Schistosomiasis is a tropical parasitic disease infecting over 200 million people. Treatment relies on a single drug, praziquantel (PZQ). In the absence of back-up drugs with PZQ's therapeutic spectrum, the risk of resistance to PZQ and eventual drug failure is a major concern. Traditional phenotypic screens, using adult- stage S. mansoni, are low-throughput and incompatible with modern high-throughput screen (HTS) systems. In keeping with the NIAID's mission, the present proposal aims to turn a newly developed, moderate- throughput phenotypic screen (MTS), into a fully automated, quantitative HTS to accelerate drug discovery for this infectious disease. The proposal involves three PIs with ongoing collaborations and respective biological, screening-technology and bio-computational skills who are focused on just this goal. As a first research track for this proposal, we will utilize in-house automation and a high-content screening (HCS) system to significantly increase throughput over our published MTS approach. The proposal will involve;expanding robotic plating of the parasite, developing protocols for bright-field and fluorescence-based microscopy and adapting commercial image-analysis software to identify (segment), quantitatively describe and track the motion of parasites with a view to prioritizing compounds for further pre-clinical development. Because commercial HCS analysis tools are not likely optimized for recording the complex and dynamic phenotypes displayed by this multicellular parasite, we will also pursue a second and parallel track of research. Specifically, we will develop de novo an automated image-analysis screening technology to define, identify, and quantify the range of phenotypic responses (morphological and behavioral) possible in this parasite. Ultimately, both the experimental and computation tracks will together produce a standardized HTS protocol and a comprehensive, quantitative suite of image-analysis programs to categorize parasite phenotypes. Such rigor will facilitate the screening of large numbers of potential compounds and their prioritization into the secondary and tertiary screening assays available in-house. We also intend to make the algorithmic framework including its methods and implementations, publicly available. PUBLIC HEALTH RELEVANCE: The major goal of the project is to turn a moderate-throughput phenotypic screen (MTS) system for schistosomiasis, into a fully automated, quantitative high-throughput screen (HTS). By so doing, the rate of discovery of drugs to treat this global tropical disease will be increased.


Breast cancer is a heterogeneous disease that can be subclassified into several tumor types. Inflammatory breast cancer (IBC) is the most aggressive form of breast cancer. IBC is a locally advanced breast cancer that affects women at an earlier age than other types of breast carcinoma. The disease is characterized by a high tumor microvessel density, fast disease progression, and by extremely poor survival. Although the frequency of IBC among breast cancer cases in the US is only 3-5%, it appears to be much more common in other geographic areas, such as North Africa. The causes of IBC are unknown and both environmental and genetic risk factors are thought to be involved. The early onset of the disease, the strong angiogenicity and propensity of IBC to invade vessels, and the large disparity in the IBC incidence among various populations are indicators of genetic predisposition and inherited susceptibility. Both low- and high-risk cancer susceptibility genes may determine the risk for IBC. Two genes, WISP3 and RhoC GTPase, have recently been identified that are differently expressed in IBC when compared with non-IBC breast cancer. WISP3 is a putative tumor suppressor gene for the disease, and may regulate the insulin-like growth factor pathway. RhoC GTPase is an overexpressed oncogene in IBC, and modulates the induction of angiogenic factors in breast cells. As part of our ongoing breast cancer case-control study, we collected seven specimens from inflammatory breast cancer (IBC) patients, 20 specimens from non-IBC breast cancer patients with poor survival and 20 specimens from non-IBC breast cancer patients with good survival. Serial sections of OCT-fixed tissues have been prepared from the 47 fresh-frozen tumors, and ten normal breast tissues from breast reduction surgery. The first slide of the serial sections has been formalin-fixed and stained with hematoxylin and eosin to identify tumor and surrounding normal tissue. The slides have been reviewed by a pathologist for the presence of tumor cells. The sections are currently being microdissected. We hypothesize that IBC has a distinct expression profile in tumor and normal surrounding tissue that predicts the risk of metastasis and poor survival. Our objective is to identify genes that are preferentially expressed in tumors and surrounding normal tissue of patients with IBC by comparing the IBC gene expression profile with the profile of non-IBC breast tumors. This is a novel approach. IBC is a rare disease, and it is difficult to collect flash-frozen IBC specimens that are suitable for array-based gene expression analysis. We have a particular interest in those genes that promote metastasis, and are expressed in the surrounding normal tissue, but not in cancer cells. To date, no report describes an expression profile in surrounding normal tissue that is associated with tumor metastasis and poor survival in breast cancer. However, a recently reported molecular signature of metastasis in primary breast tumors contained genes that are thought to be expressed in normal tissue, and not by cancer cells. Specific aims 1) Array-based gene expression profiles will be generated for all breast tumors and the surrounding normal tissues, and for ten disease-free breast reduction tissues. We will use laser-captured microdissection to separate tumor from normal tissue. Supervised methods for class comparison and prediction will be used to establish gene expression profiles that distinguish 1) IBC tumors from surrounding normal tissue, 2) IBC tumors from non-IBC tumors, and 3) surrounding normal tissue of IBC tumors from surrounding normal tissue of non-IBC tumors. Non-IBC tumors will be subclassified into patient groups with either good or poor survival, and will be analyzed as separate groups. 2) Candidate marker genes for tumor progression and survival will be validated by Taqman, and further studied by immunohistochemistry, Western blotting, and in appropriate experimental models.


The Clinical Trials Office of the UNMC Eppley Cancer Center participates in the activation and conduct of clinical cancer research at the Cancer Center. This includes assistance with protocol development, coordination of initial and periodic reviews submitted to the Institutional Review Board, data coordination and collection, and coordinates supervision of appropriate clinical trials by the Audit Committee and the Data and Safety Monitoring Committee. Since the last review (7/1/99 through 6/30/03), a total of 3171 patients have been entered onto Cancer Center clinical research protocols, of which 1054 patients have been entered onto therapeutic trials and 2117 patients were entered onto correlative clinical research studies, companion studies, and chemoprevention clinical research protocols. During the past four years (7/1/99 - 6/30/03) 385 patients were entered onto investigator-initiated clinical trials. During the past year (7/1/02 - 6/30/03), 41 Cancer NCI-A 40 2 P30 CA036727-20 COWAN, K Center members have utilized this facility, of which 26 had external funding, and 156 clinical research protocols were open to accrual during this period. In the last year (7/1/02-6/30/03), 558 patients were entered onto Cancer Center clinical research protocols, of which 310 patients (or 56% of the total patients) were entered onto therapeutic trials. Over the last year, the Cancer Center has undertaken several initiatives to enhance clinical research activities by increasing the support of the administration of the Clinical Trials Office, consolidation of the administrative staff of Clinical Trials Office in a central location in the Lied Transplant Center and the establishment of a Data and Safety Monitoring Committee.


Selenium has a role in preventing heart disease and is rapidly becoming recognized as a chemopreventive agent against cancer. The beneficial affects of this element are due, at least in part, to its presence in selenoproteins as the amino acid, selenocysteine (Sec). Our program, therefore, focuses on the means by which Sec is incorporated into protein and the role of specific selenoproteins in human health. As Sec tRNA is regarded as the key molecule in selenoprotein biosynthesis, we have produced transgenic mice encoding as few as 2 and as many as 20 extra copies (transgenes) of the Sec tRNA gene to examine effects of its overexpression. We introduced a silent mutation in the transgene that enables us to distinguish its expression from that of the corresponding wild type gene by primer extension. The transgenes and the wild type genes contribute equally to the total Sec tRNA population, but the level of expression is not linear with gene copy number, strongly suggesting that gene expression is regulated by a feedback mechanism. Overexpression does not appear to affect selenoprotein synthesis or protein synthesis as a whole. This observation, coupled with our earlier findings of normal selenoprotein synthesis in cells containing only ? the Sec tRNA population, demonstrate that Sec tRNA is not a limiting component in selenoprotein translation. We have also sequenced the Sec tRNA gene in zebrafish and Chinese hamsters. Zebrafish contains two functional copies of this gene whereas all other higher and lower animals examined to date contain only one functional gene. Chinese hamsters contain three pseudogenes whereas all other organisms examined to date contain only one or no pseudogenes and since the Chinese hamster gene encodes two pyrimidine transtions compared to all other mammals, it seems likely that these transitions occurred by editing of a transcript and re-insertion into its genome. In our studies on examining the role of specific selenoproteins in human health, we have focused on a recently discovered selenoprotein, designated as the 15 kDa protein. We have shown that its levels are normally elevated in prostate as compared to other tissues, but are reduced substantially in prostate cancer. Since selenium is known to have a chemopreventive role in prostate cancer, it would seem that the 15 kDa protein may have a role in preventing malignancy in this tissue. We, therefore, have made the appropriate constructs encoding the 15 kDa protein to examine the affects of its overexpression in mammalian cells in culture and in transgenic mice. These studies are in progress. We have confirmed the occurrence of two polymorphisms in the 3-untranslated regions of the 15 kDa protein gene and 1 of the polymorphisms may be involved in regulating protein expression. There was statistically significant differences in allele distribution between head and neck cancer tissues and controls. In a related study on the effects of selenium on human health, a previous member of our group (V.N.G., see collaborators below), had shown earlier that the incorporation of selenium into protein is diminished in HIV infected cells. We have subsequently shown that the HIV encoded TAT protein binds to the SECIS element which is a specific stem-loop structure in all selenoprotein mRNAs required for insertion of Sec into protein; but TAT also binds to most double stranded RNAs examined and thus shows no specificity. We are presently using in vivo experiments to determine if specificity occurs in selenoprotein synthesis. We have discovered two new human selenoenzyme thioredoxin reductases and characterized the role of a previously identified thioredoxin reductase in redox regulation of cell signaling. In addition, we identified a thioredoxin reductase in C. elegans and this appears to be the only selenoprotein in this organism. Our study on examining the Sec tRNA population in Drosophila has been completed as well as our study on examining the role of hypermodified bases in the anticodon loop of specific tRNAs on ribosomal frameshifting. - Genes, Gene Targeting, Gene expression, Post-transcription regulation, Selenium, Selenocysteine, Transgenic mice, Translation, - Neither Human Subjects nor Human Tissues


