Aggression is a complex behavior expressed in many different social contexts. Often abused, yet essential for the survival of organisms, little is known of the biological roots of this behavior. While model systems may not duplicate aggression in all of its many human manifestations, they can be highly informative about: the relative importance of genes, hormones and experience in laying down patterns of aggression in nervous systems;detailing the neural circuitry involved;and addressing questions of how animals choose among the varied complex behaviors available within their behavioral repertoires. It is the last two matters that are the major focus of the present application. In recent years, the Kravitz laboratory has established a fruit fly model of aggression making this important behavior available for experimental analysis in an organism that is highly suitable for genetic analysis. With fruit flies aggression is complex and is shown by both males and females, the animals engage in many other inter-related behaviors, and the behaviors can be quantitatively analyzed. In addition, however, the genome has been sequenced and incredibly powerful methods are available that allow experimental manipulations to be performed with fruit flies that cannot yet even be approximated in most other animal model systems. Recently, it has been found that the same gene that determines who flies court also determines whether flies fight like males or females. The present application extends these studies and has the following Specific Aims: I. To explore decision-making between courtship and aggression, two sexually dimorphic patterns of behavior in flies, by mapping and manipulating the fruitless/doublesex-associated circuitry within the subesophageal ganglion (SOG), the taste center in fruit fly brains;II. To map the distribution of receptors for amines and peptides associated with the SOG circuitry;and III. To introduce genes into neurons that can alter the function of these nerve cells in behaving animals and to begin to address the question of how the same neurons differ in male and female animals. These studies use a wide variety of state-of-the-art genetic, behavioral and anatomical methods in exploring these Aims. For example, in collaborative studies with the Chiang laboratory in Taiwan, neuronal maps will be made that will allow examination in great detail of the possible connections between individual neurons involved in courtship and aggression. The focus will be to define the brain circuitry important in aggression and courtship at much higher levels of resolution than are now available, and then to ask how that circuitry gets established in fly brains and how animals choose between behavioral responses when conflict situations arise. PUBLIC HEALTH RELEVANCE: All organisms, including humans, must be capable of rapidly evaluating social situations and selecting proper responses from what may be a wide variety of possible behavioral choices. Such selections must be made correctly in order to allow the survival of organisms both as individuals and as species. How organisms make such choices and how patterns of innate behavior of great complexity get established in nervous systems are not well understood, and are the theme of this application.


Basic science investigations have identified an increasingly large number of genetic factors important in cancer. By communicating this genetic information to high risk individuals, we can facilitate informed decision- making about participation in cancer prevention and early detection programs. The purpose of the proposed program is to improve adherence to cancer control regimens among persons with a genetic susceptibility to develop cancer. Subjects for the primary project will be 750 women aged 40-65 who have a positive family history of breast cancer in at least one first-degree relative. A randomized factorial design will be used to evaluate the impact of Breast Cancer Risk Counseling (BCRC), a form of genetic counseling for women at high risk for breast cancer. In addition, two different styles of presenting cancer control recommendations in BCRC will be tested. The BCRC intervention will be based on the traditional medical genetics counseling model, but will concentrate on breast cancer surveillance, rather than on reproductive decisions. Telephone interviews will be conducted prior to the interventions to collect familial and medical data to calculate individualized breast cancer risks and to assess baseline risk perceptions, psychosocial functioning, and breast screening patterns. Post-intervention and follow-up surveys will be used to assess the short- and long-term impact of the interventions on these outcomes. Also, subgroups of women who are most and least likely to benefit from the different BCRC interventions will be identified, based on their precounseling psychosocial characteristics and demographic backgrounds. The information obtained from this primary project will lay the foundation for other studies to improve adherence to cancer control practices in diverse high-risk populations. The interventions proposed for these studies are replicable, and, if effective, can be administered by nurses and physicians in medical practice.


Cell migration is essential for cellular morphogenesis and tissue repair, as well as a number of other important biological outcomes, and has a critical involvement in various developmental disorders and disease states. Of particular interest have been the regulatory events underlying cell migration as they relate to cell invasiveness, metastasis, and cancer progression. Tissue transglutaminase (tTG), a GTP- binding protein which also possesses an enzymatic transamidation activity that allows it to cross-link proteins, has been shown to play an important role in cell migration and invasion. As a signaling protein downstream of the epidermal growth factor (EGF) receptor, tTG becomes activated and is recruited to the leading edges of cells in response to EGF. In addition to its role in cell migratin and invasion, tTG has been shown to be important for Ras-driven transformation. The primary focus of this application is to learn more about how the activation and localization of tTG to the leading edges of migrating cells participates in the enhanced cell migration and invasive activity exhibited by human cancer cells and contributes to Ras- mediated cellular transformation. The following lines of investigation will be carried out to achieve this goal. 1) Determine the signalig mechanisms responsible for localizing tTG to the leading edges of actively migrating cells. EGF receptor signaling through Ras to JNK is important for tTG activation and localization. This aim will involve identifying the substrates of JNK that mediate these effects. In particular, I will determine whether tTG or Hsp70 can serve as a substrate for JNK or whether other known substrates (i.e. paxillin) are important for regulating tTG activation and localization. 2) Determie how the chaperonin function of heat shock protein 70 (Hsp70) helps target tTG to the leading edges of cells. This line of study stems from our recent findings that Hsp70 and tTG interact and co-localize along the leading edges of cells and this localization is blocked in the presence of inhibitors of the ATP hydrolytic activity of Hsp70, myricetin, methylene blue, and VER 155008. This aim will involve identifying the client proteins of Hsp70 which help target tTG to the leading edges of cells. 3) Determine how tTG works with Ras to promote cellular transformation. EGF signaling through Ras results in activation and localization of tTG to leading edges and Ras needs tTG for its transforming potential. In this aim, I will determine whether tTG- promoted Ras transformation occurs as a result of tTG's ability to enhance Ras activity or the activation of one of its downstream effectors, or whether tTG activates a Ras-independent signaling event that synergizes with Ras signaling to drive malignant transformation. These studies should provide insight into the significance of coupling tTG activation and localization to the leading edges of cells and how tTG is contributing to cell migration and invasion as well as cellular transformation.


A necessary operation for language comprehension and production is coreference, which involves keeping track of concepts in memory, updating them, and forming correct links in the mind between different coreferring forms, often over long distances. The process of coreference in text comprehension has been well studied, while the contribution of discourse and prosodic cues relevant for the speech and gesture modalities has received considerably less attention. This proposal seeks funding to investigate the process of coreference in speech-gesture comprehension and production in order to model the organization and mechanisms of the adult, end-state, language system more completely. Four hypotheses will be tested. (1) Prosodic stress accesses and makes use of the same underlying memory mechanism(s) and organizing principles as found for text comprehension. (2) Gesture can be used to mark antecedents and this coreferent gesture is co-expressive with speech. (3) Coreferring gestures also make use of the same underlying memory mechanism(s) and organizing principles as prosodic stress and text comprehension. (4) Gesture codes antecedent representations in a motor-kinetic format.


According to the Guide to Clinical Preventive Services, the common form of screening for thyroid abnormalities, neck palpation, lacks the sensitivity (33%) for detecting thyroid cancer, and ultrasound has too great a false positive rate to avoid invasive tests such as biopsy, to rule out cancer. It is the aim of this work to provide sensitive ultrasound tests to non invasively diagnose cancer in the thyroid earlier and more accurately than current imaging techniques. Elastography, which images the stiffness properties of tissues, and parametric backscatter imaging that displays the frequency dependence of ultrasound reflections offer novel signal processing approaches that provide new information about tissues. These advanced ultrasound techniques will be incorporated into a new, software-controlled research ultrasound platform. Phase I efforts will tune the platform so that it provides data and software control needed to fully develop and evaluate these novel imaging methods. Further work will evaluate algorithms for producing elastograms using raw radio frequency echo data available on the research scanner. It also will develop acoustic parametric images that are based on the frequency dependence of scattering and on attenuation, offering modes that will compliment conventional amplitude mode processing. A software development kit (SDK) that accesses fast digital signal processing (DSP) boards on the scanner will also be developed. Quantitative images of thyroids will be derived to demonstrate the new modes. Phase II will implement elastograms in real time and provide other parametric images during image freeze. Real time elastography will be achieved through careful evaluation of different algorithms for use on the DSP circuit. Algorithms will be assessed for the quality of elastograms produced and the computational overhead incurred in their generation. Phantom and preclinical tests will be done to select the most effective for thyroid imaging. Parametric images based on attenuation and the frequency dependence of scattering, characteristic of the scatterer size will be developed. Novel features will be to incorporate reference phantom data for calibration directly into the scanner memory to facilitate rapid processing, and to apply compound scanning for reducing statistical fluctuations in quantitative images. The system will be evaluated by testing in patients with thyroid nodules and in patients with proven breast masses.


Alcohol abuse can have devastating impacts on the brain and behavior and is a big burden on public health. However, the molecular underpinnings of alcohol induced neural cell injury are not fully understood. Recent advances in functional genomics suggest that long non-coding RNAs (lncRNAs) may play critical roles in alcohol- induced neural cell death. The recent work from our laboratory support this hypothesis. Therefore, a comprehensive screening system of lncRNAs related to alcohol-induced neural cell death is of significant benefits for identifying potential therapeutic targets. Genetic editing tools such as Zinc Finger Nuclease (ZFN) and Transcription Activation-Like Element Nuclease (TALEN) have great potential for the functional study of genes or the application of gene therapy through knockout or knockin techniques. A new type of genetic editing tool based on bacterial Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR?Associated System [Cas]) has been successfully used in various organisms from bacteria and yeast to mammals. Relative to ZFN and TALEN, CRISPR/Cas is advantageous because it only requires changing the sequence of the guide RNA (gRNA) and it can also be directly delivered into embryos to generate gene-modified organisms. Furthermore, the multiplexing capability of CRISPR/Cas makes it possible to target multiple genes simultaneously. In this study we will use our recently developed dual guide CRISPR/Cas approach to generate an lncRNA knockout (gRNA) library in SH-SY5Y cell model. We will then perform a genome-wide screening for lncRNAs involved in alcohol-induced cell death pathways. Our preliminary data indicated that alcohol increased the expression of a nuclear paraspeckle lncRNA in SH-SY5Y cells. Our previous work have demonstrated that alcohol activates the oxidative stress-apoptotic cell death pathway in a dose dependent manner to reduce neuronal cell viability and increase cellular oxidative stress. We expect that the application of the high throughput screening platform in alcohol-induced neuronal death system will allow for the accurate identification of all other specific lncRNAs as well as their potential roles in mediating alcohol-induced neurodegeneration. The generated lncRNA gRNA library in this study can also be available for the characterization of cell toxicity induced by other neurotoxins or other substance abuse. Thus this research has a potential to reduce the health impacts of alcohol abuse and also has benefits for other substances abuse related public heath burdens.


