PI: Ganda, Kanchan
Title: Ryan White C.A.R.E Act HIV/AIDS Dental Reimbursement Program
Abstract: Tufts University School of Dental Medicine is located in downtown Boston and is committed to providing treatment to all patients requesting services. This is accomplished in the teaching programs through fourteen weekly clinical sessions scheduled both day and evening for the convenience of patients. In addition, several special patient care units, a faculty practice and a major program for the handicapped attract with special needs from throughout the region. A minimum of 700 patients receive dental treatment each day. A significant increase in the number of patients suspected or confirmed with infectious diseases has developed in recent years. The most dramatic increase is presented by the HIV population. In order to provide high quality service to this special needs group, a physician appointed to the faculty oversees their dental treatment and provides medical referral services. The dental health care program in preventive dentistry has been expanded to accommodate HIV patients and they benefit remarkably from this service. The School of Dental Medicine is the second largest dental educational institution in the United States and is responsible for the education of 60% of the dentists in New England. An extensive continuing education program provides more than 85 courses each year for alumni and dentists in the Northeast region. In recent years greater emphasis has been placed on infection control, the identification of symptoms related to HIV, management of oral lesions associated with HIV and enhancement of oral health of HIV patients. In addition, over 65 courses have been presented externally to dental professionals by members of the faculty in the diagnosis, oral manifestations and management of HIV patients. Thus, the School is dedicated and committed to serving this population group and helping to train professionals to provide this service.

PI: Garlick, Jonathan
Title: The Impact of the Tumor Stroma on Progression of E-cadherin-Deficient, Squamous Cell Carcinomas in 3D, Human Tissue Models
Abstract: Cellular signals that control the progression of oral cancer cells are not well understood.

However, it is still not clear how loss of E-cadherin causes these tumor cells to switch to this more aggressive behavior. One factor that may contribute to cancer becoming more highly invasive when E-cadherin is lost could be the connective tissue stroma surrounding invading cancer cells. While it is known that stromal cells such as fibroblasts, endothelial cells, and immune cells make factors that communicate with invading cancer cells, no one knows which molecular and cellular factors are important in stimulating cancer progression in cells that have already lost cell-cell adhesion. I hypothesize that the properties of invading tumor cells that have previously lost cell-cell adhesion, are driven by signals from the surrounding stroma. To test this hypothesis, I will combine two state-of-the-art technologies including:

1. Fabrication of engineered human three-dimensional (3D) tissues that mimic squamous cell carcinoma:  I will grow in vitro and in vivo 30 tissues to simulate the features of squamous cell carcinoma as it occurs in humans. These tissues will contain cells in which E-cadherin's adhesive function is suppressed, to determine which molecular signals from the adjacent stroma may critically activate the more highly aggressive tumor cell behavior previously seen in research in my mentor's lab.

2. Laser Capture Microdissection (LCM):  I will use LCM to isolate cells from both stromal and carcinoma cells in 3D tissues to analyze their global patterns of gene expression in two ways:
   1. Expression in stromal cells adjacent to E-cadherin-deficient and -competent cancer cells
   2. Expression in E-cadherin-deficient and -competent cancer cells in different stromal environments

The goal of my research is to further understand how cross-talk between the stroma that surrounds squamous cell carcinoma cells that have lost E-cadherin-mediated cell-cell adhesion can activate their invasive properties. If I am successful in this project, I hope that future research will be able to develop new treatments to change these factors and to block the spread of invading cancer.

PI: Georgakoudi, Irene
Title: CAREER: Non-Invasive Modalities for Optical Imaging of Cell-Matrix Interactions
Abstract: Interactions between cells and the surrounding extra-cellular matrix dictate the fate of tissue development in the context of both normal tissue generation/regeneration and disease initiation/progression. In this proposal, we seek to merge advances in biophotonics and regenerative medicine to gain new insight into these important interactions between cells and matrices.

To address this challenge, we plan to develop and optimize optical spectroscopic imaging methods to non-invasively monitor cell-matrix interactions in dynamic bioengineered tissues grown in vitro. Of interest will be optical signals associated with matrix remodeling, cell differentiation and viability and their relationship to tissue functionality. Importantly, the initial baseline understanding of the applicability of these new tools to this complex system of matrix and cells offers a broader platform for the study of how changes in matrix composition, chemistry or structure impact cell biology.

Our ultimate goal is to demonstrate that optical methods can advance the understanding of dynamically changing cell-matrix interactions during tissue development in health and disease. We expect that this knowledge will have a significant impact as it may lead to the optimization or development of novel, non-invasive technologies to:

1. Monitor in real-time engineered tissue development and functionality in bioreactors and in vivo for regenerative medicine applications
2. Detect diseases, such as atherosclerosis or cancer, characterized by altered cell/matrix interactions
3. Monitor the efficacy of standard and novel treatments aimed at modifying these interactions

The strategy that we will employ involves the confluence of three types of research activities, with the central theme that of optical tools to interrogate structure and function.

Our first goal is to develop instrumentation that is optimized for non-invasive imaging of engineered tissues. We will use methods based on fluorescence and light scattering as endogenous sources of contrast, including confocal microscopy, multi-photon excited fluorescence, second harmonic generation and light scattering spectroscopic imaging. These methods will yield information on both the biochemical/physiological (fluorescence) and morphological/organizational (light scattering) status of tissues.

Our second goal will be to characterize the light scattering and fluorescence properties of isolated cellular and matrix components at different stages of development that we will encounter in the third phase of the project. We will specifically consider:

1. Human mesenchymal stem cells at various stages of differentiation into osteoblasts
2. Protein-based degradable scaffolds useful in bone tissue engineering scaffolding

Our third goal will be to monitor non-invasively the fluorescence and light scattering properties of developing engineered bone tissue from the silk, collagen and cell systems, and to attribute optical changes to specific interactions between these components. The proposed work will be performed in collaboration with the Tissue Engineering Resource Center at Tufts, where the tissue engineered bone systems are already available.

While the focus of this proposal is to monitor bone tissue engineering, we expect its scope and impact to be extended by collaborations with affiliated investigators working on tissues, such as cartilage, heart, liver and skin. These research efforts will be highly integrated with the educational component of this project, which involves activities from the K to graduate level. A number of students will be engaged to carry out the proposed studies in the context of PhD, master’s and senior theses, University-wide supported undergraduate research initiatives and a highly innovative undergraduate curriculum in biomedical engineering with research-centered coursework that begins during the sophomore year.

Directly relevant to the research will also be the development of a new course on optical methods in Bioengineering. Outreach efforts involving the development of simple demonstrations of optical principles for K-12 students and training for high-school science teachers will be coordinated through the Center for Engineering Educational Outreach at Tufts.

Finally, the PI is committed to mentoring and recruitment of women and underrepresented minorities and will continue to serve as an active mentor of the Computer Science, Engineering, and Mathematics Scholars at Tufts, a program that promotes the academic advancement and degree achievement of undergraduate students from potentially low income backgrounds, with a particular emphasis on minority and female students.

Thus, the broad impact of the proposal relies on its strong interdisciplinary training and mentoring component for students from the Tufts and the broader community, and the development of methodologies that will become available to the scientific community and may impact significantly human health by inspiring advances in regenerative medicine and medical optical diagnostics.

PI: Georgakoudi, Irene
Title: Light Scatter in Vivo Flow Cytometry to Monitor Cancer
Abstract: The main goal of this combined R21/R33 proposal is the development and evaluation of light-scattering based in vivo flow cytometry (LSIVFC), a novel method for the quantitative monitoring of different circulating blood cell populations, including cancer cells and white blood cells.

If successful, LSIVFC will have significant applications in the clinical management of cancer patient populations, as a non-invasive method for:

• Detecting circulating cancer cells, which may be particularly useful in the detection of minimal residual disease after cancer treatment

• Monitoring the effects of chemoprevention, photodynamic treatment and bone marrow transplantation to optimize treatment

• Improving our understanding of the relationship between circulating cancer cells and the metastatic or relapse potential

To achieve this, the project involves close collaborations between scientists and clinicians with expertise in biomedical instrumentation and spectroscopy, tumor biology, tissue engineering, regenerative medicine and hematology.

In the R21 phase, we will concentrate on in vitro studies, aimed to establish the wavelength- and angle-dependent light scattering regions that optimize detection of distinct blood cell populations of interest, such as cancer cells and white blood cells (WBCs) (Aim 1).

The capabilities of this method will be evaluated using in vitro models of blood microcirculation (Aim2).

Using the information acquired from these in vitro studies, we will perform measurements with a small number of animals to illustrate the feasibility of LSIVFC studies for monitoring circulating cancer and WBCs (Aim 3).

In the R33 phase, we will develop and optimize this technology for in vivo data acquisition and analysis, and we will illustrate the potential clinical and basic research applications of LSIVFC in the context of detection of circulating prostate cancer and leukemic cells during disease progression and in response to treatment, bone marrow transplantation and WBC activation.

This proposal is highly relevant to the improvement of public health and to the care provided to cancer patients in particular. The development of a non-invasive method for monitoring circulating white blood and cancer cells would provide a non-invasive, painless and potentially more sensitive method for detecting cancer and monitoring closely the response of individual patients to a given treatment.

PI: Gianinno, Lawrence
Title: The Role of Family and Community-Related Experience in the Development of Young People's Economic Understanding
Abstract: I plan to recruit children and parents from two different groups, and form two corresponding samples. These groups will be identified and chosen based on their subscription to particular social relationships, moral and other normative rules, and responsibilities and obligations in economic activities involving the family and community in which the children grow up.

Control variables for the two groups will include socioeconomic status, race, and ethnicity/nationality. Religion will be a key variable distinguishing between these two groups. In the proposed research, this contrast will be operationalized by studying children and families from the same ethnic group who are adherents of either of two religions, for example, Muslim and Christian youth, within the greater Boston area. Considerable evidence indicates that individual religious denominations promulgate the moral acceptability and unacceptability of particular economic practices, in the process addressing issues central to the interests of this research (Gonzalez & Maison, 2004; Kuran, 2004; Kurien, 2004; Schmidt, 2004). Such issues concern, for example, social relationships, responsibilities, and obligations in economic transactions, as well as the appropriateness of women engaging in work that puts them in contact with men. (For this reason, gender is another important variable I will assess in this study.) If feasible, I will control for degree of religiosity among the recruited parents. 

If it is also necessary (as I expect will be the case) to recruit children of immigrants and their parents for this study, then I will also control for the length of time the parents, in particular, have been in this country. I will study one age group of children in order to carefully begin to address the central question of this project.

Children in this study will be 11-12 years of age. My prior work on this topic (Gianinno, 1999, 2004) indicates that by this age children have an intuitive understanding of economic transactions that is similar if not equivalent to that of their parents—except, perhaps, in regard to the world of work. This particular age also marks the time when children’s explorations and choices related to their vocational identity become more adult-like (Gotifredson, 2002; Savickas, 2002; Vondracek, Lerner, & Schulenberg, 1986), incorporating considerations of social class and personal ability into their self-concepts, narrowing their ideas of what are acceptable and attainable occupations, all of this suggesting that they are becoming increasingly aware of and sensitive to contextual information about the work world. 

As noted above, gender is an important variable in this research, due to the rules of some religions concerning the role of women in the workplace. I estimate I will need to recruit a sample of at least 60 boys/girls in each group to provide sufficient statistical power (.80) to detect proportional differences of .25 or greater (two-tailed alpha = .025). The 120 children in each group will each be joined by one of their parents. The total number of interviews across both groups will be 480.

PI: Gorbach, Sherwood
Title: Center for Metabolic Research on HIV and Drug Use
Abstract: The aim of this Center is to provide scientific resources for studying the nutritional, endocrine, and metabolic aspects of HIV infection in chronic drug users of different ethnicities (Caucasian, Hispanic, and African-Americans) who are using various types of illicit drugs. HIV-positive drug users face a unique set of issues and challenges that may exacerbate their illness, including poor nutrition, inadequate medical care, difficulties adhering to complicated therapeutic regimens, and the use of specific drugs known to impair metabolic function. Investigation of the overall nutrition, endocrine, and metabolic effects of HIV infection in the general HIV-positive population, however, seldom extends to the drug using population, a group in which 50% of new HIV infections occurs. This Center will consist of 6 Cores: Administrative, Developmental, Drug User Resources, Nutrition and Metabolism, Endocrine, and Epidemiology/Biostatistics. The Center will build upon an existing infrastructure of studies in drug users at four East Coast institutions (Tufts University School of Medicine in Boston, Massachusetts; Brown University School of Medicine in Providence, Rhode Island; Johns Hopkins Medical Institutions in Baltimore, Maryland; and the New York Academy of Medicine, in New York City, New York). Tufts has developed programs in specific nutrition and metabolic aspects of HIV infection and has a research program in Hispanic drug users with and without HIV. Brown University has developed expertise in conducting research among specialized populations, including injection drug users, substance abusing women, and incarcerated drug users. Johns Hopkins has research in endocrine disorders among drug users with HIV, and has active longitudinal studies of African-American injection drug users. The New York Academy of Medicine's Center for Urban Epidemiologic Studies has several ongoing research studies related to HIV infection in drug using populations. The proposed center will combine the complementary resources and expertise that exist at each of the collaborating sites to develop a new research program that will focus on the nutritional, metabolic, and endocrine abnormalities in HIV-positive and HIV-negative drug users.

PI: Gorbach, Sherwood
Title: HIV Infection in Drug Users in Two International Sites 
Abstract: We propose to conduct longitudinal observational studies in two countries - India and Vietnam - each with significant problems of drug abuse and increasing incidence of HIV. In both countries the HIV epidemic is rapidly emerging as a direct result of injection drug use. Furthermore, the governments of both countries have recently initiated programs to provide combination antiretroviral therapy to HIV-infected patients.

An important goal of this proposal is to initiate research in these countries to establish baseline nutrition and metabolic status of HIV-infected patients prior to widespread access to HAART and to follow participants who start on a first-line HAART regimen to document the natural history of treated HIV infection in these countries. This is an important window of opportunity to conduct the proposed studies as, over time, access to patients who are HAART-naive may be diminished and regimens will become more complicated. Our specific aims are as follows:

1. To characterize and compare HIV-negative and HIV-positive (HAART and non-HAART) injecting drug users (IDUs) in terms of:
   a) Types, frequency and patterns of illicit drug use
   b) Co-morbidities
   c) Nutritional and metabolic abnormalities
   d) Clinical outcomes

2. To examine the independent contributions of adherence to HIV medications, psychosocial factors (e.g. depression), and socioeconomic factors (e.g. food insecurity) to the development or persistence of nutritional and metabolic abnormalities

3. To establish an infrastructure and ongoing presence in these countries so that with the increasing availability of interventions for HIV, we will have populations on which to build future studies

Results from these international studies will not only provide important data for HIV-infected patients within these countries, but will also serve as comparisons for U.S. based studies. Comparison of findings between U.S. and international studies will help expand our understanding of the causes and consequences of metabolic complications in chronic HIV infection in the U.S. Furthermore, the results of this project will help to identify generalized malnutrition or specific micronutrient deficiencies in the local populations that may be amenable to nutritional interventions for delaying HIV disease progression. These might include vitamin supplementation and/or dietary counseling to improve the quality of food intake.

