PI: Gallagher, Kevin
Title: New Models for Global Economic Governance: A Research Program on Globalization and Sustainable Development
Abstract: Since 1999, the Global Development and Environment Institute's Globalization and Sustainable Development Program has examined the social and environmental impacts of economic integration in the Western Hemisphere. GDAE researchers have partnered with researchers and advocates in Mexico and across the Americas to provide and disseminate empirical research on the region's experience with rapid economic integration. GDAE has documented many of the social and environmental impacts of free trade in the hemisphere, giving voice to Latin American researchers, helping to enhance public understanding of how the U.S. relates to the world regarding the governance of global economic integration, and helping to provide new information and analysis in the search for sustainable approaches to the globalization process.

From the beginning, GDAE's goal has been to draw out lessons for domestic policy- making in the region, regional negotiations in the hemisphere, and for the current round of World Trade Organization negotiations. With key negotiations entering decisive stages, the next two years will prove critical for shaping the outcomes of these efforts. GDAE's current project, "New Models for Global Economic Governance," builds on the institute's success and its growing reputation and policy impact. GDAE's unique combination of rigorous university-based empirical research and direct engagement with the advocacy and policy processes has proved effective in bringing new information and analysis into the policy process, elevating the debate, and improving policy outcomes. In the next two years, GDAE will continue to broaden its focus from the environment to the more comprehensive analysis of sustainable development while expanding the scope of its analysis from trade agreements in the Americas to global governance through the WTO.

Institute researchers will contribute to the policy process through their own empirical research, continued collaboration with Mexican colleagues, expanding work with specialists from Latin America, and collaboration with global experts on key WTO issues. The project will focus on the particularly sensitive issues of liberalization in agricultural trade and foreign investment. In agriculture, GDAE and its collaborators are examining the implications of trade liberalization on the sector, with particular attention to the impacts on small-scale farmers. In investment, GDAE is studying the economic, social, and environmental impacts of foreign investment to determine the conditions that optimize these impacts and identify the policies that can foster sustainable industrial development.

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: Giannino, 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 Developing World
Abstract: The Global Development And Environment Institute at Tufts University is assembling a "Social Science Library" on CD, and will send copies of it, free of charge, to all university libraries in the developing world.

The CD, Titled Frontier Thinking in Sustainable Development and Human Well-Being, will contain about 300 megabytes (the equivalent of300 books, or 4,000 articles) of materials from books, journals, and other sources. It is being designed to offer a broad social science perspective with an emphasis on ways of thinking about human well-being, and about how to improve it in a sustainable manner.

The Social Science Library ("SSL") will be presented in well-structured, user- friendly, easily copied CDs. It is designed for use by teachers, researchers, students, and other citizens in developing countries. It will provide excellent examples of what the disciplines of anthropology, economics, history, law, management, planning, political science, sociology, or social psychology can contribute to thinking and teaching on matters of urgent practical and theoretical importance.

The project is designed to further the development of the social sciences in the developing nations, most immediately in terms of academic research, writing and teaching. It is intended to ensure that global debates on the future of the humaJ:l species will increasingly include social scientists from developing countries -while those voices will also be better positioned to influence their own local policies.

PI: Gordon, Leslie
Title: The Progeria Research Foundation Cell and Tissue Bank
Abstract: You are being asked to donate your blood and/or tissue to The Progeria Research Foundation (pRF) Cell and Tissue Bank. You are being asked to take part in this study because you are the parent or sibling of a child with Progeria. This cell and tissue bank exists because currently there is no known treatment for Progeria. We want to provide cells and tissue to researchers so that they can study Progeria and find out more about the possible causes of Progeria so that potential treatments may be developed in the future.

