly used to help the estimation of the parametric image of the other modality. Therefore, there are inter- connections between these two modalities that we can use to develop elegant methods of joint reconstruction. We will first take advantage of the simultaneous acquisition of PET/MR to develop a static image reconstruction with anatomic prior derived from MR images, and to develop methods to jointly reconstruct gated PET images using a motion field computed from MR images. We believe in both cases, the quality of PET images will be significantly improved compared to traditional approaches. For PET/MR, there are many novel ways to jointly model the dynamic PET and MR images. We will thus develop an alternating direction method of multipliers (ADMM) to directly estimate the voxel-wise kinetic parameters of dynamic PET and dynamic MR together from raw data. This will achieve the maximum signal noise ratio of parametric images for both dynamic PET and MR. We will also investigate novel approaches to parametric imaging of non-stationary kinetic modeling in which not only the images are estimated but also the uncertainty on those estimates of the parametric images. The knowledge of uncertainty is important when making decisions about progression/regression of the disease, signal detection, etc. We will use a method developed in our laboratory in which the noise in raw PET data will be transferred to parameter images using origin ensemble algorithm.


The Program Project Application is concerned with basic phenomena of conception and early pregnancy in mammals, including man. The general objectives are: (1) To study in depth the stages of conception and early pregnancy in mammals and man whose failure has been identified as possibly leading to infertility, or whose vulnerability may lead to new methods of controlling fertility. Since it is virtually impossible to study in detail these phases of human reproduction, we propose to study mechanisms which are specific to the reproduction processes of mammals, and pursue their regulation in depth. Where possible, critical experiments will be done on human material. (2) The topics to be investigated include some aspects of spermatogenesis, oogenesis, fertilization, cleavage and implantation. Emphasis is placed on the kinetic aspects of early development in the mammal and the interactions of the preimplantation stages with the local environment where the preimplantation embryonic stages develop within the female genital tract. BIBLIOGRAPHIC REFERENCES: Biggers, J.D. (1975) Development of the mammalian oocyte in vitro. In Vitro 10:362. Ducibella. T., Albertini, D.F., Anderson, E. and Biggers, J.D. (1975) The preimplantation mammalian embryo: characterization of intercellular junctions and their apperance during development. Devel. Biol. 45: 231-250.


A major responsibility of cardiac care unit (CCU) nurses is to monitor patients following coronary angioplasty for signs and symptoms of cardiac ischemia which may signal the complication of sudden coronary artery reocclusion with subsequent acute myocardial infarction. The rationale for interventions to re-establish blood flow following reocclusion is that patients who develop extensive infarction often have numerous repeated hospitalizations for congestive heart failure and chronic, debilitating symptoms such as shortness of breath, inability to perform daily activities, and fatigue. A noninvasive technique more sensitive than the patient's symptoms for detecting recurrent cardiac ischemia is the patient's 12-lead electrocardiogram (ECG). However, because the ECG abnormalities are transient, cardiac ischemia is often missed by the patient's daily 12-lead ECG. The proposed study seeks to determine the sensitivity and accuracy of continuous bedside cardiac ischemia ST segment monitoring using a "derived" 12-lead "ECGD" compared to the routinely- monitored dual-lead method for detecting recurrent cardiac ischemia following coronary angioplasty. A secondary aim is to determine whether there are gender differences in the sensitivity and accuracy of differences between the 2 lead methods in these patients. 416 subjects will have 12-lead ECGDs recorded with balloon inflation, during coronary angioplasty to record the patient's ischemic pattern during "controlled" ischemia. Information will be elicited from the patient at the same time as to their symptoms of cardiac ischemia. Patients will serve as their own controls and be monitored with both lead methods in the CCU following angioplasty. The sensitivity will be analyzed using a 2-factor repeated measures analysis of variance, where the dependent variable is defined as the proportion of true ischemic events detected by each method. To compare the accuracy of the 2 lead methods, 2 nurse experts will independently determine whether both methods contain the same pattern of ST elevation, depression, or isoelectric ST segments compared to corresponding leads of the patient's 12-lead ECGD recorded during coronary angioplasty. Nurse expert ratings will be placed into a 3 X 3 contingency table, where the table rows will contain "same," "related" and "different" ratings for Method I (routinely-monitored dual leads) and table columns will contain ratings for Method II(12-lead ECGD). The contingency table will be analyzed under a non-parametric "correlated proportions" statistical model. The Stuart extension of the McNemar test will be used to test for the equality of the correlated marginal probabilities.


