PI: Orians, Colin
Title: Vascular Architecture And the Response of Forest Tree Species To Environmental Heterogenity
The goal of my research program is to understand how vascular architecture affects the movement of resources in different plants species and to explore the consequences of environment heterogenity to growth of trees in Northeastern forests. Two methods are employed to study the movement of chemicals within plants and the responses of plants to environmental heterogeneity: split root techniques and the use of isotopes. For our purposes, we will split roots of seedlings into two pots, the main root into one pot and a lateral root from one IPU into the other. We can then apply nutrients or isotopes to the lateral root. Nutrient additions can be used to track the ecological effects of vascular control of nutrient transport, and 15N can be used to directly map the flow of nutrients.

PI: Prevelakis, George
Title: Hellenic & Southeastern European Studies
In our globalized world, Diasporas exert a powerful influence on regional issues. It is therefore very mportant to act inside a crossroads of Balkan Diasporas (like the Boston area) to promote international ooperation. In addition, the discussion of problems of Greece in Diaspora (and especially in the 'avorable intellectual environment of the Boston area) offers a way out of the rigidities that characterize similar debates inside Greece. The Constantine Karamanlis Chair considers itself as a hinge between the various communities of Balkan iescent in the wider Boston area (among which the Greek-American community) and members of the Academia (students and professors) interested in Southeastern European issues. The interaction of the two spheres can create innovative ideas and contribute in deconstructing negative stereotypes.

PI: Pokras, Mark
Title: Seabird Ecological Assessment Network (SEANET)
Six primary SEANET objectives are as follows: 1) Continue to coordinate a web of experts on marine birds, environments, and issues. 2) Expand regular beached bird surveys conducted by an Atlantic coastal network of trained volunteers and students to collect and contribute data on seabird mortality. 3) Strengthen a bycatch recovery effort, in collaboration with the US National Marine Fisheries Service, to develop a descriptive pathology for such birds, and for baseline data on levels of disease, comtaminants, and biotoxins in a wide range of species. 4) Produce an Atlantic guide to beached birds, and important resource for everyone involved in data gathering in the field, in collaboration with Bird Studies Canada and the Coastal Observation and Seabird Survey Team (COASST). 5) Continue construction of web-based, searchable databases and interactive GIS maps for the assessment of risk factors and mortality patterns of seabird populations, in collaboration with the National Biological Information Infrastructure at the United States Geological Survey (USGS) and the US EPA. This infrastructure will house a web-based reporting system, allowing volunteers to enter data directly. 6) Use spatial statistical analyses, to reveal 'hot-spots' of concern, e.g., where high incidence of seabird mortality may result from high contaminant load.

PI: Reed, Michael
Title: Evaluating Hawaiian Moorhen Survey Methods and Reproductive Success
Hawaiian Moorhens (Gallinula chloropus sanvicensis) are an endangered waterbird found on O'ahu, Kaua'i, and fonnerly on Maui, Moloka'i, and the Big Island. It is the most secretive of the endangered waterbirds, and population size and trends are unknown. Because of its secretive nature, its nesting biology and movement patterns are poorly understood, particularly in natural wetlands and in those managed specifically for endangered waterbirds. Along with limited habitat availability, it is thought that poor nesting success because of predation might be an important factor affecting Moorhen persistence. Consequently, we are interested in monitoring nesting success, improving nesting success, and developing an effective method to census Hawaiian Moorhen. We are seeking funding for the first year of study on two important questions for the Hawaiian Moorhen: (1) we propose to evaluate a variety of census methods, particularly those being used currently on rails, another secretive marsh bird, to determine what might be most effective for Moorhen; (2) we propose to detennine if Hawaiian Moorhen will use nest boxes for breeding, and how nesting success compares to typical nesting in the same area. In England, a different subspecies of Moorhen is known to breed regularly in nest boxes. Although nest boxes are used extensively in duck conservation, it has never been investigated as a tool for Moorhen conservation. Survey methods and nest boxes will be tested initiallyon Oahu on the National Wildlife Refuges. After initial trials, census methods will be tried other places on Oahu and on Maui. If a more effective survey method is found, our goal would be to use it method statewide to supplement the biannual waterbird surveys. If nest boxes are successful, we would share methods and data with any group or individual interested in Hawaiian Moorhen conservation as long as they have the appropriate federal and state authority to do so. Methods would be applicable statewide, and could fonn the basis for survey and management of other endangered Moorhen species.

PI: Roffler-Tarlov, Suzanne
Title: Albinism: Defects in Tyrosinase, Amines or Melanin
This proposal requests funding for examination of the hypotheses that 1) tyrosinase activity in the embryonic eye results in the formation of developmental signals, that 2) these signals, perhaps amines, direct the generation of ganglion cells in the embryonic eye and that 3) the signals made through the activity of tyrosinase are transient and are made before tyrosinase switches to the synthesis of melanin in the pigment epithelium. The effects of amines on spatiotemporal features of neurogenesis would in turn lead to designation of the crossed and uncrossed projection of retinal ganglion cells. Our studies will focus on two lines of mice that are genetically identical except for a mutation at the tyrosinase locus. One line is pigmented (C57B16 Tyr+), the other, the albino (C57BI6Tyrc2j) carries a point mutation in the gene that codes for tyrosinase. The albino is known to have aberrant ganglion cell projections with the ipsilateral pathway reduced by half. Lack of functional tyrosinase in the albino is also correlated with changes in the timing of generation of retinal ganglion cells. Using histological and biochemical techniques, we will determine whether catechol or other amines are formed transiently in the developing eye, as is the case in peripheral tissues. We will test whether these substances are formed as a consequence of tyrosinase activity, again, as occurs in peripheral tissues. We will examine developing eye to see if there is a switch from the formation of developmental signals by tyrosinase to the formation of melanin, as also may be the case in peripheral tissues. We will correlate these events with the birth and differentiation of retinal ganglion cells. If we do find candidates for developmental signals for generation of retinal ganglion cells, we will test their effectiveness as signals in retina in vitro, and in vivo using tissues from albino mice including the OA1 knockout in which a G protein coupled receptor within melanosomes is mutated. Ultimately, if such signals deriving from tyrosinase-driven pathways unrelated to melanin production, are found, this research will open doors to therapeutic intervention in individuals carrying the OA1 gene.