The E3 ubiquitin ligase Mdm2 acts as a key factor in the degradation of p53 tumor suppressor in human cancer cells and inhibition of Mdm2 activity is a validated approach to activate p53 function in cancer therapy. Nevertheless, inhibitors of Mdm2 such as Nutlin-3 has many limitations, suggesting that additional targets in this pathway need to be further elucidated. The proposed studies aim to dissect the mechanisms of two newly identified ubiquitin ligases critically involved in ARF/p53-mediated tumor suppression and provide the unequivocal evidence for proof of concept to develop the inhibitors of these two potential critical targets in cancer therapy. ARF was originally identified as an alternative transcript of the Ink4a/ARF tumor suppressor locus. It is well accepted that ARF plays a major role in activating p53 function, specifically, in respond to oncogenic stress. Surprisingly, by usig a mouse model in which p53 status can be reversibly switched in vivo between functional and inactive states, it has been found that the protection from tumorigenesis is absolutely dependent on ARF-mediated p53 activation in vivo. Thus, ARF apparently plays a much more important role in suppressing tumorigenesis in general, than originally anticipated. Restoration of the ARF-p53 function remains an important goal in the quest for more effective cancer therapeutics. Indeed, like ARF, Nutlin-3, a small molecule inhibitor of Mdm2, is able to activate p53, and exhibits antitumor efficacy in cancer cells that express wild-type p53. Nevertheless, although Nutlin-3 can effectively block the interaction of Mdm2 and p53, Nutlin-3 is also very effective at increasing the synthesis of Mdm2 mRNA and protecting Mdm2 from degradation. These effects of Nutlin-3 on Mdm2 lead to a rapid restore of cancer cell growth upon temporary removal of the compound, sabotaging the efficacy of nutlin-3 in cancer therapy. Thus additional cancer targets aiming at this pathway are clearly needed for more effective therapeutic purpose. Our preliminary studies reveal that targeting two novel ubiquitin ligases (ARF-BP1 and ULF) in the ARF-p53 pathway represents a particularly attractive approach to activate ARF-mediated tumor suppression in cancer therapy. The central hypothesis to be tested here is that inactivation of ARF-BP1 or ULF can effectively activate ARF-mediated function and suppress tumorigenesis in vivo. It includes the following two specific aims. In Aim 1, we will investigate whether inactivatin of ULF activity is sufficient to suppress tumorigenesis by using mouse models. In Aim 2, to validate whether ARF-BP1 is indeed a potential cancer target, we will use the ARF-BP1 mutant mice to examine whether inhibition of ARF-BP1 suppresses tumorigenesis in vivo in a manner reminiscent of ARF activation.


Synaptic connections are formed during development but are continually modified through experience and learning and memory. Any alteration to the normal connectivity leads to behavioral and cognitive changes that underlies psychological and neurodegenerative disorders. Therefore, understanding how functional networks of synaptic connections are established is critical for gaining insights into normal brain function. A mutant screen for defects in synapse in Drosophila has led to the discovery of a mutant line that shows aberrant synapse formation. Named as baha (bad hair day), homozygous mutant photoreceptors and motor neurons project to their correct target area in early developmental stages but subsequently fail to maintain contacts or form mature and functional synapses. Preliminary work thus suggests that baha may function as a critical factor in synapse formation. To investigate baha's role in synaptogenesis, I propose to perform further characterization of its synaptic phenotype, to identify and clone the baha gene, and to investigate the molecular mechanisms of its action. Cloning and characterization of baha gene may thus lead to identification of components of synapse formation, a process that remains poorly understood.


The objective of our Research is to elucidate the mechanism of electron transfer and proton translocation in the cytochrome b-c1 region of the mitochondrial respiratory chain. During the forthcoming project period our approach will be to test which electron transfer reactions in the b-c1 region take place in the absence and presence of the "g equals 1.90" iron-sulfur protein, which we have recently purified in a reconstitutively active form from heart mitochondria.


Obstetric practices, evaluative tools, and treatments have been sometimes passed down by tradition or introduced without rigorous study to evaluate their impact on maternal, fetal, and/or neonatal outcomes. Long-term maternal and infant outcomes are particularly understudied, and surrogate markers of morbidity are commonly substituted in studies of smaller size. The impact of pregnancy complications can be devastating, affecting decades of life. The health and financial impacts of adverse maternal and infant outcomes on the family are potentially staggering. With millions of births in the United States each year, even uncommon complications can have a profound societal impact. Since its inception, the Eunice Kennedy Shriver NICHD Maternal-Fetal Medicine Units (MFMU) research network has conducted clinical trials and observational studies that have brought important understanding regarding prediction and prevention of preterm birth (Preterm Prediction Study, 17-OH progesterone in singletons, twins, and triplets, omega-3 supplements, antibiotics for asymptomatic bacterial vaginosis and T. Vaginalis), treatments for those at imminent risk (antibiotics for preterm labor and premature rupture of the membranes, repeated antenatal corticosteroids, magnesium sulfate neuroprotection), cesarean delivery and VBAC, prevention of preeclampsia, and treatment of gestational diabetes, among others. This network played a key role in evaluating technologies used in obstetric practice (ultrasound cervical length, fetal fibronectin, home uterine monitoring, fetal pulse oximetry). In this application, we demonstrate that the Department of Reproductive Biology at Case Western Reserve University (CASE) investigators have the ability to conduct collaborative research, and have successfully participated in and are qualified to continue in the MFMU network. Our investigators have strong backgrounds in clinical and multicenter studies, those involving neonatal and long term infant follow-up, and in the above listed network studies. CASE investigators provide leadership in network study development and administrative committees necessary to study completion and dissemination of their results. There is a strong administrative and research infrastructure to support the MFMU network, including the CASE-Clinical & Translational Science Collaborative. CASE offers the strengths needed to successfully lead and participate in NICHD-MFMU network research which will directly influence obstetric practice and improve pregnancy outcomes nationally and internationally. PUBLIC HEALTH RELEVANCE: Research performed by the CASE Department of Reproductive Biology, within the Eunice Kennedy Shriver NICHD-MFMU network, will inform and change obstetric practice. This research will identify interventions and tools to predict, prevent or mitigate pregnancy complications such as preterm birth, preeclampsia, fetal growth restriction and perinatal infections, among others. Focusing on significant perinatal and long-term outcomes, we will bring to an end procedures and practices that are ineffective or incur undue risks.


Alzheimer's disease (AD) is a progressive neurodegenerative disease characterized by gradual synapse and memory loss. While significant progress has been made in understanding Alzheimer's disease (AD) pathology, treatment options for AD remain limited. Therefore, further characterization of signaling pathways involved in the pathology of AD is important in facilitating the identification of new therapeutic targets. Emerging evidence postulates that epigenetic changes play critical roles in dementia. Consistent with this, inhibitors of histone deacetylases (HDACIs), such as SAHA (suberoylanilide hydroxamic acid; vorinostat), enhance neuroplasticity and memory-associated gene expression in the brain. These findings suggest that HDACIs may be novel agents in treating AD. Recently, a drug discovery team at Georgetown University has developed new Class II-specific HDAC inhibitors (mercaptoacetamide-based 6MAQH (W2), and hydroxamide-based H6CAHA (I2)), which have improved solubility and pharmacokinetic properties compared to HDACI SAHA. We have exciting preliminary data suggesting that 1) W2 decreases Ab levels in stably overexpressed APP N2A cells, 2) W2-injected hAPP 3xTg AD mice (9-10 month old, moderate Ab production) decreases Ab levels compared to control treatment, 3) W2 treatment decreases mRNA levels of A synthesis enzymes and increases mRNA levels of A degradation enzymes (MMP2), and 4) W2-injected hAPP 3xTg AD mice (9-10 month old, moderate memory impairment) rescued memory deficits compared to control treatment as measured by Morris Water Maze. Based on these observations, we hypothesize that Class II HDACIs (W2, I2) regulate AD pathological processes and cognitive functions by modulating the gene transcription of A synthesis and degradation enzymes. To test this hypothesis, we will examine the molecular mechanisms by which W2 and I2 regulate AD pathological processes (Aim 1). We will examine the role of Class II HDACIs W2 and I2 on rescuing cognitive deficits in 3xTg AD mice (Aim 2). Results from our study will provide the molecular mechanisms of action of Class II HDACIs on Ab production, Ab plaque formation, and cognitive function for future development of AD therapeutics.