PROJECT SUMMARY/ABSTRACT The goal of this R35 proposal is to uncover novel genetic discoveries and biological mechanisms underlying association with devastating cardiovascular diseases. This proposal builds on strengths in high-throughput genetics and genomics and development and application of innovative computational and statistical methods and genomics technology to maximize the benefits of genetic studies of cardiovascular disease. We will continue our discovery efforts to uncover genetic variants associated with a variety of cardiovascular diseases including atrial fibrillation, aortic aneurysm and dissection, and myocardial infarction and coronary artery disease. Building on our previous work where we identified a number of new genes for coronary artery disease and lipids, we also propose to uncover the mechanisms underlying association at known loci using genetics and epigenomics. We propose to assess the phenotypic impact of the ~19 million variants and 20k indels and SVs identified from whole genome sequenced samples by imputing them into 70,000 new GWAS samples with many cardiovascular phenotypes. We will perform integrated analyses with epigenomics data to highlight clusters of loci with related function. We also propose to perform targeted sequencing of 300 genes in 30,000 CAD cases and controls to search for loss of function variants at CAD loci that implicate CAD genes. We will continue to search for mechanistic insight by performing a PheWAS for all CAD-associated variants identified, disentangling multiple independent signals and correlated traits and clinical endpoints using conditional testing. Completion of these studies will provide new insights into disease mechanisms that have the potential to catalyze breakthroughs in cardiovascular disease prevention, treatment, and diagnosis.


Project Summary/Abstract Despite significant efforts, our understanding of clinically relevant mechanisms underlying human pituitary tumorigenesis remains ashamedly meager. My lab has focused on defining the precise molecular mechanisms by which ubiquitous components of growth factor/Ras/MAPK signaling pathways result in pituitary lactotrope cell-specific transcription responses. We discovered an elegant tri-partite molecular code, involving the protooncoprotein c-Ets-1, the POU-homeodomain protein Pit-1, and a composite DNA element containing an Ets binding site adjacent to a Pit-1 site. Pit-1, Ets-1 and this composite site were shown to be required not only for the oncogenic V12Ras response of the rat (r) prolactin (PRL) promoter, but also to reconstitute lactotropespecific rPRL promoter activity in HeLa nonpituitary cells. During the past funding cycle we made important advances, showing that Ets factors, particularly GABP, are critical for basal and growth factor-induced endogenous rPRL gene expression in GH4 cells and lactotrope ontogeny in transgenic mice;in dissecting the TAD subdomains of Ets-1 and Pit-1;and in identifying co-activators and co-repressors governing basal and Ras-regulated PRL gene expression. In this application, we propose to move our Ras work to a more translational level, and address the role of Ras/MAPK in pituitary prolactinoma tumor formation, and whether PTTG-1 and Menin act as downstream effectors in this pathway. Thus, the main goal of this proposal is to develop pre-clinical GH4 cell line and transgenic mouse models that target V12Ras to pituitary lactotropes, to determine the functionally relevant role of persistently-activated pMAPK and downstream effectors (eg, Menin, PTTG-1 and Mediator components) in regulating lactotrope gene expression, cell proliferation and tumorigenesis. Our unifying hypothesis is that persistent MAPK activation in lactotropes regulates specific Mediator components leading to the stimulation of prl and pttg-1 gene expression, and inhibition of menin gene expression, which leads to increased cell cycle progression, cell proliferation and tumorigenesis. Specific Aims: (1) To use Dox-inducible V12Ras to determine the role of distinct levels of activated MAPK in mediating cell proliferation and differentiation in GH4 somatolactotropes. (2) To use ChIP analysis to define the MAPKdependent chromatin alterations on the PRL, GH, PTTG-1 and Menin promoters and on recruitment of specific Mediator components to these genes. (3) To use shRNA and over-expression to identify the key MAPK downstream effectors required for MAPK-regulated promoter activity and GH4 cell proliferation. (4) To target a V12Ras-IRES-Luc transgene to pituitary lactotropes and determine whether persistent MAPK activation results in tumorigenesis and if drug induced lactotrope hyperplasia enhances tumor formation. Knowledge gained from these studies will provide critical insights into the mechanisms by which pMAPK links to the Mediator complex, and how this link regulates prl, pttg-1 and menin gene expression, thus providing a mechanistic framework for how the MAPK pathway intersects with clinically relevant proteins to contribute to lactotrope tumorigenesis


