This proposal outlines an experimental strategy aimed at understanding age-dependent dysregulation of stem cell function in the Drosophila model. The adult Drosophila intestinal epithelium is maintained by resident stem cells which contribute to all gut epithelial cell types. These intestinal stem cells (ISCs) divide in response o tissue damage and aging, but appear to lose control of their proliferative limit. Specifically, agig leads to ISC hyperproliferation, which is in itself similar to pathological conditions we find in mammalian models of cancer. Thus, understanding the mechanisms for age-associated stem cell dysfunction may illuminate on the mechanisms of cancer in higher organisms, such as humans. We will begin filling gaps in our current understanding of stem cell aging by addressing two relevant problems in the field. In the first aim, we will attempt to understand the dynamics of gut aging, specifically focusing on the contribution of cell autonomous and cell non-autonomous aging cues on ISC aging and hypeproliferation. We will utilize well established methods in Drosophila system which will allow us to genetically manipulate the ISCs and their lineage in the gut. We will also use antibodies that reveal the rate of gut cell aging. Our second aim will expand on the new techniques in metabolite profiling to characterize the endogenous small molecule content of the aging environment in flies. We will then use computational methods and stringent statistical analysis, which efficiently identifies the most probable longevity candidates and will test their role on lifespan and stem cell aging. While our metabolite profiling may offer structural information of small molecule regulators of the stem cell aging, the complementary gene expression studies may illuminate on upstream genetic circuitries which can be functionally analyzed in cell-specific manner in the fly gut.


This proposal addresses the need for definitive evidence on the role of Foxp3+/T regulatory cells in the acceptance of physiologically relevant full MHC incompatible organ grafts in various settings of operational tolerance. In the initial year of funding (ARRA), spontaneous acceptance of MHC-mismatched mouse renal allografts was shown to depend exquisitely on Foxp3 cells using diphtheria toxin (DT) to transiently deplete circulating and intragraft Foxp3 cells in B6.Foxp3DTR mice bearing life sustaining DBA/2 renal allografts. The accepted grafts had distinctive nodular perivascular infiltrates of Foxp3 cells and dendritic cells (DC). Deletion of foxp3 cells caused dissolution of the nodular aggregates and widespread infiltration of the cortex, tubules and blood vessels by T cells, typical of acute rejection, and manifested by a rise in BUN over 7 days. Similar nodular aggregates of Foxp3 cells occur in grafts of patients who have accepted MHC mismatched kidneys on mixed chimerism tolerance induction protocols. Specific aim 1 will extend these observations to other forms of induced tolerance, mixed chimerism and neonatal tolerance, some of which are expected to be Foxp3 dependent;those with deletional mechanisms are expected to be Foxp3 independent. We will seek distinctive pathologic features in the graft, indicating Treg activity, such as the co-expression of latency associated protein (LAP) and Foxp3 or DC expression of indoleamine 2,3-dioxygenase (IDO), that might be used to distinguish foxp3 dependent from foxp3 independent forms of graft acceptance. The donor reactivity and regulatory potential of the cells in grafts and in lymphoid organs will be tested by ELISPOT and compared between the different forms of acceptance and acute rejection. The dependence of systemic tolerance on the intragraft infiltrates will be tested by graft nephrectomy in recipients with accepted kidney and skin grafts that have a remaining native kidney. Specific aim 2 will seek to identify the molecular and cellular basis for the dramatic strain differences in susceptibility to spontaneous graft acceptance, comparing BALB/c (rejected) and DBA/2 (accepted) kidneys (both H-2d) in B6 recipients. We will test for strain differences in DC function, cytokine/chemokine/complement mediators, minor histocompatibility antigens and ability to generate Foxp3 cells. Follow-up experiments seek to alter identified factors to promote acceptance of BALB/c kidneys. Specific aim 3 visualize interaction of Foxp3+ cells with other T cells, recipient and donor dendritic cells and tubular cells in accepting and rejecting renal grafts using a custom real time confocal, multiphoton endomicroscope. This system permits observations of the same graft over time, live video recording, delineation of 3-4 different cell types and computer assisted measurement of cell motility and interactions. These studies are designed to reveal the significance and in vivo function of Foxp3+ Treg in murine renal allograft acceptance and provide insights potentially applicable in clinical renal transplantation. PUBLIC HEALTH RELEVANCE: The mouse kidney transplantation system offers an optimal combination of fidelity to human pathological processes, a rich spectrum of outcomes without drugs, and the ability to manipulate and observe the components of the immune response for mechanistic studies. These studies will provide insights into Treg physiology in renal allografts that will clarify their therapeutic relevance and reveal features that distinguish pathologic from beneficial intragraft infiltrates, of considerable importance in clinical transplantation and tolerance induction studies.


The primary goal of this study is the exploitation of the guinea pig inclusion conjunctivitis animal model for studies referable to human chlamydial infections. Thus, attempts will be made to determine the pathogenic events involved in the transmission of chlamydiae from the eye to the lung and gastrointestinal tract and to assess the clinical and pathologic significance of chlamydial infection in these sites. In addition, efforts will be made to study the mechanism of resistance or immunity to infection with Chlamydia trachomatis resulting from previous exposure and to determine the potential role of previous exposure in heightening the disease response from hypersensitivity.


The overall goal of this competing renewal application (Years 16-20) is to train postdoctoral physician fellows for successful research careers in academic infectious diseases. Support is again requested for 3 fellows/year for 5 years. During the current funding cycle, 6 fellows were appointed to our Research Training Program (2/year), including 2 from underrepresented minorities. Two fellows have completed the Training Program and are in academic or public health positions, and the 4 other appointees remain in training (2 in their 3rd year and 1 each in their 2nd and 1st year of training). Collectively, these trainees have published 20 papers (16 first authored) in peer-reviewed journals. Each year, we have carefully assessed the recruitment efforts and performance of the trainees and mentors in our Program and have implemented important changes to strengthen it. We will offer training in the 3 areas of greatest mentoring strength in infectious diseases at the University of Pittsburgh: HIV/AIDS, Molecular Epidemiology and Microbiology, and Sexually Transmitted Diseases. There is a critical need for well-trained physician investigators in each of these areas. The mentoring faculty, consisting of 6 MD and 9 PhD scientists from the Schools of Medicine and Public Health, have exemplary records of research accomplishment, funding and training of postdoctoral fellows. Physicians who have completed the first clinical year of infectious disease fellowship will be eligible for the Research Training Program. Appointees are selected by the Program Executive Committee, consisting of the PI and senior mentoring faculty. Appointees develop a three-year research Training Plan with their mentor for approval by the Executive Committee. The performance of the trainees is carefully reviewed by the mentor (weekly), the PI (quarterly), and the Executive Committee (biannually). The latter occurs at Fellows Research in Progress Meeting (July and January), during which all trainees present their work and are challenged to defend their research findings and plans. Issues related to responsible conduct of research are discussed throughout the Training Program. The goal of the Program is for each trainee to develop expertise in an area of infectious disease research that will serve as the foundation for a successful investigative career. Our Program will increase the capacity for research that is highly relevant to the NIAID mission.


[unreadable] [unreadable] This conference grant is for the 7th biennial workshop on "Molecular Aspects of Myeloid Stem Cell Development and Leukemia." This 7th workshop in a series held since 1995 is scheduled for May 13-16, 2007 in Annapolis, MD. The workshop hosts ~50 speakers, with a total of ~100 participants. Leukemia is a disease of hematopoietic stem cells that undergo a block in differentiation and a deregulated expansion. The workshop focuses on genes located at translocation breakpoints in leukemia such as AML1, TEL, RARalpha, and MLL, and on genes aberrantly expressed due to epigenetic alterations or function abnormally due to mutations. Mutations or abnormal levels of these factors are causally related to leukemogenesis. Murine gene-targeting studies and sequence analyses of patient samples show the critical importance of these same genes for normal hematopoiesis. The functions of these factors are combinatorial in hematopoietic development, and the complexity of interactions within overlapping networks makes it difficult for single investigators to gain wide scientific and clinical perspective to understand their roles in normal hematopoiesis and leukemogenesis. The goal is to bring together investigators with expertise in complementary aspects of stem cell biology, normal myelopoiesis and leukemia. Basic and clinician-scientists from the USA, England, Europe and Japan come together to discuss their latest findings on stem cells and hematopoietic regulators, e.g. transcription factors, including those who work on leukemia mechanisms and therapeutics, signal transducers and cell cycle components dysregulated through genetic mutations or epigenetic modifications. The aim is exchange of information through informal presentations and discussions. Young investigators (graduate students, postdoctoral fellows) comprise ~50% of participants and have the opportunity to present their work as posters or oral talks. Participants achieve greater understanding of the critical factors that regulate lineage specification and malignant hematopoiesis, and their relevance in the clinic in terms of leukemia. The workshop uniquely brings together basic scientist, clinician-investigators, and junior investigators in a manner that truly promotes communication and open exchange of ideas, unpublished results, cross-fertilization and collaborative research between scientists working on normal and leukemic stem cells and myeloid development, along with translational leukemia research and leukemia therapeutics. [unreadable] [unreadable] [unreadable]


The work carried out this year represents a continuation and extension of earlier studies on the role of carbohydrates in biological systems. Two separate areas are covered in this report. (a) The previously described findings on the hormonal regulation of sialylation in rat thyroid cells have been examined in human thyroglobulin isolated from patients with Graves disease and from patients exhibiting endemic goiter. In contrast to normal controls or material from endemic goiters, the thyroglubin recovered from patients with Graves disease was characterized by severe hypersialylation and aberrant localization of the sialic acid residues within the carbohydrate core. (b) Techniques designed to isolate and characterize individual nuclear protein fractions have revealed the presence of enzymatically degradable glycogen in the pore- lamina fraction of the nucleus and the presence of one or more complex glycoproteins within the nuclear matrix.


