This application's major objective is to test the efficacy of a potential microbicidal agent to prevent transmission of HIV to women. Such an effective agent is urgently needed to combat the predominant rise globally of new HIV infections in women. Based on promising preliminary results in vitro, the specific aim of this application is to test in vivo the efficacy of glycerol monolaurate (GML) to prevent intravaginal transmission of simian immunodeficiency virus (SIV) to rhesus macaques, a highly relevant nonhuman primate model of sexual mucosal transmission of HIV. Treated or control monkeys will be infected and biopsy and necropsy specimens will analyzed by immunohistochemical and in situ hybridization methods and viral loads in blood to assess the efficacy of GML in preventing transmission or altering viral replication in cervicovaginal and [unreadable] lymphatic tissues. [unreadable] [unreadable] [unreadable]


Previously, we reported the alternative splicing of gc pre-mRNA as a new mechanism to downregulate surface gc protein expression and to produce a new bioactive molecule in the form of soluble gc proteins. Notably, we found that increased expression of sgc resulted in impaired generation and differentiation of thymic NKT cells, which critically depend on IL-15 signaling. Interestingly, generation of IL-2-dependent Foxp3 T regulatory cells or IL-7-dependent CD8SP thymocytes were not affected, indicating distinct sensitivity of different gc cytokine receptors to sgc proteins. Altogether, these results demonstrated a previously unappreciated role for sgc in downregulating surface gc expression and in dampening gc cytokine signaling in thymocytes. Realizing such impact of alternative splicing on cytokine receptor expression, we shifted our focus to other receptors of the gc cytokine family. Among others, we found soluble IL-7Ra proteins in serum of both human and mice suggesting a potential role for these protein products in controlling T cell immunity. In humans, soluble IL-7Ra has been previously described and it is known that they are produced by alternative splicing of the IL-7Ra pre-mRNA. In marked contrast, so far, soluble IL-7Ra proteins has not been reported in mice. In fact, there is no molecular evidence reported for an alternative IL-7Ra mRNA splice isoform in mice, so that soluble IL-7Ra has been considered not evolutionally conserved. However, here we found that mice do express soluble IL-7Ra. By establishing a sensitive ELISA assay to serum IL-7Ra proteins, we could detect soluble IL-7Ra proteins in serum of wildtype mice. In humans, soluble IL-7Ra is produced by alternative splicing that omits exon 6, which encodes the entire transmembrane region. However, we were unable to detect such alternative transcripts in mice, suggesting that the mechanisms to produce soluble IL-7Ra proteins differ between humans and mice. Cloning of IL-7Ra mRNA species from mouse T cells showed that alternative splicing of IL-7Ra pre-mRNA utilized a distinct mechanism from human T cells in that is use intron-retention, instead of exon exclusion, for alternative splicing. Thus, we identified the molecular basis of soluble IL-7Ra proteins in mice. Nonetheless, we do not exclude the possibility of membrane protein shedding to produce soluble IL-7Ra proteins, and we are also investigating other post-transcriptional mechanisms of IL-7Ra production. Whether soluble IL-7Ra proteins are involved in controlling T cell immunity and T cell differentiation is an important question that is currently under investigation. In parallel to these studies, we used fluorescent beads to quantify the absolute number of gc cytokine receptors on cell surface of T cell subsets with the aim to gain better understanding of the expression under steady state condition and during T cell activation/differentiation. We found that IL-7Ra proteins outnumbered gc molecules at a ratio of four to one on surface on resting naive CD4 T cells. On the other hand, other gc cytokine receptors such as IL-4Ra or IL-2Rb were expressed at significantly lower numbers than gc. These results indicated that the availability of gc proteins is limited for association with IL-7Ra, and consequently IL-7 signaling is curtailed on CD4 T cells. However, this is not the case of other gc cytokines, such as for IL-2, IL-15 and IL-4, where gc expression and availability are not limiting to initiate cytokine receptor signaling. To understand the quantitative effects of cytokine receptor availability and signaling, we have generated a series of new experimental models that include enforced expression of gc or IL-7Ra proteins on T cells. The downstream biological effects of altering gc availability is currently under investigation.


The purpose of this proposal is to develop an Implementation Science Resource Core to support the research efforts of several CHAART consortia, building upon existing infrastructure within these consortia. The current proposal would create the infrastructure to close the loop of the research cycle for NIAAA-funded research. ORCAAA (The Operations Research Collaboration for Alcohol Abuse and AIDS) would be a resource core using implementation science to inform decisions important to investigators, public health authorities, and clinician/patient dyads. In particular, ORCAAA will create 3 different PODS (Portals Of Decision Support) that will function as catalysts of collaboration, providing CHAART investigators with tailored decision support in order assure that research will be harnessed most effectively to improve population health. In particular the ORCAAA resource core will implement the following aims: Aim 1: Facilitate implementation of CHAART HIV/AIDS interventions targeting highest risk populations; Aim 2: Increase capacity for implementation science to impact CHAART study design; Aim 3: Increase capacity for evaluating effect of CHAART alcohol interventions on HIV progression


ABSTRACT Therapeutic interventions that favorably impact the untoward natural history of heart failure (HF) either slow or reverse left ventricular (LV) remodeling. In some patients with HF with a reduced ejection fraction (HFrEF) reverse LV remodeling is associated with freedom from future clinical HF events (?myocardial recovery?), whereas in the great majority of patients the initial stabilization of LV structure/function is followed by progressive LV remodeling and untoward clinical outcomes (?myocardial remission?). Thus, although recovery of LV structure and function are associated with stabilization of the clinical course of HF, as well as reversal of many aspects of the HF phenotype, it is not associated with freedom from future HF events. Understanding the clinical and biological features of ?compensated? HF patients, who have normalized or partially normalized LV structure and function, but remain vulnerable to hemodynamic/neurohormonal stress, represents a major unmet need in the field of heart failure. The long term goal of this research initiative is to delineate the mechanisms responsible for the functional instability of hearts that have undergone reverse LV remodeling with recovery of LV function, and to develop new therapies that will address this unmet clinical need. To explore the biological basis for the clinical phenomenon of ?myocardial remission,? we have developed a clinically relevant pre-clinical model of ?reversible heart failure? that combines moderate aortic constriction (TAC) and distal LAD ligation (MI), which are the major comorbidities that cause HFrEF in industrialized nations. To ?reverse? the HF phenotype, the TAC + MI mice are hemodynamically unloaded by surgically removing the aortic constriction, resulting in the normalization of LV structure and function. Germane to the present proposal, when the de-banded TAC + MI mice (?HF-DB? mice) are exposed to a neurohormone stress, they develop increased LV hypertrophy and increased LV dysfunction, analogous to what is observed in HFrEF patients who develop functional instability following recovery of LV structure and function. Based on our preliminary observations that the autophagy-lysosome system is dysregulated during the development and recovery from HF, we prose to test the following three hypotheses: (1) autophagic flux is impaired during the development of HF and, although flux is relatively improved following hemodynamic unloading, flux remains ?inefficient? (Aim1); (2) autophagic flux is required for effective reverse LV remodeling (Aim 2); and (3) insufficient autophagic flux is responsible, at least in part, for the functional instability that develops in reverse LV remodeled hearts that are exposed to neurohormonal stress (Aim 3). Specific Aims 1-3 will provide definitive information with respect to the potential role of autophagy in the recovery of LV structure and LV function following hemodynamic unloading, as well as the functional instability of reverse LV remodeling in a pathophysiologically relevant model of reversible HF.


We plan to isolate and characterize as many enzymes of the pyrimidine-synthesis system of Crithidia fasciculata as is possible. We will use standard procedures of purification including ammonium sulfate precipitation, heat treatment, acid and alkali treatment, Sephadex and ion exchange chromatography and electrofocusing. All enzymes will be tested for inhibition by natural end products as well as metabolic analogs. Special attention will be given to those enzymes which differ in control from those of mammalian systems.


This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The SSRL BTP staff provides user hands-on training for the effective use of the SMB SAXS/D instrument. Users first receive a brief safety orientation specific to BL4-2 and updates on recent equipment changes. Users receive a short tutorial on Blu-ICE used for the majority of non-crystalline scattering and diffraction experiments. They also receive training on the use of MarParse, the data processing program for solution scattering developed recently by the BTP staff, as well as its GUI version MarParseDlg. Users are instructed on the basics in solution x-ray scattering data analysis, such as inspecting data for possible radiation-induced aggregation, subtracting the background, and calculating radii of gyration. The data processing program Fit2D, developed at the European Synchrotron Radiation Facility, as well as Primus from EMBL-Hamburg are also covered upon request. Introductory tutorials to the sophisticated solution scattering data interpretation software packages, developed by Dr. Svergun (EMBL-Hamburg), are covered. Users are advised on practical experimental aspects on experiment geometries, sample-handling equipment, and data collection strategies especially for minimizing radiation damage to help improve data quality. These are given to new user groups and have been found useful also for experienced users.