The long-term objective of this proposal is to provide new and meaningful structural-functional correlative data as it relates to solute transport along the mammalian renal tubule. The experiments described focus our attention primarily on the collecting duct and place a major emphasis on the role of angiotensin II (All) in the regulation of intercalated cell structure and function and specifically in the control of transepithelial H+ and HCO3- transport. The proposal includes four sections with clearly delineated specific aims and hypotheses to be tested. In Specific Aim I we will examine the effect of All on the ultrastructure of intercalated cells to identify specific intercalated cell subpopulations that respond to All, and determine the effect of All on the subcellular distribution of H+ATPase by immunogold cytochemistry. Hypothesis to be tested: Specific intercalated cell subtypes are responsible for the effect of All on H+-ATPase activity and acid-base transport in the collecting duct. In Specific Aim II the effects of All on H+-ATPase activity and the signalling transduction pathways involved in mediating these effects will be studied in microdissected collecting duct segments. Hypothesis to be tested: The inhibitory effect of All on H+-ATPase activity in the collecting duct is mediated via specific AT1 receptors and activation of distinct signalling systems which will be identified. In Specific Aim III we will determine the expression and cellular location of All receptor isoform mRNA in the collecting duct by quantitative RT-PCR and in situ hybridization and determine the effect of acid-base perturbations on All receptor mRNA expression. Hypothesis to be tested: All receptor mRNA expression varies between different populations of intercalated cells and is influenced by acid-base perturbations. In Specific Aim IV we will determine the effect of All on proton and bicarbonate transport in the isolated perfused rabbit cortical collecting duct and identify the subtype(s) of intercalated cells that responds to All. Hypothesis to be tested: All stimulates HCO3 secretion by type B intercalated cells and inhibits H+ secretion by type A intercalated cells in the cortical collecting duct. The experiments described in this proposal are intended to continue our long-term commitment to delineate the structural and functional characteristics of the renal tubule and specifically to establish the role of intercalated cells in the "fine-tuning" of acid-base regulation by the kidney. A better understanding of the mechanisms that control H+ and HCO3, transport in this region of the renal tubule will enable physicians to manage complicated clinical problems of acid-base balance that are often life-threatening with greater expertise and success.


The proposed research explores a synthetic strategy which will provide access to analogs of the tumor promoter thapsigargin (1). New adaptations of two metal-mediated synthetic methodologies will be examined. First, the synthesis of cycloheptene template 10 will extend available protocols for the selective functionalization of the known chiral allylmolybdenum complex 2. Second, palladium-catalyzed cycloisomerization of enyne 10 will be investigated as a means for forming a highly substituted bicyclo[5.3.0]decane system. Functional group manipulation of the resulting product will be pursued to provide tricyclic compound 21. The specific goals for the postdoctoral appointment are the acquisition of this synthetic target, as well as the validation of the methods mentioned above. However, the enantiomerically enriched target 21 represents a potentially general precursor for the preparation of analogs of thapsigargin and other guaiane natural products.


