PI: Garlick, Jonathan
Title: Pathways and Mechanisms Controlling Early Oral Neoplasia
Abstract: Oral cancer begins as a premalignant lesion in which a small nest of dysplastic cells expands while in contact with normal cells. During the recent grant period, unique models were developed which mimic the human mucosal and cutaneous microenvironment in premalignant disease and which showed that direct cell-to-cell contact between neighboring keratinocytes is crucial in controlling the development of invasive cancer from a premalignant lesion. It is known that loss of cell-cell contact mediated by intercellular adhesion is a central factor leading to the progression of advanced cancer and metastasis. However, the role of changes in intercellular adhesion in progression of early neoplasia in stratified epithelium is not clear.

The objective of this application is to discover the intercellular pathways, which direct this, control it, or cause it to be lost. Because cadherins and catenins integrate cell adhesion with growth signaling they are excellent molecular candidates to regulate cell-cell interactions in premalignant disease. The experimental plan in this proposal is to perturb cadherins and catenins models of premalignancy and to monitor intraepithelial tumor cell expansion in vitro and invasion in vivo.

The Principal Investigator hypothesizes that cadherin/catenin-mediated cell-cell interactions can control premalignancy and that changes in adhesions can activate pathways leading to cancer. He will test if overexpression of E-cadherin (Aim 1) or overexpression of alpha-catenin (Aim 2) will increase adherens junctions and limit neoplastic progression. The Principal Investigator will then determine if decreased adhesive interactions will trigger cancer progression by overexpressing dominant negative forms of E-cadherin (Aim 3) and desmosomal cadherins (Aim 4) to disrupt adherens junctions and desmosomes, respectively. He will test his hypotheses in tissue models, which mimic premalignant human stratified epithelium. Adenoviral vectors will be used to express these exogenous genes in short-term, in vitro studies and retroviral vectors will be used for long-term, in vivo studies using our novel human skin/nude mouse chimera. He expects to find cadherin-catenin-mediated pathways and mechanisms that will drive or arrest early neoplastic progression. This insight will be an important step towards finding new therapies to block premalignant disease progression and prevent cancer.

PI: Gianinno, Lawrence
Title: The Role of Family and Community-Related Experience in the Development of Young People's Economic Understanding
Abstract:  What predicts what children understand about the everyday economic world? Clearly, cognitive developmental variables play a necessary role. However, such cognitive developmental change must occur within a social ecology that provides information about the economic life of families and communities. Specifically then, we may ask what is the role of children’s family and community-related experience in the development of their economic understanding? 

This proposal relates to the first of the Foundation’s stated foci, youth development and how various contexts such as families, programs, and policies affect their development. Economic life and exchange, broadly construed, is vital for the survival and healthy development of young people and their social worlds; such exchange represents a ubiquitous target of both private and public sector interest. Accordingly, for basic and applied research reasons, I want to understand what kinds of individual, family and community-related experience have a strong bearing on what children expect to occur in transactions involving the exchange of goods and services and the exchange of wages and labor, and on how they justify those expectations. 

The role of such experience in the development of children’s understanding of the economic world has been suggested, but not studied. To address this gap in the literature, I will look at the self-reported role of particular activities, knowledge, and beliefs and attitudes in this developmental process, including, for example: economic activities that may have particular salience and importance in the parents’ environment; parents’ occupations or trades; the content and frequency of parents’ conversation with their children about their work experiences; the children’s knowledge of the parents’ occupations or trades; parents’ economic values and attitudes; the ethnic composition of parents’ and children’s social networks; family social resources relevant to the economic world; and the children’s own work experience. 

A critical variable in this study is religion because it enables us to identify and recruit different groups of children and parents based on their subscription to particular social relationships; moral and other normative rules such as transcendent concerns about social justice and the social good; and certain responsibilities and obligations to self and others, with specific interest in how these various considerations may relate to economic activities involving the family and community in which the children grow up.

PI: Goodwin, Neva
Title: Social Science Library for the Third World
Abstract:  Third World libraries are especially deficient in social science materials. The social sciences — including anthropology, economics, law, planning, political science, sociology, and social psychology — are for the most part relatively young compared to the natural sciences and humanities. Their contribution to human well-being in the industrialized world has been uneven, but, overall, importantly positive. The potential for the Third World also to benefit from these areas of human inquiry, and to contribute to their further development, will be greatly hastened by wide access to good social science writings. 

This proposal describes a project that began with an ambition to disseminate to Third World university libraries an outstanding social science resource (a combination of encyclopedia and “reader’s digest”) that was developed by GDAE, between 1993 and 2001, under the title Frontier Issues in Economic Thought (henceforth, “Frontiers”). Funding for the initial stage of the project has been received, but the project has meanwhile expanded, with the goal of including 50 times as much material as is contained in the original six Frontiers volumes. The estimated budget for the expanded project is about $240,000. The scope of the proposed expansion is, however, quite elastic; it can shrink or grow, depending on resources. We are in the fortunate position of knowing that the original plan — dissemination of the Frontiers material on CD-ROM — will be a significant contribution in itself; every addition to it will be important, but there is no set amount that has to be reached in order for the outcome to be highly worthwhile.

PI: Gordon, Leslie
Title: The Effects of Altered Lamin A on Disease Process in Hutchinson-Gilford Progeria Syndrome
Abstract: Hutchinson-Gilford Progeria Syndrome (HGPS) is a rare and uniformly fatal syndrome of previously unknown inheritance affecting 1 in 4 to 1 in 8 million live births. Recently we have determined that HOPS is an autosomal dominant disease and that the gene defect responsible for HGPS is a single base change from C>T in position 608 of the LMNA gene. The base change produces a silent amino acid mutation, where GOC and GOT both code for glycine. However, the base change activates a cryptic splice site and results in a protein product lacking 50 amino acids towards the C-terminal end of the Lamin A protein. For clarity of reviewer reading, I will henceforth refer to the HOPS Lamin A protein as Progerin. Although the genetic basis for disease is now determined, definitive cellular disease phenotypes and the biochemical basis of disease are undetermined and warrant intensive investigation. This proposal explores the hypothesis that Progerin is primarily responsible for changes in cellular phenotypes that characterize the HGPS disease process.  The specific aims are:
1. To definitively characterize HGPS cellular phenotypes that may be responsible for disease phenotypes. Based on our preliminary data, we hypothesize that:
a) Premature senescence and premature apoptosis are consistent characteristics of HGPS fibroblasts. In vitro, there are conflicting reports on the growth potential of HOPS fibroblasts but data from our laboratory have shown that, of seven different Progeria skin fibroblast lines cultured past passage 5, six have senesced completely or almost completely between passages 6 and 18, as indicated by increases in doubling time, changes in morphology, and other criteria (see preliminary data). This is less than half of the growth potential of normal skin fibroblasts from age-matched or older donors. 
b) Nuclear blebbing increases with passage number and will precede or correlate with cellular senescence and/or apoptosis.
c) HA-PCM size and HAS expression correlates with senescence and/or with donor age in senescing Progeria and normal dermal fibroblasts. Our preliminary data indicate that synthesis of the cell-associated and extracellular polysaccharide, hyaluronan, is decreased and that assembly of hyaluronan dependent pericellular matrices (HA-PCMs) is defective in these senescing HOPS fibroblasts.

Hyaluronan-cell interactions have been shown to promote cell growth and survival signaling pathways. We will extend our preliminary findings to examine whether reduction in size of HA-PCMs is associated with senescence and apoptosis in HOPS fibroblasts, normal fibroblasts, or both. The presence or absence, and the relative sizes, of HA-PCMs will be analyzed in standardized cultures from early passage to senescence in Progeria cells, normal age-matched and old-donor fibroblasts Progression to senescence will be assessed by cellular staining for senescence-associated 3-ga1actosidase (SA-13 gal) activity, H thymidine uptake, and population doubling time-move this. To eliminate Progerin production by HGPS fibroblasts and evaluate attainment of “normal” fibrobast phenotypes. We will extend our preliminary studies using double stranded RNA into retroviral RNA inhibition. We will use RNA inhibition to disrupt Progerin production in both primary and hTERT immortalized HGPS fibroblasts, without interrupting normal Lamin signaling. All of the phenotypes described in specific aim 1 will be evaluated. To produce the HGPS gene defect in fibroblasts, endothelial and smooth muscle cells and investigate phenotypic changes that may lead to ECM defects and atherosclerosis in children with HGPS. We will use directed mutagenesis and transfect human fibroblasts, EC, and VSMC with Progerin vectors. In an attempt to understand the potential mechanisms of HGPS disease phenotype and potential differential effects of Progerin expression on cellular phenotype, all of the phenotypes described in specific aim 1 will be evaluated.