The specific molecular mechanisms and nuclear events involved in leading to the transcription of pathological growth factor and inflammatory genes under diabetic conditions are not fully resolved. Circulating monocytes in diabetic individuals would be continuously exposed to hyperglycemic conditions. Monocyte activation, adhesion, transmigration and foam cell formation are key events in the pathogenesis of atherosclerosis. There is evidence of increased leukocyte-endothelial interactions in diabetic animals and with monocytes from human diabetic subjects. However, the behavior of monocytes cultured under high glucose (HG) and diabetic conditions have not been well studied. Much less is known about in vivo transcription mechanisms leading to the regulation under diabetic conditions of inflammatory cytokine and chemokine genes such as tumor necrosis factor- alpha (TNF-alpha), monocyte chemoattractant protein-1 (MCP-1) and cyclooxygenase-2 (COX-2) that are implicated in monocyte activation and atherosclerosis. The hypothesis is that diabetic conditions lead to in vivo nuclear chromatin remodeling and key alterations in the nuclear transcriptome machinery. This induces the transcription of NF-kB-regulated inflammatory genes in monocyte/macrophages and leads to enhanced monocyte activation and adhesion. The Specific Aims are: 1. To determine whether HG conditions increase interactions between NF-kB p65 transcriptionally active subunit and key transcriptional coactivators with histone acetyl transferase activity in monocytes. 2. To examine in vivo nuclear transcription and novel chromatin remodeling mechanisms by which HG and advanced glycation end products (AGEs) lead to inflammatory gene transcription in monocytes. 3. To evaluate the functional relevance of NF-kB activation under HG and AGE treated conditions by adopting state-of-the-art RNA-interference techniques to silence NF-kB p65. The project is supported by our preliminary data that human monocytes under HG conditions, as well as monocytes from diabetic patients produce significant amounts of TNF-alpha and MCP-1 in an oxidant stress and NF-kB-dependent manner. Our new data also shows evidence of chromatin remodeling under HG conditions. This project adopts several innovative approaches including in vivo chromatin screening and gene silencing, and could lead to novel new therapies for diabetic complications. [unreadable] [unreadable]


The histone genes of the sea urchin provide one of the best systems for the study of gene regulation in eukaryotic organisms. These genes represent as much as 0.3-0.5% of the sea urchin DNA as there are as many as 1000 copies of each histone gene in the genome. The genes are organized into a repeating unit of 6-7 kb, which contains the genes for the five histones interspersed with five spacer regions. The availability of gradient-enriched histone DNA as well as cloned histone DNA recombinants provides suitable nucleic acid hybridization probes to investigate both the nature of the genes and the presence of complementary transcripts. Our objectives are twofold: a) To learn more about the organization of the histone genes - the extent of length heterogeneity and where it exists, and the identity and organization of the genes coding for the sequence variants of H1, H2A, and H2B histones. b) To study the regulation of histone synthesis during development, including the basis of control of transcription of these genes. The first set of studies will employ the techniques of restriction enzyme mapping, Southern transfer hybridization, electron microscopic visualization of DNA heteroduplexes, and recombinant DNA techniques. The second objective will be pursued by the use of cell free translational systems and RNA-DNA hybridization to assay for content, rate of synthesis, and turnover times of each histone mRNA. An in vitro system will be used to assay the factors involved in the control of gene transcription. Our results should provide significant information on the maintenance, organization, evolution, and regulation of the histone genes.


Despite our understanding that consolidation of long-term memories (LTM) require new protein synthesis, the specific molecular substrates of translation are yet to be understood in detail. Recent studies have described abnormal protein synthesis in several neuropsychiatric disorders. Importantly, therapeutic potential of inhibition o translation in specific brain regions for treating Post-traumatic stress disorder (PTSD) have been described. Thus, better understanding of the molecular substrates of translation will help develop novel therapeutic treatments for disorders such as PTSD. The central hypothesis of this proposal derived from our own studies is that activation of translation of specific mRNAs in specific compartments of pre and postsynaptic neurons is required for establishing LTM. To test this hypothesis, we will identify molecular substrates of translation in specific neuronal compartments of pre and postsynaptic neurons by exploring the advantages of recently described tagging of polyribosomes (TRAP: Translating ribosome affinity purification) and well- characterized neural circuitry of gill-withdrawal reflex (GWR) of marine snail, Aplysia californica Specifically we will (1) isolate and characterize tagged polyribosomes immunoprecipitated from somatic and synaptic compartments of pre and post synaptic neurons of GWR that are exposed 5HT, a neurotransmitter important for learning in Aplysia and (2) characterize their function in memory storage by knockdown of specific candidates and electrophysiological analysis. We anticipate that these studies will provide the first ever description of molecular substrates of translation in specific pre and postsynaptic compartments of a neural circuitry and their temporal regulation during memory storage. Furthermore these studies will bring novel insights into dynamics of translation during LTM. These studies will have an important positive impact on our fundamental understanding of neural circuit function and memory storage and likely lead to identification of new targets for preventive and therapeutic interventions of memory disorders.


The cause of syphilis, Treponema pallidum, has never been cultivated in vitro. The inability to grow this spirochaete in large numbers free from tissue has prevented successful studies leading to the development of a useful vaccine, studies on the mechanism of disease production, and the solution of complex immunologic problems associated with this disease. We have shown that this organism is capable of obtaining energy from glycolytic pathways, terminal electron transport to 02 and coupled oxidative phosphorylation. We propose to obtain additional physiological information on its biosynthetic capabilities, transport problems, control mechanisms and lytic phenomena, with the aim of applying such information to the in vitro cultivation of this spirochaete.


Five patients with malignant melanomas (4 choroidal and l of the ciliary body) were treated with proton irradiation at the Harvard Cyclotron during the period from July l975 to January l976. The patients were given a tumor dose of 5000 to 6000 rads Cobalt 60 equivalent delivered in 5 fractions over a period of 8 to 9 days. All patients tolerated treatments quite well without adverse effects. Response of tumor to therapy could't be evaluated at completion of treatment since there was no evidence of any reaction at the tumor or the surrounding retina. No definite regression is yet observed in any patient but a change in the color of the tumor has been seen in the patient with the longest follow-up. No further growth of the tumor occurred in any patient. No complications were noted on the follow-up visits except minimal epilation (loss of few eyelashes) in one patient and a small erythema of the lid which disappeared gradually without any sequelae. An accurate method of aiming the proton beam within the eye has been developed. Four to five tantalum rings 2 millimeters in diameter are sutured to the sclera at the edges of the tumor, which are localized by indirect ophthalmoscopy and transillumination. The rings are used as markers for stereotactic radiography in order to align precisely the tumors with the proton beam. Monkeys irradiated two to three years ago have been followed with general eye examinations, color fundus pictures and fluorescein angiography. This should be continued for another two to three years in order to be sure that all delayed effects of irradiation have been observed. A new on-axis TV viewing system with a camera of higher sensitivity and resolution (greater than 500 TV lines at 4.5 ft. c) has been worked out and is expected to allow monitoring of eye movements continuously throughout all phases of preparation and treatment, eliminate parallax and improve monitoring at lower levels of illumination with reduced eye discomfort for the patient.


After decades of a protein-centric view of gene regulation, it has become clear that the control of gene expression by regulatory RNAs is equally important (1-2). New small and large noncoding RNA molecules continue to be discovered at a staggering rate in bacterial model organisms as well as in the transcriptomes of bacterial communities (3-5). Newly discovered structural and functional aspects of such RNAs have reached a degree of breadth that requires a meeting with a strong focus on bacterial RNA research to fully address the diversity of these new regulators of gene expression and bring together the scientists involved in these studies. Regulating with RNA in Bacteria will be the first conference dedicated to this topic and will be a premier forum for the presentation of cutting-edge advances and the latest perspectives in the areas of discovery, mechanisms and structure of bacterial riboregulators. Funds are requested to provide partial support for this meeting, which will be held on March 7-11, 2011, in Cancun, Mexico. The conference is sponsored by the American Society for Microbiology (ASM), which provides both management expertise and financial support. We have carefully chosen 31 invited speakers with high international visibility, known affability and effectiveness to maximize the breadth and timeliness of presented work. Our program will cover prominent and emerging roles of RNAs in regulatory networks, and on current understanding of mechanisms of action for diverse RNAs. One session will be dedicated to other molecules that impact sRNA function, including ribonucleases, which are both friend and foe of regulatory RNAs, as well as RNA-binding proteins. Another session will focus on structural analyses of RNAs, which have lagged behind protein-related work. Systems and synthetic approaches to studying and using riboregulators, as well as emerging mechanisms of RNA-based immunity against invading genetic elements will complete the program. In addition to the invited scientists from around the world, the program will include 20 speakers selected from submitted abstracts to provide flexibility in programming to include cutting-edge advances and to promote high visibility of rising young investigators. The main goal of the meeting is to bring together researchers that use different approaches to study different aspects of RNA regulation in divergent bacterial systems, thus facilitating cross- fertilization of ideas among investigators, postdoctoral fellows and graduate students studying a variety of bacteria. PUBLIC HEALTH RELEVANCE: Bacteria constitute the largest class of infectious agents, causing devastating diseases in humans, animals and plants in both the developed and developing world. Our understanding of control of bacterial gene expression, which forms the basis for successful prevention and treatment strategies, until recently has neglected the many roles that RNA plays in pathogens as well as commensal organisms. Recognizing the high pace of discovery in RNA research and its great potential for therapeutic interventions, this conference will foster a better understanding of how regulatory RNAs might be exploited as new drug targets in order to treat and prevent disease at a particularly critical time when the pipeline of conventional antibiotics is waning.


Epidemiological and clinical studies have established an association between periodontitis and Type 2 Diabetes Mellitus (T2DM)/obesity. However, the causality and mechanisms linking periodontitis to T2DM are not known. We have established animal models of induced periodontitis to study the causality underlying this association. Using Zucker Diabetic Fatty (ZDF) rats, we demonstrated that periodontitis accelerates the development of insulin resistance (IR) and T2DM in high fat (HF), but not low fat fed animals. Since certain bacterial lipopolysaccharides (LPS) from periodontal lesions and free fatty acids (FFAs) from HF diet promote inflammation through Toll-like-receptor 4 (TLR4), and inflammation in general promotes insulin resistance, we have recently explored the role of TLR4 in mediating periodontitis-enhanced IR using mice with TLR4 loss-of-function (LOF) mutation and wild-type (WT) controls. Our results demonstrate that insulin signaling is impaired in the livers of WT TLR4 vs. TLR4-/- animals with periodontitis and furthermore, the plasma glucose level improves in TLR4-/- animals with periodontitis. Insulin functions to maintain whole body glucose levels in a narrow ideal range by balancing production of glucose by the liver and glucose uptake by skeletal muscle and adipose tissue. We therefore hypothesize that: 1) periodontitis affects insulin target organs (liver, muscle and adipose tissue) by suppressing insulin signaling via TLR4, leading to an alteration in whole body glucose homeostasis, 2) LPS triggers hepatic IR, 3) periodontitis/LPS affects insulin secretion via a TLR- dependent mechanism by influencing beta-cell mass and/or glucose-stimulated insulin secretion. To test these hypotheses, we will 1) determine the effects of periodontitis/LPS on insulin target organs via TLR2 & TLR4 by a) identifying specific sites of inhibition of insulin signaling, and b) determining the expression of key molecules involved in hepatic glucose production (G6Pase, PEPCK) as well as peripheral glucose uptake (AS160) by skeletal muscle and adipose tissue using whole body TLR4 and 2&4 knockout mice, 2) determine the pathways involved in periodontitis-induced hepatic IR and altered glucose homeostasis via TLRs using TLR2&4 adoptive bone marrow chimeric mice, 3) identify mechanisms by which periodontitis influences pancreatic beta-cells by modulating beta-cell compensatory alterations (insulin secretion, beta-cell hyperplasia & apoptosis) via TLRs. The impact of the proposed studies will: 1) advance our understanding of how periodontitis directly influences distant organs/tissues involved in glucose homeostasis, and 2) identify for the first time, specific TLR-mediated effects of periodontitis on insulin sensitivity and glucose homeostasis. The results will impact future treatment modalities, especially for subjects who are consuming a high fat diet where inflammation/LPS resulting from periodontitis may play a central role in IR and diabetes.