This application requests support for the 11th Symposium on Cochlear Implants in Children. The symposium will be held April 11-14, 2007, in Charlotte, North Carolina. This symposium has been held biannually since 1986. The meetings have focused on numerous pediatric cochlear implant topics that are of importance to surgeons, audiologists, speech-language pathologists, hearing scientists, engineers, and educators. This ongoing series of symposia affords a valuable mechanism to keep the field abreast of advances in research and technology, and provides perspectives from a wide range of relevant fields. The symposium will be sponsored by the University of North Carolina at Chapel Hill and will highlight a number of topic areas that are of current importance to pediatric implantation. Areas of focus will include: 1) cochlear implantation for the very young child; 2) surgical, habilitation and candidacy issues related to auditory neuropathy; and 3) issues that are related to bilateral cochlear implantation and the provision of binaural hearing cues to deaf children. These areas of focus will be explored in the morning sessions via thirteen invited presentations, contributed talks from the scientific community, panel discussions, and audience participation. In addition to these focus areas, a range of other topical pediatric cochlear implantation issues will be examined in afternoon contributed oral and poster sessions. Based upon recent symposia in this series, submission of approximately 250 poster presentations and 75 oral presentations is expected. Pediatric cochlear implantation is greatly benefiting the life possibilities and expectations of children with profound hearing losses; however, we are still far from realizing all of the potential benefits of this approach to the treatment of childhood deafness. Continued progress hinges upon continued interaction and exchange of ideas among surgeons, hearing scientists, engineers, audiologists, speech/language pathologists, and educators. The purpose of this symposium is to continue this necessary and productive interaction. [unreadable] [unreadable] [unreadable]


The human interleukin-2 receptor is being studied to understand critical components of the T cell immune response in normal and neoplastic cells. Following T-cell activation, IL-2 and IL-2 receptors are induced; the magnitude and duration of the T-cell immune response is controlled by the amount of IL-2 produced, the levels of receptors expressed, and the time course of these events. Three chains of the IL-2 receptor exist, IL-2Ralpha, IL-2Rbeta, and gamma-c, with IL-2Ralpha and IL- 2Rbeta being significantly regulated at the level of transcription. The laboratory has focused primarily on the types of signals induced by IL-2, particularly the activation of STAT proteins, and the mechanism of regulating IL-2Ralpha in response to mitogen and IL-2. The group previously identified two enhancer-like regions regulating the IL-2Ralpha gene, and shown that proteins binding to these regions could physically interact. A critical element binds the Ets family protein Elf-1, which associates with NF-kappaB p50, c-Rel, HMG-I(Y), and TFIIB. Based on a binding site selection analysis, it was reported that the optimal site for ElF-1 binding is to an A(A/t)(C/a)CCGGAAGT(A/S) motif, a motif which interestingly is not found in any Elf-1 regulated genes. Instead, Elf-1-regulated genes, including IL-2Ralpha gene, have lower affinity sites, thereby allowing efficient association of Elf-1 only in the context of accessory proteins, and allowing for finer regulation of these genes. Considerable progress has been made in analyzing the STAT proteins (signal transducers and activators of transcription) induced by IL-2. IL-2 can activate both Stat5a and Stat5b (two closely related proteins with>90% amino acid identity) in fresh peripheral blood lymphocytes (PBL) and additionally activates Stat3 in PBL preactivated with phytohemagglutinin. Both human Stat5a and Stat5b have been cloned and it was reported that the genes encoding these two proteins are located on chromosome 17. It was also reported that IL-2-induced Stat5a and Stat5b binding activity could be reconstituted COS-7 cells. Interestingly, however, IL-2-induced transcriptional activation of a Stat5-responsive reporter construct was not induced, suggesting that either a lineage-specific modification or accessory factor was required for activation. This system should prove valuable in clarifying what additional components are required for successful reconstitution. An IL-2 response element was also delineated in the 5' regulatory region of the IL-2Ralpha gene. This element contains binding sites for Stat5 as well as an Ets family protein and provides an understanding of how the IL-2Ralpha gene can be stimulated by antigen and mitogen (via the two enhancer-like element previously described) or by IL-2 (via a mechanism requiring this newly identified IL-2 response element). Thus, these studies provide a molecular basis for IL-2 upregulation of the IL-2Ralpha gene. We have produced Stat5a and Stat5b protein in baculovirus so that the requirements for binding and activation of these proteins can be studied with purified reagents, and have performed the yeast two hybrid analysis to identify factors that can interact with Stat5. Overall, these findings extend our understanding of IL-2 receptor gene regulation as well as other T-cell activation genes regulated Elf-1 and Stat5.