DESCRIPTION: (Applicant's Abstract) Opioids have been demonstrated to alter immune functions. The natural opioid peptide methionine enkephalin (Met-ENK) has been reported to boost T-lymphocyte numbers and function in a cytokine-like manner. Thus, this peptide has been suggested as an adjunct to antiviral chemotherapy in the treatment of acquired immunodeficiency syndrome (AIDS). The combination of antiviral and immunostimulatory substances has been under serious evaluation for effectiveness in treating AIDS for several years. However, this approach has been limited by the need to be conservative in designing clinical protocols. The current proposal uses a murine retrovirus-induced immunodeficiency model to study various combinations of the anti-retroviral drug azidothymidine and Met-ENK. Initial studies indicate that Met-ENK can significantly enhance protection against the mouse retrovirus infection, as measured by reduced splenomegaly and increased survival. The use of the murine model, which uses various numbers of subjects treated by drug combinations, is advantageous in allowing studies that fully evaluate the interaction between these agents. These studies will focus on determining: 1) the optimal drug combination in dosing, time, and route of administration; 2) whether or not the Met-ENK effects are via opioid receptors; 3) if the effects of Met-ENK result in a cytokine pattern (type I cytokines) that favors resistance to disease; and 4) whether therapy reduces viral load temporarily or long term, and whether viral infection is actually cleared from the mice. This model has excellent potential for establishing whether the therapeutic combination of antiviral and immunostimulatory agents may be applied in the future to human AIDS or other retroviral infections.


Our proposed research is aimed at providing a systematic approach and an eventual mechanistic understanding of the kinetic factors associated with cholesterol gallstone dissolution in bile from the physical chemical viewpoint. It involves the experimental and theoretical evaluation of the influences of the principal constituents of human bile by conducting dissolution rate studies of cholesterol gallstones and cholesterol monohydrate "model gallstones" in chemically defined media and comparing these results with those obtained with human gallbladder bile. Emphasis is being directed toward the assessment of the importance and the understanding of the interfacial resistance factor which rate limits stone dissolution in vitro and, probably, in many in vivo situations corresponding to certain bile compositions. Results of current studies will be used to examine in vivo dissolution data from patients being treated for stones with orally administered chenodeoxycholic acid. Also the present results are being used to provide an explanation of why certain formulations for T-tube infusion treatment of retained common duct stones appear to be more clinically effective than others. Efforts are being put into seeking out and studying conditions, procedures, and chemical agents that may, from a kinetic viewpoint, significantly accelerate cholesterol gallstone dissolution. An animal model will be studied for testing in in vitro findings with regard to effectiveness.


Approximately 5-20% of psychiatric patients hospitalized in public facilities receive long-term, high-dose antipsychotic treatment. Most of these patients &e prescribed the high-dose treatment because they have severe symptoms of schizophrenia. This treatment, associated with higher risk for side effects, was not demonstrated to be therapeutically more effective than the (lower) standard dosage regimens. Plasma level of 10 ng/ml of haloperidol (approximately 18 mg/day by mouth) was demonstrated to provide sufficient antipsychotic effects in schizophrenic patients. The main goals of the current proposal are to identify schizophrenic inpatients receiving long-term high dose haloperidol treatment; to reduce their plasma levels gradually and under controlled conditions to 10 ng/ml, and to determine the effects of that reduction. The subjects will be 290 schizophrenic inpatients whose treating psychiatrists prescribed oral doses of haloperidol yielding plasma levels greater than 15 ng/ml. Patients will be randomly assigned to one of two groups: level reduction or level continuation. The patients in the first group will have their doses reduced over a period of 12 weeks; the target plasma level will be 10 ng/ml. They will be maintained on that level for the subsequent 16 weeks. The patients in the continuation group will be maintained on their level for 28 weeks. Any relapsing patients will have their medication adjusted. Comparison of these groups outcomes will show whether the high doses were needed. A subgroup of patients who need the high-dose treatment may be identified and characterized. Proposed 5-year study will provide a rational basis for the dosage regimens of typical neuroleptics in the treatment of severe forms of schizophrenia.


The proposed research involves the collection of quantitative and qualitative data on past reproductive behavior and the prospective follow-up of the demographic dynamics of a population of about 13,000 people in rural Cambodia. The first aim of this study is to provide the accurate and timely demographic indicators much needed for development planning in Cambodia. To date, the 1998 census and recent surveys have yielded conflicting results on current levels of fertility and child mortality. The data collection proposed here and extant techniques of demographic analysis will provide several indicators of the quality of the existing census and survey data. The population follow-up will provide reliable and timely indicators of current demographic levels and trends. The second aim is to document how the dramatic events of the 1970s have shaped the survivors' reproductive system, both at the community and the family level. A study of post-war reproductive behavior will combine analyses of our individual interviews and focus group discussions with males and females at different stages of their reproductive careers, with recent census and nationally representative survey data. The unusual features of recent Cambodian demography can provide unique insights on the long-standing theoretical debate on the impact of mortality changes on marriage and reproduction. The third aim of this study is to lay the foundations for longitudinal studies of social change in this population and, most immediately, of the demographic and social impact of the HIV epidemic. The study of the impact of HIV will be initiated during the two years of the follow-up but the bulk of the impact is likely to be felt in the later part of this decade. During the initial two-year period, however, we will be able to test a light structure for demographic surveillance that could be maintained indefinitely at low cost. The existence of this long-term observatory will complement and enhance future substantive research in this population.


We have initiated the development of assay techniques that will allow us to understand the sulfation code in chondroitin glycosaminoglycan (GAG) chains. These assay techniques take advantage of specific chromatography techniques (ion exchange, hydrophilicity) to separate the different disaccharides and monosaccharides that comprise the GAG chain. This is the only technique capable of doing this. We have also initiated mass specrtrophotometric analysis of these separated GAG chains to begin to determine the sequence of sulfations on the different parts of the GAG chain. We have continued experiments to modify the biological activity of GAG chains by altering the level or activity of enzymes that either synthesize or degrade sulfated chondroitin. In particular, we are developing methods to specifically alter the sulfation of GAG chains in particular positions in order to evaluate if they alter the biological activity of the GAG chain. We showed that a specific reduction in 4-sulfation abolishes the biological activity of CSPGs, thus suggesting that sulfation on the 4 position of GalNAc is essential for biological activity. We have started experiments to evaluate novel intracellular signaling mechanisms in response to GAG chains. Growth cones turn in response to bound GAG chains. We have used this property of growth cone turning to assay the activity of drugs and other compounds. In particular, we have determined that the activity of particular intracellular molecular motors and other components of the cytoskeleton are essential for this growth cone turning. Modification of their activity by drugs may ultimately prove to be a useful therapeutic approach for treatment of brain injury. Because many of these mechanisms are also found in injury to heart and blood vessels, these approaches may have a more general applicability.


Virginia Commonwealth University (VCD) and the University of Chile will jointly offer translational research training in reproductive biology to junior scientists from throughout Latin America. Potential trainees will submit applications to the University of Chile, which will be responsible for screening, interviewing, and selecting program participants. Eligibility will be based on completion of doctoral degree requirements (plus clinical training for MD candidates) within the past 5 years; area of research interest (relevance to public health priorities in reproductive medicine in their home country); commitment to return to home country to establish research program; and letters of reference. Trainees will complete 1-2 years of coursework and laboratory training at the University of Chile before coming to VCU to conduct a 1-3 year intensive mentored research project. Each year, 2 trainees will be supported in Chile and 1 in the US. While at VCU, trainees will complete an NIH grant-writing workshop tailored to the needs of international researchers that will be open to all Fogarty population science training program participants; this workshop will be converted to an online distance education course. Additional distance education offerings led by presenters from VCU and Chile will cover research areas covered by training mentors, including polycystic ovary syndrome and insulin resistance, preterm birth, pre-eclampsia, fetal membrane rupture, cell signaling, gonorrhea vaccine development, steroidogenesis, and promotion of healthy pregnancy. Training in the responsible conduct of research will be provided at both the University of Chile and VCU, and practical scientific management skills (Making the Right Moves) will be taught so trainees can understand how best to partner with researchers at US institutions and how they can apply these skills in their home setting. Other enrichment activities include Endocrine Grand Rounds, Journal Club, Clinical Conference, and lab brainstorming on a weekly basis; a monthly Visiting Professor series; and annual seminars on basic endocrinology research and women's health research. A detailed evaluation plan with measurable outcome indicators will be used to track the success of the program, of trainees who participate, and of research mentors involved. Trainees who complete their mentored research at VCU will be eligible for a re-entry grant of up to $5000 per year for up to 3 years (continued funding contingent on completion of productive research in their home country).