Adult dental caries, including both coronal and root caries, is an infectious disease that afflicts the majority of Americans aged 55 and older and is the most common chronic disease at midlife. In addition, adult caries exacts a significant economic toll, and this economic toll continues to grow. Despite the high prevalence and bacterial pathogenesis of caries, no FDA-approved anti-microbial treatment for caries is available to the American dental professional. The Prevention of Adult Caries Study (PACS) is a randomized, double-blind, placebo-controlled Phase III clinical trial conducted under FDA Investigational New Drug license #45,466. This study is designed to evaluate the efficacy of a topical, temporary, 10% w/v chlorhexidine dental coating in reducing caries increment in at-risk adult dental patients. This study will follow 1000 patients over a 13-month study period at four centers with vastly different populations. The primary endpoint for this study will be caries increment. The centers participating in this proposed research are: Center for Health Research, Kaiser Permanente Northwest, in Portland, Oregon;Tufts University Dental Clinic in central Boston, Massachusetts;Dental Service of Massachusetts Clinical Center in Southborough, Massachusetts, and;the dental clinic of the Tuba City Regional Health Care Corporation in Tuba City, Arizona. Kaiser Permanente's Center for Health Research in Portland, Oregon will act as the data-coordinating center, and Tufts University will act as the Administrative Center. This application from the Kaiser Permanente Portland Clinical Center is part of the coordinated set of applications in support of the PACS study. Please refer to the Administrative Center Application for a complete description of the study Protocol and Manual of Procedures.


Telomeres are the natural ends of linear chromosomes and crucial for genome stability, cellular viability and chromosome integrity. Telomeres shorten during each cell division, representing a cellular clock and limiting the replicative lifespan. To overcome this limit, cancer cells have to activate telomere maintenance mechanisms, which counteract telomere shortening and endow the cells with immortality. Ninety percent of cancers do so by activating telomerase, a reverse transcriptase complex that adds telomeric repeats to chromosome ends. The remaining 10% of cancers take advantage of a recombination-based mechanism for telomere length maintenance, called ALT (Alternative Lengthening of Telomeres). While telomerase activation is the more frequently used mechanism and widely investigated, it is becoming clear that ALT pathways can be activated upon telomerase inhibition, emphasizing that both telomere maintenance mechanisms have to be understood before telomere elongation can be successfully targeted as cancer therapy. ALT-dependent telomere lengthening is based on recombination between long and short telomeres, but the mechanisms are not currently understood. ALT has long been considered the result of defects in cellular recombination pathways, but despite intense efforts no deficiencies in recombination regulators have been identified in ALT-cells. Recent data suggest the hypothesis that ALT relies on aberrant recombination pathways needs to be reexamined and is likely incorrect. We discovered that ALT is likely a consequence of poor histone placement at repetitive regions, such as telomeres. We can induce ALT dependent telomeric recombination by suppression of isoforms of the histone chaperone Asf1. Upon Asf1 down regulation all characteristics of ALT emerge in primary and transformed cells, which include telomere sister chromatid exchange, telomere length heterogeneity, the formation of single stranded telomeric C-circles and the colocalization of PML, RPA and TTAGGG repeats in ALT associated PML bodies. The three intellectually connected but independent aims of this proposal are designed to investigate our novel concept for the ALT mechanism, which suggests that improper nucleosome placement at telomeres leads to single stranded loops at telomeres, which then readily recombine with each other. In AIM1 we will investigate telomere structure in cells with suppressed Asf1, as well as nucleosome placement and DNA damage signaling at telomeres and throughout the nucleus in Asf1 suppressed cells. We will also address whether Asf1 dependent ALT activation is capable of long-term telomere maintenance. AIM2 is designed to investigate why Asf1 suppression has telomere-specific effects and whether it leads to changes in telomeric chromatin in primary and transformed cells, therefore defining an ALT specific epigenetic signature. In AIM3 we will investigate the Asf1 status of ALT tumor cells, whether ALT can be suppressed by Asf1 expression, and whether ALT is an epigenetic state that can be induced by constitutive Asf1 suppression.


Ongoing comparative analysis of the protein compositions of the pigment epithelial (PE) cell plasma membrane from normal and dystrophic (RCS) rats will be extended in greater detail. The goal of this research has been to determine if the RCS mutation results in retinal degeneration by affecting the production of a PE plasma membrane protein that is involved in the recognition and phagocytosis of shed rod outer segment (ROS) membranes. Two dimensional (2D) electrophoretic analysis of the protein composition of plasma membrane enriched fractions from normal and dystrophic rat PE suggests that a 183,000 MW glycoprotein is underglycosylated in the RCS PE. In order to elucidate the chemical nature of this apparent decreased glycosylation, the carbohydrate compositions of the normal and RCS 183,000 MW proteins will be analyzed by sequential degradation with exo- and endoglycosidases, gel filtration chromatography and high performance liquid chromatography (HPLC). Cultured rat PE cells will be assayed for enzymes commonly found in the plasma membrane of other absorptive epithelia. Electroblots of 2D maps of PE cell membranes will be stained with antibodies specific for enzymes that test positive in the preliminary assays. Alternatively, subunits for some of the enzymes will be identified by use of specific radioactive affinity labels or histochemical stains. In this manner, specific functions will be identified for proteins previously characterized by molecular weight only in 2D electrophoretic analysis of PE plasma membrane proteins. This information will be valuable for narrowing down the number of spots in the complex 2D map that may be involved in recognition/phagocytosis of ROS membranes by the PE. Another important component of the PE cell plasma membrane, the receptor for serum retinol binding protein (RBP), will be detergent solubilized from swine PE. An assay for detergent solubilized receptor will be devised, and subsequently employed in purification of the receptor by lectin affinity chromatography, gel filtration chromatography, and HPLC. Antibodies will be prepared to the purified receptor. The anti-RBP receptor antibody will be used to determine if distribution of the receptor in the plasma membrane is altered as a secondary effect of retinal degeneration in the RCS rat. The long term goal of this research project is to elucidate, on a molecular level, the cause of inherited retinal degeneration by determining how alterations in the proteins of the PE plasma membrane are involved in both primary and secondary effects of inherited retinal degeneration.


The recent release of the Surgeon General's Call to Action to Promote Sexual Health and Responsible Sexual Behavior evidences the continued interest and concern regarding sexual behavior and its outcomes, particularly among adolescents. Research and great deal of resources on the prevention of negative sexual outcomes such as unintended pregnancy and sexually transmitted infections (STI), with only mixed results. The Theory of Planned Behavior (TPB) has been used to study a wide range of health behaviors, including safer sex behaviors such as condom use. Comprised of various sociocognitive elements (attitudes, norms, and perceived behavioral control), the TPB postulates that these elements predict intentions to perform a behavior, and that these intentions, in turn, predict the actual behavior. Despite the proliferation of the TPB in studies of sexual behavior, there remain several compelling issues worthy of study. This research aims to explore four different issues relating to the TPB and adolescent sexual behavior: 1) The predictive value of past behavior has received some attention in the literature on the TPB, but the issue of whether or not past behavior should be included in the model remains unresolved; 2) In addition, a growing body of research on attitudes toward sexuality suggests that having a "sex-positive" attitude, one that endorses sexuality and sexual behaviors as positive aspects of one's life and personality, increases one's ability to advocate for themselves in the sexual arena, such as for the need for safer sex. This suggests a new course for preventive interventions for teens, and calls for further empirical consideration; 3) Third, although the TPB has many strengths, it fails to consider the influence of macrocontextual factors on behavior. Gender differences in social power, for example, may hinder the ability of an adolescent girl to use safer sex if her male partner is unwilling, regardless of her personal perceptions of behavioral control ; 4) Last, as advocated in the Surgeon General's report, it is necessary to consider physical, sexual, and mental health as interrelated rather than discrete domains, and to fully explore the connections among them. In order to accomplish these objectives, the TPB will be applied to data from the National Longitudinal Study of Adolescent Health (Add Health). Add Health's size, sample diversity, breadth of focus, and longitudinal design each enable researchers to tackle a wide range of issues, including those mentioned above. This proposed work strives to contribute to existing knowledge by exploring emerging areas of interest in the field of adolescent sexuality while still remaining grounded in a well-established theory of health behavior prediction.


Our long term goal is to develop immune therapies for disease caused by Human Papillomavirus (HPV). While the field of cancer immunotherapy has shown proof of principle in humans, namely, that established malignancies can be recognized and eradicated by a tumor-specific adaptive T cell response, reproducibly achieving this effect has been difficult. One reason may be that therapeutic vaccines tested to date have not been immunogenic enough to eliminate established disease. Another reason may be immunologic suppression directly mediated by developing and progressing tumors, which are operative in the lesion microenvironment. This project will test the hypothesis that HPV-specific adaptive T cell responses can be elicited by therapeutic vaccination in subjects with high grade cervical dysplasia (CIN3), and that the application of a topical TLR7 agonist, imiquimod, directly on the lesion, will enhance access of CD8+ T cells to the lesions. CIN3 lesions are relatively accessible, and some of them do regress within our 15-week study window. The disease provides rational, non-self antigenic targets for therapeutic vaccination. This project will test a heterologous DNA prime, recombinant vaccinia-based boost regimen to enhance the immune response against HPV16 E6 and E7, which are expressed in a functionally obligate manner in cervical cancer and its precursor lesion, high grade cervical dysplasia (CIN3). This project also provides the opportunity to analyze target lesions directly to determine if we can identify evidence of mechanisms that would mitigate the function of immune effector responses, namely, impairment of homing and function of immune cell subsets. In fact, we found that CD8+ T cells accumulate at CIN3 lesions, but in the case of persistent disease, fail to access lesional epithelium. While patterns of immune cell infiltration have been identified to predict clinical outcome in the clinical setting of invasive disease, to our knowledge, they have not been identified early in disease, before development of an overtly invasive phenotype. This work is an opportunity to study immune responses that are localized to incipient neoplasia, and immune homeostatic mechanisms which could be locally uncoupled to enhance both access and function of effector immune cells to eliminate disease.


