PI: Lee, David
Title: Self-Assembly of Linear Viral Arrays
Abstract: The broad long-term objective of the research program described here is to understand how molecular features and hierarchical structure collaborate to establish the properties of a material or device. It is now well known that novel properties and phenomena can emerge from ordering at the nanoscale. The great immediate challenge facing the field is the construction of designer structures that enable one to test the effects of nanoscale organization. The Lee laboratory at Tufts is exploiting biological molecules to create such model systems. Specifically, tobacco mosaic virus is modified with biotin in a regiospecific fashion and using tetrameric streptavidin to drive the organization of the virus into a 1-dimensional array. The advantage of such a system is that its dimensions and its molecular properties can be easily tailored in a systematic way, through molecular biology and chemical approaches, to suit the needs of the researcher. The specific objectives are:

1. to optimize conditions for the assembly of the array
2. create arrays that span the length scales of interest to nanoscience, from tens to over 100 nm
3. assemble quantum dots on the array

The intellectual merit of this approach is that this model will enable fundamental studies in the nanosciences by permitting one to study the effects of nanoscale architecture in a systematic fashion. Such a construct will permit the researcher to 1) array molecules and other nanometer-sized objects at the nanoscale, and (2) vary the dimensions of the array on the nanometer size scale. It is expected that this ability to systematically change various properties of a highly organized nanostructure will have a major impact on the field. This proposal has broad impact at several levels. First, it will help advance the goals of the National Nanotechnology Initiative, thereby helping to keep the United States at the cutting edge of nanoscience and competitive in the global race to develop nanotechnologies. For example, the research described here is a starting point for a collaboration with others to create nanoscale photonic devices that may have value in the further miniaturization of computers. Second, the project will provide many opportunities for interdisciplinary training of both students and postdoctoral fellows. Third, the scientific background of this proposal serves as a springboard for the development of laboratory experiments that will be developed for students at the high school and undergraduate level. These experiments are designed to be in the context of issues of interest to the students (eg. curing disease) but teach basic concepts. The interdisciplinary nature of this proposal also permits the development of a new course, framed in the context of real world topics, which explores how science is actually practiced and brings together concepts and techniques from biology, chemistry, and nanoscience. These pedagogical efforts will be backed up by a panel discussion that explores how people get inspired to take up science.

PI: Lee, Mary
Title: Strengthening Educational Capacity at Christian Medical College
Abstract: Tufts University and Christian Medical College (CMC) propose to build upon their existing partnership that has introduced the use of Tufts University Sciences Knowledgebase (TUSK) to CMC. The aim of this project is to strengthen medical education at CMC through the capabilities offered by TUSK.

CMC is a one hundred-year-old national medical school known for the quality of its undergraduate and postgraduate medical education, clinical services, and commitment to support health services through its network of over 200 secondary hospitals located across rural parts of India. CMC along with Tufts University has piloted the use of TUSK by developing locally appropriate content, supporting specific courses, and performing curriculum management functions. This new project will enable scaling the pilot to expand the development of learning content relevant to the Indian setting, the use of e-learning methods both within and outside the classroom, more efficient management of the curriculum, and increased capacity among the faculty to maintain and use the e-learning system.

The goal of this project is to strengthen medical education at CMC through the use of TUSK. Our objectives towards achieving that goal are to:

1. Develop and deliver high-quality curricula that are appropriate and relevant to the health care needs in India.
2. Increase CMC faculty’s capacity to manage both the technical and content creation components of TUSK.
3. Share these resources with secondary hospitals networked to CMC so as to reach underserved areas nationwide and also to disseminate this work to other medical colleges in India.
4. Work towards self-sufficiency through the resources and capacity that have been created through the project.

PI: Lerner, Richard
Title: 4-H Study of Positive Youth Development
Abstract: American youth, and the nation that seeks to nurture and support them, face a set of problems of historically unprecedented scope and severity that, together, limit the opportunities for active and constructive participation by young people in community life and civil society. For example, estimates are that 50% of American youth engage in two or more of the major four categories of risk behaviors (substance use and abuse; crime and violence; unsafe sex and teenage pregnancy and parenting; and school failure) and that 10% engage in all four sets (Dryfoos, 1990, 1998). This level of comorbidity places American youth at unprecedented risk. Moreover, since the 1980s there continues to be a strikingly alarming rate of youth poverty--involving a fifth of the children and adolescents in the United States. Rates of all of the above-noted problem behaviors are higher among poor youth (Huston, 1991). 

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

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

If Health Rocks! as it is implemented within 4-H is effective in delivering such resources to young people, then not only should instances of the above-noted risk behaviors be reduced significantly but, as well, several positive developmental outcomes should develop among youth. These outcomes can be summarized by the “five Cs” of positive youth development: Competence, Connection, Character, Confidence, and Caring (or Compassion) (Carnegie Council on Adolescent Development, 1989; Hamilton, 1999; Lerner, 1995; Little, 1993). These five attributes represent five clusters of individual attributes--for example, intellectual ability and social and behavioral skills (competence); positive bonds with people and institutions (connection); integrity and moral centeredness (character); positive self-regard, a sense of self-efficacy, and courage (confidence); and humane values, empathy, and a sense of social justice (caring/compassion), respectively. When these five sets of outcomes are developed in youth, civil society is enhanced through the intergenerational effects these youth initiate in regard to the rearing of subsequent generations of citizens, that is, through the parenting of their own children. As such, a sixth “C” will emerge: Contribution. Youth who possess the attributes of positive youth development will develop into members of society capable of and committed to contributing to civil society. 

Do young people involved in Health Rocks! develop these attributes of positive development? Does participation in Health Rocks! enhance the development of these attributes in a manner beyond levels of positive development produced by participation in 4-H programs per se? As compared to youth who do not participate in 4-H (and thus experience neither its general approach to community youth development nor, specifically, the Health Rocks! program) does participation in 4-H alone, or both 4-H and Health Rocks!, result in greater levels of positive youth development? 

Answers to these questions will provide information about the efficacy of Health Rocks! in meeting its ambitious goals for diminishing risks and promoting positive youth development. However, much more will be learned by research that answers these questions. Knowledge will be gained about the contributions of 4-H more generally to the healthy and positive development of America’s young people and to the enhancement of community life and civil society in our nation. Research that addresses these questions will constitute a test of the 4-H theory of change—of the model that 4-H uses to promote positive youth development through healthy engagement with the community. The research we will conduct will provide data uniquely able to answer these questions, ones critical to not only Health Rocks! and 4-H but to the welfare of our nation and its youth.

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: Lichtenstein, Alice
Title: Nutrition and Cardiovascular Disease Predoctoral Training Program
Abstract: The objective of this proposal is to obtain funds for the training of highly qualified PhD scientists committed to a research career in the area of nutrition and cardiovascular disease (CVD) at a basic, clinical, epidemiological, and/or translational level. Despite advances in the past two decades, CVD is still the leading cause of disability and death in the U.S. It costs the nation approximately $432 billion annually, a figure in excess of 20% of the total national healthcare expenditures. Given the continued demographic shift in the U.S. towards the older age groups, this burden is predicted to increase. Lifetime risk for CVD and median survival rate is strongly associated with risk factor burden at age 50 years. Understanding the underlying mechanism(s) of this association and refining optimal approaches to minimize CVD risk factor burden is critical to advancing approaches to decreasing CVD incidence and improving prognosis.

The rational for this proposal is based on the firm belief that nutrition is the most significant modifiable lifestyle behavior that can alter CVD risk. This proposal seeks funds to train the next generation of researchers to address CVD genesis, prevention, and treatment at a molecular, cellular, whole organism, and population level. Continued support is requested for the four predoctoral training slots for each of five years. All trainees are first admitted to the PhD program at the Friedman School of Nutrition Science and Policy. After one or two years of coursework (depending on prior training) students are eligible to be admitted to the Training Program. Acceptance is predicated upon outstanding academic and research achievement, and establishing a doctoral research project in the area of nutrition and CVD. Training Program faculty have a proven record in their ability to provide exemplary training to predoctoral students.

Critical components of the Training Program include didactic training in nutrition, basic sciences and epidemiology; independent mentored research in the area of biochemical and molecular nutrition or nutritional epidemiology (doctoral thesis); preparation and submission and/or publication of research in peer-reviewed journals; oral and poster presentation of research in public forums; NIH-style research proposal preparation and defense; and training in scientific ethics and research responsibility. Training Program administration and trainee supervision will be the responsibility of the program director and Steering Committee, which will meet semi-annually to review and discuss trainee selection and progress, adequacy of mentoring, and transition of trainees to independent researchers.

We believe the training environment at the Friedman School and Tufts Univeristy provides an outstanding opportunity for future researchers in the area of nutrition and CVD and continued support in both the scientific future of the trainees and the public health of the nation is a good investment.

PI: Lichtenstein, Alice
Title: Evaluation of Glycemic Index to Assess Diet-Associated Chronic Disease Risk
Abstract: The objective of this proposal is to investigate the intra-individual reproducibility (within the same individual, when repeatedly measured) and inter-individual variability (among individuals) of glycemic index (GI) and glycemic load (GL) value determinations for individual foods and food combinations. The specific aims to accomplish this objective are to evaluate reproducibility and variability of GI value determinations in volunteers differing in biologic characteristics - body mass index (BMI), age, and gender; assess the effect of macronutrient amounts and combinations, and fiber on variability of GI and GL value determinations; assess the effect of prior meal macronutrient composition ('second meal' effect) on GI value determinations; and relate these data to chronic disease risk factors monitored prior to and during the intervention period. These aims will be accomplished by assessing intra-individual reproducibility and inter-individual variability of repeated GI value determinations for white bread, commonly used as a reference food, relative to glucose, in volunteers selected to represent a range of BMI's (18.5-24.9, 25-29.9, ≥30) and ages (18-49.9, 50-85 y), and on the basis of gender, and relate these data to body composition and insulin sensitivity (Phase I).

This work will then be extended to address issues related to variability potentially introduced by differences in macronutrient and fiber combinations and loads (Phase II), and finally by 'second meal' effects (Phase III). Prior to each set of food challenges (glucose and test food[s] in random order) volunteers will be characterized on the basis of fasting HbA1c; fructosamine; lipids and lipoproteins; insulin, glucose and C-reactive protein. During the 5-hour challenge (sampling at 0, 15, 30, 45, 60, and every 30 minutes thereafter) volunteers will be monitored for changes in blood glucose, insulin, triglycerides, total and high density lipoprotein cholesterol and non-esterified fatty acid levels. The concepts of both GI and GL are in the public domain and it has been suggested that the concepts be incorporated into U.S. federal dietary guidance (U.S. Dietary Guidelines and Dietary References Intakes [DRI]) formulated to promote health and reduce chronic disease risk. This proposal addresses some of the understudied areas for which additional information would be useful in order to determine whether GI and GL should be used to classify foods on an individual basis, as has been suggested, and when formulating dietary guidance for the general population. 

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: Lischko, Amy
Title: Using Geographic Variation to Identify and Reduce Over-treatment in Massachusetts
Abstract: Total health care expenditures in Massachusetts reached $43 billion in 2004, representing an increase of 35 percent from 2000 and an increase of 73 percent from 1995. Historically, Massachusetts has had the highest per capita personal health care expenditures of any state. Massachusetts has also positioned itself to be a leader in comprehensive health insurance reform by passing landmark legislation in 2006 aimed at insuring nearly all state residents. This reform, however, is at risk of long-term sustainability due to rising health care costs. The next phase of health care reform in Massachusetts is focused on developing strategies for constraining the growth of health care costs. One cause of rising health care costs is over-treatment which is defined here as the overuse or misuse of medical procedures, tests, and specialist care. The specific aims of this project are:

1. to identify over-treatment through variations in inpatient and outpatient care provided by Massachusetts hospitals and physician groups,
2. to enumerate and map the various structural elements of primary, specialty and diagnostic care capacity across the Commonwealth of Massachusetts; and,
3. to develop strategies that policymakers and regulators can use to constrain health care costs through the reduction of over-treatment.

This research will use the state’s new all-payer claims files covering the period from 7/1/06-9/30/07. Algorithms will be developed based largely on the work established by Wennberg et al. regarding geographic variation used for the Dartmouth Atlas. The focus of this work, however, will be to identify variation for over-treatment for the under-65 population in Massachusetts. First, a list of preference-sensitive and supply-sensitive treatments will be identified for analysis. Analysis of the claims data will proceed by using geographic variation of the identified treatments across the Commonwealth. Estimates of the cost of over-treatment for each of the conditions will also be calculated. The top 10-12 inpatient and outpatient procedures, tests, and/or care patterns with the largest variability and highest costs across regions of the Commonwealth will be identified for further analysis at the provider-level. The third phase of analysis includes identification and mapping, using GIS tools, the supply of health care resources — such as free-standing imaging centers, hospital beds including emergency department beds, intensive care beds, etc., number of primary care physicians, numbers of particular specialists related to the procedures and tests identified, and the number of ambulatory surgical centers. 

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, and
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: Mason, Joel
Title: Doctoral Program in Human Nutrition and Metabolism
Abstract: The Postdoctoral Training Program in Human Nutrition and Metabolism combines outstanding resources for nutrition research with a uniquely qualified faculty of scientists in human nutrition to train physicians, other graduate-level health professionals, and selected Ph.D.s to conduct research in human nutrition, metabolism, and the biological sciences basic to nutrition. Participating institutions include the U.S.D.A. Human Nutrition Research Center on Aging at Tufts University, Tufts-New England Medical Center, the Tufts University School of Nutrition Science and Policy, the Tufts University School of Medicine and the Sackler School of Graduate Biomedical Sciences. The Boston Obesity Research Center complements these institutional resources. There are 37 faculty members in this program, 28 of who are preceptors and 9 of who are supporting faculty. The preceptors serve as principal investigators for over 20 NIH grants and have trained over 215 pre- and post-doctoral trainees over the last 15 years. Faculty research ranges from molecular and cellular biology to epidemiology and policy in areas of nutritional science relevant to the prevention and treatment of diseases such as cancer, heart disease, diabetes, hypertension, stroke and osteoporosis. The program primarily recruits physicians, although other graduate-level health professionals and selected Ph.D.s are also recruited, and the program has actively sought out minority trainees. Renewal of four funded slots is requested. Trainees pursue an academically rigorous three year training program in which they have the additional options of obtaining either a Ph.D. in nutrition or an M.A. in clinical care research. Progress in the program is monitored by a Steering Committee that includes the trainees' preceptors or thesis advisors. Research education is complemented by a rich schedule of research conferences and seminars and, for trainees with health professional degrees, by clinical training in an inpatient nutrition support service, and by rotation in outpatient nutrition subspecialty clinics. Trainees acquire a broad background in basic nutritional science and nutrition research, familiarity with the current literature in nutrition and disease, training in the use and interpretation of methodologies to assess the nutritional status of individuals and populations, and a sophisticated understanding of the applications and limitations of research design and biostatistical methodologies. 