Group of 12 weanling Osborne-Mendel rats were fed diets containing 0, 0.1, 021, 5 or 56% sucrose. Each rat was caged singly with a donor rat that was infected with Streptococcus mutants 6715-15. The recipient and donor were swabbed daily and the bacteria were cultured on Mitis Salivarious agar. Recipient rats fed 56 and 5% sucrose rapidly became infected with high levels of S. mutants. Recipients receiving low sucrose diets remained free of infection for much longer and when infected harbored lower numbers of organisms. The donor rats fed no sucrose failed to retain infection with S. mutants.


The purpose of this study is to 1) compare glycolytic muscle metabolism in patients with chronic fatigue syndrome (CFS) with normal humans, 2) test the feasibility of using near-infrared spectroscopy (NIRS) measurements to evaluate patients with chronic fatigue syndrome, and 3) to determine if exercise training improves muscle metabolism in CFS patients. To address these aims we tested 27 subjects controls and patients with CFS as identified by Dr. Natelson's research group. The medial gastrocnemius was studied and repeated plantar flexions were performed using a isokinetic device. CFS subjects had oxidative capacity (Vmax determined from PCr recovery) compared to control subjects, consistent with our previous studies. Seven of the 21 CFS patients had abnormal rates of H+ production during the 64 s maximal test. Three had slower H+ rates and four had faster H+ rates. NIRS measurements of the rate of oxygen resaturation showed close agreement with PCr recovery rates in control subjects (32.5 + 11 for NIRS compared to 29.9 + 4.8 s for PCr). However, NIRS recovery after exercise was slower the CFS patients than PCr recovery (48.2 + 21 for NIRS compared to 35.4 + 14 s for PCr). In addition, NIRS recovery of oxygen saturation was slower after cuff ischemia in CFS patients compared to controls (20.8 + 13 for CFS compared to 11.3 + 2.8 s for controls). Four CFS subjects have completed the training and testing protocol. Future studies will continue to test CFS patients and control subjects to complete our aims. We plan to continue testing of CFS patients and normal subjects who are enrolling in the exercise program. We also plan to follow up the NIRS findings of reduced oxygen delivery by measuring muscle blood flow in CFS patients with alternative means (Duplex Doppler flow measurements). In one CFS patient with slow NIRS recovery, Doppler measures showed normal peak blood flows, but reduced sensitivity to submaximal hyperemic stimuli.


The field of dermatology is riddled with many problems that expect solutions from modern biomedicine. Several biologicals/biotherapeutical approaches such as anti-TNFalpha antibodies have already made their impact in the daily treatment of dermatology patients. However, many challenges are still ahead. We still have to fill many gaps in our understanding of biological processes in the skin. For example until recently, an entire class of small regulatory RNA molecules, microRNAs, had remained undetected. Their emergence as important players in virtually all tissues and signaling pathways has led us to explore their significance for skin biology. In our proposal we will focus on one microRNA, miR-31, with an exceptional expression pattern in many skin diseases. This finding prompted us to address the functional significance of miR-31 in epidermal homeostasis, aberrant growth control, wound healing, stress response and hair growth. Its involvement in the regulation of major skin signaling pathways such as TNFa, TGFb and BMP signaling makes it a central node for the control of epithelial-mesenchymal transitions. We have identified a crucial role of miR-31 in hair folliculogenesis, hair growth, nail growth, and the response to wounding and the phorbol ester, TPA. TNFa, EGF and FGF7 can induce miR-31 expression and TGFb stimulates the expression of miR-31 precursors. Based on our existing mouse model of miR-31 overexpression and our preliminary data, we have developed a research plan aimed to establish miR-31 as an important regulator of such processes as EMT, proliferation and motility in keratinocytes. Thereby, we will be able to judge the potential of miR-31 as a therapeutic target for skin diseases with defects in these processes. We plan to use our existing mouse model and establish novel tools in this proposal to apply to our career-long goal to translate this knowledge into therapeutic potential of miR-31 using miR-31 inhibitors in vivo. In Specific Aim 1 of this current research plan, we will test our hypothesis that miR-31 is a key regulator of hair growth and the response of the skin to challenges such as wounding. In Specific Aim 2, we will address the important issue of identifying miR-31 target genes to understand miR-31 mediated processes. And in Specific Aim 3, we will test our hypothesis that miR-31 regulates epithelial-mesenchymal interactions and transitions, EMI and EMT. Furthermore, we will test whether miR-31 buffers certain expression patterns and signaling pathways against unwanted fluctuations and define which pathways exactly are influenced by miR-31 in addition to EMT and hair growth regulating signaling networks.