The recent availability of a genetic test (homozygosity for the C282Y mutation in the HFE gene) for the diagnosis of hereditary hemochromatosis (HH) has focused renewed attention on this disorder and has resulted in the diagnosis being made in younger subjects with smaller iron burdens. However, phlebotomy therapy in HH remains hampered by lack of simple, physiologic laboratory monitoring guides. In addition, phlebotomy therapy has been perceived as wasteful because the blood obtained has not been not used for allogeneic transfusion although many HH subjects meet standards for allogeneic blood donation. Recent regulatory changes now allow increased flexibility in establishing policies for transfusion of blood obtained from HH subjects. In studies performed over the last 12 years, we have developed a simple, physiologically based phlebotomy guide as the basis for a protocol to utilize blood from appropriate hemochromatosis patients for allogeneic transfusion. Prospective studies in the DTM have shown that the red cell mean corpuscular volume (MCV), a physiologic, precisely measured indicator of erythopoietic iron availability, when measured in conjunction with the hemoglobin, provides all the information necessary to manage patients during induction as well as maintenance phlebotomy. Phlebotomy was started weekly and transitioned to less frequent intervals when the MCV decreased to 5 to 10 percent below baseline and the hemoglobin and MCV were decreasing n concert. During maintenance phlebotomy, the hemoglobin was targeted at greater than 13 g/dL and the MCV was kept at 5 to 10 percent below baseline (generally 85 to 90 (3). A stable, asymptomatic, iron-depleted state was obtained in all patients after a median of 38 phlebotomies and removal of 9.0 grams of iron. The MCV was stable for a prolonged period during induction therapy, and then decreased in a smooth, consistent manner, indicating the onset of iron-limited erythropoiesis. Transferrin saturation varied considerably during phlebotomy, but remained below 40 percent during stable MCV-targeted maintenance. Ferritin values were not useful to assess the pace of iron depletion or maintenance phlebotomy. The median stable maintenance interval was 7.5 (range 6 to 16) weeks, corresponding to an average daily iron removal of 35 to 67(g/kg. The data in this study indicate that the red cell MCV is a physiologic, inexpensive guide to phlebotomy therapy in patients with HH. In contrast to standard, more expensive measurements such as ferritin levels, the MCV accurately predicts iron depletion, prevents excessive rebound transferrin saturation and guides maintenance according to each patient?s individual iron absorption. The MCV-guided phlebotomy guideline, along with extended rheumatologic and hepatic evaluations, forms the core of a large, omnibus protocol being developed to optimally manage phlebotomy in hemochromatosis patients while utilizing blood from appropriate patients for allogeneic transfusion.


Project Summary The successful management of chronic pain is inadequate in many patients and has contributed to the abuse of and addiction to opioids, a continuing major public health crisis in the United States and worldwide. The development of non-addictive pain therapeutics can help counter opioid addiction and benefit patients, including those who suffer from neuropathic pain, and in particular diabetic neuropathic pain (DNP). Our project?s goal is to develop a safe, efficacious and non-addictive small-molecule drug that activates Kv7 voltage-gated potassium channels to address overactive neuronal activity in DNP. The first specific aim is discover Kv7 activators that favor Kv7 isoforms altered in DNP and found in dorsal root ganglia, namely the Kv7.2/3 isoforms. Through iterative lead optimization studies on our novel series of Kv7 activators, we are targeting Kv7.2/3 activation with selectivity over both Kv7.4 channels and another off target of other Kv7.2/3 activators, GABAA receptors. This approach is expected to decrease off-target side effects observed with the use of earlier non-biased Kv7 activators including urinary retention (mediated by Kv7.4), and somnolence and dizziness (mediated by enhancement of GABAA receptor function). This phase also includes optimization of absorption, distribution, metabolism, excretion, and toxicity profiles and building correlations between in-vitro activities to in-vivo efficacies in a neuropathic rat DNP model. A second animal model, the L5/L6 spinal nerve constriction model, will also be used to test the ability of candidate compounds to generalize to other forms of neuropathic pain. The second specific aim will be to further characterize two to four advanced compounds by assessing additional pharmacological properties including CYP450 induction/time dependent inhibition and in-vitro safety/selectivity panels. The third aim is to select a candidate for study in non-GLP toxicology and pharmacokinetic studies in rodent and non-rodent species. Specific aims four and five involve completing the studies needed to prepare an Investigational New Drug (IND) application. These studies include Chemical Manufacturing Controls activities such as formulation studies and Good Manufacturing Practices synthesis of drug candidate followed by cardiovascular and safety pharmacology studies, and 28-day Good Lab Practices toxicology studies in two animal species. This screening paradigm is intended to establish a clinic-ready, well-tolerated and widely effective product to treat neuropathic pain.


We have previously demonstrated that beta-arrestin1 and 2 form complexes with various prostaglandin receptors during mouse skin tumor development (Chun et al., Carcinogenesis, 9:1620, 2009). Therefore, the goals of the present study were to elucidate the effects of beta-arrestin1 or 2 deficiency on mouse skin tumor development using the multi-stage mouse skin tumor model. Surprisingly, beta-arrestin 1 decreased the number and size of skin tumors using this model; whereas the deficiency of beta-arrestin2 did not effect the number of tumors but in fact significantly increased tumor size. Furthermore, while beta-arrestin1 deficiency decreased p-ERK1/2 and COX-2 induction, beta-arrestin2 deficiency increased p-ERK1/2 but had no effect on COX-2 expression. Further studies are underway to better elucidate the effects of beta-arrestin1 and 2 deficiency on the signaling pathways we have previously demonstrated to be involved in mouse skin tumor development. In the ovarian cancer studies, our results to date indicate that COX-1, but not COX-2, deficiency reduces mouse ovarian cancer development by about 50%.


Project Summary Significant progress in human cancer therapy in the last decade has been driven by conceptionally new approaches to targeting cancer, including cancer immunotherapy, cancer nanotherapy, or new types of biologics and small molecules. Both my dissertation and postdoc research will be focused on the development of fundamentally new approaches to targeting cancer. My dissertation research is focused on the development of photoswitchable lipids for the optical control of lipid metabolism and function. In addition to targeting specific receptors, ion-channels, or enzymes, light-induced structural changes in a lipid nanoparticle (LNP) could serve as trigger for the release of encapsulated drugs. Triggered release could markedly improve the efficacy of clinically approved LNP-based cancer therapeutics, which include Doxil/Caelyx, DaunoXome, Myocet, Lipo-Dox, or Marqibo. I seek to design and synthesize photoswitchable lipids for `photoactivable lipid nanoparticles', herein termed paLNPs, that allow for effective light- triggered release of encapsulated cancer drugs. Two complementary approaches will be developed for small molecule drugs and RNA-based therapeutics and the pharmacological properties of paLNPs will be systematically investigated in vitro and in cell culture. In my postdoctoral research I seek to use my acquired knowledge in chemistry, lipid-biology, and medicine to develop lipid-drug conjugates for biological targets that function at plasma-membrane signaling hotspots. The initial target will be the mutated oncogene KRAS G12C, which is ideally suited for this new approach. Conjugating selective covalent modifier of this oncogene with a lipid will attach an additional lipid tail to the surface of KRAS that could largely alter its membrane-protein interaction and in the best case completely inhibit its function. This could markedly increase the efficacy of the covalent pharmacophores currently in clinical trials. I seek to synthesize and systematically study these lipid-drug conjugates in vitro and in cell culture.


This proposal will investigate virulence mechanisms of cytoadherence/sequestration using Babesia bovis as a natural host model system. Virulent B. bovis infection is associated with development of severe clinical disease and neurological manifestations similar to those observed in human patients with severe malaria caused by virulent strains of P. falciparum. The proposed unique model enables in vivo study of virulence mechanisms using defined and reproducible virulence phenotypes. In this model, the role of cytoadhesion/sequestration in determining virulence and whether specific gene products mediate these phenotypes can be determined. Published and preliminary data from three virulent and derived attenuated B. bovis pairs strongly supports the role of VESA1 proteins in virulence, and the overall hypothesis that differential VESA1 protein expression alone may be responsible for mediating virulence. Clonal lines of virulent and attenuated strain pairs will be used and their differential phenotypes confirmed. The specific VESA1 surface protein dimer associated with sequestration and expressed by virulent versus attenuated strains both in vitro and in vivo will be identified using monoclonal antibodies. Direct demonstration that a specific VESA1 dimer is associated with sequestration and virulence will be determined by complementation and expression of cytoadhesion and virulence associated ves1 gene(s) in the attenuated T2Bo clonal line, followed by an in vivo study to demonstrate acquisition of the virulence phenotype. If the hypothesis is correct, and if similar mechanisms are used in other pathogens, molecular targets for intervention to prevent severe clinical manifestations and death associated with hemoparasite infection can be further examined. Future directions will be to determine specific domain(s) of VESA1 proteins required for virulence, determine whether these domains are present in other VESA1 proteins and if so whether these proteins are also associated with virulence. Once this is established, the design and testing of virulence directed interventions such as peptide analogues or vaccines to reduce the morbidity and mortality associated with hemoparasite infection in animals and humans will be possible. PUBLIC HEALTH RELEVANCE (provided by applicant): By expanding our understanding of mechanisms used by pathogens to cause disease (virulence mechanisms), advances in targeted therapeutics and vaccines to significantly reduce morbidity and mortality will be made. For blood-borne parasites such as malaria and babesiosis, both of which contribute very significantly to the global health burden in resource poor countries, cytoadherence is a virulence mechanism which leads to severe clinical signs associated with increased mortality. This project will use Babesia bovis infection of cattle as a natural animal model of cytoadherence and virulence, and will address a significant knowledge gap to demonstrate the causal relationship of specific parasite derived gene products to virulence.


16.0 Abstract: Hormone Responsive Cancers Program The overarching goal of the Hormone Responsive Cancers (HRC) Program of UMGCC is to reduce morbidity and mortality from cancers of hormone-responsive tissues. To achieve this mission, the program focuses on three specific themes and related aims: Theme 1: Therapeutic strategies against hormone-responsive tumors?identify new agents to target malignancies that remain responsive to hormone manipulation; Theme 2: Mechanisms of innate and acquired hormone resistance?identify and target the mechanisms that confer de novo or acquired resistance to hormone manipulation; and Theme 3: Invasion and metastasis? identify the mechanisms that promote tumor dissemination and identify therapeutic strategies to target these mechanisms. The HRC Program has 47 members representing 16 academic departments and 5 schools/colleges of the University of Maryland. Members of the program conduct cancer-focused research that receives $7.1 million total annual funding, including $3.4 million from NCI and $2.6 million from other peer-reviewed sources. In addition, HRC Program members receive over $1 million annually from non-peer-reviewed funding sources. Between January 2010 and December 2014, HRC members authored 407 cancer-related publications, of which 30 percent resulted from intraprogrammatic and 23 percent from interprogrammatic collaborations. Twenty-two percent of the publications represent collaborations with external investigators. Research efforts of HRC faculty are supported by extensive use of the BSS, FCSS, GSS, ISS, PBSS, and TLSS.