The overall objectives of the proposed research are: 1) Prediction of safe exposure to noxious vapors and gases, with respect to exposure schedule, physical status of the exposed subject, physico-chemical properties of inhaled substance, and the effect of other chemical agents; and 2) design of an effective system for monitoring biological samples for evaluation of occupational exposure. Mathematical modeling will be used as a tool to achieve these objectives. Compartmental models will be used, because they afford insight into the effect of physiological parameters and physico-chemical properties of the substance on uptake, distribution, and elimination of inhaled vapors. These insights are crucial to the prediction of bio-equivalent exposures and to the monitoring and evaluating of exposure from biological samples. In our attempt to explore mathematical modeling to generate information applicable to inhalation toxicology (with special emphasis on evaluation of occupational exposure), our research is taking four courses: 1) Refinement of animal models to generate basic data, and to corroborate experimentally the predictions made by the mathematical model; 2) refinement of the existing mathematical models to accomodate the complexity of occupational exposure, and use of the model to evaluate the effect of individual factors on uptake, distribution, and elimination of inhaled vapors; and 3) simplification of the mathematical model and programming of the mathematical solution of the model for a programmable pocket calculator or microcomputer to make modeling accessible to toxicologists and hygienists for design, monitoring, and evaluation of occupational exposure; and 4) obtainment of insight on kinetics of inhalation of mixtures of vapors.


The effect and mechanisms through which lipoproteins regulate MVA metabolism and act as competence factors for cultured cells will be analyzed using as models normal diploid cells (capillary endothelial cells (CE), vascular smooth muscle cells) and an established cell line (MDCK cells). The ability of very low density lipoproteins (VLDL) which are LDL precursors to support cell shape and proliferation will be determined using cultures exposed or not to compactin. Their effects will be compared to that of HDL. As a specific VLDL and HDL metabolic response, their effect on HMG-CoA reductase will be measured and considered as a reflection of substrate flux into sterols and nonsterol metabolites. We will determine which key nonsterol products resulting from MVA metabolism would be relevant to the control of cell proliferation and cell shape and would explain the ability of lipoproteins to act as competence factors. Our attention will be focused on the synthesis of terpenes which modify specific polypeptides post translationally. The effect of HDL, VLDL and isolated components (phospholipid liposomes, apolipoproteins) on terpene synthesis and their incorporation into polypeptides as well as the effect of b FGF ( a progression factor for CE cells) on such a process will be examined using as models sparse CE cells exposed or not to compactin.


(A) Study pathogenesis and pathophysiology of high biocontainment viral pathogens utilizing molecular technologies including reverse genetics systems: Bunyaviruses: We are currently in the process of establishing the first nonhuman primate model for Crimean-Congo hemorrhagic fever virus (CCHF) utilizing Cynomolgus macaques. The model will be extremely valuable for pathogenesis studies and countermeasure development. (studies ongoing) Filoviruses: The Syrian hamster model for Ebola virus infection displays almost all hallmark features of human disease. Using this model, we have investigated the mechanisms in which hamsters are protected from wild-type Ebola virus infection and determined that a CD4+ T cell response, facilitating a neutralizing antibody response, is responsible for natural immunity. (studies ongoing) We continued to utilize the Collaborative Cross (CC), a panel of reproducible, recombinant inbred strains spanning the genetic breadth of three murine subspecies, to identify host factors important for Ebola virus disease progression. Transcriptomics revealed that the host responses dependent on genetic background determine susceptibility to infection independent of virus replication. (studies ongoing) We have studied the virulence of several Ebola-Makona isolates from the West African epidemic in macaque models and could show that these isolates are slightly attenuated compared to the Ebola-Mayinga prototype strain. More interesting, these new isolates did not cause lethal infection in the IFNR-/- mouse model, an established rodent model for non-rodent-adapted Ebola isolates. We have started to utilize humanized mice to study Ebola virus pathogenesis and immune responses. (studies ongoing) (B) Study immune responses to infection and vaccination of high containment viral pathogens and develop new vaccine candidates: Arenaviruses: We continued a project focused on the elimination of Lassa virus from the reservoir species, Mastomys natalensis. For this, we used two vaccine platforms, a recombinant attenuated strain of Salmonella and a recombinant cytomegalovirus vector, to immunize the peri-domestic reservoir with the hope to block Lassa virus transmission among rodents and to humans. (studies ongoing) Bunyaviruses: We have continued to characterize and optimize the adenovirus based vaccine for CCHF. Ongoing transfer and depletion studies using the adenovirus-based vaccines expressing the CCHF nucleoprotein or glycoproteins indicated that antibodies are the mechanism of protection. We also developed VSV-based vaccine vectors. Efficacy testing in the mouse model has shown that the glycoprotein itself is insufficient to protect animals. Filoviruses: The VSV vaccine efforts are reported under the 'Trivalent Filovirus Vaccine' project. In the meantime, we have proceeded with the development of alternative vaccine candidates for filoviruses. One approach is targeted towards wildlife vaccination, in particular the great apes, using the concept of a disseminating vaccine vector. The idea is the introduction of a recombinant CMV expressing the Ebola virus glycoprotein. Proof-of-concept studies have been successful in the mouse and rhesus macaque model using a murine and rhesus CMV vector, respectively. WE have also continued to investigate a vaccine based on Modified Vaccinia Ankara (MVA) expressing filovirus-like particles; initial rodent and nonhuman primate studies have revealed very promising results. (C) Study vector/reservoir transmission of high containment viral pathogens using appropriate animal models: Arenaviruses: We have studied infection kinetics of different Lassa virus in the Mastomys reservoir utilizing a unique colony established here at RML. The animals support virus replication and shedding for several weeks before Lassa virus gets cleared. The model will allow for important transmission studies. (studies ongoing) (D) Utilize in vitro and in vivo systems to study the interactions between viral pathogen or viral components and host cells and develop new antiviral strategies: Arenaviruses: Previously we had tested the efficacy of two antivirals, T-705 and ribavirin, in the guinea pig model of Lassa virus infection. In particular, T-705 showed promise as a treatment for Lassa virus with protection even when treatment was started post-disease onset. More recently, we were able to demonstrate efficacy of T-705 against Lassa infection in the nonhuman primate model. (studies ongoing) Filoviruses: Ribavirin can effectively extend the time-to-death of hamsters infected with Ebola virus, but resistance will rapidly develop. Surprisingly, T-705 protected hamsters against Ebola virus infection when animals were treated for two weeks beginning the day after infection. This promising treatment option is currently being followed up. We have also identified a few promising broad spectrum antivirals against Ebola virus, which are in development as treatment options for other RNA viruses. Further confirmation is underway. In contrast, treatment with anti-malaria drugs, including chloroquine, failed in Ebola rodent models and should not be further considered. (studies ongoing) (E) Study the epidemiology and ecology of high biocontainment pathogens utilizing newly developed rapid, sensitive and specific diagnostic test systems including those that can be applied under field conditions: Filoviruses: From August 2014 through May 2015 we have operated a diagnostic laboratory in Monrovia, Liberia to support the international response to the West African Ebola epidemic. We have started to analyze data for scientific purposes under approved protocols. So far we have demonstrated the importance of performing on-line clinical chemistry and malaria differential diagnostics. Most importantly, we have identified the interesting observation that plasmodium co-infection increases the survival rate of Ebola-infected patients. Future studies will be designed to test this observation in animal models and to understand the underlying mechanism.


Although much information has become available concerning the biochemical changes taking place during the chronic administration of morphine, it still is not clear which of these changes are related to the development of tolerance and addiction to the narcotic agents. A different approach to these studies may be useful at this time. Animals made thiamine deficient by feeding diets deficient in the vitamin will be used to study the development of morphine tolerance and various associated biochemical changes. The effect of thiamine deficiency on catecholamine and serotonin turnover with morphine will be studied by monitoring the incorporation of C14-tyrosine or C14-5- hydroxytryptophan into the amines, since synthesis is decreased in the deficiency state. The mechanism of decreased motor activity in deficient animals has been investigated using agonists, antagonists and precursors of the catecholamines. The effect of morphine on the various thiamine-dependent systems will be examined in tolerant and non-tolerant animals, as well as in vitro. Studies will be conducted to determine whether morphine will cause the release of thiamine from nerve membrane preparations and whether it will either inhibit or enhance the release of thiamine induced by other agents.


There has been a huge increase in demand for comprehensive quantitative analysis of neurovascular imaging data produced in the clinical setting for diseases such as multiple sclerosis, traumatic brain injury, stroke and dementia. Our objective in this project is to design and develop advanced image processing software that can rapidly and accurately analyze such data. To achieve this objective, we propose a range of novel algorithms to process data from the following MR imaging sequences widely used in the aforementioned applications: time resolved 3D contrast enhanced MR angiography (CE-MRA) for the assessment of vascular anatomy, time resolved 2D phase contrast flow imaging (PC-MRI) for the evaluation of vascular hemodynamics, susceptibility weighted imaging (SWI) for quantifying iron deposition in the brain, and fluid attenuated inversion recovery (FLAIR) imaging for the detection of white matter hyperintensities (WMH) and lesions. A variety of tools will be designed and implemented to tackle these problems including: tissue similarity mapping and active shape models to segment the vasculature in both CE-MRA and PC-MRI images; automatic tissue segmentation in the basal ganglia and thalamus for a two-region of interest analysis for iron quantification with SWI; and finally adaptive approaches incorporating fuzzy C-means, shape factor analysis, compactness and fractional anisotropy to quantify lesions and WMHs. To exploit the advantages provided by different imaging sequences, co-registration algorithms will be used to improve segmentation of vessels between CE-MRA and PC-MRI, and between 3D T1 weighted imaging and SWI. Upon finishing this project, we expect a multi-fold increase in processing efficiency and a significant increase in accuracy will be achieved. The resulting software will not only help the growth of our company, but also improve the diagnosis and treatment of neurovascular diseases. PUBLIC HEALTH RELEVANCE: The huge increase in demand for a more comprehensive and accurate analysis of the vast amount of clinical MR imaging data for neurovascular diseases such as multiple sclerosis, traumatic brain injury, stroke and dementia is the driving force for th development of more advanced image processing software in our company. In this project, we propose an integrated approach to develop a set of processing software for imaging sequences that target the assessment of both anatomy and function of the neurovasculature system. The results will lead to a better access to quantitative data about the brain's vasculature, flow, hemodynamics and iron content present in neurovascular diseases. The completion of this project will not only help the growth of our company by increasing processing throughput and accuracy, but also improve the diagnosis and treatment of patients with neurovascular disease.