An identified class of neurons in the cerebral cortex respond characteristically to both acetylcholine and norespinephrine. The increase in cell firing produced by acetylcholine is closely mimicked by cyclic GMP while the depression of cell firing produced by norepinephrine was mimicked by cyclic AMP. Other nucleotides (e.g. 5'AMP and adenosine) also produce profound depression of these same neurons which can, however, be selectively blocked by theophylline. In addition, simultaneous application of both norepinephrine and either 5'AMP or adenosine by microiontophoresis result in a synergistic action of both depressants, suggesting that these two substances (norepinephrine and 5'AMP' or adenosine) have an interaction in the cerebral cortex which may be quite important in modulating the output of these neurons. BIBLIOGRAPHIC REFERENCE: T.W. Stone and D.A. Taylor: Microiontophoretic studies of the effects of cyclic nucleotides on the excitability of neurones in the rat cerebral cortex. J. Physiol. 266: 523-543, 1977.


The objective of this research is twofold: first, to analyze the mechanisms by which an adenovirus positive regulatory element activates the expression of viral genes, and second, to see how these regulatory elements influence the initiation and maintenance of cellular transformation. Methods for introducing mutations into early region I, the transforming region of Adenovirus 5, have been developed. One temperature-sensitive mutant has not been isolated, and we plan to isolate others. These temperature-sensitive mutants will be used in pulse labeling experiments after shifting from the permissive to the nonpermissive temperature. We hope to determine from these experiments whether early region functions are continuously required for expression of other viral genes. The mutants will also be used to transform rodent fibroblasts at the permissive temperature. We will determine if the mutant function is required for maintenance of the transformed state by shifting to the nonpermissive temperature and analyzing cellular properties. Those cellular properties that regress will be considered under continuous control of the temperature-sensitive viral product.


This proposal outlines a multidisciplinary effort by four principal investigators to continue development of a series of cyclic and linear conformationally constrained peptide analogues which have high receptor specificity high agonist or antagonist biological activities, high stability in vivo, and prolonged biological activity at kappa (kappa) opioid receptors. This multidisciplinary approach combines modern computer assisted molecular modeling, organic amino acid and peptide chemistry, conformational analysis and molecular mechanics and dynamics calculations, and biochemical, biophysical, physiological and pharmacological studies. The specific aims of this investigation include: a) continues development in the design, synthesis, and evaluation of novel peptides and peptide mimetics derived from dynorphin and related peptides which possess high kappa opioid receptor potency and specificity; b) comprehensive examination of the potency and specificity of all new ligands for kappa vs. mu and delta opioid receptors using radioligand binding techniques. The most selective and potent ligands will be radiolabeled to high specific activity and used in extensive radioligand binding studies and for receptor localization using autoradiography; c) opioid agonist or antagonist activities will be evaluated in vitro using the GPI, MVD, LVP and HVD assays to establish receptor selectivity and potency; d) in vivo kappa receptor properties will be evaluated with the most selectivity compounds using among other assays, the mouse abdominal stretch test following intrathecal administration, and the standard mouse hot plate test; 5) careful examination of the conformational and dynamic properties of the most potent and selective analogues using 1D and 2D nuclear magnetic resonance spectroscopy, molecular mechanics calculations, and other biophysical techniques; and 6) using all of the above results of the design and synthesis of novel compounds with more potent and selective biological activities, and to test conformational models proposed for kappa receptor selective ligands. The long term goal of this research is to develop an understanding of the physiological roles of the kappa opioid receptor in comparison to other opioid receptors, and to develop ligands for the kappa receptors that can be used for the treatment of disease.