Abstract While inflammation is a normal, protective response to injury, it is becoming increasingly clear that uncontrolled inflammation is integral to most disease processes including cancer and heart disease, the leading causes of death in the United States. The resolution phase of inflammation is an active process involving downmodulation of proinflammatory mediators and clearance of dead or dying cells. Understanding the molecular mechanisms involved in the resolution of inflammation will provide a framework from which to design directed therapeutics to prevent or diminish aberrant inflammation. Rapid and efficient removal of apoptotic cells is critical to the resolution of inflammation. CD93 is a cell surface molecule required for ingestion of apoptotic cells in vivo and is expressed by myeloid cells, platelets and endothelial cells: those cell types principally involved in regulation of inflammation. It belongs to the Group XIV family of transmembrane C-type lectin-like domain (CTLD) containing glycoproteins with shared functions in adhesion, migration and inflammation. We have recently identified a soluble form of CD93 that is elevated in inflammatory fluids. This proposal tests the hypothesis that CD93 regulates monocyte/macrophage activation extracellularly via its membrane tethered or soluble ectodomain, as well as intracellularly via protein and lipid interaction with the intracellular tail. Specifically, the role of CD93 in regulating inflammation in vivo will be assessed in a sepsis model using both CD93-/- mice and mice double deficient in CD93 and TM-D1, a CD93 homologue that downregulates inflammation in sepsis. Soluble CD93 will be tested for its ability to enhance ingestion of apoptotic cells and gram negative bacteria by primary human and mouse phagocytes. Anti-inflammatory properties of sCD93 will be investigated and compared to TM-D1. Membrane tethered CD93 regulates phagocytosis and adhesion in vitro, critical processes in the regulation of inflammation. Previous studies that have identified specific CD93 cytoplasmic tail interacting molecules known to be involved in cytoskeletal dynamics, such as those required for phagocytosis and adhesion, will be extended. Localization and function of these molecules during the phagocytic process will be assessed. These studies will contribute to our understanding of inflammation, and may lead to CD93 directed therapeutics to regulate this process.


Liver is the primary site of alcohol metabolism and is the primary organ that is injured by alcohol. Though it has long been clear that ethanol is metabolized to acetate in hepatocytes, it is known that the acetate isn't burned as fuel in the liver or directly converted to fat in the liver. The knowledge that the calories from ethanol are burned in the muscle may have inhibited people from considering whether any further metabolism of acetate occurs in the liver and whether this could be linked to alcohol hepatopathology. Recently, liver mitochondrial protein acetylation has been described in response to alcohol ingestion. This means that liver mitochondrial proteins are tagged with the exact same molecule that liver produces when we drink alcohol. We also know from genetics that many mitochondrial proteins with the acetylation tag are inhibited by this adduct and that mice that have this modification accumulate in mitochondrial proteins get fatty liver. In this proposal, a straightforward mechanism from ethanol to acetylated liver mitochondrial proteins is proposed and will be tested. Moreover, nicotinamide riboside, a vitamin that has the property of increasing NAD+ in mitochondria, is proposed as an agent to protect against the development of alcohol-derived mitochondrial protein hyperacetylation and alcoholic fatty liver.


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. . " Glyceollins as novel targeted therapeutics for the treatment of metastatic triple negative breast cancer." Dr. Collins-Burow obtained her Ph.D. from Tulane in 1998. She then went to medical school at Tulane and carried out her residency and fellowship in hematology/oncology at Tulane as well. Although she stayed in touch with the laboratory somewhat and carried on some research, she needs time to get her laboratory work going again. Thus, we decided that she would be an alternate rather than a PJI and would receive pilot funding from the Cancer Center to begin to establish her model systems. Her outstanding fit in terms of animal models of carcinogenesis, as well as cell survival would make her an outstanding replacement for a graduating PJI when her work will be able to take off. Dr. Collins-Burow has demonstrated in the MDA-MB-231 cell line that glyceollin inhibits proliferation/survival of these cells in clonogenicity assays. Utilizing an in vivo xenograft mouse model she has shown the ability of glyceollin to suppress tumor growth of the MDA-MB-231 cells. A very exciting finding on follow-up of this study was a decrease in visceral lung metastasis in the animals treated with glyceollin. Based upon these preliminary data she proposes to test the overall hypothesis that glyceollins function as a novel targeted agent in triple negative breast cancer suppressing tumorigenesis and metastasis. She propose the following specific aims: Specific Aim #1, To test the hypothesis that glyceollins block triple negative breast cancer migration/invasion and chemokine signaling in vitro. Specific Aim #2, To test the hypothesis that glyceollin regulates key clusters of gene expression associated with a metastatic phenotype and chemokine function in triple negative breast cancer cells. Specific Aim #3, To test the hypothesis that glyceollins suppress in vivo tumorigenesis and metastasis of triple negative breast cancer cells. We believe that the glyceollins represent the parent structure for a new class of anti-cancer agents that can target tumorigenesis and metastasis of triple negative breast cancer while enabling us to begin to elucidate the distinct molecular targets of this disease which will ultimately be translated to a clinical trial for triple negative breast cancer.


The goal of this proposal is to address the growing problem of prescription opioid abuse by examining the effects of oxycodone in a laboratory setting in the presence of clinical pain and following significant pain relief following maintenance on the buprenorphine / naloxone combination (Bup/NX). Participants diagnosed with moderate pain initially will be admitted to hospital and transitioned from their baseline prescription opioid to a standing daily dose of Bup/NX. During the Bup/NX maintenance periods, participants will have the opportunity to self-administer oxycodone and subjective responses to oxycodone as well as analgesic, physiological and performance effects will be measured. In the second phase of this study, the same patients who participated in the inpatient phase will be followed on an outpatient basis while maintained on Bup/NX. Primary outcome variables during the outpatient phase will include percentage of negative urine toxicology samples for opioids and number of weeks in treatment. A major goal of this proposal is to determine which variables collected in the laboratory most reliably predict subsequent relapse to opioid abuse. In addition, the utility of Bup/NX in treating patients diagnosed with both chronic pain and substance abuse will be assessed. Despite the significant increase in prescription opioid abuse over the past 5-10 years, very few studies have systematically examined the issues surrounding prescription opioid abuse in the population of individuals with chronic pain. To our knowledge, no studies to date have examined opioid self-administration in persons with pain who have a history of opioid abuse. Specifically, the current proposal aims to 1) characterize the behavioral effects of oxycodone in Bup/NX-maintained pain patients who are currently abusing their prescription opioids; and 2) examine the relationship between behavioral effects of oxycodone as measured in the laboratory setting, and subsequent relapse to opioid abuse. These studies will provide important information about prescription opioid abuse liability in pain patients and will yield a laboratory model for predicting likelihood to relapse to prescription opioid abuse. These questions are of immediate clinical relevance to the treatment of chronic pain with opioid therapy.


This core provides in vivo model support for the three research projects. Specifically: 1. Support for Projects 1 and 2: Tumor growth delay studies will be carried out in male nude mice bering subcutaneously implanted human prostate carcinoma (PC-3, DU-145, LnCaP or CWR22) or human bladder carcinoma (RT4 or 5637). The animals will be treated with local hyperthermia to the tumor-bearing limb via water bath heating in the presence or absence of thermal sensitizer administration and/or fractionated radiation therapy (daily for 5 days by external beam). The results of these studies will provide an indication of therapeutic efficacy for the findings of Project 2 and provide a basis for translation of potential thermal sensitizes into Project 1. 2. Support for Projects 1 and 3: Large animals (pigs) will be used as a preclinical in vivo system to test the characteristics of new insonation portals using FSUM spherical applicator and urethral thermometry techniques. These animals will be fully instrumented for acquisition and analysis of all temperature and perfusion data, thus allowing calculation of ultrasound power fields, temperature fields, and thermal dose data. The results of these studies will translate into continued instrument development in Project 3 and into clinical protocol development in Project 1.


Enzyme mechanisms are investigated with small molecules which are designed to resemble altered substrates in the process of being transformed into products. The additional purposes of this work are to provide a basis for the rational design of potent antimetabolites, and to determine the dynamic role of enzyme structure in catalysis. Enzymes currently under investiation include asparaginase, papain, cytidine deaminase, adenosine deaminase, and pyruvate kinase.


This research program will study the regulatory mechanisms which control the different functional levels of the microcirculation in skeletal muscle. This work is based on the general concept that different functional levels in the microcirculation are involved to different degrees in the control of oxygen delivery to, and carbon dioxide removal from peripheral tissues, and that a few stimuli principally govern this control in the normotensive animal. The general concept forms the basis for the second more important hypothesis that at least one functional level for control of oxygen delivery is altered through changes in the type of stimuli which provide control of oxygen delivery in the hypertensive animal. This research program will investigate the relationships among oxygen and carbon dioxide content of inspired air, and small artery and vein phenomena for the microvasculature in skeletal muscle. The relative significance of the effects of innervation, intravascular pressures, and blood gas chemistry on small artery and vein diameters will be determined in normotensive rats and in renovascular and spontaneous hypertensive rats. These studies will utilize techniques which include closed-circuit television microscopy and on-line electronic analysis of video signals in order to obtain measurements of small artery (30-120 microns) and small vein (50-200 microns) diameters.