PI: Gordon, Leslie
Title: The Progeria Research Foundation Cell and Tissue Bank
Abstract:  PRF is the only organization dedicated to the rare disorder Hutchinson-Gilford Syndrome, a premature aging syndrome. Its mission includes initiating, accelerating and funding promising areas of research, which could provide information about the root causes, potential treatments and cure for Hutchinson-Gilford Syndrome, also often referred to as Progeria. Patients die prematurely of cardiovascular disease or stroke at an average age of thirteen years. The syndrome is very rare, with an incidence of approximately 1/8,000,000 live births. Hence, there is a severe lack of resources with which to conduct scientific research on HOPS. A potential consequence of our mission is the increased need for biological samples for research. 

We at PRF also want to protect affected individuals from being asked to donate to multiple research projects and from a loss of confidentiality when donating. By centralizing, coding, and making confidential the samples from affected individuals and their relatives, there will be a substantial asset for the initiation and support of research projects. In addition, many researchers could have access to samples they might not otherwise have the resources to collect. Molecular studies, mutation analysis, genotype/phenotype correlations and animal model research would all benefit from increased availability of cell lines and DNA samples. 

PI: Greenberg, Andrew
Title: Perilipins and Lipolysis
Abstract: Adipocyte lipolysis contributes significantly to the pathogenesis of obesity-associated diseases by increasing levels of circulating free fatty acids (FFA). FFA promote insulin resistance and type 2 diabetes. My laboratory's long-term goal is to elucidate molecular mechanisms of lipolysis regulation. The proposed studies will investigate structure/function relationships of Perilipin A (Peri A), a lipid droplet- associated phosphoprotein that regulates lipolysis mediated by hormone sensitive lipase (HSL) and non-HSL lipase(s). Peri A acts dually as a suppressor of basal lipolysis (in the absence of hormonal stimulation) and as a potent enhancer of protein kinase A (PKA)-stimulated lipolysis (in the presence of hormonal stimulation). Despite its important regulatory role, the primary sequences and the mechanism(s) by which Peri A regulates lipase actions have not been determined. Our preliminary studies indicate that Perilipin regulates lipolysis via multiple regulatory domains, which exhibit surprising lipase specificity.

The proposed studies will (1) identify the minimal domains of Peri A that modulate basal and PKA-stimulated lipolysis by HSL and non-HSL lipase(s), (2) determine the relative role of PKA phosphorylation sites in PKA- stimulated lipolysis by HSL and non-HSL lipase, and (3) define the in vivo effects of altered Peri A expression, Peri A truncations and Peri A PKA site mutants using Peri A transgenic and Peri null mice.

Our adipocyte and systemic studies will measure basal lipolysis, lipolytic response to beta-adrenergic agents, and antilipolytic response to insulin. These studies will provide in vivo proof of concept tests of how Peri A expression levels, regulatory domains, and phosphorylation sites regulate basal and stimulated lipolysis. These data will be directed to the prevention and treatment of diabetes, hyperlipidemia and other obesity - associated disorders.

PI: Greenblatt, David
Title: CYP3A Function in Aging African-Americans
Abstract: The Cytochrome P450-3A (CYP3A) drug-metabolizing enzymes are responsible for the biotransformation and clearance of a large number of drugs used in contemporary clinical therapeutics. Individual variation in the expression and activity of CYP3A, both in the liver and gastrointestinal (GI) tract mucosa, appears to underlie much of the large individual variability in pharmacokinetics and response to therapeutically-administered medications that are CYP3A substrates. Many clinical studies demonstrate that age, gender, and ethnicity (race) may account for components of this variation in predictable ways. For example, some data suggest that clearance of certain CYP3A drugs: (a) Becomes reduced in old age; (b) Is higher in women than in men; (c) Is greater in African-Americans than in Caucasians. However the available data are not by any means consistent. Furthermore, variants in the CYP3A4, CYP3A5, and Pregnane-X receptor genes may modulate age, gender, or ethnicity-related variations in CYP3A function in ways that are not understood.

This study will prospectively evaluate cohorts of young (18-45 years), "young" elderly (60-69 years) and "old" elderly (>70 years) volunteers, consisting of African-American and Caucasian men and women. The study paradigm will assess: (a) Hepatic blood flow (HBF), based on clearance of low-dose intravenous lidocaine; (b) Pharmacokinetics and pharrnacodynamics of intravenous and oral midazolam, a "pure" CYP3A substrate; (c) The prevalence of variants in the CYP3A4, CYP3A5, and pregnane-X receptor genes, using molecular genetic techniques; (d) Plasma concentrations of biologically active testosterone. From a and b, it is possible to estimate midazolam clearance by both routes, net oral bioavailability, and bioavailability attributable to hepatic and gastrointestinal presystemic extraction. With appropriate statistical techniques, the contributions of age, gender, and ethnicity to overall variance can be determined, as well as modulation of the relationships by genetic CYP3A variants and by biologically active testosterone. This study should provide important mechanistic information on the role of age, gender, and ethnicity as sources of variability in CYP3A-mediated drug metabolism and response.

PI: Holcomb, Phillip
Title: The Cognitive Neuroscience of Becoming Bilingual
Abstract: In most of the world bilingualism is the norm. Even in the US, a primarily monolingual society, there is a growing awareness that knowledge of a second language is essential to our competitiveness in an increasingly interactive world. However, there are a number of issues concerning the cognitive and neural systems that underlie monolingual and bilingual language use that remain unresolved. This proposal focuses on one specific, but critical component of the mechanisms involved in becoming bilingual - the cognitive and neural processes involved in acquiring and using a vocabulary in a second language (L2). Using both behavioral and electrophysiological (ERPs) techniques, our primary aim is to plot the cognitive and neural consequences of vocabulary acquisition in a foreign language by examining various stages of L2 language learning in both cross-sectional and longitudinal samples of foreign language learners. Reaction time and error data collected in the proposed experiments will allow us to link our data with the large extant literature from prior behavioral studies. The ERP data will help us follow both quantitative and qualitative changes in the processing of L2 words as a function of proficiency. Moreover, by employing this cognitive neuroscience approach it will be possible to more closely tie the cognitive and perceptual processes involved in second language vocabulary acquisition to their underlying neural mechanisms. An important and unique aspect of this proposal is the plan to test two complementary populations of bilingual participants: English native speakers learning French, and French native speakers learning English. This approach will allow unconfounded comparisons of performance in L1 and L2.

A major aim of the present project is to test a new model of L2 vocabulary acquisition (the developmental interaction activation model), which predicts three major developmental consequences of second language acquisition in terms of the (re) structuring of form and meaning representations of words in L1 and L2. The three categories of proposed experiments (15 in all) are designed to investigate the developmental trends predicted by this model: (1) unprimed single word recognition experiments manipulating orthographic neighborhood, concreteness, and cognate status of translation equivalents; (2) masked priming studies used to probe the evolution of L2-L1 lexical links and L2 form-concept links; and (3) language switching studies used to probe the evolution of control over the relative activation of words in each language.

PI: Holcomb, Phillip
Title: The Cognitive Neuroscience of Comprehension
Abstract: The primary goal of the proposed research is the study of language comprehension from a cognitive neuroscience perspective. The experiments outlined in the current proposal are focused on providing answers to questions about the sequence and timing of neural events that underlie the perceptual and cognitive processes involved in visual word comprehension. The proposed approach entails the recording of event-related brain potentials (ERPs) while subjects perform tasks designed to tap specific aspects of visual word processing. The proposed research has three specific aims. The first is to test and elaborate on predictions of the Bi-modal Interactive Activation Model (BIAM) of word comprehension in competent adult users of language. While models like the BIAM have been touted as neurally plausible, relatively little work using cognitive neuroscience methods has actually attempted to test the predictions of such models.