PI: Rosenblatt, Michael
Title: Nature of PTH/PTHRP - Receptor Bimolecular Interactions
This research proposal is focused on elucidating the interactions of parathyroid hormone (PTH) with its receptor (Rc, the hPTH1-Rc). Formation of the complex between PTH and the hPTH1-Rc leads to a sequence of events, namely hormone binding, Rc activation, and signal transduction, which culminate in expression of hormonal bioactivity. The impetus for this research program comes from a desire to understand: (1) the fundamental nature of molecular recognition between a peptide hormone and its G protein-coupled Rc; (2) the mechanism of action of the hormone (PTH) responsible for minute-to-minute regulation of calcium levels in blood; and (3) the differences in Rc states (conformations) which translate into hormone agonism, antagonism, inverse agonism, etc. The introduction of PTH as a major new agent for treatment of osteoporosis also focuses attention on the mechanism of anabolic action of this hormone. By gaining insight into the nature of the hormone-Rc complex, the discovery of small molecule PTH-mimetics may be facilitated by structure-guided design in the future. During the previous grant award period, by integrating photoaffinity scanning, molecular biology, pharmacology, and structural biology (conformational studies of hormone and Rc, and molecular modeling), we succeeded in generating an advanced experimentally derived model of the PTH-hPTH1-Rc bimolecular complex that provides structural detail and reveals some of the dynamics of hormone-Rc interaction. We are now positioned to take the next major step in mapping the interface of PTH and its Rc, and to extend our studies to identify the shifts in Rc conformation associated with activation. Specifically, we plan to: (1) improve the resolution of the map of the hormone--Rc interface; (2) study the interaction of PTH ligands covalently tethered to the Rc; (3) investigate the ability of dual-reactive analogs to simultaneously make contact with two sites in Rc; (4) perform "reverse" crosslinking from Rc to PTH; (5) prepare Rc and constitutively active Rc mutants on a large scale for structural studies of antagonist and inverse agonist interaction; (6) use disulfide bridge formation as a probe of Rc states; and (7) integrate all the above efforts in a molecular modeling initiative.

PI: Roubenoff, Ronenn
Title: TNF Blockade in Rheumatoid Cachexia
A major metabolic consequence of rheumatoid arthritis (RA) is an elevated resting metabolic rate, lower physical activity, and accelerated muscle protein catabolism, which contribute to a loss of body cell mass (BCM). We have previously termed this loss of BCM, rheumatoid cachexia (RC). RC leads to muscle wasting, weakness, and decreased functional capacity. Consequently, it is believed to exacerbate the disability caused by direct joint damage in RA, and may also contribute to the accelerated morbidity and mortality of RA. We have previously shown that RC is associated with increased production of tumor necrosis factor-a (TNF) by peripheral blood mononuclear cells (PBMC) and skeletal muscle in RA. However, until now, it has not been possible to prove a causal link between TNF and RC. In addition, recent evidence suggests that TNF is an important mediator of insulin resistance (IR) in humans, an effect associated with increased fat mass, and comorbid conditions such as diabetes, atherosclerosis, and hypertension. The relationship between excess TNF production and IR has not been examined in patients with RA, but is important insofar as TNF mediated IR may contribute to cachexia, excess fat deposition, and accelerated morbidity and mortality in RA. With the advent of anti-TNF therapy such as etanercept (Enbrel), it is now possible to test whether TNF is in fact the major cause of RC in RA by examining whether TNF inhibition can block the metabolic abnormalities of RC, including attenuated insulin action. In a recently funded AF Clinical Science Grant, we hypothesized that TNF inhibition would be significantly more effective in (a) improving protein kinetics; (b) reducing insulin resistance; and (c) preservingBCM; compared with non TNF inhibitory treatment. In this proposal, which will serve as an adjunct to the original AF-funded Clinical Science Grant: TNF Blockade in Rheumatoid Cachexia, we hypothesize that TNF inhibition will be significantly more effective in (a) reducing serine phosphorylation of skeletal muscle insulin receptor substrate-l (IRS-l); and (b) reducing skeletal muscle TNF expression. We propose to achieve the following novel aims, in addition to those previously proposed and funded in the AF Clinical Science Grant: 1. To assess the effect of 12 weeks of TNF blockade on serine phosphorylation of skeletal muscle IRS-l in patients with RA beginning etanercept therapy; 2. To evaluate the effect of 12 weeks of TNF blockade on skeletal muscle TNF expression (mRNA and protein) in patients with RA beginning etanercept therapy; and 3. As proposed in the previously funded parent AF Clinical Science Grant: To compare the effect of etanercept to two control groups: (a) patients with RA on stable treatment with slow-acting anti-rheumatic drugs (SAARD's) such as gold and methotrexate; (b ) patients with RA beginning treatment with leflunomide (Arava), an agent which suppresses T -cell function without direct TNF inhibition. Both groups will be studied at weeks O and 12 to facilitate direct comparison with the etanercept group.