Lead poisoning, particularly in children, remains a major health problem in the United States today. Nearly 19% of the children in a recent series of screening programs from 1969-1971 had blood levels of lead of 60 micron g/100 ml or higher, amounts that are generally accepted by physicians as being severe enough to require treatment. Lead intoxication is known to affect the erthropoietic system, producing anemia. One possible mechanism for the production of anemia is inhibition of heme biosynthesis in erythropoietic tissue. A dialyzable factor, identified as a polyglutamated folic acid derivative (pteroylpolyglutamate), has been discovered that protects against lead inhibition of uroporphyrigogen I synthetase, an enzymatic step of the heme biosynthesis in erythropoietic tissue. A dialyzable factor, identified as a polyglutamated folic acid derivative (pteroylpolyglutamate), has been discovered that protects against lead inhibition of uroporphyrinogen I synthetase, an enzymatic step of the heme biosynthetic pathway. This pteroylpolyglutamate has also been found to be an activator of the enzyme which indicates that such a pteridine derivative functions as a coenzyme for this enzymatic reaction. The objectives of the proposed research are to elucidate the role that pteroylpolyglutamates serve in the regulation of inhibition of uroporphyrinogen synthetase activity by lead and other inhibitors, to learn how drugs, hormones and pteridine derivatives regulate this enzyme and the synthesis of heme, to extend these studies to extrahepatic tissues, such as the brain, kidney, bone marrow and endocrine system, and to the neonate. The results of these investigations will provide valuable information on the mechanism of the conversion of porphobilinogen to uroporphyrinogen and the synthesis of heme, and its regulation by lead and other inhibitors. This information will contribute to our knowledge of lead and drug toxicity and its treatment.


Abstract. In the past two years, elevated HEF1 expression have been shown to induce glioblastoma invasion and lung cancer and melanoma metastasis in both animal models and in humans: our preliminary studies have now suggested an equally important role for HEF1 in breast cancer progression. Our studies of HEF1 since we first described this protein in 1996 have elucidated a complex function. HEF1 acts to assemble signaling complexes at focal adhesions in interphase cells, at the centrosome in pre-mitotic cells, and at the cilium in cells emerging from quiescence. Taken in sum, these data suggest that deregulation of HEF1 may represent a point of cellular vulnerability based on coordinate dysregulation of these discrete signaling complexes. If so, restriction of HEF1 action may offer a useful therapeutic approach. The goal of this proposal is to better understand the mechanisms by which the HEF1 scaffolding protein controls both cell cycle progression and cell attachment signaling. Our work in past cycles of RO1 CA63366 defined interactions of HEF1 with the oncoproteins Src and FAK as critical for HEF1 action in promoting cell migration and invasion. Subsequently, we defined HEF1 interactions with Aurora-A, Ajuba, and HDAC6 as essential for regulation of mitotic progression and ciliary disassembly. These two sets of signaling relationships have been considered in isolation. However, accumulating evidence suggests roles for Src in mitosis, and for Ajuba and HDAC6 in interphase cells. We propose that HEF1 is a core component of a signaling module that functions both in mitosis and interphase cells, and timed migration of HEF1 and its partners between intracellular compartments allows coordinated regulation of cell division and cell migration/invasion. We propose deregulated HEF1 expression most commonly marks later rather than earlier stages of cancer development because prior lesions inactivating mitotic checkpoints allow cells to tolerate HEF1 scaffolding defects. This proposal has 3 aims. In Aim 1, we will assess the factors regulating HEF1 movement from focal adhesions to centrosomes, allowing HEF1 activation of AurA at mitotic entry. We will determine whether Src and FAK contribute directly to this process. In Aim 2, we will define the role of Aurora A, Ajuba, and HDAC6 in allowing HEF1 to return to focal adhesions at cytokinesis, and the importance of interactions between HEF1, Ajuba, and HDAC6 in interphase for HEF1-dependent cell migration and invasion. In Aim 3, we will use murine knockout and transgenic models to study HEF1- dependent breast cancer development, and to analyze the relationship between deregulated HEF1 expression, genomic instability, and metastasis.


Multiple sclerosis (MS) is a chronic autoimmune disease characterized by the degradation of CNS myelin. The etiology of the disease is still unknown;both genetic and environmental factors have been implicated. Oligodendrocyte apoptosis may represent an initiating event in MS, with improper removal of apoptotic cell bodies and released myelin fueling the immune response. Low density lipoprotein receptor-related protein-1 (LRP-1) is a cellular receptor, which functions in endocytosis and cell signaling. We have demonstrated that LRP-1 is a key endocytic receptor for myelin in vitro. LRP-1 also inhibits the inflammatory response by controlling the NF?B pathway. We, therefore, hypothesize that LRP-1 could be directly involved in regulation of the development and/or progression of MS. We will test this hypothesis by a combination of in vivo and in vitro experiments. As complete LRP-1 deficiency is lethal, the role of LRP-1 in a mouse EAE model will be determined using mice with conditional LRP-1 deficiency in the myeloid lineage. Our preliminary results suggest that LRP-1 deficiency in this model leads to a significant increase in EAE symptoms. Effector functions of LRP-1- deficient macrophages will be characterized. Finally, we propose to identify LRP-1 ligands in myelin preparations, using a proteomics discovery approach. This research grant proposal should provide the basis for understanding the function of LRP-1 in MS and for potential development of novel therapeutics for MS. PUBLIC HEALTH RELEVANCE: Multiple sclerosis (MS) is a chronic disease characterized by the immune system attack on myelin sheath, which is necessary for proper brain function. This proposal is focused on a cell protein, LRP- 1, which we show is important in the clearance of degraded myelin, and controls of the immune response. We hypothesize that LRP-1 regulates the sequence of events leading to MS development. We aim to identify the mechanisms by which LRP-1 functions in MS, with potential of discovering ways leading towards development of novel treatments for MS patients.


Inactivation of p53 is the most common genetic alteration detected in human cancers. Underscoring the importance of p53 in preventing tumorigenesis, mice in which p53 has been disrupted invariably develop lymphomas and other cancers. The overall goal of this proposal is to expand our understanding of the p53 tumor suppressor pathway, including upstream effectors and down-stream mediators, and the mechanisms by which it prevents pediatric cancers. Individuals diagnosed with Li-Fraumeni Syndrome (LFS) generally carry a germline p53 mutation and are remarkably predisposed to cancer at an early age. The mutations are highly penetrant and are associated with brain, breast, bone and adrenal cancers. We have recently identified a group of unrelated pediatric patients with adrenocortical carcinoma (ACC) in which 35 or 36 patients have a identical germline p53 mutation (R337H). Analysis of intragenic polymorphisms eliminate a founder effect. Tumors deleted the wild-type allele and express high levels of mutant p53 in the nucleus, indicating that p53 is functionally inactive in vivo. However, these patients are not from cancer-prone LFS families and over-expression of 337H in cell culture-based assays fail to detect a functional defect. Our hypothesis is that 337H is functionally inactive under physiologic conditions and that this mutation contributes to th4e development of ACC through a loss of p53-mediated cell cycle arrest and/or apoptotic activities. To test this hypothesis, we propose to address the following specific questions: 1) Does the 337 H mutation disrupt p53 biological activity under physiological conditions?; 2) Does the 337H mutant have altered biochemical and biophysical properties?; 3) Does the 337H mutation contribute to tumorigenesis? These studies should establish a new class of p53 mutation, a low penetrant allele, that would previously go undetected but nevertheless, significantly contributes to the development of human cancers.


This goal of this proposal is to uncover the molecular mechanisms for sensing and regulating intracellular iron in the model eukaryote S. cerevisiae. To maintain optimal intracellular iron levels, iron transport and storage is tightly regulated in al eukaryotic cells ranging from yeast to humans. However, there are significant gaps in our understanding of iron regulation mechanisms at the cellular and molecular level. We will address these gaps by teasing out the molecular details of iron regulation in yeast and defining the roles of each component in the iron signaling pathway. In yeast, the monothiol glutaredoxins Grx3 and Grx4, the BolA- like protein Fra2, and the aminopeptidase P-like protein Fra1 function together in an iron-responsive signaling pathway that controls nucleocytoplasmic shuttling of the iron-responsive transcription factor Aft1. Under iron replete conditions, this pathway induces dimerization of Aft1 (and presumably its paralog Aft2), favoring their localization to the cytosol. We have demonstrated that Fra2 forms [2Fe-2S]2+-bridged heterodimers with Grx3 or Grx4 and characterized the Fe-S coordination chemistry of these complexes. In addition, we have strong evidence that [2Fe-2S] Fra2-Grx3 transfers a [2Fe-2S] cluster to Aft2, facilitating Aft2 dimerization. Aft1/2 dimerization, in turn, is proposed to inhibit activation of the iron regulon. Despite our significant progress in defining the molecular interactions between several components in this signaling pathway, some key aspects of the iron sensing and regulation mechanism remain unresolved and will be addressed in this proposal. We will uncover the mechanistic details of Fe-S transfer from Fra2-Grx3/4 to Aft1 and Aft2 and determine the impact of Fra1 on this process by using mutagenesis, biochemical analysis, and biophysical spectroscopy to examine the kinetics and efficiency of cluster transfer to Aft1 and Aft2 and identify residues in Grx3/Grx4/Fra1/Fra2/Aft1/Aft2 that are critical for both donor-target recognition and Fe-S transfer (Aim 1). We will test whether the Fra-Grx complex transfers an Fe-S cluster to Aft1/2 and induces dimerization in vivo by determining how mutations in Fra2, Grx3/4, or Fra1 affect protein-protein interactions within the iron signaling pathway, Fe binding to Aft1/2, and Aft1/2 subcellular localization and dimerization in vivo (Aim 2). Finally, we will elucidate the mechanism by which Fra-Grx mediated Aft1/2 dimerization inhibits activation of the iron regulon by testing if Fra-Grx-mediated dimerization of Aft1/2 disrupts movement of Aft1/2 to the nucleus, binding of Aft1/2 to its DNA targets, or recruitment of transcriptional co-activators using both in vivo and in vitro protein-protein and protein-DNA interaction assays (Aim 3). Since several key proteins in this pathway are conserved in humans and essential for viability, exploiting the yeast system to define their functional and physical interactions will provide a fundamental understanding of their roles in human iron metabolism. PUBLIC HEALTH RELEVANCE: Both iron deficiency and iron overload are significant human health issues: iron deficiency is the most common and widespread nutritional disorder in the world, while iron overload disorders are some of the most common genetic disorders in the U.S. This proposal is designed to define the sensing and control mechanisms for maintaining optimal intracellular iron levels using bakers' yeast as a model system.