Currently, failure to persist after transfer into cancer patients is a factor that limits the effectiveness of adoptive cell transfer of T cells genetically modified to express a tumor-reactive T cell receptor. The goal of this application is to modify th human TCR TIL 1383I (melanoma-reactive) by appending the cytoplasmic signaling domains of DAP10 to the end of the cytoplasmic domains of the - and - chains of the TCR. We believe that the TCR/DAP10 will directly activate the DAP10 pathway upon TCR engagement, and that these unique signals will enhance the survival of transferred T cells. Preliminary data: We have demonstrated in CD8+ T cells that signaling through the [naturally expressed] activation receptor complex NKG2D enhances memory development. Our data show that NKG2D signaling in TCR-transduced CD8+ T cells augments their anti-tumor potency and in vivo persistence. These data argue that signaling through NKG2D in CD8+ T cells is a viable approach to overcoming the limited survival of TCR- Td T cells. We will couple TCR ligation and activation of downstream NKG2D signaling. Because NKG2D is unable to signal by itself, we will use the signaling domain of DAP10, the adaptor molecule that mediates signaling downstream from NKG2D in CD8+ T cells. We will modify the human TCR TIL 1383I (tyrosinase- reactive) by appending the cytoplasmic signaling domains of DAP10 to the end of the TCR cytoplasmic domains of the - and - chains. Hypothesis: We hypothesize that the TCR/DAP10 will directly activate the DAP10 stimulatory pathway upon TCR engagement, and that these unique signals will enhance the survival of adoptively transferred T cells. Strategy: The retroviral vector containing TCR/DAP10 construct will then be transduced into human T cells. Human T cells expressing TCR/DAP10 will be examined for their signaling pathways, capacity to survive and ability to mediate the regression of established human cancer using a humanized model of melanoma. Immune deficient A2/NSG mice and human melanoma cell lines will be used for anti-tumor experiments. We will test our hypothesis through the following specific aims SA1. Develop TCR/DAP10 constructs to induce and dissect DAP10 signaling. SA2. Study the signaling pathways utilized by TCR/DAP10 and their cellular consequences in human T cells. SA3. Test T cells expressing TCR/DAP10 against human melanoma. The significance and innovative character of our strategy would be that these engineered T cells will have specificity for tyrosinase, and engagement of their TCR will activate the DAP10 costimulatory pathway as well as downstream TCR signaling.


The core resources include the computer hardware and support personnel required to supply the computational needs of the component projects. The core will also provide the administrative personnel for the visiting scientist program for the program project and for the organization of meetings.


In the first 3 quarters of FY 2010 , the FACS Core Lab provided service to 24 NCI labs or branches and 2 other institutes. From these labs, there were 68 principal investigators using the FACS Core Facility. These labs, with the number of principal investigators and the number of individuals using the FACS Core in parentheses, are: CCBB (5 principal investigators,18 individual users), Dermatology Branch (1,1), Genetics Branch (3,11), HAMB (1,2), LBMB (3,5), LCB (1,3), LCBG (7,21), LCMB (6,15), LCO (3,8), LCP (1,1), LEC (2,10), LHC (3,15), LICB (3,14), LM (3,6), LMB (4,13), LMP (5,11), LRBGE (1,3), LTIB (2,2), MBTL (3,6), Medical Oncology Branch (3,9), Pediatric Oncology Branch (1,2), Radiation Oncology Branch (1,3), Surgery Branch (3,4), NCI-Frederick (1,1), NHLBI (1,1), and NHGRI (1,1). From these labs, 186 scientists have used the FACS Core in the first 9 months of FY 2010 and of these, 60 were new to the Core lab this past year and received training from the core staff. 163 people used the flow cytometers and sorting was provided by the core lab staff to 84 users. Identifying and studying cancer stem cells is one of the major research areas of the FACS Core Lab users. A number of NCI labs are using flow cytometry and fluorescence-activated cell sorting to identify and sort the cancer stem cell by membrane antigen expression using monoclonal antibodies or with a functional assay involving active membrane substrate transport. Investigators in CCBB, MBTL, LEC, LHC, and LCBG are studying cancer stem cells from breast, ovarian, hepatic, thyroid, pancreatic, and lung carcinomas. The LSRII flow cytometer, the FACS Vantage cell sorter, and the new special order FACS Aria, because each is equipped with a UV laser, are frequently used for these assays. Transfection of cells with genes expressing fluorescent reporters is a technique used by the majority of the labs using the FACS Core. The FACS Core cytometers and cell sorters have been equipped with specific lasers to allow detection and sorting of cells labeled with any of the green, yellow, blue, red, and UV fluorescent proteins or with combinations of these fluorescent reporters. Sorted transfected cells are used to prepare protein, DNA, and RNA that can be used in Western blotting and microarrays. Sorted cells are also used to determine effects of siRNA, to look at signaling proteins, or may be further passaged to create stable cell lines. Fluorescent reporter proteins may also be linked to luciferase. Tumor cell lines have then been sorted based on their expression of green or red fluorescent protein to establish cell lines with high levels of luciferase. These cells have then been used to establish tumors in mice and to image metastasis. In addition to the 3 co-authored publications, 12 additional publications this year have included work done in the FACS Core and, of these, 7 acknowledged the assistance of the FACS Core staff. These publications were authored by investigators in CCBB, LBMB, LCBG, LHC, LICB, LMB, and ROB.


The distant objectives of this study are the evaluations of thermodynamic characteristics associated with selected nonhelical and aberrant structures of DNA, some that may provide specificity for the attraction of enzymes for replication, transcription, etc. Quantitative appreciation of the forces that induce different modes of dynamic behavior in DNA should help explain subtle effects on gene expression of small changes in sequence in noncoding regions, as well as the evolution of DNA sequences. The near-term goal is to measure the energetic costs of interrupting the helix and maintaining nonhelical loop structures, including the shear forces needed to separate opposing chains over short lengths in internal stretches of helix. Initially it is intended that the loop forming specimen be short lengths of oligo(A degrees T) N, where 50 greater than N greater than 10 bp, sandwiched between stable boundaries of (G degrees C) 40-80 degrees. The specimen will be synthesized as a recombinant in the Pst I site of pBR322 and excised for the physical studies. Thermodynamic characteristics are to be extracted from observations of the helix-nonhelix equilibrium monitored by high resolution optical melting at several wavelengths simultaneously by the digital-difference approximation method. Later studies will take up the question of loop formation in domains of different base compositions and sequence features. In a correlated study the thermodynamics associated with each of the ten unique stacking interactions will be evaluated from the stabilities of specific, well characterized domains producing the multitransitional melting curve of pBR322. Nearest neighbor frequencies, to which the ten stability constants are related, will be assigned to specific subtransitions that reveal themselves by changes in melting temperature when the domains responsible for the subtransitions are cut by restriction enzymes. In a separate study, the precise amounts and level of sequence variation will be determined for the 1399bp satellite I and 46bp satellite II repetitive sequences from the bovine genome. Sequence variations in these satellites will be determined from the increase in the range of temperature of the subtransition for the population of repetitive sequences over that of single cloned specimens.


High fat diet and enduring obesity pose a more dangerous threat in lupus patients than in general population. Obese lupus patients show much higher inflammation markers and risk of heart attack. The link between high fat western diet and accelerated atherosclerosis is clinically well established. Recent studies also show evidence that healthy eating patterns enhance immune function and reduce inflammation in obese patients. The inability of a large population to eat healthy signifies the need to explore medical interventions for diet-induced cardiovascular diseases in lupus patients. However the molecular and cellular mechanism of how high fat diet dysregulates the immune system is still unclear and thus hindering efficient therapy. B1 B cells reside in the peritoneal cavity and omentum, in direct contact with accumulated and inflamed visceral adipose tissue. B1 B cells are the source of natural IgM antibodies that protect from atherosclerosis through masking oxidized LDL, inhibiting uptake by macrophage and preventing foam cell formation. Dietary effect on B1 B cells that may lead to accelerated atherosclerosis has never been studied. Based on our preliminary study, this proposal aims to test the hypothesis that high fat western diet and chronic obesity may over-stimulate and disarm B1 B cells. Even worse, it may transform protective B1 B cells into self-attacking B cells. The result of this study will reveal new molecular and cellular mechanisms underlying diet-induced arthrosclerosis and provide new avenues for future design of B cell-targeted immune therapy. INNOVATION: This is a novel study where B1 B cells are for the first time investigated as a link between diet-induced obesity and atherosclerosis. The novel concept that high-fat-diet-stimulated B1 B cells may alter the balance of inflammatory and regulatory T cells will be tested using a novel animal model. A newly developed assay will replace traditional flow cytometry-based analysis of B1 B cells that will revolutionize B1 B cell study. Short-term high fat western diet might activate B1 B cells to produce atheroprotective IgM, cytokines and promote Treg cell differentiation. However chronic western diet consumption and obesity would switch protective B1 cell HYPOTHESIS: functions to pro-inflammatory response leading to the aggravation of atherosclerosis. SPECIFIC AIMS: Aim #1: Determine the extent to which diet-induced obesity dysregulates B1 B cell functions~ Aim #2: Determine how B1 cells from obese gld/ApoE-/- mice may affect Treg vs. Th17 balance. IMPACT: We propose to investigate an important yet unexplored immunologic component that may be the key intermediary between diet-induced obesity and heart disease in lupus patients. The result of this study will unravel unique aspects of B1 B lymphocytes in the abdominal cavity and omental milky spots and will provide a novel mechanism, as well as a non-static view of B cell-mediated protection and pathogenesis. The knowledge obtained from this study will be fundamental for the future design of B cell subpopulation-tailored risk-assessment, prevention and therapeutic intervention at different disease stages. PUBLIC HEALTH RELEVANCE: Obesity poses a severe life threat in lupus patients by escalating inflammation and increasing the risk of heart diseases. This proposal aims to test the hypothesis that chronic consumption of high fat western diet and consequently, the deposition of abdominal visceral fat may over- stimulate B1 B cells in the abdominal cavity and as a result, lose their atheroprotective immunologic functions. The result of ths study will provide a rationale for future design of B cell- targeted immune therapy to reduce inflammatory heart diseases in lupus patients.