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. Objective: To convert human pluripotent stem and somatic cells into hematopoietic stem cells (HSCs) and self renewing cardiac progenitor cells. The Midwest Progenitor Cell Consortium is a collaboration between the UW-Madison and the University of Minnesota, which will serve as a research hub within the NHLBI's Progenitor Cell Biology Consortium. The goal is to develop new strategies to convert human pluripotent stem and somatic cells into hematopoietic stem cells (HSCs) and self renewing cardiac progenitor cells through identification of genetic program leading to formation of pre-HSCs and cardiac progenitors and activation of self-renewal program in lineage restricted cells. Dr. Slukvin's involvement in the consortium relies on WNPRC Stem Cell Resources.


Nocturnal cyclic intermittent hypoxia, such as experienced by patients with obstructive sleep apnea (OSA) is thought to alter vascular tone and function leading to peripheral vasoconstriction and consequent arterial hypertension. Studies in patients indicate that sleep apnea results in diminished reactivity to endogenous vasodilators, and altered sensitivity to some endogenous vasoconstrictors. Furthermore, OSA patients demonstrate an augmented pressor response when exposed to hypoxia and fail to decrease forearm vascular resistance (FVR) when exposed acutely to progressive isocapnic hypoxia as do non-apneic volunteers. Our own preliminary data, collected in normal volunteers and OSA patients, suggests, however, that intermittent hypoxic exposure may lead to vasodilation rather than vasoconstriction. Normal volunteers exposed to intermittent hypoxia for 14 nights have no change in vascular resistance despite increased sympathetic activity and also fail to vasodilate when acutely exposed to isocapnic hypoxia after the repetitive exposure. Based on these and other observations we propose three hypotheses. First, we hypothesize that hypoxic exposure causes either sustained vasodilator release (e.g., epinephrine, NO) that persists during normoxia or altered sympathetic transduction with diminished vasoconstriction (e.g., altered receptor density or transmitter release), or both. Second, we speculate that an acute re-exposure to hypoxia after a prior intermittent exposure results in an abrupt increase in sympathetic nervous system activity but little further increase in vasodilator release, thus resulting in impaired vasodilation. Finally, we hypothesize that maximum vasodilator release declines over time with continued hypoxic exposure, resulting in a gradual increase in arterial pressure. To test these hypotheses we plan a series of investigations in normal volunteers before and after an exposure to cyclic nocturnal hypoxia for 28 nights, and in OSA patients before and after 30 days of monitored therapy with nasal CPAP. These studies will use selective intra-arterial infusions of specific pharmacological agonists and antagonists to assess the roles of endogenous vasodilators and vasoconstrictors in altering vascular tone following an exposure to hypoxia. We anticipate that these studies will significantly enhance our understanding of how intermittent hypoxia and obstructive sleep apnea influence vascular tone and function.


Two genetic loci, one dominant, Ir-Z1, the other recessive, ir-Z2, controlling immune responsiveness to aqueous solutions of Beta-galactosidase have recently been found. It is proposed to characterize the function of these genes. First, the location and the number of genes involved will be investigated in hybrid and congenic strains. Subsequently, cell-to-cell interactions will be studied by adoptive transfer experiments and by in vitro synthesis of antibodies. The methodology developed for studying immune responsiveness to Beta-galactosidase is such that activating antibody can be detected at the single molecule level and antigen-binding cells can be easily enumerated. The objective is to establish the type of cell in which the product of the Ir-Z genes are expressed and ultimately, to isolate and characterize the gene products.


Human health depends strongly on adequate nutrition. Symbiotic fixation of atmospheric dinitrogen by legume rhizobia is the major source of nitrogen in the human diet, as well as the major factor limiting legume crop yield. This project aims to characterize the alterations entailed in differentiation of the alfalfa symbiont Rhizobium meliloti from free-lving bacteria, which do not fix nitrogen, to the nitrogen-fixing bacteroid form. Three interrelated studies will be carried out. (1) Fix genes, required for differentiation, will be identified by transposition mutagenesis with a Tn5-lac promoter probe, which will allow characterization of regulatory circuitry. (2) Changes in the outer and inner bacterial membranes will be characterized as a function of bacterioid differentiation. (3) The heat shock response, which depends on a specific RNA polymerase sigma subunit, will be characterized in R. meliloti, as a possible genetic approach to identification of their sigma subunits suspected to be involved in differentiation. These studies should begin to define the molecular changes required for differentiation of the bacteria to a form that can fix nitrogen. Such information will ultimately be necessary for genetic engineering of nitrogen fixation, with benefits to human health and agronomy.


This is the firs application from a new investigator with the overall objective to investigate the regulation of caudal-related homeobox (CDX) proteins in normal colon and colorectal cancer. CDX1 and CDX2 are transcription factors that are involved in regulation of proliferation and differentiation of the intestinal epithelial cell. Additionally, expression of replacement of CDX gene expression in colon carcinoma cell lines dramatically inhibits cellular proliferation by cell cycle arrest in G1. Thus, altered regulation of CDX genes in colonic epithelial cells is involved in the development of the neoplastic phenotype. Therefore, this proposal is based on the hypothesis that an understanding of the regulation of CDX genes in both normal and neoplastic cells will elucidate pathways that control the phenotype in cancer cells. The goal of this project is to elucidate the normal regulation of CDX1 and CDX2 and to determine changes that occur in the colonic neoplasia. Preliminary are presented that demonstrate variable of CDX genes in colorectal cancer cell lines and that this differential expression is regulated the level of gene transcription. To accomplish this goal, four specific aims are proposed: 1) To elucidate the transcriptional regulation of the human CDX1 gene in normal and neoplastic colonocytes using DNase I hypersensitivity. mapping and transfection and DNA-protein interaction studies. Preliminary data shows that there is a specific hypersensitive site in a cell line that expresses CDX1 that is absent in non-intestinal cell lines and intestinal cell lines and intestinal cell lines that do not express CDX1. The function of this site will be explored and the remainder of the gene will be studied for additional sites. 2) To elucidate the transcriptional regulation of CDX2 in normal and neoplastic tissue. Similar to CDX1, we have mapped and characterized and characterized the CDX2 gene. Transfection studies suggest that elements for directing intestine-specific expression are outside the immediate upstream region of the gene. DNase I hypersensitive site analysis will also be employed to identify potential regulatory regions. 3) To study the regulation of CDX genes in transgenic mice. These studies will validate the function of regulatory elements identified in Aims 1 and 2 in the whole animals. 4) To examine cellular signaling pathways that regulate CDX genes in colon cancer cells. Once regulatory elements are defined, the cellular pathways that regulate expression in normal and neoplastic cells will be studied. Completion of these studies will provide a detailed picture of the transcriptional regulation of CDX genes in normal and neoplastic cells. This will provide the basis for understanding the network of cellular pathways that control these important cell specific transcription factors and how they are altered in neoplasia.


Lactase non-persistence (adult-type hypolactasia) results in digestive system malabsorption of lactose, the carbohydrate macronutrient present in milk. Human DNA sequence variants associated with intestinal lactase persistence and non-persistence have recently been identified. This research project focuses on characterization of the genetic determinants of intestinal lactase persistence in humans. We have previously characterized molecular mechanisms regulating intestine-specific expression of the rat lactase gene in cell culture and in living animals. We will now direct attention to characterization of the molecular mechanisms mediating human lactase gene persistence and non- persistence and the temporal decline of lactase in rodents. Preliminary studies have shown that DNA sequences in the region of lactase gene polymorphisms can bind nuclear proteins and enhance lactase promoter activity. We hypothesize that DNA polymorphisms associated with lactase persistence function to regulate lactase gene transcription in adult human enterocytes via differential interaction with specific nuclear protein transcription factors. Research objectives are therefore aimed at defining the mechanistic roles of genetic determinants of human lactase persistence. In Aim 1, we will characterize a functional role for DNA polymorphisms associated with persistence of lactase gene transcription in humans. Functional characterization of lactase gene polymorphisms will be investigated in cell culture and in vivo in transgenic mice. In Aim 2, we will define the molecular mechanisms regulating human lactase persistence gene transcription. The goals of this aim will be to identify and characterize nuclear proteins capable of interacting with DNA sequences in the region of lactase gene polymorphisms associated with lactase persistence and to characterize whether the nuclear proteins function to regulate lactase transcription. In Aim 3, to complement our investigation of the mechanism of lactase persistence in humans, we will further elucidate mechanisms regulating the temporal decline of lactase in rodents. Knowledge of mechanisms regulating human lactase gene expression may provide future novel diagnostic, therapeutic or prognostic strategies for gastrointestinal disorders that result in loss or inappropriate gain of epithelial cell digestive function. Our combination of in vitro and in vivo experimental approaches will elucidate roles for the genetic determinants associated with lactase persistence and non-persistence. PUBLIC HEALTH RELEVANCE: Adult-onset hypolactasia, lactase non-persistence, renders much of the world's adult human population intolerant of excessive consumption of milk and other dairy products. In some adults, however, high levels of lactase enzyme activity persist in adulthood presumably due to inheritance of a genetic mutation that prevents the normal maturational decline in lactase expression. Knowledge of mechanisms regulating expression of the digestive lactase gene may provide novel strategies for enhancing expression of digestive enzymes in intestinal diseases and will have broad implications for understanding regulation of other genes with aging.