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 promotes 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
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:

1. Hepatic blood flow (HBF), based on clearance of low-dose intravenous lidocaine
2. Pharmacokinetics and pharrnacodynamics of intravenous and oral midazolam, a "pure" CYP3A substrate
3. The prevalence of variants in the CYP3A4, CYP3A5, and pregnane-X receptor genes, using molecular genetic techniques
4. 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: Greenblatt, David
Title: Grapefruit Juice Drug Interaction
Abstract: There continues to be uncertainty regarding the following critical issues with regard to the basic and clinical research available to date identifying a number of natural substances present in GFJ that may account for interactions involving GFJ and certain prescription drugs:

1. The concentrations of these identified potential inhibitors in GFJ, and variations in content attributable to factors such as season, site, and brand.
2. What other significant inhibitors may be present in GFJ.
3. The anticipated contribution of the various inhibitors to clinical drug interactions (that is, which inhibitors are important and which inhibitors are unimportant).

PI: Griffiths, Jeffrey
Title: Water Test to Capture Cryptosporidium Oocysts
Abstract: 1000 L of MWRA water are filtered through a filter system designed to capture Cryptosporidium oocysts each week during this project period (May 2001 – April 2005). The compressed filter is transported to the laboratory at Tufts University via an identified chain of custody. Materials captured in the filter are then released via elution from the filter and concentrated via centrifugation. Cryptosporidium oocysts are then captured via the use of immunomagnetic beads, and are then fluorescently labeled using commercially available anti-Cryptosporidium antibodies. Each sample is then analyzed via direct visual detection using an Olympus fluorescent microscope equipped with a MagnaFire camera and imaging system. Cryptosporidium oocysts are identified using criteria identified in EPA Method 1622 and 1623. All positive samples are documented via a digital image, which is saved both on the analysis computer and at a distant site.

PI: Griffiths, Jeffrey
Title: Air Pollution and Respiratory Disease Traineeship
Abstract: Acute respiratory infection (ARI) is responsible for 19% of all deaths worldwide among children less than five years old. Environmental factors such as air pollution have been hypothesized to increase the risk of incidence for ARI in young children. Malnutrition is also known to suppress the immune system that renders a child more susceptible to ARI. This five-year traineeship aims to link environmental pollutants, while controlling for host nutritional factors, to an overall increase in the incidence of respiratory diseases in Ecuadorian children. Air pollution in Quito, Ecuador will be closely monitored by the City of Quito and using mobile handheld monitors to allow a time-series and geographical analysis of respiratory disease in a cohort of children already enrolled in an NIH-funded study. These data will be used to statistically analyze adverse respiratory health outcomes in variably malnourished Ecuadorian children as it relates to air pollution. In addition, molecular and microbiologic analysis will establish a spectrum of pathogens that cause disease.

PI: Griffiths, Jeffrey
Title: Innovative Water and International Research Curricular
Abstract: This application proposes to support two interdisciplinary research curriculum objectives. The first is to devise and implement a 'Water and Health' interdisciplinary course at Tufts University, which has recently started an interdisciplinary, all-University program focusing on drinking water. This program is a partnership of the Tufts University Schools of Arts and Sciences; Engineering, the Fletcher School of Law and Diplomacy, the School of Veterinary Medicine, and the School of Medicine.

In order to meet the challenge of developing an integrated approach across disciplines to understand and solve global water issues, Tufts has launched the "Water: Systems, Science, and Society" PhD and MS Program. The purpose of the program is to provide the interdisciplinary tools and perspectives need to manage water-related problems. This program enjoys the highest level support at Tufts, including the participation of the President as a WSSS faculty member. The second objective is to make concrete an internet based, interdisciplinary curricular linkage between Tufts' Graduate Program in Public Health, and specific East African centers of excellence in public health training and research. The PI has cultivated educational and research linkages at both the University and individual faculty level in East Africa since the fall of 2000 in pursuit of this objective. The purpose of this objective is to develop interdisciplinary shared courses or modules that will lend themselves to the teaching of research methods, and to the building of research networks.

The applicant is ideally situated to develop and implement these innovative curricula. He is the Director of the Graduate Programs in Public Health at Tufts University School of Medicine, whose MPH program was ranked #1 in the US in 2004, and a seasoned educator. He is also an active biomedical researcher with 6 active grants (5 NIH, 1 USAID), 5 of which are active in Kenya and Ecuador, most of which have a 'water' focus, and several of which are highly interdisciplinary. As such, he is an integral part of Tufts' research and academic programs, and is qualified to provide leadership in innovative interdisciplinary research curricula. Innovative aspects of this proposal include the support of student research projects within the context of courses in both specific aims. Strong institutional commitment to these objectives has been, and is, present.

PI: Griffiths, Jeffrey
Title: A Global Health Framework at Tufts University
Abstract: Tufts University as a whole has an extensive portfolio of Global Health related education, scholarship, and research. However, these activities have not to date been overtly linked. We propose to build an administrative framework for this Global Health program at Tufts University. It will be housed in the Department of Public Health and Family Medicine at Tufts, which is also the seat for the public health programs at Tufts. We also propose to develop an interdisciplinary Global Health curriculum at Tufts. In this endeavor, we will utilize not only the wisdom, expertise, and resources existing at Tufts, but will also include those of partner organizations in Africa, India, and elsewhere.

This project will in particular leverage the strengths of new and innovative internet based educational efforts at Tufts that are supported by NIH Roadmap Curriculum development funding. This project enjoys the highest level of support at Tufts. As the core administrative part of this endeavor, we will open an interdisciplinary Global Health concentration in the Tufts MPH program, the courses of which will be open to all students at Tufts. The public health programs have ongoing collaborative joint degree programs with most of the Schools at Tufts, including the Schools of Veterinary Medicine, Nutrition, Engineering, and the Fletcher School of International Law and Diplomacy. Through the mechanism of this concentration, we will engage relevant faculty, staff and students at these collaborating Schools in a variety of venues so as to build interdisciplinary education and research in the field of Global Health. Tied to the development of this administrative and educational core function is the development of a Curriculum in Global Health, which will be developed in conjunction with faculty from the Schools of Nutrition, Engineering, Veterinary Medicine, and the Fletcher School, as well as our partners overseas. Global Health is the term we use to describe the body of knowledge relating to medicine and public health all around the world. It includes subjects such as development economics, disaster assistance, malnutrition, and the treatment of water so it is safe to drink. Our project will support the education of students, and new research that combines all of these knowledge areas.

PI: Griffiths, Jeffrey
Title: Transmission and the Respiratory Tract in Cryptosporidiosis
Abstract: Cryptosporidiosis is a clinically significant cause of diarrhea. The infection can be life-threatening in individuals with immunodeficiencies because of the severity and chronicity of the diarrhea, and lack of effective therapy. Cryptosporidiosis is usually considered to be wholly confined to the gut, and the mode of transmission to be exclusively fecal-oral. This proposal will explore the contribution, if any, of Cryptosporidiosis to respiratory tract infections. We hypothesize, based on a wealth of data (circumstantial information, human case reports and case series, animal studies, human population-based studies, and human volunteer studies), that respiratory infections with Cryptosporidium occur frequently. However, to our knowledge, no study has ever been conducted to prospectively study this possibility. We have designed a rigorous study of children in Uganda that should either convincingly confirm, or refute, this hypothesis. The outcome of this study will have significant clinical, biological and epidemiological implications, which could be important, given the lack of therapy. For example, the finding of respiratory Cryptosporidiosis may lead to improved clinical care (respiratory support, such as oxygen), and new measures to interrupt transmission (since pulmonary secretions [sputum] may be a source for transmission). How common—and consequently how significant—are infections of the respiratory tract, is unclear.

In this revised application, we have strengthened and focused on the clinical study in Uganda, and dropped a prior specific Aim designed to explore the question of respiratory transmission in piglets. Should our project in Uganda show that respiratory Cryptosporidiosis occurs in children, then we will again address that question in a next-step application. The project will focus on attempts to recover Cryptosporidium oocysts from induced sputum of children with acute or persistent diarrhea concurrently experiencing cough, with or without fast breathing. These studies will be performed in children presenting to Mulago Hospital in Kampala, Uganda with acute or persistent diarrhea and cough with or without fast breathing. Mulago Hospital is a site where we have had an ongoing program on diarrhea and malnutrition in children aged 3-59 months for several years, which resulted in several publications. In this resubmission, we have strengthened the study by increasing the sample size, and by proposing to perform more complete genetic analysis of the parasite isolates. This study is important to public health because it may help us to understand if the parasite also causes a lung infection in children.

PI: Harris, Jonathan
Title: Can Economic Growth be Sustainable?
Abstract: This grant supports a conference, research, and wide dissemination of results, addressing the macroeconomic questions:

• What would a SAEJAS economy look like?
• What plausible path can be described that can take us from our present system to a SAEJAS economy?

Much good work has been done on aspects of these questions by some research think tanks and civil society groups. But these questions have not yet been established as an important part of societal and academic discussions about macroeconomic issues of production, growth, consumption, and jobs creation. This is what the Global Development and Environment Institute has uniquely positioned itself to do during the course of its twelve-plus years of intensive research and writing. 

This project will consolidate and build upon aspects of the Institute’s ongoing work, and integrate them with new elements, during the two periods for which we are requesting funding. During the first phase (March through June 2006) we will prepare a discussion paper summarizing issues and current research (including extensive on-going research at GDAE) on the interrelations between economic growth and (SAEJAS) sustainability. We will communicate widely with both academics and activists who are deeply concerned about these issues. We will seek out especially talented and creative people, including, among academics, junior scholars who may be encouraged to make consideration of these issues an important part of their life work. We will issue invitations to a fall conference, and solicit papers to be written for it. 

The second phase (principally September through December 2006) will include the conference, integrating its findings into our ongoing work, and initiating dissemination of the results. (Work on collecting and finalizing papers for publication will continue into spring 2007.)

The intended results may be generalized thus:

1. The project will advance understanding of the macroeconomic goals that must be set in response to environmental challenges, and of the macroeconomic changes required to move from the current situation toward a SAEJAS macroeconomy.

2. Actions will be taken to move this improved macroeconomic understanding into the realms of public knowledge, including, importantly, through the teaching of economics, in the United States and in the Global South.

PI: Hata, Akiko
Title: Mechanisms of TGFB Signal Transduction
Abstract: Bone morphogenetic proteins (BMPs) represent the largest subset of the TGFß family of growth factors, with actions that range from osteomorphogenesis to ventralization of mesoderm during early development. The mechanisms by which BMPs exert such diverse effects and tissue-specificity are poorly understood. The long-term objective of this proposal is to understand molecular mechanisms by which BMPs are able to generate specific gene expression events by exploring spatial and temporal effects of BMPs.

We recently cloned a novel transcription factor for BMP-mediated genes, called OAZ. Preliminary data support that OAZ regulates different genes by distinct protein interactions with at least two partners, Smads1/4 or Olf-1/EBF. The specific hypothesis to be tested is that OAZ interactions with such distinct proteins leads to assembly of unique transcriptional complexes and differential gene regulation. New results show that OAZ activates the Smad6 gene, a well-recognized antagonist of BMP signaling, supporting a potential novel negative feedback loop for BMP signaling via OAZ.

In Specific Aim 1, we will test this by:

• Confirming the sequence element in the mouse Smad6 promoter to which OAZ binds
• Examining the functional significance of Smad6 induction by OAZ with time-course studies of Smad1 activation by BMPs and wild type or dominant negative OAZ expression
• Identifying novel mammalian target genes for OAZ

We have identified two novel cofactors of OAZ, Asc-1/TRIP4 and Parp1 in yeast two-hybrid screens using OAZ as a bait.

In Specific Aim 2, we will study the functional significance of the OAZ-Asc-1 complex by:

• Elucidating a physiological role of Asc-1 in activation of Xvent-2 by inhibiting Asc-1 expression by overexpression of antisense-Asc-1 construct
• Determining the functional role of Asc-1 as a transcription partner of OAZ
• Characterizing whether the OAZ-Asc-1 complex assembles on known BMP target gene promoters (Smad6, Msx-1, Msx-2, and Id1)

Finally, in Specific Aim 3, we will study a physiological role of poly (ADP-ribose) polymerase, Parp1, as a cofactor of OAZ by studying:

• The mechanism by which Parp1-mediated poly-ADP-ribosylation positively regulates transcription of Xvent-2 gene
• Whether or not known proteins of the OAZ transcriptional complex (OAZ, Smads, and/or Asc-1) are poly-ADP-ribosylated by Parp1
• Whether Parp1 is involved in the regulation of known BMP target gene promoters (Smad6, Msx-1, Msx-2, and Id1)

These studies should provide important insights into TGFß-mediated regulation of gene expression, which is of direct relevance to both normal physiology and a variety of disease states.

PI: Hata, Akiko
Title: Role of BMPRII Mutants in the Pathogenesis of Pulmonary Hypertension
Abstract: Pulmonary hypertension (PH) is characterized by an increase in pulmonary vascular resistance that impedes ejection of blood by the right ventricle, leading to right ventricular failure. Primary PH (PPH) is a rare but progressive disease with a mortality of 30 percent over 4 years. Recently germline mutations in bone morphogenetic protein receptor type II (BMPRII), a member of the transforming growth factor a (TGF a) receptor family, have been found in over 50 percent of familial PPH patients and in 30 percent of sporadic cases of PPH. Mutations have been found in the extracellular, ligand binding, and cytoplasmic serine/threonine kinase domains, as well as the long carboxyl-terminal region (tail domain). The long-term objective of this application is to understand the molecular mechanism(s) by which BMPRII mutations contribute to the pathogenesis of pulmonary arterial hypertension. We found that BMPs promote, apoptotic cell death in normal human pulmonary artery smooth muscle cells (PASMCs). BMP-mediated apoptosis in PASMCs is associated with activation of caspases-3, -8, and -9, cytochrome c release, and down regulation of Bcl-2. PASMCs expressing mutant forms of BMPRII identified in PPH patients are resistant to BMP- mediated apoptosis.

The specific hypothesis to be tested is that mutation(s) in the BMPRII disrupts BMP-mediated apoptosis in PASMCs, which is required for maintenance of normal cell number in the pulmonary vasculature. In Specific Aim 1, we will characterize the apoptotic-signaling pathway mediated by BMP7 in PASMCs. In Specific Aim 2, we will characterize the biological activities of BMPRII mutants found in PPH and generate transgenic mice expressing a BMPRII tail domain truncation mutant gene in smooth muscle to examine the role of this mutant BMPRII in vivo. We recently discovered that neuronal cell death-inducible putative kinase (NIPK) interacts with the tail domain of BMPRII in mammalian cells. NIPK contains a ser/thr kinase-like domain. Therefore, in Specific Aim 3 we will focus on the functional role of the interaction between NIPK and the tail domain of BMPRII in the regulation of BMP-mediated signaling pathways. These studies will elucidate the mechanism of BMP-dependent apoptosis in PASMCs and the role of the tail domain of the BMPRII in the regulation of the BMP signaling pathway, which is of fundamental importance to understanding the molecular mechanisms underlying the pathogenesis of pulmonary hypertension.

PI: Hatini, Victor
Title: Cellular Interactions in Patterning and Morphogenesis
Abstract: During development, cell fates are established by signals that emanate from specialized "organizer" regions. How these signals direct cells to differentiate migrate, and alter their shape during development is a fundamental yet poorly understood question. The analysis of Hedgehog (Hh) and Wingless (Wg) signaling in Drosophila embryonic epidermis is contributing novel insights into these fundamental developmental problems. Preliminary work identified three conserved genes drumstick (drm), lines, and bowl. Their gene products interact physically to mediate patterning by the epidermal organizer. In imaginal discs, these gene products control epithelial morphogenesis by signals distinct from Hh and Wg. Genetic and biochemical experiments suggest the following model to be tested in Aims 1 and 2: Lines targets Bowl for degradation via the ubiquitin-proteasome pathway; Drm inhibits Lines to shield Bowl from degradation; Drm accomplishes this function by localizing Lines to the cytoplasm.