The second complementary aim focuses on improving the precision of ERP measures of word processing by providing a better understanding of their relationship with the perceptual, cognitive and linguistic processes they are hypothesized to reflect. The third aim seeks to improve our understanding of word comprehension by examining how comprehension processes develop over time. The 25 experiments that are proposed to achieve these aims are organized into three groups of studies. The first group, which includes 18 masked priming experiments to be conducted in young adults, uses our short interval repetition priming paradigm. In pilot work this paradigm has allowed us to isolate four temporally overlapping ERP effects that we have hypothesized are sensitive to a cascade of word comprehension processes.

The experiments outlined in this section of the proposal will test predictions about the processing nature and timing of these ERP effects and use these ERP effects to test predictions generated by the BIAM. The second group includes four single word experiments, also in young adults, where a number of variables will be manipulated that have previously been suggested to influence early visual word comprehension processes. We hypothesize that previous failures to see effects with these variables were due to lack of power. We will increase the power of our designs and determine if effects early in the time-course of word processing can be obtained. The final group of studies will include three word comprehension experiments modeled on those proposed in the young adult subjects but run in five groups of children between 7 and 11 years of age. These experiments will allow us to examine the development of visual word comprehension over time and thus better characterize changes in some of the neural/cognitive processes involved in reading. One long term goal is to extend these studies to children and adults with word processing and other language/cognitive deficits.

PI: Huber, Brigitte
Title: B Lymphocytes: Differentiation and Triggering (Merit Award)
Abstract: This competitive renewal application is based on the discovery that the env gene of HERV-K18 (Human Endogenous Retrovirus) encodes a superantigen, which is transcriptionally activated by EBV (Epstein-Barr Virus) and IFN-alpha (type I interferon). The working hypothesis is that the T cell stimulation elicited by the superantigen is not only essential for establishing life-long persistent infection with EBV in healthy individuals, but also plays a crucial role in EBV-associated diseases and malignancies. The following specific aims are proposed to test this model:
I) The control of HERV-K18 expression will be defined. The role of CD21 engagement in the initiation of superantigen expression will be analyzed, based on the observation that IFNalpha and EBV infection lead to superantigen expression, both acting through CD21 on resting B cells. Furthermore, the role of EBV LMP-2A in sustained superantigen expression will be evaluated, based on the finding that this EBV latent gene product is sufficient to transactivate HERV-K18 env, leading to superantigen activity.

II) HERV-K18 superantigen-reactive cells will be delineated in vivo and in vitro. For this purpose, an HLA.DR/HERV-K18 Env tetramer will be constructed to identify and stimulate superantigen-reactive T cells. The recent discovery of the murine TCR (T cell receptor) Vbeta specificity for the human superantigen will be exploited to define and map the TCR-superantigen interaction site. In addition, the role of CD48 in the superantigen-induced T cell activation will be tested, since EBV upregulates expression of this co-stimulatory molecule.

III) The role of the HERV-K18 superantigen in EBV-infection will be determined, because the central dogma of this proposal is that the superantigen activity is required for the successful EBV life-cycle in the human host. In vitro and in vivo EBV-infection/ outgrowth/ persistent latency/lytic cycle/reactivation models will be used to address this aim.

IV) The DNA of patients suffering from EBV-associated diseases will be typed for preferential expression of certain HERV-K18 alleles, compared to their respective healthy controls. This aim is based on the observation that the 3 HERV-K18 env alleles defined so far are unequally distributed in the Caucasian population and differ in their superantigen expression level. Collectively, these studies will further the general understanding of how viruses exploit the immune system of their hosts to their own advantage.

PI: Huber, Brigitte
Title: Chronic Lyme Arthritis: Is this Autoimmunity?
Abstract: Lyme disease is a multifaceted illness, initiated upon infection with the spirochete Borrelia burgdorferi (Bb). One manifestation of the disease is arthritis that can become a debilitating, chronic disease in genetically susceptible individuals. During the tenure of this grant, a candidate auto-antigen, hLFA-1, was identified which may elicit an autoimmune response in T cells by molecular mimicry with outer surface protein A (OspA) of Bb. These data form the basis for the current proposal.

(I) The analysis of the inflammatory T cell response will be expanded and refined by: a) single cell sorting of OspA-reactive T cells from Lyme arthritis patients, using cytokine secretion/capture assays to identify and clone OspA-reactive T cells, regardless of epitope fine specificity. The T cell response of patients in the acute and the chronic phase of the disease will be compared that will provide insights into the mechanism of treatment resistant Lyme arthritis. b) Analyses of OspA responses in HLA.DRB1 transgenic mice. DRB1 0401 and 0101 are associated with susceptibility to chronic Lyme arthritis, while the presence of 1101 seems to protect from this disease. To study the underlying mechanism, the OspA immune response in Bb infected C3H mice expressing these human DRB chains will be mapped and compared. c) testing hLFA-1/DRIB 1*0401 transgenic mice as an animal model for chronic Lyme arthritis. These mice will be infected with Bb and screened for the development of a chronic arthritic disease.

(II) The contribution of Abs to the inflammatory joint disease will be assessed. A prominent OspA-specific Ab response is observed during the chronic phase of the disease, correlating with the onset of prolonged bouts of arthritis. These Abs will be tested for an autoimmune reaction, and their involvement in joint inflammation will be determined by: a) the identification of immune complexes in synovial lesions of Lyme arthritis patients; b) the use of OspA Abs from chronic Lyme arthritis patients as probe for autoantigen; c) Bb infection in FcRn-deficient, hLFA-1/ DR*0401 transgenic mice: FcRn-/- mice clear serum IgG fast and are protected from Ab-mediated autoimmune diseases.

(III) A new candidate for molecular mimicry of OspA, hMAWD-BP, will a) be tested for cross reaction with OspA specific T cells and Abs, b) its aa sequences in the OspA-homologous regions of human and mouse will be compared; c) if significant differences are observed, hMAWD-BP transgenic mice will be prepared; finally, d) Genebank searches will be carried out for the identification of other human homologues of bacterial OspA. These experiments are designed to elucidate the mechanism of treatment resistant Lyme arthritis.

PI: Islam, Shafiqul
Title: A Diagnostic Study of Possible Enhancement of the Hydrologic Cycle
Abstract:  Recent modeling studies have suggested that a likely manifestation of a possible climate change would be an enhancement of the global water cycle. Such an enhancement is expected to result in increased precipitation, faster evaporation, and more frequent extreme hydrologic events. There is growing evidence, however, that the natural fluctuations intrinsic to precipitation processes may mask a simple response to anthropogenic greenhouse forcing. 

We will use two different types of diagnostic analyses to quantify natural fluctuations and presence of possible trends in precipitation. Our first research question is: Can we identify trends in storm events based precipitation statistics as a function of location, season, and time periods? We will characterize the storm event based precipitation statistics to evaluate how much of any precipitation increase (decrease) is attributable to changes in frequency, duration, and intensity of various precipitation events as a function of time period, season, and location. 

Our second question is: Can we identify the influences of climate intrinsic oscillatory processes on possible enhancement of precipitation pattern in space and time? Important specific issues include: (a) Can we distinguish natural fluctuations in precipitation from presumed anthropogenic or external effects? (b) Are the time intervals of steepest temperature change coherent with analogous changes in precipitation? (c) How much of an identified trend in precipitation is attributable to low frequency climate oscillation? We will use discrete wavelet transforms, multiple-taper time series method with singular value decomposition, and multiscale analysis of space-time correlations to identify and quantify possible presence of scale dependent trends in spatiotemporal precipitation signals. 

In this collaborative proposal between the Tufts University and Massachusetts Institute of Technology, we will determine from the data itself the timescales of possible changes to the signals and the time intervals during which trends are evident. Instead of interrogating the precipitation anomaly data set for known or suspected linear trends, we will systematically explore observed and proxy precipitation records at different space and time scales to find evidence of trends and determine their coherence with known oscillatory climate processes for different geographical and climatic regions. We will quantify spatiotemporal coherence between temperature and precipitation signals over the United States using frequency dependent correlation analysis. We will also compare and contrast identified trends in precipitation from atoll rain gages to those of satellite derived outgoing long wave radiation trends over the central tropical Pacific.

