The principal aims of this project are to 1) identify state-independent alterations in corticotropin releasing hormone (CRH)-mediated physiological processes that can be utilized in the identification of biologically vulnerable individuals as an essential step for future molecular genetic approaches to unipolar depression, and; 2) to determine abnormalities in the regulation and actions of CRH in depression that are relevant to the susceptibility and intrinsic pathogenesis of this disorder, as well as to abnormalities in growth, reproduction, metabolism, and immunity, and hence to long-term health. In our search for a state-independent marker, we employ three protocols that intensively study the HPA axis; a) assessment of HPA responses to a naturalistic stressor (exercise at 70% and 90% V02max [Ex90, Ex70]); b) deconvolution techniques to assess instantaneous ACTH and cortisol production rates; and c) analysis of pulsatile hormonal secretion utilizing non-linear dynamics and low dimensional stochastic chaos. Our assessment of HPA responses to Ex90 in patients with seasonal affective disorder revealed a state-independent hypothalamic CRH deficiency similar to what we have previously observed in other syndromes of atypical depression. We have identified a sub-group of controls that shows HPA hyper-responsivess and GH hyporesponsiveness to Ex90, and increased trait anxiety. In a study of 20 consecutively admitted patients with affective disorder, we found a clinically significant decrease in the density of trabecular bone in association with decreased plasma osteocalcin levels (a potent osteoblastic factor), as well as a significant increase in intra-abdominal fat mass. These defects often persisted for months or years after resolution of the depressive episode and are potentially attributable to CRH mediated alterations in pituitary-adrenal, growth hormone, and gonadal steroid secretion. We also found that core CRH responsive elements in the POMC promoter have high homology to key elements of the genome of viruses (human immunodeficiency virus and cytomegalovirus) as well as to oncogenes and genes involved in the encoding of immune mediators such as IL-1 converting enzyme; thus, CRH could potentially influence susceptibility to infectious, neoplastic, or inflammatory diseases. In experimental models of cerebral ischemia in the rat, we have also found that CRH acts as an endogenous neurodegenerative agent that enlarges the area of infarct, while the IL-1 receptor antagonist serves as an endogenous neuroprotective agent potentially protecting the organism from the consequences of ischemia or other profound stressors.


The general transcription factor P-TEFb, consisting of Cdk9 and cyclin T, strongly stimulates RNA polymerase II elongation. It is also a host cell cofactor for Tat activation of HIV-1 transcription. Accumulating evidence suggests that Tat and the TAR RNA, located at the 5'end of all viral transcripts, not only recruit P-TEFb to the HIV-1 LTR but also cause the activation of Cdk9 kinase. For general transcription of cellular genes, data obtained during the current funding period indicate that P-TEFb is recruited to chromatin templates by the bromodomain protein Brd4. In addition, a major reservoir of nuclear P-TEFb is sequestered in the inactive 7SK snRNP. Further analyses indicate that in response to Ca2+signaling, P-TEFb is released from 7SK snRNP upon the dephosphorylation of the conserved Cdk9 T-loop by PP11 and PP2B. The dephosphorylated P- TEFb is preferentially bound by Brd4, which recruits it to the transcription pre-initiation complex. As the phosphorylation of Cdk9 T-loop is essential for P-TEFb activity, the T-loop is expected to undergo rephosphorylation by an as yet unidentified Cdk activating kinase (CAK) at a later stage in order to restore full activity to P-TEFb. Given that P-TEFb is essential for productive HIV-1 infection, the objective of this proposal is to examine how the various modes of P-TEFb regulation exerted by its associated factors, a putative Cdk9-specific CAK and the HIV-1 Tat/TAR will impact HIV-1 transcription and replication. Proposed are experiments to investigate whether the expression and activity of various P-TEFb-associated factors can be manipulated to control HIV-1 replication and latency. A combination of targeted investigations and comprehensive, unbiased screens will be employed to identify the Cdk9-specific CAK and elucidate the mechanism and functional significance of its phosphorylation of P-TEFb. To determine the mechanism of Tat/TAR activation of P-TEFb, the impact of Tat/TAR on phosphorylation status of the Cdk9 T-loop at different stages of HIV-1 transcription, the possible existence of novel components within the Tat-TAR-P-TEFb complex, and the ability of TFIIH and TAF7 to inhibit P-TEFb activation will be examined. These experiments will offer an exciting opportunity to identify novel factors that contribute to the activation of P-TEFb and HIV-1 transcription and provide fresh insights into how P-TEFb stimulates transcription of both HIV-1 and cellular genes. A better understanding of the mechanism by which P-TEFb controls HIV-1 replication and latency and the versatility of Tat/TAR in modulating this process will be informative toward the identification of new targets for anti-HIV therapy. PUBLIC HEALTH RELEVANCE: The Cdk9-cyclin T1 heterodimer of P-TEFb is a host cell cofactor for Tat activation of HIV-1 transcription. The proposed experiments will allow us to investigate how the P-TEFb-dependent HIV-1 transcription and replication can be controlled by: (1) the various P-TEFb-associated cellular factors;(2) a putative Cdk9 activating kinase;and (3) the HIV-1 Tat protein and TAR RNA that can do more than simply recruiting P-TEFb to the viral LTR. These experiments will lead to the identification of novel factors that contribute to the activation of P-TEFb and HIV-1 transcription and provide fresh insights into how P-TEFb controls transcriptional elongation of both HIV-1 and cellular genes. A better understanding of the mechanism by which P-TEFb regulates HIV-1 replication and latency and the versatility of Tat/TAR in modulating this process may lead to the identification of new targets for anti-HIV therapy.


A collaborative project between the Microbial Interactions Section and the Department of Anaerobic Bacteriology at Virginia Polytechnic Institute and State College was initiated April 1990. The subgingival floras of 20 patients exhibiting frank AIDS and 20 HIV seropositive patients suffering from gingivitis are being enumerated and identified to determine whether: (1) any changes in the normal flora occur following the viral infection; and (2) the gingivitis is associated with a typical gram negative flora, respectively. Twelve subjects, six from each category, have been examined thus far. Qualitatively, the microbial flora appears to be much the same as that seen in HIV uninfected individuals, with slightly more Gram positive bacteria present. However, both the Gram positive and Gram negative bacteria present do not exhibit the same growth characteristics observed previously. In general, they are more difficult to cultivate and show abnormally long lag periods, or refuse to grow at all, when transferred from medium to medium. Internal controls reveal that this is a reproducible trait of oral bacteria from HIV-infected individuals.


Terminated, 9/12/2001. Survival following radiotherapy for glioblastoma remains limited to 9 to 12 months. Agents which inhibit DNA repair represent a novel class of radiosensitizers which are currently being explored in the treatment of Glioblastoma. Hydroxyurea (Hu) is a recognized radiosensitizer which is not commercially available in an iv preparation for continuous infusion. In vitro data supporting the various possible mechanisms of radiosensitization including DNA repair inhibition, suggest that this agent would be most effective if a treated tumor were exposed to steady rather than bolus concentrations. In addition, its rapid half life and clearance would require frequent oral dosing to achieve steady state concentrations. We are thus administering Hu as a continuous IV infusion. Hu is synergistic with other repair inhibitors such as Ara-c, Ara-a, and Pentoifylline (PTF). PTF is a methylxanthine analog with hemorrheologic activity which is a potent radiosensitizer and repair inhibitor in vitro. In this study, Hu is being given via continuous infusion with PTF in a phase I dose escalation of PTF to patients receiving twice daily radiotherapy for glioblastoma.


Biophysical studies are planned on human and porcine neurotubule assembly and disassembly. Kinetics will be characterized in vitro by electron microscopy and optical mechanism of assembly. Assembly and disassembly of twisted tubules, which have been found only in the abnormal human brain, will be studied using protein isolated from autopsied brain from patients with senile dementia, Alzheimer's disease or Guam- Parkinsonism dementia. Major goals are to understand the mechanism of action of colchicine and vinblastine on microtubules, the possible regulatory role of Ca and Mg ions on microtubule assembly and the difference between neurotubules and twisted tubules.


Catecholamine systems, operating at the interface between the mind and the body, exemplify three ways the brain regulates homeostasis -- via neurotransmitters (norepinephrine, NE), hormones (epinephrine, EPI), and autocrine/paracrine factors (dopamine, DA). We conducted investigations in the areas of clinical neurocardiology, catecholamine neurochemistry, and novel catecholaminergic systems. 6-[18F]fluorodopamine ([18F]-6F-DA) positron-emission tomographic (PET) scanning led to a new pathophysiological classification of primary chronic autonomic failure. Patients with pure autonomic failure (PAF) or parkinsonism and autonomic failure had cardiac sympathetic denervation, whereas patients with the Shy-Drager syndrome had decreased or absent post-ganglionic sympathetic neural outflows to intact nerve terminals. 13N-ammonia and [18F]-6F-DA PET scanning detected perfusion or sympathoneural abnormalities in the affected limbs of patients with sympathetic dystrophy (RSD). Patients prone to neurocardiogenic syncope had decreased cardiac NE spillover, and patients with hypertrophic cardiomyopathy (HCM) had intact sympathetic innervation of the hypertrophic myocardium. Clinical neurochemical studies linked specific catecholaminergic phenotypes with genotypic abnormalities in Menkes' disease, familial dysautonomia (FD), and L-aromatic-amino-acid decarboxylase (LAAAD) deficiency. Treatment with L-DOPA as a dopaminergic pro-drug produced beneficial natriuretic responses in patients with congestive heart failure. Plasma DA-sulfate levels were found to depend on dietary factors and on production of endogenous DA in the gastrointestinal tract.