Specific Aim 1 is designed to test how Lines decreases the abundance of Bowl. A combination of genetic, molecular, and pharmacological approaches in cell culture and in vivo will link the activity of Lines to proteasomal, or non-proteasomal pathways.

Specific Aim 2 is designed to test whether Drm localizes Lines to the cytoplasm to stabilize Bowl and whether Drm accomplishes this function by decreasing Lines-Bowl interaction. Molecular genetic experiments suggest that Lines blocks epithelial folding in imaginal discs, whereas Drm inhibits Lines, and thereby activates Bowl, to initiate epithelial folding.

Specific Aim 3 utilizes a combination of loss- and gain-of-function experiments in mosaic clones to test the model.

The proposed experiments will help explain how primary organizing signals direct cells to differentiate and alter their shape during development. Insight from this work may apply to vertebrate development where parallel pathways operate. In addition, the proposed experiments will provide essential insights into the regulation of the ubiquitin-proteasome system (or another proteolytic system) in a developmental setting. Insight from this work may suggest new strategies for reversing diseases that arise from constitutive degradation of key regulatory proteins.

PI: Heldwein, Ekaterina
Title: Structural Determinants of Membrane Fusion by HSV-1 gB
Abstract: Herpes simplex viruses infect their hosts for life, causing cold sores (HSV-1), eye and genital infections (HSV-1 and -2), and encephalitis. Furthermore, they can wreak havoc in immuno-compromised individuals or in newborns. Host cell entry by HSV-1 requires four viral envelope glycoproteins: gB, gD, and gH/gL complex. Binding of gD to a cell-surface receptor, which triggers fusion events, has been studied crystallographically. By contrast, no structural information is available on the other three proteins, and little is known about how they participate in cell entry. Knowing the structures of all the components of the membrane fusion machinery is a necessary step in unveiling the mechanism of HSV cell fusion. Our long-term goal is to elucidate the mechanism of Herpes virus entry into host cells at the atomic level and to use this knowledge to design effective inhibitors of viral entry into cells for preventative and therapeutic purposes. In preliminary studies, we have crystallized the ectodomain of gB and are currently determining its structure. In this application we propose biochemical and structural studies of additional domains and larger forms of gB, in order to build a more complete picture of its activities and interactions The three specific aims are:

1. To determine the structure of the cytoplasmic domain of gB
2. To produce pure, soluble forms of gB encompassing multiple domains for structural studies
3. To study the mechanism of the antiviral effect of a G-quartet oligonucleotide crystallographically

This structural information will provide the framework for elucidating the function of gB in cell entry. For example, known functional mutations will be mapped onto the structure. Moreover, strategies developed for producing soluble multidomain forms of gB for structural studies will allow us to approach gH/gL and, eventually, to assemble multi-protein complexes.

PI: Herbert, Jean
Title: Resumed Education for Adult Learners (REAL) Program
Abstract: Since 1970, Tufts University has offered adults the opportunity to continue their undergraduate education. The Resumed Education for Adult Learners or REAL Program enables highly-motivated adults to earn an undergraduate degree at a selective, private institution. Instead of a separate or extension program for adults, REAL students attend the same courses and earn the same degree as other Tufts undergraduates, and participate in every aspect of campus life - clubs, studying abroad, tutoring opportunities, internships and joining sports teams - while also receiving individual attention from admission to graduation to help meet their unique academic needs. 

The Resumed Education for Adult Learners Program is one of the few adult education programs that offer financial aid to qualified candidates. Currently, the average financial aid award is $20,000, which is $1,000 more than the average undergraduate award. Out of an average class size of 30 students, all but 2 or 3 receive financial aid. Because the need has become greater than available aid, awards are now determined by ranking all qualified candidates and accepting just the top students. This has caused fewer REAL students to be admitted and a significant decrease in the number of REAL students on aid over the past couple of years from 35 (2002-2003) to 20 (2005-2006). There have been instances where more aid was given - when 2 students just needed $10,000 each instead of one student needing $20,000 - but those instances are few and far in-between. Also, if students decrease their work hours, the amount of aid needed is impacted - usually increasing. 

The current budget for REAL is $400,000, all of which is used for financial aid packages. Because of its commitment to REAL, Tufts University absorbs all other costs associated with the program. Therefore, a $50,000 grant from the Bernard Osher Foundation will have an immediate impact, enabling us to offer two additional - and possibly three - financial aid packages to deserving qualified REAL candidates.

PI: Herman, Ira
Title: Regulating Retinal Microvascular Morphogenesis
Abstract: Retinal microvascular morphogenesis is a complex and highly coordinated process, which occurs during embryonic development, post-natally and in association with several visually-impairing diseases, including retinopathy of prematurity (ROP), age-related macular degeneration and diabetic retinopathy. Recent work carried out in the principal investigator's laboratory has revealed that isoactin-based cytoskeletal remodeling is integral to the microvascular migration and proliferation observed during developmental and pathologic angiogenesis, including the pericyte-based remodeling seen during retinal microvascular maturation. In a focused, inter-disciplinary research plan that will take advantage of in vitro and in vivo models using a spectrum of well-established molecular, cell biology-based and molecular genetic approaches, we aim to reveal the molecular mechanisms and the isoactin-based signaling cascades regulating (i) retinal endothelial migration driving normal and pathologic angiogenesis, and (ii) pericyte-based control of endothelial proliferation and capillary contractility. Quantitative analyses of retinal microvascular endothelial cell cultures will be performed in conjunction with experiments aimed at over-expressing the novel beta-actin specific binding and filament capping protein, betacap73, discovered in the lab. To reveal the molecular mechanisms driving isoactin-based control of developmental and pathologic angiogenesis, we will take advantage of a 'two-mouse' transgenic approach, where we will specifically induce betacap73 over-expression within the post-natal vascular endothelium. These combined results, revealing alterations in endothelial motility and impaired angiogenesis, will serve to guide cDNA expression array analyses aimed at identifying key signaling effectors controlling these pivotal microvascular events. Further, to reveal the molecular signaling mechanisms regulating pericyte contractility and retinal endothelial growth, we will characterize the role that Rho GTPase family members play in signaling vascular cytoskeletal remodeling and isoactin dynamics during microvascular morphogenesis. Based on the preliminary data recently obtained and the experimental approaches proposed, we anticipate that our research plan will provide important new insights into the molecular mechanisms regulating retinal microvascular morphogenesis during normal development and in association with the pathologic angiogenesis accompanying diabetic retinopathy.

PI: Hess, Andrew
Title: Armenian Economic & Diplomatic Summer Training Program 2006
Abstract:

I. CURRICULUM REQUIREMENTS

A. ECONOMICS AND INTERNATIONAL BUSINESS

1. International Trade and Investment

The first portion of this course will investigate why nations trade, what they trade, and the distribution of the gains from trade. Topics include trade and economic growth, technology, the product cycle, multinationals, international labor integration, tariffs, regional economic integration, dumping, and international competitiveness of firms and nations.

The second portion of this study of trade deals with trade policy reform and institutions. Topics include subsidies, agriculture, market access and reciprocity, the WTO Doha Development Round, preferential integration, dispute settlement, World Bank and IMF trade policy measures, trade and income distribution, and trade and the environment.

2. Empirical Topics in Globalization

This course is an examination of the evidence for the extent of globalization as well as its consequences. Is there an unprecedented expansion of global trade, and has it produced economic growth, expansion of labor markets, an increase in inequality and a substantial change in environmental conditions, all the while encouraging greater capital market liberalization?

3. Global Financial Intermediation Corporate Accountin2 and Capital Markets

The course will employ lectures and case studies for the purpose of understanding international corporate finance. Emphasis will be placed upon an analysis of balance sheet structure and cash flows, including operating and project finance proposals for developing countries. Students will identify major financial actors in the global arena and explore their interests in the economies of developing countries. Finally, Armenian policy makers will assess the ability of Armenia to access international financial markets.

4. International Marketing

This course introduces students to the fundamental issues faced by international marketers. The offering addresses the multi-faceted problems of marketing across national boundaries as well as the numerous challenges of marketing within foreign countries and coordination of global marketing programs. It covers the problems encountered by all firms as they operate in an international or global environment. The full range of marketing activities faced by exporters, licensors, joint ventures, and the multinational firm is covered: marketing research, product policy, pricing, distribution, promotion, planning, organization, and control.

While internationally oriented in nature, the course will also bring exposure to classic and fundamental marketing management principles when appropriate. Non-traditional aspects of international marketing will be considered for a variety of constituencies as well. From this course students should learn how to study foreign markets and identify business opportunities: how to enter foreign markets; how to devise appropriate product pricing and promotional strategies for foreign markets; how to fight product piracy and protect intellectual capital; how to cope with high levels of risk and uncertainty in emerging economies; how to adapt and integrate business practices an the marketing mix in light of new technologies and media such as the Internet; and how to deploy proper organizational and human resources to plan, manage, and control international marketing activities.

B. INTERNATIONAL LAW

1. Law and Development

The goal of this course is to define the role of law and the legal system in the economic and social development of developing nations, emerging markets, and countries in transition, such as Armenia. It first explores the nature of law, the nature of development, and the theoretical relationships of law to the development process. It then studies the practical links between law and development through a series of case studies addressing such crucial issues as land tenure, foreign investment, the environment, governance, corruption, judicial reform, private enterprise, and the rule of law. The course concludes with a discussion of the role of foreign technical assistance in bringing about legal change.

C. GLOBAL DIPLOMACY FOR POLICY MAKERS

1. Seminar on Development Policies for Armenia

This seminar will conclude the Armenian Program. It will take place under the direction of Professor Graham Bird. He will assist the Armenian group in the preparation of a policy statement on Armenian development. The details and format of this work will be determined by the students in consultation with Professor Bird and will reflect a blending of their own experience in Armenia and the knowledge the students have acquired over the course of the three month program at the Fletcher School.

2. Thinking About Global Change for Armenian Policy Makers

The course establishes a basis for understanding the modern turbulence associated with contemporary governance in Central Asia and the Caucasus through the application of concepts derived from new theories about contemporary global change. This study will begin with a major effort to apply new thinking about the reasons for the collapse of the Soviet Union. For the post 1991 period the course will cover the rise of ethnic and sectarian conflict, the impact of accelerated technological change, the dynamics of oil politics, the rise of criminal activity, fundamentalism and institutional instability. The main theme for the course is the need for policy makers to understand the complexities of a global politics that both interlinks and divides the states and societies of the Caucasus and Central Asia with the rest of the world.

3. Development Countries and the Global Politics of Sustainable Development the premise of this course is:

   1. That despite the problems of definition, a global politics of sustainable development is emerging which not only includes the intersection of environment and development but also embodies a growing set of concerns related to social development, human security and global governance

   2. That the developing countries of the South Caucasus and Central Asia have embraced this new global politics as a way to rearticulate their longstanding desire for systemic international reform

   3. Tn doing so, they have not only changed the global environmental discourse but have themselves changed

   The course will explore how the interest in sustainable development has shaped, and been shaped by, multilateral environmental negotiations, institutions for global enviromnental governance, and the rise of non-governmental actors over the last thirty years. We will focus on the negotiation behavior of the G77, an unlikely group of oyer 130 developing countries that have dramatically different economic, ideological, political, and environmental conditions but have demonstrated remarkable resilience in the face of internal divisions as well as external threats.

PI: Holcomb, Phillip
Title: The Cognitive Neuroscience of Word 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: 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

3. Language switching studies used to probe the evolution of control over the relative activation of words in each language

PI: Huber, Brigitte
Title: B Lymphocytes: Differentiation and Triggering
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:

1. 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.

2. 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.

3. 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.

4. 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: QPP: Protease that Prevents Apoptosis in Quiescent Cells
Abstract:  QPP (Quiescent cell Proline di-Peptidase), also termed DPP7, is a protease hypothesized to control the survival of lymphocytes and neuronal cells that are in the G₀ stage of cell cycle, because QPP inhibitors induce apoptosis in these cells. We have carried out a detailed analysis of QPP at the cellular, biochemical and molecular levels, forming a solid basis for this competitive renewal. We now propose to directly test our working hypothesis and to define the mechanism(s) of QPP-mediated survival of quiescent cells:

1. Construction of QPP mutant mice. The most direct approach to define the functional significance of QPP in vivo is the construction of QPP ko mice. Four systems will be used which allow a broad analysis of the role of this enzyme in various cell types and stages of development in the mouse.

1. Conventional QPP^-/- mice: We have already obtained one chimeric QPP+/- founder that has germline transmission of the mutant QPP gene. More founder mice will be produced, bred to homozygosity for the mutant QPP gene and analyzed for defects in the development of the lymphoid system and other organs.

2. Fetal liver transfer into RAG-2^-/- mice: If the QPP^-/- genotype is embryonically lethal, then we will test lymphoid development by transferring QPP^-/- fetal lymphoid stem cells into RAG-2^-/- mice.

3. RAG-2^-/- blastocyst complementation: If embryonic lethality occurs before lymphoid stem cell development, we will generate chimeric mice that are QPP-deficient only in the lymphoid lineage.

4. Conditional QPP^-/- mice: To define the role of QPP in mature cells and individual organs, we will use the Cre-loxP recombination system that provides control of expression of the homozygous mutation.

2. Inhibition of QPP expression in vitro. To test the hypothesis that QPP is required for the G₀ program in human lymphocytes, we will block expression of this protease in vitro. Four systems will be employed:

1. Dominant negative forms of OPP: Since the QPP protease activity requires dimerization, enzyme active site mutants will be tested in transfection experiments for suppression of the wildtype QPP enzyme.

2. RNAi: RNA interference is an efficient mechanism to silence specific transcripts that was pioneered in C. elegans and has recently been shown to be highly efficient also in mammalian cells. Thus, we will select appropriate QPP sequences for the generation of siRNA duplexes that should silence QPP mRNA.

3. Antisense Oligos: An antisense oligo binds the sense mRNA and prevents translation. Coupled with a new delivery method that allows highly efficient endocytosis of the polar oligo, this provides the system of choice to block QPP protein expression in primary lymphocytes and neuronal cells.

4. Antisense cDNA: Full length antisense QPP cDNA in a recombinant adenovirus will be introduced into human and mouse cell lines and primary cells to block expression of QPP.

3. Characterization of QPP substrate(s). To fully understand the mechanism and/or pathway of QPP-mediated survival of G₀ cells, it is essential to define the physiological substrate(s) of this protease.

   1. Direct screen of potential substrates: Based on our working hypotheses, we will test LKLF and neurotrophins, as well as novel candidates, for susceptibility to cleavage by QPP in vitro, and if so, whether the modification changes the functional activity of these molecules.

   2. Localization of QPP substrates: To confirm that cleavage of a candidate substrate by QPP in vitro is functionally significant, we will test whether the two molecules co-localize in vivo, either in a specific vesicular compartment or as secreted proteins.

Collectively, these studies will lead to a better understanding of the constitutive G₀ survival program in eukaryotic cells. In addition, they will provide insights into the default apoptosis pathway of quiescent cells.

PI: Huber, Brigitte
Title: Multidisciplinary Biodefense Training Program
Abstract: This is a new application to establish a biodefense training program to produce predoctoral trainees who will aid the nation in combating the new challenges of the bioterror threat. The program has a faculty of 19, drawn from the Genetics, Immunology and Molecular Microbiology Graduate Programs of the Sackler School of Graduate Biomedical Sciences of Tufts University School of Medicine and New England Medical Center Hospitals. Focused training in the pathogenesis and genetics of microorganisms that are relevant to biodefense will be integrated with training in understanding host responses to these microorganisms. In this way, our trainees will be prepared to attack biodefense-related problems with an in-depth understanding of pathogen-host interaction. A newly developed curriculum integrates courses offered by Genetics, Immunology and Molecular Microbiology. In addition, a newly-developed course presents the pathogenesis of the most potent biological agents together with scientific, medical, social, and political issues relevant to bioterrorism and biowarfare.