PI: Roy, Ananda
Title: Potential Role of TFII-I in Immunodeficiency
TFII-II is an important multi-functional transcription factor that links events to transcription in several genes. TFII-I is constitutively associated with Bruton's tyrosine kinase (Btk), a non-receptor tyrosine kinase that is essential for normal B cell function, as its mutation causes X-linked agammaglobulinemia (XLA) in humans and X-linked immune deficiency (xid) in mice. We propose that TFII-I is an important and novel component in linking Btk-mediated signaling to transcription in B cells. Furthermore, the TFII-I gene gets deleted in William's syndrome (WS) which is a neuro-developmental disorder with multi-system manifestations, including supravalvar aortic stenosis, hypercalcemia in infancy, mental retardation and cognitive defects. Thus, TFII-I appears to be involved in two genetic disorders: William's Syndrome and X-linked agammaglobulinemia (XLA). Knowledge gained from these studies may help us better understand a critical Btk dependent pathway that links B cell receptor mediated signal transduction to B cell specific transcription. These studies may also ultimately help identify potential target gene(s) that are affected by mutations in Btk. Importantly, these studies may establish possible connections between the neuro-developmental disorders (as in WS) and immuno-developmental disorders (as in XLA). Toward a better understanding of TFII-I function in Btk mediated immune response, we will first map the region(s) in TFII-I important for its physical and functional interactions with BTK. We will determine by deletion and point mutation the region(s) in TFII-I that is important for its interaction with Btk, followed by mapping the sites in TFII-I that are tyrosine phosphorylated by Btk in vitro and in vivo by a combination of site directed mutagenesis, phosphopeptide, finger printing, and mass spectrometric analysis. We will also analyze these mutants in functional transient transfection assays. To determine the functions of TFII-I and its biochemical interactions with Btk in B cells, we will employ in vivo transcriptional analysis. To determine the functions of TFII-I and its biochemical interactions with Btk in B cells, we will employ in vivo transcriptional analysis followed by the interaction studies by co- immunoprecipitation and ectopic expression of mutant forms of TFII-I in B cells. We will also stably express wild type and mutant forms of TFII-I, and Btk in B cell lines, and genetically delete TFII-I from chicken B cells. Finally, to ascertain the localization of TFII-I in the absence and in the presence of non-activated versus activated Btk, first, we will co-express various mutants of TFII-I with Btk in COS cells. Subsequently, we will employ freshly isolated primary splenic B cells derived from wild type, xid and Btk-/- mice and study the localization and tyrosine phosphorylation of TFII-I in the absence and in presence of B cell receptor signaling.

PI: Roy, Ananda
Title: Molecular Mechanisms of Acute Promyelocytic Leukemia
The promyelocytic leukemia (PML) gene codes for a tumor suppressor protein that is associated with distinct subnuclear macromolecular structures called the PML bodies. The PML gene is frequently involved in the t (15;17) chromosomal translocation of acute promyelocytic leukemia (APL). The translocation results in a fusion gene product, PML-RARalpha, in which the PML gene fuses to the retinoic acid receptor alpha (RARalpha) gene. PML-RARa behaves as a potent transcriptional repressor for apoptotic genes and disrupts the architecture of PML bodies, a phenotype reversed by treatment with all trans retinoic acid (ATRA). Besides its role in APL, PML bodies have also been linked to viral infection. A variety of viruses targets the nuclear bodies and often causes their disruption. Moreover, upon interferon treatment of normal cells, PML is induced and the number of PML bodies increases dramatically, suggesting that PML may function as a mediator of interferon function and behaves as an immune surveillance factor. Although the activities of several transcription factors are modulated by virtue of physical association with PML bodies, the molecular mechanisms of PML or PML-RARalpha-mediated gene regulation remain elusive. Given the biological importance of PML and PML-RARalpha proteins, it is critical to ascertain their functional role and mechanisms of action. The TFII-I family of multifunctional transcription factors is activated in response to growth factor and antigenic signals to regulate growth-controlling genes, thus linking signal transduction events to transcription. We show a novel association of TFII-I family of factors with PML bodies. We further show a previously unknown function for PML-RARalpha it hyper-activates c-fos promoter in response to growth factor signaling. Based on these and other results, we hypothesize that the growth-regulatory and antigenic signals are processed through PML bodies in normal and APL cells to activate genes that control cellular growth or death via TFII-I family proteins. We propose to elucidate this novel pathway that will lead to a better understanding of PML and PML-RARalpha function.

PI: Rozanski, Elizabeth
Title: The Effect of Obesity of Airway Function in Retrievers
The objective of this project is to evaluate the effect of obesity, as measured by the Purina Body Condition Score (BCS), on pulmonary function. Our hypotheses is that obesity will impair pulmonary function and thus result in increased airway resistance (Raw), specific airway resistance (sRaw), and alveolar-arterial gradient (A-a gradient) and in lower functional residual capacity (FRC), diffusion capacity (DLCO) and oxygen saturation (SPO2).

PI: Schaefer, Ernst (Ann McDermott)
Title: Genetics, Lifestyle and Obesity
Better understanding of how societal, behavioral, and environmental conditions interact with diverse genetic backgrounds will allow at-risk populations with an increased susceptibility to obesity to be targeted for preventive measures. Also, the prescription of customized obesity treatment assignments will increase the likelihood of successful interventions and the restoration of health. In addition, the reduction of cardiovascular risk factors will improve morbidity and mortality in this population, and result in a reduction in the cost of health care. This study aims to examine and compare lifestyle, cardiovascular risk, health status, and genetic background in two populations for a total of 2000 individuals: half with BMI >40 kg/m2 and half <30 kg/m2. Diet, body composition, lifestyle, cardiovascular risk factors, and the prevalence of polymorphisms in genes known to affect body habitus, energy intake or expenditure will be assessed and compared. High throughput genotyping will allow the examination of DNA and the determination of polymorphism prevalence in this racially and ethnically diverse cohort. Obese subjects will then undergo a one year period of treatment at a obesity clinic with a protocol selected by the patient and their attending physician, response to treatment monitored, and variability in response examined for the influence of genetic factors. This research project attempts a collaborative, multidisciplinary approach linking biomedical, social, and behavioral science in order to clarify the potential role of genetic-environmental interactions in obesity treatment.