PI: Maxwell, Daniel
Title: Livelihoods Change Over Time
Abstract: Three objectives are proposed for the scoping mission. These include:

1. To gain a better understanding of the livelihoods context, the main institutional issues, and the humanitarian programs being implemented, to flesh out research questions and methods.
2. To understand partners’ objectives and programs and to ensure the research team/agency partnership will succeed.
3. To gather information on the logistics and cost of a 3-5 year study
1. Identify major constraints
2. Develop a full research proposal and protocol
3. Develop an understanding between Tufts University and the cooperating agency 

Three distinct activities are proposed for the scoping mission.

1. Background literature review prior to field trip.
2. Three-week field trip to conduct necessary consultations.
3. Write up the results of field trip into a full draft of research protocol, proposal and budget. 

The main activity of the scoping mission would be a field trip, involving a member of the Tufts research team and at least one staff member of the cooperating agency to conduct consultations with stakeholders. Prior to a field trip, some desk review of existing literature would be conducted. A number of issues would need to be addressed with the cooperating agencies prior to the desk review. These include confirming the proposed geographic location of the proposed study and the humanitarian programs being conducted in study area; and organizing the logistics of the scoping mission (travel to the location and meetings with partners). Following the field visit, all of the information gathered would be written up to form the full research proposal and protocol. 

PI: Mazurana, Dyan
Title: Ambiguity Gender and Generational Analysis of Conflict in Uganda and South Sudan
Abstract: Over the past 2.5 years with support from IDRC/CIDA, our teams have documented, analyzed and reported on a series of grave crimes and human rights violations committed by all sides of the conflict in northern Uganda against women and girls, in particular, and to a lesser extent against men and boys. It now appears likely that the 21 year war will reach an end in northern Uganda with the parties signing a peace agreement within the next year. In depth work by the team with key actors involved in the Juba peace process and key actors in Kampala and northern Uganda suggests that the most difficult issues to come out of these processes will be regarding accountability and justice for the grave crimes and rights violations suffered by civilians, in particular young women and girls, at the hands of the LRA and, to a lesser extent, by the UPDF, militias and SPLA. 

PI: McVey, Mitch
Title: Error-Prone Repair of DNA Double-Strand Breaks and Aging in Drosophila
Abstract: One feature of aging cells is a gradual decline in their ability to repair DNA damage, particularly double-strand breaks. Numerous studies have demonstrated that double- strand breaks are primarily repaired by two competing pathways: non-homologous end joining and homologous recombination. Of the two pathways, end joining is typically more error-prone and therefore mutagenic. This is especially true for “backup” end- joining pathways that operate with slower kinetics in both yeast and mammals. Although recent data indicate that these alternate forms of end joining are particularly relevant to the aging process, their molecular mechanisms remain largely uncharacterized. 

In order to address this, we have developed Drosophila transgenic systems that enable us to create double-strand breaks at specific genomic locations, quantitate the contributions of various repair pathways, and recover individual repair products. Using these systems, we have recently obtained compelling evidence for a ligase TV-independent form of end- joining that operates robustly in mitotic germline cells. These preliminary data strongly suggest that this pathway may comprise the Drosophila equivalent of backup end joining. Interestingly, it often creates repair products that have large deletions and insertions of templated nucleotides, similar to those observed in many human leukemias and lymphomas. Our lab is currently determining the prevalence of backup end joining in different repair contexts and mutant backgrounds. These studies will allow us to test the hypothesis that aging in Drosophila is accompanied by a decreased ability to repair double-strand breaks through error-free mechanisms. Our ultimate goal is to identify targets for potential therapeutic interventions to prevent the decline in DNA repair fidelity in aging individuals. 

One hallmark of aging is a gradual decline in the ability of cells to repair DNA damage. Experiments in mice and humans have shown that aging cells accumulate somatic mutations and have increased mutation rates, suggesting a general loss in DNA repair efficiency in older cells. Of the many types of DNA damage, double-strand breaks are likely to be particularly relevant to the aging process. In support of this, cancer incidence in mammals increases with age, and chromosomal translocations or deletions caused by faulty repair of double-strand breaks are often the precipitating event in cancer development. Furthermore, several human diseases characterized by cancer predisposition and premature aging are caused by mutation of genes that likely play roles in double-strand break repair, most notably Werner Syndrome. 

Double-strand breaks can be repaired by homologous recombination (HR) or non-homologous end-joining (NHEJ). HR, which uses a homologous donor template such as a sister chromatid or homologous chromosome, is considered error-free. In contrast, NHEJ does not use a repair template and is considered error-prone, often resulting in addition or deletion of nucleotides at the repair site. Investigations into repair of double-strand breaks induced by X-rays or topoisomerase II inhibitors in higher eukaryotes have revealed the existence of at least two mechanistically distinct end-joining pathways. The dominant pathway displays rapid kinetics (t112 in minutes) and requires DNA-PKcs, the Ku70/80 heterodimer, and the ligase IV protein. The other pathway, termed backup NHEJ (BNHEJ) is slower (t112 of several hours) and independent of both Ku and ligase IV. Ku-independent end joining mechanisms have also been reported in S. cerevisiae, although their dependence on ligase IV is less clear in this organism.  B-NHEJ often results in large deletions and appears to be utilized to a greater extent in cells nearing replicative senescence. While the mechanism of the rapid NHEJ pathway is fairly well-understood, little is known in any organism about the proteins involved in B-NHEJ. A system in which B-NHEJ is the major end-joining pathway for double-strand break repair would facilitate the elucidation of its mechanism and promote greater understanding of its role in the aging process. 

Recently, we have shown that end-joining repair of both P-element and ionizing radiation- induced double-strand breaks in Drosophila melanogaster is largely independent of ligase IV. This is true both in HR-competent and HR-deficient (rad5l mutant) backgrounds. Although it remains to be tested whether this end-joining pathway is also Ku-independent, our data suggests that the predominant form of end joining in Drosophila may be B-NHEJ. Thus, our system provides a unique opportunity to study the genes involved in B-NHEJ in a multicellular eukaryote. Intriguingly, the repair products that we recover from both wild-type and lig4 mutant flies frequently have small deletions and insertions at their junctions (Table 1). Such repair junctions are similar to those found at chromosomal translocations in human leukemias and lymphomas. Thus, further study of end joining in Drosophila may provide insight into mutagenic repair processes that contribute to gene dysregulation, cancer, and the aging process.

PI: McVey, Mitch
Title: CAREER: Interactions between Error-Prone and Error-Free DNA Double-Strand Break Repair Pathways in Drosophila Melanogaster
Abstract: Precise and controlled repair of DNA double-strand breaks is crucial for cell survival and genomic integrity. Double-strand breaks can be repaired by a number of both accurate and inaccurate mechanisms. Although accurate repair pathways have been extensively studied, error-prone double-strand break repair is not well characterized at the mechanistic level. This CAREER project will elucidate the genetic requirements for error-prone break repair and characterize the dynamic interactions between various double-strand break repair pathways in the multicellular eukaryote Drosophila melanogaster. To accomplish these objectives, teams of graduate and undergraduate students will utilize reporter systems that measure the relative use of repair pathways to study how developmental and tissue-specific cues affect repair pathway choice. They will employ web-based wikis to promote effective communication within the teams and to share ideas and protocols with the larger scientific community. In addition, the research will serve as the basis for a new seminar course that engages students in critical analysis of primary literature articles within the field.

This project will further our understanding of a crucial question in molecular biology-how various DNA repair pathways are coordinated to ensure genomic stability in a constantly changing multicellular environment. In addition, it will provide insight into the mechanisms by which error-prone DNA repair pathways can promote genetic diversity. While conducting research, students will participate in mentoring workshops and outreach activities designed to develop skills required for successful research careers. These experiences will encourage them to take ownership of their research and career paths at an early stage in their training. The project should therefore have a broad impact on the retention of students in research-oriented scientific fields, particularly those students from traditionally underrepresented groups.

PI: Mecsas, Joan
Title: Inhibitors of Type III Secretion and Translocation in Yersinia
Abstract: Many gram negative bacterial pathogens, including Yersinia, Salmonella, Pseudomonas, Shigeila, enteropathogenic E. coil, Chylamydia, and Burkhoideria, use type III secretion systems (TTSS) to translocate effector proteins from the bacterial cytosol into mammalian cells. Translocated effector proteins, called Yops in Yersinia, subvert normal host processes to promote the survival of the pathogen, and thus TTSS play an essential role in the infectious process and virulence of these bacteria. TTSS are comprised of a base, which spans the inner and outer membranes of the bacteria, a needle, which extends from the base to the host cell, and a translocon, which is inserted into host cell membranes and through which Yops are thought to travel to reach host cell cytoplasm. In Yersinia, the needle is primarily composed of one 7kD protein, YscF, which polymerizes to form a long tube. The needle is thought to be a conduit for the passage of Yops from the bacteria to the translocon and is thought to conduct signals from the host cell membrane to the TTSS base when the pathogen comes in contact with a host cell. These signals trigger Yop translocation. A tip protein, LcrV, is found at the distal end of the needle. One function of LcrV is to position, assemble, and/or insert the translocon, into plasma membranes, which is essential for Yop delivery into cells. Two proteins, YopB and YopD comprise the translocon. The overall goals of this proposal are to understand the molecular interactions between TTSS components that are required for translocating Yops upon contact with mammalian cells. We have identified 3 dominant negative YscF alleles and 8 small molecules that inhibit Yop translocation. These unique mutants and compounds will be studied intensively as probes to understand the required molecular interactions for Yop translocation into host cells. Our working hypothesis is that our inhibitors interfere with critical interactions between YscF, LcrV, YopB and/or YopD. Understanding the mechanism of translocation and needle assembly will support the development of new strategies that interfere with these processes and thus neutralize the bacteria’s pathogenecity.  

PI: Meydani, Simin
Title: Age-Related Changes in the Proteome and Lipodome of the Immunological Synapse
Abstract: Age-associated declines in T cell function contribute to the higher incidences, longer recovery periods, and elevated morbidity and mortality from infectious and neoplastic diseases in the elderly. Impaired T cell function, as measured by IL-2 production and cell proliferation, contributes significantly to immune senescence. Age-related reductions in IL-2 production are associated with defects in the recruitment of signaling molecules to immune synapses, the adhesive junction that forms between a T cell and an antigen-presenting cell. This has been demonstrated by using immunofluorescence microscopy to monitor the translocation of individual proteins to the immune synapse. Accumulating evidence indicates that the plasma membrane is laterally compartmentalized into small lipid microdomains that coalesce around the activated T cell receptor (TCR), where they facilitate the recruitment of signaling proteins required for IL-2 production. The resulting structures incorporate both lipid rafts and protein scaffolds, and exist as small ~200nm “signalosomes” within the immune synapse. Age-related differences in overall composition the immune synapse have not been evaluated in a systematic manner. Furthermore, the mechanisms responsible for the impaired recruitment of proteins to the immune synapse remain unknown. Changes affecting the composition of the plasma membrane, particularly changes affecting the sphingolipid metabolite ceramide, have significant impacts on lipid raft structure, and could influence the assembly of signalosomes. Age-related changes in the sphingolipid composition of the plasma membrane have not been examined. However, our preliminary data indicate that T cells from old mice have significantly higher levels of specific sphingolipids. These changes in cellular composition may contribute to the reduced ability of aged T cells to form effective immune synapse.

We hypothesize that TCR-induced signalosomes exhibit age-related differences in their patterns of protein and lipid recruitment, and that changes in the sphingolipid composition of aged T cells impact the downstream signaling cascades that regulate IL-2 production. We will test this hypothesis using an enhanced magnetic immunoisolation procedure in conjunction with state-of-the-art proteomic and lipidomic methods. We will further test this hypothesis by inducing age-related changes in cellular sphingolipids and measuring immune synapse formation and T cell activation.

These studies will provide the first comprehensive investigation of the protein and lipid content of the signaling complexes formed in response to antigenic stimulation. The information obtained from these proteomic and lipidomic analyses of the immune synapse will form the basis for constructing a “map” of age-related changes in T cells. This map will enhance our understanding of the molecular bases of immune senescence, and will promote the development of strategies to reverse the detrimental changes associated with aging. The age related decline in T cell (immune cells important for fighting against pathogens and tumors) function is an important determinant of the health and quality of life in elderly. The underlying mechanisms of these defects are not completely known. This project will utilize state-of-the-art methods to construct a “map” of the key age-related changes in lipids and proteins of T cells. This map will enhance our understanding of the molecular bases of immune senescence, and will promote the development of strategies to reverse the detrimental changes associated with aging.

PI: Miczek, Klaus
Title: Behavioral Neurobiology of Aggression: Alcohol, GABA, and 5-HT
Abstract: The proposed research will increase our understanding of the neural mechanisms via which alcohol escalates aggressive behavior in some individuals but not in others. Violent outbursts are one of the most costly, horrifying and damaging consequences of alcohol consumption, representing one of the most significant problems for the public health and criminal justice systems. The overarching hypothesis is to assess how escalated aggression, particularly under the influence of alcohol, is a function of dysregulation of serotonergic activity in the raphe cells by feedback via somatodendritic autoreceptors and by GABAergic and glutamatergic influences, especially by feedback from the prefrontal cortex, and by CRF input. We propose that the dysregulation of feedback control on serotonergic neurons projecting to prelimbic, infralimbic and orbitoventral regions of the prefrontal cortex characterizes those individuals who engage in escalated aggressive behavior after alcohol consumption. Specifically, experiments in mice and rats are designed to answer the following questions:

1. How is the activity of serotonergic projections from the dorsal raphe n (DRN) to the prefrontal cortex (PFC) regulated in individuals who engage in escalated aggressive behavior? To which extent is the expression of 5-HT receptor subtypes in the prefrontal cortex critical for escalated aggressive behavior? Is gene expression for the 5-HT1 and 5-HT2 receptor families in the prefrontal cortex suppressed in animals that engage in alcohol-heightened aggression? What is the respective role of presynaptic receptors in the PFC terminals relative to somatodendritic autoreceptors and SERT in gating serotonin transmission in highly aggressive individuals, particularly after alcohol consumption?

2. Are glutamatergic and GABAergic influences on the 5-HT cells in the DRN critical for the display of escalated aggressive behavior, particularly after alcohol self-administration? Do these signals originate from GABAergic interneurons? How significant is the glutamatergic feedback from the PFC? Which subunits in GABA-A receptors are essential for the aggression- heightening effects of alcohol? Are NMDA glutamate receptor subtypes more selective in their modulation of escalated aggression after alcohol self-administration than AMPA receptors?