The Biodefense Training Program also interfaces with the newly conceived Study Center for the Immunogenetics of Infectious Disease (SCIID), an organization that focuses on genetic, immunological and microbiological aspects of infectious disease. The SCIID will be located in the newly constructed Jaharis Family Center, and will provide an intellectual and physical home for our trainees. This Center will also give students the opportunity to work at the BL-3 level of containment, use gene array technology to study host and pathogen gene expression, and have access to BL-3-level cell sorting. Predoctoral students enrolled in the Genetics, Immunology or Molecular Microbiology Graduate Programs may apply to enter the Biodefense Training Program after their first year of graduate school. Selection for our program will be based on high achievement in lab rotations and coursework during the first year, selection of one of the Biodefense Training Program faculty as a thesis mentor, and commitment to work on a dissertation topic relevant to biodefense concerns. Three students will be selected for two years of support (six positions). Benefits of our program include:

1. Students are already enrolled who are working on biodefense related issues
2. A highly experienced training faculty is in place
3. A strong curriculum already exists
4. Development of the SCIID shows institutional commitment to this program and provides an outstanding training setting

PARTICIPATING FACULTY: The 19 training faculty include 11 professors, 3 associate professors, and 5 assistant professors. Six of the training faculty are women. Fourteen of the training faculty have primary appointments at Tufts University and five training faculty are located at the New England Medical Center Hospital. The primary areas of research include genetics, immunology and molecular microbiology.

PI: Huber, Brigitte
Title: HERV-K18 as a Risk Factor for CFIDS
Abstract: The etiology of CFIDS is far from understood and is likely due to multiple genetic components. Infection with Epstein-Barr virus (EBV) and treatment with interferon-alpha (IFN-fl) have been implicated in the pathogenesis. Our laboratory has shown that EBV infection, as well as exogenous IFN-0, activate transcription of the env gene of a Human Endogenous Retrovirus, HERV-K18. This retroviral gene encodes a superantigen, which is a class of proteins that is capable of deregulating the immune system.

Three alleles of HERV-K18 env have been documented, K18.1, K18.2, K18.3, and the gene products from all three alleles have superantigen activity. The three alleles are predicted, however, to have different biochemical and functional characteristics. The purpose of this investigation is to establish whether a differential distribution of HERV-K18 allele and genotype frequencies exists in CFIDS patients, compared to healthy controls.

We have developed a novel technique for genotyping HERV-K env. This genotyping procedure can be performed in a reasonable time frame and is relatively inexpensive, allowing efficient genotyping of large numbers of samples. During the past year, we have accumulated genotyping data on over 500 healthy individuals of Caucasian decent. This database will be used to look for deviations in the allele and genotype frequencies in CFIDS.

We are particularly interested in analyzing the cohort who suffers from IFN-associated fatigue, as observed by the CDC/Emory University Collaborative Group. In addition, the collection of patients from the Wichita clinical studies will be included in our investigation. If a differential distribution of HERV-K18 alleles and/or genotypes is seen in patients suffering from CFIDS, compared to the healthy control group, this would open new avenues for the development of clinical treatment of this chronic disease.

PI: Hyde, James
Title: Health Literacy and Informed Consent
Abstract: There is a growing body of literature showing that subjects who consent to participate in research studies do so without the opportunity to make truly informed decisions. It may also be that potential research subjects may refuse participation in clinical research studies because they are unable to obtain satisfactory answers to questions and concerns. One of the important and documented barriers to obtaining informed consent from subjects is the disparity between the health literacy skills and abilities of subjects and the complexity of the language, both written and oral, used to communicate with them. Data from the National Adult Literacy Survey (1993) suggest that as many of 90 million Americans, 47% of the adult population, may have difficulty accessing and using health information.

We propose a pilot intervention study to address the disparity between the health literacy level of subjects, clinical researchers, and the documents and tools that they use to communicate with one another. While previous studies have focused on either subjects or researchers independently, the proposed approach addresses both. We will develop and test an adult informed consent template, fine-tuned to reach those with limited health literacy skills, in conjunction with a web-based educational program intended to enhance the ability of clinical investigators to communicate with low literacy populations. Because the consent process also serves the dual purpose of protecting the institution and researchers from legal liability, we have taken special care to honor this dual function in the design of our intervention. We will evaluate the effectiveness of this approach using surrogate research subjects evaluating their response to a mock research study. Outcome measures include level of comprehension, satisfaction and rate of subject accrual. The research team includes a multi-disciplinary group of clinical researchers, health communication, legal and public health scholars. The work will take two years to complete.

PI: Imanishi-Kari, Thereza
Title: The Role of Interon Type I, TLR and BCR Engament in the Activation of Immature Self Reactive B Lymphocytes
Abstract: The specific aim of this grant proposal is to study immunoglobulin (lg) repertoire development. The primary goal of this project is to accomplish the following aims:

1. To determine the genetic contribution of specific transgene and background genes in the regulation of transgene expression and selection.
2. To determine what stage of B cell development and how early in the ontogeny of B cells is somatic hypermutation taking place.
3. To determine whether somatic mutation is a consequence of negative selection followed by positive selection.

These studies are important for our understanding of B cell development and mechanisms regulating the primary B cell repertoire. It will provide insights into the question of whether there are crucial developmental checkpoints involving interactions between surface immunoglobulin molecules and specific ligands at each one of these stages.

PI: Isberg, Ralph
Title: Molecular Basis of Yersinia-Host Cell Interaction
Abstract: The primary goal of this project is to investigate the molecular mechanism of Y. pseudotuberculosis entry into cultured cells and to analyze the role played by β1 integrins in bacterial uptake:

1. We are testing a model for the binding of a single invasin molecule to multiple host receptors. The model proposes that receptor multimerization is a central determinant in bacterial uptake.
2. The region of the integrin heterodimer involved in invasin binding is being determined, by isolating mutations in the integrin that have altered specificity for invasin derivatives.
3. The role in bacterial uptake of the β1 integrin chain cytoplasmic domain is being investigated, by constructing hybrid proteins containing maltose-binding protein fused to carboxyl terminal derivatives of the β1 integrin chain. Mutations previously shown to alter the efficiency of bacterial internalization will be introduced into these hybrid proteins to test for their ability to associate with cytoplasmic factors.
4. Mammalian cell cytoplasmic components that are necessary for uptake are being identified using our recently developed perforated cell assay.
5. The role of invasin in intestinal infections is being analyzed. Kinetic experiments are being performed to determine if failure of Y. pseudotuberculosis inv mutants to localize in mesenteric lymph nodes is due to reduced viability or a failure to translocate from the lumen of the intestine.

The long-term objective, that of determining how bacterial- and host-encoded factors promote cellular penetration, is part of our general goal of understanding how an enteroinvasive microorganism causes systemic disease.

PI: Isberg, Ralph
Title: Molecular Analysis of Microbial Pathogens
Abstract: This application requests support for continuation of a rigorous predoctoral training Program that focuses on the molecular analysis of microbial pathogens. The Program is interdepartmental and is centered around the Department of Molecular Biology and Microbiology, with members being drawn from the Departments of Biochemistry, Medicine, Rheumatology, and Pathology. All investigators have either a common interest in pathogenic microorganisms, in basic processes performed by such microorganisms, or have complementary expertise in molecular biology. The varied research interests of the group include:

• Bacterial pathogenesis, including the study of colonization, intracellular growth, toxin expression and development of tools to study microbial genes expressed during animal infections
• Viral pathogenesis and replication
• Sporulation as model regulatory and pathogenic system
• Protein secretion and the analysis of microbial surfaces, including those of pathogenic bacteria and yeast
• Regulation of gene expression and cell growth in microbial model systems
• Analysis of developmental stages in fungal pathogens

The members of this Program use genetic and biochemical strategies to analyze microbial pathogens, as well as animal infection models. This Program has a long history of having a strong collaborative spirit of learning and research among faculty and students. Recruitment and admission strategies have been highly successful, with an excellent minority recruitment program, with 20% of the students being members of underrepresented minority groups, as defined by NM guidelines. The overwhelming majority of the 113 PhD graduates of the Department since 1964 are currently employed in research positions in academics and industry. The Program is overseen by the Training Committee, a group of internationally recognized bacteriologists and virologists who participate in the graduate education of all trainees. All faculty members of the Program have individual NIH grants or other forms of support.

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 (El) and crop water stress (CWS) over a range of spatial and temporal scales. Operational methods of El 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 El 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 El 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:

1. Develop and refine El and CWS estimation algorithms for large heterogeneous areas by using mainly remotely sensed data
2. Test and validate the proposed methodology for different regions in the United States
3. Compare and contrast uncertainties in the estimates between the proposed method and currently available methods
4. Develop a user-friendly estimation tool for agricultural, water resources, and forest management applications using routinely available remote sensing data

In this collaborative project among the Tufts University, Penn State University, and Purdue University, we will use readily available remotely sensed data to deliver a methodology for the estimation of El and CWS over large areas. We have developed partnerships with the North Carolina Agricultural Research Services, Cooperative Extension Services, Water Resources Task Force and the South Florida Water Management District to apply the results from this research for agricultural and water management applications. 

The goals and objectives of this project address issues directly related to Natural Resources and Environmental Quality theme. Expected results from the proposed research would aid crop yield monitoring and modeling, irrigation recommendations, assessment of water use and water stress under various water management, vegetation management, and soil water availability conditions.

Results from this research would have the potential to be translated to remote regions where very little or no surface based measurements are available. We will work closely with our partners and end-users to integrate these findings into an operational El and CWS estimation and monitoring system for various agricultural and water resources management and decision making over large areas.

PI: Jackson, Rob
Title: Role of Andante/CKIIB in the Drosophilia Circadian Clock
Abstract:

Specific Aim 1. Which transcription unit within chromosomal interval 10E corresponds to Andante?
As indicated in previous progress reports, this specific aim was finished during year one of the grant. Behavioral studies of mutants and genetic rescue experiments demonstrated that Andante is a mutation of the Casein Kinase 2 (CK2) regulatory subunit (the β subunit), implicating CK2 activity in clock function. This work was published in Nature Neuroscience (Akten et al., 2003) and is described in detail in Dr. Akten's Ph.D. thesis (Akten, 2005).

Specific Aim 2. How does the Andante mutation affect CK2 function?
Our DNA sequence analyses showed that the Andante mutation is a single-nucleotide change resulting in an amino acid substitution in a region of CK2P that is evolutionarily conserved (from flies to humans; see Akten et al., 2003). Published results from another group (Rasmussen et al., Mol. Cell. Biochem. 274: 151, 2005) indicate that the Andante mutation does not interfere with α::β interactions nor affect enzymatic activity in vitro. Thus, we conclude that the mutant subunit is less stable, in vivo, and this is predicted to result in reduced enzymatic activity. Consistent with this explanation, we previously showed that there are decreased levels of CK2β in the Andante mutant.

Specific Aim 3. Where is Andante (CK2β) protein required within neural tissues for normal rhythmicity?
Our studies have shown that CK2α and CK2β can be detected within the PDF-containing clock cells of the fly brain (Akten et al., 2003 and unpublished results), although the β subunit has a broader pattern of localization. We employed RNA interference (RNAi) methods to determine cellular requirements for the CK2 subunits. This analysis showed that both CK2α and β are required within the PDF cell population; i.e., knockdown of either in this cell population leads to long-period rhythms (V. Sundram, unpublished results). Furthermore, behavioral rhythms of the Andante mutant can be rescued by expression of the P subunit in PDF neurons. Thus, it would appear that CK2 is required only within the PDF population even though the β subunit has a wider pattern of localization (it is found in other cell types including cells of the adult mushroom body). A manuscript describing this work is in preparation for publication.

Specific Aim 4. What is the mechanism of action of CKZP(Andante) in the clock system?
As described in previous progress reports, our studies show that Andante flies have constitutively higher levels of the clock proteins PERIOD (PER) and TIMELESS (TIM). The same studies have also shown that the nuclear translocation of PER and TIM proteins is delayed by ~2 h in the mutant. The latter result completely explains the long-period phenotype of the mutant and suggests that delayed nuclear translocation and decreased transcriptional repression of the per and timgenes may be responsible for increased PER and TIM levels. Consistent with this idea, per mRNA is constitutively higher in the Andante mutant. Obviously, both PER and per mRNA continue to show molecular oscillations in the mutant, as expected, since Andante causes a long-period phenotype, not arrhythmicity.

The S6KII kinase cooperates with CK2 to regulate circadian period
Studies funded by this grant have identified another kinase known as S6KII (mammalian Rsk1) that cooperates with CK2 in regulating circadian period. Our collaborators in this work, Drs. Eike Jauch and Thomas Raabe (University of Wurzburg), have shown that CK2 subunits (both α and β) can physically interact with S6KII in cell-based assays. Their work indicated that S6K2 can interact with a dimerization-defective version of CK2β and an Andante mutant version of CK2β. Thus, the interactions do not require dimerization of the β subunit, unlike the interaction of CK2α with CK2β, which requires dimer formation.

Specific Aim 5. Do changes in CK2α subunit abundance and activity result in an alteration of behavioral rhythmicity?
As indicated in our previous progress reports, the group of Ravi Allada (Northwestern University) showed that CK2α mutations cause long-period circadian rhythms, similar to our documented effects of Andante (a CK2β mutant). Thus, it is clear that both CK2β and CK2α are required for rhythmicity and our recent results with RNAi transgenes indicate both are required within the PDF cell population for normal locomotor activity rhythms.

PI: Jackson, Rob
Title: TUSM/T-NEMC Center Core for Neuroscience Research
Abstract: This application requests funds to establish a NINDS Center Core to augment the research capabilities of NINDS and other neuroscience investigators at Tufts School of Medicine (TUSM) and the affiliated medical center Tufts-New England Medical Center (T-NEMC). Within the research labs of TUSM and T-NEMC, there are 17 NINDS-funded research projects, spanning most areas of contemporary neuroscience research. Most of our Tufts NINDS investigators are members of the Sackler School Graduate Neuroscience Training Program, a highly collaborative group of 28 neuroscientists from TUSM and T-NEMC. This is a rapidly evolving program that includes 6 new neuroscience departmental faculty and a total of 12 new TUSM or T-NEMC neuroscientists hired within the last 6 years.

Due to the rapid growth of our program, we have become concerned with providing adequate research core facilities to neuroscience investigators, and have held planning meetings to determine the need for additional or expanded core facilities. The award of NINDS Center Core funds will permit us to integrate faculty and research facilities from TUSM and T-NEMC, with the primary intent of providing needed services to NINDS investigators. A related goal is to foster collaborative research among the large collection of NINDS investigators at TUSM and T-NEMC. Importantly, the TUSM/T-NEMC NINDS cores will be available to other investigators at TUSM, T-NEMC, and Tufts University. The establishment of these cores is greatly aided by the expressed support of Tufts University, TUSM, and T-NEMC, who are providing assistance, funds and space for the establishment of a NINDS Center Core. The administration of the new center core will be based in the TUSM Department of Neuroscience. Supervision for the center core will be provided by the P.I., Dr. F. Rob Jackson, and an advisory committee including core directors and outside scientists with relevant expertise.