The focus of the experiments of this proposal are the processes by which pigeons learn matching to sample (MTS) and the conditions that lead to different ways that pigeons learn MTS. A newly developed technique will be used to measure if-then rule learning in the MTS task. Another newly developed technique will be used to measure relational-concept learning during acquisition of the MTS task. Tests are proposed to manipulate and confirm the presence of configural learning when evidence indicates that the whole MTS display is learned as a configural pattern. Other evidence suggests that in certain conditions the MTS task may be learned by combinations of these different types of learning. Enhancing and inhibitory interactions between these different learning processes in MTS will be studied. Configural learning will be made more difficult by increasing the number of displays to be learned and by making the display less likely to be configured as a unitary pattern by the pigeons. These manipulations of configural learning. Other manipulations will test the durability of relational-concept learning and explore the type of learning that replaces it. This research will extend our knowledge of how tasks are learned, problems solved, and conditions that lead to either higher-order concept learning or stimulus-specific learning.


Fluorescence Spectroscopy Core (Millar. Director; Pond. Manager) Overview The Fluorescence Spectroscopy Core provides state-of-the-art instrumentation and expertise to support a wide range of fluorescence spectroscopic measurement techniques, including steady-state, time resolved and single-molecule methods. These resources will support the development of new imaging technologies to visualize the assembly and disassembly of virus-host complexes and to identify homogeneous complexes suitable for structure determination.


If more were known about the role of diet in the cause of colon cancer, then specific dietary interventions could be recommended that would reduce the incidence of colon cancer. It has been observed that hyperplasia of colon epithelium precedes neoplasia and that the cell proliferation rate of colonic epithelium is correlated to carcinogen-induced neoplasia. Thus, nutritional modification of cell proliferation rate in the colon is hypothesized to modify the carcinogen-induced progression of normal eptihelium to the neoplastic state. To test this hypothesis we have designed experiments to give answers to the following specific questions regarding the progression of 1,2-dimethyl-hydrazine (DMH) treated colon eptihelium to the overt neoplastic state. 1. Is the reported prevention of DMH-induced hyperplasia in the colon, which was caused by administration of total parenteral (intravenous) nutrition ITPN) at isocaloric intake levels, permanent or reversible? If hyperplasia does not return after TPN treatment, is colon tumor incidence reduced by such TPN treatment? 2. Does a hypercaloric diet given to DMH-treated rats enchane colonic hyperplasia, and increase tumor incidence? 3. How does the manipulation of the non-nutritive fiber content in the diet, the caloric content of the diet, the chemical composition of the diet, one at a time while holding all other variables constant, affect interactions between such nutritional factors and carcinogen-included colonic neoplasia? Briefly, the experiments call for dietary modification either during, or after, or during and after an eight-week course of DMH treatment to rats. Groups of DMH and non-DMH treated rats will be sacrificed at times after the beginning of the carcinogen administration. To monitor the progression of colon carcinogenesis, the epithelium will be examined for: cell proliferation activity and crypt structure using histologic examination, intracellular content of sodium using X-ray microanalysis procedures, and also for the incidence and size of all visible tumors. The data will be subjected to appropriate statistical analysis. The findings should give insight into the interactions between nutritional factors and colonic carcinogenesis and should lead to nutritional concepts of prophylaxis against colon cancer. We will be on schedule with our original proposed experimental protocol by the end of this year and will follow the original proposed schedule of experimentation closely this next year.


The major allergen (designated as Par h 1) was purified from the pollen of Parthenium hysterophorus (a major cause of allergic rhinitis) using anion exchange chromatography and HPLC. The purified protein was homogenous on SDS-PAGE and revealed a single N-terminal amino acid (lysine) upon Edman degredation. Par h 1 was a glycoprotein, rich in glycine and proline, and with an estimated molecular weight of 31 kd. Isoallergens were observed with pI values from 4.3 to 5.2. The carbohydrate moieties on Par h1 appeared related to its IgE binding ability. The N-terminal 91 amino acid sequence of Par h 1 shows 81% identity with a protein from sunflower anther and the hydroxyproline-rich region of Par h 1 is 30-40% identical to similar stretches in extensins. IgE antibodies in the sera of Parthenium allergic individuals cross-reacted with a 50 kd hydroxyproline-arabinose-rich extensin from potato tuber. Thus, it appears that a group of soluble plant glycoproteins related to extensins, have carbohydrate-containing IgE binding epitopes that may contribute to allergenic cross-reactivity among specific pollens and foods.


The overall goal of the Administrative Core (AC) is to provide oversight, coordination and integration of Center activities. The specific aims are to: 1) Coordinate and integrate the scientific aims of the Projects and Cores, track and evaluate progress and outputs, and ensure successful completion of all Center aims; 2) Manage resources to assure the needs and priorities of every Project and Cores are met; 3) Act as coordinating center by convening meetings of Project, Core, and subcontract investigators, organize External Advisory Committee, ensure timely translation of research findings, prepare Center-wide reports, and interface with NIEHS and EPA project officers; 4) Evaluate the progress and success of the career, development plan, with specific focus on the career development of Faculty Development Investigator; 5) Ensure quality control of all stages of research namely study design, establishment of standard operating procedures and study protocols, secure sample and data transfer and management, data analysis, and accurate interpretation and translation of research findings to academic, government, healthcare and community stakeholders. The AC will operate under the direction of the Core co-Leaders, Consultant, and Pediatric Health Specialist in collaboration with Leaders of each Project and Core. The AC will ensure coordination of Center activities, manage resources across Projects and Cores, identify the most efficient use of infrastructure and communication and resolve needs as they arise, in order to increase the efficiency and productive output of the Center. AC Leaders will interact with the External Advisory Board to ensure the merit and value of all UM-CEHC elements to accomplish the overall Center aims. The Center Manager will prioritize coordination with the Community Outreach and Translation Core to optimize outreach and research translation to community stakeholders. Finally, the AC will oversee career development and training of New Investigators, particularly the UM-CEHC Co-investigator designated as the Faculty Development Investigator. Career development and training activities will draw upon extensive institutional resources and will be fully integrated with the Center's Research Projects and Cores.


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. There has been no change in the scope of this project. The success of islet transplantation is hampered by the high rate of islet cell death and dysfunction after isolation. Therefore, the repair of islet damage from the isolation process and the opportunity to maintain islets long term in vitro as a new islet resource would represent significant advances and lead to a more widespread use of islet cell transplantation. Successful utilization of bone marrow in repairing skin, neuron, heart, and muscle injury led us to propose that bone marrow could offer a potential solution to these challenges. In our preliminary studies using co-cultures of whole bone marrow with islet, bone marrow was shown to increase islet function/survival (more than six months), stimulate islet growth and generate long-term insulin producing tissue in vitro. We hypothesize that specific subpopulations of marrow cells may be responsible for these findings. We have also hypothesized that extracellular ATP, ATP receptor (purinoreceptor P2XR), and interleukin 1beta (IL-1beta) are involved in bone marrow-induced repair of islet injury. In this project, we plan to identify whether multiple or single specific lineage marrow cells contribute to islet reconstitution. We will examine whether these reconstituted islets have sufficient function and vascularization in vivo as determined by transplantation into NOD/SCID mice. Finally, we will investigate whether bone marrow modulates ATP, its receptor P2XR, IL-1[unreadable] and its downstream pathways. This project will have benefits for current islet transplantation protocols and will provide insight into the mechanisms of islet cell death and regeneration.


Of the body's fat depots, visceral adipose tissue (VAT) specifically has been shown to strongly correlated to metabolic disorders and diseases such as hypertension, diabetes and heart failure as compared to subcutaneous adipose tissue (SAT) or overall obesity. Furthermore, reduction of VAT is associated with a reduction of metabolic risk factors. Determination of VAT is therefore of high clinical importance, however, current methods to determine VAT are inaccurate, costly, or inconvenient. To meet the need for a more accurate, low cost, easy to use, portable device suitable for the convenient determination of an individual's VAT and obesity in general, PhiloMetron proposes to utilize ultra wideband radar (UWR). Accordingly the objective of this Phase 1 proposal is to assess the ability of UWR to determine the amounts of abdominal body adipose tissue, in particular to quantify SAT and VAT, in model systems. Upon successful completion of these Phase I studies, planned Phase II studies will include validating the use of the technology in the determination of abdominal fat, including VAT, in obese or overweight individuals. PUBLIC HEALTH RELEVANCE: Most of the major chronic diseases impacting the US healthcare systems are being driven by the obesity epidemic. There is a need to create a clinically effective weight management program that can be used to lose and maintain a person's weight to limit the long-term economic and societal impact. These weight management plans need efficacious diet and exercise plans to successfully lose and maintain weight. Currently, weight is the most often used physiological measurement to determine success of the patient's diet and/or exercise, but is a grossly insensitive measurement. More importantly, determination of regional body fat depots is showing increasing importance in obesity and wellness management however weight determinations are incapable of distinguishing between these fat depots. Of the body's fat depots, visceral adipose tissue (VAT) specifically has been shown to strongly correlated to metabolic disorders and diseases such as hypertension, diabetes and heart failure as compared to subcutaneous adipose tissue (SAT) or overall obesity. Furthermore, reduction of VAT is associated with a reduction of metabolic risk factors. Determination of VAT is therefore of high clinical importance, however, current methods to determine VAT are inaccurate, costly, or inconvenient. The objective of this Phase I proposal is to establish a foundation for the use of a potentially breakthrough technology for the measurement of VAT.