3. How critical is the modulation by CRF of serotonergic projections to the PFC in individuals who engage in escalated aggressive behavior? Can the respective role of CRF 1 and 2 receptor subtypes be defined for the intensification or attenuation of alcohol-heightened aggressive behavior? Are the CRF receptors on serotonergic cells the critical population that is pivotal for escalated aggressive behavior after alcohol self-administration?

The experimental work relies on quantitative ethological methodology for the analysis of species-normative and escalated forms of aggression, voluntary alcohol self-administration, real time PCR, in situ hybridization histochemistry, genetic point mutations, in vivo microdialysis and HPLC, and intracerebral microinfusions. The anticipated outcome will identify targets for therapeutic interventions. The rationale for the proposed research is readily translated to one of the most significant problems for the public health and criminal justice system, namely to understand why alcohol escalates aggressive behavior in some individuals but not in others. Violent outbursts are one of the most costly, horrifying and destructive consequences of alcohol consumption. The proposed research will assess how escalated aggression, particularly under the influence of alcohol, is a function of dysregulation of serotonergic activity in the raphe cells by feedback via GABAergic, glutamatergic and CRF modulation.

PI: Mistry, Jayanthi
Title: Massachusetts Healthy Families Evaluation - 2
Abstract: Our first-cohort evaluation of HFM (MHFE-1) was composed of three overlapping components:

• the Process Study, which investigated HFM program operations;
• the Outcome Study, which investigated the effects of the program;
• the Ethnography, which informed both other components. 

MHFE-1 employed a quasi-experimental design, relying on other sources of comparison data, such as state and nationwide historical data on key indicators and extant data from studies of adolescents and young parents. Using a mixed methods approach, data were collected from a sample of 361 HFM participants, at six-month intervals, at four different time points over a period of 18 months. The ethnographic substudy, conducted in three communities, explored participants’ beliefs about parenting, childrearing, and help-seeking, and the extent to which HFM services were consonant with those beliefs. 

MHFE-1 yielded provocative findings pertaining to the operations of HFM, and to its perceived and observed effects among the young families it served. Our plan for a second-cohort evaluation (MHFE-2), detailed in this prospectus, is built both on the MHFE-1 findings and our experiences conducting that study, and on lessons learned over the past decade from other home visiting program evaluations.

While the findings from the first evaluation phase were promising, the quasi-experimental design precluded our being able to definitively attribute positive outcomes to the HFM program. For the second evaluation, the Tufts team is proposing a more rigorous, and more sophisticated, research approach than was possible in the first-cohort impact investigation. This proposed six-year evaluation study consists of two components: an Impact Study and an Integrative Study. 

PI: Moore, Claire
Title: The Coupling of mRNA Transcription and 3' End Formation
Abstract: The emerging model of eukaryotic mRNA synthesis is that transcription and mRNA processing events are carefully orchestrated in vivo by a physical association of the different machineries. For example, RNA polymerase II affects the efficiency of 3’ end processing, and processing factors affect the efficiency of transcription termination downstream of poly(A) sites. We are interested in the precise molecular mechanisms involved in the coordination of these two events and have identified several new points of interaction between transcription and cleavage/polyadenylation factors. These findings suggest that the presence of processing factors at the promoter might affect the efficiency and/or specificity of transcription initiation and facilitate recycling of RNAP II back to the promoter for another round of transcription. This may serve as a mechanism insure the proper loading of processing factors onto the transcriptional complex, and in turn, the subsequent polyadenylation of the transcript, which is essential for optimal export, translation, and turnover of mRNA. To investigate this issue, we propose the following specific aims: 

1. Can the activity of Ssu72 in transcription be separated from its role in 3’ end cleavage? We have found that Ssu72, previously identified as a protein affecting initiation, is directly involved in mRNA 3’ end cleavage. We analyze an existing collection of Ssu72 mutants to try to separate the cleavage activity of Ssu72 from a function in transcription initiation and develop new assays to help discriminate these functions. 

2. What is the functional significance of the interactions of Sub1 and Ssu72 with Pta1, and how are these interactions regulated? Ssu72 and Sub1 were initially identified based on genetic interactions with TFIIB. We have found these proteins genetically interact with the Pta1 subunit of Cleavage/Polyadenylation Factor (CPF). Moreover, they physically bind Pta1 in a mutually exclusive manner. We will test the hypothesis that sequential interactions of Pta1 with Ssu72 and Sub1 are important for efficient initiation and/or cleavage of pre-mRNA. 

3. Does Swd2 function in mRNA synthesis as part of CPF? This protein is intimately associated with CPF and Set1 histone methylase. However, Swd2 depletion has no effect on 3’ end processing, but causes inefficient transcription termination and reduced mRNA levels. We will test the hypothesis that Swd2 affects termination recruiting Set1 to the transcription complex. We will identify the contact point of Swd2 with CPF and examine how disruption of this interaction affects mRNA synthesis. A genetic screen will be used to identify important functional interactions with Swd2. 

PI: Moore, Claire
Title: Training in Education and Critical Research Skills
Abstract: This is a resubmission of our application to establish the Training in Education and Critical Research Skills (TEACRS) Program at Tufts University in response to the Institutional Research and Academic Career Development Award Program. Our program is based on the need for university faculty that are prepared to meet the multiple challenges faced by young Assistant Professors pursuing their first independent position. We will provide our postdoctoral trainees with the career skills they will need to succeed in an academic research environment by integrating three training components: rigorous bench research; mentored teaching experience; and career development skills. By partnering with two local minority serving institutions, Roxbury Community College and the University of Massachusetts, Boston, our trainees will also acquire a greater knowledge and sensitivity to diversity issues and provide enriched course material to institutions serving a minority population. Our faculty of 42 is committed to research excellence and has a strong training record. Research opportunities focused on multiple biomedical problems with special emphasis on basic biological problems, cancer, neuroscience and infectious disease will be available to trainees. Research committees and research advisors will monitor progress and career development with respect to research. The teaching component uses workshops, discussion groups and tutorials to prepare trainees to develop and present a course of their own design at one of the minority serving institutions. A series of career building workshops, along with checkpoints that provide mentoring and constructive feedback round out the program. Our goal is to produce the academic scientific leaders of the future. University faculty must develop independent research programs, an activity that requires creative thought, high intellectual capacity, outstanding facility with oral and written communication and considerable experimental skill. These activities must be sustained while faculty participate in classroom teaching, serve on a variety of committees and mentor trainees. Success also requires additional skills in interpersonal relations, budgeting and time management. The TEACRS program will provide trainees with the research portfolio needed to compete for academic positions and the skill set that postdoctoral fellows need to meet the challenges they will face as they enter academic careers. 

PI: Moore, Claire
Title: High Throughput Screening for Polyadenylation Inhibitors Using the MLSCN Library
Abstract: Accompanying the development of advanced medical techniques, candidiasis has emerged as a significant nosocomial infection that causes considerable morbidity and mortality among immunocompromised patients. Candida blood stream infections are increasing in frequency and are associated with high mortality. Oral candidiasis is an extremely common opportunistic infection in AIDS patients. Despite the prevalence of these infections, treatment options are limited. With the exception of the newly developed echinocandins, the antifungal drugs currently in use are limited by toxicity and natural or acquired resistance. Therefore, development of new antifungal drugs is of great importance. Our long-term goal is to develop new drug therapies for fungal infections. The difficulty in achieving this goal is that fungi use mechanisms for gene expression and cell growth that are similar if not almost identical to those used by mammalian cells. An essential process shared by all eukaryotes is the modification of the 3’ ends of mRNAs by cleavage of longer precursor molecules and the subsequent addition of a tract of adenosine residues. Acquisition of this poly(A) tail is important for accumulation of mature mRNA, its export from the nucleus, its utilization in translation of protein, and its removal when the mRNA is no longer needed by the cell. In the last few years, our research and that of others has identified most, if not all, of the subunits of this processing complex and revealed a remarkable conservation between the yeast Saccharomyces. cerevisiae and metazoans. However, we have also found significant species-specific differences, suggesting that inhibitors uniquely interfering with fungal mRNA 3’ end formation could be found. 

In this study, we will screen the MLSCN Small Molecule Repository for inhibitors of mRNA polyadenylation. This screen utilizes an assay in which defects in 3’ end processing in S. cerevisiae lead to production of a reporter required for cell growth. We have adapted this assay to a 384-well format that can be analyzed by an automated plate reader, and it gives robust performance in pilot screens. To assess the spectrum of activity of our hit compounds, we will test them for growth inhibition of mammalian cells and fungal pathogens. We will also use in vivo and in vitro assays for polyadenylation as secondary screens to confirm that hits are indeed targeting mRNA 3’ end formation. Finally, we will work with the MLPCN Center to design and synthesize derivatives with increased potency and specificity. We expect that this study will yield a novel class of anti-fungal drugs and thus address the pressing need for additional inhibitors of pathogenic fungi. An added benefit will be the discovery of chemical probes to help us understand the molecular mechanism of eukaryotic mRNA polyadenylation. 

PI: Moore, Claire
Title: High Throughout Screening for Anti-fungal Drugs that Inhibit mRNA Polyadenylation
Abstract: Accompanying the development of advanced medical techniques, candidiasis has emerged as a significant nosocomial infection that causes considerable morbidity and mortality among immunocompromised patients. Candida blood stream infections are increasing in frequency and are associated with high mortality. Oral candidiasis is an extremely common opportunistic infection in AIDS patients. Despite the prevalence of these infections, treatment options are limited. With the exception of the newly developed echinocandins, the antifungal drugs currently in use are limited by toxicity and natural or acquired resistance. Therefore, development of new antifungal drugs is of great importance. 

Our long-term goal is to develop new drug therapies for fungal infections. The difficulty in achieving this goal is that fungi use mechanisms for gene expression and cell growth that are similar if not almost identical to those used by mammalian cells. An essential process shared by all eukaryotes is the modification of the 3’ ends of mRNAs by cleavage of longer precursor molecules and the subsequent addition of a tract of adenosine residues. Acquisition of this poly(A) tail is important for accumulation of mature mRNA, its export from the nucleus, its utilization in translation of protein, and its removal when the mRNA is no longer needed by the cell. In the last few years, our research and that of others has identified most, if not all, of the subunits of this processing complex and revealed a remarkable conservation between the yeast Saccharomyces. cerevisiae and metazoans. However, we have also found significant species-specific differences, suggesting that inhibitors uniquely interfering with fungal mRNA 3’ end formation could be found. 

In this study, we will develop a high throughput assay to screen S. cerevisiae for small-molecule inhibitors of mRNA polyadenylation. This assay will be based on an existing reporter construct used in our laboratory to detect defects in mRNA 3’ end processing. Once the screen is optimized for a 384-well format, we will use it to screen a collection of 140,000 chemicals available through the Broad Institute of Harvard and M.I.T. We will use several in vivo and in vitro assays as secondary screens to confirm that hit molecules are indeed targeting mRNA 3’ end formation. We will then determine if the candidate molecules inhibit growth and mRNA 3’ end formation in the pathogen C. albicans, and construct a reporter for polyadenylation inhibitors that can be employed in additional large-scale screens directly in Candida. We expect that this study will yield a novel class of anti-fungal drugs and thus address the pressing need for additional inhibitors of pathogenic fungi.

PI: Morgan, John
Title: Fernald Dental Program - Comprehensive Oral Health Care for Individuals with Mental Retardation and Development Disabilities
Abstract: The Tufts Dental Facilities (TDF) of Tufts University School of Dental Medicine (TUSDM) proposes an evolutionary continuation of the program which in partnership with the Department of Public Health and Mental Retardation, has served persons with developmentally disabilities since its inception in 1976. The current proposal has taken into consideration several significant trends in the dental health care environment that have taken place in the past several years. These include:

• Local and national shortages of dental care providers, especially those with the specialized skills and professional dedication to treat persons with developmental disabilities. This has made recruitment of dentists and hygienists to treat this deserving population extremely competitive.

• Patients treated at the TDF clinics are primarily adults who rely on MassHealth for reimbursement for their dental care expenses. Current reimbursement rates for adult dental care offered by MassHealth: 1) are lower than the fees for children; 2) are not competitive with usual and customary fees charged in dental practice; and 3) have not been increased to meet the rising cost to deliver dental care. This has led to below average reimbursements as nearly every cost to deliver dental care to the target population has significantly increased.

• Although the recent (July 1, 2006) reinstatement of adult MassHealth dental benefits is encouraging, the lack of these benefits for the previous three years resulted in many special needs patients losing their dental providers in settings outside the Tufts system. From April 2, 2002 through June 30, 2006, patients with disabilities were required to submit special documentation of their condition to qualify for dental benefits through the MassHealth program. Many dental practitioners were not able to accommodate this special circumstance requirement. As a result, the TDF statewide clinical network was inundated with patients who no longer had a dental provider. Tufts responded by increasing their dental and hygiene providers as tolerated by budgetary constraints.

Basic to our proposal is that TUSDM shall continue to deliver a quality, state of the art program of comprehensive dental care. Tufts University fully understands and will continue to meet and exceed state and federal health care regulations. However, in order to maintain the level of service Tufts currently provides, and to accommodate the vast increase in request for services that has occurred over the past several years, the payment for these services must realistically reflect the cost of providing them. Patients with developmental disabilities have great difficulty accessing dental services. The budget in this response is the realistic cost to continue the program at its current level of service. If additional funds required to maintain the program at its current level are not made available, TUSDM will work with DPH to redesign a program that is more realistic in meeting the financial realties of the University and DPH, while still meeting the goals of the RFR. 

PI: Moss, Stephen
Title: The Role of Ubiquitination in Regulating GABA_A Receptor Functional Expression
Abstract: γ-aminobutyric acid (GABA_A) receptors are key sites of synaptic inhibition in the brain and are critical drug targets for therapeutic agents including benzodiazepines, barbiturates, general anesthetics and neurosteroids. Moreover compromised GABA_A receptor function is central to a number of CMS pathologies including epilepsy, anxiety, sleep disorders, addiction, autism, mental retardation, depression and schizophrenia. Ubiquitination of lysine residues is a commonly used cellular mechanism to regulate both protein half-life and the endocytic fate. This accepted paradigm together with our preliminary studies has led us to formulate a central hypothesis driving the experiments described in this proposal: GABA_A receptors are subject to direct ubiquitination of lysine residues with the intracellular domains of individual receptor subunits, a process that is subject to dynamic modulation by neuronal activity. Depending on the subunit ubiquitinated this covalent modification acts to decrease receptor half-life within the secretory pathway or enhances lysozomal targeting, thereby modulating GABA_A receptor cell surface stability and the efficacy of synaptic inhibition. Our efforts will center on four complementary but distinct experimental goals: 

1. To test the hypothesis that GABA_A receptor β3 subunit ubiquitination regulates receptor cell surface stability by modulating insertion at the plasma membrane 
2. To test the hypothesis that GABA_A receptor g2 subunit ubiquitination regulates receptor cell surface stability by modulating lysozomal targeting 
3. To test the hypothesis that ubiquitination modulates the efficacy of synaptic inhibition mediated by GABA_A receptor 
4. To test the hypothesis that neuronal activity regulates GABA_A receptor cell surface stability by modulating receptor ubiquitination 

Together, our approaches will provide a more thorough understanding of the primary determinants that regulate accumulation of GABA_A receptors at synaptic sites. The results of these studies will have the potential to make significant contributions to the development of novel therapeutic strategies for such debilitating disorders as epilepsy, anxiety, sleep disorders, addiction, autism, mental retardation, depression and schizophrenia. 