Broad, long-term objectives and career goals: To acquire the knowledge and skills needed for independent patient-oriented research concentrated on improving the diagnosis and management of pulmonary malignancy through imaging and image analysis techniques. Health relatedness: About 1.2 million cases of invasive cancer are diagnosed annually in the United States. About 500,000 Americans die annually from cancer, and bronchogenic cancer is the leading cause of cancer deaths. Early diagnosis of pulmonary malignancy, comprised primarily of bronchogenic cancer and metastatic disease, is important for early treatment and patient survival. Early primary lung cancer and metastatic disease to the lung parenchyma commonly manifest as indeterminate nodules, meaning nodules that are too small to be characterized as benign or malignant by other methods, on diagnostic and, more recently, screening computer tomography (CT). Other than calcification, morphologic characteristics have not been shown to differentiate benign from malignant nodules. Change in volume over time has been used as a marker of malignancy; however, for small nodules in particular, subtle volume changes are difficult to detect and quantify consistently using current techniques. Specific aims: The immediate career goal is to obtain knowledge and research skills through a structured career development program in a mentored research environment. This entails multidisciplinary didactic training in digital imaging, image segmentation, image analysis, epidemiology and mechanisms of tumorigenesis, and clinical study design, and conducting the proposed research in a mentored environment. The overall aim of the proposed research is to facilitate the CT diagnosis of malignant nodules by the development of precise quantitative techniques for the measurement of pulmonary nodule volumes. Our central hypothesis is that early nodule growth can be detected on CT using computer-assisted techniques and be correlated with biologic indicators of preneoplasia and neoplasia. The proposed method will enable early recognition of nodule growth and, therefore, the early diagnosis of malignancy.


Self-control may be vital when individuals cut back or cease drinking. To regulate their alcohol consumption, individuals must resist temptation and restrain their desire to drink. Resisting the temptation to drink should require self-control. Recent research suggests that the exertion of self-control is costly, as it requires and consumes a limited resource (self- control strength) that may be critical to the success of self-control. Individuals who previously exerted self-control may have less self-control strength and therefore perform more poorly on tests of self-control than individuals who did not exert self- control. Self-control strength may be particularly important when individuals are regulating their alcohol intake. Specific Aim 1 will help establish whether self-control strength is consumed when heavy drinkers resist the temptation to drink. After sniffing alcohol in a cue exposure paradigm, heavy drinkers should perform more poorly on subsequent tests of self-control as compared with their performance after sniffing water when contrasted with the performance of light drinkers. Negative mood, frustration, or arousal should not mediate the decline in self-control Performance after sniffing alcohol. In addition, resisting a temptation should not influence performance on a task that does not require self-control. Comparisons between light and heavy drinkers after sniffing alcohol and water will help establish whether self-control strength is required for, and consumed in the process of resisting the temptation to drink. Specific Aim 2 will explore whether proximal and distal risk factors related to self-control may influence the risk of violating alcohol consumption limits. Any factor that reduces self-control capacity may increase the likelihood of drinking limit violation. To test the relationship between limit violations and self-control demands, heavy drinking participants who were tested in the cue exposure paradigm will call an interactive voice response (IVR) system daily for 3 months to report on their drinking behavior, self-control demands, and self-control strength. Participants who experience an increase in self-control demands or a reduction in self-control strength may be more likely to violate their drinking limit. Survival and multilevel analyses will be used to determine whether excessive drinking is related to changes in day-to-day levels of self-control strength, effort exerted to resist the temptation to drink during experimental chase of the study, and individual differences in trait self-control. The present study has the potential to elucidate the self-control processes needed to enhance the maintenance of limits on alcohol intake.


Although a number of studies have been conducted on the efficacy of various medications in the treatment of alcoholism, opiate antagonist compounds appear to show the most promise. There is increasing evidence that the endogenous opiate system is involved in the experience of alcohol effects and in the maintenance of alcohol consumption. Several opiate antagonist drugs which have reported utility in the treatment of alcoholism have different opiate receptor binding characteristics. While both naltrexone and nalmefene are predominantly mu opiate antagonists, nalmefene binds to the delta receptor with 2 times the affinity of naltrexone. Given this difference and preclinical reports of grater potency of delta antagonism on alcohol consumption, nalmefene may have more pronounced affects on acute alcohol reactivity in humans. While prospective pharmacologic treatment trials in alcoholism are the only definitive method for evaluating the utility of medications for the treatment of this condition, these trials are costly, time consuming, and put patients at risk for adverse events. Laboratory paradigms which examine the effect of medications on acute alcohol reactivity and consumption in alcoholics may be able to identify compounds which would be the most efficacious to employ in treatment trials. The proposed study will be a randomized placebo controlled comparison of the ability of two opiate antagonist drugs, naltrexone and nalmefene, to alter the reactivity of alcoholics and social drinkers to an acute administration of alcohol and to consumption of alcohol in a "free choice" limited access paradigm. Non-treatment seeking individuals with alcohol abuse or dependence (N-135) and social drinkers (N=135) will be randomly assigned to take placebo, naltrexone, or nalmefene for 7 days. On the eighth day each individual will receive their study medication in a laboratory setting and will ingest a fixed dose of the beverage of their choice followed by a limited access alcohol consumption period. Reactions to alcohol presentation (anticipatory), consumption (pharmacologic), and free choice ingestion (cognitive control and craving), will be measured utilizing subjective, cognitive, physiologic and biologic assessments. Subjects will be paid for their participation and alcoholic individuals will undergo a brief motivational counseling session at athe end of the protocol to educate them about the risks of heavy alcohol consumption and motivate them to seek treatment.


Factors and processes influencing entry into alcohol treatment programs will be investigated among population-based samples of alcoholics and first admissions to alcoholism treatment agencies. The research will be guided by a comprehensive model of factors predicting entry into an alcohol treatment program. The model includes state-of-the-art conceptualization of biopsychological factors thought to influence the severity and chronicity of alcohol abuse and dependence; a comprehensive assessment of the severity and chronicity of alcohol problems, including blood and urine tests; informal pressure by family and friends and formal pressure by employers and the legal system to seek alcohol treatment; health beliefs; and stage of change in addictive behavior. In addition, factors associated with unassisted or natural recoveries will be investigated among population-based alcoholics. Population-based alcoholics will be recruited from two prospective alcohol epidemiologic studies and from the household sample employed as controls for cases of coronary heart disease and lung cancer in Research Components 2 and 3. It is estimated that 795 individuals having a lifetime DSM-III diagnosis of alcohol abuse/dependence will be available for study; a subset of 398 will have had symptoms in the past year. Samples will be available for study; a subset of 398 will have had symptoms in the past year. Samples will be merged and post-stratified on sex, race, education, and heavy drinking. A representative samples of Erie County residents entering alcohol treatment programs for the first time(N-616) will be recruited using data on number of previous admissions to alcohol treatment, county of residence, and key study factors. These data are available from the NYS Office of Substance Abuse Service Information System for clients seen in all 50 alcohol treatment agencies in Erie County, making the recruitment of this sample feasible and cost-efficient. Analyses will employ simple and multivariate logistic regression and structural equation modelling techniques, where appropriate. A better understanding of alcohol treatment seeking behavior and unassisted recovery is critical to the improvement of current efforts to intervene more effectively and efficiently at earlier stages of alcohol abuse and dependence.


Endometrial epithelial cells undergo dramatic remodeling during the peri-implantation period. Such changes include alterations in cell-cell adhesions, in cell-matrix interactions, modified apical-basal polarity, and, in some species, cell-cell fusion. We have documented the expression of an mRNA encoding a transmembrane protein rbMDC9, a member of the ADAMs gene family with potential cell binding, cell-matrix interactions and fusogenic properties. RbMDC9 expression is up-regulated in rabbit endometrium during hormonal preparation for implantation, and expression is further augmented by blastocysts. Preliminary evidence suggests a similar up-regulation in mouse and human uteri. We will test the hypothesis that MDC9 in rabbits serve as an integrin-binding adhesion molecule between epithelial cells and, in doing so, also functions in the redistribution of junction and cytoskeletal proteins. Furthermore, we hypothesize that ADAMs family proteins participate in cell-matrix interactions and in the fusions between adjacent epithelial cells and between trophoblast and epithelial cells during implantation, and in the ectopic attachment and invasion of endometrial tissue during endometriosis. Aim of the project will be 1) to determine if domain specific-targeting and post-translational processing regulate the function of epithelial cell rbMDC9 in peri-implantation-stage endometrium, 2) to determine if rbMDC9 ligand interactions are required for uterine epithelial junction or cytoskeletal protein modifications during implantation, 3) to define the function of rbMDC9 in implantation-specific cell-cell adhesion and/or fusion processes, and determine whether its expression of processing are regulated by blastocysts in vitro, and 4) to determine if MDC9 is expressed in a cycle-specific pattern in endometrium of women with and without endometriosis, and to analyze its regulation and potential roles in the adhesion and invasion of ectopic endometrium in an experimental model of endometriosis.