The overall objective of the Clinical Laboratory Core will be to provide support for all laboratory needs of the clinical trials described in Projects 1 and 2 of this Program Project. This will include the collection of biological specimens from study participants, facilitating clinical laboratory testing needed for patient management and assessment of drug safety and tolerability, routine storage of biological samples for future use, malaria specific laboratory testing from blood specimens, and the collection and process of placental samples including histopathology. The clinical laboratory core will also be responsible for supporting the collection of biological samples for the immunology studies described in Project 3 ofthe Program Project. Performance sites will include a phlebotomy center and clinical laboratory within our study clinic located on the Tororo District Hospital (TDH) campus, a placental histopathology laboratory located near our study clinic on the TDH campus, and the Joint Clinical Research Center (JCRC) referral laboratory where clinical laboratory testing will be performed. The specific objectives oithe Clinical Laboratory Core will be as follows: Objective 1: To collect, transport and store all biological specimens from study participants. Objective 2: To perform all malaria diagnostic testing. Malaria microscopy will be performed by experienced technicians including double reading of blood smears for quality control purposes. Malaria microscopy results will be available in real time for patient management. A PCR based assay will be available for the detection of asymptomatic parasitemia using dried blood spots collected on filter paper. Objective 3: To collect placental specimens and perform histopathology studies. The gold standard for classification of placental malaria is based on histopathological assessment of placental tissue collected at the time of birth. Our research center has established local capacity for the collection, processing, and staining of histopathology specimens for the diagnosis of placental malaria, one of the primary outcome measures for the studies outlined in this Program Project. RELEVANCE (See instructions): The success of the proposed Program Project, which includes two randomized clinical trials and supporting immunology studies, requires a strong Clinical Laboratory Core that includes proper collection of biological specimens, transport of specimens to our reference laboratory, and performance of on-site laboratory testing to ensure that study participants are managed properly and study outcomes accurately assessed.


The objectives of the Developmental Projects Program are to ensure a continual renewal of high-quality scientific endeavors in the DF/HCC Prostate Cancer SPORE and to fund efforts that will complement or enhance the overall quality of the DF/HCC Prostate Cancer SPORE. In general, the Developmental Projects Program has funded established investigators. Based on our review, we will be mindful of including more junior investigators. This Program will rely on the infrastructure created by the Administrative, Evaluation, and Planning Core (Core A) to: 1. Solicit applications and/or identify novel prostate cancer research projects 2. Evaluate these projects for funding 3. Fund innovative developmental projects 4. Re-evaluate projects for possible transition into full project status 5. Evaluate the success of the program


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. To reduce high blood glucose levels and sustain glycemic control levels among low-income, inner city adults receiving care at a local, federally qualified community health center Objective: To measure six quality indicators to assess quality improvement including four performance measures: HbA1c testing, lipid profiles total cholesterol, LDL, HDL, and triglycerides, retinal examination, and monitoring nephropathy and two quality indicators- glycemic control HbA1c level 7% and lipid control LDL 100 mg/dl. Key Findings Process measures are not being documented as stringently as they should be. This makes it difficult to determine whether or not a provider is giving adequate care and attention to disease management in this population of patients. Our main outcome measure is glycemic control. The majority of patients for each audit had an HbA1c value above 9% at the time of testing. This suggests to us that the efforts to reduce HbA1c levels could be improved. Challenges: There are some inherent challenges with the progression of this project. They are listed as follows: West End Medical Center is currently undergoing the implementation of an EMR system. The implementation process is projected for completion late September, 2006. Inevitably, this will cause some of the timelines for the audits and nurse-mediation component to shift if the implementation process does not go smoothly. Information entered into the EMR system will be provider driven. Consequently, this will affect the audit and feedback component of the project due to the fact that not all of the providers have decided on how much information should be entered into the system. Fortunately, the project can proceed without the aid of the EMR. We will proceed as if the EMR were never to have been implemented and the clinic was operating with a paper based medical record system. Change in investigators with Dr. Mayberry's departure.


The purpose of this core is to provide a centralized facility in which all of the various animal procedures outlined in the Program Project may be performed. Personnel experienced in implementing murine models of cardiovascular disease in a rigorous and reproducible manner will staff the core. The main responsibilities of the core will be the performance and analysis of the following murine models: 1. Myocardial infarction 2. Peritonitis 3. Carotid arterial injury 4. Bone marrow transplant


This study will investigate the histopathology and histochemistry of normal human orbicularis muscle and orbicularis muscle injections of botulinum toxin, to weaken the muscle on a temporary basis. This will extend previous research in the histopathology of vertebrate extraocular muscles injected with botulinum toxin to human muscles and provide data correlating clinical and electromyographic effects of botulinum toxin injections with histology.


PROJECT SUMMARY/ABSTRACT Melanoma is the deadliest form of skin cancer and is one of the most difficult cancer to treat. Therefore, the underlying mechanism of melanomagenesis and melanoma development demands intensive study. Recent findings identified that palmitoylation of melanocortin-1 receptor (MC1R), primarily mediated by the protein-acyl transferase (PAT) ZDHHC13, is essential for activating MC1R signaling and prevent melanomagenesis. Preliminary studies also revealed that APT2 is the major depalmitoylating enzyme of MC1R and is harmful for metastatic melanoma patients. In this proposed study, I aim to elucidate the in-depth molecular mechanism by which ZDHHC13/APT2-regulated palmitoylation repress melanoma development and progression, and test the hypothesis that ZDHHC13/APT2-regulated palmitoylation is essential for melanomagenesis and melanoma metastasis in vivo. During the mentored K99 phase, I will elucidate the underlying regulatory mechanism of ZDHHC13 in melanocytes by using new developed ZDHHC13 transgenic mice. I will also characterize the effects of depalmitoylation inhibition in melanomagenesis by targeting APT2. During the independent R00 phase, I plan to determine the role of ZDHHC13/APT2-regulated palmitoylation in melanoma metastasis in vivo and identify whether inhibition of depalmitoylation improves survival by suppressing metastasis. Furthermore, I will need additional trainings during the award period in melanoma mouse model, targeted therapy, as well as professional skills which will contribute to my long term career goal. In summary, successful completion of my proposed studies will bring novel insights into the roles and molecular mechanisms of ZDHHC13/APT2-regulated palmitoylation in melanoma development and progression, which can be translated into new intervention and treatment strategies. Receipt of this award will allow me to expand my research plan and serve as a platform for me to receive additional trainings, thus prepare myself becoming an independent principal investigator in the field of melanoma research.


The goal of this project is to map and identify genes for herditary deafness. Linkage analyses are being conducted in several large pedigrees segregating non-syndromic and syndromic forms of deafness. If linkage to the known syndromic, DFNA (dominant) and DFNB (recessive) loci in large families are excluded, we initiate genome-wide screens. This strategy has allowed us to map new deafness loci such as DFNA20, DFNA27, DFNA28 and DFNA36. The chromosomal map locations of these novel deafness genes are then refined prior to initiating positional cloning strategies to identify the genes responsible for the hearing loss. Recently we have identified genes for DFNB12, DFNA28, Usher 1D and usher 1F. Additional families with dominant and recessive modes of inheritance with profound congenital or progressive hearing loss are being ascertained with the goal of mapping and cloning additional novel genes that are necessary for hearing and/or maintenace of the auditory system. We are also asscertaining families, mapping loci and identifying genes for Usher syndrome. The defining clinical features of Usher syndrome are hearing loss and progressive retinopathy.


The purpose is to measure neutrophil production and function in healthy, young and elderly subjects in their normal basal state and after stimulation with two doses of granulocyte colony stimulation factor.


I am a surgical oncologist dedicated to caring for patients with complex malignancies and to developing novel immunotherapeutic approaches for metastatic liver cancer. Early in my medical training, I developed a passion for immunology and host-tumor interactions. After graduating from the New York University (NYU) School of Medicine with the top academic record in my class, I trained in general surgery at NYU and Bellevue. I spent two years as a research fellow with Dr. Ronald DeMatteo at Memorial Sloan-Kettering (MSK), where I gained valuable experience in immunologic research and published papers on liver immune cells in Hepatology and the Journal of Immunology. During my clinical surgical oncology fellowship at MSK, I studied immune infiltrates in liver metastases, work published in the Annals of Surgical Oncology and featured in Nature Reviews Clinical Oncology. I was recruited to Roger Williams to join a strong immunotherapy program and build a lab focused on investigating how suppressive liver immune cells may contribute to the development and progression of liver metastases. My K08 proposal is a critical step in my career development as the project represents a fusion of my basic liver immunology background with a therapeutic platform, genetically modified or designer T cells (dTc), we are presently using in clinical trials at Roger Williams. The associated training plan, as noted below, will provide me with important educational opportunities to facilitate my transition toward independent funding. Environment - The Roger Williams Medical Center (RWMC) is an ideal environment for my academic growth and development. Dr. Richard Junghans, my mentor, is a well established immunologist with a rich experience with immunotherapy trials and dTc. I also work with Dr. Vincent Falanga, the RWMC Chief of Dermatology, as he shares valuable resources obtained through their COBRE grant. As the RWMC is of modest size, my laboratory and career development are top priorities for the institution. I receive a tremendous level of support from th administration and my Division Chief and Cancer Center Director, Dr. N. Joseph Espat. This enables me to have the necessary protected time and maintain a high level of focus on my laboratory work. Development and Training Plan - I have assembled a mentoring team of well respected experts to monitor my progress, support my research, and promote my career development. Each member of the team has successfully competed for NIH funding and offers a particular area of expertise that meshes well with my proposal. Dr. Junghans will be the primary mentor, and his experience with immunotherapy and T cell biology will be invaluable. The three co- mentors will make important contributions as well. Dr. Espat, an authority on liver metastases, will ensure that the work and publications maintain a translational focus. My mentor from MSKCC, Dr. DeMatteo, will provide important insight and critique from the vantage point of an expert in liver immune cell biology. Dr. Alfred Ayala at Brown, with whom I have recently begun to collaborate, is another well respected expert in liver immunobiology. I have also included translational medicine and immunology course-work at Brown to expand my knowledge base in critical areas. Research Plan - Based upon the work I have published to date, I hypothesize that suppressive liver immune cells limit the effectiveness of T cell based therapies for eradicating liver metastases. T regulatory cells (Treg) and myeloid derived suppressor cells (MDSC) are likely contributors to liver tolerance. Our lab has spent over one year optimizing our murine model of CEA+ colorectal liver metastases and the immunologic assays described in the proposal. My first specific aim focuses on elucidating the mechanisms by which Treg and MDSC may limit anti-CEA dTc function in the liver. I have designed the experiments to focus on each component of the model separately, including the Treg or MDSC, dTC, and immune receptor express by dTc. In addition, contact-dependent mechanisms such as the programmed death-1 pathway and secreted factors will be studied. For the second specific aim, specific strategies for blocking the suppressive interactions between Treg or MDSC an dTc will be tested in vivo. We hope some of our immunologic manipulations will be translatable into the clinical arena.