PI: Moss, Stephen
Title: Constructing Inhibitory Synapses
Abstract: GABA_A receptors (GABA_AR) are heteropentameric Cl^- selective ligand-gated ion channels that mediate the majority of fast synaptic inhibition in the brain. They are key therapeutic targets for benzodiazepines, barbiturates, general anesthetics, and neurosteroids. Changes in GABA_AR functional expression are of significance in a plethora of neuropsychiatric disorders including autism, anxiety, addiction, cognitive deficits, depression, epilepsy, and schizophrenia. A critical determinant for the efficacy of neuronal inhibition is the specific accumulation of GABA_AR subtypes at the appropriate postsynaptic sites. Benzodiazepine-sensitive synaptic GABA_AR subtypes are largely assembled from α1, 2, 3, β and γ2 subunits, but how they are concentrated at these subcellular specializations remains poorly understood. One protein consistently implicated in this process is gephyrin. While this multifunctional protein is enriched at central inhibitory synapses, how it facilitates GABA_AR accumulation at these structures remains obscure. These phenomena together with our preliminary studies have led us to formulate a central hypothesis driving the experiments described in this proposal: That gephyrin binds directly to conserved amino acid motifs within the intracellular domains of selected GABA_AR α subunit isoforms, a process that is critical for their accumulation at inhibitory synapses and for the efficacy of neuronal inhibition. Moreover, phosphorylation of specific amino acids within the intracellular domains of a subunits by casein kinase 1 (CSK1) provides a dynamic mechanism to regulate gephyrin binding and the number of GABA_ARs at synaptic sites. Our proposal will center on four specific aims: 

1. To test the hypothesis that gephyrin binds directly to conserve motifs within the intracellular motifs of selected GABA_AR α subunit isoforms. 
2. To test the hypothesis that blocking the interactions of GABA_AR α subunits with gephyrin reduces the accumulation of GABA_A receptor subtypes at synaptic sites. 
3. To test the hypothesis that inhibiting the interaction of gephyrin with GABA_ARs modifies the efficacy of neuronal inhibition. 
4. To test the hypothesis that phosphorylation of GABA_AR α subunits by casein kinase 1 acts as a molecular switch to regulate their interaction with gephyrin and synaptic clustering.
   

PI: Moss, Stephen
Title: Modulation of GABA_B Receptor Signaling
Abstract: GABA_B receptors are the major sites of slow synaptic inhibition in the brain. Changes in GABA_B receptor function play key roles in epilepsy, nociception, depression, addiction, mental retardation and neuroprotection. The number of GABA_B receptors expressed on the cell surface of neurons is dependent on the formation of heterodimers between GABA_BR1 and R2 subunits. Recent studies have demonstrated that once at the cell surface GABA_B receptors are stable entities, which do not undergo agonist-induced internalization. Therefore primary determinants of the efficacy of slow synaptic inhibition mediated by GABA_B receptors will be the production of receptor heterodimers, the trafficking of assembled heterodimers to the cell surface, and their coupling to the appropriate effectors. Here we hypothesize that GABA_B receptor activity is regulated by the activity of AMP-dependent protein kinase (AMPK), which phosphorylates serine residues within both receptor subunits. Phosphorylation increases GABA_B receptor cell surface expression levels by enhancing receptor trafficking and assembly in the secretory pathway. In addition, phosphorylation by AMPK enhances receptor-effector coupling by reducing desensitization. Therefore AMPK activity provides a dynamic mechanism for regulating the efficacy of GABA_B receptor signaling. We will use a combination of cell biological, biochemical and electrophysiological approaches to carry out three independent but related specific aims:  

1. To test the hypothesis that GABA_B receptor are dynamically phosphorylated by intimately associated AMPK.  
2. To test the hypothesis that direct phosphorylation of GABA_B receptor by AMPK regulates receptor cell surface stability and effector coupling.  
3. To test the hypothesis that AMPK activity modifies GABA_B receptor membrane trafficking and assembly.  

Together, our approaches will provide a more thorough understanding of the cell surface stability and function of GABA_B receptors. The results of these studies will have the potential to make significant contributions to the development of novel therapeutic strategies for such debilitating disorders as epilepsy, nociception, depression, addiction, and mental retardation. 

PI: Moss, Stephen
Title: The Regulation of GABA_A Receptor Cell Surface Stability
Abstract: GABA_A receptors (GABA_ARs) are heteropentameric Cl^- selective ligand-gated ion channels that mediate the majority of fast synaptic inhibition in the brain. They are key therapeutic targets for benzodiazepines, barbiturates and neurosteroids. Changes in GABA_AR functional expression are implicated in a plethora of neuropsychiatric disorders including autism, anxiety, addiction, cognitive deficits, depression, epilepsy, and schizophrenia. Synaptic GABA_AR subtypes are composed of α, β and γ2 subunits and undergo significant rates of clathrin-dependent endocytosis, a process that is facilitated via the direct binding of the intracellular domains of individual receptor subunits to the µ2 subunit of the AP2 adaptin (µ2-AP2), a critical regulator of endocytosis. In vitro binding studies together with crystallography have revealed that µ2-AP2 binds with nanomolar affinity to an amino acid motif centered on tyrosine 365 and 367 (Y365/7) thin the γ2 subunit. Binding of µ2-AP2 to the γ2 subunit is negatively regulated via the phosphorylation of Y365/7 but the significance of this process for the efficacy of synaptic inhibition remains to be determined. These phenomena together with our preliminary studies have led us to formulate a central hypothesis driving the experiments described in this proposal: Phosphorylation of Y36517 in the γ2 subunit acts as a dynamic mechanism to modulate GABA_AR endocytosis, accumulation at inhibitory synapses and the efficacy of synaptic inhibition. Our experiments will focus on four specific aims: 

Specific Aim 1: To test the hypothesis that phosphorylation of Y365/7 acts as a “molecular switch” to regulate the binding of AP2 and Fyn to GABA_ARs. 

Specific Aim 2: To test the hypothesis that mutation of Y365/7 to phenylalanine residues increases the cell surface stability and accumulation of GABA_ARs at inhibitory synapses. 

Specific Aim 3: To test the hypothesis that mutation of Y365/7 in the γ2 subunit increases the efficacy of synaptic inhibition. 

Specific Aim 4: To test the hypothesis that increased expression levels of the γ2 subunit in Y365/7F+/- animals leads to reduced tonic inhibition. 

Results of the experiments proposed here will provide mechanistic insights into the cellular processes that control the cell surface accumulation of GABA_ARs and the efficacy of neuronal inhibition. Given the critical roles that GABA_ARs play in regulating neuronal inhibition our studies have the potential to make contributions to the development of novel therapeutics to alleviate autism, anxiety, addiction, cognitive deficits, depression, epilepsy, and schizophrenia. 

PI: Must, Aviva
Title: Severe Early Childhood Caries: Role of Dietary Cariogenicity and Eating Patterns
Abstract: Early childhood caries is a serious pediatric health problem that disproportionately impacts children of lower socioeconomic status. In the U.S., early childhood caries affect 2 to 20% of pre-school children. Despite the general appreciation for the magnitude of childhood dental problems and the role of dietary sugars in the development of caries, there are critical gaps in our knowledge regarding how best to characterize the cariogenicity of child diet, what aspects of child diet are most important in the development of caries, and how dental treatment influences subsequent dietary patterns. The proposed exploratory/developmental research makes use of a rich complement of dental and dietary data being collected prospectively on 900 children as part of project within the Northeast Center for Research to Evaluate and Eliminate Dental Disparities. Low-income predominantly non-white children ages 2 to 6 years are being studied at three time points over a 12-month period. Equal numbers of children with severe early childhood caries and caries-free control children have been enrolled. A cariogenicity classification system will be developed based on foods identified by 24-hour diet recall interview and food frequency questionnaire, and used to systematically develop and test a pediatric “dietary cariogenicity index” to represent total dietary cariogenicity based on both the physical form and timing of food consumed. Secondarily, we will explore comprehensively the impact of S-ECC on dietary patterns, and evaluate changes in dietary patterns and dietary cariogenicity following comprehensive dental treatment. Collectively, these activities will improve our ability to characterize child diets with methodology relevant for oral health, and to inform prevention efforts and our ability to intervene in children with early childhood caries.

PI: Olum, Ken
Title: Does General Relativity Permit Exotic Phenomena?
Abstract: Does semiclassical gravity permit stable wormholes, faster—than—light travel, or the construction of time machines? To rule out such exotic phenomena requires energy conditions: restrictions on possible stress—energy tensors that can act as sources for gravity. The averaged null energy condition (ANEC) requires that the total energy seen by an observer traveling on a null geodesic be nonnegative. It would be sufficient to rule out the exotic phenomena above, but it is not universally obeyed in quantum field theory. In previous work, we discussed the “self—consistent achronal averaged null energy condition”. It requires that ANEC hold only on achronal geodesics and only considers spacetimes which are self—consistently generated by their stress—energy tensors. The self—consistent achronal ANEC is sufficient to rule out exotic phenomena, so we will attempt to show that this condition (or a similarly powerful one) holds for quantum field theory in curved spacetime. The main obstacle to be overcome is the presence of the scale anomaly, which introduces extra, potentially ANEC—violating terms, when a system is rescaled. It is this anomaly which necessitates the self—consistency condition. 

PI: Ordovas, Jose
Title: Gene/Diet Effects on Plasma Lipoprotein Levels
Abstract: Coronary heart disease (CHD) remains the leading cause of death and disability in our society. Every year, approximately 500,000 deaths in the U.S. and 7.2 million globally are caused by this disease. As many women die from CHD as do men. The opportunity to reduce the major causes of CHD is at hand. By focusing on prevention, we can have a major impact on people’s health. However, major gaps exist in our knowledge about nutritional adequacy, nutrient-disease interactions, and effective strategies to implement the current recommendations, which have the widely recognized potential to decrease the disease burden of the American population. Moreover, we know little about specific genes and genetic variations that affect risk directly and indirectly by the way they interact with nutrients. Therefore, one of the goals specified by U.S. Health Organizations is to characterize the numerous genes that likely play a critical role in the causation of major diseases or the protection of individuals from such diseases and to investigate their interactions with nutrients. Similarly, the roles of specific polymorphic forms of genes that influence individual susceptibility to specific dietary factors must be identified.

Hence, the primary aims of this proposal are:

1. To evaluate whether polymorphisms at the proposed candidate genes [Peroxisome Proliferator-Activated Receptors (PPARA and PPARG), Estrogen Receptor (ESRA), Sterol Regulatory Element-Binding Proteins (SREBP1 and SREBP2), SREBP cleavage-activating protein (SCAP), 3-Hydroxy-3-Methylglutaryl-CoA Reductase (HMGCR), Scavenger Receptor Class B-I (SRBI), Intestinal Fatty Acid-Binding Protein (FABP2), and ATP-Binding Cassette 1 (ABC1)] are associated with plasma lipid variability and CHD in a free-living population, and
2. To examine interactions between those gene variants and dietary factors. Information about such gene-diet interactions will provide significant clues about the mechanisms by which dietary factors regulate lipid metabolism.

The candidate genes proposed contribute to variation in plasma low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C) and triglyceride (TG) levels. The study population consists of subjects participating in the Framingham Offspring (FOS) and OMNI (Minorities) studies (n~3, 180). The primary outcomes are LDL-C, HDL-C, TG, remnant-like particle-C levels, lipoprotein particle size, and measures of LDL oxidation. Secondary outcomes are carbohydrate metabolism-related measures (i.e., glucose and insulin levels). Our main hypothesis is that genetic variants at these loci are associated with variability of plasma lipid measures, thus determining CHD risk. These effects are modulated by dietary and behavioral factors. This knowledge will facilitate the generation of genetic screening panels that will assist in individual assessment of CHD risk and dietary therapeutic counseling. 

PI: Papas, Athena
Title: Prevention of Adult Caries (PACS) - TUFTS CLINICAL CENTER
Abstract: Adult dental caries, including both coronal and root caries, is an infectious disease that afflicts the majority of Americans aged 55 and older and is the most common chronic disease at midlife. In addition, adult caries exacts a significant economic toll, and this economic toll continues to grow. Despite the high prevalence and bacterial pathogenesis of caries, no FDA-approved anti-microbial treatment for caries is available to the American dental professional. The Prevention of Adult Caries Study (PACS) is a randomized, double-blind, placebo-controlled Phase III clinical trial conducted under FDA Investigational New Drug license #45,466. This study is designed to evaluate the efficacy of a topical, temporary, 10% w/v chlorhexidine dental coating in reducing caries increment in at-risk adult dental patients. This study will follow 1000 patients over a 13-month study period at four centers with vastly different populations. The primary endpoint for this study will be caries increment. The centers participating in this proposed research are: Center for Health Research, Kaiser Permanente Northwest, in Portland, Oregon; Tufts University Dental Clinic in central Boston, Massachusetts; Dental Service of Massachusetts Clinical Center in Southborough, Massachusetts, and; the dental clinic of the Tuba City Regional Health Care Corporation in Tuba City, Arizona. Kaiser Permanente's Center for Health Research in Portland, Oregon will act as the data-coordinating center, and Tufts University will act as the Administrative Center.  

PI: Papas, Athena
Title: Prevention of Adult Caries (PACS) - TUFTS ADMINISTRATIVE CENTER
Abstract: Adult dental caries, including both coronal and root caries, is an infectious disease that afflicts the majority of Americans aged 55 and older and is the most common chronic disease at midlife. In addition, adult caries exacts a significant economic toll, and this economic toll continues to grow. Despite the high prevalence and bacterial pathogenesis of caries, no FDA-approved anti-microbial treatment for caries is available to the American dental professional. The Prevention of Adult Caries Study (PACS) is a randomized, double-blind, placebo-controlled Phase III clinical trial conducted under FDA Investigational New Drug license #45,466. This study is designed to evaluate the efficacy of a topical, temporary, 10% w/v chlorhexidine dental coating in reducing caries increment in at-risk adult dental patients. This study will follow 1000 patients over a 13-month study period at four centers with vastly different populations. The primary endpoint for this study will be caries increment. The centers participating in this proposed research are: Center for Health Research, Kaiser Permanente Northwest, in Portland, Oregon; Tufts University Dental Clinic in central Boston, Massachusetts; Dental Service of Massachusetts Clinical Center in Southborough, Massachusetts, and; the dental clinic of the Tuba City Regional Health Care Corporation in Tuba City, Arizona. Kaiser Permanente's Center for Health Research in Portland, Oregon will act as the data-coordinating center, and Tufts University will act as the Administrative Center. 