Project Summary/Abstract Persistent HIV health disparities impacts African Americans and are a public health priority. Despite declining HIV rates nationally, African American communities continue to experience disproportionate HIV burden and increasingly account for the largest share of HIV-related morbidity and mortality in the United States. These concerning disparities necessitate research to inform robust responses that target priority subgroups within this population. High rates of incarceration in many African American communities contribute to high HIV risk environments, where the risks persist after release from the criminal justice system. Formerly incarcerated African American youth (FIAAY, ages 18-24 years) in particular are at heightened risk in terms of sexual behavior, illicit drug use, and challenged rates of HIV testing behavior after release. A prominent barrier to HIV testing and timely diagnosis in this and other vulnerable populations is HIV-related stigma. Thus, more research needs to not only examine stigma-related factors that reduce HIV testing behavior, but also create a basis for interventions that can reduce stigma and increase HIV testing and HIV preventive behaviors in priority populations, such as FIAAY. Towards these goals, my proposed study focusing on FIAAY in Louisville Kentucky will allow me to: 1) develop a risk profile of HIV-negative or HIV status unknown FIAAY at greater likelihood of having stigmatizing beliefs related to HIV infection and longitudinally examine the likelihood of HIV testing based on this profile and 2) delineate mediating and moderating mechanisms by which stigma reduces HIV testing in this population. These actions will help generate knowledge for the development of a multilevel intervention targeting individual and structural factors to reduce HIV-related stigma and increase HIV testing and prevention among this population of vulnerable youth. This Career Development Award will provide the necessary knowledge and skills to conduct this and future research with criminal justice-involved populations. This entails enhancing my skills in advanced statistical methods such as survival analysis and structural equation modeling. In addition it will foster content expertise in decision science and the criminal justice system, which will help inform intervention development. Study methods will include longitudinal survey data collection (baseline, 12-months, 24-months) from 274 FIAAY in Louisville, KY. This research will inform the development of stigma-related HIV-risk profiles for FIAAY who are less likely to test and delineate mediators and moderators of the stigma-HIV testing relationship. If a relationship between stigma and testing is not detected, other psychosocial factors will be examined to determine mediators and moderators of HIV testing. This study has important downstream benefits for HIV prevention and treatment in this highly vulnerable population.


Solving the structure with a view to understanding the function of membrane proteins remains one of the grand challenges in all of Biology. Given that a third of the human genome codes for membrane proteins, many of which serve signal transducing, structural and transport roles and are targets of disease causative and treatment agents, the health consequences of meeting the challenge are great. A multifaceted approach will be taken to advance our understanding of membrane protein function by producing structure grade crystals for use in macromolecular crystallography. Emphasis is placed on crystallization in lipidic mesophases by what is referred to as the 'in meso' or cubic phase method. This is proving to be a generally useful approach for membrane protein structure determination. With this method crystallization takes place in a membrane environment that is likely to be favored by the target membrane protein. We have built a unique, state-of-the-art robot that performs in meso crystallization in high-throughput fashion and that requires miniscule amounts of protein, lipid and precipitant. It will be used in the current application to produce 3-D crystals for the high-resolution structure determination of a select group of membrane proteins. The target group covers a broad range of membrane protein types including eukaryote and prokaryote, integral and peripheral, monomeric and multimeric, as well as protein-protein and protein-peptide complexes. Some of the target proteins will be produced in-house, some will be provided by individual collaborators, while others will be supplied through the NIH Structural Proteomics Initiative. [unreadable] [unreadable] In line with the NIH Structural Biology Road Map, and in parallel with the proposed structure study, effort will be devoted to improving crystallogenesis and to broadening the range of membrane protein targets that yield to structure determination. This will be achieved by implementing a synthesis program whereby lipids with desirable physico-chemical characteristics are produced for use in crystallization trials. The molecular mechanism of crystal nucleation and growth will be studied too with a view to more rational and successful crystallogenesis. Success in obtaining crystals, and ultimately the atomic structure of membrane proteins, will enhance our understanding of some of the most fundamental processes underlying cellular function that are integral to human health. [unreadable] [unreadable] [unreadable] [unreadable]


Dietary restriction (DR) is the only experimental manipulation known to retard aging in mammals; however, the mechanism responsible for the life prolonging action of DR is unknown at the present time. One attractive explanation is that the effect of DR arises at least partially through changes in gene expression. Using the heat shock gene hsp70 as a model system to elucidate the mechanism by which aging and DR alter gene transcription, we have found that the changes in hsp70 transcription that occur with age and DR are correlated to changes in a specific transcription factor: HSF (heat shock transcription factor). In higher eukaryotes, HSF is found in non-stressed cells in an inactive (i.e., non- DNA binding) monomeric form in the cytoplasm. An increase in temperature, or other stress, results in the activation of HSF to an oligomer that binds the heat shock element (HSE) in the promoter of the hsp70 gene. We have shown that age and DR alter the activation of HSF. The objective of the research described in this proposal is to elucidate the molecular mechanism whereby aging and DR alter the activation of HSF. Specific Aims: 1. To determine if the changes in HSF activation are due to an alteration in HSF expression. The protein and mRNA levels of HSF will be measured as a function of age and DR. 2. To determine if the changes in HSF activation arise from alterations in the oligomerization of HSF. The oligomerization, phosphorylation, and translocation of HSF will be measured in cell extracts obtained from rats of various ages fed ad libitum and a DR diet. 3. To determine if the changes in HSF activation can be correlated to alterations in the physiochemical properties of HSF. The hydrodynamic properties of HSF, the affinity of HSF for the HSE, and the interaction of HSF with the hsp70 promoter will be studied as a function of age and DR. 4. To determine if the changes in the activation of HSF with age and DR arise from changes in the levels/activities of factors that inhibit the activation of HSF. A novel system has been designed for screening an expression cDNA library from the liver of old rats for the presence of cDNAs coding for inhibitors of HSF activation. 5. To demonstrate that HSF is responsible for the changes in the induction of hsp7o transcription. The ability of recombinant HSF to reverse the changes in the in vitro transcription of an hsp70-promoter/reporter template will be studied, and transgenic mice will be used to determine if the overexpression of HSF can reverse the age-related decline in hsp 70 transcription.


PROJECT SUMMARY/ABSTRACT The C9ORF72 GGGGCC repeat expansion associated with familial and sporadic amyotrophic lateral sclerosis (c9ALS), frontotemporal lobar degeneration with TDP-43 inclusions (c9FTD) and combined presentations of ALS with frontotemporal dementia (c9FTD/ALS) is the most common cause of familial ALS, ALS-FTD and FTD identified to date, and is reported in about 6% of sporadic ALS and FTD. The causes of phenotypic variability in c9ALS are not established. Wide ranging clinical expression of c9ALS is a challenge to elucidating the pathogenesis of the repeat expansion, as genotype-phenotype correlation calls for molecular genetic data and correlative clinical data in manifesting carriers of the repeat expansion and asymptomatic mutation carriers. Similarly, studies of disease pathogenesis using patient-derived biospecimens, including skin fibroblasts and assessment of postmortem neuropathology, require detailed clinical phenotypic data in order to associate biospecimen-derived data with clinical expression of disease. Studies in this P01 to elucidate the molecular pathogenesis of c9ALS will require ascertainment of patients with c9ALS, their extended family members who carry the C9ORF72 repeat expansion, and appropriate comparison groups with collection of clinical data, biospecimens and post mortem tissue. In order to identify symptomatic and asymptomatic C9ORF72 repeat expansion carriers for collection of longitudinal clinical data and biospecimens for Project 1, to provide clinical data and biospecimens of c9ALS and ALS patients with normal C9ORF72 repeat length for in vitro studies of disease mechanisms in Project 2, and to provide clinical data and post mortem tissue for investigation of the neuropathology of c9ALS in Project 3 the goals of Core B are: to identify for Project 1 at least 50 probands with c9ALS, and at least 10 ALS patients with normal C9ORF72 repeat length (c9(-)ALS) (Aim 1); through genealogic studies of c9ALS probands and c9(-)ALS patients ascertained in Aim 1 identify for Project 1 the following 3 categories of first, second and third degree blood relatives without symptoms of ALS or dementia: 1) at least 50 c9ALS relatives carrying the C9ORF72 repeat expansion; 2) at least 5 c9ALS blood relatives who do not carry the repeat expansion; and 3) at least 5 blood relatives of c9(-)ALS patients (Aim 2); collect as called for in Project 2 single time point biospecimens including skin tissue from 1) at least 10 patients with c9ALS and 2) at least 10 c9(-)ALS patients ascertained through Aim 1 (Aim 3); and, offer all study subjects participation in our deeded autopsy program, and for those who die during the course of the project, conduct autopsies to harvest brain, spinal cord and other tissues in support of Projects 1 and 3.