PI: Jackson, Rob
Title: Circadian Control of Behavior
Abstract: Drosophila locomotor activity is regulated by the circadian clock, and it provides a convenient assay for identifying and analyzing the factors downstream of the clock, that function in the circadian control of behavior. The work proposed in this application seeks to advance our understanding of circadian control mechanisms. It represents an extension of our earlier NSF-funded work on ebony mutants (DCB-8613798; 1987-1990) and recent molecular studies showing that ebony RNA abundance is under circadian control. Together, these data implicate ebony products in the circadian control of adult locomotor activity.

The ebony gene encodes N-ß-alanyl-dopamine synthetase (BAS; reviewed in 30). We have recently shown that ebony product is expressed in a restricted population of neural cells (see Project Description), in which it is postulated to regulate dopamine (DA) and/or ß-alanine (BA) pools. Of relevance to the present proposal, our previous work showed that ebony mutants exhibit severe circadian behavioral defects (22). That work demonstrated that ebony mutations cause either arrhythmic activity patterns or complex and abnormal patterns of activity, and we attributed the behavioral defects to an altered clock regulation of locomotor activity.

The results of a recent molecular analysis are consistent with the idea that the ebony gene product may function in the circadian control of behavior. In a gene chip screen for “rhythmic RNAs”, the ebony RNA was identified by Claridge-Chang et al. (3) as one of many head RNAs showing robust circadian oscillations in abundance. Interestingly, the peak phase of ebony RNA abundance is near the beginning of the day (i.e., the beginning of the photoperiod), and well correlated with the initiation of locomotor activity, which is predominantly restricted to the subjective day in wild-type individuals. An independent gene chip study showed that RNA from another gene known as black, which like ebony may regulate DA or BA pools in a restricted neuronal population, oscillates in a circadian manner (18). Interestingly, the peak phase of black RNA expression is also at the beginning of the day.

Thus, both ebony and black gene products may function as components of a circadian control mechanism. Although we are interested in how DA and/or BA might regulate activity, that is not the focus of this revised application. Instead, this proposal has the aim of determining where (in which cell types) ebony is required for its circadian function, and identifying the cellular pathway(s) through which the clock regulates ebony.

In addition, we will behaviorally characterize mutants of the black gene, as black RNA also exhibits circadian changes in abundance. In the proposed work, we will: 1) Determine the neuronal localization, rhythmic properties and cellular requirement for ebony product. These studies will utilize immunostaining procedures and cell-specific expression (Gal4/UAS) techniques to determine where ebony is required for the circadian control of activity rhythms.
2) Anatomically map connections among the Drosophila clock cells, ebony-containing cells, and downstream neurons of the cellular pathway regulating locomotor activity. This analysis will use existing reagents, including an antibody against ebony protein, to delineate the cellular output pathway from the clock to the neural loci controlling activity.
3) Characterize the role of the ebony cell population for behavioral rhythmicity. We will utilize genetic techniques to selectively ablate or physiologically perturb ebony-containing cells, in order to verify that this population is critical for the circadian control of locomotor activity.
4) Explore the circadian function of the black gene. Similar to ebony, black mRNA exhibits circadian changes in abundance.

Because of the functional relationship between the two genes, we will examine circadian rhythms in flies carrying black null mutations and in flies doubly mutant for black and ebony, using equipment and methods similar to those employed for our published analysis of ebony mutants.

PI: Jacque, Laurent
Title: Feasibility Study and Marketing Plan for a Proposed Master of Science Degree in International Management
Abstract:  The Fletcher School is seeking to expand its current International Business Program and create a new Master of Science degree in International Management (MScIM) that would leverage Fletcher’s strengths in international affairs and provide graduates with a truly interdisciplinary cutting-edge business management education. As identified in a strategic planning process, this new program is of highest priority for Fletcher and will serve to increase its competitive advantage and leadership in the international business education field. A key to the Program’s long term success however, will be Fletcher’s ability to effectively market the program, recruit the best students at the outset and place them in private sector leadership positions at time of graduation. As such, the Fletcher School is seeking funding to support the research and development of a comprehensive recruitment strategy and actionable marketing plan for attracting students and placing graduates.

PI: Jay, Daniel
Title: Proteome Signatures and Target Validation in Lymphomas
Abstract: To address tumor diversity and their varied responses to chemotherapy, we require the ability to custom design treatment for each tumor. The goal of pharmacogenomics is to generate gene expression signatures for tissue using microarrays that are then correlated with prognosis or response to therapeutics. However, much of the complexity and diversity of response may be due to proteomic differences. Thus far, probing for proteomic diversity and linking this to therapeutic efficacy has been difficult. Our overall goals are to develop surface proteome signatures (SPS) and perform functional proteomic target validation analysis directly on primary tumor tissue. There is a great need to assess the efficacy of different chemotherapy combinations directly on patient tissue samples.

A major difficulty is this respect is our inability to assess the small amounts of primary tumor tissue available from patient-derived samples. As part of our previous IMAT-funded research, we developed a library of single chain (scFv) phage display antibodies that recognize approximately 500 components of the surface proteome. In this work we also developed high-throughput methods for immunocytochemistry (ICC) using scFvs and apoptosis assays that use small number of cells (<500) per assay. Together, these developments allow us to generate SPS and measure apoptosis after chemotherapy treatment using small amounts of primary tumor tissue. Cells will be tested with a battery of drugs alone and in combination and analyzed for apoptosis. Thus, a differential response to therapeutics will be correlated with a SPS. We will develop and test these assays in the R21 phase using thymic lymphoma mouse cell lines derived from three mouse models: transgenic MyrAkt and two genetic deletions, PTEN-/+ or p53-/-. This is an ideal model system, as the primary tumors are genetically defined by single oncogenic mutations. We will establish SPS for cell lines derived from thymic lymphoma lines from these mice.

We will test for the efficacy of different drug regimens to obtain the optimum combination for inducing apoptosis of the cells. We will then test these drug combinations in primary tumor tissue from MyrAkt mice. We will also address the causal link between SPS and drug response and will test the functional role of scFvs that are biomarker candidates. In the R33 phase we will test the predicted optimal drug regimen on thymic lymphomas in the three mouse models, examining tumor load and survival. We will also characterize ten of the scFvs as potential biomarkers or targets for drug discovery. Our studies complement pharmacogenomics and provide a novel route to pharmacoproteomics.

PI: Kaplan, David
Title: Silk Protein Polymer Designs for Improved Expression and Processing
Abstract:  Our objective in the present proposal is to determine the relationships between genetic/protein block designs gleaned from our recent domain mapping studies of all silk proteins, coupled with the limitations imposed by an all aqueous processing environment based on our recent models of how silk proteins are assembled in solution toward gel states and then spinning. These issues are addressed in concert with codon optimization, expression in thermophilic hosts that can accommodate GC-rich genes and glycine/alanine overproducing mutants. The rational design of silk encoding genes, borrowing from the more generic designs identified in Nature, in combination with the processing constraints for these proteins in Nature, highlight the novel and important design limitations and benefits offered by these intriguing protein spinning systems. The general rules of construction for silk proteins based on these observations should provide a useful guide to how Nature has solved the problem of processing hydrophobic proteins in water, and how this process can be copied industrially. The sign of the proposed studies is that by employing these design rules there should be improved expression, recovery of soluble protein and control of processing into high solids solutions and gels leading to spinnable dopes for fibers, films or other material outcomes. The insights to be gained from the proposed studies have implications in fundamental structural biology as well as direct utility toward improved options in silk-based polymer synthesis, processing and materials fabrication.

PI: Kaplan, David
Title: Silk Polymer Models for Structure-Function Relationships
Abstract: Silk proteins provide a useful model system for the study of novel functional properties derived from hydrophobic polymers in aqueous systems. The impressive mechanical properties of spun silk fibers rest with the unusual control of structure development in glands in silkworms and spiders, all achieved through control of water content in combination with appropriate sequence chemistry. Our objective is to understand the mechanistic basis for silk protein assembly, through control of water content in silk solutions, as a route to new and improved processing options for hydrophobic polymers in general. This insight has strong implications for: (a) new materials engineering from silks, and (b) for new polymer chemistries/sequences that mimic silk in that the designs (chemical sequences) must consider processing environments as well as functional outcomes. We propose a systematic investigation into the formation and structural features of silk gels containing high concentrations of protein achieved via osmotic stress, and subsequent transitions and structural and morphological features induced through chemical and mechanical factors.