PI: Shuster, Louis
Title: The Role of NMDA Receptors in Stereotypic Behavior Disorders
Aim 1: Elucidate the role of NMDA receptors in stereotypic behavior disorders. Aim 2: Define the contribution of genetic determinants of several different neurotransmitters (NMDA, serotonin, dopamine and opioid) in models of compulsive and self-injurious behaviors. Aim 3: Develop effective treatments for some of these disorders.

PI: Skelly, Patrick
Title: Gene Silencing in Schistosomes Using RNAi
Schistosomes are extracellular blood worms that infect over 200 million people globally and cause several thousand deaths annually. Large-scale genome sequencing projects for each of the three major human schistosome species: Schistosoma mansoni, S. haematobium and S. japonicum, are currently underway. Despite the wealth of new data to be generated by all of these undertakings, there is still no routine technique available that allows us to exploit the data through manipulation of the schistosome genome. Because of this, our understanding of the molecular and cellular biology of schistosomes has been severely hampered and lags behind that of most other important human pathogens. In this application, we propose to utilize a relatively new technology in gene manipulation called RNA-mediated interference (RNAi) to examine gene function in schistosomes. In preliminary experiments, we have achieved considerable inhibition of select genes involved in nutrient acquisition by the parasites. We plan to build on this success by first optimizing the protocol for gene suppression using RNAi in different schistosome life cycle stages by varying the amount and nature of the dsRNA employed, the mode of delivery and the duration of exposure. Such a systematic assessment of the relative importance of the factors that impinge on the phenomenon could have wider applicability for researchers using RNAi in other biological systems. Next we will utilize the optimized protocol to test specific hypotheses concerning an important area of schistosome biology: The involvement of proteases in hemoglobin degradation and nutrient acquisition. The work proposed here is designed to provide a simple, powerful and widely applicable protocol for the schistosome research community to facilitate functional schistosome genomics. In addition, our application of the technology is designed to provide significant new information about schistosome metabolism and biology.

PI: Soto, Ana
Title: Prostate Cell Proliferation in Cancer and Aging
Prostate cancer is the most common cancer in American men. These cancer cells initially respond to androgen(A)-withdrawal, undergoing apoptosis and decreased cell proliferation. Later on, they overcome this inhibition and relapse. However, in addition to A-dependent proliferation and a total lack of A response, there is a prostate cancer phenotype whereby proliferation is inhibited by androgens (androgen-induced shutoff). Our research objective is to understand the molecular mechanisms underlying this phenomenon. The AS3 gene was identified as a candidate because its pattern of expression was consistent with that of a mediator of the proliferative shutoff. Functional evidence obtained using expression vectors containing AS3 demonstrated that AS3 triggers a proliferative shutoff. Conversely, antisense AS3 blocks the expression of the A-induced shutoff. The AS3 sequence seems to be a transcription factor with trans-activating protein recognition, and DNA binding domains; it also has a protein kinase motif. Alternatively, AS3 may behave by activating other proteins through phosphorylation, and/or by binding to them. The Specific Aims of this application are designed to elucidate the mechanisms by which AS3 inhibits cell proliferation. Aim #1: Exploring the hypothesis that AS3 is a transcription factor (identification of the downstream mediators of the proliferative shutoff). Aim #2: Investigating the hypothesis that AS3 activates effector proteins by phosphorylating them. Aim #3: Mapping the AS3 pathway. Testing the role of the putative downstream mediators identified in Aim #1 in the shutoff effect. Aim #4: Testing the hypothesis that AS3 expression arrests tumor growth by inoculation of tetracycline-regulated AS3 transfectants into nude mice. We expect that this research will lead to the development of markers to identify this phenotype, and to the development of specific therapeutic strategies.

PI: Souvaine, Diane
Title: Problem Solving and Critical Thinking with Discrete Mathematics
Students in Massachusetts' high-need districts are not as successful in mathematics as students in the general population. Research shows that the key factor in improving student achievement is professional development for teachers. What teachers need in addition to content knowledge is a different perspective on what mathematics is how it is learned, and a different understanding of what their students can accomplish. ,
This project addresses the needs stated by the partner school districts for high quality professional development in mathematics. Tufts University and Framingham State College in conjunction with its school partners -the Arlington, Boston, Dedham, Everett, Framingham, Marlborough, Natick, Revere and Somerville Public Schools -and the nationally recognized Leadership Program in Discrete Mathematics (LPDM) will strengthen teachers' skills in both content and instructional strategies, and help develop mathematics leadership capacity in the individual schools and districts. Through this effort, these partners will work toward achieving the following objectives: 1. to strengthen teachers' knowledge base in discrete mathematics. 2. to improve teachers' attitudes towards mathematics. 3. to model sound pedagogy and strengthen teachers' effectiveness for teaching math to diverse populations. 4. to develop future teacher leaders in mathematics, who will mentor other teachers and serve leadership roles in their schools, districts, and beyond. We will assess the impact of the program and its components in a variety of ways, including: data collected from pre- and post-institute tests; participant journals; and pre- and post-institute surveys to measure the impact on the participants; post-institute participant portfolios and presentations; site visits and classroom observations.