In addition to long known contributions from particular MHC class II molecules, there is growing appreciation that in both humans and NOD mice some MHC class I variants also play an essential role in autoimmune type 1 diabetes (T1D) development by mediating pathogenic CD8 T-cell responses. The overall goal of this renewal application continues to be dissection in NOD based mouse models of the mechanistic basis for MHC class I restricted diabetogenic CD8 T-cell development, and use of this information to identify potentially clinically translatable means to attenuate such effectors. While the H2g7 MHC haplotype encoded Kd and Db class I molecules are essential to T1D development in NOD mice, they are common variants also characterizing many non-autoimmune prone strains. This suggested H2g7 MHC class I molecules aberrantly mediate diabetogenic CD8 T-cell responses in NOD mice through interactions with some of the many other disease susceptibility (Idd) genes characterizing this strain. Aim 1 will test the hypothesis based on preliminary mRNA transcript profiling and congenic truncation analyses that a hyper-expression variant of the NFkB inhibitory Nfkbid gene located within the previously identified Idd7 locus is an important contributor to the failure of diabetogenic CD8 T-cells to undergo thymic negative selection in NOD mice. Similarly, epidemiological studies indicate that in humans certain common class I molecules such as HLA-A2.1 can aberrantly contribute to T1D development also likely through a genetically contextual process. Indeed, we found that when expressed in the context of the NOD genome, human HLA-A2.1 molecules mediate diabetogenic CD8 T-cell responses. HLA-A2.1 restricted diabetogenic CD8 T-cells in this NOD background stock primarily recognize two peptides each derived from the pancreatic ss cell proteins insulin (INS) and islet specific glucose-6-phosphatase catalytic subunit related protein (IGRP). Immunological tolerance can be efficiently induced to antigens bound to autologous leukocytes by the cross-linking agent ethylene carbodiimide (ECDI), and such an approach is in a clinical trial as a possible multiple sclerosis intervention. However, there are many hurdles to cell based therapies, and possible T1D intervention approaches can only be considered in humans already at a late prodromal stage of disease development. Therefore, to broaden potential clinical translation, Aim 2 will test the possibility supported by new preliminary data that treatment with synthetic microparticles bearing appropriate ECDI coupled INS and/or IGRP autoantigenic peptides can exert late disease stage T1D protective effects in NOD-HLA-A2 mice, and/or enables reversal of established disease by pancreatic islet transplantation. Finally, epidemiological evidence implicates B39 as a potentially highly potent diabetogenic HLA class I variant in humans. Thus, Aim 3 will assess whether transgenically expressed B39 molecules mediate diabetogenic CD8 T-cell responses in NOD mice, and if so, identify cell autoantigens displayed by this class I variant, and test their capacity to serve as broadened disease intervention reagents.


PROJECT SUMMARY The overarching hypothesis of this proposal is that kisspeptin may explain many of the changes in glucose metabolism in pregnancy: high levels of kisspeptin during normal pregnancy amplify insulin release to maintain normoglycemia, and relative hypokisspeptinemia may underlie the pathophysiologic defects in gestational diabetes mellitus (GDM). The initial steps to examining this overarching hypothesis are to 1) define the effect of kisspeptin (at pregnancy levels) on insulin physiology in non-pregnant women and 2) to determine if kisspeptin levels predict the development of GDM. Given the complexity of factors in pregnancy that regulate metabolism and insulin physiology, these initial studies are designed to isolate the effect of one factor: kisspeptin. This proposal utilizes gold standard (hyperglycemic and hyperinsulinemic euglycemic clamps) and physiologic (mixed meal tolerance test) methods in randomized, placebo-controlled and blinded clinical trials to assess kisspeptin?s impact on insulin secretion and insulin sensitivity in non-pregnant women. Going further, the grant utilizes comprehensive specimen banks with longitudinal pregnancy samples and patient outcome data to measure kisspeptin levels across pregnancy and to determine if kisspeptin levels early in pregnancy can predict the development of GDM. This grant will refine our understanding of the impact of hyperkisspeptinemia of pregnancy on insulin and incretin physiology and development of GDM. This application details a comprehensive five-year training program for mentored career development in patient- oriented research. The Applicant proposes research, including independent clinical trials, specifically constructed to provide focused training pregnancy physiology and in the mechanisms of insulin physiology. To achieve this goal, she has chosen mentors with complementary expertise: Dr. Stephanie Seminara is an expert in kisspeptin physiology with a strong background in human physiology research, and she is Chief of the Reproductive Endocrine Unit at Massachusetts General Hospital (MGH); Dr. Patrick Catalano is an expert in pregnancy physiology and gestational diabetes, and he is a Professor of Obstetrics and Gynecology at Tufts University School of Medicine and Principal Investigator in the Mother Infant Research Institute; Dr. Jose Florez is an expert in physiological mechanisms in the development of diabetes, and he is Chief of the Diabetes Unit at MGH. This mentoring team will position the Applicant well to launch a successful independent investigative career in metabolism, with a focus on pregnancy and the influence of reproductive hormones, a key NICHD research priority area. The Applicant's career development plan entails rigorous coursework and seminars, hands-on practical experience, and close guidance from scientific advisors with diverse scientific expertise. Collectively, the experience gained during this award will serve as the foundation for the Applicant's independent, academic career as a physician-scientist with expertise in translational research in human metabolic disease.


This is a multi-institutional, Intergroup III trial examining the potential chemopreventative effect of Finasteride for prostate cancer. Study subjects who accrue can have no evidence of prostate cancer clinically. Enrolled participants randomize to either placebo or the study drug (proscar 5 mg daily), which is administered in a double-blind fashion. Participants are followed with yearly digital rectal exams and serum PSA values. All PSA samples are sent to a central lab for processing, and elevated values are evaluated with biopsy. any cancer detected during the study results in patients being taken off study. The follow-up period will continue for 7 years, after which all study participants will undergo trasnrectal unltrasound-guided biopsy, and tiddues form the biopsy will be processed at a central pathology site. Differential rates, if any in the prevalence of prostate cancer between the two study groups will be recorded.


D-alanyl-D-alanine peptidases catalyze the crosslinking of peptidoglycan inthe final stages of bacterial cell wall biosynthesis. The DD-peptidases are the targets for B-lactam antiobiotics(penicillins and cephalosporins). The structure of the enzyme from Streptomyces R61 has been determined to 1.6A resolution (Crystallographic R-factor = 0.153). We are now studying complexes of the native enzyme with drug molecules and substrate analogs. These studies will be facilitated by access to the Cambridge Structural Database.


This project follows our discovery of ganglioside presence in the nuclear envelope (NE) of neural cells, and the functional association of GMtwith a Na-Ca exchanger in the same membrane. In potentiating the exchanger activity, GM1 was shown to bind to this protein with unusually high affinity, a phenomenon that was not shared by these molecules in the plasma membrane. One goal of the proposed studies is to elucidate the molecular basis of this unusual association by determining which of the several known exchanger isoforms has the ability to (a) traffic to the NE and (b) bind GM1. Various isoforms of the exchanger will be transfected into Jurkat cells, which we have found do not normally express the GM1-exchanger complex in the nucleus, and the NE examined for evidence of such expression. Exchanger isoforms having this ability will be compared for common structural motifs that appear to promote high affinity binding (likely to include basic amino acids); the structures will be altered through site-directed mutagenesis to determine whether such exchangers, following transfection, have lost the ability to sequester GM1 in the NE. We will test the hypothesis that the GM1-exchanger complex occurs in the NE in association with ganglioside GD1a and neuraminidase, both of which were also shown to occur in the NE. The presence of these 2 molecules could constitute a supply mechanism to ensure sufficient GM1 for potentiating the exchanger. The functional role of the GM1-exchanger complex, postulated to remove elevated nuclear calcium, will be tested by comparing nuclear calcium changes in cells which do and do not have the GM1-exchanger complex. The functional role will also be tested in vivo with knockout mice lacking GM1, which were found to be highly susceptible to kainate-induced seizures. Demonstrated attenuation of such seizures by administered LIGA-20, a membrane-permeant derivative of GM1, will be further studied by determining the role of nuclear GM1/exchanger in modulating the seizure mechanism.


Exercise Testing & Training Facility utilization only. Epidemiological studies suggest that low levels of physical activity and physical fitness are strong and independent risk factors for cardiovascular disease, cancer and all-cause mortality. Inactivity in the United States appears to be a major health problem that is of comparable magnitude to cigarette smoking, obesity, high blood pressure, and high blood cloesterol. There are over 40 million adults in the US whose sedentary habits put them at considerably increased risk of morbidity and mortality from several diseases. Exercise has been found to be a very strong predictor of successful weight management once weight loss has been achieved. No studies have compared changes in risk profiles and body composition in people treated by diet plus lifestyle activity vs. diet combined with a traditional exercise program. We hypothesize that patients who are randomized to diet-plus- lifestyle activity will maintain both weight losses and improved risk profiles significantly better than the subjects who receive the diet plus traditional exercise training because the former participants will continue to implement their increased lifestyle activity strategies into their daily schedules. The purpose of this study is to examine whether a lifestyle activity program helps patients maintain weight losses and adhere to the activity program after a supervised weight loss program. Fifty obese women will be randomly assigned to either: 1) control group, 2) diet plus lifestyle activity, or 3) diet plus traditional exercise, i.e, combined strength training and aerobic exercise in a supervised setting. We hypothesize that the women who receive the lifestyle treatment will achieve better weight maintenance and continue to pursue lifestyle activity after the supervised program is over.