We will investigate the factors regulating the subcellular distribution of the high conductance, calcium- and voltage-gated BK channel, a critical ion channel that regulates neuronal firing output in health and disease. Despite its powerful role in modulating excitability, experimental evidence indicates that it is sparsely distributed at the plasma membrane, a phenomenon that is regulated via interactions with the brain-specific accessory subunit, ?4. Conventional methods to study membrane protein localization have relied heavily upon overexpression of tagged proteins, a method that can significantly alter protein distribution by changing the stoichiometry of the target with its regulatory factors. To accurately determine how BK channels are distributed across the cell, it is important to be able to determine the location of individual molecules at endogenous expression levels to preserve critical concentration-dependent interactions with regulatory partners. We have developed a novel protein/dye tag with high-fluorescence emission that enables single-molecule detection, for both high- and low-abundance proteins. To preserve normal channel expression levels, we will generate a transgenic mouse where this tag has been inserted into the endogenous BK channel gene. The localization of this channel in primary neurons derived from these animals will be evaluated, and its accessory subunit and activity-regulated surface distribution will be determined.


The relationship between aging and carcinogenesis was explored by transforming cultured cells derived from various sources with oncogenes. To simulate the in vivo state, we prepared primary culture cells from lung and skin tissues. Young and old rats were mainly used as donors. For in vitro studies, fibroblasts from human fetal lung and rat whole embryos were passaged to appropriate population doublings. Cells were transfected with the calcium phosphate precipitated oncogene DNA, then subjected to a focus assay. The number of foci, which indicate the number of transformed cells, were examined to determine if older cells respond differently to the transfected oncogenes than younger cells. Our results indicate that cellular aging alters the intrinsic susceptibility to oncogene induced neoplastic changes at the cellular level.


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. As part of its dissemination strategy, BioCAT takes part in any facility wide workshops that are organized by the APS and other institutions that covers techniques supported by BioCAT. The main purpose of the National School on Neutron and X-ray Scattering is to educate graduate students on the utilization of major neutron and x-ray facilities. Lectures, presented by researchers from academia, industry, and national laboratories, will include basic tutorials on the principles of scattering theory and the characteristics of the sources, as well as seminars on the application of scattering methods to a variety of scientific subjects. As part of this activity Prof. Irving gave a lecture on non-crystalline diffraction in the formal sessions. As part of the school students conducted six short experiments at Argonne's Advanced Photon Source and Oak Ridge's Spallation Neutron Source and High Flux Isotope Reactor facilities to provide hands-on experience for using neutron and synchrotron sources. As part of this activity BioCAT hosted 14 graduate students to provide training in small angle x-ray scattering techniques. The admission process to the school is highly selective and so we view this as a highly effective way of reaching the next generation of researchers.


More than 200,000 patients are treated every year for peripheral nerve injury. Approximately 90% of patients never recover full movement of an injured limb. Slow axon regeneration and/or misdirected axon targeting are most often blamed for these poor functional outcomes. Increased neuronal activity, such as through exercise or electrical stimulation, enhances axon regeneration following peripheral nerve injury. We have shown that brain derived neurotrophic factor (BDNF) expression in the neurons whose axons are regenerating is required for this enhancement, but also that signaling through androgen receptors is required in both males and females. The source of the ligands for these receptors, androgens, is not known, especially in females. We hypothesize that their availability could be altered by the effects of neuronal activity on steroid converting enzymes. By measuring the expression of these enzymes in both neuronal and non-neuronal cells in response to increased neuronal activity, we anticipate that we can identify the sources of androgen that promote enhanced axon regeneration. The targets of androgenic activity required to promote axon regeneration also are poorly known. By evaluating the effects of increased neuronal activity on axon regeneration in mice in which the androgen receptor gene is knocked out in a cell-type specific manner, we will be able to identify the cellular requirements for androgen receptor-mediated enhancement of axon regeneration. In this proposed study, we will use a combination of newly available technologies to investigate these roles. Clinically, androgen therapy is already in use. Knowledge of androgen production and signaling is needed to tailor this potential therapy for peripheral nerve regeneration and could be applied to other neurodegenerative diseases.


Schizophrenia is currently thought to be a complex genetic disorder, with altered neuronal development, perhaps very early in life, important in determining vulnerability. Neurogenesis and neurodevelopment occur in the adult olfactory epithelium, and probes of the olfactory system may provide insights into the neuro-biological basis of altered neurodevelopment in this disorder. Indeed, studies of patients with schizophrenia have demonstrated robust behavioral, structural, and functional impairments of the olfactory system. Our work, along with others', has indicated that olfactory brain regions are affected by both neurodegenerative and genetically-mediated neurodevelopmental processes. This project represents the only systematic effort to examine the underpinnings of these chemosensory impairments from a life-span perspective. To date, our efforts have established that: 1)pervasive and stable olfactory deficits exist across the lifespan in schizophrenia;2)no significant medication effects are seen;3)similar psychophysical, structural, and functional deficits exist in first-degree relatives of patients;and 4)underlying facial structures such as nasal and palate volumes are abnormal. Over early fetal life, cerebral morphogenesis proceeds in embryological intimacy with craniofacial morphogenesis. As such, quantitative analysis of craniofacial and cerebral dysmorphology along with a detailed psychophysical assessment of the olfactory system may provide important information concerning the developmental origins of schizophrenia. In this application, we propose an in-depth assessment of the structural and functional abnormalities of the olfactory system from an early neurodevelopmental perspective. We will study 40 schizophrenia patients, 40 otherwise healthy first-degree family members, 40 unrelated healthy controls, and 40 high-risk subjects who present with early symptoms of psychosis. New methods will be utilized to examine the neurodevelopmental contributions to chemosensory dysfunction in patients including nasal/palate volume, structural MRI, and quantitative examination of facial morphology. Predictive utility of these impairments will be probed by examining subjects at increased risk for the development of psychosis. Our working model is that olfactory impairment reflects developmentally disturbed processes of neurogenesis and synapse formation and that these measures may represent specific markers of embryological dysmorphogenesis underlying schizophrenia. Schizophrenia is currently thought to be a complex genetic disorder, with early developmental abnormalities in brain structure and function, being important in determining vulnerability to illness. This study will examine changes in smell abilities in schizophrenia along with detailed measurements of the nasal and oral cavities and mapping of facial and brain structure and topography. We believe that robust olfactory impairment seen in patients reflects a disturbance of an early developmental process and that the analysis of these functions and structures may provide important information concerning the developmental origins of schizophrenia.


Clinical-grade T cells rendered specific for CD19 have demonstrated anti-tumor activity. We are now proposing a translational study to investigate the temporal-spatial biodistribution and microenvironment associated with adoptively transferred CD19-specific T cells as achieved using positron emission tomography (PET). To target aggressive B-cell malignancies, we have initiated two clinical trials to infuse autologous and allogeneic T cells that have been genetically modified to express a CD19-specific chimeric antigen receptor (CAR) which recognizes CD19 on the cell surface, independent of MHC. This new R01 grant application establishes an inter-disciplinary (chemistry, imaging, biostatistics, bioinformatics, nuclear medicine, gene therapy, and immunology) and multi-institution (MDACC and TMH) team, partnering with industry (CellSight Technologies, Inc.) to investigate a platform for imaging infused CAR+ T cells by PET. This will be accomplished by coexpressing a mutant of herpes simplex virus-1 thymidine kinase (sr39tk) with the CD19-specific CAR in T cells using the Sleeping Beauty (SB) transposon/transposase system which we have adapted for clinical translation. We will synchronously electro-transfer two DNA plasmids expressing the SB transposons (i) CAR and (ii) sr39tk, using a new method we dub ?double transposition?. Aim #1 seeks to determine if non-viral gene transfer will produce T cells that co-express CD19-specific CAR and sr39tk under control of constitutive and conditional promoters. The sr39tk reporter gene will be fused to hygromycin phosphotransferase (Hy) and thus CAR+sr39tk+ T cells will be selectively propagated in presence of cytocidal concentration of hygromycin B on γ- irradiated artificial antigen presenting cells that co-express CD19 along with desired T-cell co-stimulatory molecules. Aim #2 seeks to undertake longitudinal μPET imaging of infused human CAR+sr39tk+ T cells with the reporter probe [18F]FHBG in immunocompromised mice to assess biodistribution and sensitivity of detection. T-cell activation status will be imaged by comparing (i) conditional expression of sr39tk under control of NFAT promoter with (ii) the new PET probe [18F]F-AraG developed at CellSight. T-cell hypoxia will be assessed by introducing a molecular sensor for oxygen to test whether sr39tk can report low oxygen tension. Aim #3 seeks to translate these pre-clinical data to a new clinical study infusing CAR+sr39tk+ T cells in patients undergoing gene therapy with CD19-specific T cells. This trial will be a companion study to our existing trial (IND# 14193) infusing CAR+ T cells after autologous hematopoietic stem-cell transplantation for research participants with advanced B-lymphoid malignancies. The PET probe [18F]FHBG, marketed by CellSight Technologies, will be manufactured for clinical imaging at TMH, per IND #61880. In aggregate, these studies will test the central hypothesis that CD19-specific CAR+sr39tk+ T cells can be imaged in humans using PET. These studies will establish principles and practices for translating PET-based imaging of CAR+ T cells and provide the first human imaging data on the biodistribution of genetically modified T cells.