PI: Stadecker, Miguel
Title: Immunoregulation in Schistosomiasis
Schistosomiasis continues to be a major parasitic disease suffered by millions of people throughout the world. The underlying potentially lethal immunopathology in schistosomiasis is a granulomatous inflammation around parasite eggs, which is mediated by the host s T cells sensitized to parasite egg antigens. The proposed studies are based on the concept that such pathogenic T cell responses are amenable to down-regulation by immunotherapy, thereby resulting in the prevention of amelioration of disease. This approach has been referred to as anti-pathology vaccine. Specific T cell hybridomas will be used as probes to identify and isolate the major sensitizing egg antigens. Antigens will be cloned and their dominant T cell epitope peptide(s) will be synthesized. The type of elicited T cell response will be characterized and the genetic restriction of the T cell response will be determined. The major schistosomal egg antigen Sm-p40 will be the subject of close analysis by investigating the interaction of its dominant epitope 13mer peptide with the MHC class II molecule I-Ak, and by assessing its intrinsic pathogenicity. A broad range of experiments will test selected strategies involving specific homologous or altered peptides for the purpose of down-regulating the pathogenic T cell response. The long-term goal of this project is the achievement of effective and lasting specific T cell tolerance by way of a suitable biological vector capable of delivering the peptides into the host suffering from, or susceptible to, disease.

PI: Tang, Guangwen
Title: Retinol Equivalency of Plant Carotenoids in Children
This project is to determine the vitamin A value (equivalence) of dietary provitamin A carotenes from spinach, Golden Rice, and pure Beta-carotene (B-C) in oil. These experiments will be conducted in children (ages 6-8) with/without adequate vitamin A nutrition. As plant provitamin A carotenoids are a major and safe vitamin A source for a vast population in the world, it is essential to determine the efficiency of provitamin A carotenoid (mainly B-C) conversion to vitamin A. By introducing B-C into rice endosperm, Golden Rice may directly benefit consumers by providing vitamin A nutrition. Our investigation uses hydroponically grown, decadeuterium labeled spinach and Golden Rice, synthetic Beta-C-d10 and a vitamin A isotope reference, decadeuterated retinyl acetate (RAc-d10), to evaluate the bioavailability and the bioconversion of plant provitamin A carotenes to retinol as compared with B-C in oil capsules in vivo. Our objectives will be to test the following hypotheses and to make the following determinations: (1) The absorption and bio-conversion of provitamin A carotenes taken by children are different between spinach, Golden Rice, and B-C in oil capsules. (2) The absorption of provitamin A carotenes and their bioconversion to vitamin A are different in children with or without adequate vitamin A nutrition. (3) To define the vitamin A equivalence(s) of dietary spinach, Golden Rice, and a B-C in oil dose by using an isotope reference method in children with or without adequate vitamin A nutrition and to compare those values with values derived from model based compartmental analysis. (4) To determine the number and time of blood samples needed for future studies in various field settings on the retinol equivalence of a large number of plant sources. Seventy-two children each will take two meals, lunch and supper, containing equal amounts of B-C in labeled spinach (along with white rice), or Golden Rice (along with light colored vegetables), or B-C oil capsules (along with white rice and light colored vegetables), every day for 7 days. Before the two meals, the volunteers will take a breakfast with a RAc-d10 dose as a reference for 7 days. The enrichment of labeled B-C and labeled retinol in human circulation will be determined using advanced liquid chromatography / mass spectrometry and gas chromatography / mass spectrometry. Through the applications of these novel technologies, we will be able to determine the relative biological activities of endogenous carotenoids; that is, the vitamin A value of spinach, Golden Rice, and B-C in oil capsules for children with/without vitamin A malnutrition. This study will be of importance in planning vitamin A deficiency prevention strategies and also will provide useful information regarding the potential efficacy of a bioengineered crop to provide vitamin A nutrition.

PI: Theoharides, Theoharis
Title: Stress Induces Skin Mast Cell Activation and Vasodilation
Mast cells are located perivascularly close to nerve processes and can secrete many vasoactive and proinflammatory molecules. In addition to allergic triggers, mast cells can also be activated by direct nerve stimulation, by neuropeptides, and by acute immobilization stress through the local release of corticotropin releasing hormone (CRH) or urocortin (Ucn), which is more potent than CRH. Intradermal injections of CRH or Ucn induced rat skin mast cell activation and increased vascular permeability, both of which were inhibited by pretreatment with neutralizing antiserum to CRH or the CRH-receptor antagonist antalarmin. CRH or acute stress-induced skin vascular permeability measured with Evans blue extravasation was absent in W/W (v) mast cell deficient mice, but was present in their wt controls indicating it is mast cell dependent; this finding was supported by the fact that vascular permeability was also blocked by the "mast cell stabilizer" disodium cromoglycate (cromolyn). Similar results were obtained in rats and mice in response to acute immobilization stress. The in vivo, but not in situ, CRH action was absent in rodents treated with capsaicin to deplete sensory nerve fibers of their substance P (SP) content and was also inhibited by the neurotensin (NT) receptor antagonist SR48692. We are hypothesizing that acute stress releases CRH and/or Ucn in the skin leading, directly or through neuropeptides such as SP and NT, to mast cell activation and increased vascular permeability; leukocyte infiltration may then contribute to local inflammation and possibly to delayed-hypersensitivity (DTH) reactions. We propose to use normal and genetically deficient mice to investigate: (1) the effect of acute stress on (a) skin mast cell activation determined by image analysis, as well as residual skin histamine and mouse mast cell protease (MMCP)-6 content in CRH knock-out mice, and (b) vascular permeability quantitated by 99Technicium-gluceptate (99Tc) extravasation in CRH knock-out and W/W (v) mice and their +/+ controls; (2) any change in skin dorsal root ganglia (DRG) or spinal cord CRH, Ucn or CRH receptor expression after stress, using immunohistochemistry, Western and Northern analysis or RT-PCR; (3) the involvement of SP on stress-induced mast cell activation and vascular permeability in SP and NK-1 receptor knock-out mice, as well as the possible sequence of action of CRH or SP using combination of knock-out animals and CRH or SP-receptor antagonists; (4) the effect of CRH and Ucn on secretion of histamine, IL-6 and MMCP-6 or tryptase, respectively, from mouse purified skin and human umbilical cord-derived mast cells stimulated immunologically or by SP. These studies will help us understand how acute stress triggers skin mast cell activation, increased vascular permeability and possibly DTH. They will also help investigate the pathophysiology of skin syndromes exacerbated by stress, such as atopic dermatitis or psoriasis, especially since CRH and CRH receptors have been identified in human skin.