PI: Greenberg, Andrew
Title: Perilipiin 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: Chronic Benzodiazepines: Behavior and Neurochemistry
Abstract: Benzodiazepine agonists continue to be the principal pharmacologic option available for the treatment of anxiety, panic disorders, and insomnia. Despite an overall record of efficacy and safety that is generally favorable, concerns regarding tolerance, dependence, withdrawal syndromes, and abuse of benzodiazepines remain issues of medical and public health importance. Also of concern is the usage of these agents by the elderly, who may have increased susceptibility to adverse CNS depressant effects. There is continuing need for basic mechanistic data on the causes and consequences of tolerance and withdrawal; such data can form the basis for strategies to identify patients at highest risk, or to develop other pharmacologic interventions to minimize the risk of tolerance and dependence. We propose to continue and broaden our ongoing research program having this overall objective. The core of the model involves male CD-1 mice that receive continuous infusions of benzodiazepine agonists, or vehicle control, for up to 14 days via implanted osmotic pumps. During the period of infusion, and in the 7-day post-infusion withdrawal period, the following outcomes are determined: computerized ambulatory activity; pentylenetetrazole seizure threshold; in vivo benzodiazepine receptor occupancy; in vitro receptor binding; GABA(A) receptor function; receptor autoradiography; receptor subunit mRNA expression; and plasma and brain concentrations of infused substances. The principal research questions to be addressed include the following: a. Do benzodiazepine agonists with relative selectivity for the BZ1 receptor subtype have reduced liability to produce tolerance, dependence and withdrawal? b. Does the protein kinase C second messenger pathway have a modulatory role in benzodiazepine-associated tolerance? c. Does the excitatory amino acid (EAA) receptor system co-modulate the development of tolerance and withdrawal associated with benzodiazepine agonists, and does pharmacologic antagonism of specific EAA receptor systems modify these phenomena? d. Do aging organisms have differential patterns of benzodiazepine tolerance and withdrawal? Are such differences explained by protein kinase C or EAA regulatory systems? These studies should continue to provide mechanistic data relevant to the clinical management and prevention of tolerance and dependence problems associated with therapeutic use of benzodiazepine agonists.

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: Hassoun, Soha
Title: Tools for Designing and Integrating Configurable Components
Abstract: System design automation tools will be key to the successful development of next generation ubiquitous, communication-savvy, devices. To meet stringent cost, performance, power, and adaptability constraints during short design cycles, these tools must take a truly system view of the design. To help designers realize an optimal design, these tools must aid designers in evaluating design tradeoffs. The tools must also significantly facilitate and automate integrating system components.

Configurable (i.e. reconfigurable or adaptable) components have emerged within the past decade as a powerful and post-fabrication adaptable system component. While traditional Field Programmable Gate Array (FPGA) performance and capacity flourished due to advances in fabrication technology and FPGA-specific design improvements, their fine granularity results in poor performance for highly regular computations. Substantial research efforts investigated coarser granularity programmable data paths. These architectures vary in proximity to a controlling processor, in configuration abilities, and in their underlying computational model. The practical potential use, however, of these architectures and related compilers in a system environment remains unexplored.

This research proposal addresses the creation and usage of configurable adaptable components as Intellectual Property (IP). The research proposes a novel composition for a minimal adaptive component (MAC). The MAC is comprised of a single or hybrid configurable fabric, and a run time manager. This manager provides on-demand services, such as security checks, request buffering, and configuration caching, to clients (algorithms) wishing to use the configurable component. The manager also provides an API that facilitates using the configurable component. The research proposes novel algorithms for (1) efficiently selecting an optimal configurable component composed of one or more configurable architecture, and for (2) creating hardware-independent run time managers. This research will potentially enable system designers and CAD developers to assess trade offs of using different configurable components. It will also facilitate integrating configurable components within a system. Finally, the research provides an impetus for the development of IP usage models.

The educational plans of this CAREER proposal include integrating different aspects of system design into the Computer Engineering curriculum at Tufts University. In addition, the PI proposes a variety of activities for enhancing students' learning experiences and integrating research and education within the department. Finally, the PI outlines a plan for developing a valuable educational CAD program at the national level.