Human milk oligosaccharides (HMOs) have been thought to play a role in the development of specific intestinal flora in breast-fed infants for many years. Nowadays it is known that they are also potent inhibitors of bacterial adhesion to epithelial surfaces (initial stage of the infection process). Oligosaccharides are not hydrolyzed in the upper small intestine and reach the large intestine intact, where they serve as substrates for bacterial metabolism. Thus, HMOs are considered as the ''dietary fiber'' of human milk. Another characteristic of oligosaccharides is their proposed ''anti-infective effect''. This role is achieved thanks to their capacity to inhibit the adhesion of bacteria to the epithelial surfaces, thereby playing an important protective role against infection in the gastrointestinal, respiratory and urogenital tracts by direct and indirect mechanisms. Therefore, HMOs have antimicrobial activity and may be useful in treating and/or preventing specific enteric bacterial and viral infections. However, the road to convert HMOs into pharmaceuticals or nutritional substances has been blocked by the lack of pure, single component oligosaccharides from human milk in quantities large enough for scientific investigation, as well as preclinical tolerance and safety studies and for safety and clinical testing in populations that are exposed to gastrointestinal pathogens. Therefore, this proposed research program aims to develop practical processes to produce HMOs on multi-gram to kilo-gram scales. Since it was repeatedly reported that 2-linked fucosyloligosaccharides exhibited more antimicrobial activity than non-2-linked fucosyloligosaccharides, we will choose five 2-linked fucosyloligosaccharides such as 2'-FL, LNF-I, 2H-antigen, LDFH-I and Ley as our main targets. Moreover, non-2-linked fucosyloligosaccharides LNF-II and LNF-III will provide us the opportunity to confirm the observation of higher antimicrobial activity for 2-linked fucosyloligosaccharides. In addition, the non-fucosylated oligosaccharide LNT and LNnT will provide control experiments to evaluate the effect of fucose in oligosaccharides. Over the past 14 years, the Wang lab has been developing enzymatic oligosaccharides synthesis. We have invented and further developed the superbeads and superbug technology for large scale oligosaccharide production. The most efficient approach for oligosaccharide synthesis is to follow the natural carbohydrate biosynthetic pathway where oligosaccharides are assembled together by specific glycosyltransferases using individual sugar nucleotides as building blocks. These building blocks are themselves biosynthesized and recycled from individual monosaccharides through a series of biosynthetic enzymes. For small to medium scale synthesis of oligosaccharides, Wang has developed simple solid phase synthetic systems by immobilizing all the necessary biosynthetic enzymes onto a so-called superbeads. These beads function as stable and versatile synthetic reagents, which can be used to synthesize a variety of glycoconjugates in cell-free systems. For large-scale production, Wang superbug essentially transfers the entire natural biosynthetic pathway into an E. coli strain. The approach includes cloning each enzyme along the biosynthetic pathway and connecting the genes of these enzymes together to produce an artificial gene cluster. A recombinant E. coli transformed with such a gene cluster is then used to produce the oligosaccharide through fermentation and purification. Thus, the superbeads and superbug approaches will be used in this program to produce the 9 oligosaccharides. Specifically, there are four aims: Aim I: Production of HMOs by immobilizing multiple enzymes (superbeads). This involves investigation on the necessary microbial glycosyltransferases, development of superbeads for UDP-GlcNAc, UDP-Gal and GDP-Fuc production, and combination of the glycosyltransferases with sugar nucleotide production to produce oligosaccharides. Aim II: Production of HMOs by recombinant E. coli (Superbug), which involves combination of the biosynthetic pathways of these HMOs into one or several recombinant E. coli strains. Aim III: Production of HMOs by GRAS (Generally Recognized as Safe) yeast cells. This new system will provide safer production system for HMOs synthesis. Aim IV: Characterization of the oligosaccharides through systematic biomedical and microbiome approaches in collaboration with other specialized laboratories, also in our own lab, with the advantage of multi-gram or kilo-gram scale neutral human milk oligosaccharides produced from this project. It is expected that the biosynthetic technology developed in Aim I - III will be transferred to biotech company(s) (such as the biotech startup Carbogene USA LLC which specializes in large scale oligosaccharide production) and GMP processes will be developed to produce the oligosaccharides in quantities large enough for preclinical studies of tolerance and safety, as well as for safety, dose-ranging, and efficacy trials in infants and children who are at high risk of exposure to gastrointestinal pathogens.


Responsibility for Planning and Evaluation is vested in the Georgetown Lombardi Comprehensive Cancer Center (LCCC) Director and Executive Committee (EC). Since the last review, LCCC has undertaken an extensive review and overhaul of its planning and evaluation processes and refined its overall Strategic Plan. Director Weiner, Deputy Director Atkins, 6 Associate Directors (ADs), and a MedStar Georgetown Cancer Network Representative form the EC, which oversees Center-wide strategic and operational initiatives, discusses overarching goals of LCCC related to mission and comprehensive cancer center status, reviews metrics for each area overseen by the ADs, approves membership, oversees Developmental Funds, and fosters collaborations across Research Programs and disciplines. An Internal Advisory Committee (lAC) meets semi-annually to provide advice on integration with other components of the University and MedStar Health. The annual External Advisory Committee (EAC) meeting is supplemented with individual EAC members' guidance via conference calls and on-site visits. In-depth external reviews have evaluated progress of specific aspects of LCCC (clinical research, Shared Resources, Administration, biostatistics and investigator-initiated protocols). Research Program Leaders meet monthly to review Program themes, vet new and continued Program membership, and discuss needs for Shared Resources and promising opportunities for collaborative interprogrammatic research. Program meetings allow in-depth research discussion, refinement of program themes and goals and progress toward meeting them, discussion of correlative science for clinical trials and encourage incorporation of health disparities into ongoing research. Identification of opportunities for collaboration (multi-investigator studies, translational and transdisciplinary projects) is high priority for all planning and evaluation activities. Within the Shared Resources, individual Advisory Committees meet annually to review metrics and update and prioritize goals. User satisfaction is monitored by surveys. Frequent clinical leadership meetings ensure metric review and update. Administration is monitored through external review, a faculty liaison group and user surveys. Retreats have focused on health disparities; the population science merger; development of therapeutic trials; transdisciplinary, translational projects; and Administration. These actions have led to LCCC's strategic objectives to 1) advance transformative cancer research, 2) reduce the impact of cancer and diminish disparities in our catchment area, 3) lead high-impact clinical research, and 4) ensure long-term growth, vibrancy and stability ofthe research enterprise.


The long-range goal of this project is to increase the percentage of Hispanic women who receive cervical cancer screening and who are in compliance with recommendations for follow-up after an abnormal screening. The objective of this proposal is to integrate existing theoretical models in the development and pilot-testing of a scale measuring how Hispanic women construct meaning regarding cervical cancer prevention screening. In addition to this scale, demographic and psychosocial information will also be included. The rationale for the proposed research is that this theory-driven and culturally-appropriate questionnaire can be used as a tool to determine factors that predict cervical cancer screening among Hispanic women, which, in turn, will assist in the development of tailored interventions to increase cervical cancer screening in this population. We are well suited to undertake the proposed research because we have assembled a multidisciplinary team with the scope and breadth of expertise in the areas of public health, health psychology, cancer control, Hispanic culture, measurement, and qualitative research. Given our long-standing relationship with a Hispanic grassroots organization and other agencies serving the Hispanic population in the Memphis area and our experience working with this population, we feel confident that we can recruit a large number of Hispanic women. Once we complete the work proposed in the R03 application, we expect to be well-positioned to develop and submit an R21 application to propose the validation of the questionnaire, as well as pilot-testing and feasibility-testing of a theory-driven, culturally appropriate cervical cancer prevention intervention targeting Hispanic women. The specific aims are: (1) To test and integrate components of the existing theoretical models (primarily the PEN-3 model and components of the Theory of Planned Behavior and Social Cognitive Theory) to determine contextual variables associated with cervical cancer screening among Hispanic women of reproductive age through a series of 20 focus groups with Hispanic women between the ages of 18 and 42; (2) To develop a theory-driven and culturally appropriate questionnaire to assess contextual variables (and their social constructions) that contribute to cervical cancer prevention screening among Hispanic women based on the results of Aim 1; (3) To psychometrically evaluate the culturally appropriate questionnaire in a random sample of Hispanic women (N= 300) based on the results of Aim 1 and 2.


The aims of this project are to (1) to understand the molecular mechanism for depression of synthesis of enzymes involved in polysaccharide synthesis that occurs in capR, capS, and capT mutants (2) to understand the molecular mechanism of control of cell division as it is specifically related to the point mutations in the E. coli K12 gene designated capR (1on). The capR mutants, besides overproducing polysaccharide synthesizing enzymes, are also sensitive to UV, X-rays and ozone. Upon UV or x-ray exposure they form long filaments without crosswalls and these forms are nonviable. The capR ion gene specifies a protein (as determined by genetic studies) that appears to be a repressor or controls repressors that bind to many sites on the E. coli chromosome to stop synthesis of enzymes of polysaccharide synthesis. The capR ion protein may function similarly to control UV and x-ray sensitivity. We intend to isolate the capR ion protein. We have obtained the capR gene on a tetracycline resistance plasmid using tetracycline as a selective marker and combining DNA molecules in vitro with restriction enzymes and DNA ligase. We can amplify the capR ion protein in each cell to permit us to isolate the protein. Using genetic techniques we will also identify the postulated DNA site (the "UV operon" for structural genes) of capR ion protein action that is responsible for causing a UV and x-ray sensitive phenotype in capR (ion) mutants. Recent results indicate the gal operon, in addition to being controlled negatively by the classic galR repressor and positively by cyclic-AMP and cyclic AMP receptor protein, is also independently regulated negatively by the capR protein. This is the most complex control system available that can readily be investigated at the molecular level and we will pursue this subject in conjunction with those described above. The capR gene also effects bacteriophage lambda transcription and committment to lytic growth, and we are pursuing this fact.