PI: Perrin, Mercio
Title: Neuron Survival in Chagas Disease
Abstract: Patients with Chagas' disease, caused by Trypanosoma cruzi infection, exhibit neural degeneration in the acute phase, neuroregeneration in the chronic indeterminate (asymptomatic) phase, and degeneration of neurons in the chronic symptomatic phase. Most patients remain asymptomatic and with signs of neuronal regeneration for years or decades. Some asymptomatic patients progress to the chronic, fatal chronic disease stage. It remains unknown why patients can remain asymptomatic and with signs of neural regeneration for decades while others develop neurodegeneration later in life.  

Recent studies demonstrate that T. cruzi expresses surface membrane-bound and shed parasite-derived mimic of neurotrophic factors (PDNF), formerly known as neuraminidase/trans-sialidase, that potently promotes survival of several types of neurons, Schwann cells, and astrocytes. A major and wide audience-reaching finding in the previous five years was that PDNF induces survival and differentiation of neurons by binding and activating TrkA nerve growth factor (NGF) receptor tyrosine kinase. This unique result opens up many interesting avenues to understand the molecular basis of a microbial invader of the nervous system, specifically whether T. cruzi, via PDNF, like authentic host growth factors, recognizes other neurotrophin receptors, triggers off neurotransmitter rate-limiting enzymes through Trks receptors, bear neurotrophin motifs that can be exploited as synthetic peptides to reverse neuronal and glial damage, and elicit autoimmune responses to Trks that might activate or inhibit receptor function.  

The project will utilize a combination of cell biology, biochemistry, genetics, and immunochemical approaches to identify and characterize the interaction of TrkA and other receptors with PDNF and the novel Trk autoantibodies. Most likely, the outcome of the proposed studies will provide insights into the pathogenesis of Chagas' disease, particularly neuroregeneration, and lead to the development of compounds, principally NGF-like peptidomimetics, to treat not only Chagas' disease but also other neurodegenerative disorders such as Parkinson's disease. 

PI: Perrin, Mercio
Title: Receptors for Neuron and Glia Survival in Chagas Disease
Abstract: Chagas’ disease, Caused by the protozoan Trypanosoma cruzi, progresses through 3 stages:

1. acute disease, characterized by robust parasite growth, immunological disturbances, and peripheral neural degeneration;
2. chronic indeterminate phase or asymptomatic, featuring extensive regeneration of neurons; and
3. chronic symptomatic phase, characterized by immunological alterations and pronounced degeneration of neurons in the heart and gastrointestinal tract.

Most chagasic patients remain asymptomatic and with signs of neuronal regeneration for decades while, for unknown reasons, some of these patients (<10%) undergo extensive degeneration of neurons as well as immunological and vascular disturbances to progress to the fatal chronic disease. 

T. cruzi encodes a surface-bound and shed neuraminidase that mimics the mammalian neurotrophic factors by promoting differentiation and survival of glial Schwann cells and neurons exposed to various insults, including neurotoxins that cause Parkinson’ disease. Thus, the neuraminidase has been renamed PDNF (parasite-derived mimic of neurotrophic factor). PDNF is active at extremely low concentrations (low picomolar range). In conjunction with NS42960, PDNF has been found to recognize and activate nerve growth factor (NGF) receptor TrkA. In addition, a null mutation in the neurocytokine ciliary neurotrophic factor (CNTF) appears to be increased many-fold in female patients with megacolon and megaesophagus, raising the possibility of CNTF-/- to be a risk factor for Chagas’ disease. 

This proposal attempts to extend these novel and unique findings by testing three hypothesis:

1. Tyrosine kinase receptor TrkA is a cellular receptor for T. cruzi invasion of the nervous system.
2. A null mutation in the human CNTF gene underlies chagasic enteric pathology.
3. Deletion of the CNTF gene underlies pathogenesis in experimental Chagas’ disease.

The outcome of these studies could provide insights into the pathogenesis of Chagas’ disease and may lead to the development of drugs to prevent or treat Chagas and other motor neuro diseases, particularly Parkinson’ disease. 

PI: Perry, Ronald
Title: Shear Bond Strength of a New Bonding Agent (Bond Force) 24-hour Bond Strength
Abstract: The objective of this study is to compare the shear bond strength of a new bonding agent (Bond Force) versus 5 other different adhesives systems when bonded to dentin and enamel. 

PI: Poltorak, Alexander
Title: Hyper-Responsiveness to TLR Agonists in Wild Derived Mice
Abstract: The release of pro-inflammatory cytokines is a critical early event in host pathogen defense, often limiting the initial spread of infectious agents through innate mechanisms and activating the adaptive response for more long-term control or complete elimination of infection. Because these immunologic processes can be damaging to host tissue, the amount and time course of cytokine release must be finely controlled and numerous pathologies are associated with dysregulated cytokine activity. The molecular mechanisms used in fine-tuning this process are therefore the subject of active investigation. Here we propose using a forward genetic analysis of inbred (C57BL/6J) and wild derived (MOLF/Ei, Czech/Eill, MSM/Ms) mouse strains that secrete different amounts of IL-6 in response to TLR stimulation as a novel approach to characterizing the regulation of early cytokine release. Initial studies from a panel of backcross mice have identified two loci on chromosomes 6 and 9 that make major contributions to the trait. Further, these two loci have a significant epistatic interaction (p<10^-6, LRS 48.1). Our mapping indicates that these loci contain candidate genes not previously implicated in TLR-mediated signaling. We propose to map these genes via meiotic recombination (and test the hypothesis that allelic variation at these loci contributes to the phenotype?). We also propose to establish the mechanistic basic for their observed epistatic interaction. This proposal aims to approach the problem through more refined genetic analysis as well as biochemical and molecular characterization of the candidate genes. We believe that we will reveal important novel mechanisms of TLR signaling. 

PI: Pothos, Emmanuel
Title: CNS Dopamine as Marker of Obesity Predisposition: Link to Food and Drug Reward
Abstract: Dietary obesity is a fast growing epidemic worldwide and effective approaches for addressing this public health problem are necessary. The involvement of hedonic aspects of feeding in the obesity epidemic is still unclear and its study may reveal common mechanisms that mediate susceptibility or resistance to natural and drug rewards. This proposal focuses on understanding the link between dietary obesity predisposition and central dopamine systems implicated in food and d-amphetamine rewards. We hypothesize (and so far have confirmed) that the inbred obesity-prone (OP) phenotype in the rat is characterized by low central dopamine tone and reduced dopamine response to stimuli like food and d-amphetamine. The reduced dopamine response may lead to compensation with increased laboratory chow or amphetamine intake. Proteins that downregulate the dopamine tone are potential novel markers of obesity predisposition. Appropriate interventions may alter the susceptibility of the OP phenotype to indulgent stimuli (like high-energy diets or psychostimulants) by fine-tuning central dopamine exocytosis. The specific aims of this proposal include considering:

1. how obesity predisposition is linked to dopamine release kinetics before and after body weight differences arise and before and after a meal or an amphetamine challenge
2. whether regulatory proteins of central dopamine exocytosis are markers for obesity predisposition
3. developmental and molecular interventions that alter the OP phenotype  

Inbred OP and obesity-resistant (OR) rats will be used throughout as they develop a weight difference while on the same diet and allow for distinction from diet history-related effects. OP rats closely model human obesity predisposition through polygenic inheritance favoring weight gain. Following a multi-level approach, we propose to profile in vivo dopamine release and behavior; real-time dopamine exocytosis in the acute slice and primary cell culture; and mRNA and protein levels of regulators of dopamine exocytosis. Preliminary results show that adult outbred obese and inbred OP rats had lower basal, meal-challenged and amphetamine-challenged extracellular dopamine levels. Tyrosine hydroxylase mRNA levels were low in OP adults, while the vesicular monoamine transporter (VMAT2) protein levels were low in OP neonatal rat dopamine cell cultures. Proposed interventions to alter the OP phenotype and associated central dopamine neurotransmission include placement of OP litters with OR dames at day PO, VMAT2 over expression in the nucleus accumbens of OP rats, and pair feeding of OP and OR rats.

PI: Ramsburg, Andrew
Title: Collaborative Research: Exploration of the Interplay between Contaminant Partitioning and Biotransformation in Multiphase Porous Media
Abstract: Biological technologies aimed at cleaning up aquifers contaminated by nonaqueous phase liquids (NAPLs) frequently produce multiple degradation products which may or may not represent an overall reduction of the risk posed to human health. Understanding the fate and transport of these degradation products within a multiphase subsurface environment is critical given the interest in applying bioenhanced dissolution for remediation of sites contaminated by chlorinated solvent NAPLs. Assessing the fate and transport of biological degradation products in multifluid subsurface environments requires knowledge of the interplay between physical, chemical, and biological processes. At the center of this interplay is bioavailability of degradation products influencing contaminant fate. Bioavailability is influenced by the kinetics of mass transfer processes active in multiphase subsurface environments.

This research project integrates laboratory experiments and mathematical modeling to explore how kinetics of interphase mass transfer influences rates of biodegradation and degradation product accumulation in NAPL source zones containing chloroethenes. Specific objectives are:

1. identify the capacity of chloroethene NAPLs to reversibly sequester cis-dichloroethene and vinyl chloride,
2. investigate the rates of mass transfer of these degradation products within heterogeneous porous media containing chloroethene NAPLs, and
3. understand the significance of these processes in the context of field-scale, bioactive, NAPL source zones.

PI: Ramsey, Norman
Title: ITR Collaborative Research: A Reusable, Extensible, Optimizing Back End
Abstract: A computer program is written in a high-level programming language, but to be run, it must be translated into a machine language. A translator is difficult and expensive to build, and using today's techniques, each programming language needs its own translator. For example, it is nearly impossible to use the same translator for both Java and C++. The project is developing techniques by which a “back end,” which understands machine language, can serve many different “front ends,” each of which understands a different programming language. Combining such front and back ends can produce a translator much more cheaply than is possible at present. The major new idea is to use three interfaces. The C++ language is an interface that enables the front end to tell the back end what a program should do. The C++ run-time interface reveals decisions made by the back end so that the front end can support such services as garbage collection and threads. The Cobalt language is an interface that enables the front end to tell the back end what special translation techniques are needed for efficiency in a particular language. These interfaces are supported by new, automatic techniques for eliminating potential errors in translation. 

PI: Rauch, Annette
Title: Professionalizing Shelter Medicine: A New Model to Expand Training and Education in Shelter Medicine within the DVM Curriculum
Abstract: The mission of the TCSVM Shelter Medicine Program is: 

• To improve the quality of life for homeless, displaced, abused or neglected animals through the education of veterinary students, veterinarians, and veterinary para-professionals in the principles, core competencies, and best practices of shelter medicine; 

• To develop a community that is more capable of meeting the needs of at-risk animal populations: shelter animals, animals displaced by natural and manmade disasters, and animals belonging to underserved populations, such as low-income pet owners. This community will become well-versed in animal welfare issues; and

• To instill in our veterinary students a sense of urgency and obligation, driven by compassion, to give back to their communities, by sewing as expert community resources on animal policy issues for vulnerable populations.  

PI: Rios, Maribel
Title: BDNF and TrkB-Containing Neuronal Circuits Mediating Energy Balance
Abstract: Obesity is a risk factor for the development of type 2 diabetes, cardiovascular disease and other afflictions. This is troubling as close to 30% of the population in the United States is obese and 64% is overweight. The study of brain-derived neurotrophic factor (BDNF) and its receptor, TrkB, provides a new and promising avenue in obesity research. They mediate neuronal survival, differentiation, function and plasticity in the developing and mature brain. The relevance of this signaling pathway in the etiology of energy balance disorders is highlighted by the dramatic obesity exhibited by mice with brain-specific targeting of the Bdnf gene. Mutants display hyperphagic behavior and become hyperleptinemic, hyperinsulinemic and hyperglycemic, indicating that this neurotrophin is an essential regulator of food intake acting through as yet unknown mediators in the central nervous system. Human studies also show links between genetic alterations in the TrkB gene and hyperphagic behavior and obesity. 

This proposal outlines studies designed to ascertain the molecular and cellular mechanisms facilitating the satiety effects of the BDNF/TrkB signaling pathway in the adult animal. For this, the effects of energy signals on the expression and activity of BDNF and TrkB in distinct appetite-modulating regions of the brain will be evaluated. Moreover, the Bdnf gene will be targeted in adult animals in a site-specific manner in hypothalamic and caudal hindbrain nuclei associated with these processes. The impact of these manipulations on energy intake and expenditure will be evaluated. Finally, as BDNF has emerged as a prominent facilitator of synaptic plasticity, we will investigate whether it is required for synaptic plasticity-related processes in the hypothalamus thought to influence energy homeostasis. Together, these studies will distinguish developmental roles of BDNF from ones in the mature brain that influence body weight. This analysis will help clarify disease mechanisms and critical periods of intervention for obesity disorders. Moreover, they will bring us closer to novel strategies for the treatment of obesity and its associated syndromes. 

PI: Rios, Maribel
Title: Examination of the Role of Brain-derived Neurotrophic Factor in Binge Eating Behavior
Abstract: A recent national study revealed that within the US population, binge eating disorder (BED) afflicts 3.5 and 2.0% of women and men, respectively, and that bulimia nervosa (BN) affects 1.5% of women and 0.5% of men. Effective treatment strategies for affected individuals are greatly needed. However, the neuromolecular mechanisms contributing to the etiology of these disorders remain largely unknown. Defective brain-derived neurotrophic factor (BDNF) signaling through the tropomyosin related kinase B (TrkB) receptor emerged recently as a candidate mechanism. Human association studies suggest a link between BED and BN with the Val66Met polymorghism in the Bdnf gene, which impedes regulated secretion of BDNF. Furthermore, mice (BDNF^2L/2LCK-cre) in which we deleted Bdnf across the brain, exhibited dramatic hyperphagic behavior, reminiscent of bingeing behavior in humans. This proposal aims to elucidate the role of deficient BDNF signaling in binge eating behavior, a feature of both BED and BN. We propose behavioral studies to ascertain whether BDNF^2L/2LCK-cre mutants exhibit increases in food reward under baseline conditions and following food restriction and stress. Moreover, we will conduct a systematic analysis of the mesolimbic dopaminergic system in BDNF mutants as this pathway mediates reward and motivated behavior, including consumption of palatable food. Analysis will include measurements of dopamine signaling, synthesis and secretion in wild types and BDNF mutants. The effect of palatable food ingestion on TrkB signaling within the mesolimbic system will also be determined. Finally, to pinpoint regions of the brain that are essential suppliers of BDNF for the regulation of motivated eating, we will evaluate the effect of selectively deleting Bdnf in the ventral tegmental area (VTA) and medial prefrontal cortex (mPFC), which are the chief sources of BDNF within the mesolimbic system. Collectively, these studies will determine whether the BDNF/TrkB pathway is a viable target for the treatment of BED and BN. 