ABSTRACT / PROJECT SUMMARY Dysregulated bone or dentin resorption is associated with a host of oral diseases including periodontitis and external root resorption, to name a few. Currently, standard diagnosis of these conditions relies on radiographic findings or computed tomography (CT) scans. Nevertheless, these methods usually detect the problem at an advanced stage where significant tissue damage had occurred,oftentimes leading to tooth loss. Moreover, imaging techniques may not indicate if `clastic' cell activity is ongoing or historical. Exosomes are small vesicles of endosomal origin that are released by different cell types after fusion of multi-vesicular bodies with the plasma membrane. Due to their protein, lipid and nucleic acids contents, which closely reflect the nature and state of their parental cells, exosomes are considered an important source of information. Here we present preliminary data demonstrating that 1) there is a difference in composition of exosomes shed from clastic cells resorbing bone (osteoclasts) vs. clastic cells resorbing dentin (odontoclasts), and 2) exosomal proteins involved in clastic cell activity can be identified in gingival crevicular fluid (GCF) collected from resorbing teeth. Thus, we hypothesize that there is a difference in the biochemical composition of exosomes released during the processes of bone and dentin resorption that would allow for distinction between odontoclastic and osteoclastic function. We will test this hypothesis by pursuing the following two specific aims: 1) To Isolate and characterize exosomes derived from osteoclasts and odontoclasts in vitro, and 2) To establish the clinical feasibility of an exosome-based assay in the detection of oral conditions where upregulated bone or dentin resorption is present. In summary, the overall goal of this project is to optimize procedures for exosome analysis while at the same time testing the capability of oral-fluid derived exosomes to distinguish osteoclast-active sites from odontoclast-active sites in vivo. The study will provide a basis for the development of new, safer and less expensive tests to diagnose and monitor the progression of periodontal disease and dental root resorption.


Administration Over the past 16 years, the UNC Lineberger's administrative responsibilities have grown dramatically. Physical space has doubled. Dollars directly administered have increased eight-fold from $15.1 million in 1993 to $127.3 million in 2009 -2010 (including the Center's $50M/year University Cancer Research Fund). Over the next five years the Center will continue to grow in space, funding, and personnel. Growth will follow a Research Strategic Plan that proposes continued recruitment, new research initiatives. and development/expansion of core facilities. In all these efforts, the UNC Lineberger must continue to develop and improve its administrative, technology, and communication infrastructure/operation to facilitate cancer research. Cancer Center Administration (62 FTE. $4 million/year) supports the research activities of all 295 members. Administration provides direct grant, fiscal, and other administrative services to 93 Cancer Center members and 18 core facilities. Administrative support includes: laboratory, office, and conference space;common equipment;grant and fiscal management, human resources and other administrative services, computing infrastructure / networking, communications. and imaging / printing / web services. Center support alos includes In addition, the Center provides support by organizing recruitment, developmental pilot funding, seminars, symposia, and retreats. For Year 36, the Center requests a committed level of $489,049 to support 5.80 FTEs plus costs for supplies, travel, and other operational expenses. The proposed Year 36 budget is approximately 3% higher than the current budget. The requested CCSG Administration budget represents 12.5% of the UNC Lineberger's total Administration budget and 8% of the overall CCSG budget request.


As a cardiologist interested in the basic mechanisms of myocardial ischemia, my primary career objective is to gain an understanding of the role of disturbed endothelial function in atherosclerosis and how it contributes to abnormalities of vasomotor tone and to thrombosis. My dual training in cell biology and clinical research places me in an advantageous position in this regard, since few cardiologists have developed the necessary skills to apply new insights derived from vascular biology to patients with cardiovascular diseases. This application comes at a crucial point in my career as I complete my transition toward fully independent research status. For this burgeoning interest in closing the gap between basic and clinical research to continue to evolve will require well structured and protected time. The institutional environment will be conducive to this strategy for career development provided that my salary support comes from research sources. The studies presented here focus on defining how the presence of atherosclerosis in the coronary arteries of patients effects endothelium-dependent vasodilator and vasoconstrictor responses. A program of research is proposed that utilizes new insights from the experimental laboratory to address three specific aims. The first will test the hypothesis that endothelial vasodilator dysfunction in human coronary arteries leads to constrictor hyperresponsiveness to catecholamines. The second specific aim will test the hypothesis that endothelium-derived constrictor factors (the polypeptide endothelin, as well as prostanoid factors) are secreted from human atherosclerotic arteries in inappropriately large amounts in response to stimuli known to cause enhanced vasoconstriction and thereby contribute to the development of myocardial ischemia. The third specific aim will examine in patients the concepts derived from experimental studies that aggressive lowering of cholesterol can improve endothelium-dependent vascular function apart from important changes in vessel luminal size. These studies should expand our knowledge of the mechanisms and treatment of abnormal constriction of human coronary arteries, and facilitate clinical attempts to control active ischemia.


Convergence disorders and refractive amblyopia are among the most common and treatable vision problems seen in general optometric clinical practice. Oce- and home-based vision therapy (OBVT and HBVT, respectively) are standard treatments for such disorders. Recent advances in computerized video therapies that emulate vision therapy exercises performed in the optometrist's oce provide a lower-cost and more convenient alternative to OBVT. The downside of current commercial HBVT systems is that the recommended eye exercises are highly repetitive, monotonous, and uninteresting, which leads to high levels of patient noncompliance. This presents a problem since consistent performance and proper technique are key to achieving therapeutic benet. An alternative approach, which oers strong potential to increase the success of HBVT, is to incorporate vision therapy into fun and interactive computer games that patients will nd more engaging. To address this need, Barron Associates proposes to develop a novel computerized virtual reality HBVT system called iCare that will overcome the limitations of present systems by addressing three obstacles to successful home treatment: (1) patient compliance;(2) procedural accuracy;and (3) the ability of the optometrist to monitor training. The proposed approach takes advantage of advanced human-interface devices (HIDs) (e.g., Wii Remote Controllers) that have been developed for the video gaming market. Due to the popularity of video gaming, HID technology that would have been prohibitively expensive in the recent past is now widely available at low cost (e.g., <$40). These o-the-shelf HIDs permit the creation of virtual three-dimensional (3-D) environments on computer monitors (for \near-point" vision therapy) or plasma displays (for \distance" vision therapy). Traditional 3-D graphics technology based on \3-D glasses" (i.e., red-blue anaglyphs) is static with respect to the frame of the viewing device;that is, the image is the same regardless of the viewer's position relative to the monitor. The proposed iCare technology exploits motion parallax via low-cost tracking of the patient's head position and orientation, creating a dynamic visual 3-D environment. Visually, the technology allows the viewer to perceive objects as if they are coming o of, or receding into, the screen. This enables the creation of virtual xation objects in a nearly innite 3-D space. Users can even see \behind" and manipulate virtual objects. Under this SBIR Fast Track grant application, the proposing Team will develop and test prototype computer gaming environments for HBVT using the technology outlined above to improve treatment eectiveness. Phase I therapy will be aimed at treating refractive amblyopia in children 8-12 years of age;Phase II will focus on treating refractive amblyopia in teens and adults, as well as convergence insuciency in both populations. These disorders are clearly amenable to vision therapy and are common in clinical practice. For both vision problems, clinical studies of the comparative eectiveness of the most popular computerized HBVT programs vs. the iCare system's games will be conducted in children to assess the ecacy of the iCare system. PUBLIC HEALTH RELEVANCE: Convergence insuciency is one of the most common binocular vision disorders, and refractive amblyopia a vision disorder for which a very-high percentage of patients seek treatment;both disorders aect millions of U.S. children and adults. Such vision anomalies can cause a variety of chronic symptoms which, left untreated, often degrade academic and work performance;they may even result in learning disabilities in some cases. Computerized home-based vision therapy (HBVT) provides a relatively low-cost and convenient treatment option;however, the available commercial HBVT programs are highly repetitive and dull, leading to poor compliance. The goal of this research is to make HBVT a more eective alternative to oce-based vision therapy by incorporating the eye exercises into fun, interesting, and interactive computer video games that patients will nd more engaging, which should lead to better compliance and improved therapeutic outcomes.


The long-term objectives of the proposed project are to determine whether and how modulation of gap junctional intercellular communication is involved in the process of carcinogenesis. The specific aim of the project is to test the hypothesis that blocking of communication between an initiated cell and surrounding normal cells is an important determinant in clonal expansion of the initiated cell, i.e., the tumor promotion process. Changes in intercellular communication will be examined during the process of cell transformation by measuring intercellular transfer of fluorescent dye which is microinjected into individual cells. Cell cultures, mainly BALB/c 3T3 and C3H 10T1/2 cell transformation systems, will be employed during the earlier phase of the project. Experiments are designed to answer following specific questions: (i) whether phorbol ester-mediated enhancement of cell transformation is related to the block of intercellular communication by the phorbol esters; (ii) whether so-called "initiated cells" in which promotion is suppressed by retinyl acetate communicate with surrounding non-transformed cells, (iii) whether transformed cells communicate with each other and/or with surrounding non-transformed cells, and (iv) whether blocked intercellular communication also occurs during the process of promotion that is not triggered by phorbol esters. Variant cell lines that are sensitive or resistant to TPA-mediated enhancement of cell transformation (Syrian hamster embryo cells) and those that have different susceptibilities to UV- and chemically-induced transformation (BALB/c 3T3 cells) will be utilized to pursue these goals. The possibility of using the block of intercellular communication to screen tumor promoting agents will be examined. We plan to study molecular and biochemical mechanisms in detail. Our approach includes studies on (i) the molecular mechanisms by which phorbol esters inhibit the communication, with special emphasis on possible involvement of protein-kinase C activity, (ii) biochemical analysis of the factors involved in cell-cell recognition that influence gap-junctional communication, and (iii) possible oncogene involvement in blocking intercellular communication. Similar approaches will be applied to an in-vivo study using a rat-liver multistage carcinogenesis model. The microinjection technique coupled to electrophysiological measurements will be used to determine intercellular communication between preneoplastic nodules and/or hepatoma and surrounding normal cells.