Approximately two thirds of patients with intrahepatic cancer present with unresectable disease. Our previous studies show that high dose conformal radiation combined with chemotherapy appears to prolong the survival of patients with unresectable intrahepatic cancers. However, attempts to increase radiation dose still further have been limited by the development of radiation-induced liver disease (RILD). The pathology of RILD is veno-occlusive disease, which is characterized by thrombosis within the central veins of the liver producing post hepatic congestion. In the past, efforts to develop models to estimate the likelihood of developing RILD have been based primarily on the planned radiation dose distribution for the normal liver. These analyses have demonstrated that increasing mean liver dose correlates with the likelihood of developing RILD. While these models have permitted the safe delivery of far higher doses of radiation than have previously been possible, they also suggest that there is a broad range of individual patient sensitivity that is not reflected by predictions made solely based on the physical dose distribution or general clinical features. As the basic pathophysiology of RILD is venous occlusion, we develop the hypothesis that early monitoring of portal venous perfusion and hepatobiliary function would have the potential to predict liver function after irradiation, thereby permitting to safely deliver the higher dose to the tumor without an increase of complications. Our preliminary data provide evidence to support this hypothesis. In this R01 application, we propose to develop a model to predict liver function after the completion of radiation therapy based upon the radiation treatment plan, and the values of portal venous perfusion and hepatobiliary function assessed by DCE MRI and SPECT prior to and during radiation therapy. Also, we assess individual and regional radiation sensitivity during radiation therapy using the liver dose response function that we have developed, for prediction of liver function after irradiation. Our proposed approach is highly innovative and represents a new paradigm to investigate radiation toxicity in the liver. It has potential to be a tool for individualized therapy.


Tuberculosis (TB) continues to be an important public health problem, with an estimated 9.4 million cases and 1.7 million deaths in 2009. Multidrug resistant TB (MDR-TB, defined as resistance to at least isoniazid and rifampicin), and HIV-associated TB pose important threats to TB control. The high case fatality rate of HIV- associated MDR-TB has major implications for sub-Saharan Africa. The lack of laboratory capacity for Mycobacterium tuberculosis culture and drug susceptibility testing (DST) in resource limited settings poses important challenges to the fight against MDR-TB. In 2008, only 7% of the estimated 440,000 MDR-TB cases worldwide were detected. Xpert MTB/RIF, a novel rapid diagnostic that simultaneously detects M. tuberculosis and rifampicin resistance within two hours, was recently (Dec. 2010) recommended by the WHO as the initial test in those suspected of MDR-TB or HIV-associated TB. The assay detects rifampicin resistance with 95.1% sensitivity and 98.4% specificity, high negative predictive value (99%) but relatively low positive predictive in people with a low pre-test probability of MDR-TB. While rapid diagnosis of rifampicin resistance could revolutionize the fight against MDR-TB, this technological advance in rapid diagnosis will only result in improved patient outcomes if the rapid diagnosis is linked to rapid access to optimal treatment. In 2008, only 11% of MDR-TB cases were appropriately treated. To ensure optimal treatment outcomes and avoid amplification of resistance, knowledge of the complete resistance profile is required. Using Xpert MTB/RIF, clinicians will need to make a treatment decision based on the knowledge of rifampin resistance only, and thus risk the initiation of a suboptimal regimen. To guide the initial standardized management of rifampicin resistance detected by Xpert MTB/RIF, we will comprehensively characterize the phenotypic and genotypic resistance profiles in a large (n=474) number of patients with rifampicin resistant TB. To evaluate the impact of Xpert MTB/RIF on patient outcomes, we will perform implementation research nested within the phased implementation of Xpert MTB/RIF by the South African Department of Health (DOH). We will document treatment decisions made in a cohort of rifampicin resistant TB cases, and compare outcomes (time to culture conversion, amplification of resistance, drug toxicity, and survival rates) during the first 6 months of treatment between 237 patients diagnosed with rifampicin resistance on Xpert MTB/RIF and 237 patients with rifampicin resistance detected on culture based-DST (patients without routine access to Xpert MTB/RIF). The proposed implementation research aims to change the current paradigm for screening, diagnosis and treatment of MDR- TB, by building a robust knowledge base on the resistance profiles of patients diagnosed with rifampicin resistant TB on Xpert MTB/RIF, by quantifying the impact of the assay on amplification of resistance and treatment outcomes in patients with drug resistant TB. The evidence generated will contribute to the successful implementation of this novel assay into routine practice in resource limited, high TB/HIV burden countries.


DESCRIPTION: (Applicant's Description) The broad long-term goal of this revised research program is to produce an educational program to train cancer registrars (CR) to code co-morbidity information for inclusion into cancer registries. There are three specific aims: 1) to teach CR to code co-morbidity information with the use of a valid co-morbidity instrument; 2) to assess accuracy, consistency, and efficiency of co-morbidity coding, and 3) to develop an educational program consisting of written materials and a videotape to train cancer registrars at other hospitals. Cancer patients often have other non-neoplastic diseases or co-morbidity. These other diseases can be so severe as to impact on prognosis, treatment selection, and outcomes. At present, data collected by cancer registries include demographic descriptions of the patient and morphologic descriptions of the tumor. However, no information about co-morbidity is presently collected. Several valid measures of co-morbidity exist and their inclusion in cancer registries would improve the scientific accuracy of cancer statistics and evaluation of treatment. This project will be a two-year, prospective, longitudinal study with three distinct phases. Phase I will include education of CR on the importance of co-morbidity to the pretreatment evaluation and classification of cancer patients. Phase II will include coding the medical records of 400 newly diagnosed cancer patients and assessing the accuracy, consistency, and efficiency of coding. To test the generalizability of the instrument, one CR from three of the BJC-affiliated hospitals, will be asked to use the instrument to code co-morbidity for 100 consecutive cancer patient records. Phase III will include the preparation of the educational material. This work is significant because if cancer registrars are shown to be able to code co-morbidity accurately then this information can be included into standard oncology data collection efforts. Improved accuracy of cancer statistics and evaluation of treatment effectiveness will decrease mortality and improve quality of life for cancer patients.


Examination of the potential therapeutic effect of 4-ASA on patients with ulcerative colitis. This is an FDA "Orphan Drug" sponsored double blind randomized 6 week controlled trial that includes state of the art assessmen of the severity of the disease.


We have examined various aspects of hormonal control of adipocyte metabolism with isolated rat epididymal adipocytes as a model system. (A) In 32-Pi-loaded cells, the phosphorylation of a 65 KDa protein, found associated with the lipid fraction of adipocyte homogenates, was examined under conditions of unchanging, steady- state cAMP-dependent protein kinase (A-kinase) activity. Steady- state phosphorylation was achieved following a pulse, or overshoot, of phosphate incorporation, indicating that increases in A-kinase are accompanied by increased phosphate activity, and that the concerted action of both kinase and phosphatase provide the cell with a means to produce graded responses to graded increases in cellular cAMP. In a manner independent of A-kinase activity. Insulin also leads to the removal of phosphate from this protein. Insulin stimulates the phosphorylation of an abundant 62 KDa protein, also located exclusively in the lipid fraction: phosphorylation of this protein is abolished by increased A-kinase activity. Such data reveal a tight interplay, or crosstalk, between adenylate cyclase-linked receptors and the insulin receptor. (B) A high affinity antibody against hormone-sensitive lipase (HSL) was raised and used to probe a fat cell cDNA library. Also, a probe for the HSL gene has been produced from a peptide sequence derived from HSL purified in this laboratory. (C) Analysis of fat cell G proteins showed that fat cells contain 3 different subspecies of Gi and 2 different Gs proteins, all located primarily on plasma membranes. However, intracellular membranes contain a large "Gs-like" protein of 55 KDa that is ADP-ribosylated by cholera toxin, both in vivo and in vitro, and recognized by affinity purified antibodies against Gs.


Proteomics is a rapidly expanding field but current methodologies remain inadequate for achieving its full potential. The most basic enzymological tool for characterizing proteins is the protease. Proteases are already an essential part of proteomic analysis but more sophisticated tools are needed to identify low abundance proteins in highly complex samples. We have engineered prototype restriction proteases which are active in denaturing conditions and which cut specifically at well-defined cognate sequence motifs. Our basic innovation would be the ability to parse a proteome into sequence-edited slices and to detect these edited portions with high resolution and high sensitivity. In Phase I we tested the prototype restriction protease for suitability in proteomic analysis and implemented a novel directed evolution methodology for the selection of proteases that cut new sequence motifs. The Phase II objective is to develop a sophisticated set of protease tools to facilitate proteomic analysis in the way restriction endonucleases have facilitated genomic analysis. PUBLIC HEALTH RELEVANCE: The complex and dynamic nature of proteomes make them rich with useful information but difficult to characterize. Our long-range objective is to develop a sophisticated set of protease tools to facilitate proteomic analysis in the way restriction endonucleases have facilitated genomic analysis. Better proteomic tools will lead to earlier detection of disease states, better treatments, better predictability of the effects of various treatments, and the development of individualized therapies.