PI: Robbat, Albert
Title: Chemical Analysis Proposal
Abstract: The purpose of this research is to investigate the sensory and chemical nature of essential oils, botanicals and their use in distilled beverages. Past research has lacked a systematic approach to identify chemical constituents in these mixtures. Typically, researchers focus on compounds that are easy to detect and identify, but may not be important contributors to flavor or aroma. Low level compounds are often ignored by traditional methods of chemical analysis because they are buried in chemical noise or are below the detection limits of standard chemical instrumentation. 

Critical to improving manufacturing processes, developing quality control standards, and understanding public perception of finished products is the ability to find those compounds that contribute from a sensory perspective. Another aspect of the work may be to identify where in the product manufacturing process unwanted chemicals may be formed. Also of interest may be those compounds that are formed as product ages. If the goals are to better flavor “freshness” and extend product shelf-life, research aimed at detecting, identifying, and quantifying compound contributors is of great importance. 

This research has several objectives:

• To profile chemical constituents in essential oils, botanicals, and finished products.
• To determine differences in finished products when botanicals are left out of the manufacturing process and to assess whether missing or enhanced components can be added by other means.
• To develop a database of flavor active chemical compounds in these extracts.
• To develop a mass spectral library that can be used to identify key ingredients in other finished products.
• To develop an understanding of the flavor active chemistry that is formed in the manufacturing process. 

PI: Rogers, Beatrice
Title: Training Program in Water and Health (RMI)
Abstract: In 2002, Tufts University created an interdisciplinary doctoral program to train students to work with a global view toward assuring water security for the protection of health, the environment, and human livelihoods. The program is called Water: Systems, Science, and Society (WSSS), and includes six schools of the university: Medicine, Veterinary Medicine, Nutrition, Arts and Sciences, Engineering, and the Fletcher School of Law and Diplomacy. WSSS is designed to assure that students acquire interdisciplinary knowledge and approaches, even as they develop a specialization. With this application we propose to support students in WSSS with a concentration in Water and Health. Problems of water needs, use, and quality are pervasive throughout the world as well as the US, and are considered important enough by the National Institutes of Health to be mentioned specifically within the goals of Healthy People 2010. The Water and Health training program will support both HP 2010 goals and the New Roadmap goals of creating a new kind of interdisciplinary professional. Integrated, interdisciplinary analysis is necessary to understand and manage complex water related problems. Our vision is to educate professionals skilled in health-related disciplines who can use multiple disciplinary perspectives and tools. Water and Health graduates will be able to respond to the interdisciplinary, integrated water-related research challenges. Engineers will understand the principles of Public Health. Nutrition students will understand the basics of hydrology. Policy students will understand the fundamentals of epidemiology. These new professionals will be able to participate in the analysis of water problems and work to develop solutions jointly with colleagues from many specialties, using the rigorous methods and tools of the relevant disciplines. 

PI: Rogers, Chris
Title: Using Wide-Spread Collaboration to Motivate Innovation and Creativity
Abstract: The goal of this project is to develop a new environment for collaboration in creative projects and to promote creativity and innovation in IT problem solving among disadvantaged youth. The new environment is a wiki-based authoring toolkit, called the Briefcase, that will guide students through the use of various software packages and allow them to share and discuss with others locally and over the Internet. The intent of the Briefcase concept is to extend the functionality of the RoboBook product currently under development at Tufts to include the scalability of a Wiki-like backbone. Once the software has been designed, we will develop instructional content for it as well. The idea is to provide students with an authoring environment (like Word or PowerPoint) that helps students:

1. Learn how to use various software packages;
2. Keep track of what they are doing through blogs, pictures, and movies;
3. Discuss and team with others (locally and remotely) to problem solve; and
4. Share what they develop with others around the world.
   

Further, the Briefcase will allow the Learning Centers to recruit excellent mentors that are not necessarily software experts. The idea is that a user could “Check out a Briefcase” for a project in a software package (e.g., Scratch). She opens the Briefcase to an instruction manual, complete with movies, pictures, and interactive pages. She then starts to move into the open-ended design of her project, with the included design brief providing scaffolding for her design. At this point the tool becomes more of a diary and note-taking environment (where notes can include pictures, voice, and movies). As she completes her design, she creates her final presentation within the Briefcase that is then shared with others on the web.

The hybrid technology being delivered to children in different cities in different socioeconomic conditions, can make a real impact in improving science education opportunities for disadvantaged youth. This work could also inform new directions for textbooks of the future. 

PI: Romero, Michael
Title: Physiology of Stress in Wild Animals
Abstract: All organisms are faced with noxious stimuli from the environment (such as predators or storms). Vertebrates activate a suite of physiological mechanisms to protect themselves from these stimuli, collectively called the stress response. A vital part of the stress response is the release of the glucocorticoid steroid hormones, but even after more than 60 years of studying stress we still have only a rudimentary idea of how glucocorticoids help wild animals survive. Earlier work indicated that several free-living bird species seasonally regulate glucocorticoid release by varying concentrations of the hormone throughout the year. This proposal builds upon these results to ask the following questions: why do animals seasonally regulate glucocorticoid levels?; is there a physiological consequence?; and does it affect their susceptibility to chronic stress?

Answering these questions will require an integrated approach of both laboratory and field studies, such as the one proposed here. We will capture wild free-living house sparrows (Passer domesticus) and test how glucocorticoids help regulate blood glucose and triglyceride levels at different times of the year. We anticipate that glucose and triglyceride regulation will depend upon seasonal changes in glucocorticoid levels. We will also bring house sparrows into the laboratory to test whether physiological responses to chronic stress vary seasonally. We anticipate that the magnitude of the negative consequences of chronic stress will depend upon the time of year. These results will help define the physiological consequences of seasonal glucocorticoid release during stress in wild animals, thus having a far-reaching impact on our basic knowledge of the ecological validity of stress responses. This research will also provide important training for students. Both graduate and undergraduate students will be exposed to, and learn, many important research skills in both the laboratory and field. These findings will be valuable to researchers in diverse fields including Conservation Biology, Endocrinology, and Population Biology, just to mention a few. 

PI: Rouzine, Igor
Title: Working Models of HIV Persistence and Evolution
Abstract: HIV infection involves many different cell types (infected cells, uninfected cells permissive for virus replication, HIV specific immune cells, and others) which interact with each other and whose numbers, in general, depend on time. Because of interaction among its parts, the system automatically arrives at an approximate steady state coincident with the asymptomatic phase of an HIV infection. The replicating virus population remains quite constant but exhibits considerable genetic variation in time (and among sampled sequences).  

Our long-term goal is to use mathematical modeling to aid in understanding how such a system can work. Our approach is to (i) select mathematical models of HIV infection based on the criterion that all their predictions agree with the known features of HIV pathogenesis in representative individuals and (ii) formulate predictions for new experiments to test the models. This strategy of "multiple match" has been successfully used in the physical sciences for dealing with complex systems of unknown structure, but it is only being implemented in HIV research. We will apply this strategy to develop models of virus-immune cell interaction in vivo and explain the mechanism of steady state, and to understand the principal factors of evolution of drug resistance and antigenic escape. At this stage of our research, we will put emphasis on follow-up tests of our models in collaborating groups and design of new tests of updated models. 

PI: Roy, Ananda
Title: Transcription Regulation of B-Cells by TFII-I
Abstract: TFII-I is a multi functional, signal dependent transcription factor that is implicated in the regulation of many genes. The purpose of this work is to determine its role in the immune system specifically in the Immunoglobulin heavy chain (IgH) locus of B-cells. It was previously shown in vitro that two TFII-I family members bound to a region of the IgH promoter named Downstream Immunological Control Element (DICE). However, these family members, TFII-I and BEN, have opposing transcriptional functions. Specific goals of the project include; determining how TFII-I and BEN transcription factors are recruited to the rearranged IgH promoter in vivo. This will be accomplished by the use of EMSA analysis with TFII-I and BEN mutant proteins, chromatin immunoprecipitation of the DICE region using TFII-I and BEN antibodies and functional assays to determine the potential role that these factors play. Because the IgH locus is also regulated at the level of subnuclear positioning, by virtue of their distinct function and subnuclear localization, TFII-I and BEN may be involved in this movement. If so, then 3D-FISH analysis of both knockdown and wild type B-cells might determine their potential role on the movement of the locus. 

PI: Ruskai, Mary Beth
Title: Quantum Information Theory
Abstract: This research project is concerned with mathematical problems that arise in the description of noisy quantum systems. Some of the work addresses the question of when entanglement can enhance the capacity of a noisy quantum channel. This includes work on longstanding conjectures and related questions about operator spaces. The project also investigates aspects of quantum error correction beyond the familiar stabilizer codes. Finally, the work addresses several mathematical questions that arise in adiabatic quantum computation and may shed light on the controversial issue of whether or not adiabatic quantum optimization can solve hard problems in polynomial time. 

Physicists have made considerable progress in demonstrating that quantum systems can be used for new methods of cryptography, communication, and computation. Further development of efficient, practical quantum communication systems requires theoretical investigations analogous to the fundamental work in classical communication theory on channel capacity and error correction. This project investigates several such mathematical issues directly connected to the practical implementation of quantum information processing.

PI: Rybak-Akimova, Elena
Title: Mechanism-based Design of Biomimetic Green Oxidation Catalysts
Abstract: The long-term goal of the proposed project is to develop efficient and selective catalytic systems for green oxidations using hydrogen peroxide or dioxygen as oxidants that do not generate environmentally harmful wastes. Dioxygen and hydrogen peroxide are ideal oxidants because they are readily available and environmentally clean, producing water as the only byproduct. These reagents allow for the most efficient, atom-economy approach to oxidative functionalization of organic molecules, thus saving energy in chemical synthesis. Non-toxic, biocompatible iron complexes will be used as catalysts acting similarly to natural iron-containing oxidative enzymes. Identification of kinetically competent intermediates (transient, very reactive species) is critical for the synthesis of useful, selective and efficient oxidation catalysts utilizing dioxygen and hydrogen peroxide as terminal oxidants. We propose detailed kinetic and mechanistic studies that will allow us to watch the intermediate formation, and their subsequent reactions with substrates by time- resolved spectrophotometry on a millisecond time scale. In order to obtain mechanistic information relevant to rapid catalytic reactions, the individual reaction steps should also be rapid. Thus, multi-mixing stopped-flow technique is applicable to the systems of interest, and low temperatures will help uncovering the details of formation and reactivities of short-lived, unstable intermediates. This program will provide much needed information about the details of individual reaction steps in oxygen activation. 

The project will focus on both previously known and new iron complexes with aminopyridine ligands that generate iron-peroxo or high-valent iron-oxo species upon reactions with O₂, H₂O₂, and other oxygen donors. Specific reactions include new aromatic hydroxylation recently discovered in the group, and olefin epoxidation catalyzed by a new family of mononuclear iron macrocyclic complexes. Both reactions, which are highly selective, utilize H₂O₂ as an oxidant, and are amenable to mechanistic studies. Proposed project is aimed at establishing the scope and mechanisms of these reactions, with an emphasis on distinguishing between monometallic and bimetallic peroxide activation. These studies will allow for rational, mechanism-based reagent and catalyst design. Additionally, a family of iron complexes with amide-containing macrocycles will be developed for dioxygen binding and activation. 

As an outcome of the proposed studies, detailed picture of the preferential reactivity of various iron-peroxo and high-valent iron-oxo intermediates will emerge. This knowledge will allow researchers to rationally select a suitable intermediate for a specific organic transformation (as exemplified by epoxidations and aromatic hydroxylations in the proposal). The ligand structural features that stabilize particular intermediates will be determined, along with the chemical ways of activating less reactive, yet possibly more selective intermediates. The understanding of reactivity profiles of various iron-based intermediates will help to interpret the mechanisms of known catalytic oxidations, as well as provide guidance in future catalyst design.

PI: Sacheck, Jennifer
Title: Impact of Physical Fitness and Overweight on Inflammation in Children
Abstract: The majority of U.S. children do not meet national recommendations for physical activity and physical fitness and that affects their health adversely. Physical fitness is inversely related to systemic low-grade inflammation and altered blood lipids in children and adults but whether this is through its effects on enhancing muscle quality and/or decreasing adipose tissue is unclear. Whether physical fitness can counteract the underlying inflammation associated with excess body weight in children remains unknown. The central hypothesis of this scientist development grant is that adequate physical fitness contributes to whole body health in children. I propose to examine this concept in children in a community setting using annual school-based fitness testing and BMI screening and a fasting blood draw to assess a child’s biological health. My working hypotheses and specific aims are: 

1. Physically fit children have healthier blood lipid profiles and less inflammation than children who are unfit, independent of weight status. I will determine whether school children who are physically fit have lower levels of blood lipids and circulating inflammatory biomarkers independent of body mass index (BMI). A blood draw in one community will be used to measure students’ blood lipid profiles and low-grade systemic inflammation as measured by serum IL-6 and C-reactive protein (CRP). Lipid and inflammatory data in approximately 300 students will be combined with the annual fitness testing and BMI screening.

2. A decline in physical fitness predicts the incidence of overweight in children. I will examine longitudinally in normal weight physically fit children, whether becoming physically unfit predicts becoming overweight. Data collected in two communities will be used to examine any changes in fitness and weight status over 3 years in over 2000 physically fit and normal weight children. 

If indeed loss of fitness predicts overweight and fitness overrides some of the negative inflammatory effects of overweight, these findings would further emphasize the importance of maintaining or enhancing physical fitness levels in school-age children to protect against future cardiovascular risk. Such findings will guide the development of evidence-based recommendations for policies around physical fitness and testing in school-age children. 