(This application was written in response to the NIH Announcement for the Availability of Recovery Act Funds for Competitive Revision Applications (NOT-OD-09-058). The experiments outlined in this application were designed to expand the scope of the specific aims, research design, and methods of the parent grant (RO1 EB007357) entitled "Image-guided Hydrodynamic Gene Delivery". The objective of the parent grant is to develop an image-guided hydrodynamic gene delivery system for site-specific gene delivery to the liver. Since the start of the project last year, we have established the procedure using pigs as an animal model and demonstrated that the image-guided hydrodynamic gene delivery is highly effective and safe for liver gene delivery. In this competitive revision application we propose to expand the study by including the non-human primate as an additional animal model for further development. We plan to systematically assess the effectiveness of the image-guided hydrodynamic gene delivery in baboons in order to establish the hydrodynamic parameters that can be used for development of a computer program for gene delivery in humans. The specific aims are: (1) To study the effect of selected injection pressures and volumes of DNA solution on gene delivery efficiency to baboon liver, and (2) to assess the persistency of transgene expression and the long-term effect of hydrodynamic procedure on the transfected animals. In compliance to Acceleration of the Tempo of Scientific Research and/or Allow for Job Creation and Retention, We are going to hire two full time employees for the proposed study. In addition, the use of animals will also create job for those who are animal caring professionals. The reagents used in the proposed would also help in creating or retaining people who work in research reagent companies.


SUMMARY Glial cells play an essential role in defending brain health and managing neuronal stress and damage. Neurodegeneration triggers robust glial immune responses, including changes in cytoskeletal dynamics, glial cell migration, and increased phagocytic activity. Timely removal and degradation of degenerating axons and neuronal debris by glia confers neuroprotection in the brain. Despite the importance of glial responses to axon injury, we still know surprisingly little about how damaged neurons invoke immune reactions in glial cells. What signals are released from degenerating neurons? What prompts the release of these injury cues? Finally, how are these signals translated by glia to carry out efficient immune responses to damage? We are using the fruit fly Drosophila melanogaster as a tractable model to investigate the immune communication relays that exist between neurons and glial cells in vivo. The fly nervous system contains distinct glial subtypes that are molecularly and functionally similar to vertebrate glia. Moreover, well-established axotomy assays in the adult olfactory system and the adult wing reveal that Drosophila axons undergo a classic Wallerian degeneration (WD) program, which includes increased intra-axonal calcium waves, axon fragmentation, and subsequent clearance by phagocytic glia. Notably, our lab has recently shown that axon degeneration triggers activation of the insulin-like signaling (ILS) pathway in reactive ensheathing glia, which, in turn, elicits essential glial immune responses, including transcriptional upregulation of immune genes (e.g. the engulfment receptor Draper) and phagocytic activity. We hypothesize that neuropeptide-containing dense core vesicles (DCVs) are broadly released from severed axons to trigger immune responses in local glial cells. Here, we propose to use static and live confocal imaging, transcriptional profiling, and newly developed in vivo reporters to investigate how neuropeptide signaling between neurons and glia informs glial immune responses to nerve injury. Specifically, we will 1) monitor DCV dynamics and release in adult severed nerves, 2) utilize novel single transcript labeling methods to visualize local translation of immune mRNA transcripts in glial extensions at sites of injury, and 3) determine how neuropeptide signaling between discrete glial subtypes ensures that glial responses to degenerating axons are properly carried out. Together, these findings will offer exciting molecular and cellular insight into how neuropeptide signaling between neurons and glia govern immune responses in both acute and chronic degenerative conditions.


Pulmonary arterial hypertension (PAH) in its familial form (FPAH) is a heritable autosomal dominant disorder that, in the majority of patients, results in progressive right heart failure and death within five years of diagnosis. Bone morphogenetic protein receptor (BMPR) 2 haploinsufficiency occurs in over 70% of the FPAH patients, but intriguingly, only 20% of mutation carriers get clinical disease. This proposal aims to uncover the mechanism underlying the reduced penetrance of BMPR2 mutation in causing FPAH using patient-specific induced pluripotent stem cells derived endothelial cells (iPSC-ECs). Understanding the molecular mechanisms underlying the protective phenotype in those BMPR2 mutation carriers without FPAH could lead to novel therapeutic approaches for familial and non-familial forms of PAH as they all share reduced expression or function of BMPR2. These studies also hold the key to understanding penetrance in other genetic conditions related to PAH, such as the caveolin 1 (CAV1) mutation. Studies carried out during Dr. Gu's AHA postdoctoral fellowship utilized iPSC-ECs from three sets of FPAH patients and from their family members with the same BMPR2 mutation but without disease. Dr. Gu uncovered a compensatory p-p38 signaling pathway leading to preserved adhesion and survival of iPSC-ECs from the unaffected mutation carriers. The mechanism accounting for the preserved p-p38 signaling appears to differ among the families. The first aim (K99 phase) of Dr. Gu's proposed studies is to extend the findings described above, through gain and loss of function studies to pursue the mechanism of `carrier compensation'. She will also determine whether correction of the BMPR2 mutation by CRISPR/Cas9 technology restores BMP signaling pathways and EC functions in FPAH iPSC-EC from all three families. The second aim (K99 phase) will determine how the transcriptome and epigenome explain the protective phenotype in the unaffected mutation carriers. RNA-Seq was carried out on iPSC-ECs from all family members (n=11). Seventy-one differentially expressed genes were identified by comparing iPSC-ECs from controls and mutation carriers versus FPAH patients, and four genes of interest have been verified by qPCR. This aim is strengthened by a collaboration with Dr. Michael Snyder's lab, to help relate gene expression changes with alterations in histone marks identified by ChIP-Seq and polymorphisms called by whole genome sequencing. The third aim (R00 phase) will extend studies in Specific Aims 1 and 2 to novel mutations associated with FPAH, such as caveolin1. In the fourth aim (R00 phase), Dr. Gu will establish a high-throughput platform for personalized drug screening using iPSC-ECs as a continuous cell source. These studies will help Dr. Gu to launch a research program that optimizes the use of iPSC-derived vascular cells and integrative `omic' technologies to model vascular disease pathophysiology and to develop personalized treatments using either a bioinformatics' approach or high throughput screening to repurpose FDA approved drugs that activate the protective pathway.


Many geriatric patients are being treated for extended periods of time with neuroleptic and antidepressant drugs. The degree and type of therapeutic and side-effects of these drugs may be different in the elderly than in younger patients. Recent evidence suggests that many of the therapeutic and side-effects of these psychotropic drugs may be mediated by their interaction with neurotransmitter receptors in the brain. We are proposing to study the effects of older age on the changes in receptor function in the brain induced by chronic administration of neuroleptic and antidepressant drugs. We will use an animal model to study their effects in the rat brain. Four different age groups of rats ranging from young adult to senescent ages, will be chronically administered a neuroleptic or an antidepressant drug. Radio-ligand binding techniques will be used to assay dopaminergic, adrenergic, and cholinergic receptors in several regions of the rat brain both during, and after termination of chronic drug administration. Questions we will attempt to answer include: Do old-age rats develop quantitatively greater or lesser degrees of changes in receptor binding on these ligands during or after chronic drug treatment than younger animals? Is there a different pattern of changes in receptor binding in old rats than in young rats? The results of the proposed research may provide an animal and biochemical model to determine the differences in the therapeutic and toxic effects of neuroleptic and antidepressant drugs given to elderly patients; it may help provide an explanation for the much greater prevalence in the aged of one important side-effect of neuroleptic drugs, tardive dyskinesia.


ONE OF THE AIMS OF OUR LAB IS TO MAKE AN EQUIVALENT CIRCUIT MODEL OF THE MUDPUPPY, NECTURUS MACULUOSUS, GASTRIC EPITHELUM. MUCH OF OUR PRIOR WORK ON THE EQUIVALENT CIRCUIT HAS ALREADY BEEN COMPLETED (DEMAREST AND MACHEN, 1985). WE HAVE YET TO DETERMINE THE MAGNITUDE OF THE PARACELLULAR SHUNT. THE MUDPUPPY HAS SEVERAL ADVANTAGES FOR DOING ELECTROPHYSIOLOGICAL STUDIES, MAKING IT MUCH EASIER TO USE THAN THE STANDARD MAMMALIAN MODELS. THESE INCLUDE LARGE CELLS AND A GASTRIC MUCOSA THAT READILY LENDS ITSELF FOR USE IN A TRANSEPITHELIAL VOLTAGE CLAMP. THIS PROJECT IS CURRENTLY IN PROGRESS. THE ACID-SECRETING REGION OF THE STOMACH OF VERTEBRATES IS A COMPLEX EPITHELIUM, SINCE IT IS COMPOSED OF SURFACE EPITHELIAL CELLS AND GASTRIC GLANDS. IN THE GASTRIC GLANDS ARE CHIEF CELLS AND PARIETAL CELLS IN MAMMALS AND OXYNTIC CELLS IN OTHER VERTEBRATES. BECAUSE OF THE PRESENCE OF MULTIPLE CELL TYPES, THE STANDARD MACROSCOPIC TRANSEPITHELIAL VOLTAGE CLAMP TECHNIQUES CAN NOT BE USED TO DETERMINE THE SHUNT RESISTANCES OF THE DIFFERENT CELL TYPES. THE VIBRATING PROBE, HOWEVER, CAN BE SITUATED ABOVE OPENINGS OF THE GASTRIC GLANDS AND ABOVE THE SURFACE EPITHELIUM IN THE REGION OF EACH OF THESE CELL TYPES. USING PHARMACOLOGICAL TOOLS, THAT INCLUDE AMILORIDE, CIMETIDINE, AND ION REPLACEMENTS, WE ARE ABLE TO DISSECT THE CURRENTS AND RESISTANCES DUE TO THE PARACELLULAR AND TRANSCELLULAR PATHWAYS.