Specifically, we plan the following aims: (a) to compare structure development in aqueous systems by systematic control of water content, including comparisons between native gels from silkworm glands and reconstituted silkworm silk; and (b) to study the role of specific environmental factors (e.g., pH, divalent cations, temperature) on rates and nature of structure and morphology development at different water contents.

To address these questions, we will characterize our model materials using spectroscopic techniques (Fourier Transform infrared, FTIR; Raman), scattering techniques (wide and small angle X-ray scattering, WAXS/SAXS; small angle light scattering, SALS), and imaging techniques (atomic force microscopy, AFM; scanning electron microscopy, SEM; optical ellipsometry; differential interference contrast microscopy, DIC) to assess morphologies and structures at a variety of length scales. These assessments will permit the formulation of phase diagrams for quiescent silk gels. Then, to approximate the step of fiber formation we will study the effects of stress (using tensile deformation and shear) on structure evolution and the corresponding mechanical properties of reconstituted silks. The planned studies build off our recent discoveries on the solution behavior of silk proteins and control of this behavior.

The proposed experimental studies represent an interdisciplinary approach to the study of this unique polymer, including a biochemist (David Kaplan) and a physicist (Peggy Cebe). The graduate and undergraduate students involved will gain direct insight from both perspectives during their research, while also contributing to new directions in polymer science and engineering. The research approaches and outcomes will be exported into classroom settings in a number of ways for a broader audience of students including lecture modules in both undergraduate and graduate courses, windows on research laboratory experiences for undergraduate students, and specific programs for deaf and hearing impaired students.

PI: Kaplan, David
Title: Tendon Formation Mediated by SMAD8 Signaling Pathway
Abstract:  Tendon and ligament tears present a major clinical problem in orthopedic surgery, resulting in morbidity and function loss to the inflicted patients. The repair of torn tendons encounters major difficulties, and often results in impaired healing and function loss. Tissue engineering, which aims to construct three-dimensional tissues available as a source for implantation and tissue replacement is a novel approach in regenerative medicine. We have been studying novel biomaterials and ex vivo culture systems for ligament tissue engineering, while the Israeli Pt has identified a novel pathway in mesenchymal stem cell (MSCs) differentiation to ligament tendon tissues mediated jointly by SMAD8 and BMP2 genes. Thus, in the present application we propose to promote tendon tissue formation by combining the two strategies: MSCs expressing novel signaling molecules and designated scaffolds. We therefore hypothesize that in vivo tendon repair could be achieved by combining specially designed silk scaffolds with mesenchymal stem cells co-expressing SMAD8 and BMP2 genes.

In order to explore our hypothesis the following specific aims will be pursued: Specific Aim 1: In vitro culture of MSCs over expressing SMAD8 BMP2 genes on silk scaffolds. Genetically engineered MSCs will be cultured on silk scaffolds in vitro. Differentiation will be monitored and characterized on various hierarchical scales (gene protein expression and structure).

Specific Aim 2: Functional evaluation of engineered tendons in vivo. Engineered tendons will be tested in vivo to evaluate their phenotype stability, and the capacity to function and remodel under the conditions of physiological loading. Silk scaffolds seeded with genetically engineered and non-engineered MSCs will be implanted ectopically and in a tendon injury site. Tendon tissue engraftment, survival and regeneration will be evaluated. 

Finally we believe that our platform for generating tendon tissues applying novel signaling pathway represents a powerful system for producing a functional biological tendon substitute. In addition, our approach should pave the way for novel modalities in designing biological grafts available for implantation worldwide.  This research will be done primarily in Israel at the Hebrew University of Jerusalem in colaboration with Gazit Dan as an extension of NIH grant ROl AR46563-O1.

PI: Kaplan, David
Title: Tissue Engineered Ligaments
Abstract: The grant proposed one specific aim, to identify optimal levels of biochemical and mechanical manipulated parameters to promote hBMSC (1) attachment and proliferation, (2) differentiation, and (3) ligament tissue development in vitro.  Due to the broad scope (spectrum) of experimentation involved with this study, the aim has been broken down into three milestones; the first, hBMSC proliferation after 1 week, was to be addressed in Year 1.  We have accomplished ~80% of this milestone.

PI: Kaplan, David
Title: Tissue Engineering - The Next Generation
Abstract: The overall goal was to identify the scientific and technological gaps between the fields of developmental biology and tissue engineering in order to guide the scientific inquiry in the field. The main goal was to begin to fill the gap between fundamental concepts in developmental biology and engineering approach to tissue formation.  At present, this gap is not even recognized by most engineers. This is in large part due to the fact that the underlying understanding of tissue development from the field of developmental biology remains untapped by tissue engineers. In addition, the gap is due to the fact that an engineering approach to tissue formation remains very empirical and highly limited in terms of the core understanding of guiding principles. It is essential to begin to find ways to bridge this gap so that the knowledge base and principles from developmental biology can help guide tissue engineering in the next generation.

Otherwise, our ability to understand and optimize tissue outcomes in vitro (during cultivation) and in vivo (following implantation) will not progress at a rate needed to have the impact in medicine. In a more general sense, we feel that this is the time to step back and critically rethink the whole field of tissue engineering.

PI: Krimsky, Sheldon
Title: The Corruption of Science: A Speaking Tour and CD Archive
Abstract:  The requested funding is for developing (1) a speaking tour focusing on the issue of the corruption of academic and government science by private interests and (2) a CD archive with Power Point slides and primary materials that can be used in courses and for general public education. (3) A webpage on “Corruption of Science” will be attached to Dr. Krimsky’s Tufts website www.tufts.edu/~skrimsky.

I will develop a set of talks for presentation at universities, professional societies, medical schools, and public venues supported by visual materials from documentary videos, investigative news stories, scholarly studies, books, and litigation discovery documents. These talks will be designed to disclose the nature of the problem of “biased” and “corporate captured” science as well as the efforts being made to address the problems.  Since the publication of Science in the Private Interest in 2003, I have come to realize the important of reaching broad audiences inside and outside the universities for discussing scientific and medical ethics of research and publication, the corporate capture of American universities, and the impact that the loss of “disinterested science” is having on American democracy and justice. 

I have been able to accept one out of 3-4 invitations to speak either because groups do not have the fees for travel or honoraria or I have teaching obligations that restrict time away. The funds for this project will allow me to subsidize organizations and institutions that wish to learn about these issues and to reduce my teaching time allowing me to make more presentations. It will also allow me to produce materials that can be used by institutions that want to incorporate the issues into research ethics courses. 

In the Fall of 2004, I was able to speak at: University of Virginia at Charlottesville, Colby College, Waterville, Maine, Notre Dame, South Bend, Indiana, University of Michigan, Ann Arbor, Union of Concerned Scientists, Harvard University, University of Nevada, Las Vegas, MIT, Cambridge, MA, and CIJNY, New York City. The demand has been far greater than that. This grant will give me the opportunity to bring the topics in Science in the Private Interest to a much wider audience, to develop a state-of-the-art CD to make the sources easily available, and to post the power point presentations on a webpage.

PI: Kumamoto, Carol
Title: Regulation of Drug Resistance Genes in C. Albicans
Abstract: This research project investigates the regulation of drug resistance genes in Candida albicans. Greater than 90% of AIDS patients suffer from oropharyngeal candidiasis (OPC). Fluconazole is the most commonly prescribed antifungal drug for these infections due to its efficacy and lack of side effects. Treatment failures with fluconazole have risen, most notably in AIDS patients with recurrent OPC that receive extended fluconazole therapy. The majority of treatment failures are due to fluconazole resistant C. albicans isolates. Although resistant C. albicans strain most often exhibit increased drug efflux due to the increased transcription of multidrug resistance pumps, little is known concerning the molecular mechanisms that lead to this increased transcription. C. albicans drug resistant mutants have been isolated that increase transcription of the multidrug efflux pumps MDR1 and CDR2.

These mutations fall into two classes: (i) transacting mutations that lead to high level expression of either MDR1 or CDR2, and (ii) cis-acting promoter mutations that lead to a more moderate, fluconazole-dependent increase in the transcription of either MDR1 or CDR2.