PI:  Sadler, Kate
Title: Community Case Management of Severe Acute Malnutrition in Southern Bangladesh: An Operational Effectiveness Study
Abstract: Malnutrition is a major public health problem throughout the developing world and is an underlying factor in over 50% of the ten to eleven million children under 5 years of age who die each year of preventable causes. In their State of the World’s Children Report 2007, UNICEF estimates that acute malnutrition, or wasting, affected 10% of children under 5 in developing countries between 1996 and 2005, and whilst it is Asia that has the highest numbers of wasted children that require treatment, Sub Saharan Africa is the only region where wasting continues to rise. The problem identified by this proposal is hindering progress towards millennium development goals one and four: the eradication of malnutrition and the reduction of child mortality.  In many areas where chronically high levels of acute malnutrition have been identified there is a dearth of feasible strategies for identifying the condition and delivering treatment within ongoing child survival programming. Severe acute malnutrition (SAM) is the severest form of malnutrition and is associated with very high rates of morbidity and mortality across the developing world. Despite this, ongoing national programs that aim to improve the nutritional status of young children, such as the integrated management of childhood illness (IMCI) and growth monitoring programs (GMP), do not include an effective mechanism of identifying or treating young children that suffer from SAM. The GMP does not include any indicator of acute malnutrition and IMCI includes only “visible severe wasting”, an indicator that can be subjective, difficult to use in practice, and unreliable. Where children suffering from SAM are identified they are referred to inpatient units for treatment. These units are often very long distances from people’s homes, involve long inpatient stays and high opportunity costs for patients and do not deliver good quality treatment according to that laid out by the World Health Organization (WHO). It is likely therefore that currently a large proportion of cases of SAM go undiagnosed and untreated. Although we now have a very good understanding of the nutritional and medical protocols needed to treat conditions such as SAM, we still have a very poor understanding of how to deliver this treatment to all those that need it. This lack of focus on delivery of basic child survival interventions has recently been implicated in the vast majority of deaths that occur among young children across the developing world. “We must move beyond ‘more studies and more data’, and enter the realm of effectiveness and operational research. Let us start with our goal of child survival and work backwards and learn by doing” Heikens et al Lancet 2008.” 

This study aims to develop the evidence base for effective community-based delivery of treatment for severe acute malnutrition in poor agrarian settings. 

The primary hypothesis of this study is:

1. Community case management (CCM) of SAM will achieve acceptable clinical outcomes (recovery and mortality) when compared with international standards, and better outcomes (recovery and mortality) than those reported by the ‘standard of care’ for SAM in Bangladesh. 

In addition there are three sub-hypotheses. These are:

1. The cost effectiveness of CCM of SAM is better than that of treating SAM by the ‘standard of care’ in Bangladesh.
2. Enabling community health workers to diagnose and treat SAM will increase utilization of SAM treatment services among children under 2 years compared to the provision of treatment through standard facility-based inpatient services.
3. Community health volunteers can treat children suffering from uncomplicated SAM as effectively as facility-based health personnel. 

PI: Schaffhausen, Brian
Title: Products of the Transforming Genes of Polyomavirus
Abstract: Murine polyomavirus provides an important model for neoplastic transformation. The virus causes a broad spectrum of tumors. Conclusions reached in the laboratory can be readily tested in animals. Studies on polyoma have repeatedly provided insight into basic mechanisms of cellular growth regulation. Tyrosine kinase and phosphatidylinositol 3-kinase (PI3K) signaling are two fundamental mechanisms uncovered from studies on polyoma. Transformation results from the action of three viral gene products: large T (LT), middle T (MT) and small T (ST) antigens. There are four specific aims that will provide new insight into how they work:

1. The first specific aim concerns large T. Patterns of host cellular RNA expression that result from different large T pathways will be determined. The mechanism will be established by which LT regulates genes containing CREB/ATF sites. Because acetylation has been connected to transcriptional regulation, the sites of acetylation on LT will be established and their function tested by site-directed mutagenesis. Recent results show that LT causes G2/M arrest. Experiments will be carried out to determine the mechanism by which LT causes this arrest. We have identified two new LT-binding partners, Pin1 and Bub1, which are known regulators of G2/M. Their role in LT function will be probed.

2. The second specific aim concerns small T. Important differences in function have been detected between polyoma and SV40 small T. Expression profiling will be used to identify the extent of, and the basis for, the differences between them. New polyoma small T partners will be sought by tandem affinity purification and mass spec. Protein phosphatase 2A is a critical small T target. Experiments will be performed to distinguish between different mechanisms by which small T targets PP2A.

3. Human mammary epithelial cells provide a useful model of human cancer. The third specific aim will use the model to probe the function of middle T and small T. The MT signaling pathways required for transformation will be determined. Small T mutant in binding novel partners will be tested in these cells.

4. Our fourth specific aim involves structural studies using NMR. The structure of the N-terminal domain (NT) of LT will be determined. This domain is sufficient to promote cell growth, to cause apoptosis and to regulate cellular RNA transcription. We will also initiate studies to carry out structure determination of polyoma small T.

PI: Schnitzler, Gavin
Title: Do Chromatin Changes Control Polymorphic Expression of Drug Metabolizing Genes?
Abstract: The CYP3A4 cytochrome p450 and UGT1A1 and UGT1A6 UDP-glucuronosyl transferases are some of the most important enzymes for the metabolism of drugs, exogenous carcinogens and endogenous compounds such as steroid hormones. The activities of these enzymes vary up to 40-fold between individuals, and this can have a major effect on drug efficacy or toxicity and cancer risk. Genetic polymorphisms in the promoters of these genes can significantly upregulate or downregulate their expression. In many cases, however, there is no obvious explanation for these transcriptional effects, since the polymorphisms often do not alter known transcription factor binding sites. We hypothesize that these polymorphisms may regulate transcription factor binding indirectly, by altering the positions of promoter nucleosomes. DNA sequence can affect nucleosome positions by creating low energy, default nucleosome binding sites, and our initial computational predictions indicate that CYP3A4, UGT1A1 and UGT1A6 promoter polymorphisms can significantly alter default nucleosome positions. Our preliminary results have also revealed that DNA sequence determines the locations of nucleosomes moved and/or structurally-altered by the chromatin remodeling complex human SWI/SNF, and that these positions differ from the default positions. Despite the importance of CYP3A4, UGT1A1 and UGT1A6, there is currently no detailed information about the chromatin structures of their wild type or variant promoters. In the work proposed here, we will examine the default and hSWI/SNF-remodeled chromatin structures of these promoters in both a defined in vitro system and in vivo, in a human liver cell line (using RNAi to control hSWI/SNF levels). We will then test the hypothesis that regulatory polymorphisms alter this chromatin structure by comparing the results of wild-type and variant promoters in vitro and in vivo. This work is ideal for the PA-06-149 R21 grant mechanism because it will explore a novel hypothesis which may provide essential insights into the origins of hepatocellular variability in drug metabolizing gene expression, and because it will develop novel techniques for studying the effects of chromatin structure on transcription. 

PI: Schwartz, Judah
Title: The Fulcrum Institute for Education in Science
Abstract: Tufts University is working in close partnership with TERC, Malden Public Schools (of Malden, Massachusetts), and Boston’s Mission Hill School to develop leaders of Education in Science for the coming generation of K-8 students. The proposed five-year grant envisions creation of the Fulcrum Institute for Education in Science. The Institute is a two-year graduate program for experienced K-8 teachers that is designed to produce a pivotal group of Educators in Science uniquely qualified to implement and lead research-centered science learning and teaching in their schools and districts. Participants advance their professional knowledge and status through the Institute’s credit-bearing, three-course sequence. The Institute will comprise advanced graduate work focusing on Physics for accomplished educators: online courses, summer workshops, and intensive work in each participant’s respective school. Participants will share data and information from their ongoing work with students and other educators. In this way, the institute will support rather than interfere with teachers’ work in schools and help them place their work in broader perspective. 

PI: Schwob, James
Title: Gene Expression Profiling of Adult Olfactory Stem and Progenitor Cells
Abstract: The capacity of the olfactory epithelium (OE) to recover both anatomically and functionally after injury to near-normal, a capacity that extends throughout life, is unique in the nervous system. However, we know next to nothing about the molecular phenotype of the olfactory stems nor their downstream progenitors, an ignorance that severely hampers our ability to isolate them, understand their regulation, or use them in a controllable manner. We are proposing exploratory and developmental studies designed to address that vast gap in our knowledge. We will isolate three specific types of globose basal cells (GBCs), among whose functionally heterogeneous population, a broadly pluripotent, stem or stem-like cell is found: multipotent GBCs, sustentacular cell-forming GBCs, and transit-amplifying GBCs. The different kinds of GBCs will be defined by the timing of their re-emergence after methyl bromide lesion of the mouse OE and by their expression of one among a group of basic Helix-Loop-Helix (bHLH) transcription factors that will include Hes1 and Mash1. The bHLH TF-expressing cells will be isolated by FACS on the basis of markers on their surface that define them as GBCs and the expression of GFP in parallel with each individual TF (from either a transgen or the endogenous gene locus). For each TF-defined population we will carry out AffyMetrix microarray analysis designed to profile its pattern of gene expression. In addition, mitotically quiescent and BrdU label-retaining GBCs in the normal OE are a potential stem cell population. We will isolate them by FACS as well and subject them to gene profiling. As a consequence of these studies, we will have a comprehensive understanding of resting and activated olfactory stem cells and their downstream progenitors and will be poised to begin hypothesis-driven assessments of functional regulatory pathways. 

PI: Schwob, James
Title: Medical Scientist Training Program at Tufts University
Abstract: The MD/PhD program at Tufts University School of Medicine and the Sackler School of Graduate Biomedical Sciences is designed to train the physician scientists of the future, and provide an outstanding cadre of investigators well-versed in both the practice of medicine and the conduct of basic biomedical research. Our goal is to bridge the communication and cultural divide that too often separates physicians and scientists and educate individuals who will be firmly rooted in both cultures and achieve highly productive careers that meld these two arenas. Medical training interfaces with eight basic science graduate programs in Biochemistry, Cellular & Molecular Physiology, Cell, Molecular & Developmental Biology, Genetics, Immunology, Molecular Microbiology, Neuroscience, and Pharmacology & Experimental Therapeutics Programs that have a combined faculty of 135. The Tufts MSTP has been funded since 1991, but has undergone a major transformation in the past two years with the appointment of a new director and a new leadership team. A completely redesigned administrative structure, new admissions and advising processes, and major changes in the curriculum have been implemented. Our new curriculum effectively integrates research and clinical training. Research rotations prior to beginning medical school and research selective during the first two years of medical school allow students to make informed decisions about research mentors in a timely fashion. A new Clinical Connections program that provides an array of patient experiences during the years focused on research training has been put in place. A new course "Clinical Implications of Basic Research" that brings together MD/PhD trainees in all phases of their training to discuss the ways bench research is being translated in new clinical practices has also been added. A workshop that provides orientation and hands-on experiences for students transitioning from research to clinical education will be given for the first time in the spring of 2006. A Retreat, seminars, and a career-oriented dinner program are also built into the training program. Our program has graduated 40 trainees since its inception and 32 are currently enrolled. Based on the changes that have occurred, we are requesting funding for 16 positions in this application. 

PI: Selsing, Erik
Title: Immunity in Transgenic Mice
Abstract: Class switch DNA recombinations are important for isotype switching in B cells and also appear to be involved in chromosomal translocations of some oncogenes. However, little is known about the switching mechanism. Tandemly-repeated sequences located upstream of all H-chain antibody constant region genes have generally been thought to be important in the targeting and/or the mechanism of class switch recombination. We have used gene targeting to delete the tandem repeats from the switch (S) region associated with the Cµ constant region in mice. Surprisingly, these mutant mice are still able to undergo isotype switching, although the efficiency of the process is reduced. We also find that switching to different isotypes is differentially affected by deletion of the Sµ tandem repeats. The current proposal seeks to extend our preliminary results to analyze several aspects of class switch DNA recombination. First, the mice lacking Sµ will be analyzed to determine whether lgA-switching is essentially intact as indicated by preliminary data and to assess the effect of Sµ deletion of IgE switching. Potential molecular explanations for the different effects of Sµ on switching to the different isotypes will be investigated by characterizing switch recombination junction sites for isotypes that show the greatest and least effect due to Sit deletion. Second, double- stranded DNA breaks within the JH-Cµ intron will be analyzed in wild-type and Sµ mutant mice to assess whether DNA cleavage sites might be affected by Sµ deletion. Third, gene targeting will be used to delete additional sequences within the JH-Cµ intron to localize those sequences that are required for isotype switching. And fourth, specific DNA sequences will be introduced by gene targeting into an lgH allele that is defective for switching to investigate the minimal sequences that can restore the capacity for switching. 

PI: Slonim, Donna
Title: Evaluating Biological Networks by Mining Public Data
Abstract: A molecular understanding of biological systems is crucial for the development of innovative medical advances from novel genomic technologies. The systems annotation currently available in public pathway databases is still too limited for many of its envisioned applications in medical research. Recently, our ability to identify novel genes has outstripped our capacity to analyze these genes’ interactions experimentally. Thus, algorithms are being developed to infer biological networks by computational analysis of genomic data. A major factor limiting the integration of computationally-derived networks in pathway databases is the lack of common quantitative methods for evaluating and comparing these networks. There is currently no established standard for comparison that is objective, statistically grounded, and scalable. A network evaluation method with these properties would spur measurable improvement in our systems biology resources and a concomitant improvement in our ability to translate genomic data into practical medical knowledge. The objective of this application is to develop software and methods that use information in online databases to evaluate biological networks.

In Aim 1 we will develop, assess, and select statistical evaluation methods that incorporate probabilistic network models and reflect our confidence in the quality of the validation data. Our statistical approach will report the probability of seeing such confirmation data by chance in a random network. We will validate our methods in part using networks our collaborators have derived from unpublished data, avoiding bias since the source data cannot yet be represented in the online data sources we are mining.

In Aim 2 we will implement network evaluation software that incorporates text-based and experimental validation data mined from links in the Entrez Gene database. Our software will report the statistical significance of the input networks and will provide direct links from each network edge to the relevant supporting data used in the evaluation.

With the completion of the proposed work, we expect to offer the community an open-source computational resource for network evaluation that is quantitative, scalable, informative, and grounded in peer-reviewed published experimental findings. 

PI: Sonenshein, Abraham
Title: Molecular Genetics of Basic Cell Functions
Abstract: Continued support is requested for five predoctoral trainees per year for five years for an interdepartmental training program in genetics of basic cell functions. The emphasis is on rigorous training using the power of genetic analysis to study basic cell processes, such as chromosome replication and segregation, regulation of gene expression, cellular differentiation, and host-parasite interactions. This training program has strongly benefited from continuous support from NIGMS since 1975. Supported students are almost exclusively in their first year, allowing them to have a broad exposure to the role of genetics as a science in itself and as a tool for solving important biological problems. The training faculty, from the Departments of Molecular Biology and Microbiology, Biochemistry, Pathology, and Medicine, is highly interactive and dedicated to close, joint supervision and mentoring of graduate students. Past trainees include leading researchers in academia and industry. The training program is administered by the Graduate Program in Molecular Microbiology. Starting with a pool of 80-145 applicants, the graduate program annually admits 6-8 new students, most or all of whom are eligible for training grant support. The program has been successful in attracting a significant number of students from under represented minority groups (including 8 of the 41 current students), nearly all of whom have been supported by this training grant. Nearly all graduates of the program have obtained high-quality postdoctoral appointments and are still active as researchers, as teachers, or in allied fields. Entering students take required courses in Genetic Analysis and Biochemistry and pursue 9-week rotation projects in four different laboratories. At the end of the first academic year, they choose a thesis supervisor and begin thesis research. In the second and third years, the students complete their coursework and prepare a research proposal (unrelated to the thesis topic) as a qualifying examination. All students are required to complete a seminar course in scientific ethics. 