DESCRIPTION: This program for Ph.D. and postdoctoral students will train specialists who will be able to conduct preclinical research at levels ranging from the molecular to the behavioral on biological mechanisms underlying the development, maintenance, and elimination of alcoholism. Twenty members of the graduate faculty of the Oregon Health Sciences University will train postdoctoral research fellows and graduate students matriculating into programs in Behavioral Neuroscience or Neuroscience. Training will include firm curricular grounding in the basic sciences, specific training in the pharmacology of alcohol and other abused drugs, exposure to clinical and psychosocial aspects of human alcoholism, and extensive and continuous participation in basic research. The focus of our training opportunities is on biological processes involved in the etiology of problem drinking and alcoholism. Our general approach is interdisciplinary, emphasizing genetic, molecular, physiological, pharmacological and psychological/ behavioral processes. The research questions addressed by trainees fall into four general areas: (a) genetic bases for alcohol and drug responses, (b) learned and unlearned determinants of alcohol and drug reward, biological bases for self-administration of alcohol and drugs, and (d) stress and the biology of alcoholism and drug abuse. These areas reflect the research interests and expertise of a participating faculty using behavioral, systems-level, and cellular/molecular methods. Their shared biobehavioral perspective is consistent with growing evidence indicating that many forms of human alcoholism are best understood in terms of an interaction between genetic and environmental factors. Areas of faculty collaboration include: studies of genetic determinants of alcohol and drug responses; neuroendocrine and neuroactive steroid participation in alcohol's effects; studies of dopaminergic, gabaergic and glutaminergic systems involved in alcohol and drug effects; study of learned and unlearned determinants of responses to alcohol and other drugs, particularly their rewarding effects and self-administration; and studies of sensitivity, tolerance, and dependence/withdrawal phenomena for alcohol and all major classes of drugs of abuse.


Engagement of the T cell receptor (TCR) triggers a number of intracellular signaling events that lead to the differentiation and activation of T cells. Several studies have shown that SLP-76 plays a central role in T cell activation and T cell development, as mice deficient in SLP-76 lack mature T cells. SLP-76 has three functional domains: an acidic domain with three phosphorylatable tyrosines, a central proline-rich domain, and a C-terminal SH2 domain. Of these domains, mutation of the three N-terminal tyrosines results in the most profound defects in T cell development and function. It is hypothesized that the individual tyrosines of SLP-76 activate specific signaling cascades required for T cell development including thymocyte selection, and T cell differentiation and function. This hypothesis will be tested by 1) analyzing SLP-76 deficient Jurkat cells reconstituted with SLP-76 bearing mutations at one or multiple tyrosine phosphorylation sites, 2) analyzing SLP-76 knock-in mice expressing tyrosine mutations at particular tyrosine residues and 3) mating SLP-76 knock-in mice to T cell receptor transgenic mice to evaluate thymocyte selection and mechanisms of T cell tolerance. Understanding how T cells transmit signals to direct thymocyte development and mature T cell function is critical to understanding the mechanisms to drive several disease processes including autoimmune diseases such as arthritis. This work will be performed by Dr. Martha S. Jordan at the Abramson Institute at the University of Pennsylvania. Dr. Jordan's background in cellular immunology and current training in molecular immunology provides her with a distinct angle from which to approach questions of T cell development. This proposal will provide her with the tools and reagents required to study T cell function in vitro and in vivo, in normal and diseased states as an independent investigator at a research university. Full access to faculty and services provided by the University of Pennsylvania, especially those within the Abramson Institute, will ensure the success of this proposal. [unreadable] [unreadable] [unreadable]


Core C: Data Management and Statistics Core Project Summary/Abstract The overarching goal of the Data Management and Statistics Core (DMSC) within the UCI ADRC is to provide data management support and statistical collaboration to all ADRC investigators at all phases of scientific projects. Data management support includes comprehensive data management and dissemination of data arising from the Uniform Data Set (UDS) and all ADRC projects. DMSC personnel are responsible for the development of National Alzheimer's Coordinating Center (NACC) approved collection forms, maintenance of the ADRC database, and the timely transfer of accurate data to the NACC. Strong statistical design and efficient data analyses are also crucial components to achieving the scientific goals set forth by the UCI ADRC. DMSC members work closely with ADRC investigators to refine scientific hypotheses and develop analytic plans that are both appropriate and efficient for meeting the scientific needs of each investigator. DMSC statisticians are key collaborators in conducting studies and interpreting and reporting results. While a large portion of effort from DMSC faculty is devoted to providing collaboration and service to ADRC investigators, the DMSC also emphasizes the development of independent research programs among members of the DMSC. These DMSC-specific research endeavors not only increase the intellectual contribution of the DMSC to the scientific community at large, but will also lead to improved methods for collecting, entering, and analyzing complex data in the study of AD as well as other neurodegenerative diseases.


The principle of ALARA (as low as reasonably achievable) has been developed to reduce the risk of induced malignancy from radiation exposure, In practice this requires a rigorous quality control process for clinical operations in radiology. Currently, a critical tool (a radiographic test phantom) needed for quality control is not commercially available for computed radiology and digital radiography. The application's long-term goal is to develop a new class of phantoms and objective system performance tools for use in digital radiographic systems. These devices will be used to address emerging needs in computed radiography and digital radiography. These needs include (1) quality control procedures for daily operations, (2) quality assurance methods to evaluate products arid perform acceptance testing, (3) quality assurance methods for optimizing system operating parameters, and (4) development tools for manufacturers to assess new hardware and software products. Because neonatal chest radiography is a technically challenging procedure, with profound radiation dose implications, a prototype neonatal chest phantom was successfully developed in Phase I and is the focal point for Phase II phantom development. The neonatal chest phantom will be developed to simulate the disease states of pneumothorax and hyaline membrane disease. An observer's ability to diagnose these two disease states will test system resolution and noise, respectively. An automated objective system performance tool will also be developed to measure quantifiable parameters, such as resolution (modulation transfer function), noise (noise power spectrum), and detector efficiency (detective quantum efficiency). Phase II has two Specific Aims: 1) Clinical Validation of the Neonatal Chest Phantom in Normal and Disease States; 2) Clinical Validation of the Objective System Performance Tool. Successful neonatal chest phantom design will be determined by the appearance and histogram analysis of images; the attenuation values of anatomic structures; and the diagnostic performances of radiologists for detection of disease states within the phantom. Successful objective system performance tool design will be determined by agreement with established methods of calculating quantitative parameters. The commercial applications are driven by the phantom's clinical significance, which is to assure optimal equipment performance and minimize x-ray exposure, thereby reducing the concomitant risk of induced malignancy to neonates.


This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. The LTQ-Velos ion trap mass spectrometer has recently been modified to allow performing higher-energy collision-induced dissociation (HCD) within the first quadrupole (Q00) in the intermediate vacuum region of the instrument. The new instrument is called the LTQ-Velos-Pro. HCD is a collision-based fragmentation technique which uses electric potentials to drive ions through a collision gas, in this case air. This beam-like fragmentation technique produces fragmentation spectra that are qualitatively similar to fragmentation patterns observed in triple-quadrupole instruments. We investigate the utility of performing HCD on the LTQ-Velos Pro for proteomics. In particular, we compare HCD and resonant CID, using several different experimental configurations, comparing the methods in the same LC/MS run and between different runs. We also evaluate the utility of HCD in producing library spectra that are well-matched to triple-quadrupole data and are thus well-suited to providing a transition from discovery-based proteomics to a more targeted approach.


This application requests funds for travelling expenses of speakers and session chairmen participating in the 31st Gordon Research Conference on Cancer. The conference will be held at Colby-Sawyer College, New London, New Hampshire during the week of August 22-26, 1977. The pattern of the conference will follow the format utilized in the previous conference, which in general has been considered very successful. Admission will be limited to approximately 150 persons with participation of academic, clinical, industrial and governmental scientists. The topic of the conference will be "Regulation from within and from without of the Tumor Cell Phenotype." The initial session will be devoted to the operation and definition of the tumor cell phenotype as delineated by in vitro and in vivo parameters, with later sessions exploring the various mechanisms and methods by which this phenotype can be controlled or reversed. The selection of the mechanisms to be discussed will obviously be limited to those which at present appear more promising. Whenever possible, in vitro approach will be correlated to in vivo examples or experimentation. It is conceivable that some of these methodologies could eventually be useful as adjunctives to other therapeutical approaches.


A murine model of sdisseminated histoplasmosis has been established in this laboratory to facilitate studies of immunoregulatory distrubances occurring in the disease. To foster these investigations, the proposal attempts to compare the agbsolute and relative numbers of the T cell subsets in the various lymphoid organs of normal and infected animals. Studies to determine whether these subsets retain their normal function during the course of infection will also be undertaken. Attempts will be made to clone a T cell capable of modulating the response to Histoplasma antigens in vitro and in vivo. The surface phenotype of such a cloned cell will be determined. The possible production of soluble factors by these cloned cells will be studied. Finally, attempts will be made to produce anti-idiotypic antibodies capable of blocking/modulating the effects of thse cloned cells both in vitro and in vivo.