The proposed research will analyze these mutations at the molecular level, determine the mechanisms that lead to increased transcription of these drug resistance pumps, and study the influence of drug selection regimens on the acquisition of these mutations. The long-term goal of these studies is to contribute to a more informed use of fluconazole with respect to prophylaxis, drug dosage regimens and the development of fluconazole resistant strains.

PI: Kumamoto, Carol
Title: Regulation of Fungal Invasive Growth
Abstract: This research project investigates the regulation of invasive growth in fungal organisms. In mammals, the ability to invade normal tissue barriers is an important attribute of metastatic cancer cells and of cells in the developing embryo. In contrast, in the adult regulatory interactions with the substratum control cell proliferation and apoptosis. This regulation by substratum is commonly lost upon malignant transformation.

The goal of this research is to understand regulatory interactions with substratum. The fungi Candida albicans and Saccharomyces cerevisiae will be used as model systems because fungal cells also interact with substratum. In the opportunistic pathogen, C. albicans, interactions with the substratum may be important in promoting invasive growth of the organism within the tissues of a host. Thus, studies of C. albicans invasive growth will also contribute to the understanding of a process that plays an important role in disease. C. albicans responds to the presence of substratum by producing invasive filamentous hyphae, which penetrate into the matrix. Culture of C. albicans cells within surrounding matrix promotes rapid production of hyphae. Genetic analysis of this process has led to the identification of two gene products that are needed for normal hyphal production in response to surrounding matrix. S. cerevisiae also responds to the presence of substratum by undergoing invasive growth, although, in this organism, invasion is not associated with a dramatic change in morphology. S. cerevisiae homologues of the C. albicans genes described above are needed for normal invasive growth.

Therefore, studies in S. cerevisiae will be performed in order to develop detailed molecular hypotheses for the function of the genes. Studies in C. albicans will test the generality of the hypotheses developed in S. cerevisiae. Experiments are proposed to identify interactors that bind to the gene products of interest and to elucidate the pathways in which these gene products participate.

PI: Kumar, Krishna
Title: Ion Channel Design Using Unnatural Amino Acids
Abstract: Nearly a third of completed genomic sequences and almost half of all receptors that are likely to be targets for drug design are integral membrane proteins. Understanding the structure and energetics of membrane proteins is therefore a key and unsolved problem in structural biology. In contrast to proteins soluble in aqueous media, the primary interactions that contribute to the stability and specificity of membrane protein structures are poorly understood. Soluble proteins display a bipartite architecture with hydrophilic exteriors and hydrophobic interiors. This kind of binary patterning is not observed in membrane proteins, rendering design a difficult problem. The challenge is one of control over structure in nonpolar surroundings. The broad long term objective of this proposal is the development of design elements for membrane proteins. Our approach relies on the unique phase separation properties of highly fluorinated materials. The proposed studies will make use of folding driven by phase separation in the non polar membrane environment of appropriately positioned fluorinated side chains to deliver predetermined structural and functional ensembles.

The specific aims of this proposal are:
(1) To develop methodology for synthesis of enantiomerically pure fluorinated amino acid analogues;
(2) To probe the three dimensional structure of fluorous peptides using biophysical techniques;
(3) To study the effect of stability of fluorous phases in protein environments by selectively replacing core hydrophobic residues;
(4) design and synthesis of membrane spanning and pore forming peptide ensembles based on phase separation within the membrane;
(5) biophysical characterization of the peptide-lipid interactions in lipid vesicles and planar lipid membranes using a combination of fluorescence and CD spectroscopy, differential scanning and isothermal titration calorimetry;
(6) characterization of the influence of peptides on lipid morphology and direct visualization of pore formation by scanning force microscopy and
(7) investigation of channel activity using single channel conductance and fluorescence assays probing dye or H+ release.

Ultimately the design and characterization studies proposed here should facilitate the construction of effective membrane transport agents that will add to currently available antibiotics and help in combating the ever- increasing resistance of bacteria to current therapeutic drugs.

PI: Kuperwasser, Charlotte
Title: Breast Cancer Stem Cells in the Formation of Bone Metastasis
Abstract: The research to be performed at the Raymond and Beverly Sackler Foundation Laboratory, Tufts New England Medical Center will center on the use of a novel xenograft model system to identify the genetic and stromal changes that mediate breast cancer progression. Using this model, two broad areas will be investigated.

Specific Aim 1) To identify the genetic requirements for in vivo breast cancer development.  The genetic requirements for a normal human cell to become transformed in vitro have been well characterized but whether the same set of genes are relevant and disrupted in vivo in order to form a breast cancer is unclear. More importantly, the sequence of genetic events that occur during tumor progression has not been established for breast cancer progression. The working hypothesis based on preliminary data is that the alteration of relatively few genes results in precursor lesions, and perhaps full transformation in vivo. In addition, experiments will explore the hypothesis that hepatocyte growth factor (HUF) or transforming growth factor beta (TGFβ), two factors produced by the stroma, can promote precursor lesions into frank carcinoma.

Specific Aim 2) To identify whether the stromal requirements for in vivo breast cancer development are mediated by transcription factors snail or slug.  It is known that HGF and TGFβ are able to promote occult breast cancer cells to proliferate in a neoplastic fashion. This suggests these factors are specifically required for revealing this neoplastic growth, or that the downstream effects of the growth factors could do so. However, it was puzzling as to why two very differently acting factors (HGF and TGF could elicit such a similar outcome. Preliminary experiments as well as published work by others (Janda et al., 2002) have demonstrated that both of these factors can induce an invasive phenotype in initiated breast epithelial cells. Both of these factors are known to activate downstream transcription factors snail and slug and therefore, the laboratory will examine the effects of these stromal cells on the induction of snail or slug promoters in vivo to determine when and where during cancer progression these factors are induced.

PI: Lamon-Fava, Stefania
Title: Estrogen, HDL, and Coronary Heart Disease in Women
Abstract: Coronary heart disease (CHD) is the leading cause of death and disability in postmenopausal women in the United States. Low plasma levels of high-density lipoprotein cholesterol (HDL-C) are a well-established risk factor for CHD. Elevated plasma triglyceride (TG) levels are also a risk factor for CHD in women. HDL particles are heterogeneous in composition (containing apo A-I only, LpAI, or apo A-I and apo A-II, LpAIAII) and charge and size (preBeta1, preBeta2, alpha1-3, preAlpha1-4). Different HDL subpopulations have different physiological functions and therefore may vary in their anti-atherogenic potential. Changes in alpha1 HDL subpopulations are a predictor of coronary disease progression in men. Hormonal replacement therapy (HRT) increases plasma levels of HDL-C, but has adverse effects on TG and CRP levels. While observational studies had indicated a protective role of HRT in CHD, recent intervention studies have shown no CHD protection with the use of HRT. Our preliminary data indicate that there is a large inter-individual variability in HDL subpopulations and TG-rich lipoprotein remnants response to HRT.

The purpose of the current application is to clarify the effects of estrogen, with or without progestin, on HDL and its subpopulations and on lipoprotein remnants. This application will also examine the impact of changes in HDL subpopulations and in lipoprotein remnants during HRT on progression of coronary atherosclerosis. We will conduct these studies in participants in the Estrogen Replacement and Atherosclerosis (ERA) trial, a randomized, placebo-controlled study of HRT and progression of atherosclerosis in postmenopausal women with CHD (n=309), in whom baseline and follow-up angiographic measurements of coronary artery diameter have been obtained.

We propose to measure the following HDL parameters: preBeta1, preBeta2, alpha1-3, preAlpha1-4 HDL subpopulations by 2dGE, LpAI and LpAIAII in plasma and apo C-III in HDL and total plasma by immuno-electrophoresis, lipoprotein remnants by an immunoseparation method, and polymorphisms at gene loci involved in HDL metabolism (lipoprotein lipase, hepatic lipase, cholesteryl ester transfer protein, scavenger receptor B1, and ATPA1 receptor).

Our hypotheses are: 1) these HDL parameters and lipoprotein remnants will be significantly associated with severity of CHD at baseline; and 2) HRT-related changes in these parameters will predict coronary atherosclerosis progression in the ERA participants. The proposed analyses provide a unique opportunity to shed light on the physiological role of HDL subpopulations and HRT on CHD in women.