Research Project 1 Summary HIV infected individuals have heightened cancer risk. Particularly for those caused by human oncogenic viruses such as EBV, HPV and KSHV. AIDS Malignancies are a sanitary problem for Latin American Countries as Argentina, even in the post HAART era. There is scant information regarding cancer among HIV-infected individuals in Argentina. Moreover, translational research in AIDS malignancies is an area traditionally neglected where most Argentinean clinicians and oncologists rarely collaborate. The recent convergence of efforts of Dr. Mesri laboratory at University of Miami Miller School of Medicine's Sylvester Comprehensive Cancer Center (UMMSM/SCCC), Dr.Omar Coso at IFIBYNE ? UBA ? CONICET and Dr. Pedro Cahn from Fundacin Husped the most prestigious ONG devoted to AIDS studies, diagnosis and prevention have converged in this consortium proposal under the U54 format, including other Senior Prestigious Researchers and Junior Scientists in every Consortium Component. Project 1 aims to ?Define oncogenic signaling networks in AIDS-associated viral cancers as targets of chemoprevention and treatment.? The proposal will make use a variety of strategies at the molecular, cellular level and even through animal models to identify molecules (mainly proteins and miRNAs) implicated in the onset of viral cancers and investigate its relationship with HIV infection, using different approaches and targeting different molecules. Putative Molecules identified as responsible for the onset of cancer by laboratory approaches will be studied in Human Samples with the aid of Project 2. Relationship with the OGB Core will allow enhancement of the research capabilities a significant step further by using unbiased genomic analysis, as well as IHC techniques, to obtain high throughput data that will validate data obtained in the laboratory and prompt new questions into the bench making possible to address research questions that were unimaginable only a few years back. Mentoring is of paramount importance to this project. Dr Coso will lead the studies in KS related molecular signaling working as a PI to Project 1. On the other hand and combining with his role in the CE Core, he will supervise Dr Ana Raimondi, exploring the possibility of targeting mTORC1 in HPV models. Dr. Coso will also team with his former trainee Dr Julian Naipauer, who has developed a new cell and animal model of KSHV oncogenesis, conducting experiments in the laboratory and mentoring for the future development of his scientific career as a viral oncologist upon return to Argentina.


The Scientific Review Committees (SRC) provide thorough scientific reviews of all protocols to be activated at the Moffitt Cancer Center. Support of the SRC process is the primary focus of the staff within the Protocol Review and Monitoring (OPRM). Specific goals of the PRMS are: ? Appropriate and thorough scientific and statistical review of Moffitt protocols ? Efficient processing and accurate tracking of Moffitt clinical research protocols, and ? Monitoring of scientific progress for ongoing clinical research trials. OPRM support staff process the documents necessary for the review committees and facilitate timely communication of SRC actions between the committee and investigators. All correspondence is saved electronically and noted within the research database (Oncore?). Support staff members also assist investigators in the development of protocol-specific data and safety monitoring plans. The OPRM office maintains an accurate protocol-tracking database for use by the regulatory staff, principal investigators, study teams, and committee members to monitor protocol progress through the review processes, including SRC and IRB review, budgeting, and contracting as appropriate, and an operational review process to allow ancillary departments to review and sign off on protocol feasibility. Other OPRM staff members assist Moffitt investigators with efficient, accurate review and processing of research protocols and submission to the IRB for human subject approval and work with monitors in the data and safety monitoring process. Moffitt has committees that perform scientific review, each meeting monthly. Two committees review clinical research proposals, while the third committee reviews tissue and data studies. Meeting dates are staggered to allow for SRC meetings approximately every 10 days. The three SRCs provide the initial scientific peer review of the protocol and monitor these protocols throughout the term of the study for scientific progress (e.g., meeting accrual goals) and for changes that might impact the objectives of the study. The PRMS requests CCSG Support of $247,671, which is 28% of its operational budget.


The Cancer and Stem Cell Biology (CSCB) Program Area was established in 2010, in response to increasing evidence that the concepts and tools of stem cell biology can be used to gain new insights into cancer biology, and to take advantage of the tremendous expansion and productivity of the stem cell research community at UCLA. The primary goal of the CSCB Program Area is to link basic and translational investigators interested in the unique biological processes shared by malignancy and stem cells. The fundamental biology that characterizes these two fields can be viewed as the control of the opposing processes of self-renewal and differentiation. The balance of these processes is, in turn, determined by cell intrinsic and micro-environmental cues. These common mechanisms are studied by CSCB investigators using normal stem cells and their malignant counterparts from two experimental platforms: Hematopoiesis and Epithelium. Similarly, clinical research projects in CSCB focus predominantly on hematologic and genitourinary malignancies. Scientific interactions between the two platforms are driven by three main integrating themes that are relevant to the study of malignancy and stem cell biology irrespective of the specific tissue/cell source: 1) the regulation of growth and differentiation in tissue-specific stem cells during malignant transformation and normal development; 2) the role of microenvironment in tumor formation and stem cell regulation; 3) the use of pluripotent stem cells as a model to study basic mechanisms of self-renewal and transformation. The CSCB Program Area is comprised of 31 members, representing three schools and twelve departments; six members are


We propose to carry out the detailed structure analysis of selected nucleotides, nucleotide analogs and modified nucleotides of transfer RNA to obtain further support for the established rules of the stereochemistry of nucleic acid constituents as well as gain additional insights into the conformational perturbations on nucleotides wrought by base and sugar modifications. The strain energy incurred by a nucleotide when it assumes the unfavorable conformations will be studied by obtaining precise information on bond distances and bond angles of cyclic nucleotides. Structural studies on metal salts of nucleotides typically contain a large number of water molecules. Hence, these studies will be aimed at providing information on the preferred binding sites and coordination schemes of the metals with the nucleotide base, sugar and phosphate groups as well as the role of water molecules on nucleotide conformations. Our continued studies of the nucleotide antibiotics is beginning to disclose some of the relevant structural features for antibiotic action. Whenever possible we focus our attention on studies of oligonucleotide structures. These studies which are being performed in conjunction with theoretical potential energy calculations are designed to uncover the broad as well as the intricate rules that govern the structure of nucleic acids. BIBLIOGRAPHIC REFERENCES: Nygjerd, G., McAlister, J., Sundaralingam, M. and Matsuura, S., "The Crystal and Molecular Structure of the Fluorescent Base Yt (1H-4, 6-Dimethyl-Imidazo (1,2-a) Purine-9-One) in tRNA." Acta Cryst., B31, 413 (1975). Prusiner, P. and Sundaralingam, M., "Molecular and Crystal Structure of the Modified Nucleoside 2' -O-Methyladenosine. A Novel 2'-exo-3'-exo (2T3) Sugar Pucker." Acta Cryst., in press (1975).


ABSTRACT Chronic neuroinflammation of the olfactory epithelium, as occurs in chronic rhinosinusitis (CRS), results in the loss of the sense of smell, due in part to cytokine-mediated neuronal death and inhibition of olfactory regeneration. Within the scope of the parent grant, the exploration of the effect of chronic neuroinflammation on the olfactory system extends to the axonal projections of the olfactory epithelium to the olfactory bulb (OB). In the brain, including the OB, neuro-immune interactions are increasingly believed to play an important role in the pathogenesis of Alzheimer's disease (AD) and its related dementias. Neuroinflammation is a prominent feature of AD that is highly correlated with disease escalation. The precise etiologic relationship between inflammation and AD histopathology is uncertain, but recent evidence increasingly suggests that systemic inflammatory disease is a risk factor and exacerbator of ADRD. The OB, which is an early site of AD histopathologic change, is connected and in close proximity to the olfactory epithelium. Directly exposed to the outside environment, the delicate olfactory neuroepithelium is subject to microbial exposures and local immune responses. In chronic rhinosinusitis, the olfactory epithelium becomes infiltrated with inflammatory cells producing a variety of pro-inflammatory mediators. A case-control study has linked dementia with a history of chronic rhinosinusitis, suggesting that longstanding nasal inflammation may propagate effects to the brain. The major goal of this project is to use a mouse genetic model of chronic olfactory epithelial inflammation to investigate whether neuroinflammation spreads to the OB over time, either driving deposition of amyloid plaques and neurofibrillary tangles, or exacerbating disease histopathology in mouse AD models. We further plan to investigate the role of the MAP kinase JNK in AD pathophysiology, which is an inflammatory signaling mediator that we have been studying as a therapeutic target for olfactory neuroprotection in CRS. Using genetically-modified mouse strains and pharmacologic agents, we will explore the roles of specific cytokines implicated in ADRD in mediating olfactory inflammation-induced AD-like changes in the OB, and whether these can be modulated by inhibiting JNK activity. The experiments described in this proposal will afford new insights into neuroinflammatory mechanisms that may trigger early stages of ADRD.


Project Summary In the past decades, extensive research has aimed to identify risk factors, causative agent, treatments and preventive strategies for Alzheimer's disease (AD), the most common type of dementia. These efforts have predominantly focused on proximate behavioral and biological factors, but the upstream social determinants of cognitive decline and AD and related dementias (ADRD) are not well-understood. The overall objective of this proposed project is to ascertain how marriage, an important but overlooked social risk/protective factor, is linked to trajectories of cognitive decline and risk of developing dementia in late life. Marriage has long been identified as the most important type of social relationships that holds the greatest significance for health during adulthood. However, surprisingly, scientists know little about whether and how marriage influences cognitive decline and progression to ADRD. Rapid social changes in marriage, family formation, and family dissolution mean that an increasing number of older Americans are entering late life with complex marital histories, which reshapes key aspects of their lives, and may in turn affect cognitive function over the life course. We hypothesize that the complex life-course traits of marital relationships such as current marital status, histories of marital dissolutions, and marital strain, will influence trajectories of cognitive decline and dementia risk over one?s life course via multifaceted economic, psychosocial, behavioral, and biomedical pathways. This project breaks new ground with an innovative interdisciplinary life-course research design that conjoins data from three NIH-sponsored, national, longitudinal datasets: (1) Health and Retirement Study (HRS), (2) National Health and Aging Trends Study (NHATS), and (3) National Social Life, Health and Aging Project (NSHAP) to examine a full life course picture of marital relationships including marital status, marital biography and marital quality linked to cognitive trajectories and dementia. We will apply advanced statistical methods to systematically examine economic, psychosocial, behavioral and biomedical mechanisms through which marriage influences cognitive trajectories and dementia risk over the life course, and further assess potential gender differences in the processes. The interdisciplinary research team is characterized by a complementary set of talents and experience in population health, cognitive aging, marriage and social relationships, neurology, neuropsychology, bio-demography, epidemiology and quantitative methodologies that uniquely position them to carry out this innovative project.