[unreadable] [unreadable] The ability of a drug to reach and penetrate its intended target within the body is critical to its success in treating disease. However, drug efflux proteins such as p-glycoprotein (pgp) actively pump hydrophobic drugs away from target tissues and are linked to low oral absorption and multidrug resistance in chemotherapy. Protein pumps are of increasing interest to the pharmaceutical industry, most importantly based on new draft FDA guidelines requiring knowledge of whether a drug candidate is a substrate or inhibitor of pgp. Current pgp assays are cumbersome, expensive and unreliable. Our overall goal is to prepare and validate a novel assay reagent - Fluorosome-trans-pgp - which capitalizes on the Fluorosome Technology that we have developed for measuring passive permeability of small molecules through lipid membranes. Fluorosome-trans-pgp will provide a direct, reliable, simple, rapid and inexpensive assay to determine if a drug is a pgp substrate or inhibitor. Toward this goal, in phase I of this project we have: cloned and expressed the human pgp construct containing a 10His tag in HEK293 cells. isolated and purified the protein "pgp 10His" in lipid micelles. demonstrated verapamil-stimulated ATPase activity in the purified pgp 10His lipid micelles. reconstituted the purified pgp 10His into liposome membranes. developed the methodology for the manufacture and assay of Fluorosome-trans-pgp. validated the test systems that will be used to screen potential pgp substrates and inhibitors. Phase II of this project will bring the Fluorosome-trans-pgp assay to the state of an important commercial product. To complete this development, the Specific Aims of phase II are to: 1. complete Fluorosome-trans-pgp production and validation. 2. scale up Fluorosome-trans-pgp production to commercial levels. 3. demonstrate the utility of Fluorosome-trans-pgp with representative pgp substrates and inhibitors. 4. optimize the properties of the Fluorosome-trans-pgp system. 5. develop data analysis software. Our overriding goal in Phase II is to bring Fluorosome-trans-pgp and accompanying software to market. Successful completion of phase II will satisfyi the increasing need for a reliable and economical method to measure a drug's susceptibility to efflux by the pgp pump or to act as a pgp inhibitor. This project also lays the foundation for the design of Fluorosome-based systems to test for the susceptibility of drug candidates to other drug transport proteins. Markets include pharmaceutical and biotechnology companies, contract research organizations, and in-house fee-for service assays. This project completes the development of a test which determines if drugs will be extruded from their target tissue by biological pumps. The test thereby allows the pharmaceutical industry to evaluate, at an early stage, the suitability of drug candidates for continued development. The test is reliable, simple, rapid, inexpensive and amenable to robotics. [unreadable] [unreadable] [unreadable]


The integrated, transformational strategies of the proposed Orange County Center for Community Health Research (OCCCHR) will potentially have a broad impact on health research and in the reduction of health disparities. The OCCCHR will utilize a state-of-the-art web-based resource portal and videoconference technology to improve information dissemination, technical assistance in, networking, training and mentoring between the Academic Health Centers (AHCs) and community based organizations. The web-based video conference training component builds on UCI's considerable experience with training students, faculty, and community providers. Prior projects have demonstrated that the bi-directional web-based training is an accessible, convenient and cost effective approach to improving participation and suggest that the OCCCHR's videoconference training component will be a powerful tool for increasing community and researcher capacity to engage in research. Furthermore, the annual planning retreat and team building workshops will utilize collaborative models that have proven to be successful planning strategies in Orange County. It will focus the collaborative strengths of the community agencies with the AHCs toward the development of an academic-community partnered health agenda and research projects. The proposed infrastructure should have a major public health impact as a model for information dissemination, resource sharing, web-based training and collaboration. Further, the proposed infrastructure provides the opportunity to improve utilization of health data to develop evidenced-based community research priorities, and increase community research capacity to engage in community-based participatory research. This model can be used to engage communities nationwide to improve the quantity and quality of academic-community partnered research that substantially accelerate the diffusion scientific advancements into the community, thereby reducing health disparities. PUBLIC HEALTH RELEVANCE: The integrated, transformational strategies of the proposed Orange County Center for Community Health Research (OCCCHR) will potentially have a broad impact on health research and in the reduction of health disparities. The OCCCHR will utilize a state-of-the-art web-based resource portal and videoconference technology to improve information dissemination, technical assistance in networking, training and mentoring between the two AHCs and community based organizations. This model can be used to engage communities nationwide to improve the quantity and quality of academic-community partnered research that substantially accelerate the diffusion scientific advancements into the community, thereby reducing health disparities.


Abstract Despite the crucial importance of the inter-follicular epidermis (IFE) for the essential body barrier function, molecular characterization of the stem cells (SCs) within the basal layer has not been achieved. This impairs our ability to study the mechanisms that specifically control IFE SCs for proper homeostasis and wound healing and to understand how these mechanisms may be affected in skin disease and aging. The IFE SCs have been traditionally identified in vivo as DNA label retaining cells (LRCs) while transit amplifying (TA) or progenitor cells were considered non-LRCs. However, LRC and non-LRC markers to unambiguously test this model in vivo had been lacking. Using our H2B-GFP pulse-chase transgenic mouse system, we label IFE LRCs and non-LRCs, define mRNA expression profiles, and find that these cells are molecularly distinct. In our first set of preliminary data, we demonstrate that, contrary to the expectation that SC are frequently dividing cells, both of our IFE cellular subsets of LRCs (marked by Dlx1CreER) and of non-LRCs (marked by Slc1a3CreER) act as two independent SCs in long-term lineage tracing. Collectively, our data support a model in which the IFE is a heterogeneous tissue, being composed of molecularly distinct domains or territories, which are spatially patterned relative to each other and to skin landmarks. These territories are enriched in LRCs and non-LRCs, regenerate at different rates, and contain distinct SCs and differentiated cells that can be defined as molecularly discrete IFE cellular subsets. We provide LRC and non-LRC-enriched markers and genetic labeling tools that define novel IFE cellular subsets, which will enable us to rigorously examine the newly uncovered IFE heterogeneity. The specific objectives are to: (i) examine the organization of IFE territories throughout life, and determine relative SC potential of newly uncovered IFE cellular subsets; (ii) validate and refine markers of IFE heterogeneity from our newly uncovered undifferentiated and differentiated IFE cellular subsets; and (iii) unravel mechanism of IFE heterogeneity by focusing on two transcription factors we identified in our LRC versus non-LRC IFE subsets. Collectively, our data will constitute a key for understanding previously un-recognized cellular and molecular heterogeneity within the IFE and provide a new entry point into SC regulation in this essential, yet poorly understood skin compartment.


This education program will develop, implement, evaluate and disseminate a comprehensive integrative medicine curriculum to allopathic and CAM medical students and health professionals. This longitudinal curriculum will enable students and practitioners to acquire the knowledge, skills and attitudes to practice evidence-based medicine that appropriately incorporates best practices from both conventional and alternative medicine. Core competencies in CAM will be taught using web-based, face-to-face, and interprofessional curricula that takes full advantage of the rich CAM resources in the Bay Area. The curriculum also incorporates a student wellness program to help them maintain their own health and promote wellness as a model for their patients.This program will be designed and executed by faculty and staff at the Osher Center for Integrative Medicine in collaboration with CAM community experts under the guidance of an independent Advisory Committee, composed of allopathic and CAM practitioners, educators, and researchers. The program is unique in its emphasis on self-directed evidence-based learning, case-based instruction, and mix of web-accessible modular learning, 'face-to-face' experiential curricula, and collaborative programs with community CAM institutions.The process and outcomes of this program will be rigorously evaluated and the content continuously updated. Because of its web-based interface, this curriculum is highly adaptable and portable to other academic institutions. Dissemination of the curriculum will also be accomplished by CD-ROM, implementation manuals, lectures, and workshops


A Fully Automatic System For Verified Computerized Stereoanalysis SUMMARY The requirement for a trained user to interact with tissue and images is a long-standing impediment to higher throughput analysis of biological microstructures using unbiased stereology, the state-of-the-art method for accurate quantification of biological structure. Phase 1 studies addressed this limitation with Verified Computerized Stereoanalysis (VCS), an innovative approach for automatic stereological analysis that improves throughput efficiency by 6-9 fold compared to conventional computerized stereology. Work in Phase 2 integrated VCS into the Stereologer", an integrated hardware-software-microscopy system for stereological analysis of tissue sections and stored images. Validation studies of first-order stereological parameters. i.e., volume, surface area, length, number, confirmed that the color-based detection methods in the VCS approach achieve accurate results for automatic stereological analysis of high S:N biological microstructures. These studies indicate that fully automatic stereological analysis of tissue sections and stored images can be realized by elimination of two remaining barriers, which will be addressed in this Phase II Continuation Competing Renewal. In Aim 1, applications for feature extraction and microstructure classification, developed in part with funding from the Office of Naval Research, will be integrated into the VCS program. The new application (VCS II) will use these approaches to automatically detect and classify polymorphic microstructures of biological interest using a range of feature calculations, including size, color, border, shape, and texture, with support from active learning and Support Vector Machines. Work in Aim 2 will eliminate physical handling of glass slides during computerized stereology studies by equipping the Stereologer system with automatic slide loading/unloading technology controlled by the Stereologer system. This technology will approximately double the throughput efficiency of the current VCS program and support "human-in-the-loop" interaction for sample microstructures on the border between two or more adjacent classes. The studies in Aim 3 will rigorously test the hypothesis that fully automatic VCS can quantify first- and second-order stereological parameters, without a loss of accuracy compared to the current gold-standard - non-automatic computerized stereology, e.g., manual Stereologer. If these studies validate the accuracy of VCS II, then commercialization of the fully automatic program will facilitate the throughout efficiency for testing scientific hypotheses in a wide variety of biomedical research projects;reduce labor costs for computerized stereology studies;hasten the growth of our understanding of biological processes that underlie health, longevity, and disease;and accelerate the development of novel approaches for the therapeutic management of human disease. Solid evidence that the SRC and its strategic partners can effectively commercialize this technology is demonstrated by their worldwide sales and support of the Stereologer system for the past 13 years. Key personnel and participating institutions: 7 Peter R. Mouton, Ph.D. (PI), Stereology Resource Center, Chester, MD. 7 Dmitry Goldgof, Ph.D., University of South Florida Coll. Engineering, Tampa, Fl. 7 Larry Hall, Ph.D., University of South Florida Coll. Engineering, Tampa, Fl. 7 Joel Durgavich, MS, Systems Planning and Analysis, Alexandria, VA. 7 Kurt Kramer, MS, Computer Programmer, University of South Florida, Coll. Engineering, Tampa, Fl. 7 Michael E. Calhoun, Ph.D., Sinq Systems, Columbia, MD PUBLIC HEALTH RELEVANCE: Many fields of scientific research require a trained expert to make tedious and repetitive measurements of microscopic changes in animal and human tissues. This project will produce a computer program that performs these measurements with equal accuracy to a trained expert, but with dramatic savings in time and costs. Allowing scientists to complete more research in less time will accelerate our understanding of the factors that promote health and longevity, and hasten progress toward the development of new treatments for human diseases.