PI: Jacob, Michele
Title: Regulation of Neuronal Synaptic Components
Abstract: Nicotinic acetylcholine receptors (nAChRs) function at key inter-neuronal 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 inter-neuronal 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:

Aim 1: Directing nAChR transport to and/or stabilization at postsynaptic sites

Aim 2: Directing the alignment of pre- and postsynaptic specializations by anchoring retrograde signaling complexes at sites of nAChR accumulation

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: Jacob, Robert
Title: Reality-Based Interaction: A New Framework for Understanding the Next Generation of Human-Computer Interfaces
Abstract: The state of practice in human-computer interaction today is the graphical user interface (GUI) or direct manipulation interaction style. The project seeks to identify an emerging next generation of user interaction from a range of seemingly disconnected current research and to develop a framework for unifying them. These include: virtual reality, augmented reality, ubiquitous, pervasive, and handheld interaction, tangible user interfaces, lightweight, tacit, or passive interaction, perceptual interfaces, affective computing, context-aware interfaces, and speech and multi-modal interfaces. Ubiquitous, computing, tangible interfaces, and the spread of computers into a wide range of products and objects are changing interacting with computers from a specialized activity segregated from daily life to becoming more and more a part of the real world. At the same time, as computers are becoming more a part of the "real world," user interfaces seem to be evolving to behave more and more like the real world, for example in virtual reality. These can be connected through the idea of "reality-based interaction" by focusing on the ways in which interfaces that are based on reality exploit users' built-in abilities. This idea provides the leverage to attempt to tie them together and define a new generation of user interfaces and to see whether this naive notion can be built into a 11 Seful theoretical framework.

The goal is to begin to develop and test a framework or theory to connect developments in next generation user interfaces. The project starts from the idea of natural or "reality-based" interfaces. These interfaces gain their strength because they exploit abilities that their users already possess. The project will thus formalize this notion (if the learned knowledge vs. the "reality-based" skills needed to use a system). The project will develop it into a theoretical framework and flesh it out more formally. It will also identify some specific open issues for investigation below. It will then modify or reinvent the initial approach as needed. It will test the theory, first, by applying it against a range of published results. Next, it will devise experiments specifically for testing, where aspects of a user interface can be selectively manipulated for the experiment. The project will build and test new interaction techniques as needed for the experiments, using the workbench and infrastructure package to be developed for dissemination, as described below. The final stage of the project will design, implement, and evaluate some selected new interaction techniques representing gaps or opportunities suggested by the framework, again using and adding to the infrastructure package.

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: Jalal, Ayesha
Title: Religion and Revolution Amongst Modern Bengal Muslims, 1917-47
Abstract: I plan to research how religiously informed visions of a new, postcolonial society among Bengal Muslims influenced their participation in anti-colonial movements between 1917 and 1947.

In this period, Bengali Muslim politicians, activists, and writers debated issues related to the concept of a new, postcolonial society, including the ownership of land, the treatment of tenants by landlords, and the relationship between Muslims and non-Muslims. These issues are often defined as ‘secular”, but in this period, these issues were often considered within the framework of religion. Many Muslims, prompted by various strands of politics that invoked the idea of a Muslim ummah, or community of believers, perceived issues such as land ownership and tenancy in the context of Islam and its teachings about society.

However, the majority of scholarship on this period focuses either on politicians and movements in the public sphere or the world of religious preachers, missionaries, and madrassas (Muslim schools) and rarely examines the interrelation of these spheres. I seek to contribute to our understanding by attempting to find where religious and secular politics, relating to the imaginings of a postcolonial India, may have intersected for Bengali Muslims. 

The question driving my research regards the success of the political party, the Muslim League, and its slogans for a “Pakistan,” in Bengal. How did this allegedly “religious” party gain such overwhelming support in the 1940s for a separate “Muslim” state? This question arises because the Muslim League failed to garner much support at all in urban or rural locations of Bengal until the mid 1940s.

The 1947 creation of a two-part nation-state of Pakistan consisting of a portion of Punjab in the West, along with other northwestern parts of British India, and a portion of Bengal in the East, ranks among the most important events in twentieth century South Asia. The creation of the nation-states of India and Pakistan in 1947 and Bangladesh in 1971 shapes a great amount of contemporary discourse about nationalism and religion in the region. 

As a case study for South Asia, as well as for comparisons with other Muslim societies, Bangladesh serves as a fruitful place to conduct such a study as it is a country of 140 million people, 123 million of whom are Muslim, making it the third largest Muslim country in the world. I hypothesize that ideas related to land ownership and tenancy which grow in this period resonated with the growing world of modern, leftist conceptions of justice propounded by Communists and Marxist activists, I seek to research not only how and where religious ideologies and modern, left oriented ideas connected but also to analyze why this connection existed.

PI: Jay, Daniel
Title: Inhibiting Surface HSP90 To Limit Metastasis
Abstract: The vast majority of breast cancer-related deaths are due to secondary tumors after metastasis. There is currently no effective therapy to limit metastasis. Our long term objective is to develop new therapies that reduce cancer invasion, a critical first step in metastasis.

This proposal is based on our observations that a novel extracellular form of the molecular chaperone hsp90a is required for cancer invasion by the activation of the matrix metalloproteinase MMP2. Hsp90 has been implicated in cancer, and hsp90 inhibitors with anti-tumor activity are currently in clinical trials. These drugs may be problematic in that they interfere with the many intracellular functions of hsp90. Our findings suggest our main hypothesis that inhibiting extracellular hsp90a will decrease invasion and thus limit metastasis. This presents an opportunity for novel anti-cancer therapy by inhibiting invasion without interfering with the myriad intracellular functions of hsp90.

To support this idea, we will address three specific aims.

Aim 1: We will determine the mechanism of how hsp90a functions on the outside of cancer cells.

Aim 2: We will then use this information to develop and test extracellular hsp90 inhibitors. We already have one impermeant hsp90 inhibitor in hand and several candidates for neutralizing single chain antibodies from our collaborators at NCI and Xerion Pharmaceuticals.

Aim 3: Finally, we will test the best of these inhibitors of extracellular hsp90a for their ability to limit metastasis in a new model developed by our collaborators at Tufts using human breast cancer cells metastasizing to human bone explants in immunocompromised mice.  

Thus, these experiments take us from an initial discovery of hsp90a function with cell-based assays to in vivo animal models taking an interdisciplinary approach to address a key issue of human health: limiting metastasis to improve breast cancer prognosis. These studies aim to expedite the translation of our basic research into a potential therapy. If successful, the proposed work would provide data for developing future clinical studies and thus impact human health.

PI: Joseph, Hugh
Title: New Entry Sustainable Farming Project – Farmer Training Program
Abstract: In this renewal application to OASDFR, the New Entry Sustainable Fanning Project (NESFP) proposes to improve and expand outreach, training, technical assistance and farmer leadership activities that are being implemented with OASDFR funds during the current (2003-2004) award period. The goal of this program is to engage socially disadvantaged immigrants with agricultural backgrounds in farm enterprise development and ownership. Associated objectives include:

1. Expanding partnerships with community organizations and social service agencies to
1. Increase enrollment by immigrants and refugees
2. Secure in-kind resources
3. Promote cross-cultural awareness among project partners and farmers
2. Conducting broad-based outreach to identify farmland
3. Providing comprehensive technical assistance to participating farmers to address all aspects of farm enterprise development
4. Revising and expanding NESFP formal training programs
5. Strengthening participating farmers' roles in project planning and implementation
6. Linking project farmers to federal (USDA), state and local programs and services
7. Involving NESFP staff, partners and farmers in regional and national immigrant farming initiatives

Program implementation includes recruiting and evaluating qualified participants, conducting needs assessments, and organizing an 18-week off-season farming course, involving USDA, state, and county farm organizations and community organization in training and technical assistance activities (T&TA); providing in-season T&TA on our farm training sites; providing one-on-one assistance; coordinating farmland identification; developing a research and demonstration plot; supporting farmer committees; and providing direct marketing assistance. OASDFR funding will assist up to twenty socially disadvantaged farmers per year to become independent farmers, and to assist them and many additional participants in the region to access USDA and other farm sector services.

PI: Kaplan, David
Title: Silk Scaffolds
Abstract: Silks represent an important advanced biomaterial due to their unique and impressive mechanical properties, versatility in processing to control structure and morphology, degradability, biocompatibility and genetic tailorability. While silk has a long term historical use in sutures, recent studies have expanded both fundamental and applied aspects of this protein in many areas related to advanced biomaterials. The hypothesis for this proposal is that silk protein bioengineering, with appropriate consideration for processing and chemical modifications, can be employed to modulate the rate of 3D scaffold degradation and tissue-specific outcomes suitable for bone growth and repair. Reprocessed native silkworm silk formed into 3D scaffold porous structures, in combination with cytokine and adhesion sites, will be used in perfusion bioreactor systems and in vivo rat models.

Our goal is to understand the relationships between silk scaffold structure and function related primarily to degradation and bone-like tissue outcomes in vitro and in vivo. The outcome of the proposed study will be an understanding of fundamental relationships between silk structure and morphology with function (mechanical properties, rates of degradation, cellular responses related to bone tissue formation). The outcome of the proposed studies will be a roadmap of the impact of processing and modification factors on silk structure and function for this family of advanced biomaterials. In part, by mimicking the native processing of silk, we envision a range of possible outcomes in terms of structures and functions based on silk scaffolds that will provide useful interactions with cells toward bone-specific needs. The three specific aims will address:

1. Relationships between silk crystallinity and porosity in 3D scaffolds on mechanical properties and rates of enzyme degradation in vitro.

2. How scaffold structure, morphology and surface chemistry (RGD, BMP-2 decoration) impact bone-like tissue formation from Saos-2 (human osteosarcoma cells) and MSCs (human adult bone marrow stem cells). Cellular and genetic assessments of bone-related outcomes, spatial and temporal deposition of calcium using non-destructive imaging methods (mCT), and changes in structural, mechanical and morphology features of the scaffolds will be assessed.

3. Biocompatibility of the matrices in vitro and in vivo. The data from the proposed study will improve the understanding and utility of silk-based advanced biomaterials for a wider range of biomedical applications.

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 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 collaboration with Gazit Dan as an extension of another NIH grant.

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:

• New materials engineering from silks
• 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:

• 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
• 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: Tissue Engineering Resource Center
Abstract: The field of tissue engineering has been propelled in recent years by advances in cell and molecular biology, biomaterials science and engineering and bioreactor design and function. With the resulting avalanche of information, the complexity of the interactions needed to achieve desired tissue outcomes in vitro to adequately address clinical needs in vivo represents a growing challenge. It is difficult for any one laboratory to deal with all of the scientific and technological issues involved. The proposed Tissue Engineering Resource Center (TERC) will integrate cell biology, biomaterials and bioreactor systems, built upon strong core knowledge in each of these areas, to provide a systems approach to the field of tissue engineering and the associated service to address laboratory and clinical challenges. The core research projects in the Center will focus on:

1. Stem/progenitor cells - stem cell biology characterization expansion differentiation

2. Bioinductive scaffolds - structurally and functionally tailored

3. Advanced bioreactors - with enhanced environmental controls and a capability for nondestructive real time assessments

A unique Center will be established and hosted by an academic consortium led by Tufts University, MIT and the University of Toronto. The Center will couple the capabilities of these laboratories through a Core Service Lab operated at Tufts to provide outside researchers full access to the latest techniques integrated in one location to solve complex challenges in the field. The Service Core will focus on the integration of scaffolds, stem cells and complex reactors to achieve new fundamental insights for use in the field, targeted tissues for clinical needs, and general service to support investigators. The Center will also interface with and complement other Centers with relevant components in tissue engineering - such as the Imaging Center at Harvard-MGH and Biomaterials Center at the NIST. The Center will host a number of collaborations with other laboratories related to specific enhancements of the core projects, such as tissue engineering of human ligaments using transfected stem cells. Information on scientific and technological advancements will be actively communicated through workshops, courses, symposia and educational outreach by the core at Tufts with additional outreach through MIT and Toronto. The Service Core will also provide essential support to help new industries in the field move ahead in the current challenging economic climate.

PI: Kaplan, David
Title: Bioemulsifying Vaccine Delivery System for Immunomodulation
Abstract: In our recent studies we have demonstrated that the biopolymeric emulsifier, emulsan, possesses exciting potential with regard to dual function (direct emulsification and immunomodulation). In particular, a compelling set of features including structural tailorability, innate ability to carry proteins, easy and large scale synthesis, lack of toxicity (to the extent tested thus far), and strong indication of vaccine efficacy and immunomodulation prompt the plans in the present proposal. The structural features of these novel biopolymeric systems can be 'tailored' through a combination of physiological control and/or genetic engineering and we have demonstrated that alteration in structure of these bioemulsifiers directly relates to changes in solution properties (e.g., emulsification, surface tension) and biological function (e.g., macrophage activation such as TNF). In vivo, vaccine efficacy has been demonstrated in a number of animal studies including with a DNP-hapten carrier, Lyme and Yersinia.

Based on these prior data, the hypothesis in this proposal is that structural variants of these microbial exopolysaccharides can provide new insight into our understanding of mechanistic features of the polymer interactions with the innate immune system and lead to the identification of promising candidate vaccine adjuvants. These data would lead to new and more effective vaccine adjuvants, an area of strong clinical need. The unique and powerful dual function of emulsification properties of these polymers combined with strong structural tailorability, suggests that new and useful functional adjuvants can be obtained. The studies planned will focus on biological preparation and assessment of these bioemulsifiers. An interdisciplinary team of scientists with expertise in biopolymer engineering, immunology and disease models, will conduct the proposed studies and has also been involved in the collection of the Preliminary Data. The specific aims include: (#1) synthesis and characterization of emulsan structural variants, (#2) assessments of innate immune recognition of the structural variants, (#3) assessment of the variants in an in vivo vaccine disease model based on the utilization of emulsan with a recombinant Yersinia pseudotuberculosis vaccine to induce protective immunity in a murine disease model (Aim #3), and the efficacy of emulsans in intranasal delivery of the vaccine formulation (Aim #4). The proposed studies will provide a solid foundation upon which to correlate emulsan structure and cellular responses leading to specific levels and types of immunological activities. Furthermore, lead adjuvant candidates will result from the study in preparation for formulation studies and clinical trials in future work.

PI: Kauer, John
Title: A Novel DNA-Based Detector Library in an Artificial Nose
Abstract:

Specific Aim I: To approach the first hypothesis related to examining the mechanisms by which ssDNA-Cy3 sensors respond to odors.

Early experiments using circular dichroism showed conformational changes in aqueous DNA-Cy3 with exposure to liquid phase odors in solution. Fluorescent Resonance Energy Transfer (FRET) investigations showed a correlation between conformational changes of solid-state DNA-Cy3 sensors and odor responses. In addition, experiment excluded the following potential hypothesis for odor response:

1. Resonance energy transfer between DNA and Cy3 or Cy3 and odor
2. Change in Stokes shift
3. Proton transfer (pH)
4. Dynamic quenching by odorant
5. Static quenching by odorant
6. Covalent or ionic binding of odorant to ground state

A second important factor in sensor-odor interactions was discovered. Cy3 aggregation occurs in the processing of sensors and effectively quenches some dye molecules in the H-aggregate. Odors and sequences able to manipulate the percentage of stacked dye molecules would affect dye fluorescence. A particular case of this occurring with dC(21) was studied exhaustively in solution. The data showed that in the presence of acids, a parallel stranded double helix formed bringing the two 5' dyes into close proximity resulting in quenching. This establishes another type of odor sensor interaction in DNA-Cy3 odor sensing.