PI: Thorley-Lawson, David
Title: Host Immunity to EBV Infection in Vitro and in Vivo
The long term objective of this study is to develop a deeper understanding of persistent infection with Epstein-Barr virus (EBV). EBV has the capacity to drive the proliferation of resting B lymphocytes and this makes it a risk factor for human cancers such as Hodgkin's disease, Burkitt's lymphoma, immunoblastic lymphoma and nasopharyngeal carcinoma. However, the virus is able to persist in a quiescent state in vivo where it specifically targets resting memory B cells. By understanding how EBV can persist in most individuals without causing disease we hope to gain insight into what goes wrong when the virus does cause neoplastic disease. This study wilt employ sophisticated cell fractionation techniques and quantitative RealTime DNA and RT PCR assays to address four unresolved issues around EBV persistence. 1. Does acute EBV infection, infectious mononucleosis (AIM), represent a disordered state of EBV infection or simply an amplified version of the stable, long term carrier state? 2. Does EBV, like other herpesviruses, shut off the expression of all protein coding genes when it reaches its final site of persistence - long lived memory B cells in the peripheral blood? 3. What is the nature and origin of the latently infected memory cells proposed as the site of EBV persistence? Are they bona fide memory cells? Does antigen play a role in the production and/or maintenance of these memory cells or do latent proteins perform these functions? How rapidly do the infected cells turn over? 4. Are epithelial cells of the nasopharyngeal lymphoid system e.g. tonsils infected with EBV in vivo or infectable in vitro? Previous studies have analyzed EBV infection of epithelial cell lines and tissues from sites other than the site of persistent infection - the nasopharyngeal lymphoid tissue. However, epithelial tissues are biologically diverse so we will focus our studies on the biologically relevant epithelium from the tonsil.

PI: Trimmer, Barry
Title: Central and Peripheral Actions of Nitric Oxide
Although much is known about the cellular actions and developmental functions of nitric oxide (NO) in the central nervous system (CNS), relatively little is known about how this signaling system coordinates behavior. This proposal will build on results from the previous funding period to examine how NO controls motor functions through both central and peripheral mechanisms. These studies take advantage of the well-characterized NO/cGMP system of the insect Manduca sexta to identify NO-regulated responses. It will use physiological and pharmacological methods together with our newly developed RNA interference (RNAi) procedure for inhibiting nitric oxide synthase (NOS) expression in intact larvae. The long-term goal of these studies is to develop a comprehensive understanding of the specializations and limitations of NO in its role as a behavioral signaling molecule.

PI: Tzipori, Saul
Title: Slt Specific Human Monoclonal Antibodies for HUS Prophylaxis
This proposal is the second revised competing renewal for continuation of NIH Award #2 R01-A1-41326. Our objective is to develop an immunotherapeutic formulation for the treatment and prevention of complications associated with Stx-producing Escherichia coli (STEC) infections. Clinical isolates of STEC are known to predominantly produce Stxl, Stx2, and/or Stx2c. Children are particularly susceptible to development of Stx-mediated HUS. Our hypothesis is that administration of Stx-specific antibodies will prevent or modify the outcome of infection for individuals at risk of developing HUS. In the earlier awards, we have generated a panel of human monoclonal antibodies (Hu-mAbs) specific for Stx 1 or Stx2. Using the gnotobiotic piglet model, we have shown that Stx-specific Hu-mAbs neutralize Stx and prevent development of Stx-mediated complications. We now wish to determine which Hu-mAbs should be included in a formulation suitable for clinical evaluation. Based on superior efficacy, four Hu-mAbs specific for Stx2 (3 against the A subunit and 1 against the B subunit), and 2 for Stxl (both against B subunit) have been selected as candidates. The next step is to determine which combination of Hu-mAbs, is both compatible and highly effective. In this proposal we plan to define the structural and functional characteristics, which facilitate protective efficacy of Stx-1 and Stx2-specific Hu-mAbs (Specific Aim 1). Affinity and efficacy of each HumAb will then be studied against their respective toxin (Specific Aim 2). The efficacy of protection of a given antibody dose will then be determined in terms of time after bacterial infection (Specific Aim 3). Finally, combinations of Hu-mAbs specific for B subunit of Stx 1 and A or B subunits of Stx2, will be examined for relative efficacy and compatibility, to determine which is the most effective and thus suitable for clinical evaluation (Specific Aim 4). At the conclusion of these experiments we will have determined the components, and optimized the formulation of Hu-mAbs which will be recommended for testing in human patients. The Hu-mAbs will first be characterized and ranked according to their efficacy, affinity and compatibility with each other. The optimal amount of each Hu-mAb in the formulation required to provide the longest protection after bacterial infection will also be established. This is not a hypothesis-driven proposal, but an essential segment for the characterization of a promising therapy for HUS, against which currently there is no effective treatment.