PI: Holcomb, Philip
Title: Plausibility and Syntactic Processing Load
Abstract: The focus of this study is the process that underlies sentence comprehension. Sentences convey information about how the meanings of words are related to one another --which entities are performing which actions, which entities are having which actions performed on them, what properties hold of which entities, etc. This information, known as the "propositional content" of a sentence, is largely determined by the syntactic structure of the sentence. For instance, in the sentence The boy who chased the girl fell down, it is the boy, not the girl, who fell down, because the boy is the subject of fell, whereas the girl has no syntactic relation to fell.

Listeners and readers compute syntactic representations and use them to determine propositional meaning rapidly as they encounter and recognize the words of a sentence. This process is more or less difficult depending on the representations to be constructed and the cues available as to what they are. For instance, the sentence in (I) is nearly impossible to understand. However, the same propositional content can be expressed in a much simpler syntactic form, as in (2), making it much easier to understand. Similarly, if the pragmatic plausibility of the syntactically-specified meaning relations can restrict likely alternatives, a sentence is easier to understand. (3) has the same syntactic structure as (1) but is easier to understand.

(1) The boy who the mother who the girl saw chased fell down.

(2) The girl saw the mother who chased the boy who fell down.

(3) The boy who the rock that the girl threw hit fell down.

In this project, we propose to investigate this second type of influence on the ease/difficulty of assigning syntactic structure and determining propositional meaning. We will primarily examine cases involving ambiguity.

PI: Holcomb, Philip
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 project 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 Ll and L2. A major aim of the present project is to test a new model ofL2 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 Ll 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-Lllexical 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: HUBER, BRIGITTE T.
TITLE: QPP: Protease that Prevents Apoptosis in Quiescent Cells
ABSTRACT: Not Listed

PI: Islam, Shafiqul
Title: Estimation of Evapotranspiration and Crop Water Stress Over Large Areas Using Remote Sensing Observations
Abstract: Many water resources, agricultural, and forest management applications require estimates of evapotranspiration (ET) and crop water stress (CWS) over a range of spatial and temporal scales. Operational methods of ET and CWS estimation using standard meteorological input variables have been developed, refined, and tested over the last several decades. While these approaches have shown a varying degree of success, they require surface and meteorological observations that are not easily available over large areas to provide spatially distributed estimates.

Satellite remote sensing provides an unprecedented spatial coverage of critical land surface and atmospheric data that are logistically and economically impossible to obtain through ground based observations. We propose a methodology to estimate ET and CWS using primarily remote sensing information over large heterogeneous areas. A key motivation is to develop a systematic methodology for ET and CWS estimation that can be easily implemented with routinely available remote sensing observations over different types of terrain and climate conditions. The proposed estimation methodology provides a new paradigm for the space-time estimation of ET and CWS over large areas. Our proposed algorithm will provide a temporally continuous and spatially consistent map of ET and CWS over large areas. Our approach is based on a contextual relationship between remotely sensed surface temperature and vegetation index. Principle objectives are to (a) Develop and refine ET and CWS estimation algorithms for large heterogeneous areas by using mainly remotely sensed data, (b) Test and validate the proposed methodology for different regions in the United States, (c) Compare and contrast uncertainties in the estimates between the proposed method and currently available methods, and (d) Develop a user-friendly estimation tool for agricultural, water resources, and forest management applications using routinely available remote sensing data.