Conformational changes are expected to lead to changes in interstrand and intrastrand redox reactions between bases and Cy3 that result in sequence specific alterations in fluorescence. In addition, odor induced, or DNA conformation induced changes in Cy3 aggregation would also lead to alterations in fluorescence. The former is a type of dynamic interaction where fluorescence lifetime is altered by the microenvironment of the dye. The latter is a static interaction where the fluorescent population is changed without changing the lifetime of any of the fluorophores. I propose that the interactions between these two prorecesses result in the DNA-Cy3 odor response.

Specific Aim IB: Examine temperature and solvent polarity effects on ΔFI in DNA-Cy3 sensors exposed to odors in order to test the hypothesis (1) that odorant molecule diffusion into a residual solvent layer is part of the mechanism of odor sensing in these detectors.

FTIR experiments were unable to finally determine the etiology of the DNA-odor interaction. Despite developing techniques for using FTIR to assay the DNA-odor interaction in the solid state with vapor phase odorants, single-stranded DNA does not offer characteristic peak changes when its hydration state is altered, although double-stranded DNA does. Thus the effect of odor on the hydration state of the DNA remains hypothetical. However it is still possible from the data collected in FRET experiments to draw the conclusion that for all polar odors tested, the FRET distance (a surrogate for the length of the DNA-Cy3 sensor) increased and for all nonpolar odors, the FRET distance decreased. Charged odors like the amines did not follow this trend. A reasonable hypothesis given these data is that there is a direct, charge-based interaction between the amines and the DNA backbone and that all other odors have interactions with DNA that are related to their polarity, possibly through affecting the hydration state of the DNA. The similarities between the FRET responses for dried and hydrated sensors and the FRET responses for sensors exposed to nonpolar or polar odors support this hypothesis.

Specific Aim IC: Perform DNA base swapping experiments and DNA truncation experiments to test the hypothesis that sequence dependent tertiary structure modulates the ΔFI of DNA-Cy3 sensors responding to odors. Truncation and base-swapped sequences were constructed and tested, but the conclusions were ambiguous. It appears that both proximal and distal bases are likely to be important in the overall conformation of the DNA-Cy3 sensor. In addition, bases on neighboring strands or aggregation of the Cy3 dye may play important roles in DNA-Cy3 sensor odor responses.

Specific Aim II: To approach our second hypothesis related to finding the appropriate DNA-Cy3 sensors for a defined health-related odor set, I will:

Specific Aim IIA: Develop a library of thousands of random DNA-Cy3 sensors and a high-throughput screening for testing our second hypothesis related to finding appropriate sensors to detect health-related volatile compounds. This library and screening process will be used in IIB to search for sequences that detect health-related odors.

In early experiments I developed microarray tools to test large numbers of DNA-Cy3 sensors for odor responses and developed an E. coli based library system for making sensors. Most of this work is now being conducted at Cogniscent, Inc. using tools developed in this research in order to allow my work to concentrate on the hypothesis based basic research of understanding the DNA-Cy3 sensor-odor interaction. Progress was identifying sequences that detect important health-related odors. DNA-based sensors that detect DNT (a component of landmines), DMMP (a component of sarin nerve gas), ethanol (a common drug of abuse), molds, and acids have been found.

Specific Aim IIB: Perform a high-throughput screening of a random library of DNA-Cy detectors in order to test the hypothesis (2) that a set of solid-state DNA-Cy3 sensors may be found that has sufficient diversity and combinatorial complexity to detect important health-related compounds.

Techniques for screening a random library have been developed using microarray-related techniques such as robotic spotting and array reader hardware. Using a library of 32 synthesized sequences, these techniques have been tested and appear promising. While some troubleshooting remains to improve system variability, out best data show a 0.972 Pearson correlation coefficient for identical sensors. For random sensors, 12 out of 30 correlate at less than 0.9 with some as low as 0.1. This indicates that sequence variability can lead to response variability, supporting in principle our hypothesis that sets of DNA-Cy3 sensor will have diverse responses in detecting odors. In addition, DNT (a component of landmines), DMMP (a component of sarin nerve gas), and ethanol (a common drug of abuses) are among the odorants tested. Sensors have been found that have responses to each of these compounds.

Specific Aim IIC: Optimize the health-related compound detection thresholds for sensors chosen in (IIB) by testing differing buffers, and humidity conditions. In the case of DNT, it is preferred to achieve detection levels below 10 ppb and most preferred below 1 ppb. For other health-related odors, the preferred levels of detection may be different.

Progress was made in this area. Sequence LAPP1 was discovered to detect DNT at 6 ppb. This is within the detection levels we had hoped to achieve and shows the potential for beneficial applications of this technology.

PI: Kehayias, Joseph
Title: Monitoring Sarcopenic Obesity in the Elderly
Abstract: Obesity in the elderly can be masked by body composition changes that result in a net weight loss. Sarcopenia, the loss of muscle and function with age, is universal in the elderly. The reduced protein stores inherent in sarcopenia leave the elderly with decreased ability to cope with illness or injury. The combination of increased % fat and sarcopenia results in a body composition status described as "sarcopenic obesity." This condition cannot be assessed by anthropometry or other traditional indirect body composition methods. The purpose of the proposed research is to develop and test a portable and relatively inexpensive instrument (X-ray absorptiometer) for carrying out body composition analysis of soft tissue in the field. These measurements are important for providing an index of nutritional status of individuals and will be used to evaluate the benefits and risks of anabolic treatments, nutritional support and the management of obesity in children and adults. Being portable, inexpensive, and simple to use, the instrument will permit the evaluation of nutritional health status of individuals in their own environment. It will be particularly beneficial to those for whom access to hospitals is difficult or limited (e.g. elderly in nursing homes or assisted living) or in underdeveloped nations, where aggressive nutrition support programs are being evaluated. The design is based on single and dual energy X-ray absorptiometry (DXA), optimized for soft tissue. The instrument is a hand-held caliper with a collimated X-ray source and a detector. The use of a simple detector array will facilitate the positioning of the caliper. The instrument will be evaluated for both single and dual energy mode and will be tested against mid-thigh CT and reference whole body composition techniques. The outcome of this research will be a fully developed and tested instrument and a method for its use in the field. This portable instrument will be particularly useful for monitoring sarcopenic obesity in the elderly where existing simple methods (such as Body Mass Index) fail to identify the true level of adipose tissue and frailty.

PI: Kilmer, Misha
Title: Collaborative Research: Turning Libraries to Effectively Exploit the Memory Hierarchy
Abstract: Sparse matrices and irregular codes introduce complexity into effectively utilizing a hierarchical memory system. As the gap between processor and memory speeds continues to grow, the importance of effectively utilizing hierarchical memories becomes increasingly important. We need to consider new code transformations that can enable optimizing compilers to map sparce or irregular data structures to more spatially local structures. While compilers can perform aggressive optimizations to map data structures and code to the target hierarchical memory systems, they typically do not consider the interaction of the application with precompiled library code.

In this project we propose to develop new source-level and binary-level transformations that improve the usage of the memory hierarchy by sparse and irregular codes. Our work focuses on multiple levels of cache, main memory (and TLB layout), and disk storage mapping. We will develop novel profile-based algorithms that can improve layout of both code and data by profiling execution behavior. Our profile-guided approach will also be applied to library codes. We will utilize binary editing to provide for increased spatial locality when considering the application codes and library codes. Our target applications include:

• Numerical codes
• Sorting and join algorithms
• Search and query
• Iterative solvers (e.g., Krylov methods)
• Finite difference methods

The impact of this research will be:

• To develop new source-level transformations that identify poor locality and perform source-to-source transformations to remedy this poor locality
• To develop transformations to multi-pass algorithms with better latency tolerance
• To develop analytical performance models that allow for accurate prediction of elapsed time for memory-bound and disk-bound computations
• To take greater advantage of available parallel resources (multiple disks and CPUs) to overcome latency through greater bandwidth

The broader impacts of this project will be to provide research experiences for both undergraduate and graduate students. This research should also greatly benefit society, providing a new approach to addressing important classes of problems in imaging and scientific software systems. This project will also work to insure that underrepresented groups are involved in this research project through targeted recruiting and information dissemination. Specifically, we will work with the Women in Engineering and Multicultural Engineering offices at Northeastern University to insure that the educational opportunities provided by this project will include participation by female and minority engineering students. We will specifically target undergraduates supported through the NSF REU program, which we have successfully pursued in past NSF-funded research. We will also work to attract summer interns from the Boston public schools to participate in this project.

PI: Kos, Claudine
Title: The Role of the Extracellular Calcium-Sensing Receptor in Dietary Calcicum Chemoprevention of Colon Cancer
Abstract: Colorectal cancer is one of the main causes of cancer mortality in Western societies. Dietary calcium has been shown to have a chemopreventive effect on colon tumorigenesis in both humans and mice. Although there is strong evidence that dietary calcium decreases colon tumors, the molecular mechanism by which calcium leads to chemoprevention is unknown. There are two hypotheses for how increased dietary calcium lowers the risk of colon tumors. One is that calcium binds excess ionic lipids (which are potentially toxic to colon) in the lumen and causes them to be excreted. The second is that calcium acts directly via the G-protein-coupled extracellular calcium sensing-receptor (CaSR) to decrease colonic cell proliferation. We will test directly this second hypothesis by comparing the chemopreventive effects of a high calcium diet in azoxymethane carcinogen-induced colon tumorigenesis in 140 calcium-sensing receptor deficient and control mice. The findings of these studies will demonstrate to what extent CaSR is a specific molecular target responsible for mediating the effect of dietary calcium chemoprevention. This work will determine whether or not CaSR is a novel signaling molecule in colonic epithelial cell development and whether or not CaSR is related to the positive impact of nutritional calcium on digestive disease. In addition the study will further characterize the rescued CaSR deficient mouse and determine whether it can be further utilized as a novel animal model of colorectal cancer.

PI: Kounaves, Samuel
Title: Measurement of Total Organic and Inorganic Carbon on Mars
Abstract: In support of the TIC/TOC science investigation and in close collaboration with investigators at NASA's JPL and Ames research centers, Professor Kounaves (as the Tufts PI), will be responsible for developing, evaluating, and implementing, the electrochemical oxidation system for use with the TIC/TOC, per the statement of work below.

Year 1: The first generation electrochemical TIC/TOC system will be designed, assembled, and evaluated. Analysis of the generated CO2 will initially be accomplished by measurement of the remaining organics. During year one, we will also evaluate and select a MS-based CO2 monitoring system and integrate it with a gas sampling, purging, and calibration manifold. A Tunable Diode Laser (TDL) spectrometer will also be evaluated. Specifically the following tasks will be accomplished:

1. A prototype reaction chamber will be manufactured out of chemically inert materials. This chamber will contain rudimentary sensors for temperature, pH and redox potential and the boron doped diamond electrodes. By the end of year-one the system will be interfaced to a mass spectrometer CO2.
2. Electrochemical oxidation efficiency will be studied as a function of sample type, solution concentration, time, added reagents, and temperature.
3. A standard TOC analyzer will be used to evaluate the electrochemical oxidation efficiency.

Year 2: The following tasks will be performed in conjunction with JPL/ARC:

1. We will optimize the first generation system, determining (for example) the efficiency of the purging and gettering system for removing background CO2. We will determine sensitivity limits on both TIC/TOC analysis and isotopic ratio determination.
2. We will analyze geologically relevant samples, normalizing the first generation system to the Total Carbon Analyzer, and evaluate particle size effects. We will determine if contextual information (e.g. specific surface area) will need to be acquired in situ for complete interpretation of the TIC/TOC data.
3. We will integrate the electrochemical oxidation system with the existing RCAL platform and evaluate its performance.

Year 3: In conjunction with JPUARC the following tasks will be performed:

1. We will develop calibration procedures for the first generation instrument and measure final detection limits and isotope ratio sensitivity. We will develop operational protocols for a flight instrument and fully document its use in a "legacy review."
2. We will procure a dedicated RCAL unit, integrating it with the electrochemical oxidation system. We will evaluate this second generation system, comparing its performance to the first generation platform. Pending the result of complementary proposals we will attempt to integrate the unit with a flight-like CO2 detector.

PI: Kounaves, Samuel
Title: Microbial Detection Array (MDA): Unambiguous Detection of Extraterrestrial Microbial Metabolic Activity Using Differential Electrochemical Detection
Abstract: The ideal microbial life detection instrument for use beyond Earth must be based on absolutely minimal assumptions about the nature of the organism. For example, the organic chemistry, internal structure, and internal components of microbial life might be significantly different from terrestrial microorganisms and cannot be accurately predicted. However two properties of microbial life that are likely to be universal and require no prior assumptions of specific properties are:

1. An ability to reproduce itself in a self-regulated form
2. The ability to maintain some degree of isolation of its internal compartment from the surrounding environment

In addition, we will assume that any microbial life will require water and carbon. During the process of reproduction, the organism’s metabolism, mediated by its membrane processes, will, by necessity change the surrounding chemical and redox environment.

Thus, an appropriate instrument must be capable of detecting such changes, rapidly and free of extraneous or non-biogenic interferences. We propose to demonstrate a new detection technique via a prototype instrument dubbed the Microbial Detection Array (MDA). A substantial portion of the MDA, the sensor array, is heritage-based and already space qualified.

The MDA is designed to provide a response to minute chemical and physical changes occurring in one of two identical chambers via differentially monitored electrochemical sensor arrays. Minimal metabolism will alter the physico-chemical steady state in one chamber such that a difference between the sensor arrays will result in a signal. This detection system makes minimal assumptions about the nature of the microorganism, assuming only that after addition of water it will reproduce and in the process cause changes in its immediate surroundings by consuming or generating, metabolizing, and transporting in both directions, a number of molecules and ionic species.

The experiment begins by placing a homogenized split-sample of soil into each chamber, adding pure water, sterilizing at a high temperature incompatible with any form of terrestrial life, and zeroing. In the absence of any metabolically active organism in either chamber, no signal will be detected. The “inoculation” of one chamber with a minimal of viable microorganisms, which proceed to multiply, will produce a significant metabolically generated disequilibrium in the system (compared to the control) to provide a detectable signal.

Replication of the experiment and positive results would lead to the conclusion of bio-logically induced changes. Changes resulting from non-biological chemical reactions of whatever type are canceled out by the control. The replication of the procedure, split sample, and minimal inoculation protocol, eliminate non-biogenic causation. In addition to detecting microbes, the sensor array will also characterize the chemical composition and electrochemistry of the sample.

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. This is in contrast to 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: 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:

• Transacting mutations that lead to high level expression of either MDR1 or CDR2
• 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: 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

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: Kumar-Singh, Rajendra
Title: Gene Therapy for Retinitis Pigmentosa
Abstract: Retinitis Pigmentosa (RP) is one of the most frequent causes of hereditary blindness in the United States, affecting approximately 1 in every 3000 individuals. There is currently no treatment available for this disease. The broad, long term objectives of this study are to develop a therapy for RP. Some forms of RP in humans, dogs and mice (rd) are caused by mutations in the beta subunit of cGMP phosphodiesterase (bPDE).

In order to deliver a normal cDNA encoding bPDE to the rd retina, we have developed a novel class of safer and less-toxic adenovirus vectors termed Encapsidated Adenovirus Minichromosomes - (EAMs or 'gutted' vectors). EAMs have a 36Kb cloning capacity, allowing insertion of large upstream regulatory elements for regulated transgene expression and/or inclusion of multiple therapeutic transgene cassettes. We have used EAMs to temporarily rescue retinal degeneration in rd1 mice by approximately 10 weeks, which would otherwise be complete by 3 weeks postnatal.