PI: Vogel, Richard
Title: Balancing Human and Environmental Water Needs
The Nature Conservancy (TNC) and others working at the federal, state and local level to manage and protect water resources have identified the alteration of natural hydrologic regimes in rivers and streams as one of the major threats to aquatic biodiversity in the Northeast. In response to the growing understanding of the importance of this issue states throughout the Northeast are engaged in developing stream flow protection policies and water quantity management strategies. The purpose of this project is to demonstrate the use of ecological streamflow components as the basis for managing community water supply systems. The work is anticipated to be integrated in the Water Evaluation And Planning tool (WEAP) developed by the Stockholm Environment Institute (SEI). This project will require working with The Nature Conservancy (TNC) and SEI project managers and other experts to conduct hydrological and ecological analyses that will define critical ecological flow components of Northeastern streams and rivers. The project will also require the development ofwatershed-based models for how these components can be used to maximize environmental protection and meet reasonable water supply needs.

PI: von Moltke, Lisa
Title: Regulation of Cytochrome P450-3A Activity in the Elderly
Pharmacologic treatment is an important part of the therapy of mental disorders in the aged population. Central nervous system (CNS) concentrations of these drugs are determined in part by plasma concentrations, which show increasing variability with age. These changes however, may not fully explain the dynamic differences and seemingly increased sensitivity that has been observed in the elderly population. One particularly important class of drugs where decreased clearance in aging individuals has been documented are those that are substrates for the cytochrome P450 3A (CYP3A) subfamily of enzymes. Clinical pharmacologic studies and in vitro studies using human microsomes have not yet established the mechanism of this kinetic variability. Of interest is that a striking number of substrates and inhibitiors of CYP3A enzymes are also substrates and/or inhibitors of P-glycoprotein (P-gp), a multi-drug transport protein encoded by MDR1 (mdr1 a/b in rodents) found in many tissues, including intestine and the blood-brain barrier (bbb). The function of P gp in the intestine and the bbb have not been clearly define, but indications are that drug bioavailibility and elimination as well as CNS permeability to particular substrates may be importantly influenced by the presence and/or activity of this protein. Hence, the cause of the differences in clearance and sensitivities observed when psychotropic CYP3A substrates are administered to the elderly should be examined for both CYP3A and P-gp components. Although in vitro studies using human microsomes have proven invaluable in elucidating metabolic pathways and predicting drug interactions due to inhibition, the variability in the tissue procurement process and clinical situation preceding tissue donation can make it difficult to use the in vitro methodology to isolate the single variable of age and confidently attribute differences in metabolic activity to that factor alone. Consequently, appropriate animal models may be a more reliable system to study the kinetic and dynamic models of aging. Fischer-344 rats have been shown to handle a number of CYP3A substrates in a manner qualitatively similar to humans. Using this experimental system, dynamic, kinetic and molecular information can be simultaneously obtained to evaluate animal age and gender effects on: hepatic and GI CYP3A expression and function, and response to chemical induction; GI and bbb expression of P-gp; and systemic kinetics and CNS dynamic of a CYP3A-substrate benzodiazepine agonist.

PI: Waldor, Matthew
Title: Molecular Biology and Virulence of CTX Phage
CTXphi is a filamentous bacteriophage that encodes cholera toxin. This is the principal virulence factor of Vibrio cholerae, the Gram-negative bacterium that causes the severe diarrheal disease cholera. CTXphi is the first filamentous bacteriophage shown to mediate the horizontal transfer of a virulence gene. CTXphi integrates into the Vibrio cholerae chromosome and, in the lysogenic state, most CTXphi genes are not expressed due to the activity of the CTXphi repressor, RstR. Generally, the integrated form of CTXphi is found as part of tandem arrays of prophage DNA interspersed with the related genetic element RS1. RS1 encodes a protein, RstC, that can counter RstR repression and lead to markedly enhanced expression of CTX prophage genes including ctxAB, the genes encoding cholera toxin. The long-term goal of this work is to understand the molecular events in the life cycle of CTXphi and the role that this phage plays in the pathogenesis of cholera. The proposed studies will explore 3 processes central to the phage life cycle: i) the site-specific integration of phage DNA into the bacterial chromosome; ii) the repression of most phage gene expression following integration; and iii) the activation of phage gene expression and virion production by environmental and genetic stimuli. Experiments in Aim 1 to identify the mechanism and factors that mediate the integration of CTXphi DNA into the V. cholerae chromosome will reveal how the chromosome encoded recombinases XerC and XerD interact with phage and chromosome sequences to accomplish CTXphi integration. These studies will elucidate a novel mechanism of phage integration and may shed light on the mechanism of ctxAB amplification as well. Experiments in Aim 2: to characterize the regulation and mode of action of RstR will clarify how CTXphi can be maintained in a quiescent state. rstR autoregulation and modulation of RstR levels by environmental factors will be explored. RstR's binding to its unusual operators will also be studied. Experiments in Aim 3 to determine the mode of action of RstC-will explore how RstC can inactivate RstR-mediated repression. RstC's ability to bind to either RstR and/or RstR's binding sites will be investigated and the expression of rstC during infection will be measured. All of these studies will yield insights into fundamental aspects of phage biology. In addition, they may reveal ways in which changes in phage gene expression or copy number can contribute to the pathogenicity of V. cholerae.