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. We propose to study a Drosophila gene, ebony, that is known from recent molecular studies to be under clock control, and we postulate that it is important for the circadian patterning oflocomotor activity. The ebony gene encodes N-alanyl-dopamine synthetase (BAS; reviewed in 30). This enzyme is expressed in the adult brain, and is required for the regulation of dopamine (DA) and/or _-alanine (BA) pools; in brain and other tissues, BAS catalyzes the conjugation of BA and DA to produce N-_alanyl dopamine (NBAD; Fig. I in Project Description). Not surprisingly, it has been shown that ebony mutations are associated with increased levels ofDA and p-alanine. Of relevance to the present proposal, our previous work has shown that ebony mutants exhibit severe circadian behavioral defects (22). We previously demonstrated that ebony mutations cause either arrhythmic activity patterns or complex and abnormal patterns of activity, and we attributed the behavioral defects to elevated DA and/or BA levels. Those studies suggested that changes in biogenic amine levels may be relevant for the clock control 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 oflocomotor activity, which is predominantly restricted to the subjective day in wild-type individuals.
An independent gene chip study showed that RNA ITom another gene known as black, which is required for the synthesis of BA, oscillates in a circadian manner (18), and 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. The aggregate of the behavioral and molecular data on ebony strongly suggests that the circadian regulation of BAS activity, and the consequent regulation of dopamine and N-alanine levels, may be important for the rhythmic patterning oflocomotor activity. Indeed, dopaminergic control of motor activity has been observed in both invertebrates and vertebrates (e.g., ref 4). The work proposed in this application seeks to advance our understanding of ebony and black functions. The aims of the research are to:

1. Determine the neuronal localization and rhythmic properties of ebony RNA and protein. These studies will provide important insights about the neural centers controlling activity rhythms.
2. Anatomically map connections between the Drosophila clock cells and neurons containing ebony protein. This analysis will allow us to delineate the cellular output pathway from the clock to the neural loci controlling activity.
3. Characterize the circadian control of activity in flies lacking the ebony neuronal cells. We will utilize the GaI4/UAS binary expression system with the cell death gene reaper to genetically ablate cells expressing ebony, in order to verify that these cells are critical for the circadian control of rhythmicity.
4. Explore the circadian function of black and other potential biogenic amine mutants.

PI: JACKSON, F. ROB.
TITLE: Role of Andante/CKIIB in the Drosophila Circadian Clock
ABSTRACT: Not Listed

PI: Jacob, Michele
Title: Regulation of Neuronal Synaptic Components
Abstract: Nicotinic acetylcholine receptors (nAChRs) function at key interneuronal and central sensory organ synapses. Their activation mediates excitatory transmission, reinforces nicotine addiction, increases memory formation, and regulates the sensitivity of hearing. Malfunction of cholinergic synapses has been implicated in developmental and neurodegenerative disorders such as Alzheimer's disease, schizophrenia, nocturnal frontal lobe epilepsy, and autoimmune autonomic neuropathies. Despite the physiological importance of nicotinic synapses, little is known about the molecular mechanisms that direct their assembly during development. Further, proper synapse formation and function require precise alignment of pre- and postsynaptic specializations, but the underlying mechanisms are poorly understood. Our recent studies identify adenomatous polyposis coli (APC) as a key molecular player in interneuronal cholinergic synapse assembly in vivo. We show that APC is essential for localizing a3-nAChRs to postsynaptic sites, and thereby identify APC as the first non-receptor protein to function in nAChR targeting to neuronal synapses. We propose that APC has two key synapse organizing functions: (1) directing nAChR transport to and/or stabilization at postsynaptic sites (Aim1) and (2) directing the alignment of pre- and postsynaptic specializations by anchoring retrograde signaling complexes at sites of nAChR accumulation (Aim2). Further, we posit that APC'S interactions with three essential postsynaptic components: microtubule plus end binding protein-1 (EB1), beta-catenin and postsynaptic density protein-93 (PSD-93) mediate these essential aspects of synapse formation. We will use loss-of-function and gain-of-function strategies to test the specific roles of these APC interactions and binding partners in organizing cholinergic synapses in vivo. We will test APC'S role at two different nicotinic preparations: a3-nAChR-containing peripheral ciliary ganglion neuronal synapses and a9-nAChR-containing central efferent olivocochlear synapses on sensory hair cells of the inner ear. The experiments will use genetic, molecular, morphological, biochemical and functional approaches. The studies will provide important new insights into molecular interactions that direct the assembly and function of cholinergic synapses. Further, the studies will determine whether the organizational mechanisms are shared between peripheral neuron and central sensory nicotinic synapses.