PI: Lang, Kenneth
Title: Very Large Array Observations In Support Of Hessi, Soho, Trace And Yohkoh
Abstract:  The first edition of Sun, Earth and Sky was published in early 1995, when it immediately became a very popular book, translated into German, French, and Japanese. This popularity resulted from its comprehensive treatment of all aspects of the Sun and its interaction with the Earth, as well as its light and friendly writing style, with apt metaphors, similes and analogies, and the use of numerous images from spacecraft and even a few works of modern art.  But Sun, Earth and Sky was published before the launch of the Solar and Heliospheric Observatory, abbreviated SOHO, on 2 December 1995, and before the Ulysses spacecraft had completed its first orbit over the solar poles — crossing the south solar pole in July 1995. In the ensuing decade, SOHO has revolutionized our understanding of the Sun, extending our gaze from the visible solar disk to down within the hidden solar interior and out in all directions through the Sun’s tenuous million- degree atmosphere and solar winds. When combined with the results of other modern solar spacecraft, SOHO has provided more new information about the Sun then perhaps the entire century of previous observations of our home star.  SOHO's recent insights have been complemented and extended by other solar spacecraft, including Ulysses, which sampled the solar wind during two complete circuits around the Sun, and Yohkoh, which completed a decade of observations of solar X-rays.

The Transition Region And Coronal Explorer, abbreviated TRACE, which was launched in April 1998, provided additional information on the heating of the million-degree outer atmosphere and the ubiquitous magnetic loops that it is made of. The Ramaty High Energy Solar Spectroscopic Imager, or RHESSI for short, launched in June 1998, has now revealed new aspects of the energetic explosions on the Sun known as solar flares. These spacecraft have together provided more new information about the Sun then perhaps the entire century of previous observations of our home star.  In addition, massive subterranean neutrinos detectors, the SuperKamiokande and Sudbury Neutrino Observatories, have revealed new physics of the ghostly neutrino, which has a tiny mass and changes form as it moves from the Sun to Earth, thereby solving the solar neutrino problem. 

All of these recent discoveries will be included in the proposed Second Edition of Sun, Earth and Sky, which Springer-Verlag has agreed to publish. It will describe how recent NASA solar missions have provided new insights to the Sun’s fusion reactions, internal motions, magnetic fields, explosive activity, space weather, variability and winds, as well as the Earth’s climate, ozone depletion and global warming.

PI: Lee, Kyongbum
Title: Adipose Metabolic Profiling for Obesity Drug Targeting
Abstract: The three major aims of this project, as actually funded, are as follows: (1) to quantitatively profile metabolic transitions underlying adipose growth; (2) to determine the effects of adipose growth modalities – precursor cell differentiation and progressive lipid loading- on intracellular metabolic fluxes; (3) to identify metabolite and reaction markers common and unique to normal and obese adipose growth modalities.

PI: Lee, Mary
Title: Tufts University Open Courseware Project - Phase II: Trial Period
Abstract: Annually, so that additional effort to contribute to OCW is minimized.  Regarding content, while MIT has documented high user interest for its science and engineering content, it is not yet known whether there will be the same interest or feasibility in sharing graduate health sciences and international affairs content. However, through a recent visit to Tufts by a delegation from Binzhou University, China, we glimpsed the scale of educational challenges faced elsewhere that could be aided by Tufts OCW. Binzhou is considered a “small” university in relatively remote northeast China. Binzhou reaches one million students, with five thousand enrolled in their medical doctor degree program alone (compared to less than 700 M.D. degree students at Tufts, which is considered a fairly large U.S. school). The delegation was highly excited about the prospect of having access to any of our digital content, which would not even require translation since they (and many other international medical schools) use English-based resources. In addition, over the next few months, three Tufts faculty are volunteering to go to Eritrea, Africa, to help develop their first medical school (25 years after gaining independence from Ethiopia). They are bringing donated books and other materials, but the Eritrean school would also greatly welcome digital materials. 

We anticipate that Tufts OCW can provide high quality content that has strong applicability to an international audience, thereby showing the tremendous value and impact of OCW and hopefully attracting others to contribute. However, that content must be offered quickly enough and with wide enough breadth to capture interest and demonstrate feasibility.

During Phase I of Tufts OCW, we encountered the expected challenges of starting a complex project, particularly one involving five schools and multiple departments across the University. While every team representing a working stream remained committed to bring Tufts OCW to our Phase I launch despite adding time to their “day jobs,” we realized that we will need a few personnel dedicated to Tufts OCW if we are to progress effectively beyond the pilot phase—for project management, content development, programming, and evaluation. Associate Provost Lee will remain Project Steward, but Nancy Wilson is unable to remain as Project Director after this fall due to her responsibilities to The University College. Our program coordinator has been “loaned” to us by University Advancement for Phase I, but is eager to continue with the project as project manager.

PI: Lemire, Joan
Title: Developing a Smooth Muscle Cell Model for the Study of Hutchinson-Gilford Progeria Syndrome: Is Aggrecan a Significant Component of the Phenotype?
Abstract: HOPS has been considered a disease of aging and there are pathological changes in a number of tissues. Death, however, results from cardiovascular disease.

We have chosen to focus this application on one type of vascular cell, the arterial smooth muscle cell (ASMC), for 2 reasons. First, smooth muscle cells are present in the arterial intima as part of the early stages of atherogenesis, and produce the extracellular matrix in which lipids are trapped and into which macrephages and lymphocytes invade. Second, the only information we have about molecular changes in HOPS is derived from studies of fibroblasts, and smooth muscle cells are the component of the arterial intima that is most similar to the fibroblast. For example, both normal ASMC and normal dennal fibroblasts produce the proteoglycans versican, biglycan, and decorin. Thus molecules that are over or underexpressed in HOPS fibroblasts might show a similar pattern in patients ASMCs. ASMC from HOPS patients would be ideal for study but are unavailable. The recent discovery of the mutation responsible for HOPS, a mutation in lamin A, has given us a tool by which we can hope to make a reasonable facsimile of an HOPS smooth muscle cell, by introducing the mutant form into normal cells. 

We hypothesize that ASMC expressing progerin will also express some of the phenotype characteristic of HOPS fibroblast, including the expression of the proteoglycan aggrecan (A. Weiss, personal communication, and this application). Aggrecan is not usually produced by either fibroblast or ASMC. We hypothesize that aggrecan expression by ASMC contributes significantly to the phenotype, and thus blocking expression of aggrecan will revert progerin-expressing ASMC to a more normal phenotype. We further hypothesize that aggrecan contributes significantly to both arterial lumen narrowing and lipid deposition, and we will describe plans to address this question in future studies. 

Aim 1. Do human fibroblasts and ASMC expressing progerin show similarities in phenotype to fibroblasts from IIGPS patients, including the expression of aggrecan? If the expression of the lamin A mutant form, rather than a reduction in the amount of normal lamin A, leads to the HOPS phenotype in patients fibroblasts, then we would predict that transfection of normal flbroblasts, containing normal alleles of lamin A, with progerin cDNA would lead to expression of the HOPS phenotype. We will introduce the progerin cDNA into fibroblasts from normal children and ask if aggrecan is overexpressed and whether levels of decorin and matrix metalloprotease 2 (MMP2) are decreased. 

Aim 2. Characterize progerin-expressing ASMC. There are no ASMC cultures from HOPS patients and no description of changes at the molecular level in patients’ arteries. Characterization of progerin-expressing ASMC will provide us with a molecular phenotype to target. The same progerin sequence be introduced into human ASMC and we will determine whether aggrecan expression is induced. We will specifically evaluate progerin-expressing ASMC for the expression of extracellular matrix components of the HGPS-fibroblast phenotype: reduced levels of decorin proteoglycan and MMP2. We will develop a detailed description of the alterations seen in progerin-expressing ASMC by performing a microarray analysis, and examine this information for the overexpression of molecules which are believe to play a role in vascular disease. 

Aim 3. Determine whether blocking aggrecan in progerin-expressing ASMC blocks other components of the progerin-ASMC phenotype.  SiRNA will be used to block the expression of aggrecan in progerin-expressing ASMC. The molecules to be measured will be chosen based on the characterization performed in Aim 2.