PI: Wang, Xiang-Dong
Title: Alcohol Intake and Retinoid Metabolism and Signaling
Our long-term objective is to study the chemopreventive effect of retinoids on chronic and excessive alcohol related carcinogenesis in the liver and peripheral organs. The present grant proposal focuses on cell proliferation, which plays a central role in hepatic carcinogenesis in both the initiation and promotion stages, particularly when chemical carcinogens are involved. Retinoic acid plays an important role in controlling carcinogenic progression in a variety of cancers, including liver cancer. One of the chemopreventive effects of retinoids is thought to be mediated through control of proliferation via delaying progression of damaged cells into S phase, which allows for DNA repair and induction of apoptosis, thereby reducing the risk of carcinogenic initiation. However, long term and excessive ethanol intake reduces hepatic retinoid levels. The observation that retinoid concentrations are decreased in both plasma and cancerous liver tissues of hepatocarcinoma patients, suggests a role for retinoid depletion in hepatocarcinogenesis. However, it is not known 1) whether chronic ethanol-induced hepatocellular proliferation (which could convert hepatocytes from a state of resistance to a carcinogen to a state of susceptibility) is due to alcohol-impaired retinoid metabolism and signaling, and if so, 2) whether restoration of retinoid status by either inhibiting ethanol-induced retinoid catabolism or supplementing retinoic acid can suppress both ethanol-induced cell hyperproliferation as well as ethanol-promoted (diethylnitrosamine induced) hepatocellular carcinogenesis. We will investigate the possible role of diminished retinoid signaling and/or the up-regulation of the Jun N-terminal kinases-dependent (JNK) signaling pathway by chronic ethanol treatment on alcohol induced hepatocellular cell proliferation as well as alcohol-promoted hepatocellular carcinogenesis (induced by diethylnitrosamine). Simultaneously, we will test whether treatment with either chlormethiazole (an inhibitor of retinoic acid catabolism) and all-trans retinoic acid in ethanol-fed rats can inhibit alcohol-induced hepatocellular cell proliferation as well as alcohol-promoted hepatocellular carcinogenesis via either restoring normal retinoid signaling and/or inhibiting the JNK dependent signaling pathway. This study will be the first to link the regulation of retinoid signaling with Jun N-terminal kinases-dependent pathway, cell proliferation and apoptosis in an alcohol-treated, chemically induced carcinogenesis animal model, which would have implications for the prevention and treatment of alcohol related human cancers.

PI: White, Joel
Title: Characterization of Mechanisms Underlying Vapor Phase Responses of DNA-Based Sensors
We have observed that fluorescent-labeled, short, single-stranded DNA oligomers can be used as sensing materials when dried onto the detector substrates used in the Tufts Medical School artificial nose. This proposal is directed at addressing the mechanisms that govern how these molecules react to vapor phase compounds (odors). How does ambient humidity within the sensing chamber affect sensitivity and breadth of response to a define odor set? How do oligomer length, sequence, and the position of the fluorescent dye label affect response? How do different salts, buffers and substrate materials influence response?

PI: Willson, Robert
Title: Very Large Array-SOHO Investigations of Nonthermal Energy Release on the Sun
This proposal involves observations of the Sun using the unique capabilities of the Very Large Array (VLA) in support of NASA's SOHO satellite. Our main objective uses long wavelength (20 cm, 91 cm and 400 cm wavelength) VLA data to image sources of nonthermal energy release outside flares and within the low and middle corona. The VLA is the only facility in the United States that can produce the proposed images at these long wavelengths. These observations will be made in coordination with the SOHO EIT, LASCO and MDI and, when appropriate, with the Transition Region and Coronal Explorer I (TRACE) and the Reuven Ramaty High Energy Solar Spectroscopic Imager (RHESSI). The combined data will establish physical conditions in the relevant sources and the radio data will provide information about nonthermal processes and the coronal environment in which energy release takes place. Specifically, we will use the V LA at 20, 91 and 400 cm to study nonthermal metric and decimetric bursts associated with transient phenomena, including coronal mass ejections (CMEs) and evolving trans-equatorial coronal loops detected by the SOHO (EIT and LASCO) and TRACE, and the radio signatures of magnetic reconnection along the boundaries of coronal holes (EIT, CDS).

PI: Yee, Amy
Title: Mechanisms of Transcriptional Repressor HBP1 in Cancer
Breast cancer is a major killer of women in whom the etiology of tumor induction is poorly understood. The Wnt pathway has emerged as a major oncogenic pathway with a complex interplay of oncogenes and tumor suppressor genes. A key feature is the stability of f3-catenin, which functions as a transcriptional co-activator with the LEF and TCF family of transcription factors. The oncogenic phenotypes are ultimately established by the regulation of promoters for key growth regulatory genes. For example, Cyclin D1 is activated by, the Wnt-betacatenin pathway. Cyclin D1 mRNA is increased in many breast cancers, and its role in breast cell proliferation is well established. While the components and the activation of the pathway have been an area of intense study, the molecular mechanisms that inactivate the Wnt pathway in normal tissues are not well understood. These suppressive mechanisms are excellent candidates for the identification of new tumor suppressor genes. The working hypothesis of this proposal is that HBP1 is a suppressor of Wnt-beta-catenin signaling through the transcriptional repression of oncogenes and other gene targets. We had previously identified HBP1 as a transcriptional repressor and cell cycle inhibitor. Like LEF and TCF, HBP1 is an HMG box transcription factor. However, HBP1 remains one of few examples of repressors within this important transcription factor family. Recent work indicates that HBP1 is a transcriptional repressor of the Cyclin Dl promoter, which is activated by Wnt-B-catenin signaling. Experiments are specifically designed to test the role of HBP1 and transcriptional repression in breast tumorigenesis. The possible role of HBP1 as a tumor suppressor gene in human breast cancer will be tested directly. HBP1 is located in human Chromosome 7q31--a region that is frequently deleted in breast and other cancers. Deletion in cancer is a hallmark of tumor suppressor genes. The long-term goals are the mechanisms that may govern normal breast cell proliferation and that may become aberrant in tumorigenesis. This is critical to understanding tumor suppression and to how mis-regulation may lead to oncogenesis. Together with other work, the proposed studies may provide insights into new diagnostic and/or therapeutic strategies for breast cancer.


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