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 to support the research and development of a comprehensive recruitment strategy and actionable marketing plan for attracting students and placing graduates.

PI: Kaplan, David
Title: First Annual Biomedical Engineering Conference in Vietnam
Abstract: The First International Conference on the Development of Biomedical Engineering in Vietnam will be held from July 27th to July 29th 2005 in Ho Chi Minh City. This conference is jointly organized by the Ho Chi Minh City University of Technology (Vietnam) and Tufts University (USA).

PI: Kaplan, David
Title: Inducible Surface Hydrophobicity of Microbial Consortia for Biofilm Remediation
Abstract: Free-phase pollutants are persistent environmental contaminants. The feasibility of an enhanced process of in situ free-phase pollutant removal by micro-organisms that had been pre-adapted to the pollutant free-phase surface was confirmed during Phase I. The high affinity of the micro -organisms to the pollutant free-phase resulted from their increased hydrophobicity. More importantly, the inductiveness of the culture hydrophobicity by exposure to contaminants was demonstrated. It was shown in Phase I that the biofilm formation on free-phase pollutants was a two-stage process, involving first the colonization of the microbial population at the interface and secondly, a “steady" stage in which the culture continuously degraded the contaminants and the flux of chlorinated solvents into water was reduced by two orders of magnitude.

During Phase II, the pre-adapted micro-organisms will be genetically evaluated. Mechanisms of controlled microbial death upon the substrate exhaustion will assure environmental safety. The biosafety of the selected organisms will be confirmed. The microbiological products will be formulated and their stability will be evaluated for their use and commercialization during Phase III. Pilot-scale studies involving the monitoring of the distribution of the organisms and their performance after injection in a simulated soil/water environment will also be carried out.

PI: Kaplan, David
Title: Silk Protein Polymer Designs for Improved Expression and Processing
Abstract: Silk fibers formed by insects and spiders are noted for their remarkable mechanical properties as well as their durability and biocompatibility. The exceptional solubility in vivo (20-30% w/v) of these proteins is dictated by both the need to produce solid fibers with a high packing fraction and the high mesogen concentration required for lyotropic liquid crystalline spinning, while also achieving high end mechanical properties for survival (orb webs, cocoons). A further design requirement for silk proteins is the predominance of hydrophobic amino acid residues to provide for hydrophobic interactions, water exclusion, and beta-crystallite formation to produce these strong insoluble threads. Thus the domain structure of silk proteins to accommodate high solubility of hydrophobic proteins in aqueous solution but also to permit the formation of water-insoluble final products (fibers) is critical. Additionally, silk proteins need to avoid premature precipitation as β-sheets during storage and processing. Combining knowledge of the solution state behavior, protein folding requirements and silk genetic/protein designs employing complex block-copolymer attributes, offers new experimental directions in the construction, expression and assembly of silk proteins that can help overcome longstanding limitations with silk production in heterologous systems. These studies also offer unique opportunities to marry processing environment (water) with gene designs (mimickitig silk block designs) to optimize outcomes during intracellular and extracellular processing of these complex proteins. The result of this new insight has the potential to lead to substantively improved synthesis, recovery and processing of recombinant silk proteins into Useful fibers and other silk-based materials. 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 significance 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: Liquid Crystal Phase Transitions in the Hierarchical Assembly of Collagen
Abstract: Collagen is the most commonly used natural biomaterial, whether reprocessed from bovine sources, generated as genetically engineered variants, or reconfigured as gelatin-based matrices. Collagen is structurally complex due to hierarchy at different length-scales and this structural complexity forms the basis for the diverse mechanical and biological features of collagen that vary with each tissue type. There is an exhaustive literature on fibrillar collagens, including several characterized by detailed structural models. However, there is little experimental data available describing the role and mechanism of the helical segments formed from collagen proteins in controlling the size and shape of the assembled fibrils. Insight into these issues is critical in order to design biomaterials in vitro based on collagen to match mechanical and biological profiles in vitro and in vivo, and to understand collagen processing and assembly in vivo. Liquid crystallinity is the likely mechanism for the portion of the self-assembly process that occurs prior to specific recognition and binding related to fibril organization. In fact, liquid crystallinity and liquid crystal-like textures have been observed for fibrous proteins such as collagen; reported as helicoids. An in depth understanding of the forces controlling collagen hierarchical assembly will provide new avenues for the formation of collagen-based biomaterials for a variety of needs. Additionally, microgravity will provide advantages to the study of biomacromolecular hierarchical self-assembly in collagen-based biomaterials since the effects strongly influencing the liquid crystalline morphology and that of the materials fabricated from liquid crystals, such as surface anchoring and density fluctuations, can be eliminated.