Our objectives are now focused on extending this short period of rescue to one that might be more therapeutically relevant to humans. EAMs bind to their target cells by attachment of the antenna-like fiber protein to the Coxsackievirus B-adenovirus Receptor (CAR). We have determined that CAR is not significantly expressed on murine and human photoreceptors which could explain the low levels of infection by EAMs and adenovirus vectors. In order to overcome this problem, we propose to modify the structure of EAMs such that they now bind cell surface sialic acid instead of CAR. Sialic acid is abundantly present on the rod cell membrane.

We also propose to test adenovirus fibers recently shown to have significantly enhanced tropism for neurons. We will use these improved EAMs to deliver bPDE to the rd10 retina. We will measure therapeutic effects on photoreceptors by PDE assays, histology, electroretinograms and western analysis.

Upon completion of this study, we will have constructed and tested a vector which will have potential use in a Phase 1 clinical trial for treatment of RP in humans.

PI: Kumar-Singh, Rajendra
Title: VP22 and TAT Mediated Gene Therapy for the CNS
Abstract: Adenovirus (Ad) mediated gene transfer is a promising technology for the treatment of many genetic disorders of the Central Nervous System (CNS). Many of the drawbacks that exist with first-generation Ad vectors have been resolved through the use of 'gutted' or 'helper-dependent' vectors. The genetic basis of neurodegeneration is currently best understood in the retina. In addition, relative to the rest of the CNS, the physical accessibility of the eye makes the retina an excellent model system for studying gene therapy for the CNS. Upon ocular delivery, Ad vectors primarily infect the retinal pigment epithelium (RPE) and the Mueller cells of the retina.

However, the diseases that most frequently cause blindness are associated with the expression of mutant proteins in the photoreceptor neurons. In our preliminary studies we have found that adenovirus-delivered green fluorescent protein fused to the full length Herpes Simplex Virus (HSV) tegument protein VP22 can translocate from infected cells to uninfected cells in culture or from the RPE to photoreceptors in vivo. This has led to the hypothesis we wish to test in this study: Can HSV VP22 be used to deliver therapeutic proteins to photoreceptor neurons via the RPE? This will be answered using a mouse model of inherited retinal degeneration (rd).

Specifically, in this study we propose to:

1. Construct an adenovirus vector expressing the protein transduction domains (PTD) of HSV VP22 fused to GFP, and compare their ability to traffic GFP in cell culture and in murine retina

2. Construct an adenovirus vector expressing a fusion between beta PDE and the PTD of HSV VP22. Assess the ability of this virus to express a functional PDE

3. Administer a gutted adenovirus vector expressing either a VP22-beta PDE fusion to the retina of rd mice (which have a naturally occurring mutation in beta PDE) and assess the effects upon photoreceptor degeneration

PI: Kuperwasser, Charlotte
Title: Breast Cancer Stem Cells in the Formation of Bone Metastasis
Abstract: It is estimated that nearly 216,000 women will be diagnosed with breast cancer in 2004 and despite the enhanced detection methods and improved surgical treatment, breast cancer relapse and subsequent metastatic spread remains the major obstacle in the clinic. 4%-45% of patients with early stage breast cancer already have disseminated breast cancer cells that do not appear to be growing in the bone marrow by the time the primary tumors are surgically removed, and, despite the removal of the primary malignancy many years prior to relapse in these patients, metastases at distant sites such as bone and lung still develop, and the mechanism for this remains unclear.

One important property that the cancer cells must exhibit in order to form a metastasis much later after the primary tumor is removed is the ability to survive in these distant sites for years. In normal tissues, tissue stem cells are the only cells that exist throughout the life of the organism. For example, the stem cells that are the progenitors of all the blood cells in the body remain as stem cells in the bone marrow because of specific interactions with the bone cells.

HYPOTHESIS: The metastatic cells in breast cancer are the cancer stem cells. They remain as stem cells in the bone marrow because of the specific interactions with the bone cells and therefore can give rise to metastases at a later time point. The development of metastasis is due to the changes that happen in the bone marrow environment as a result of inflammation or localized bone turnover.

SPECIFIC AIMS/DESIGN: To determine if the breast cancer stem cells are the metastatic cells, several human metastatic breast cancer cell Lines will be characterized for cancer stem cells and whether these cells can metastasize compared to the non-metastatic cell lines. Specific mouse models will be used to address whether the bone marrow environment can maintain the stem cell properties of the cancer cells and to demonstrate that the formation of overt metastases is dependant on localized bone turnover or systemic inflammation, both of which induce changes in the bone marrow environment.

PI: Kuperwasser, Charlotte
Title: Use of a Novel Xenograft Model system to Identify the Genetic and Stromal Changes that Mediate Breast Cancer Progression
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 (HGF) or transforming growth factor beta (TFGβ), 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 caner 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 TFG-β) 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: Kuperwasser, Charlotte
Title: The Role of Estrogen in ER-Negative Breast Cancer Pathogenesis
Abstract: Breast cancer is a hormone-based disease with numerous factors contributing to the lifetime risk of developing the disease. While breast cancer risk is reduced by nearly 50% after one full term pregnancy, women over the age of 25 have a significantly greater risk of developing breast cancer immediately following childbirth compared to their nulliparous counterparts. It is widely presumed that the increased risk of developing breast cancer following pregnancy is due to the ability of pregnancy-associated hormones to promote the further proliferation of a pre-cancerous target cell population. It is surprising however, that the majority of breast cancers that develop during this time lack expression of either the estrogen or progesterone receptors. This important observation suggests that if hormones play a part in promoting breast cancer following pregnancy, they may not be doing so through direct binding to hormone receptor molecules expressed by breast cancer cells. It has also been accepted that removal of the ovaries prevents the formation of both ER-positive as well as ER-negative breast cancers, suggesting that even ER-negative breast cancers may be sensitive to ovarian hormones for their formation. We have observed that estrogen can promote ER-negative cancer development by recruiting cells termed "stroma" to breast cancers. Surprisingly, these cells were recruited from the bone marrow to the breast and were sufficient to promote cancer growth. Currently our goal is to determine the precise identity of the cell types in the bone marrow that are sensitive to estrogen and how they may respond to this hormone. Within the last decade, the development of superior and more effective anti-estrogen therapies have led to remarkable successes in the therapy of ER-positive breast cancers, osteoporosis, and cardiovascular disease. Therefore, it will be of great clinical importance to determine if these compounds have any effect on the development of ER-negative breast cancers or in the treatment of other cancers in pre-menopausal women. Further understanding of the molecular mechanism by which estrogen can promote ER-negative tumor growth will most likely have significant and highly relevant clinical impact in the treatment of breast cancer.

PI: Kuperwasser, Charlotte
Title: Mechanisms of Estrogen Action on Bone Marrow-Derived Stem Stromal Cells
Abstract: It is widely presumed that the increased risk of developing breast cancer following pregnancy is due to the ability of estrogen to promote the further proliferation of an initiated target cell population. However, since the majority of breast cancers that do develop during this time lack appreciable expression of either the estrogen (ER) or progesterone receptors (PR), this suggests that if hormonal changes play a part in promoting breast cancer, they may not be doing so through direct binding to hormone receptor molecules expressed by breast epithelial cells. Moreover, it is well established that ovariectomy prevents the formation of both ER-positive as well as ER-negative breast cancers in women, further highlighting the importance of estrogens in the development of ER-negative cancers. To reconcile this conceptual conflict we investigated the hypothesis that estrogen promotes the outgrowth of ER-negative cancers by influencing host cell types distinct from the breast epithelium itself. We utilized a novel xenograft mouse model in which the tumors that arise lack the expression of nuclear hormone receptors, recapitulating the clinical situation described above. Despite lacking ER expression, we showed that the tumors that develop in this model require circulating estrogens for their formation. Moreover, we demonstrated that increasing the levels of circulating estrogens is sufficient to promote the formation and progression of ER-negative cancers via a systemic increase in angiogenesis. Remarkably, the systemic enhancement of neo-angiogenesis was accompanied by a striking increase in the recruitment of bone marrow derived cells into the growing tumor mass, including endothelial and stromal cells. Based on our evidence, we now propose to determine the mechanism by which estrogen effects bone marrow cell recruitment, angiogenesis and tumor promotion. To this end, we aim to determine whether ER expression by the host is necessary for the stromal effects mediated by estrogens, and whether these actions occur through the genomic or non-genomic actions of ER signaling. In addition, we also aim to determine if bone marrow mesenchymal stem cells are the targets of estrogen-mediated angiogenesis and tumor promotion. This will be investigated through the use of ERKO mouse models, bone marrow cell fractionation, and in vivo and in vitro functional assays to estrogen signaling. Most recently, superior and more specific endocrine therapies targeting estrogen synthesis, turnover, as well as genomic and non-genomic activities of the receptor have been developed, however they are only utilized for the treatment of ER-positive breast cancers due to the lack of evidence these compounds would work in ER-negative tumors. Thus, understanding the mechanism by which estrogen can promote bone marrow cell recruitment, angiogenesis, and tumor growth would have significant and highly relevant scientific and clinical impact for ER-negative cancers.

PI: Lamon-Fava, Stefania
Title: Fatty Acid Regulation of Gene Expression in the Reverse Cholesterol Transport Pathway
Abstract: Plasma levels of high-density lipoprotein (HDL) are inversely related to the risk of developing coronary heart disease. It is thought that HDL protect from cardiovascular disease by playing a very important role in the reverse cholesterol transport pathway, in which HDL serve as an acceptor of excess cholesterol from the peripheral tissues and then transport it back to the liver for excretion. Several proteins, enzymes, and cellular receptors participate in the complex pathway of reverse cholesterol transport, but the proteins that play the pivotal role in this pathway are apolipoprotein A-I, the major protein component of HDL, and the receptors ATP binding cassette A1 (ABCA1) and scavenger receptor class B type I (SR-BI) present on the cell membrane of both macrophages and hepatocytes. These cell receptors mediate cell cholesterol efflux and uptake, respectively.

Both epidemiological and clinical intervention studies have shown that increased intake of n-3 polyunsaturated fatty acid (PUFA) is associated with a reduced risk of cardiovascular disease. However, PUFA have been shown to have a lowering effect on HDL levels. Currently, it is not known if this reduction in HDL levels translates into a reduced efficiency of the reverse cholesterol transport. Very little is known about the effect of different types of fatty acids, including n-3 and n-6 PUFA, on the metabolism of HDL and particularly on the reverse cholesterol pathway. The objective of this proposal is to define the effects of n-3 and n-6 PUFA on the expression of the apo A-I, ABCAl, SR-BI genes in liver cells and on the expression of SR-BI and ABCAl in macrophages. We also propose to define the molecular mechanisms involved in the modulation of the expression of these genes by fatty acids, including effects on transcription factors and cell signaling. Our study will clarify the overall effect of dietary fatty acids on the efficiency of the reverse cholesterol transport.  

The current research proposal will contribute to the improvement of public health by providing important research data about the role of dietary fatty acids in the reverse cholesterol transport pathway in relationship to cardiovascular disease.

PI: Lee, Mary
Title: Tufts University Open Courseware Project – Tools for Modular and Interoperable Content
Abstract: Tufts OpenCourseWare (OCW) aims to develop cost-effective tools that will deliver content that is both modular (for easier access, searching, and modification) and interoperable (able to be used across multiple platforms such as eduCommons, Blackboard, TUSK and other IMS standard-compliant systems). These tools will be shared with others, such as our TUSK partners in Africa and the U.S., to improve access and ability to modify content to local teaching and learning environments.Another tool will display "hot content" lists that will alert OCW users to the most requested courses, documents, and multimedia cases for further viewing.

Multimedia cases with a case player will be added to the OCW site to test the value of this type of interactive content in an open environment. We will expand our high quality courses in health sciences and international affairs, while adding undergraduate offerings. With our extensive partners and networks in educational technology, institutional experience in open learning systems, strong leadership, and exceptionally robust TUSK infrastructure, we expect to reach or exceed each milestone.

Success will be measured by the delivery of functional tools as well as the value of the tools and content to external users such as our university partners in Africa.

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: Leveille-Webster, Cynthia
Title: cAMP Modulation of Bile Acid Induced Hepatocyte Apoptosis
Abstract: The long range goal of the proposed studies is to identify therapeutic strategies to prevent hepatocyte apoptosis in cholestatic liver disease. Hepatocyte apoptosis accompanies congenital and acquired cholestatic hepatobiliary disorders and is due, in part, to the retention of endogenous compounds normally excreted in bile. Bile acids are among these retained toxins. Bile acids induce apoptosis in primary cultures of hepatocytes and in hepatoma cells. Our results show that the hormonal intracellular messenger cAMP protects against bile acid induced apoptosis. The anti-apoptotic effect of cAMP is largely independent of classical cAMP signaling through protein kinase A, but requires cAMP mediated activation of phosphoinositide-3 kinase (PI3K). In our studies cAMP mediated cytoprotection and PI3K activation occurs through a novel cAMP stimulated pathway involving activation of cAMP dependent guanine exchange: factor (cAMP-GEF) which signals through the small GTP binding protein Rap. Analogue specific activation, of cAMP-GEF in hepatocytes activates Rap 1 and confers PI3K dependent protection from bile acid induced apoptosis.

An initial aim of these studies is to characterize the mechanism(s) whereby cAMP binding to cAMP-GEF activates PI3K and to demonstrate that Rap 1 activation is a downstream effector of cAMP's anti-apoptotic effect.

A second aim of these studies is to identify downstream PI3K dependent effectors which mediate cAMP cytoprotection. We will determine if cAMP/PI3K dependent survival involves activation of a PI3K/Akt/glycogen synthase kinase 3 beta pathway.

Our third aim is to identify the molecular targets of cAMP in the apoptotic machinery. Initial investigations will examine the effect of cAMP on death receptors dependent signaling mechanisms. Dissection of the signaling pathways and molecular mediators of cAMP mediated cytoprotection might lead to the discovery of signaling molecules that can be targeted for antiapoptotic drug therapy.

PI: Levinson, James
Title: Ecuador Nutrition Review: An Examination of Relevant International Experience
Abstract: The Minister of Health has established that improved nutritional status is a central goal of Ecuador's health strategy. In this context, a renewed emphasis has been given to nutrition-related programs in the 198 poorest parishes of the country.

With support from World Bank's Programmatic Human Development Reform Loan (PHDRL) series, the institutional and budgetary consolidation of programs and projects in the nutrition field has begun, under the supervision of the Health Ministry. The main institutions involved are: the Ministry of Social Welfare; the Ministry of Education, and PAN 2000, as well as the micronutrient program of the Health Ministry. Previously, the food and nutrition sector was led by the Social Welfare Ministry. To support this process, the World Bank has agreed with the Government of Ecuador (GoE) to carry out a study of nutritional outcomes in Ecuador; and of the public policies which aim to improve the situation.

The study will use a broad, multi-sector approach covering:

1. The nutritional condition of the population
2. Knowledge, attitudes and practices of the population relevant to nutritional outcomes
3. The coverage, efficacy and impact of the programs and projects funded by the Government, international agencies, and Private Development Organizations (PDOs) in this field

The study is highly complementary with the World Bank's program of investments in Ecuador, especially the following operations: the second Programmatic Human Development Reform Loan (PHDRL 11); the Universal Health Insurance Project, and the Human Development Bond Project. It is expected that the study will contribute to the consolidation of a programmatic vision that links these interventions in support of the health and nutrition sector.

The study will be implemented between October 2005 and May 2006, by a multidisciplinary team of Bank technical staff, together with international and national consultants. The study director is Ian Walker and the World Bank team from Washington also includes Alessandra Marini and Leonardo Lucchetti. Nelson Gutierrez and Carmen Tene from the Bank's Quito office will also participate in the study.