DESCRIPTION (provided by candidate): A Scientist Development Award for New Minority Faculty is being requested to support five years of career development and research activities in developmental psychopathology (substance use and violence) among African American adolescents of maternal substance users. The K01 will provide 80% release time from an academic appointment at UM-St. Louis to collaborate with a three-mentor panel available locally. The panel and consultants will provide mentoring and consultation in developmentally psychopathology, clinical observation, advanced statistical analysis, the responsible conduct of research, and guided research activities. The career development activities will be enhanced through coursework in developmental psychology and qualitative research, training in assessment of adolescent psychopathology, and workshops specific to adolescent substance use and violence. The K01 fuses two research focal points that have dominated the candidate's research career: substance use and violence in African American adolescents and psychopathology in adult African American female substance users. The purpose of the K01 award is to prepare the candidate for an independent research career in African American adolescent mental health. The goal of research activities is to collect pilot data that will provide the basis for an R01 study that longitudinally examines the intervening role of maternal substance use on adolescent normal or abnormal development. The research will occur in two phases to maximize research and career development experiences. Phase I of the research will qualitatively assess the role of fathers and the extended family network in adolescent development and in the home environment from a sample of adolescents of maternal substance users. Phase II of the research will gather pilot data on 150 African American adolescents (15-17 years of age) of active substance using mothers (and 50 demographically matched comparisons). Mentors and consultants will provide expertise and oversight in all career development and research activities.


Cognitive decline is a common, but not inevitable, accompaniment to aging. While much research is currently being directed at Alzheimer' s Disease -- one of the most severe expressions of cognitive decline -- relatively little work is currently aimed at identifying risk factors and mechanisms associated with early (i.e., subclinical) cognitive decline, even though this may be the stage most amenable to prevention. An important issue that needs to be clarified in relation to the etiology of cognitive decline is the role of environmental lead exposure and the interaction of lead with specific candidate genes. In this application we discuss how four candidate genes -- APOE, the HFE (hemochromatosis) gene, transferrin, and Tau protein -- may interact with lead burden to increase the risk for oxidative cell damage leading to neuronal cell loss and cognitive deficits. We review the last 11 years of our research on low-level lead toxicity and describe compelling preliminary data. We then propose a new study that calls for new data collection in our established Bostonarea cohorts [Normative Aging Study (NAS), Nurses Health Study (NHS), and Community Lead Study (CLS)]. Our specific aims are to test hypotheses related to the impact on cognition of lead burden and its potential interaction with our four polymorphisms of interest. We will look at cognition cross-sectionally and longitudinally, using a validated battery of telephone cognitive function assessment tools as well as a battery of in-person cognitive tests that we have been administering in person since 1993. Results of this research promise to shed further light on lead's potential impact on society and may also give rise to tools for identifying susceptible individuals as well as early brain effects.


The Vector Core is a critical resource to members of the Institute for Human Gene Therapy providing access to a wide array of gene transfer technology. This dedicated facility is run by Dr. Guang-ping Gao, who has extensive academic based experience in recombinant viruses. The Core has assembled a comprehensive inventory of plasmids, cell lines, and viruses useful to the development of vectors. More comprehensive service is provided in the design, creation, production, and analysis of recombinant viruses. A number of vector systems are available through the Core, including murine-based retroviruses, adenoviruses, and adeno-associated viruses (AAV). The Vector Core maintains strong links to key laboratories in the Institute for Human Gone Therapy involved in novel vector development. Relevant innovations are quickly brought into the Vector Core, where they are validated and distributed to faculty. In the context of this P01, the Vector Core will play an important role. Methodology for creating chimpanzee adenoviruses will be developed in Project 1 and transferred to the Core who will provide services in terms of vector creation and expansion for all four projects.