This project will develop X-ray tubes generating mono-energetic radiation in the energy region useful for mammography (17-30 keV). Earlier work has demonstrated significantly reduced dose in such examinations by using fluorescent radiation at higher energies than the 17.5 keV characteristic radiation of molybdenum. By proper choice of energy, adequate contrast can be maintained while the dose is substantially reduced. However, to date the production of clinically useful intensities has been hampered by thermal loading of the fluorescent anode. This problem will be overcome through the use of new materials and techniques for anode fabrication. Anode properties including radiant output per unit charge, spectral distribution, loading factors, phantom penetration, etc. will be studied under operating conditions with a demountable x-ray tube and with radiation detection equipment that includes a high resolution spectrometer interfaced to a minicomputer for data manipulation. The ultimate goal will be the production of a new fluorescent-anode X-ray tube which will give substantially reduced doses in clinical mammography.


The cellular and molecular events in limb development are being studied in mouse embryos. The techniques used require both histologic analyses and biochemical assays. The primary morphological tool is Golgi staining which permits studies of cell polarity as the Golgi apparatus occupies the pole of the cell from which material is secreted. Studies to date have shown that the epithelium is always oriented away from the underlying mesenchyme. The mesenchyme beneath the epithelium shifts its polarity on days 12 to 13 (the time of chondrogenesis) to point primarily toward the epithelium. The stimulus for this shift has not been identified. The types of collagen and proteoglycans in the developing limb are also being determined. We have found that three small molecular weight hydroxyproline-containing compounds are present during limb development. These are much more abundant than large molecules like collagen which also contain hydroxyproline. These morphologic and biochemical studies are also being carried out on mouse limbs malformed by the dominant gene Dh (dominant hemimelia). It is proposed that the single mutant gene malformation will interrupt primarily one aspect of limb morphogenesis. By comparing different hereditary limb malformations one would expect to find primary effects on different aspects of the same process. Specimens from human limb malformations are being studied to see if they contain abnormal or immature types of collagen.


Summary Stroke is the leading cause of disability in the US, and, apart from physical therapy, therapeutic options to reduce this burden are non-existent. Following a stroke, cell death and glial scarring leads to the formation of a non- regenerative stroke cavity surrounded by the regenerative peri-infarct region. We are interested in understanding how bioengineered therapeutics can synergize with pro-repair programs active in the peri-infarct region to promote tissue regeneration in the stroke cavity and thereby improve neurological recovery. We have previously engineered a dual-acting angiogenic hydrogel that, when injected into the stroke cavity, can reduce glial scarring and promote vascularization to allow axonal infiltration. Although achieving brain repair in the stroke cavity is remarkable, these results were achieved in young mice with un-impaired neuroplasticity. We believe that to bring this technology closer to clinical translation, we must be able to show similar brain repair and behavioral improvement in more clinically relevant animal models, like aged mice with decreased neuroplasticity. In this application, we aim to identify our angiogenic hydrogel?s mechanism of action and optimize its formulation and to utilize this new formulation in a stroke models with decreased neuroplasticity. The hydrogel is composed of hyaluronic acid functionalized with cell-binding integrins and loaded with clustered vascular endothelial growth factor (VEGF) and heparin nanoparticles. Heparin is a known anti-inflammatory agent that potentially acts by breaking the inflammatory cycle between macrophages and astrocytes following stroke. We will use design of experiment methodology to determine the composition of these three factors (integrins, clustered VEGF, and heparin nanoparticles) that leads to substantial brain repair (Aim 1) and assess how the hydrogel components modulate the pro-repair environment by analyzing temporal changes in proteins, mRNA, and immune cell populations following stroke (Aim 2). Using the improved formulation, we will also evaluate the angiogenic hydrogel?s ability to promote neurological regeneration and functional recovery in more rigorous animal models, specifically aged mice treated immediately following cerebral ischemia and young mice with cerebral ischemia treated as the plasticity window is closing (Aim 3). Overall, we aim to deepen our understanding of how bioengineered therapeutics synergize with endogenous pro-regenerative programs and thereby improve behavioral outcomes following stoke in more difficult-to-treat cases.


Extracorporeal shock wave lithotripsy (ESWL) has been used routinely for the treatment of kidney stone patients for almost two decades. Despite the widespread clinical application, the underlying physical mechanisms of stone comminution and tissue injury in ESWL have not been fully understood yet. In fact, although some progress has been made recently on the development of better lithotripters and treatment protocols for stone fragmentation, these new machines have also resulted in an increase in both treatment imes and soft tissue injuries; leaving the original Dornier HM3 lithotripter as the gold standard for ESWL. A possible reason for this lack of improvement is that the great majority of ESWL studies have been focused primarily on experimental approaches while, in contrast, there has been a very limited development in realistic and accurate mathematical models that can capture the essential physics of shock wave propagation, focusing, cavitation, and their interactions with renal calculi and surrounding tissue. Our goal is to develop, and experimentally validate, such a comprehensive model where all the important mechanisms in ESWL will be accounted for and coupled together. The model will include effects deriving from pure shock propagation, absorption, streaming, cavitation and bubble dynamics, as well as those associated with the elastic stresses generated in the stone. It will also be fast and accurate in simulating realistic ESWL treatments without the need of supercomputers. The relevance of this research to public health is that, once developed, this model could significantly advance our basic knowledge of how ESWL works, it could provide considerable guidance for improving the treatments' efficacy and safety of existing systems, and it could aid in the design of the next generation lithotripters. [unreadable] [unreadable] [unreadable]


Better Understanding of the Neuroprotective Mechanisms of Korean Ginseng in Stroke It has been postulated that the determinants of neuronal cell death in acute and chronic neurodegenerative conditions are mediated by free radical damage. Ginseng has been reported to be neuroprotective and a potential preventive medicine, but the underlying cellular mechanisms are still unclear. Our preliminary results prompted us to focus our attention on Korean Ginseng and test the hypothesis that the transcriptional factor Nrf2 could participate in the overall Ginseng's neuroprotective function. HO, which cleaves heme (a prooxidant) to form biliverdin/bilirubin (antioxidants), carbon monoxide (a vasodilator), and iron (a prooxidant) has been shown to play a protective role in oxidative stress, ischemia, inflammation, and hypertension. Although HO2 is constitutively expressed, HO1 is inducible. Consequently, a possible way to increase HO levels to achieve neuroprotection may be to induce HO1. Of the compounds tested in our preliminary experiments in primary neuronal cultures, Ginseng was one of the most potent HO1 inducers. Our results also indicate that pretreatment of neurons with Ginseng is sufficient to provide neuroprotection, suggesting that co-treatment during oxidative stress is not necessary. This neuroprotective effect was abolished by a protein synthesis inhibitor, and was greatly reduced by an HO inhibitor. These preliminary results implied that specific induction of HO1 could be a mechanism by which Ginseng exerts its preventive neuroprotective actions and motivated us to propose that some of the neuroprotective effects attributed to Ginseng could be mediated through HO1 induction and the associated beneficial actions of heme degradation. Recently, we and others have described Nrf2 has as a key regulator of inflammation and redox homeostasis. In Aim 1, we will determine anatomical and behavioral outcomes following ischemia in wild type mice pre-treated (acutely or chronically) with Ginseng, determine whether the beneficial effect is sustained with aging, and test whether these effects are attenuated in Nrf2 knockout mice. In Aim 2, we will determine whether Ginseng induced characteristic changes in brain cells, and start addressing in which cell types these Nrf2 changes occurs. Together, these in vivo results will help us determine whether oral consumption of a standardized Ginseng extract could be beneficial, and which cells can be most associated with the Ginseng preventive brain mechanistic pathways by which Ginseng would provide the cell/brain with resistance to acute debilitating neurodegenerative conditions.


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. Apoptosis plays an essential role in the development and homeostasis of metazoans. The genetic characterization of programmed cell death (apoptosis) in C. elegans identified four proteins, CED-3, CED-4, CED-9, and EGL-1, that collectively control the onset of apoptosis. CED-3 is a caspase and its activation depends on CED-4. CED-9 antagonize the function of CED-4 through an unknown mechanism. CED-3, CED-4, and CED-9 form a hetero-oligomer. To elucidate the mechanisms of cell death control in C. elegans, we crystallized the ternary complex of CED-3/CED-4/CED-9. We plan to determine the structure by molecular replacement or by MAD.


This application establishes the Models for Integrating Juvenile Justice and Treatment (MIJJT) Research Center in collaboration with The National Criminal Justice Drug Abuse Treatment Research System (CJ-DATS) under RFA-DA-02-011. MIJJT provides the opportunity to engage in collaborative, rigorously conducted, multisite service delivery and evaluation research involving drug-abusing juvenile offenders. Empirically, the studies that have been conducted on adults in justice settings have shown that the most consistent effects occur when there is a continuum of primary treatment and aftercare. This is likely even more critical for juvenile justice clients who are generally unemancipated and present with multiple problems in addition to substance abuse. Adolescent substance abuse and the rising juvenile crime rate have been acknowledged as two of the nation's greatest concerns and, though their etiology is diverse, they are inextricably linked. Despite numerous education and prevention initiatives, adolescent drug abuse and its sequelae of juvenile crime, risk for HIV infection and school failure remain major juvenile justice, mental health, public health and public safety concerns. The MIJJT Research Center will complement the aims of the NIDA CJ-DATS by bringing comprehensive focus to the issue of adolescent substance abuse, juvenile justice and treatment by incorporating a spectrum of agencies and treatment providers in Ohio and Pennsylvania, including minimum to maximum secure facilities and community-based programs. The potential sample assures the inclusion of a broad range of youth, including females as well as males, those with co-occurring mental disorders, HIV risk populations and members of various racial/ethnic groups. The proposed studies aim to address the multidimensional needs of adolescents who enter the criminal justice system, the multivariate, interrelated structures of provider agencies that respond to these youth, and the range of services that are required to effect positive outcomes. In accordance with the CJ-DATS objective to provide a stable infrastructure for research to improve drug abuse treatment for individuals with drug abuse or addictive disorders who are incarcerated or transitioning from jail or prison to the community, the MIJJT investigative team proposes research concepts that follow the trajectory of an adolescent's juvenile justice and treatment experience. Study 1 proposes a CBT intervention in the treatment setting; Study 2 addresses the relationship of the parole and probation officer to the adolescent; and Study 3 focuses on the youth's reintegration into the community, particularly the role of the family in facilitating and participating in a successful integration. The collaborative energy that has facilitated the development of the current proposal reflects the support of the collectivity of the participants--research, administrative, and clinical.