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 PI has identified a novel pathway in mesenchymal stem cell (MSCs) differentiation to ligament/ tendon tissues mediated jointly by SMADB 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 Ion silk scaffolds. Genetically engineered MSCs will be cultured on silk scaffolds in vitro. Differentiation will I 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.

PI: Kaplan, David
Title: Tissue Engineered Ligaments
Abstract: The proposed tissue engineering studies are motivated by the medical need for biologically based, functional tissues for transplantation. An important issue with respect to engineering tissues in vitro is to understand the role of environmental factors on the process of tissue formation. Controlled in vitro studies of tissue development in three-dimensional culture can improve our fundamental understanding of regulatory signals directing precursor cell differentiation, affecting the structure and function of engineered tissues formed, such as Anterior Cruciate Ligaments (ACL). In addition, bioreactor systems which allow the stimulation of the cells growing on these matrices by physical forces that are physiological in nature (e.g., tension and torsion in the case of the ACL) could be extended to other skeletal tissues and become a valuable tool for basic biomedical research. Specifically, we propose to test the hypothesis that mechanical forces that are physiological in nature, intensity and frequency for native ACLs will direct bone marrow stem cell differentiation into ligament-forming cells and result in the in vitro formation of functional equivalents of native ACLs. Our objectives are to (a) gain fundamental insight into the relationships between mechanical stimulation and differentiation of human bone marrow stem cells (hBMSCs) into fibroblasts expressing biochemical and genetic markers, characteristic for cells in native ligaments, and (b) to engineer ligament-like structures starting from hBMSCs. In our experimental plan we will study the culture hBMCSs on 3-dimensional cross-linked collagen fiber scaffolds in a bioreactor designed to provide a highly controlled biochemical and mechanical environment.

The study will focus on elucidating the relationships between specific biochemical factors [oxygen and serum] and mechanical regulatory signals [tension, torsion, both], related to cellular differentiation and ligament tissue structure and function. We will build upon key observations in the Preliminary Data that demonstrate: (a) hBMSCs undergo selective differentiation to ligament cells due to mechanical forces in the absence of specific exogenous growth factors, and (b ) ligament structures are formed during the process. We expect that different combinations of biochemical factors and applied mechanical stress will influence the rate and extent of differentiation of hBMSCs into ligament-Iike cells leading to ligament structures in a manner dependent not only on the presence of the specific individual factors, but also on their interactions. The evaluation of the responses will be based on statistical analysis oftime-dependent (0,7, 14 and 21 day) changes in cell proliferation (DNA content), upregulation of the ligament-specific mRNA transcripts (real time RT -PCR), production of collagen type I via western blot, and the mechanical properties [ultimate tensile strength, linear stiffness, yield point, percent elongation] of the ligaments. The target mechanical properties of the ligaments are an ultimate tensile load (N) = ≥1,500 N and a linear stiffness (N/mrn) of 100 N/mrn ≤ 600N/mrn.