Schizophrenia and other psychoses are among the leading causes of the global burden of disease in low-, middle-, and high-income countries alike. As of 2006, and assuming a point prevalence of 4.6 per 1000 population [1], at least 25 million people in low- and middle-income countries (LMIC) were suffering from schizophrenia, a disorder that, according to estimates for 2004, accounted for 1.1% of all Disability Adjusted Life Years and 2.8% Years Lived with Disability [2]. Other research suggests that schizophrenia and other psychoses are associated with excess mortality, suicide in particular, as well as stigma, discrimination, and violations of human rights [2-6]. In low- and middle-income countries the burden of schizophrenia and other non-affective psychoses is made comparatively greater because of a lack of mental health resources that can provide effective treatments and, thus, decrease the symptom severity and disability associated with these conditions [7,8]. This is certainly the case in Nigeria where a recent study found that non-affective psychoses were relatively common (1.1% prevalence) but frequently went untreated [9], undoubtedly because of a scarcity of mental health resources [10]. This application for an exploratory planning grant (R21) proposes to undertake activities whose ultimate goal is the development of a package of care for individuals with psychosis and their caregivers in one catchment area of Nigeria. To achieve this goal, we propose to establish a collaboration between the London School of Hygiene and Tropical Medicine and Institute of Psychiatry, Kings College London, and the University of Ibadan. The collaboration will build on: 1) the long and impressive history of psychiatric and mental health services research in Nigeria [11-52];2) the evidence presented in the landmark Global Mental Health and Packages of Care series in The Lancet [7,8,53-56] and PLoS Medicine [57-63], respectively;and 3) the strategies for scaling up as outlined by the WHO mhGAP programme [2]. Together, the investigators who will lead the proposed project will consider, in a local Nigerian setting, the needs of persons with psychosis and their caregivers, the available mental health resources, and then formulate an evidence-based intervention package of care that will utilize non-specialist health workers (e.g., primary care physicians, nurses, and community health workers) working in partnership with the available psychiatric specialists, to create a program that will deliver services in community based settings [60,64,65]. PUBLIC HEALTH RELEVANCE: Schizophrenia and other psychoses impose a huge burden of disease in low- and middle-income countries. The aim of this exploratory project is to design an evidence-based, feasible, acceptable, and locally appropriate community-based rehabilitation package of care and that can be implemented in two local government areas of Osun State, Nigeria.


The project's objective is to develop enabling technology for a co-robotic navigation aid, called a Co-Robotic Cane (CRC), for the visually impaired. The CRC is able to collaborate with its user via intuitive Human-Device Interaction (HDl) mechanisms for effective navigation in 3D environments. The CRCs navigational functions include device position estimation, wayfinding, obstacle detection/avoidance, and object recognition. The use of the CRC will improve the visually impaired's independent mobility and thus their quality of life. The proposal's educational plan is to involve graduate, undergraduate and high school students in the project, and use the project's activities to recruit and retain engineering students. The proposal's Intellectual Merit is the development of new computer vision methods that support accurate blind navigation in 3D environments and intuitive HDl interfaces for effective use of device. These methods include: (1) a new robotic pose estimation method that provides accurate device pose by integrating egomotion estimation and visual feature tracking; (2) a pattern recognition method based on the Gaussian Mixture Model that may recognize indoor structures and objects for wayfinding and obstacle manipulation/ avoidance; (3) an innovative mechanism for intuitive conveying of the desired travel direction; and (4) a human intent detection interface for automatic device mode switching. The GPU implementation of the computer vision methods will make real-time execution possible. The proposed blind navigation solution will endow the CRC with advanced navigational functions that are currently unavailable in the existing blind navigation aids. The PI's team has performed proof of concept studies for the computer vision methods and the results are promising. The broader impacts include: (1) the methods' near term applications will impact the large visually impaired community; (2) the methods will improve the autonomy of small robots and portable robotic devices that have a myriad of applications in military surveillance, law enforcement, and search and rescue; and (3) the project will improve the research infrastructure of the Pi's university and train graduate and undergraduate students for their future careers in science and engineering.


A patient with hereditary angioneurotic edema (C1-inhibitor deficiency) also has SLE and is secondarily deficient in C4. Patients with hereditary C4 deficiency are subject to severe SLE. She was given infusion of human C1-inhibitor to prevent the destruction of C4 in her serum by the uninhibited C1. This failed to induce a remission in SLE or to elescript submitted, and treatment with C1-inhibitor was stopped.


Core D Cell Culture Project Abstract The goal of the work described in this proposal is to collect epithelial cells that line the surfaces of the nose and upper airways of human subjects with asthma or cystic fibrosis. The airway epithelial cells can be grown in the laboratory under conditions that allow large-scale expansion of cell number and frozen for storage and distribution to project investigators. Each sample can be thawed and further expanded for study and testing. Most human airway epithelial cells currently available for study are from transplanted disease lungs or lung donations not suitable for transplant. Collection of nasal and bronchial cells by brushing the epithelial surface is simple and safe, and allows us to obtain cells from a much larger pool of subjects. The ability to collect, expand, and distribute airway epithelial cells to researchers will have a positive impact on understanding and developing therapies for common airways diseases and provide an opportunity to test newly developed drugs for cystic fibrosis on rare mutations carried by only a few individuals.


Purchase of a 700MHz NMR Spectrometer for Liquid Applications Project Summary Solution nuclear magnetic resonance (NMR) spectroscopy is one of the most powerful spectroscopic tools available to the synthetic chemist for the elucidation of the structure of a molecule. Advances in computing power, high-field magnet technology, and improved electronics have dramatically increased both sensitivity and chemical shift dispersion such that detection of low natural abundance nuclei is now routine. Presently in the Chemistry department at the University of Michigan (UM, Ann Arbor) there is a rapidly growing gap between the existing capabilities offered by our aging/obsolete NMR spectrometers and the demands of our NIH-funded investigators. This demand stems from the fact that not only have the molecular systems under investigation grown in size and complexity, the questions being addressed by NMR are also more ambitious and include atomic characterization of molecular configuration, conformation, dynamics, interactions and mechanisms. The availability of a high field, multi-channel NMR spectrometer dedicated to liquid applications will have a significant impact on the investigation of complex natural products bearing multiple stereocenters, determination of relative stereochemistry of 5-membered carbocyclic ring systems such as cyclopentanes, tetrahydrofurans, and pyrrolidines possessing up to 4 stereocenters, characterization of unstable organoboron and organosilicon intermediates, and time-resolved kinetic studies of reactive organometallic intermediates formed in low concentrations. High-resolution structural information will be highly valuable in designing small molecule isoxazolidine transcriptional activators, as well as probing how enzymes use free radicals to catalyze chemically difficult transformations. Progress in these research projects and the training of chemistry students in cutting-edge NMR techniques are hampered due to the lack of an accessible modern high field NMR spectrometer. Therefore, to close this gap, we propose the purchase of a multi-channel 700 MHz NMR spectrometer outfitted with the latest in cryogenically cooled, triple resonance probe technology. Additionally, access to such a state-of-the-art instrument will be made available to NIH-funded chemistry projects at nearby institutions such as the University of Toledo, where significant benefit would be realized in the area of complex carbohydrate synthesis. PUBLIC HEALTH RELEVANCE The NIH-funded projects described herein will contribute to a greater understanding of areas that impact human health. Advances enabled by the requested instrumentation include the discovery of potential drug leads, the understanding of biological processes involved in disease progression or prevention, and the development of new strategies in synthesis that enable creative solutions to research projects of this type.


The long range goal of this research is to understand the roles played by the superior colliculus (SC) in visual function. This requires a full accounting of the functional properties of the retinal ganglion cells innervating the SC. In the cat, almost all of these cells belong to the W-cell class. W-cells comprise 50 percent of cat ganglion cells and have obvious equivalents in primates and other mammals. In addition to dominating the retinocollicular pathway, W-cells provide most or all of the retinal input to a host of other visual nuclei that subserve visuomotor reflexes. Despite the ubiquity and importance of W-cells, study of their structure and function has been severely limited by technical factors. This project will overcome these limitations by means of a novel in vitro approach, thereby shedding new light on the organization of the retinocollicular pathway. W-cells are extremely heterogeneous in morphology and function. Rather than representing a true class, W-cells apparently constitute a loose grouping of many distinct ganglion-cell types, each as different from the others as it is from the X-and Y-cell types. Work from this laboratory in past and current project periods indicates that individual W-cell subtypes may exhibit distinctive patterns of collicular projection. The retinocollicular pathway is thus far more complex that has been generally appreciated. Functionally distinct W-cell channels may be processed independently by distinct collicular microcircuits, or may undergo a highly orderly integrative recombination by collicular neurons. Clearly, a prerequisite for understanding the functional meaning of this parallel retinocollicular organization is a detailed understanding of individual W-cell types. The proposed project begins this process through an in depth analysis of a single W-cell type -- the zeta cell -- which is a dominant contributor to the retinocollicular pathway. These W-cells will be fluorescently tagged, either by retrograde transport of tracers from the colliculus or by uptake of a vital dye. The retina will then be maintained in vitro and electrodes advanced toward tagged zeta cells under visual control. The visual response properties of these cells will be studied by extracellular and intracellular recording and their morphology revealed in detail by intracellular dye injection. This approach provides the first practical method for making direct correlations between the morphology, physiology and central projections of single ganglion cells. The method will be used to analyze the stimulus selectivities of these cells, to explore the intraretinal circuitry that produces these response properties, and to characterize the anatomical mosaic through which these cells sample the visual scene. The study will expand our understanding of diversity among the output cells of the retina and the parallel channels of visual information they emit to the SC and other brain centers. It will also establish a powerful new experimental paradigm with extremely broad applicability to the study of retinal ganglion cells.