Spinal cord injury (SCI) is among the most disabling conditions affecting wounded members of the U.S. military. Unfortunately, there has been no effective treatment available for SCI patients. It is, therefore, an urgent medical need to develop novel repair strategies to mitigate the devastating nature of SCI and to translate them clinically to improve quality of life of our veterans with SCI. Recently, a novel lipid signaling pathway, namely the cardiolipin (CL)-cytochrome c pathway, that control cell death/apoptosis has been identified. CL is a structurally unique dimeric phospholipid localized in the inner mitochondrial membrane where it is required for optimal mitochondrial function. CL is a preferred oxidation substrate in neuronal injury, is the only phospholipid in the mitochondria that undergoes early oxidation during apoptosis, and is an early target of reactive oxygen species (ROS) attack. Alteration of CL has been associated with mitochondrial dysfunction in a variety of pathological conditions. Using mass spectrometry-based lipidomics for the first time in SCI, we have generated preliminary data showing CL peroxidation and loss after SCI. Remarkably, XJB-5-131 (XJB), a novel mitochondria-targeted antioxidant, administered at 30 min post-SCI significantly reduces tissue damage and improves behavioral recovery in adult rats. These data strongly suggest that CL alteration is a key mechanism that mediates injury-induced cell death and tissue damage. However, the role and mechanism of CL alteration in SCI remain unclear. Here, we hypothesize that CL alteration, including peroxidation and loss, is a central process that mediates spinal cord secondary injury, and that restoration of CL level may lead to neuroprotection and recovery of function after SCI. Using a rat spinal cord neuronal culture system in vitro and an adult rat thoracic contusive SCI model in vivo, we will determine 1) whether CL alteration induces mitochondrial dysfunction and neuronal death and whether such detrimental effects can be reversed by a novel mitochondrial targeted antioxidant XJB; 2) the molecular role of CL alteration in the signaling pathway of neuronal apoptosis after SCI and whether such CL alteration is sufficient to mediate secondary SCI; 3) whether abnormal mitochondrial dynamics also play a role in CL alteration-mediated cell death; and 4) an optimal dose and therapeutic time window of XJB on neuroprotection and functional recovery after rat contusive SCI.


The great diversity of techniques used in Cancer Research demands equally diverse reagents and facilities. While the preparation of growth media is not technically difficult, the scale of our needs requires an efficient centralized facility. The media preparation needs for the Program will be provided by the Media Preparation Facility. This facility will prepare both liquid and solid media for the growth of bacteria and yeast, plus tissue culture media, salts, and buffers. In addition, sterile glassware and supplies will be provide for the Projects in the Program.


Many factors contribute to the current epidemic of obesity. Although estrogen (E2) status is not commonly recognized as a determinant of obesity risk in women, there is strong evidence from large randomized controlled trials that E2-based hormone therapy (HT) attenuates weight gain by about 40% in postmenopausal women. Importantly, there is also strong evidence that E2 attenuates abdominal fat accumulation, a fundamental component of the Metabolic Syndrome. The global aim of the proposed studies is to evaluate potential mechanisms by which E2 deficiency accelerates fat gain and abdominal fat accumulation in women. The first aim is to determine the effects of short-term (7 d) and chronic (5 mo) sex hormone suppression on resting energy expenditure (REE) and fat gain. It is hypothesized that a) REE will be reduced in response to short-term hormone suppression, as observed in a pilot study; b) the decrease will persist with chronic hormone suppression, promoting fat gain; and c) E2 add-back therapy will mitigate these responses. Whereas a dampened REE may cause fat gain in the E2-deficient state, it would not explain the disproportionate accumulation of abdominal fat that occurs. Altered hypothalamic-pituitary-adrenal (HPA) axis activity leading to cortisol excess causes abdominal fat accumulation (e.g., Cushing's syndrome) and there is evidence that E2 attenuates stress-induced HPA axis activity. Therefore, the second aim is to determine the effects of sex hormone suppression on stress-induced HPA axis activity and abdominal fat accumulation. It is hypothesized that stress-induced HPA axis activity will be a) amplified during sex hormone suppression; b) attenuated by E2 add-back; and c) a determinant of abdominal fat gain. These aims will be accomplished by using gonadotropin releasing hormone (GnRH) analog therapy, with placebo or E2 add-back, to determine the effects of E2 on REE, HPA axis activity, and fat gain and distribution in 100 healthy young women. Abdominal obesity purportedly accounts for 20% of coronary events in men but 48% of events in women, underscoring the value of learning the mechanisms of abdominal fat gain in women. Until menopause, women are largely protected against abdominal obesity, implicating a role for sex hormones. Recent findings that benefits of HT may not outweigh the risks in some women does not negate the importance of identifying mechanisms by which E2 attenuates fat gain and abdominal fat accumulation. [unreadable] [unreadable] [unreadable]


The generation of rapid or saccadic eye movements requires a transformation from the visual input falling on the retina to the pulse of activity to the eye muscles to move the eyeball to a new position. The areas in the brain of primates (an excellent animal model of the human saccadic system) have been identified throughout regions of the cerebral cortex and brainstem so that in outline the entire pathway from visual input to eye movement output is now known. What is not know is what signals are conveyed from one region to the next and what transformations in that signal are made at each stage of processing in the brain, and until this is known, a circuit within the brain for the control of behavior can not be understood. We have addressed this issue by determining which neurons in two cortical areas, the frontal eye fields, and the posterior parietal cortex, are the output neurons to the brainstem center for the control of saccades, the superior colliculus. By electrically stimulating in the colliculus, we can activate the axons of neurons in cortex that reach to the colliculus (antidromic activation) and thereby see which neurons are the output neurons from cortex and what is the nature of the signal (visual, visuomotor, motor) that they carry. About three quarters of the antidromically activated neurons in the parietal cortex respond to the visual stimulation that could be used to guide a saccade. In addition, these neurons were active as the monkey prepared to make a saccade to a visual target indicating that the neurons going from this region of cortex to the brainstem were carrying both a visual and a movement related signal. This signal is similar to that carried by one set of neurons in the colliculus thought to be at the earliest stage of processing in that structure, the buildup neurons. In contrast, when the same experiment was done with neurons in the frontal eye field, more neurons tended to have a clear burst of neuronal activity before the onset of the saccade. These neurons were more like the neurons in the colliculus that are close to the output of that structure, the burst neurons. Thus the major output of the two areas of the cerebral cortex is different and may represent the input to different stages of signal processing in the superior colliculus, a possibility that we are now investigating. In addition to the signal going to the colliculus from cortex, we have also identified one from colliculus to the frontal eye fields (orthodromic activation), and found that all of the neurons in the frontal eye fields that receive this signal have visual activity. Thus the neurons that project out of the frontal eye fields to colliculus tend to carry a motor signal while those that receive information from the colliculus have a visual signal.


The importance of the mismatch repair system in humans is evidenced by the fact that inheritance of a defective allele in one of several mismatch repair genes will, with high probability, lead to cancer, usually colorectal cancer. There is increasing evidence that some of the most important substrates for the mismatch repair system involve damaged DNA, including types of damage likely to be produced by normal cellular processes, especially oxidative processes in the cell. The overall goal of this proposal is to examine the interactions of mismatch repair proteins with DNA lesions and mismatches likely to be found in the cell as a consequence of normal endogenous processes. These experiments will help to test the hypothesis that mismatch repair is important for preventing mutations due to damaged DNA. The specific aims of this proposal are to determine the interaction of yeast and human MutSalpha with substrates containing specific damaged nucleotides, to determine the effect of MutLalpha on the binding of these lesions by MutSalpha, and to examine mismatch repair, in vitro, of substrates containing these various lesions.


A major reason for failure of a biomarker to act as true disease surrogate endpoint is the lack of a direct association between the biomarker and the underlying mechanism of the disease. Fibrillar beta-amyloid (AB) plaques are the main pathological hallmark of Alzheimer's disease (AD), although its role as a causative mechanism in AD is controversial and continues to be disputed. The clear association of soluble AB oligomers to neurotoxicity in animal studies indicates that measuring soluble AB oligomers may hold more promise as a true surrogate biomarker for the clinical symptoms of AD. The lack of specific ligands that can differentiate soluble AB oligomers from fibrillar AB forms both in vitro and in vivo has hindered studies aimed at testing the correlation between soluble AB oligomers and plaques and/or the relative clinical value of measuring different forms of AB species. Our group discovered a fluorophore, 68B-3, that can selectively bind soluble AB oligomers and has no measurable binding to AB fibrils or monomers. The primary aims of this study are to characterize and validate our initial discovery on 68B-3, and evaluate the value of measuring different forms of AB species in clinical samples and in a transgenic model of AD. We will determine the correlation between levels and location of soluble AB oligomers to disease progression. While the affinity of 68B-3 to synthetic soluble AB oligomers is low, it may serve as a useful tool in providing novel information in biological samples. Furthermore, it may be optimized by chemistry to yield derivatives with better properties. Another aim of this study is to understand the mechanism of binding of 68B-3 to soluble AB oligomers. This is an important first step to improving its binding affinity. It is our intent to make 68B-3 available to researchers once we have validated its uses and once we have demonstrated the robustness of our preliminary data. Selective small molecule probes such as 68B-3 will not only serve as potential tracers for non-invasive imaging, but may be used in ex vivo postmortem studies to compare the regional distribution and temporal profile of soluble AB oligomers and plaques. Such information would enable the correlation of neuronal loss with the spatial location of soluble AB oligomers and thus may accelerate the establishment of a true surrogate for the diagnosis of early AD. PUBLIC HEALTH RELEVANCE: The proposed research is directed toward the validation of our initial discovery using 68B-3, a novel small molecule fluorophore that selectively targets soluble beta-amyloid (AB) oligomers and not fibrils or monomers, and evaluation of the value of measuring different forms of AB species. Completion of this study will be important to investigate the regional distribution and temporal kinetics of soluble AB oligomers compared to fibrillar AB in postmortem tissues and animal models of Alzheimer's disease. This would also provide powerful insight into understanding the role of soluble AB oligomers in the pathogenesis of Alzheimer's disease and the clinical value of measuring levels of soluble AB oligomers.