While HIV/AIDS can be managed with antiretroviral drugs, these agents do not clear the virus and require life-long administration. Recently, we discovered a completely new class of compounds that interfere with the HIV-1 virulence factor called Nef. This viral protein is critical to HIV-1 replication in vivo, immune escape of HIV-infected cells, and AIDS progression. During the past year, we successfully completed Phase I of our STTR project aimed at development of Nef antagonists suitable for clinical testing. Working with the Fox Chase Chemical Diversity Center, we evaluated 50 analogs of our original diphenylpyrazolodiazene Nef inhibitor, some of which display tighter Nef binding while retaining potent antiretroviral activity and improved ADME properties. Several analogs prevent Nef-mediated downregulation of MHC-I on HIV-infected CD4+ T-cells, resulting in activation of autologous anti-HIV CD8+ CTLs. These data suggest that our Nef antagonists may restore recognition of HIV-infected cells by the patient's own immune system as a path to functional cure. In this Phase II application, we will expand our Nef drug development efforts with the following Specific Aims: Aim 1: Perform lead optimization medicinal chemistry. Based on SAR developed during Phase I, we propose to synthesize 100-150 new Nef inhibitor analogs to find suitable compounds for in vivo testing in Aim 3 using HIV-infected humanized mice. Our approach will employ structure-based design while considering analog ADMET and PK properties in parallel. All analogs will also be tested for Nef binding affinity, effects on Nef-mediated enhancement of HIV replication and reversal of CD4 and MHC-I downregulation by Nef in HIV-infected patient cells. Aim 2: Ensure suitable drug properties via in vitro and in vivo ADMET evaluation. Up to 15 compounds per year that meet Nef-binding and functional criteria (Aim 1) will be evaluated using in vitro ADMET assays including microsomal stability, CYP 3A4 inhibition, solubility and plasma protein binding. Three to six (IV) and 2-3 (PO) compounds will be evaluated in mouse PK studies, with the 2-3 most promising compounds advancing to in vitro non-GLP safety assays. These data are essential for choosing the best compounds for in vivo testing in humanized mouse models of HIV/AIDS (Aim 3). Aim 3: Test the hypothesis that Nef antagonists can suppress HIV replication and T-cell loss in a humanized mouse model of AIDS. Humanized mice infected with Nef-deleted HIV-1 exhibit dramatically lower viral loads and substantially less T cell depletion than those infected with wild-type virus. The most promising compounds identified in the first two Aims will therefore be administered to humanized mice to monitor effects on HIV replication, CD4+ T cell loss and immune system function. Successful completion of these goals will provide a comprehensive package to support advanced development, safety testing in large animals and advance the project further toward an IND submission for Phase I safety testing in normal volunteers.


Patients with B-cell malignancies [non Hodgkin lymphoma (B-NHL), B-chronic lymphocytic leukemia (B-CLL) and acute lymphoblastic leukemia (B-ALL)] respond to T cells redirected with chimeric antigen receptors (CARs) specific for CD20 or CD19 antigens, and encoding costimulatory endodomains. Targeting these antigens, however, does not distinguish between normal and malignant B cells, so that this approach, when effective, causes profound B-cell aplasia. It is therefore important to identify targets expressed more selectively by B-cell malignancies. B-NHL and B-CLL cells express monoclonal immunoglobulins (Igs) that contain either ?- or ?-light chains. As a proof of principle, we hav implemented a phase I clinical trial in which patients with relapsed/refractory ?+ B-cell malignancies are infused with autologous T-cell products engineered to express a CAR that targets the ?-light of human Igs, and also contains the CD28 costimulatory endodomain (CAR.?.CD28TM.CD28). This CAR would target normal and malignant ?+ cells, but spares the subset of normal B cells that express the ?-light chain. This study is currently ongoing, and it enrolled 10 patients. Two patients achieved complete response and 4 patients experienced disease stabilization. Although promising, this study also shows some limitations such as the suboptimal in vivo expansion/persistence of these cells. We discovered a specific role of the CD8? stalk that when incorporated in CARs expressing either CD28 or 4-1BB signaling domains dramatically enhance their antitumor effects. Our central hypothesis is that the inclusion of the CD8??stalk in the CAR.? (CAR.?.CD8?.CD28), rather than CD28 transmembrane domain and signaling domains, will enhance the expansion and persistence of CAR-T cells in vivo and promote higher rate of clinical responses. In the proposed phase I study we will address the mechanistic role of the CD8? stalk in CAR signaling, and then conduct a phase I clinical study in which each patient will receive two T-cell products expressing either the current CAR.?.CD28TM.CD28 or the new CAR.?.CD8?.CD28 allowing us to clearly evaluate the expansion/persistence of each T-cell subset within the same patient. On completion of this first-in-man study we will know whether this novel CAR can substitute the CD19-specific CAR currently used for mature B-cell malignancies. We will also develop preclinically the specific CAR that targets the ?-chain in order to implement a strategy that covers the great majority of patients with B-cell mature malignancies.


Human granulocytic anaplasmosis (HGA) is an emerging tick-borne disease caused by Anaplasma phagocytophilum, an obligate intracellular bacterium of neutrophils. A. phagocytophilum infection impairs neutrophil function by transcriptional reprogramming, where the reprogrammed neutrophil promotes inflammatory recruitment of new neutrophils, tissue injury, ineffective regulation of inflammation, and poor antimicrobial responses. We studied altered neutrophil function with A. phagocytophilum infection and focused on how the nuclear effector protein AnkA, when delivered into the host cell where it binds to promoters of genes regulated with infection, induces epigenetic chromatin remodeling and transcriptional reprogramming. The granulocyte transcriptome with A. phagocytophilum infection shows a number of differentially transcribed genes that promote infection [3-6]. Given the meager genomic resources of A. phagocytophilum, it is difficult to explain the extent of host transcriptional change and functional reprogramming by individual translocated effector proteins. This implies that the bacterium exerts influence over global gene transcription, including chromatin and histone remodeling, perhaps by targeting conserved mechanisms of transcriptional regulation such as in cellular differentiation and neoplasia. AnkA has properties that suggest function as a matrix attachment region-binding protein that could regulate access of chromosomal territories to transcriptional modifiers, a new paradigm in bacteria-host interactions. We hypothesize that AnkA binds to promoters of some transcriptionally regulated genes and modifies or recruits modifiers of epigenetic chromatin marks or transcription factors. In addition, we hypothesize that A. phagocytophilum reprograms the global neutrophil transcriptome by altering the epigenome through AnkAs action on nuclear matrix, chromatin, and transcriptional apparatus recruitment. We propose the following aims: 1. To identify AnkA binding sites in the CYBB promoter and to define AnkA domains or motifs involved in CYBB promoter binding and transcriptional activity. 2. To determine whether AnkA affects host gene transcription through direct action at the CYBB promoter or through recruitment of chromatin remodeling or transcription factors. 3. To determine whether AnkA functions as a matrix attachment region-binding protein that tethers DNA to nuclear matrix, regulates DNA loopscape, and permits docking of other chromatin modifiers in global transcriptional regulation. The effects that bacteria have over cellular transcription are increasingly recognized. Testing these hypotheses will provide evidence of a potentially powerful mechanism for prokaryotic control over eukaryotes. The long- term goals are to develop a mechanistic understanding of how bacteria with intimate host cell associations circumvent host functions. This information could allow rational preventions and therapies for HGA, but could also span biology and medicine, since such molecules could be engineered as epigenetic tools or therapies. PUBLIC HEALTH RELEVANCE: Human granulocytic anaplasmosis (HGA) is a tick-borne disease caused by an intracellular bacterium, Anaplasma phagocytophilum that must live within neutrophils and similar cells. Interestingly, neutrophils are the major early host defense cells, and A. phagocytophilum alters their function to benefit survival of the bacteria. The altered function occurs mostly because the mRNA-producing machinery of the cell is altered by infection with this bacterium. We learned that one protein made by the bacterium, AnkA, is transported to the neutrophil's nucleus where mRNA is made, and its presence there changes how mRNA is made. In fact, the magnitude of changes in mRNA made by the cell are very difficult to explain based on AnkA altering mRNA made from individual genes. We propose to study the exact way that AnkA changes mRNA production from single genes, and to study whether it also affects the structure and function of chromosomes in a way that mRNA production is drastically altered. This information could provide evidence of an entirely new way for bacteria to control host cells, could define some aspects of normal cell function, and might provide new tools, even new drugs, for studying cells and their function in health and disease.


Huntington's Disease (HD) is an autosomal dominant neurodegenerative disorder characterized by chorea, dementia and personality disorder affecting between 5 to 10 persons per 100,000 in the U.S. Although the mean age of onset of HD is about 40 years of age, manifestations of the disease may occur as early as 2 and later than 80 years of age. There is no effective treatment for HD which is progressive and follows an inexorable course leading first to total debilitation and then death some 15 to 20 years after onset. Typically the HD patient requires several years of full time nursing care and the disease can represent an extraordinary psychological and financial burden for both their family and society as a whole. The Huntington's Disease Center Without Walls is a basic research center that was established in 1980 with clearly stated goals: the discovery of the nature of the genetic defect causing HD, the elucidation of the mechanisms whereby this defect acts to produce the clinical and pathologic features of the disorder, and the development of effective approaches to its treatment. The Center is multidisciplinary, encompassing in a single cooperative program a broad range of research approaches including genetics, epidemiology, biochemistry, and neuroanatomy. In the past grant cycle we accomplished the goal of identifying the HD mutation. The disorder is caused by an expanded, unstable CAG trinucleotide repeat in a gene encoding a large novel protein named huntington. In this renewal period we intend to study the dynamic behavior of the HD CAG repeat, its clinical correlates, factors that modify these parameters, the origin of repeat instability, the connection between CDA expansion and impairment of energy metabolism, the consequences of CAG expansion on neural development and anatomy, on structure, expression, localization and interactions of huntingtin protein and on metabolism of cultured neuronal cells. The HD Center comprises a team of highly interactive investigators with a unique combination of talents and considerable experience and dedication to HD. We are confident that the next 5 years will see tremendous strides toward achieving our goals of understanding the nature and consequences of the genetic defect, and of being able to provide an effective treatment for this devastating disorder.