PI: Kaplan, David
Title: Tissue Engineering – The Next Generation
Abstract: We intend to organize a meeting enTitled Tissue Engineering – The Next Generation, to be held May 2-4 2005 in Cambridge MA (2.5 days, Hotel Marriot at Kendall Square). Our project is motivated by the growing need to identify the scientific and technological gaps between the fields of developmental biology and tissue engineering in order to guide or define the next generation of scientific inquiry in the field and thereby facilitate the development of new treatment options that can substantially improve human health.

Our goals are (1) To rethink where the science and clinical application of tissue engineering needs to go in the coming years, (2) To identify the needs and directions in key areas (cell sources, biophysical signaling, scaffolds and bioreactor cultivation, pre/clinical studies), (3) To document these needs as a guide to the NIH for future initiatives as well as disseminate the information to the broader scientific community via a cohesive publishable format, and (4) To enhance national and international interactions in the field of tissue engineering via information exchange at and after the meeting. To meet these goals we propose a workshop format with one keynote lecture (Robert Langer: Tissue engineering 2020), a set of coordinated sessions focused on questions we will attempt to address (e.g., engineering complex and self-repairing tissues, tissue vascularization, biological inputs into functional tissue engineering, biophysical regulation of stem cell fate and tissue assembly), and a set of related panel discussions. We have invited about 50 leaders in the field from academia, industry, and NIH, from the US and abroad, and we have received an unanimously enthusiastic response about their participation and willingness to work towards the goals of the workshop. Conference co-chairs (Drs Gordana Vunjak-Novakovic from MIT and David Kaplan from Tufts University) will be responsible for the detailed planning and organization of the meeting, and coordination of the individual talks, sessions, and panels towards the preparation of the final document (summary and recommendations) and the proceedings (published in Tissue Engineering journal). The workshop will be open free of charge to young investigators (graduate students, post docs, residents). We will work with the invited faculty so that we can develop the best possible plan for the workshop, take advantage of the collective expertise of all participants and maximize the impact and dissemination of the workshop outputs.

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. Kimsky's Tufts' website www.tufts.edu/~skrimsky.

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. aibicans 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: 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: Leyland, Timothy
Title: Pastoralist Conflict Initiative (PCI)
Abstract: As of 1 May 2002, the livestock programme has two coordinators based respectively in Khartoum and Nairobi under the supervision of an overall north/south livestock programme coordinator, with approximately 20 field staff, predominantly veterinary surgeons, but also including epidemiologists and various support staff. The north/south livestock programme coordinator has also acted as the southern coordinator and was supported by an assistant coordinator. As a result of a review jointly conducted by FAO and Tufts in March 2003, FAO and Tufts will maintain their collaborative partnership and in accordance with the findings of the review the role of the Tufts input has been modified (see first and second interim reports to this Letter of Agreement). The Tufts role has in accordance with the recommendations of the FAO-Tufts review transformed over the last few months to provide greater technical, coordination and policy direction and advice and to reduce the amount of involvement in administrative and operational activities. This is seen as a means to maximize the impact of the added value Tufts brings to the southern Sudan Livestock Programme and its partnership with FAO. The livestock staff team as a whole manage the day-to-day operations of:

• disease-control procedures, including vaccination for rinderpest (should it be needed) and other important diseases such as contagious bovine pleuropneumonia, haemorrhagic septicaemia, anthrax, blackleg and contagious caprine pleuropneumonia;
• disease-investigation and surveillance to provide early warning of major disease events with facilitation of veterinary sampling and laboratory services for disease identification and control;
• provision of drugs and vaccines; .provision of training courses for Animal Health Auxiliaries and Community Animal Health Workers (CAHW);
• coordination and management of a veterinary privatization programme aimed at the provision of a sustainable veterinary service in south Sudan;
• support to the coordination of the animal health delivery system made up of more than 20 Non-governmental Organizations and various counterpart organizations.