This project will study the mechanisms responsible for microvascular thrombosis in experimental models of sepsis. Sepsis, a systemic response to an infection, is the main cause of death in adult intensive care units in the United States, and microvascular thrombosis is a severe complication of the disease. The main model we will use involves bacterial endotoxin (lipopolysaccharide, LPS), which mediates many manifestations of patients with a common form of sepsis. In addition, we will use a clinically relevant model of human polymicrobial sepsis of abdominal origin, cecal ligation and perforation (CLP). Our preliminary data demonstrate that both sepsis models enhance microvascular thrombosis in vivo; in this project, we will explore the molecular mechanisms involved. Our central hypothesis is that LPS-induced stimulation of toll-like receptor 4 (TLR4) on endothelial cells mediates microvascular thrombosis in endotoxemia, by a mechanism dependent on the platelet adhesive molecule, glycoprotein Ibct. We propose four aims: in aim 1, we will identify which LPS receptors mediate enhancement of microvascular thrombosis in vivo. In aim 2, we will determine whether bone marrow- or non-bone marrow-derived cells mediate LPS- induced responses in vivo. In aim 3, we will use an ex vivo flow system to examine the effects of endotoxemia and CLP on platelet activation and adhesion to specific adhesion molecules (e.g.-vWf, P- selectin, fibrinogen) under physiologic flow. In aim 4, we will use the in vivo model to define the platelet and endothelial adhesion molecules responsible for LPS- and CLP-enhanced microvascular thrombosis. Completion of these aims will broaden our understanding of the mechanisms of microvascular thrombosis in models of human sepsis. This will allow identification of novel therapeutic targets for microvascular thrombosis in this disease. Our long-term goal is to apply the knowledge gained from these studies to allow optimal management of patients with sepsis and their associated microvascular alterations. Relevance to public health: Sepsis, the body's response to an infection, is a major cause of death in the U.S. Our goal is to understand the causes of a severe complication of this illness, clots in tiny blood vessels. This information would help develop new treatments for patients with this devastating illness. [unreadable] [unreadable] [unreadable]


This project has the aim of understanding the basic problem of radiation induced liver damage with the ultimate goal of solving this problem in humans when radiation therapy is needed for cancer of the upper abdomen and lower thorax. Mongrel dogs are examined, quarantined and vaccinated, then are submitted to laparotomy for liver biopsy. Blood chemistries and liver scan are obtained prior to irradiation at various therapeutic dose levels. Then the liver scan, chemistries and liver biopsy are repeated to detect liver damage. Various combinations of dose and fractionation are being tested. Anticoagulant therapy will be tried with the aim of reducing the radiation induced liver damage.


While human patients and healthy subjects are being imaged at 3T, 7T, and soon, 10.5T, more data and understanding of RF safety at these ultra-high field strengths are needed. The overall objective of this renewal proposal therefore is to extend the RF heating studies in the human head of our previous grant to the investigation of RF heating in the human body for ultra-high field MRI applications. Safety will be better assured and imaging performance improved by developing the means to accurately predict and measure RF temperature contours in human anatomy. Through development of new theory, technology, methodology, and experimental approach, these means will be achieved by accomplishing the following aims. First, a more fundamental understanding of the electrodynamics and thermodynamic nature of RF induced heating and heat transfer in anatomy will be furthered through mechanistic improvement of the empirical Pennes Bioheat Equation. Second, this new theoretical model will be validated at high field Larmor frequencies by invasive, direct measurement of RF heating in anesthetized porcine models, by fluoroptic thermometry. The porcine model is required as an intermediate step toward understanding and measuring RF heating in humans. Toward this end, the third aim is to develop a noninvasive NMR thermometer that is accurately and precisely calibrated by the invasive fluoroptic measurement. Once confidence is gained in predicting and noninvasively measuring temperatures in pigs, these methods will be applied or correlated to monitoring RF heating in humans and so accomplishing the final aim of this study. With new understanding, data, and thermal measurement methods, high field human MRI will become safer.


Systemic lupus erythematosus (SLE) is a chronic life-threatening autoimmune disorder that currently afflicts up to 2,000,000 individuals within the United States each year. Therapeutic options for treating patients with SLE include agents such as steroids, cytotoxic drugs, and anti-malarials. Although these therapies can reduce disease severity, they often have deleterious side effects that limit their extended use. A better understanding of the factors that trigger disease onset could facilitate the development of drugs that specifically target the autoreactive effector cells without the debilitating side effects and general immunosuppression associated with curent treatment protocols. This proposal is based on recent studies that identify a novel pathway involved in the activation of both autoreactive B cells and (auto)antigen presenting dendritic cells. Our group has shown that chromatin-containing immune complexes (chromatin- IC), bound by either the BCR or an FcgammaR, are delivered to an internal compartment where they activate the cells via a MyD88-dependent Toll-like receptor 9 (TLR9) pathway. Thus, inhibition of the TLR9 pathway may specifically block the development and/or progression of systemic autoimmune disease without interfering with immune responses to foreign proteins. To take advantage of this potential therapeutic opportunity, it will be necessary to further delineate the mechanism and general applicability of this dual receptor paradigm. The goal can best be met by combining the research endeavors of program investigators with diverse research backgrounds - B cell regulation (Marshak-Rothstein), antigen uptake and vesicle trafficking (Corley), chromatin remodeling (Viglianti), dendritic cell activation (Rifkin), and transgenic models of autoimmune disease (Shlomchik) - to address the following questions: (a) What are the unique functional consequences of chromatin-IC engagement of B cells and dendritic cells (project 1 and 2)? (b) What aspects of chromatin structure determine immunogenicity in this system (project 1)? (c) How are chromatin-IC processed and how does TLR9 affect the persistence and site of these events (project 1)?; (d) Do TLR other than TLR9 mediate responses to non-chromatin associated autoantigens (project 2, 3,)? (e) What is the in vivo role of TLRs in systemic autoimmunity (project 3)? and finally (f) Can this pathway be specifically targeted therapeutically with agents that block the TLR9 signaling pathway (project 1,2)? The results of these studies should provide important insights that will facilitate the development of non-invasive therapies for the treatment of systemic autoimmune diseases such as SLE.


The project will investigate the structure and function of gap junctions in the lens. For structural studies, the gap junctions will be studied in normal and cataractous lenses using freeze fracture and thin section electron microscopy, and of isolated preparations using X-ray diffraction and electron micrograph image reconstruction techniques. For the functional studies, metabolic cooperation via gap junctions (the ability of adjacent cells to share small metabolites via low resistance pathways) will be studied using whole tissue and tissue culture autoradiography, correlating functional findings with junction distribution and morphology. These studies will be performed under control, and cataractogenic condition, both metabolic and hereditary. Finally, specific monoclonal antibodies will be generated against the lens gap junction principal polypeptide.


DESCRIPTION (Applicant's Description): The overall objective of the proposed studies is to obtain information regarding the role of the bestrophin gene (VMD2) in retinal degeneration. Best Macular dystrophy (BMD, also called vitelliform macular dystrophy) is an autosomal dominant form of macular degeneration. BMD, caused by mutations in the VMD2 gene, leads to a progressive loss of central vision. Little is known about the protein product of this gene, called bestrophin. In the retina, bestrophin is specifically expressed in the plasma membrane of the retinal pigment epithelium (RPE). In the following application, I plan to conduct research toward understanding the physiological and molecular role of VMD2 in the retina. The first specific aim seeks to determine if mutations in the VMD2 gene cause other forms of retinal degeneration such as age-related macular degeneration (AMD) using PCR-based single-strand conformation polymorphism and direct genomic sequencing techniques. In the second aim, I plan to ascertain the subcellular localization of bestrophin in the plasma membrane of the RPE using imunocytochemistry. In the last aim, I propose in vitro electrophysiological studies as well as in vivo studies using mice. These studies include methods such as Ussing chambers, light, electron and fluorescent microscopy, gene transfer both in vivo and in vitro, electrocculography, and electroretinography. Discovering information regarding the function of bestrophin will hopefully provide knowledge about mechanisms involved in retinal degeneration.


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. Objectives Our objective in this core is to address the practical issues of adapting, optimizing and provisioning biomedical applications in drug discovery, cellular and organ modeling, and image reconstruction based upon current and emerging computing, data and network infrastructures. At the same time, we must ensure the accessibility, reproducibility and versatility of the large ensembles of software that are required to carry out multiscale analysis. Specific Aims Aim 1: Scaling and Adapting Multiscale Biology Codes to High-Performance Clusters and Multicore Processors: To allow application codes to take advantage of a new "balance of machines where individual nodes become large-scale, shared-memory symmetric multiprocessors (SMPs). Aim2: Providing Transparent and Service-Based Access to Multiscale and Multiphysics Biomedical Codes: To hide as much complexity as possible allows scientists to reason more easily about the logic of the systems or workflows required to carry out their research [unreadable]and less about the low-level system details. Aim 3: Defining, Testing, and Supporting a Complete Cyberinfrastructure: To provide a reproducible cyberenvironment by understanding externally-supplied software, assembling into a distribution that sits on top of a standard Linux operating system, and regularly and rigorously testing. Aim 4: Integrating and Delivering Tools for Translational Research: To integrate and test selected "suites" of software application pipelines