Salmonella are one of the most important causes of gastrointestinal infections. The overall goal of our research is to understand how the innate immune response helps to protect against salmonella infections. The first aspect of innate immunity that we will study is PMNs. We previously reported that human epithelial cells make the potent chemotactic peptide IL-8 when invaded by salmonella, which implies that PMNs are important for the host defense against this pathogen. We then showed that PMNs are crucial for defense against salmonella that have virulence plasmids, but not those without virulence plasmids. This proposal focuses on the role of PMNs in protecting the intestine from salmonella infections, using a model of oral infection in mice. The role of PMNs will be established by making mice neutropenic with a monoclonal antibody (RB6-8C5) to the Ly-6G antigen that is specific for myelocytes. We will look for evidence that one or both chemokines are made in vivo in infected mice and in vitro using both cultured murine intestinal epithelial cells and colon organ culture. The second component of the innate immune response that we will investigate is lipopolysaccharide binding protein (LBP). We have confirmed the report by Jack et al (Nature, 389,742,1997) that LBP-deficient mice are more susceptible to salmonella infections, but the mechanism of action of LBP has not been elucidated in infection. We used mice that had the wild-type Nramp1 allele so are genetically resistant to salmonella infections. LBP deficient mice had lower levels of 2 ELR+ CXC chemokines. We will now study the role of CXC chemokines in host resistance to Salmonella by blocking the CXC receptor with antibody. The third goal is to determine whether complement mediated phagocytosis is critical for early resistance to salmonella infections. We will study the course of infection in mice that are deficient in C3 and determine whether neutropenia makes them more susceptible to infection. We will compare isogenic strains of salmonella that express either the group B or 0 LPS antigens in C3 deficient and neutropenic mice, in order to determine how the structure of the LPS affects virulence in mice.


The value of this study of normal and abnormal embryology as an experimental approach to determine factors leading to regeneration of flexor synovial tendons has been demonstrated in the past granting period. The failure of synovial differentiation in paralyzed chick embryos leads to the choice of early motion as an inducer of regeneration to be studied in adult chick tendons in this grant. Suturing techniques to allow early motion as well as the effects of early motion will be evaluated in animal flexor digital tendons by light and electron microscopy, microvascular studies and biomechanical testing. Both an Instron testing machine and an arthrograph will be utilized for the mechanical studies. As well as the above applied studies, basic investigations will be continued in chick embryos and tendon organ culture. In addition, collagense activity in injured tendons and organ culture will be investigated as an additional modifiable factor in tendon healing. This enzyme activity will be determined by quantitative assay and localized in tissue by immuno assay.


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. Sub-project 4 description Obesity is associated with an increased risk of cardiovascular death. One potential contributing factor in obesity-associated cardiovascular deaths may be related to the pro-thrombotic and pro-inflammatory states induced by increases in adipose mass, both of which are critical components of the pathogenesis of the clinic manifestations of atherosclerosis. Platelets play a central role in arterial thrombosis, are activated in inflammatory states, and are directly influenced by specific adipokines, and therefore have the potential to serve as an essential mediator of the cardiovascular consequences of obesity. alpha-Granules are essential to normal platelet activity. Many inflammatory factors and cytokines are stored in [unreadable]-Granules. Activated, but not resting platelets are able to alter the chemotactic properties of endothelial cells by inducing the secretion of monocyte chemoattractant protein (MCP-1). Similarly, transforming growth factor-beta (TGF-beta) is released from activated platelet alpha-granules and has been shown to augment the release of type-1 plasminogen activator inhibitor (PAI-1) from adipose tissue. Therefore, platelet secretion may play an important role in the pro-inflammatory and pro-thrombotic consequences of diet-induced obesity. The cGMP/PKG pathway plays a critical role in platelet secretion. In addition, we have recently identified a role for Src family kinses, especially the Lyn kinase, in platelet secretion. Therefore, we will manipulate these signaling pathways to identify a role of platelet secretion in obesity-associated pro-inflammatory and pro-thrombotic states.


Membrane traffic is required for a broad range of essential cellular functions, such as controlling the accessibility of cell surface receptors, the translocation of glucose transporters in response to insulin, antigen presentation, neuronal transmission and the establishment and maintenance of epithelial cell polarity. Therefore, the regulation of membrane traffic is directly relevant to a broad range of human diseases including cancer, diabetes and neural degeneration. Rab GTPases are key regulators of membrane traffic. By recruiting and activating a functionally diverse set of effectors, a single Rab GTPase can coordinate the various sub- reactions within a given stage of membrane traffic, including vesicle budding, delivery, tethering and fusion. Furthermore, our results indicate that adjacent stages of transport can also be coupled through coordinated rab regulation. We recently defined a rab guanine nucleotide exchange factor (GEF) cascade in which one rab, in its GTP-bound state, recruits the GEF that activates the next rab along the exocytic pathway. We also have preliminary evidence for a rab GTPase activating protein (GAP) cascade operating in a counter current fashion. Here the downstream rab recruits the GAP that inactivates the upstream rab. The net effect is rab conversion in which a given patch of membrane starts out labeled with one rab, but over time becomes labeled with another rab. Since each rab recruits and activates a distinct set of effectors, this leads to a functional maturation of the membrane. We will explore these two cascade mechanisms in further detail and test the physiological consequences of uncoupling adjacent stages of membrane traffic. We will test the role of a Sec4p effector in SNARE assembly and explore the roles of several new putative Sec4p effectors. Through these studies we will begin to define the exocytic pathway as a fully coordinated system, rather than as a collection of isolated sub-reactions. Membrane traffic is the mechanism by which material is transferred between different compartments within the cell and the regulation of membrane traffic is directly relevant to a broad range of human diseases including cancer, diabetes and neural degeneration. This study addresses the molecular mechanisms by which different stages of membrane traffic are coordinately regulated.


Aging and stress responses are tightly linked~ indeed, most interventions that extend lifespan appear to do so at least in part through potentiation of stress responses. Nevertheless, the genetic and mechanistic basis of this central relationship remains poorly understood. Here we will test the hypothesis that microRNAs (miRNAs) coordinate stress-responsive pathways in response to lifespan-prolonging interventions. We propose the following: To identify critical new miRNAs that link stress responses to aging in C. elegans, we profiled expression of small RNAs during development, aging and in stressed conditions (heat shock, starvation, hypoxia and oxidative stress). Since miRNAs that are differentially regulated during stress are likely to be mechanistic regulators of the stress response, we propose to characterize ten of the most differentially expressed miRNAs and determine their position in the gene-regulatory architecture of stress responses. We will also integrate our previous profiling and functional analyses of aging-associated miRNAs with these results to identify miRNAs that are associated with both stress and aging and test whether these genes provide mechanistic links between these conditions. To investigate network-level roles of microRNAs in regulation of stress and aging, we propose to elucidate the underlying regulatory network of genes and miRNAs involved in aging and stress. In order to identify critical new sets of miRNAs and pathways that link these processes, we have integrated transcription-factor binding site information, with miRNA target predictions to build a preliminary interaction network of the known regulatory relationships between transcription factors, aging- associated miRNAs, and miRNA biogenesis genes. We also propose to determine targets of key miRNAs biochemically via CLIP-seq and RNA-seq in the presence and absence of the miRNA. These data will allow us to improve the known miRNA-mRNA regulatory interaction network. Then, using this network, we will find miRNAs that comprise feedback loops and highly connected interaction nodes. We will test whether these highly connected miRNAs play critical roles in aging, stress responses, or in integrating the two. To identify miRNA mediators of lifespan extension due to dietary restriction. Our preliminary data point to miR-71 and miR-228 as key network nodes connected to pha-4 and skn-1, transcription factors critical for the response to dietary restriction and other stressors. We will characterize the roles of thes miRNAs and use our gene- regulatory network to identify other such candidate miRNAs. We also propose to identify miRNAs differentially expressed in dietary restriction via deep sequencing and include these in our network analysis above. We are uniquely well situated to carry out this work, as the Slack lab combines extensive experience in miRNAs and aging biology with leading expertise in genome-wide small-RNA characterization. MiRNA analogues and antagonists are pharmacologically tractable~ thus identifying critical aging and stress responsive miRNAs in C. elegans may lead directly to lifespan and healthspan-prolonging interventions in humans.


Cyanide is an archetypal chemical threat. This powerful natural toxin is encountered in many different settings and potential exposures range from industrial accidents to major terrorist attacks. Cyanide has devastating consequences. Higher quantities lead rapidly to seizures, cardiovascular collapse and death, while lower doses are associated with substantial long-term morbidity including debilitating central nervous system injury. Currently available antidotes simply do not match the complexity or scale of the potential mass casualty threat scenarios, and there is an urgent need to develop novel countermeasures. The major aims of this CounterACT Center of Excellence proposal are 1) to identify novel classes of cyanide countermeasure using unbiased approaches in a validated zebrafish model system, 2) to explore in vivo the structure activity relationships (SAR) of these novel countermeasure classes and using medicinal chemistry generate a series of optimized lead compounds and 3) to validate these optimized leads in established murine and rabbit models of cyanide toxicity. Together these individual interdisciplinary aims are focused around the overarching goal of this collaborative U54 application: The identification of at least 3 novel cyanide countermeasures ready to move directly to formal preclinical testing and Phase I clinical studies at the completion of the current application. Successful generation of three validated countermeasures using this pipeline will not only lay the foundation for parallel work in preclinical development (through the CounterACT Preclinical Development Facility or CPDF as outlined below) and for subsequent clinical development of these compounds (in a logical extension of the current proposal), but will also form a generalizable approach for the accelerated development of countermeasures to any existing or emerging chemical threat.


The purpose of this study is to follow children who are currently enrolled in or were previously enrolled in pediatric ACTG treatment or drug research studies to look for any late consequences of therapy in growth, neurologic and neuropsychologic function, long-term survival and quality of life.