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: Linsenmayer, Thomas
Title: Corneal Stroma: Synthesis and Assembly of Collagen
Abstract: The avian cornea provides a model which has the major features of the human cornea, including all of the distinct layers of extracellular matrix: Bowman's membrane, stroma, and Descemet's membrane. The corneal stroma develops in stages which involve the deposition, modification, and maturation of two collagenous matrices. The first of these is the primary stroma, an epithelially-derived matrix which serves as a template for the mature, secondary stroma, which is produced by the stromal fibroblasts. This application proposes to study further the development of the primary stroma and the changes in this matrix that result in formation of the mature stroma. The primary stroma is produced as a compact matrix which subsequently undergoes swelling. This swelling, which is a critical event required for subsequent development of the mature stroma, results in separation of the collagenous layers which, in turn, allows for migration of periocular mesenchymal cells into the matrix. These cells then differentiate into the corneal fibroblasts that produce the mature stroma. Our previous observations suggest a model in which the primary stroma, when newly synthesized, remains compact due to bridging/crosslinking of collagen fibrils in adjacent layers by a fibril-associated collagen (collagen type IX). Other of our observations suggest that enzymatic cleavage of this molecule allows for subsequent fibril separation, matrix swelling, and mesenchymal cell invasion, and that this cleavage most likely involves matrix metalloproteinase (MMP) activity. MMPs are a family of enzymes whose activity can be regulated at multiple levels, including positive regulation by their synthesis and proenzyme activation, and negative regulation by naturally occurring inhibitors (TIMPs). Preliminary observations also suggest that more than one of these proteinases may be involved. Studies will be done to identify which enzymes are involved, what controls their synthesis, and how their activities are regulated. These studies potentially have broad significance, since matrix proteinases, and especially the MMPs, have been implicated in many aspects of development in both ocular and non-ocular tissues, in tissue injury and repair, and in cancer metastasis.

PI: Liscum, Laura
Title: Analysis of a suppressor of the Niemann-Pick C phenotype
Abstract: Niemann-Pick C (NPC) is caused by mutations in one of two genetic loci, NPC1 and NPC2. Our focus is on NPC1 because mutations in this locus are responsible for 95% of the clinical cases. The most striking consequence of NPC1 dysfunction is aberrant cholesterol movement, which results in lysosomal storage of cholesterol and glycosphingolipids. The mechanism by which NPC1 facilitates lipid transport from endocytic compartments to other cellular membranes is unknown. Currently, there is no definitive therapy for NPC. Elucidation of cellular factors that suppress the NPC phenotype may reveal new therapeutic targets. We have isolated a somatic cell model of NPC disease with an unusual phenotype. Chinese hamster ovary (CHO) mutants 4-4-D (disease) and 4-4-S (suppressed) belong to the same complementation group as NPC fibroblasts and contain the identical base insertion in the NPC1 gene, which results in a frameshift and termination. Mutant 4-4-D shows the classical NPC disease phenotype of lysosomal cholesterol storage; however, mutant 4-4-S shows no cholesterol storage by filipin fluorescence microscopy. The 4-4-S phenotype is likely due to expression of a gene that suppresses the mutant phenotype. Surprisingly, mutant 4-4-S still shows defective low-density lipoprotein stimulation of acyl-CoA/cholesterol acyltransferase (ACAT) in the endoplasmic reticulum (ER), which is characteristic of NPC. Our hypothesis is that the 4-4-S suppressor is a protein that mobilizes cholesterol out of endosomes, but fails to deliver the cholesterol to the ER. To test this hypothesis, we will perform the following Aims. Specific Aim #1: To identify the gene product(s) that suppresses the phenotype of mutant 4-4. Specific Aim #2: To determine the fate of LDL-cholesterol in mutant 4-4-S. Specific Aim #3: To examine intracellular trafficking of glycosphingolipids in 4-4-D and 4-4-S cells. Elucidation of cellular factors responsible for cholesterol clearance from NPC lysosomes will reveal potential therapeutic targets for this devastating neurodegenerative disease.

 

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