PI: Lerner, Richard
Title: Spirituality and the Promotion of Exemplary Youth Development (Thriving): Toward a Longitudinal Investigation of Neurological, Psychological, and Contextual Bases
Abstract:  This proposal presents three phases of a plan for work that will be both “field building” and “field defining” for the study of youth spiritual development. We propose three phases of work:  Measure development, a cross-sectional research study spanning the second decade of life, and dissemination. Through this work we will enlarge the scholarly community directly involved in the study of spiritual development and, as well as, in its relation to neural growth, generosity, purpose, and exemplary healthy youth development — what we term “thriving.”

We will also create enthusiasm for this field of research among scholars and the public more generally. We will endeavor to accomplish these goals by conducting a collaborative study that provides the conceptual and empirical foundation for a national, longitudinal investigation aimed at elucidating the brain, psychological, behavioral, and ecological (family and community) bases of the development of spirituality, purpose, generosity, and thriving among adolescents in general, and among a special subset of youth we will study, that is, spiritually “gifted” youth.

The three phases of the proposed work will provide leverage for funding this subsequent longitudinal study, and for engaging a new cohort of established and developing researchers and theologians in empirically ascertaining what role youth spiritual development plays in moderating an adolescent’s generosity, purpose, and thriving. This new spiritual knowledge is critically important for ensuring both healthy individual development during adolescence and across the subsequent life span thus in building the human and social capital requisite for the healthy perpetuation of humanity.

PI: Lerner, Richard
Title: Health Rocks! Promoting Positive Development Among America's Youth
Abstract:  American youth, and the nation that seeks to nurture and support them, face a set of problems of historically unprecedented scope and severity that, together, limit the opportunities for active and constructive participation by young people in community life and civil society. For example, estimates are that 50% of American youth engage in two or more of the major four categories of risk behaviors (substance use and abuse, crime and violence, unsafe sex and teenage pregnancy and parenting, and school failure) and that 10% engage in all four sets (Dryfoos, 1990, 1998). This level of comorbidity places American youth at unprecedented risk. Moreover, since the 1980s there continues to be a strikingly alarming rate of youth poverty involving a fifth of the children and adolescents in the United States. Rates of all of the above-noted problem behaviors are higher among poor youth (Huston, 1991). 

It is clear, then, that American communities need to have greater access to existing effective youth-serving programs. Furthermore, given the unprecedented scope of the contemporary challenges to the healthy development of American children and adolescents, new efforts must be devised, studied and, if effective, sustained. 

This proposal presents the plans for a study of the impact of one such program, Health Rocks!, an initiative conducted through the auspices of the National 4-H Council and supported by the Philip Morris Company. Health Rocks! has ambitious goals. Its aims are to reduce youth smoking and tobacco use, to help build life skills that lead to healthy lifestyle choices with special emphasis on youth smoking and tobacco use prevention, to engage youth and adults in partnership to develop and implement community strategies that promote healthy lifestyle choices, and to involve youth as full partners, and to build positive, enduring relationships among widely varying communities to address youth risk behaviors. When embedded within the broader vision, values, and programmatic approach of 4-H in promoting positive youth development by engaging young people as active participants in the civic life of their communities, Health Rocks! has the potential to enhance the success of 4-H in strengthening youth and America’s communities. Both Health Rocks! and 4-H more generally may enhance “community youth development.” That is, together they provide young people with educational experiences that promote their health and life skills, offer a supportive environment for their development, provide them with caring and reliably available adults, and offer opportunities to “give back” to their communities to volunteer, serve, and lead.

PI: Levinson, James
Title: Improving Pregnancy Outcomes Through Positive Deviance In Egypt
Abstract:  Save the Children in Egypt (SC) proposes this three-year project to design and test a Positive Deviance (PD) program in Minia Governorate to improve pregnancy outcomes. The investigations on positive deviance based pcegnancy outcome activities will facilitate the extrapolation of PD results from interventions dealing with infant nutrition to pregnancy outcomes. By improving pregnancy outcomes, SC not only will reduce maternal malnutrition and depletion but also improve child survival and child growth.

PI: Linsenmayer, Thomas
Title: Corneal Epithelial Nuclear Ferritin and UV Protection
Abstract: Ultraviolet (UV) light constitutes a major environmental insult to all exposed tissues of the body, including those comprising the cornea and other underlying ocular structures. UV-light can damage a wide variety of macromolecular components including DNA resulting, for example, in cancer. This damage can be direct, or it can be indirect through the generation of reactive oxygen species (ROS).

Corneal epithelial (CE) cells, however, seem to be refractory to such damage. Cancers of these cells are rare, even though this tissue is transparent and exposed to mutagenic UV light and other sources of ROS. Previous studies suggest that one mechanism that CE cells have evolved to prevent damage to their DNA involves ferritin in a nuclear localization. This molecule seems to greatly diminish the effects of UV-produced ROS to DNA-most likely by sequestering free iron, which acts as a catalyst in generating hydroxyl radicals, the most damaging ROS. Other studies suggest that the nuclear localization of ferritin is effected by a nuclear transporter, which is termed "ferritoid". Ferritoid is comprised of two regions, one, which contains a nuclear localization signal (NLS) and is responsible for the nuclear transport, and another, which is involved in the binding to ferritin, which ferritoid subsequently carries into the nucleus. The mechanism of this interaction between ferritoid and ferritin, and the subsequent nuclear transport will be examined further. The fate of ferritoid following transport and whether phosphorylation is involved in regulating the transport will also be investigated.
The mechanisms responsible for regulating the production of ferritin and ferritoid will also be examined.

The studies will include whether the synthesis of these molecules is co-ordinate with one another and whether the synthesis of ferritin involves a unique type of translational regulation, which results in a low iron ferritin. Such a low iron ferritin may be highly efficient at iron sequestration and therefore protection against active ROS. For the synthesis of ferritoid, studies will involve whether "stress response elements" in the gene respond to ROS. Lastly, it will be determined whether the protection against damage by ROS provided by nuclear ferritin in CE-cells, can be afforded to other cell types in which ROS potentially have deleterious effects.

PI: Liscum, Laura
Title: Somatic Cell Mutants in Intracellular Cholesterol Transport
Abstract: Mammalian cells tightly regulate their cholesterol content and distribution. In the liver, the movement of cellular cholesterol to the endoplasmic reticulum (ER) is critical for its conversion to cholesteryl esters and subsequent incorporation into nascent lipoproteins. It is also essential for metabolism of hepatic cholesterol to bile acids. The route and mechanism of cholesterol transport to the ER is unknown. Elucidation of cellular factors responsible for cholesterol movement to the ER may reveal new therapeutic targets for hypercholesterolemia. We have isolated a somatic cell mutant with defective trafficking of plasma membrane cholesterol to the ER. Chinese hamster ovary (CHO) mutant 3-6 transports both newly synthesized and lipoprotein-derived cholesterol to the plasma membrane, but fails to mobilize cell surface cholesterol to the ER for metabolism or regulation of homeostatic responses. This phenotype is likely due to a change in the plasma membrane lipid composition since, despite increased levels of cholesterol in the 3-6 plasma membranes, 3-6 cells are resistant to amphotericin B, and an polyene antibiotic that forms pores in cholesterol-rich membranes.

Our hypothesis is that the 3-6 gene encodes a protein that affects the lipid organization of the plasma membrane. Loss of 3-6 activity alters the plasma membrane lipid domain structure such that cholesterol is both latent to amphotericin B and prevented from entering its endocytic pathway.

The Aims of this study are:
Specific Aim #1: To identify cDNAs that, when expressed in mutant 3-6 cells, correct the cholesterol transport defective phenotype. 3-6 cells expressing an ecotropic retroviral receptor will be transfected with a retroviral cDNA library, cDNAs that correct the phenotype will be identified. We will determine which of the correcting cDNAs encode the 3-6 protein.

Specific Aim #2: To identify proteins whose expression levels are altered by the 3-6 mutation. Comparison of CHO vs. 3-6 proteomes and gene expression patterns by two-dimensional gel electrophoresis and microarray analysis, respectively, has revealed candidate 3-6 proteins and pathways altered by the 3-6 mutation. Candidates from both approaches will be validated.

Specific Aim #3: To define the 3-6 induced changes in cellular lipid metabolism. We will determine how the 3-6 mutation alters the plasma membrane and/or ER lipid compositions. Sphingolipid trafficking will be examined in parental CHO cells and mutant 3-6. We will investigate the mechanism by which candidate 3-6 and proteins that suppress the phenotype modulate these membrane parameters.