APPLICANT'S ABSTRACT: Chronic alcoholism in humans or chronic experimental alcohol treatment in rats results in a multitude of endocrinologic, metabolic and physiologic abnormalities such as sexual and reproductive failure and hepatic dysfunction. Although there is a wealth of data available on alcohol's effects on some aspects of drug metabolism, our overall understanding of the mechanisms underlying alcohol-related hepatic changes is superficial. The overall goal of the current proposal is to distinguish the direct effects of ethanol on liver metabolism from those that are secondary to or synergistic with changes in endocrine or nutritional systems. There is much evidence to indicate that part of the induction of P-450j (IIE1) by ethanol is a direct post-tranlational stabilization of the protein. However, the nutritional deficiencies seen in alcoholic men may also play a synergistic role in the induction of this and other cytochrome P-450 isozymes after chronic ethanol abuse. In addition, ethanol has inhibitory effects on the hormones of the hypothalamic-pituitary-gonadal axis, resulting in hypoandrogenization and hyperestrogenization in men and rats. Since hormones appear to have an important regulatory role in the expression of liver cytochrome P-450 isozymes, the interaction between ethanol abuse, endocrine function and drug metabolism requires investigation. The focus of this proposal is on 5 principal areas: 1) the direct effects of ethanol on the hepatic microsomal monooxygenase system (HMO); 2) the indirect hepatic effects of ethanol secondary to alterations in nutritional or endocrine status; 3) the in vitro/in vivo correlation of ethanol induced changes in drug metabolism and activation; 4) the interaction of chronic ethanol with other inducers of the microsomal monooxygenase system; and 5) the characterization of a new ethanol induced cytochrome P-450 isozyme. Many alcohol abusers as well as social alcohol users, are taking multiple medications, and are exposed to a wide range of xenobiotics in the diet and in the environment. Although many compounds are known to induce HMO in addition to alcohol, few studies have addressed the question of how ethanol consumption might alter induction of cytochrome P-450 forms other than P-450j. Results from our studies should provide valuable molecular and hormonal information regarding the mechanisms or ethanol induced changes in liver metabolism and provide a rational clinical basis for; 1) reducing toxic drug interactions in alcohol users, and 2) optimizing nutritional support during recovery from alcoholism.


PROJECT SUMMARY/ABSTRACT The Drug Discovery, Delivery and Translational Therapeutics (DT) Program at the Markey Cancer Center (MCC) is scientifically focused on identifying novel targets and biomarkers and discovering and developing new drugs targeting these biomarkers. The MCC catchment area population has both a high cancer risk related to excessive carcinogen exposure and lack of access to cutting-edge clinical trials due to geographical isolation and poor socioeconomic status. The DT program vision is to understand the unique molecular and phenotypic markers of cancer in Kentucky as well as barriers to accessing care and integrate that knowledge to inform drug discovery, development, and delivery of early phase clinical trial efforts for a hard-to-reach Appalachian Kentucky population. MCC investigators are international leaders in biomarker discovery (Theme 1) with ongoing translational studies including more than 600 participants, evaluating the role of environmental carcinogens and identifying biomarkers of lung cancer. DT pharmaceutical scientists work to discover and develop new anticancer agents targeting identified mutations and phenotypes (Theme 2), partnering with Cancer Cell Biology and Signaling (CS) and Genomic Instability, Epigenetics, and Metabolism (GEM) program members. For example, a novel modulator of 4E-BP1 phosphorylation, a validated colon cancer target, was identified from the Appalachian natural products repository. DT investigators lead clinical trials focusing on cancers relevant to the catchment area (Theme 3) and have enrolled more than 500 patients to lung, colon and ovarian interventional treatment and diagnostic trials. They regularly partner with Cancer Prevention and Control (CP), CS and GEM program members to inform and advance MCC basic science, for example, translating early identification of the anticancer activity of PAR-4 in CS to clinical trials focused on a PAR-4 secratagogue. DT is a cross-disciplinary program of 47 investigators from 6 colleges and 18 departments who work together to develop novel anticancer therapies and translate these therapies into the clinic. This productive program has total annual external cancer-related funding of $8.5M ($5.9M annual direct costs, of which 28% is from the NCI). Members published 366 publications over the current funding period, 99 (27%) of which are inter-programmatic, 84 (23%) are intra-programmatic, and 189 (52%) are inter-institutional. The DT program Co-leaders, Drs. Jill Kolesar and Jon Thorson, have a long-standing collaboration and bring complementary expertise in biomarker discovery, drug development, and early clinical trials. Both direct key resources supporting the DT program, the MCC Precision Medicine Center and the UK Center for Pharmaceutical Innovation, respectively. Each leader brings critical strengths including local, national and international collaborations, entrepreneurial relationships, and active participation in NCI initiatives. Taken together, the DT program has a cohesive and collaborative team that translates novel biomarkers into targets for effective anticancer treatments.


This is an application for a K01 Mentored Research Scientist Award for Dr. Scott Beeman, a postdoctoral research associate in the Mallinckrodt Institute of Radiology at the Washington University School of Medicine. He has a strong background in developing magnetic resonance techniques for non-invasive measurement of diabetes-related physiologic changes. Herein, he seeks to expand his scientific foundation to include specific expertise in the pathophysiology of diabetes and clinical investigation. A primary goal of Dr. Beeman is to become an independent investigator and a leader in diabetes-related imaging research, thus he proposes a career development plan in which he will train with world's experts in metabolism and diabetes (Drs. Abumrad and Klein) and magnetic resonance and biophysics (Drs. Ackerman and Garbow). From Drs. Abumrad and Klein he will learn: (i) the molecular and cellular mechanisms which relate to type 2 diabetes and its pathogenesis, (ii) husbandry, genotyping, and diet-modification techniques used to produce mouse models of type 2 diabetes, (iii) the gold-standard histological and assay techniques used in type 2 diabetes research, (iv) the ethical and legal issues involved in clinical and translational research, (v) clinical study design and subject recruitment, and (vi) clinical techniques required to study obesity and type 2 diabetes in human subjects. From Drs. Ackerman and Garbow he will learn: (i) a deep theoretical understanding of the biophysics which underpin magnetic resonance signals, (ii) magnetic resonance pulse sequence design, and (iii) magnetic resonance hardware development, and (iv) the nuances of managing a major research laboratory. Insulin resistance is a defining feature of type 2 diabetes - an obesity-related disease with severe morbidity and mortality. Recent studies have suggested that adipose tissue hypoxia is a key step in the cascade that leads to systemic insulin resistance. Still, the longitudinal adipose oxygen partial pressure (pO2) profile during pathogenesis of insulin resistance has not yet been resolved. Characterization of adipose pO2 profile during the pathogenesis of insulin resistance would be a major advance in understanding the hypoxia-driven insulin resistance mechanism. The goals of this work are to: (i) develop methodology to non-invasively quantify in vivo adipose oxygen partial pressure (pO2) with magnetic resonance, (ii) characterize adipose pO2 longitudinally during pathogenic expansion of adipose in mice, (iii) demonstrate the hypoxia-driven insulin resistance mechanism in humans, and (iv) show that exercise intervention restores hypoxic adipose normoxia in humans. The larger goal of this proposal is to develop Dr. Beeman into a productive independent investigator. The knowledge obtained during the proposed career development and research plans will provide him the tools to secure R01 funding and thrive as an independent investigator.


Chemosensory responses are critical components in the control of several essential behaviors of insects that are vectors for pathogens responsible for many important human diseases. In particular, olfaction plays a major role in host seeking and oviposition selection behaviors of blood-feeding female mosquitoes and, as such, constitutes a critical component of the mosquito's ability to transmit diseases such as malaria, dengue, yellow fever and West Nile virus encephalitis. Within this context, and together with our colleagues, we have undertaken a molecular and cellular examination of several elements of the olfactory signal transduction cascade in the principal African malaria vector mosquito Anopheles gambiae sensu stricto and the arbovirus vector Aedes aegypti. An increased understanding of olfactory mechanisms and their underlying chemical cues in these systems may provide insight into the processes of insect behavioral responses in general and disease transmission by vectors in particular and would likely be instrumental in the development of novel mosquito control strategies. We have made significant progress in the characterization of several aspects of olfactory process in both mosquito systems leading to, among other things, the identification and initial characterizations of a family of odorant receptor proteins in An. gambiae (AgORs) and Ae. aegypti (AaORs) that lie at the heart of the olfactory signaling pathway. Building on those advances, this proposal for competitive renewal focuses on extending several elements of our ongoing program including characterization of: (1) structure/function relationships between AgORs and AaORs, (2) the role of AgORs and cognate odorant binding proteins (AgOBPs) in the context of larval olfaction and odor coding in vivo and (3) the molecular events underlying oviposition site selection by gravid adult females. PUBLIC HEALTH RELEVANCE: We have undertaken a molecular and cellular examination of elements of the sense of smell (olfaction) that largely drive several important behaviors in malaria and arbovirus vector mosquitoes. An increased understanding of olfactory mechanisms and their underlying chemical cues in these systems may provide insight into the processes of mosquito behavioral responses and disease transmission by vectors. This information would likely be instrumental in the development of novel mosquito control strategies. Thus far, we have made significant progress and now propose to extend, our characterization of several aspects of olfactory process in both mosquito systems that focus on the role and interactions of a large family of odorant receptor proteins in An. gambiae (AgORs) and Ae. aegypti (AaORs) that lie at the heart of the olfactory signaling pathway.


Cellular immunity (CI) to tumor-antigens (TA) was evaluated and monitored using microassays of direct and indirect migration inhibition (MI), isotopic footpad and Winn neutralization tests as a function of growth of mouse mammary tumor-virus (MuMTV)-induced tumors, SV-40 virus-induced sarcomas, chemically induced sarcomas and plasmacytomas, and guinea pig hepatocarcinoma. CI to TA and antigens of MuMTV was observed in tumor bearers. CI decreased as magnitude of tumor-burden increased. Immunological "eclipse" with plasmacytomas was associated with Sephadex G10 adherent cells and the effect was reversed by levamisole therapy. Direct capillary tube and indirect MI microassays demonstrated production of leukocyte inhibitory factor (LIF) of human breast and lung carcinoma, Ewing's sarcoma and melanoma patients in response to tissue culture tumor extracts, and of breast cancer patients to MuMTV. High titered LIF supernatants were generated with small numbers of mononuclear cells by PPD and lung and breast cancer extracts and MuMTV. Physicochemical separation of supernatants was successful in partially purifying LIF. Evaluation of indicator cells for indirect human and mouse MI demonstrated that some humans and mouse strains were insensitive to lymphokine. In mice this was due to suppressor cells.


Studies of the mechanism(s) by which anthracyclines are toxic to isolated heart cells have resulted in the isolation by gel electrophoresis of a protein that may be related to the phenomenon. The protein is present in mitochondrial fractions, has a molecular weight over 120,000, and covalently binds doxorubicin in a time-dependent fashion which correlates with the drug's toxicity to these cells. Efforts are underway to identify the high affinity protein, and to determine if it is involved in the development of the unusual, delayed cardiotoxicity of anthracyclines in vivo.