PI: Levinson, James
Title: Multisectoral Nutrition Project
Abstract: Despite wide agreement that multisectoral programs (or strategies) are the most effective ways to tackle malnutrition, we have only limited evidence for how to do so, with limited sharing of the experiences that do exist. It is critical to address this lacuna given continuing high levels of under nutrition in developing countries and the need to take advantage of the present development environment that encourages a multisectoral approach, with its emphasis on MDGs, Poverty Reduction Strategy Papers (PRSPs), etc.

As a complement to a three-year grant for research to support the convergence of the nutrition and the MCH agendas, the Development Grant Facility (DGF) at the World Bank provided additional funds of $100,000 to conduct a study to identify "other ways (in addition to the proposed convergence of the nutrition and maternal and child health agendas) to remove poor nutrition as an obstacle to achievement of the MDGs." This note describes how those funds will be used, within a framework for cooperation with the Tufts University (Trustees of Tufts College, Friedman School of Nutrition Science and Policy).

PI: Levy, Stuart
Title: The Multiple Antibiotics Resistance (MAR) Regulon
Abstract: Our overall goal is to understand the function of the mar locus and its regulon in the adaptation and survival of the bacterial cell.

Genetic, functional, and structural studies of the MarR and the MarA proteins.
Mutations will be made in these two regulatory proteins to identify regions involved in DNA binding. For MarR studies will also locate regions involved in binding of, and response to, inducer and in dimerization. The secondary structure of the purified proteins will be determined by circular dichroism and the tertiary structure of MarA by 2D NMR. We shall identify conditions for growing MarR and MarA protein crystals for X-ray crystallography.

Further Definition of the Mar Regulon.
Additional loci regulated by MarA will be found using translational fusions to reporter enzymes and transcriptional fusions to green fluorescent protein. Positively or negatively regulated proteins in Mar mutants will be identified by gel electrophoresis. We shall study the mechanisms for induction of mar by structurally unrelated compounds (salicylate, dintrophenol, antibiotics), and test a battery of additional stress conditions for induction. We shall measure the MarA-independent responsiveness to various environmental stresses of the unknown gene fusions which have been identified as part of the Mar regulon to gain an understanding of their function. We shall compare the relative activation of new Mar regulon loci by MarA, SoxS and Rob. We shall determine whether Mar mutants have altered transport of rifampicin.

Clinical implications of the Mar Regulon.
Since we have shown that mar mutations contribute to clinically significant resistance, and since a genetic locus of naturally-occurring strains of bacteria often has many alleles in the population, we shall screen several different wild type collections to find the frequency of Mar mutants.

To model the possibility that consumption of aspirin, a salicylate, or other related over-the-counter drugs, might lead to mar induction and subsequent selection of fluoroquinolone resistance, we shall examine the frequency of gyrA/parC/mar mutants obtained after induction by salicylate.

We have selected one-step mutants of M. smegmatis co-resistant to isoniazid, chlorarnphenicol, and/or tetracycline. The locus(i) responsible will be cloned and identified. In collaboration with J. crawford, we shall look for homologs of the locus in M. tuberculosis.

PI: Levy, Stuart
Title: Genetics of Soil Survival and Persistence of Pseudonyms Fluorescens
Abstract: This proposal addresses the USDA strategic goal of improving the nation's nutrition and health by producing fundamental data that will help in the development of more effective biological control of plant pathogens, thereby reducing the use of chemical pesticides and pesticide residues in the food supply.

Bacteria as "biocontrol" agents are promising alternatives to chemical pesticides, but their unpredictable survival hinders the widespread development and adoption of this technology. The overall goal of this work is to use genome-wide technologies and molecular and genetic techniques to understand processes affecting bacterial survival in soil. We will use parallel approaches to achieve our objective.

1. Use IVET (in vivo expression technology) to survey the Pseudomonas fluorescens PfO-I genome for genes induced in soil. In a pilot IVET screen we have identified 22 soil-induced sequences (not expressed in laboratory media), 10 of which are antisense to predicted genes in PfO-I. Beginning with the first 22 IVET-identified sequences, we will identify regulators of these soil-induced genes to explore the regulatory network controlling soil-induced sequences. To characterize the novel antisense genes uncovered using IVET, we will measure transcription by RTPCR, and translation by gene fusions.

2. AdnA is a regulator of genes involved in survival, persistence, and adhesion. We will continue our analysis of the AdnA regulon using mimarrays to identify genes controlled by AdnA. Comparison with the regulon controlled by its P. aenrginosa homolog (FleQ) will reveal unique regulated targets, which will be examined further.

Mutation of soil-induced sequences and AdnA regulon members will be used to assess their role in soil persistence, and identify genes that could be manipulated to enhance soil fitness. Over-expression of selected genes will be used to test the possibility that soil fitness can be improved by increasing their expression.

Ultimately, this project will provide important data helpful in developing biocontrol bacteria with appropriately improved and reliable soil persistence.

PI: Levy, Stuart
Title: Efflux-Mediated Resistance to Tetracyclines
Abstract: Use of tetracyclines to treat infectious diseases has been sharply curtailed because of resistance. Among two major mechanisms for tetracycline (Tc) resistance, active efflux is the most common, represented by more than 20 different Tc determinants. Since our discovery of active Tc efflux almost 25 years ago, this laboratory has focused on a prototype efflux protein, the class B Tet A (TetA(B)) protein specified by Tn10. The proposed project has three major aims directed at better understanding the molecular basis for Tet protein efflux activity.

1. Grow and analyze 2D and 3D crystals of the TetA(B) protein. This work will benefit from recent developments in membrane protein crystal structure work and improvements in the purification of Tet protein that have led to our ability to reproducibly obtain crystals.

2. Extend efforts aimed at identifying the substrate binding site(s):

1. Prepare and use photoaffinity labeled Tcs to identify particular amino acids which comprise the Tc binding site.

2. Use a FeTc complex and the Fenton reaction to cleave the protein near the binding site and so define the second such cleavage site.

3. Perform site-directed mutagenesis of residues identified in "a" to see the effect on Tc resistance and Tc transport.

3. Continue the genetic analysis of the TetA(B) protein, focusing on the interdomain loop. By suppressor mutations, amino acid replacements and cross-linking studies, we shall study the physical neighborhood and functional role of the loop in terms of efflux and substrate specificity.

Insights into the molecular structure and function of TetA protein, a prototype of an efflux protein of the Major Facilitator Superfamily, will help in understanding this common mechanism of drug resistance and suggests ways it could be overcome. This knowledge, in turn, could help lead to a renaissance for the tetracycline family of antibiotics which have a long history of being generally well-tolerated and safe. Such understanding will also enhance efforts to reverse efflux of other antibiotics and drugs used to treat infectious diseases, including bacteria, fungi and parasites, and cancer.

PI: Lewis, Sara
Title: Connecting Episodes of Sexual Selection in Bioluminescent Fireflies
Abstract: Sexual selection is a major force driving adaptive evolutionary change. However, despite exponential growth of this field, overly fragmented approaches have hindered further progress in understanding how reproductive traits evolve. To move forward, we critically need to develop an integrated view of how both pre-mating and post-mating episodes contribute to selection on mate traits and female preferences.

The proposed research takes an innovative, comprehensive approach that encompasses both pre-mating and post-mating episodes of sexual selection using Photinus fireflies (nocturnally-active, bioluminescent beetles), a fascinating and highly tractable model system. The proposed work extends a highly productive line of investigation conducted by the PI, building on our knowledge of firefly courtship signaling, nuptial gifts, and paternity patterns to examine this emerging and fundamentally important area of behavioral ecology.

This research will use laboratory experiments, field studies and signal manipulations to accomplish four major aims:

1. Determine the relationship between male courtship attractiveness and paternity success: These studies will differentiate among alternate hypotheses for how a male’s courtship attractiveness influences postcopulatory sperm use.

2. Establish how simulated male courtship signals alter sperm use: These studies will experimentally manipulate male courtship signals to determine whether paternity differences reflect female sperm choice based on male flash traits.

3. Measure selection on male traits during pre-mating and post-mating episodes and assess fitness benefits of female choice: field studies will measure selection on multiple male traits across reproductive fitness components, and laboratory experiments will measure direct and indirect benefits of female choice.

4. Identify variation in male copulatory behaviors and cuticular hydrocarbons (CHC) – These studies will examine multiple male cues that may be involved in post-mating female choice by examining the relationship between male copulatory behaviors, CHC profiles, and paternity success.

The proposed experiments will combine behavioral, anatomical, and biochemical methods into a powerful approach that will allow us to empirically test the link between male courtship signals and postcopulatory paternity success, and to determine selection on both male traits and female preferences during different episodes of reproductive selection. These studies will, independent of the results, provide important insights into how selection acts across reproductive fitness components, knowledge that is fundamental to understanding how reproductive traits and behaviors evolve.

This research will advance the field by developing a broadly applicable, integrated conceptual model of sexual selection that will ultimately lead to a better understanding of animal behavior and communication systems.

Broader Impacts: This exciting research will yield considerable educational benefits. This project will provide training for both undergraduate and graduate students, and contribute to the National Science Board’s recently stated goal of bolstering retention rates among science majors.

This project will also support Tufts University’s strong institutional commitment to making active research participation an integral part of undergraduate science education. The Department of Biology at Tufts provides a highly collaborative research environment, and this project will synergistically benefit from the Dr. Lewis’ involvement as co-PI on our recent NSF REU Site award: Mentoring Biological Research as a Collaborative Enterprise. The PI has previously mentored 30 undergraduates in research projects, many of whom have gone on to productive research careers.

Lastly, this research will promote increased public understanding of science, especially behavioral ecology: fireflies are unusually charismatic insects, and firefly research readily captures public interest. Our previous media outreach efforts have clearly demonstrated our strong commitment to effectively communicating about science to the general public.

PI: Lichtenstein, Alice
Title: Nutrition and Cardiovascular Disease Training Program
Abstract: The objective of this Training Grant application is to generate support for well qualified students to earn a doctoral degree in the area nutrition and cardiovascular disease at the Gerald J. & Dorothy R. Friedman School of Nutrition Science & Policy at Tufts University. The specific aims of the training program are to:

1. Provide trainees with rigorous didactic training in the areas of nutrition, cardiovascular disease, basic sciences and medical ethics
2. Create a supportive environment within which trainees are guided through the process of conducting independent research
3. Develop in each trainee communication skills necessary to effectively disseminate research data in both oral and written form
4. Enable the acquisition to multidisciplinary training to make each trainee competitive upon conclusion of their formal training
5. Instill the skills necessary to become an independent investigator capable of generating research funding
6. Develop a thorough understanding of what constitutes responsible conduct of research
7. Secure a postdoctoral position on completion of the program.

The Program Faculty is drawn from a broad range of disciplines; each member currently directs a vibrant research program and has an extensive history of inter-investigator collaboration. They contribute expertise in the areas of lipids and lipoproteins, genetics, folate/homocysteine (epidemiology and basic science), obesity, cardiovascular disease risk factors and mechanisms, vitamin K, immunology, vascular biology, nutrition assessment, cardiac function and oxidative stress, and atherosclerotic plaque stability. This environment is supplemented by the combined resources of the Jean Mayer USDA Human Nutrition Research Center on Aging at Tufts University, Tufts University School of Medicine, and the Sackler School of Graduate Biomedical Sciences that together provide a fertile environment in which predoctoral students can fulfill the aforementioned aims. The symposia, seminars and meetings of institutions in the greater Boston area further serve to supplement these resources.

PI: Lichtenstein, Alice
Title: Cholesterol Metabolism and Its Relation to Diet and Other CVD Risk Factors in Framingham Offspring Study
Abstract: The objective of this application is to investigate the predictive value of using measures of cholesterol homeostasis to identify individuals at high risk of developing cardiovascular disease (CVD) relative to established risk factors. The specific aims are to:

1. Quantify circulating indicators of cholesterol homeostasis [levels of phytosterols and cholestanol (surrogate measures of cholesterol absorption) and cholesterol precursors (surrogate measures of cholesterol synthetic rates)] in plasma samples from Framingham Offspring Study participants diagnosed with established CVD and/or >50% carotid stenosis (N=165) not taking lipid-lowering medication and control subjects matched for age, sex, body mass index, hypertension and smoking status (n=330).

2. Evaluate the validity of using indicators of cholesterol homeostasis to predict CVD risk in the Framingham Offspring Study-Cycle 6 participants by

   1. Establishing adult normal ranges for circulating levels of phytosterol, cholestanol and cholesterol precursor (N=3378)

   2. Defining the relationship between phytosterol, cholestanol and cholesterol precursor levels, and lipid, lipoprotein and apolipoprotein levels in plasma

   3. Defining the relationship between phytosterol, cholestanol and cholesterol precursor levels and selected dietary intake data (energy, protein, fat, saturated, monounsaturated, polyunsaturated and trans fatty acids, cholesterol, fiber and antioxidant supplements)

   4. Determining the relationship between phytosterol, cholestanol and cholesterol precursor levels and selected genotype data related to CVD risk (gene loci of apo E, apo A-IV, scavenger receptor class B type 1 [SRB1], and ATP-binding cassette [ABC] G5 and ABCG8 transporters)

3. Monitor clinical events in the Framingham Offspring Study cohort throughout a 10-year period (1995-2005) and relate these data to the phytosterol, cholestanol and cholesterol precursor levels.

Measures of cholesterol homeostasis will be quantified first in subjects identified in specific aim #1 and then the balance of subjects identified in specific aim #2, achievable now due to the development of a gas chromatographic method using a single plasma sample. These data will be assessed relative to dietary, biochemical and genotype data currently available for the cohort. The results of the proposed work will define the relationship between markers of cholesterol absorption and synthesis, and CVD outcomes; establish reference values for measures of cholesterol absorption and synthesis; and assess the predictive value of these measures to identify high risk individuals relative to established risk factors.

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: 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: Pathobiology of Digestive Diseases
Abstract: Pathobiology of Digestive Disease is a program with 20 years of experience in training predoctoral and postdoctoral scientists to work as partners with physicians in "bench to bedside" translational research on digestive diseases. To achieve this goal, we educate basic and clinical scientists on each other's language, experimental approaches, and the major questions in their field through shared didactic courses, journal club, grand rounds, and seminars. Our core Pathobiology course, co-directed by a physician-scientist and basic scientist, teaches our trainees:

1. How scientific discovery has influenced clinical practice and how clinical observation has affected scientific hypotheses
2. To critically evaluate current knowledge about diseases
3. To use clinical problems as a starting point for hypothesis-driven research

The program is led by Laura Liscum, PhD, Professor of Physiology, and funds 5 predoctoral and 4 postdoctoral trainees. Training is supervised by 21 preceptors, who are associated with the Departments of Physiology and Molecular Biology & Microbiology at Tufts University School of Medicine as well as the Department of Medicine at Tufts-New England Medical Center. Faculty members are basic scientists and physician-scientists who bridge the gap between basic cell biology and digestive disease. Their work focuses on intracellular trafficking in the liver, pancreas, intestine and kidney; cellular growth and tissue development; colon and liver cancer, and breast cancer metastasis to liver; and intestinal pathogens. Our preceptors are strongly committed to education, have experience interweaving basic and clinical sciences, and direct active, well funded research programs that address the pathobiology of digestive diseases. Our training program is enhanced by the Center for Gastroenterology Research on Absorptive and Secretory Processes (GRASP), a Silvio O. Conte Digestive Diseases Core Research Center, which promotes basic research on the normal function and diseases of the gastrointestinal tract and liver at Tufts and plays an integral role in the Pathobiology training program.

PI: Liscum, Laura
Title: Somatic Cell Mutant Affecting 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 (3,4). 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.

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.