This project involves the measurement of prostaglandins and phospholipids in adipose tissue and liver, with a special regard to the interrelationship of these compounds in lipolysis and antilipolysis. Studies are progress, studying the relationship of the levels of the prostaglandin E series (measured by thin-layer chromatography and radioimmunoassay) and the different classes of phospholipids (measured by thin-layer chromatography) in situations within the cells of increased and decreased cyclic AMP levels. Levels of cyclic AMP are increased by exposing the isolated cells to epinephrine or norepinephrine, and cyclic AMP levels are lowered by the use of the hormone, insulin. It is hoped by these maneuvers to better understand the role and interrelationship of these compounds to this metabolic process. In addition, it is hoped that a better understanding of the physiology of these compounds will also be forthcoming.


Summary of Work: Phenolphthalein, a constituent in many laxatives, showed clear evidence of carcinogenic activity in 2-year feeding studies in rodents recently completed by the National Toxicology Program (NTP). About 3% of the older U.S. population uses phenolphthalein-containing laxatives on a daily basis. We will investigate the relation of phenolphthalein-containing laxative use with risk of ovarian cancer and adenomatous colorectal polyps in humans. We have established collaborative agreements with the principal investigators for two ongoing case-control studies of risk factors for ovarian cancer. At our instigation, these investigators have inserted into their questionnaire a 2-page addition that ascertains use of specific phenolphthalein-containing laxatives. Each study will yield about 300 cases and 300 controls, and the data will be ready for analysis in 1999-2000. Data collection for two case-control studies of risk factors for adenomatous colorectal polyps have been completed. Data on laxative use were available for both studies. We have examined laxative use in relation to risk of polyps in these data. The association of polyps with use of all laxatives and phenolphthalein- containing laxatives was specifically explored after taking into account dietary and other risk factors for colorectal adenomas. Results from the two studies were inconsistent with each other but overall offered little support for an association between phenolphthalein- containing laxative use and risk of polyps. - Phenolphthalein, Laxatives, Ovarian cancer, Adenomatous colorectal polyps, Epidemiology, Case-control study - Human Subjects: Interview, Questionaires, or Surveys Only


We propose to develop a novel DNA chip specifically for high-throughput analysis of promoter hypermethylation in primary tumors. It is now clear that abnormal DNA methylation frequently occurs in multiple promoter CpG islands in cancer cells. Increased density of CpG methylation is known to alter local chromatin structure within a promoter , resulting in transcriptional silencing of the corresponding gene. At present, bisulfite DNA sequencing is considered to be the" gold standard" for marring methylated CpG sites within a gene promoter. This method, as well as other related techniques, has provided important insights into the functional relationship of promoter methylation and transcriptional silencing on a "gene-by-gene" basis. Such approaches, however, have given limited pictures of complex epigenetic alterations in cancer and are restricted in throughput for routine clinical applications. In this proposal, we build on the approach of microarray techniques by developing a novel method, called Methylation-Specific Oligonucleotide (MSO) microarray, for high-throughput methylation analysis. Our goal aims at generating MSO chips in which thousands of short oligonucleotides are tethered to glass slide surfaces. These oligonucleotides specifically designed and tested are capable of differentiating methylated and unmethylated CpG sites at the specific locations of a promoter. Test (tumor) and reference samples are bisulfite-treated, PCR-modified products that may contain different pools of DNA fragments due to the hypermethylation status of the tumor genome. These DNA samples are co-hybridized to an MSO chip and quantitative differences in DNA methylation are determined by two-color fluorescence analysis. Distinct from the existing microarray technologies, the MSO approach allows simultaneous analysis of the anatomy of multiple promoter CpG islands in reference to the role of DNA methylation on gene silencing. In addition, this MSO chip can be directly applied to determine molecular signatures of different tumor subtypes. In the pilot R21 phase, we will apply a prototype MSO chip to analyze a small panel of tumor samples and determine its reliability in detecting DNA methylation. In the R33 phase, based on the experience we gain in the pilot study, we will design a full-fledged MSO chip for a comprehensive analysis of promoter methylation in multiple tumor types. An advanced computation system will be developed to handle a large set of methylation data and patients' clinicopathological information and to support heuristic queries. The MSO assay will furnish digital profiles of promoter hypermethylation for individual tumors and offers an alternative to cDNA microarray approaches for molecular classification of different cancer subtypes.


DESCRIPTION (provided by candidate): Through this award, a researcher with a background in physics and acoustics will receive additional training and conduct research in diagnostic ultrasound. Structured training in clinical research practice will be provided through a sequence of courses, especially related to training in biostatistics and human disease. Training in current quantitative research methods will be provided through the research portion of this proposal, which examines the accuracy of nonlinear propagation computer models when applied to tissue mimicking media. Recent progress in numerical modeling of nonlinear acoustic fields has created efficient algorithms to simulate pulsed fields from array transducers. However, few results of acoustic field parameters have been reported for measurements conducted in media with tissue-like properties. A newly developed, stable tissue-mimicking liquid (TM) will be used to test the accuracy of these efficient algorithms that include nonlinear propagation effects. The hypothesis of this proposal is that numerical models that incorporate nonlinear propagation effects will provide accurate representation of the acoustic fields of diagnostic ultrasound devices. The results will be useful to improve simulations of ultrasonic fields that are used to develop and optimize ultrasonic imaging applications and in the determination of acoustic output indices. Measurements of the acoustic field amplitude propagating in TM liquid will be compared to results of numerical models to address three specific aims: 1) Comparison of numerical models and measurements in TM liquid with attenuation coefficient of 0.30 dB/cm/MHz to assess accuracy of current derating scheme. 2) Investigation of proposed methods of "linearizing" acoustic output methodology by conducting measurements at low output levels and extrapolating to high output levels to avoid nonlinear effects. 3) Assessment of accuracy of numerical methods for models related to imaging: TM liquids with greater attenuation coefficient, including 0.5 and 0.7 dB/cm/MHz, obstetrical, and other tissue models.


The specific aims of this project are: 1. To determine the feasibility of assaying the marrow and peripheral blood progenitor cell (PBPC) fractions of breast cancer patients enrolled on S9623 for the presence of malignant cells; 2. To determine the feasibility of quantitating the breast cancer cell content of the marrow and PBPC fractions using immunocytochemistry with anti-breast cancer monoclonal antibodies and reverse transcriptase polymerase chain reaction (RT-PCR) for cytokeratin-19; and, 3. To determine the true incidence of tumor cell contamination of hematopoietic fractions of Stage II/III breast cancer patients with 4-9 axillary lymph nodes. The bulk of our preliminary breast cancer detection data was generated with fresh marrow and PBPC specimens obtained from University of Colorado breast cancer patients. Over the next two years, the current study will determine the feasibility of conducting similar detection studies with patient specimens shipped overnight from the many cooperative group institutions. If this feasibility study is successful, we will be able to determine the incidence of breast cancer contamination in the hematopoietic cell fractions of patients enrolled in the first two years of the study. We will then seek funding for two additional years to continue to assay the hematopoietic cell specimens of patients enrolled on S9623 for the remainder of the study, which is projected to accrue patients for a total of four years. Clinical outcomes, including disease-free survival rates as well as sites and patterns of relapse, will be correlated with the breast cancer detection assay results. Alternatively, if we are unable to successfully assay the specimens shipped from outside institutions, such additional funding will not be sought. The major aspects of the tumor detection assays which the current feasibility pilot will address include: assessment of the quality of the assays performed on specimens shipped overnight; assessment of outside institution compliance; assessment of the true incidence of breast cancer in the hematopoietic cell fractions of Stage II/III 4-9 nod-positive breast cancer patients; and, will compare the sensitivity of the immunohistochemical and molecular assays for breast cancer.


BACKGROUND: Although about 7 million people in the US population use marijuana at least weekly, there is a paucity of scientific data on persistent neurocognitive effects of marijuana use. OBJECTIVE: To determine if neurocognitive deficits persist in 28-day abstinent heavy marijuana users and if these deficits are dose-related to the number of marijuana joints smoked per week. METHODS: A battery of neurocognitive tests was given to 28-day abstinent heavy marijuana abusers. RESULTS: As joints smoked per week increased, performance decreased on tests measuring memory, executive functioning, psychomotor speed, and manual dexterity. When dividing the group into light, middle, and heavy user groups, the heavy group performed significantly below the light group on 5 of 35 measures and the size of the effect ranged from 3.00 to 4.20 SD units. Duration of use had little effect on neurocognitive performance. CONCLUSIONS: Very heavy use of marijuana is associated with persistent decrements in neurocognitive performance even after 28 days of abstinence. It is unclear if these decrements will resolve with continued abstinence or become progressively worse with continued heavy marijuana use.


Muscle protein synthesis, muscle carbohydrate metabolism, and the effects of insulin and growth hormone in myotonic dystrophy are under study using insulin clamps, forearm perfusion, and stable isotopes.