Existing two-phase solvent systems for high-speed countercurrent chromatography cover the separation of hydrophobic to moderately polar solvent systems, but often fails to provides a suitable partition coefficient values for highly polar compounds such as sulfonic dyes, catecholamines and twitter ions. The present paper introduces a new solvent series which can be applied for separation of these polar compounds by spiral tube countercurrent chromatography. It is composed of 1-butanol, ethanol, saturated ammonium sulfate and water at various volume ratios. The system consists of 10 steps which are arranged according to the polarity of the solvent system so that the two-phase solvent system with suitable K values for the target compound(s)can be found in few steps of search. Each solvent system gives proper volume ratio and high density difference between the two phases, and provides a satisfactory level of retention of the stationary phase in the spiral column assembly. The method is validated by partition coefficient measurement of four typical polar compounds including methyl green (basic dye), tartazine (sulfonic dye), tyrosine (twitter ion) and epinephrine (catecholamine), all of which show low partition coefficient values in the polar 1-butanol-water system. Recently, the above ultra polar solvent systems (Organic/high ionic strength aqueous solvent system) has been improved by increasing the solvent composition in 20 steps (1 to 10 and 11 to 20). Using this new system, a graphic determination of two-phase systems can be done in two steps, i.e., first the sample is partitioned in system 10. If the K value is over 1, the second K determination is performed at system 1 whereas if the K value in system 1 is below unity, the second K determination is performed at system 20. By drawing the line between these two points, the optimum K value of near unity can be easily obtained from the crossing point with the K = 1 line in each case. The method was successfully applied to separation of tartrazine (K=0.77), tryptophan (K=1.00), methyl green (K= 0.93), tyrosine (0.81), metanephrine (K=0.89), tyramine (K=0.98), and normetanephrine (K=0.96). Three sulfonic acid components in D&C Green No. 8 were separated by HSCCC using the graphic selection of the two-phase solvent system This new method was also applied for separation of ultra polar samples polar compounds including nucleobases, nucleosides and nucleotides.


All organisms, from bacteria to humans, need to recognize where they are, and respond accordingly. A pathogen must sense its location in the infectious cycle, then produce factors necessary for that site while repressing synthesis of inappropriate factors. Thus, disruption of key regulatory pathways can inhibit a pathogen's ability to infect, indicating useful targets for developing novel antibiotics. In our studies of th Lyme disease bacterium, Borrelia burgdorferi, we discovered that regulation of a new DNA/RNA-binding protein named BpuR is required for mammalian infection. B. burgdorferi regulates production of BpuR, synthesizing it at low levels during mammalian infection processes, and at high levels during colonization of vector ticks. We genetically engineered B. burgdorferi to constitutively produce high levels of BpuR during murine infection, and found the mutant to be significantly impaired in its ability to colonize mice. Transcriptomic analyses (RNA-Seq) demonstrated that BpuR significantly affects expression of approximately 5% of B. burgdorferi operons, disturbing cellular levels of several known virulence factors and metabolic enzymes. We hypothesize that disruption of the processes controlling production of BpuR will significantly inhibit B. burgdorferi infection. The planned studies are designed to elucidate the mechanism by which B. burgdorferi controls expression of this critical regulatory factor, and determine how disrupting that mechanism impacts upon infection processes.


1. X-linked retinoschisis (XLRS) gene therapy clinical trial. XLRS is a genetic disease caused by mutations in the retinoschisin (RS1) gene. An absent or mutated RS1 protein in the retina of patients with XLRS leads to abnormalities in the normal laminar structure of the retina, resulting in impaired visual acuity and increased propensity to retinal detachment. In collaboration with Dr. Paul Sieving's group, we have developed and optimized a self-complementary AAV vector that is capable of mediating stable and retinal specific expression of human RS1 protein. To meet the FDA requirements for gene therapy clinical trials, we have completed a preclinical efficacy study in a retinoschisin knockout mouse model, and a vector toxicology study in both mouse and rabbit models. The investigative new drug application has been approved by FDA, and a Phase I/II clinical trial has started. A total of nine patients with XLRS will receive (or have received) the vector administration in year 2015. 2. Preclinical gene therapy studies for retinitis pigmentosa due to RPGR or RP2 mutations. X-linked forms of retinitis pigmentosa (XLRP) are relatively severe blinding disorders, resulting from progressive photoreceptor dysfunction primarily caused by mutations in RPGR or RP2 gene. Gene therapy for RPGR-XLRP has been hampered by the relatively slow disease progression in mouse models and by difficulties in cloning the RPGR-ORF15 cDNA that includes a purine-rich 3-coding region. We managed to overcome these problems and have generated AAV vectors carrying full-length mouse and human RPGR ORF15 coding sequences. We have also developed a self-complementary AAV vector carrying human RP2 expression cassette. We have completed long-term (18-24 months) dose efficacy/toxicity studies in mouse models with RPGR or RP2 deficiency. Our results demonstrate that administration of the RPGR AAV vectors at appropriate doses can significantly preserve the retinal function and delay the photoreceptor loss in the mice with RPGR deficiency. Additionally, administration of the RP2 vector with a broad dose range can remarkable maintain the function and viability of cone photoreceptors in the mice with RP2 deficiency. In order to bring the therapies to the clinic, we are currently optimizing the vectors and testing them in mice with different background/mutations. 3. Gene therapy for Leber congenital amaurosis due to CEP290 mutations. Leber congenital amaurosis (LCA) is one of the most common causes of blindness in children. People with this disease typically have severe visual impairment beginning in infancy. Mutations in the CEP290 gene account for 20-25 percent of LCA, afflicting an estimated 20,000 people worldwide. Since the size of CEP290 coding sequence (7.4 kb) exceeds the packaging limit of AAV vector (5kb), we are seeking several approaches including the use of dual-vector and functional subunit of CEP290 to deliver the therapeutic genes into the retina. We have made a series of AAV vectors and have tested them on a mouse model with Cep290 mutation. We recently identified one vector that is capable of preserving the retinal function and structure in the mouse model. A detailed study is being conducted. 4. Application of CRISPR for treating retinal degenerative diseases. We have recently initiated a new project involving the use of CRISPR technology to knockdown dominant gene mutations in the retina. We are hoping that we can develop the CRISPR technique into a treatment for autosomal dominant retinal diseases.


Trafficking of mRNAs from the nucleus to the cytoplasm is essential for gene expression and is highly regulated by pathogens and signaling pathways. To exit the nucleus, most mRNAs interact with the receptors NXF1-p15 (TAP-p15) via adaptor proteins, such as E1B-AP5. Another mRNA export factor is Rae1, which aids in docking mRNAs onto nuclear pore complex proteins, such as the nucleoporin Nup98. The mRNA export complex is then translocated through the nuclear pore complex to the cytoplasm. We have reported the interaction of viral proteins with constituents of the mRNA export pathway, which induced inhibition of Mrna export and provided insights on regulatory mechanisms of mRNA export. We showed that the vesicular stomatitis virus (VSV) matrix (M) protein binds Rae1 and that the nonstructural protein 1 (NS1) of influenza virus interacts with NXF1-p15, Rae1, and E1B-1AP5. NS1 is a major virulence factor of influenza A virus that is essential for pathogenesis. These viral-host interactions inhibit expression of antiviral proteins. However, Nup98 and Rae1 are up-regulated by interferons (IFN), which constitute a mechanism of antiviral response that can revert the mRNA export block mediated by these viral proteins. Another nucleoporin involved in antiviral response is Nup96, which is regulated by IFN and in turn preferentially facilitates expression of IFN-regulated mRNAs. Using viral proteins as tools, we propose to uncover novel molecular mechanisms of mRNA export. Our specific aims are: 1. To investigate the mechanisms through which viral proteins disrupt the mRNA export machinery. We have evidence that NS1 and VSV M proteins interact with different forms of the mRNA export complex. We have also identified novel constituents of the complex. Biochemical approaches will be used to assemble these mRNA export complexes in vitro. Functional studies will be carried out using knockdown, overexpression, and mutagenesis strategies in combination with mRNA export assays. 2. To study regulation of mRNA export by antagonists of viral-mediated mRNA export block. We have identified novel chemical inhibitors of the NS1 protein of influenza virus, some of which target constituents of the mRNA export machinery. Biochemical analyses of interactions between these inhibitors and the mRNA export machinery will be performed to investigate key regulatory mechanisms. Functional assays to assess the effect of these inhibitors on mRNA export will also be performed in vitro and in vivo. 3. To determine the role of mRNA export in antiviral response. We have reported that Nup96 is up-regulated by IFN and is involved in antiviral response to facilitate mRNA export of IFN-regulated mRNAs. Nup96 interacts with Sec13 and Seh1. To study the relationship between these Nups in nuclear transport and Nup96-mediated regulation of mRNA export and IFN response, we have generated novel hypomorphic mice and cells that allow gradual down-regulation of Sec13 and Seh1 alone or in combination with Nup96. Altogether, these studies will generate new information on basic mechanisms of mRNA export and on how viruses and host regulate this machinery to their own advantage. PUBLIC HEALTH RELEVANCE: Trafficking of molecules between the two major compartments in the cell, the nucleus and the cytoplasm, involve processes that are targeted by viruses, as they are important for antiviral defense. Our laboratory studies the mechanisms and key players of these trafficking processes. By understanding these mechanisms, we were able to design inhibitors or drugs that work against viral toxicity.