The investigations described herein are designed to provide new information on the action(s) of pituitary somatotropin (GH) and/or prolactin upon the endocrine cells of the gastroenteropancreatic (GEP) system in normal and diabetic rats by means of radioimmunoassay (RIA), quantitative light microscopic immunocytochemistry and ultrastructural immunocytochemical analysis. Both qualitative and quantitative information will be obtained on the chronic effects in vivo of exogenous FH and PRL or endogenous GH-PRL mixtures produced by transplantable mammosomatotropic tumors upon the numbers, distribution, morphology and fucntion of the insulin, glucagon, somatostatin (SRIF), pancreatic polypeptide (PP), vasoactive intestinal polypeptide (VIP), and possibly gastrin cells of pancreatic islets and the glucagon-, and GIP-producing cells of the gastrointestinal mucosa. The results of these studies correlating tissue and serum hormone levels with quantitative measurements of immunocytochemically identified endocrine cell populations within the GEP system with the diabetic status of the animals should provide further information on interactions among the pituitary, gut and pancreatic endocrine cells and, in particular, should help to elucidate the role and/or fate of the non-B pancreatic islet cells and gut hormone cells in metabolic dysfunctions characterized by either hypersomatotropism (acromegaly or giantism) or hyperprolactinemia. Since large mammosomatotropic tumors promote both an increase in B cells and a decrease in D cells in islets of tumor rats, these studies may provide insight into the mechanisms underlying the development of carbohydrate intolerance and diabetes in the above conditions, and also they may well provide a useful model for future studies of pancreatic endocrine cell interactions in normal and pathological conditions (like maturity onset diabetes).


This is a revised competing renewal of a project studying the synaptic organization of postmortem striatum in schizophrenic subjects (SZ) at the ultrastructural level. The striatum, which interacts with other brain areas to affect motor, cognitive and limbic behavior, is one of the regions affected in schizophrenia. The results of the studies in the last grant cycle indicated an increase in cortico-striatal type synapses in the caudate matrix and putamen patches, that was not caused by antipsychotic medication. The higher density of cortical-type synapses in the SZ cases than in controls suggests hyper-stimulation of striatal projection neurons. This could have several important and different downstream effects depending on the precise circuitry involved. The present application seeks to identify the specific striatal circuitry affected in SZ. SA#1) To test the hypothesis that limbic and prefrontal circuitry are perturbed at the level of the striatum, we will examine synaptic density in the subregions of the striatum that process these circuits. SA2 will examine synaptic density of striatonigral and striatopallidal neurons in the patch and matrix in select striatal territories determined in SA1. SA#2A) To test the hypothesis that striatopallidal matrix neurons in the caudate receive more excitatory inputs, the immunocytochemical localization of enkephalin, a marker of these neurons, will be performed; the number of synapses formed onto labeled spines will be compared between groups. SA#2B) Tests the hypotheses that striatonigral matrix neurons in the caudate receive more excitatory inputs, but that striatonigral neurons in the putamen patch receive normal or fewer numbers of synapses. The immunocytochemical localization of substance P, a marker of striatonigral neurons, will be performed; the number of synapses formed onto labeled spines will be compared between groups. SA#3) To test the hypothesis that typical vs atypical APDs have different effects on the patch and matrix compartment, we will treat rats chronically with APDs, process the tissue for calbindin immunocytochemistry to identify the patch and matrix and analyze EM samples obtained from each. In monkey tissue obtained from Dr. Lewis, we will examine the synaptic density (labeled with synaptophysin) within the patch and matrix compartments in chronic haldol treated animals and controls using light microscopy. The proposed experiments will: 1) distinguish between drug effects and disease related alterations in synaptic pathology; 2) will provide insight into the mechanisms of action of antipsychotic drugs; and 3) are an important initial step in identifying putative abnormal striatal circuitry that may underlie some of the psychopathology of schizophrenia. [unreadable] [unreadable]


Children with sickle cell disease (SCD) are living longer with the advent of medical advances such as prophylactic penicillin, chronic transfusion, and hydroxyurea. Despite greater longevity in SCD, the period following the transition from pediatric to adult care is critical; youth aged 18-30 years are at high risk for mortality and have high rates of healthcare utilization, leading to high healthcare costs. As such, health care transition (HCT) programs have been created to prepare patients for adult-centered care and subsequently, improve health outcomes. However, very few programs have been evaluated for effectiveness in achieving optimal health outcomes in SCD. This paucity of program evaluation is attributed to a lack of identifiable predictors and outcomes. The aims of this proposal are to: 1) identify potential predictors of successful HCT through a survey of pediatric and adult SCD providers, 2) examine the associations between predictors of successful HCT and HCT outcomes in a cohort of youth with SCD (ages 16-20 years), and 3) identify and characterize trajectories of HCT over a 24-month period and determine predictors of these trajectories. The results of this project may have significant implications for the delivery of healthcare in SCD during the period of HCT. This study will contribute to the understanding of the predictors and outcomes of HCT among youth with SCD and the long-term goal of developing approaches to best evaluate HCT interventions and identify areas of improvement. This project will provide support for Dr. Porter to establish herself as an independent investigator with a research program in HCT in youth with SCD. The short-term goals of the project are to develop her skills in systematic assessment of HCT predictors and outcomes, assist her in learning and applying more advanced methodology to examine longitudinal HCT relationships, and expand her abilities to investigate, disseminate, and translate her work into effective interventions. The goals will be accomplished via mentorship by experts in the areas of SCD and HCT, participation in didactic activities, and the conduct of a research project focused on HCT in youth with SCD. (End of Abstract)


The goal of this project is to quantify airway secretory activity in horses with recurrent airway obstruction (RAO), to investigate mechanisms underlying increased production and accumulation of airway secretions, and to assess the functional significance of this increase. Equine RAO mimics many of the changes observed in human diseases, such as chronic asthma, chronic bronchitis, and organic dust-induced airway disease, in which persistent mucus overproduction is an important component of airway obstruction. Immunochemical, morphometric, functional, and in vitro methods will be utilized to identify temporal alterations in the production, secretion, and intraepithelial storage of mucus/mucus-like material in disease-affected horses versus controls, and to assess the functional significance of any such alterations. In addition, mechanisms of increased secretion will be addressed by evaluating the effect of neutrophil elastase and endotoxin on airway secretory activity. It is my general hypothesis that equine RAO is associated with a persistent increase in mucus production that contributes to airway obstruction even during periods of apparent disease remission, that increased intraepithelial storage accompanies this persistent increase, and that increased concentrations of the inflammatory mediator neutrophil elastase or a hypersensitivity to this mediator contributes to increased secretory activity.


The long-term objective of the research described in this application is to develop new stereocontrolled methods for the synthesis of a variety of unsaturated heterocyclic systems. The vast majority of pharaceuticals used in medical practice are heterocycles. We believe by developing efficient synthetic entries to heterocyclic ring systems, we will allow promising pharacological leads in the future to be rapidly developed with potential therapeutic implications. Heterocyclization reactions are the focus of the studies we propose for the 04 to 07 project years. Cyclizations of this type are much less well developed than the corresponding preparation of carbocycles by polyene cyclizations. Our attention will continue to focus on heterocyclizations which are terminated by vinylsilanes. Since our introduction of these cyclization terminators in 1980, vinylsilanes have proven useful in a number of laboratories to terminate a wide variety of cyclization reactions. Because of the pivotal role that new initiators plan in expanding the horizons of heterocyclization chemistry, we also aim to develop new azacyclic and oxacyclic cyclization initiators. Tetrahydropridines related to the Parkinson's disease stimulaten, MPTP, will be prepared. The new antibiotic streptazolin will be prepared in enantioselective form, as will the powerful "uteroevacuant" diterpene zoapatanol. Intermediates and natural product targets will be broadly sdcrened for biological activity at the Biological Sciences Research Center of the Shell Development Co., and at NCI.


Immunosuppressants are absolutely essential for successful organ transplantation and are useful in the treatment of autoimmune disorders. Analyzing their mechanism of action can, as in the case of cyclosporine and calcineurin, yield important insights into basic features of T cell biology. We recently embarked upon an ambitious three-step project intended to use chemical biology approaches to identify unknown pathways involved in lymphocyte function. The over-arching rationale underlying the project was that compounds that inhibit T cell activation and work via unknown molecular mechanism (MMOA) could be developed into chemical probes that could be used to identify novel cellular targets, which would reveal currently-unknown aspects of basic T cell biology and might become the basis for new classes of immunosuppressant agents. The first step of the project- screening the NIH's Molecular Libraries Small Molecule Repository of ~375,000 compounds and identifying compounds with unknown MMOA- succeeded. We monitored lytic granule exocytosis using TALL-104 human cytotoxic T lymphocytes as a model, measuring externalization of LAMP-1/ CD107a using flow cytometry. Among hits with unknown MMOA was 2-N-[(2-methoxyphenyl)methyl]-4-N-[(4- propan-2-ylphenyl)methyl]thieno[3,2-d]pyrimidine-2,4-diamine, CID 49792547, which is the subject of this application. This compound inhibits lytic granule exocytosis with potency in the micromolar range, but does not work via any of the mechanisms we tested. It inhibits IL-2 production by Jurkat human leukemic T cells, confirming that has broad immunosuppressive activity. It is a drug-like molecule that is amenable to synthesis and the generation of diverse analogs. The project's intended second step was to generate analogs for structure-activity analysis, then use that information to design chemical probes to use in the third and final step, applying affinity-based and/or photo- crosslinking approaches to identify the unknown target that underlies the compound's activity. However, Chemistry Center support was withdrawn when the NIH MLPCN program ended. This application for an R03 is intended to allow us to continue to pursue the overall goals of the project by creating probes for target identification. We will create analogs of CID 49792547, then test their effects on lytic granule exocytosis, IL-2 secretion and toxicity. This information will allow us to identify the highest possible affinity analogs and will reveal sites that can be used to attach linkers which will allow the compound to be coupled to biotin for creation of an affinity matrix and to bifunctional photo-crosslinking/ click-chemistry groups that can be used to label target proteins. Future efforts will be directed towards target identification using probes developed in this application and in a companion application submitted previously focused on another compound.