Breast cancer susceptibility gene, BRCA1, has been implicated in the maintenance of genomic integrity. However, the molecular mechanism(s) by which BRCA1 plays a role in maintenance of genomic fidelity remains to be defined. Interestingly, recent studies in our group and others have demonstrated that BRCA1 can regulate the GADD45, a p53-regulated stress inducible gene that plays an important role in cellular response to DNA damage. These results have evidently linked GADD45 to BRCA1 and raised the possibility that GADD45 might be a BRCA1-downstream effector and mediate BRCA1's role in maintenance of genomic stability. Therefore, this proposal seeks to define the biochemical mechanism(s) by which BRCA1 transactivates the GADD45 gene, and to define the role of the BRCA1-GADD45 pathway in the induction of apoptosis following genotoxic stress. The long-term objective described in this application will specifically focus on three key issues: (1). To characterize the GADD45 gene as a BRCA1's downstream effector. We will define the BRCA1-regulatory elements in the GADD45 promoter and identify the proteins that modulate the BRCA1 transactivation of the GADD45 promoter. (2). To define whether the GADD45 is an essential player in the BRCA1-induced apoptosis. We will analyze the induction of apoptosis following expression of GADD45 and BRCA1. We will also examine the alterations of the BRCA1-activated apoptosis and BRCA1-induced growth suppression in GADD45- deficient cells. (3). Our most recent studies demonstrated that BRCA1 is cleaved by caspase-3 during apoptosis induced by DNA damage. This DNA damage-activated cleavage results in an accumulated 90-kDa band of the BRCA1 C-terminus. Therefore, we will first determine whether the BRCA1 cleavage is required for BRCA1-activated apoptosis following DNA damaging agents. We will also determine the functional role of the cleaved 90-kDa band of the BRCA1 C-terminus in activation of apoptosis. Finally, we will analyze the role of this cleaved product in the BRCA1 transactivation of the GADD45 promoter. The studies proposed in this application would define a novel pathway (BRCA1-GADD45) controlling apoptosis following DNA damage and provide information regarding the biochemical mechanism by which BRCA1 regulates its targeted genes. Since apoptosis is closely associated with the therapeutic sensitivity, the perspective outcome will also provide insight into the development of therapeutic agents.


In the United States, human papillomaviruses (HPVs) cause an estimated 6 million new infections annually. Some HPVs cause the most prevalent benign viral sexually transmitted hyperproliferative disease, genital warts, and are referred to as low risk (LR) HPVs (e.g., HPV 6). Others cause cervical cancer and some head and neck cancers and are, therefore, referred to as high risk (HR) HPVs (e.g., HPV 16). All HPVs are maintained as low copy number extrachromosomal genomes in the undifferentiated layer and amplify their genomes and complete their life cycles in the differentiated compartment of the epithelium where cells normally have exited the cell cycle. All HPVs have to create an S-phase environment in this latter compartment to complete their life cycle. While the HR HPVs have been extensively studied because of their ability to cause human cancer, it is imperative that we understand the mechanism of pathogenicity of the LR HPVs, clinically significant pathogens in their own right. We have been focusing on the E7 proteins encoded by LR and HR HPVs because of their pivotal role in both the life cycle and oncogenesis. This application builds on our novel observation that both LR and HR HPVs can target the pRb family member, p130/pRB2, for degradation while only HR HPVs can target other members of the pRb family. Previously, it was thought that only HR HPV E7 could target proteins for degradation. Building on our paradigm-shifting observation, the Specific Aims of this exploratory R21 application are: 1) to test the hypothesis that LR and HR HPV E7 target other (non-pRb family member) proteins for degradation and 2) to test the hypothesis that LR HPV E7 binds members of the ubiquitin ligase complex. We will use mass spectrometry to detect endogenous proteins that are differentially ubiquitylated in the presence of LR HPV 6 and/or HR HPV 16 E7. We will identify proteins that form a complex with LR HPV 6 E7 by tandem affinity purification coupled with LC MS/MS. Results will be appropriately validated. By generating the first global comparison of the substrates ubiquitylated in response to LR and/or HR E7 expression, a profile of proteins that contribute to the viral life cycle versus those involved in oncogenesis will be developed. The analysis of HPV 6 E7 binding partners, by comparison to HPV 16 E7 binding partners, will identify ubiquitin ligases that potentially mediate LR HPV 6 E7 and/or HR HPV 16 E7 targeted degradation, providing possible novel therapeutic targets for directed inhibition of such degradation. By identifying shared E7 activities in Specific Aims 1 and 2 we will gain new insight into potential biomarkers of and therapeutic targets pertinent to the life cycle of all HPVs. Subtracting out these shared activities will identify biomarkers and therapeutic targets specific to the oncogenic activity of the HR HPVs. The feasibility of these experiments is high based on data in the literature and the expertise of the two laboratories involved in their execution. PUBLIC HEALTH RELEVANCE: Human papillomaviruses, as causative agents of cervical cancer and some head and neck cancers, have a major public health impact. Although the FDA-approved HPV vaccine holds promise of preventing infection, it cannot cure existing infections. This application offers the potential of identifying new biomarkers for diagnosis and monitoring of disease status as well as potential therapeutic targets, and thereby the translation of the results from the bench to the bedside. [unreadable] [unreadable] [unreadable]


Hemagglutinin molecule is a viral attachment protein. Another antigenic protein, the neuraminidase, is also on the surface of the virus particle, in conjunction with the hemagglutinin. The examination of influenza virus with high-resolution cryo-SEM would be extremely useful to reveal the conformational change of hemagglutinin.


Compaction of the myelin membrane into lamellae is essential for its function as an insulator in axons of the peripheral nervous system (PNS). P(O), the most abundant glycoprotein in the PNS, is believed to hold these lamellae together. The long term goals of this study are to clarify in molecular terms the role of P(O) (as well as other proteins) in myelination in the hope of contributing to our understanding of the mechanisms of demyelinating diseases such as Guillain-Barre syndrome (GBS) and in neuronal regeneration in the PNS. The extracellular domains of P(O) undergo hemophilic P(O)P(O) interactions in which the carbohydrate moieties play a major role. Heterophilic interactions of P(O) with neurons are believed to promote neurite outgrowth. The overall goals of this proposal are to determine what roles various regions of P(O) play in the synthesis and compaction of myelin. Our adhesion assay specifically developed to identify extracellular regions of P(O) participating in interactions will be used to pursue the following aims. First, to determine by mutation whether a 5-amino acid sequenced in immunoglobulin-like domain of P(O) is crucial to adhesion. This sequence is also found in a flu virus vaccine which elicited GBS, therefore the results may also bear on the mechanisms of demyelination in this disease. Second, to clarify the role of the intracellular domain of P(O) in the adhesive properties of its extracellular domain and in the sorting of P(O) to myelin. Sorting will be assessed by expressing shark P(O) in rat Schwann cells, allowing myelination to occur, and determining by means of species-specific antibodies whether truncated forms of P(O) reach myelin. Third, to elucidate the interactions of P(O) with myelin associated glycoprotein, possibly in the initial stages of myelination, by mixed adhesion assays and by determining the adhesive properties of cells co-expressing both proteins. Fourth, to map the regions of P(O) that may be involved in heterophilic interactions with neurons by comparing the ability of cells expressing unaltered and mutated P(O) to promote neurite outgrowth. Accomplishment of these aims should substantially advance the long term understanding of the mechanism of myelin organization.


Objectives: The Developmental Core will play a prominent role in the revitalization of CSDA as it reorganizes in line with current budget realities. Though important to all CSDA Associates and Affiliates, the core will be instrumental in seeking to identify and incorporate promising young population researchers as they join the faculty.


The Cancer Control & Population Sciences Program (CCPS) organizes all of the cancer prevention-related research activities of the Winship Cancer Institute (WCI), which span the cancer prevention continuum from primary to secondary to tertiary prevention at the individual, select population, and societal levels. The overall goals of the Program are to reduce cancer risk, incidence, morbidity, and mortality in Georgia through describing and tracking cancer incidence and mortality trends of all cancers; conducting etiologic research in humans; conducting chemoprevention trials in Georgia and in national collaborations; applying theories of human behavior to prevent cancer through early detection; conducting multi-disciplinary research that describes, interprets, and predicts the impact of interventions and other factors on cancer outcomes important to decision makers; characterizing the scope of behavioral co-morbidities that occur in patients with cancer; and understanding relevant mechanisms and treatments of these behavioral co-morbidities. To promote the development of the different areas of research associated with cancer control and population sciences across the entire prevention continuum, and to do so in a coordinated manner, the program is organized into four general research areas, each headed by a senior investigator: 1) Epidemiology, Biomarkers & Chemoprevention (EBC); 2) Health Behavior Research (HBR); 3) Outcomes & Quality (OQ); and 4) Symptom Management & Control (SMC). The CCPS Program includes members from 17 departments across all three schools in the health sciences at Emory UniversityPublic Health, Nursing, and Medicineand the Emory College. Beginning with few members and little funding at the start of the P20, through recruitment and internal development, the Program now has 62 members, of whom 41 are Core, eight Research, eight Clinical, and five Coalition, and a portfolio of 52 NIH and other national, peerreviewed, cancer-related grants headed by 25 Core members, with a combined funding for this year of $9,931,319 in Program direct costs (out of a total of 84 externally-funded cancer research grants with $12,185,701 in Program total costs). The number of peer-reviewed cancer publications by members of the Program during the last grant period was 455 of which 22% were intra-programmatic and 7.5% were interprogrammaticproportions that are rapidly escalating in this young program.


The use of thermometric methods for analyzing serum components is being investigated. Currently three activities are in progress. First, the study of enzyme calorimetry, including creatinine phosphokinase and various transaminases. Second, the use of enzyme inhibition to monitor low levels of heavy metal ions and third, the development of a flow calorimeter for rapid analysis.


The Hematopoietic Stem Cell Facility is part of the NCI Case Western Reserve University Ireland Cancer Center. This protocol is established for obtaining blood and bone marrow samples from normal donors and patients under a wide variety of research programs at CWRU including, for the most part, the CWRU Cancer Center members and their related protocols. The bone marrow aspirations done on normal donors are conducted on the GCRC by the CAP. This facility was just refunded by the NCI.