Over the past 30 years there has been a dramatic increase in the number of harmful algal blooms (HAB's) in coastal waters throughout the world. As a result, there are more toxic algal species, more algal toxins and more geographic areas impacted than ever before. When these toxic species proliferate, they may cause massive kills of fish and shellfish, wildlife mortality, human illness and death. One of these marine organisms, pseudo-nitzchia produces a neurotoxin, Domoic Acid (DA). Most of what we know about the human health effects of Domoic Acid has been derived from a single documented outbreak in Montreal Canada in 1987. Persons who ate mussels with high levels of DA suffered serious medical illnesses, including seizures and coma, and 3 people died. Survivors were left with a profound memory disorder, Amnesic Shellfish poisoning (ASP). Based upon animal models, regulatory levels of DA in shellfish were established. Within the past 17 years, DA levels have been close to or exceeded these safety levels at razor clam harvesting beaches on Native American Reservations in the Pacific NW. Recent data indicate that this population is currently at risk for significant, but preventable, neurobehavioral impairment (ASP) from razor clam consumption, with memory problems ranging from the low average to amnesic range. There appears to be a dose- response relationship between exposure and memory problems and the base rate of persons meeting the criteria for severe memory impairment, or ASP is 4.3%. The purpose of this 5 year longitudinal cohort study is to extend these findings to establish their clinical and public health significance. A prospective longitudinal cohort design of 735 Native Americans (ages 6 months to 75 yrs) from three Tribes, with nested case-control study of identified cases of ASP will be implemented. The health impacts of chronic, low level exposures to DA over time will be determined as well as the exposure and host factors associated with DA neurotoxicity. A new model of ASP, to include the potential for delayed or latent toxicity, recovery, and recurrence will be tested with state-of-the-art procedures for assessing human exposure (mobile technology) and behavioral neurotoxicity in infants, children, adults and geriatric groups. Findings will have a significant clinical and public health impact. The diagnosis and prognosis for ASP will be defined and established safety levels for exposure will be re-evaluated to insure they are protective of persons with long term, repeated exposure to DA.


Chronic lymphocytic leukemia (CLL) is the most common type of adult leukemia and remains incurable. The introduction of the CD20 antibody rituximab combined with chemotherapy (chemoimmunotherapy) was the first intervention that has been shown to impact survival of CLL patients. Building upon the success of antibody directed therapy in CLL, we have actively pursued development of similar therapies directed at alternative antigens expressed selectively on CLL cells. CD37 is a tetraspanin protein of uncertain function that is expressed predominately on mature B-cells. Our research group has worked with Trubion Pharmaceutics, Inc/Emergent Biosolutions to develop CD37 directed TRU-016, a small modular immune pharmaceutical similar to an antibody but lacking the CH1 domain. Prior pre-clinical work demonstrated TRU-016 to have potent direct and immune based killing of CLL cells and also in vivo activity in several lymphoma pre-clinical models. A first in man phase 1 clinical trial initiated with TRU-016 in previously treated CLL has demonstrated single agent activity and combination studies have now been initiated. During this early phase 1 development of TRU-016 we have continued pre-clinical work to discern its mechanism of action. Preliminary data described in this proposal demonstrate that TRU-016 ligation of the CD37 antigen causes recruitment and phosphorylation of SHP1 via a potential ITIM domain within the intracytplasmic domain. SHP1 activation leads to decreased PI3-Kinase activity and AKT phosphorylation concomitant with FOXO3A accumulation in the nucleus and induction of the pro-apoptotic gene BIM and CLL cell death. siRNA targeting either SHP1 or BIM antagonizes death induced by TRU-016. Interestingly, the C-terminal of the CD37 protein contains ITAM like motif with associated activation function. Deletion of this motif enhances TRU-016 induced cytotoxicity suggesting a potential negative regulatory role for the C-terminal in TRU-016 cytotoxic effect. Consistent with this, inhibition of pro-survival signaling by PI3K and BTK overcame the inhibitory effects of the C-terminal domain resulting in enhanced TRU-016 induced apoptosis. Moving forward, this proposal seeks to further characterize in detail the ITIM and ITAM function of CD37 activated by TRU-016, study the mechanisms of cross talk between the activation and inhibitory signaling relevant to TRU-016 response and pursue development of afucosylated TRU-016 (TRU-016GV) leading to rational combination strategies that will facilitate enhanced TRU-016 and TRU-016GV response. Knowledge gained from this proposal has potential to impact not only CLL but also lymphoma and acute lymphoblastic leukemia patients who may also derive benefit from TRU-016 or TRU-016GV. Additionally, mechanistic study of CD37 ITIM and ITAM dual signaling has relevance to general B-cell immunology. The senior investigative team formed to perform this project includes expertise to accomplish the proposed goals and support from a large internationally known leukemia program. Collectively, TRU-016 and its application to CLL therapy have potential to transform treatment of this disease.


Defects in the core human mismatch repair (MMR) genes are the cause of Lynch syndrome (LS/HNPCC), as well as 10-40% of sporadic colorectal, gastric, endometrial, ovarian and upper urinary tract tumors. MMR recognizes and repairs polymerase misincorporation errors, suppresses recombination between non-allelic partially homologous DNA sequences during double-stranded break (DSB) repair (homeologous recombination; HEOR), and functions as a lesion sensor in DNA damage signaling. Unrepaired errors in MMR-deficient cells lead to mutations (Mutator Phenotype) that drive tumorigenesis. Importantly, MMR- deficient tumors display resistance to several common chemotherapeutic drugs. The components of MMR from numerous organisms have been purified and MMR reactions have been reconstituted in vitro. Approximately a dozen fundamental biochemical properties have been detailed that together may account for the excision of a mismatch from DNA. Based on these studies several mechanisms for the MMR reaction have been proposed. Remarkably, the biochemical, genetic and structural studies performed to date have not fuly determined the function(s) of the core MMR components or resolved the validity of the proposed mechanisms for MMR. Moreover, the vast majority of biochemical studies have been performed with naked DNA that is significantly different from the cellular chromatin where MMR functions occur. We have developed three robust real-time single molecule measures capable of visualizing and interrogating the detailed function(s) of MMR components. Using these new methodologies we propose to resolve the mechanism of MMR on naked DNA and then determine the function(s) of MMR components on physiologically relevant chromatin for comparison. In this renewal application we propose the following Specific Aims: 1.) enumerate the precise mechanism of human MMR using single molecule analysis, 2.) determine the functions of the human MMR proteins on physiologically relevant chromatin, and 3.) examine the biophysical mechanism of homeologous recombination suppression by MMR. We will apply our results to determine the functional defects associated with missense mutation found in LS/HNPCC families. These studies should provide a visual and quantitative platform for understanding the detailed function(s) of core MMR components in repair, damage signaling, drug resistance and tumorigenesis


When simian virus 40 (SV4O) is serially passaged at high multiplicity, a heterogeneous collection of naturally arising variants is generated. Some of these variant viruses become very abundant in later serial passages presumably due to their enhanced replication efficiency. Previous studies have shown that these abundant variants contain multiple copies of the SV40 replication origin, frequently as inverted repeats and in association with inserts of host DNA. These evolutionarily-selected origin regions will be cloned into a bacterial plasmid and then transfected into monkey cells constitutively producing T antigen (COS cells) in order to quantitate the effects of duplicated sequences, inverted repetitions, and insertions of host DNA on the replication efficiency of the SV40 origin region. In addition, the evolutionarily-optimized sequences will be further modified by in vitro mutagenesis in order to precisely define those features of the nucleotide sequence which affect replication. Clones from a monkey genomic library which hybridize to the variant-selected host DNA segments will also be analysed for functional activity. The proposed studies constitute a unique approach to understanding the functional aspects of a replication sequence and to understanding the regulation of replication. Both types of understanding have important implications for understanding oncogenesis and viral-host interactions.


Tougaloo College has a long history of producing graduates who have gone on to PhD program and to careers in research. The science division of the college has several programs such as the Minority Access to Research Careers program, Minority Biomedical Research Support program, the Alliance for Minority Progress program and the Jackson Heart Study Undergraduate Training Program that provide excellent research opportunities for our students. Between these programs we can provide research training experience for 25 students every year. However, we do not have enough faculty involved in research with whom the students can work. At present the students gain their experience off campus. We are convinced that our students will benefit even more if more of our faculty were involved in research. This EARDA FRESP/Phase II proposal will enable Tougaloo College to continue the task start under Phase I of increasing the number of faculty members involved in research. During the past three and a half years the PI Asoka Srinivasan's focus has been on underscoring the importance of faculty involvement in research, informing them of funding opportunities and improving t infrastructure in the preparation and submission of proposals. The objectives during Phase II will be to continue the efforts started under phase I and to focus more sharply the efforts to involve the faculty in research by funding 2 pilot research study awards per year and to formalize the establishment of the Office of Research Development. We expect at least 50 percent of the faculty receiving the pilot research study awards to be successful in obtaining research grants.


DESCRIPTION (Adapted from applicant's description): This proposal is requesting funds for a very promising and exciting new direction of research that may shed light on a controversial issue in the scientific community. The proposes to isolate and characterize a novel form of gonadotropin-releasing hormone (GnRH), that is lamprey-like, in the primate hypothalamus. She proposes to test the hypothesis that the primate brain has two hypothalamic GnRHs. This hypothesis is based on recent data from preliminary and collaborative studies that support her earlier claims of a novel ir-GnRI-I (lamprey GnRH-like) form in humans. The applicant proposes to isolate and characterize this novel form of GnRH in the hypothalamus of primates by screening the cDNA libraries to hypothalamus using oligonucleotide probes against lamprey GnRH as well as other related probes. She theorizes that an ancient form of GnRH (lamprey) as well as the more modem mammalian form of GnRH function as hypothalamic neurohormones stimulating gonadotropin release in the primate. In mammals, it has not been fully explained how one GnRH hormone differentially stimulates the release of two gonadotropins (FSH and LH). She is in a unique position to accomplish this proposed project because she has recently identified a full length cDNA encoding the lamprey GnRH-I and has an established collaboration with research in humans. The presence of multiple GnRHs in the hypothalamus could provide the answer to this question. Her specific aims include 1) screening and isolating cDNA clones encoding the precursor forms of brain GnRH(S) in primates and (2) screening genomic libraries using the newly identified cDNAs as probes to isolate the complete GnRH gene(s). She predicts that findings from these studies will contribute to our understanding of reproductive physiology and provide further information for developing medical therapies using GnRH analogs. A new hypothalamic GnRH in mammals could shed light on the formation of new GnRH analogs that may not have some of the side effects noted with the GnRH analogs currently used.