PI: Sonenshein, Abraham
Title: Regulation and Functional Analysis of Cellulose Utilization Elements in Clostridium Thermocellum
Abstract: The overall aim of this research is to study the regulation of cellulose utilization elements in Clostridium thermocellum. C. thermocellum is an anaerobic, Gram-positive, thermophilic bacterium that is capable of efficiently converting crystalline cellulose to ethanol. The proficient utilization of lignocellulose matter, the most abundant polysaccharide in nature, is of great economic potential, since it may allow the utilization of the plant cell wall material as a renewable source of energy and carbon. Although the cellulolytic system of C. thermocellum is well characterized structurally, very little is known about the regulation of the different components and their role in overall cell metabolism. Recently, a first draft of the C. Ihermocellum genome was published, allowing for the first time the identification of all of the genes of the bacterium and the construction of a whole genome DNA chip. 

In the framework of this research we will analyze the expression of cellulose utilization-related components and characterize their roles. Batch and continuous culture techniques will be used to alter physiological conditions such as growth rate, limiting growth factors and cell densities. Regulatory mutants will be prepared and characterized in order to identify their roles and mechanisms of action, as well as the mechanisms by which carbon sources and growth rate affect their expression. In addition, a whole genome DNA microarray of C. thermocellum will be used to follow expression patterns of cellulose utilization related genes. 

The successful implementation of this project is expected to reveal new regulatory mechanisms in C. thermocellum that may pave the way for metabolic engineering of this important bacterium for the development of an economical fermentation converting cellulose into ethanol. 

PI: Sonenshein, Abraham
Title: Effector Binding by Bacillus Subtilis CodY
Abstract: Bacillus subtilis employs several adaptive strategies when faced with nutrient limitation. Expression of many of these adaptive mechanisms is under regulation by CodY, a highly conserved protein in Gram-positive bacteria. CodY senses nutrient availability by interaction with its effectors, GTP and the branched-chain amino acids (BCAAs). Upon effector binding, CodY is able to bind to and repress its DNA targets. It is uncertain which amino acids in CodY are necessary for interaction with these effectors, and it is unclear how effector binding alters CodY such that it interacts with DNA. The goal of this project is to address both of these underlying questions. As CodY has been shown to regulate expression of virulence factors in several bacterial pathogens, it is an attractive target for novel antimicrobial therapy. However, development of this line of therapy will require examination of these basic issues concerning CodY activity.

The first aim of this application will identify amino acids essential for effector binding. Homology searches and x-ray crystallography studies have yielded some clues about which CodY residues may be important for GTP and BCAA binding, respectively. These residues will be mutagenized, and their impact on effector binding will be determined.

The second aim of this application will investigate how effector binding increases CodY's affinity for DNA. Partial proteolysis has indicated that a conformational change occurs in CodY upon binding to BCAAs, but no change was observed upon CodY binding to GTP. Near-UV circular dichroism, spectrofluorometry, and Fourier transform infrared spectroscopy will be used to identify whether GTP induces a subtle alteration in the tertiary structure of CodY.

Experiments in the third aim will assess the physiological impact of the loss of CodY effector binding on gene expression. These CodY mutations will be introduced into the B. subtilis chromosome, and their effects will be assayed by reporter fusion experiments and microarray analysis. The results of this work will allow us to understand how bacteria regulate gene expression in response to nutrient starvation. Disruption of this regulation may result in a novel means of treatment of certain bacterial infections. 

PI: Sonenshein, Abraham
Title: Physiological Consequences of CodY: A Master Regulator in Gram-Positive Bacteria
Abstract: Bacteria in the wild seldom find themselves in conditions that promote efficient and robust growth. Short periods of nutrient surplus give rise to even longer periods of nutrient limitation. When deprived of nutrients or otherwise favorable conditions, many bacteria attempt to adapt by deploying strategies to acquire alternative carbon and energy sources either by activating secondary catabolic pathways and transporters or by unleashing cytotoxic compounds, including antimicrobials, to ward off competition and scavenge for resources. Many of these adaptive responses are coincidently harmful to humans. The CodY protein is the master nutrient sensor and transcriptional regulator responsible for controlling the expression of this adaptive program in low G+C gram-positive bacteria, including a number of recalcitrant human pathogens. This application aims to determine how CodY processes multiple input signals for controlling transcription on a global level to alter important aspects of central metabolism. In addition, research proposed in this application will define the physiological importance of altering CodY activity under different growth conditions, and will identify additional coeffectors of CodY. The broad goal is to elucidate the molecular mechanism by which CodY functions to activate and repress transcription at numerous loci. The projects described herein take a multidisciplinary approach using genetics, biochemistry, molecular biology and structural biology to address biological questions from a variety of directions. Reporter fusions, quantitative RT-PCR and microarrays will provide local and global expression data in vivo. In vitro techniques including gel mobility shift assays, DNase I footprinting, partial proteolysis, and in vitro transcription will confirm and complement in vivo data. Collaborations with experts in X-ray crystallography (Professor Anthony Wilkinson - University of York) and bacterial physiology (Uwe Sauer - ETH, Zurich) have been established to increase the breadth of research and discovery. Dissecting this complex regulatory network and studying the physiological consequences of disrupting genetic and metabolic circuitry underlying CodY activity will reveal how bacteria decide to activate developmental and adaptive programs. Recent work has documented genes encoding virulence determinants as direct targets of CodY-dependent repression. As such, methods and bioactive compounds that can increase the capacity of CodY to maintain repression of virulence determinants can lead to new ways to control and prevent illnesses of microbial origin. 

PI: Sonenshein, Abraham
Title: Regulation of Glutamate Synthesis in Bacillus Subtilis
Abstract: Management of key metabolic intersections is a critical issue in bacterial economy. One such intersection, the interconversion of a-ketoglutarate and glutamate, is particularly important and interesting because it represents the point of coupling between central carbon metabolism and central nitrogen metabolism. Synthesis of the enzymes of the tricarboxylic acid branch of the Krebs cycle (citrate synthase, aconitase and isocitrate dehydrogenase) that produce a-ketoglutarate and the enzymes of nitrogen assimilation (glutamine synthetase, glutamate synthase and glutamate dehydrogenase) are encoded by genes that are subject to multiple forms of regulation. This project seeks to understand in detail the molecular mechanisms that regulate these genes in Bacillus subtilis, a well studied model Gram-positive bacterium, and Listeria monocytogenes, a model intracellular pathogen whose metabolic regulation is relatively unexplored. The proposal focuses on three transcriptional regulators (CcpC, GltC and GlnR) and two enzymes (glutamate dehydrogenase and aconitase) that have post-transcriptional regulatory functions.

The aims of this proposal are:

1. to determine the molecular mechanisms that control the synthesis and activity of the tricarboxylic acid branch enzymes;
2. to reveal the molecular mechanisms that control the synthesis of glutamate and glutamine; and
3. to determine the role of aconitase as a post-transcriptional regulator of sporulation in B. subtilis and of iron metabolism and virulence genes in L. monocytogenes.

These issues will be addressed using a combination of genetic and biochemical techniques. The phenotypes of mutants defective in each of the regulatory factors will be determined and the proteins will be purified and tested using in vitro transcription and DNA and RNA binding experiments. Since L. monocytogenes mutants that lack two repressors of the Krebs cycle genes have a significant defect in virulence in a mouse model, the molecular basis for this defect will be explored.  

PI: Soto, Ana
Title: Mechanism of Developmental Toxicity of Bisphenol-A
Abstract: Increased malformations of the genital tract and hormone-related cancers are significant problems in the industrialized world. Suspicions have focused on environmental estrogens as one causal agent. Among them, bisphenol A (BPA) is of particular interest due to widespread human exposure. Perinatal exposure of rodents to low, environmentally-relevant doses of BPA induces pleiotrophic effects in estrogen target tissues that manifest long after exposure has ended. In particular, altered sexual differentiation of a nucleus important for estrous cyclicity, and altered gonadotropin-releasing hormone (GnRH) neuronal activation may have repercussions on fertility and fecundity, while altered morphogenesis of the mammary gland may impair lactation. We expect that effects observed in estrogen target tissues of BPA exposed females will impair their ability to produce viable' and healthy offspring. The proposed studies will establish a causal mechanistic chain for BPA action encompassing cellular, tissue and organismal levels of organization; hence, they will be both integrative and analytical. On the integrative side, we will evaluate the reproductive success of perinatally exposed female mice. This information is essential to elucidate the physiological consequences of the molecular events described in the previous funding cycle. On the analytical side, we propose a dual approach to study how BPA alters the tissue organization of two important target tissues, the developing hypothalamus (HYP) and the mammary gland (MG). The HYP is critical to overall reproductive success, and the MG is critical to the survival of the neonates. In addition, the HYP can influence MG development by modulating pituitary gonadotropins and ovarian hormone synthesis and prolactin release.  

Aim 1: How does BPA affect the reproductive outcome of perinatally exposed females? Fertility, fecundity, and MG function will be assessed in order to define the reproductive impact of developmental low dose BPA exposure.  

Aim 2: How does BPA exposure alter tissue organization in the developing HYP? We hypothesize that BPA alters the architecture and connectivity of nuclei important for the regulation of gonadotropin release. We will examine these nuclei for: i) changes in patterns of cell survival, apoptosis, and connectivity; ii) expression of steroid receptors, enzymes of testosterone metabolism, and factors downstream of estrogen action such as glutamic acid decarboxylase and astrocyte differentiation. Completion of these studies will identify mechanisms underlying altered GnRH neuronal activation.  

Aim 3: How does BPA exposure affect gene expression and tissue organization in the MG? We hypothesize that: i) BPA acts as a morphogen directly on the MG anlagen (to be tested by QRT-PCR in MG organ culture); ii) BPA effects are mediated by ER alpha and/or beta (to be tested by QRT-PCR using null ER mice), and iii) these initial events translate into altered stroma-epithelium interactions. 

To dissect the effects resulting from BPA exposure of the MG anlagen from systemic effects due to the action of BPA on the endocrine system, the MG of BPA exposed and unexposed animals will be transplanted into exposed and unexposed hosts. To assess whether the stroma, the epithelium or both compartments are permanently altered by BPA exposure, tissue recombination studies will be performed. This Aim will begin to reveal the mechanisms by which BPA disturbs the organization and architecture of an estrogen target organ.  

The realization of this project will provide mechanistic information linking BPA action in target tissues and its organismal consequences. It will also reveal whether current levels of environmental exposure produce significant health effects in a surrogate model. This information is critically needed to develop public policy on endocrine disruption. 

PI: Sternberg, Robert
Title: A Teacher's Colleguim to Design, Implement, Evaluate, and Revised Teaching Innovations to Improve Student Learning
Abstract: The goal of the proposed initiative is to form a Teachers’ Collegium to Design, Implement, Evaluate, and Revise Teaching Innovations to Improve Student Learning. In other words, we want to invite a group of teachers to come together to:

1. learn about empirically validated best teaching practices, grounded in research on how we learn;
2. design course-based curriculum interventions building on this research;
3. implement these new teaching strategies in a course and collect data on student learning; and
4. interpret student data to revise teaching strategies as necessary.

This Collegium will be hosted and guided through the process by Tufts’ existing Center for the Enhancement of Learning and Teaching (CELT). 

PI: Swan, Chris
Title: The Role of Service-Learning: Improving Engineering Education: Attracting Women into Engineering
Abstract: Instructional strategies that use holistic, real-world applications of science and technology tend to be more effective for attracting and retaining the interest of women and underrepresented minorities. This proposal focuses on service-learning, a pedagogy which combines active citizenship with student learning. A major component of service learning is that it serves to enhance the academic curriculum as an integrated feature. At the same time, the service projects meet genuine needs of the communities in which and with whom they take place. This project will examine the outcome that participation in service-learning in schools of engineering better attracts and retains women engineers. Along with traditional assessment of success such as course grades or graduation rates, this research will measure participants' perceptions of self-efficacy, their perceptions of the nature of engineering, and their understanding of fundamental engineering concepts. The overall goals of this proposal are to measure the effectiveness of service-learning programs as pedagogical methods for teaching engineering and to examine how service-learning programs attract a more diverse set of engineering students than is currently represented in the population of engineering students. The study of three well-established service learning programs at Tufts and Purdue will use established methods to test self-efficacy and understanding of the nature of engineering and will also develop new instruments to evaluate student understanding of the concepts of engineering design.

The results of this research could have significant impact on the number and diversity of engineering graduates and on their ability to effectively address real-world problems. If service learning is shown to provide a more holistic understanding of engineering and its role in society for all students, then implementation of service learning programs will enhance the general workforce by recruiting and graduating more diverse and more competent students to meet the needs of the future. 

PI: Sykes, Charles
Title: Collaborative Research: The Structure and Chemistry of Naturally Chiral Metal Surfaces
Abstract: This collaborative research project combines experimental studies carried out in the laboratory of Charles Sykes at Tufts University, experimental work from the laboratory of Andrew Gellman at Carnegie Mellon, and theoretical studies by David Scholl and coworkers, also at Carnegie Mellon. With the support of the Analytical and Surface Chemistry Program, this collaborative team is investigating the structure and reactivity of naturally chiral metal surfaces. Copper single crystals cut to expose chiral kink sites are the substrates being examined, and the adsorption and reaction of chiral and achiral small molecules on these surfaces are studied. A combination of scanning tunneling microscopy (STM), vibrational STM, photoemission of adsorbed Xe, thermal desorption spectroscopy, and density functional calculations are the tools used to examine these reactions. A fundamental understanding of structure and reactivity of chiral surfaces helps to develop enantioselective catalytic and separation processes, of considerable value in the pharmaceutical industry. 

PI: Sykes, Charles
Title: Single Molecule Studies of Ferroelectric Self-Assembly
Abstract: In recent years the scanning probe field has advanced to the point where one can manipulate individual atoms on a surface, take vibrational spectra of single bonds and even induce a chemical reaction between just two molecules. This level of precision has enabled the investigation of a vast variety of surface phenomena with unprecedented resolution. This proposal is aimed at investigating the ferroelectric assembly, ordering and switching of individual molecules using these novel experimental tools. 

Ferroelectric materials have an interesting set of properties such as controllable polarization, piezoelectricity, and nonlinear optical and dielectric activity. As such, they have been utilized for many applications such as nonvolatile random access memories, sensors, capacitors, microactuators, and optical components. In recent years, device miniaturization has led to an interest in the development of smaller ferroelectric materials with even faster switching rates. This has raised questions about the relevant size effects that lead to deviations from bulk properties and the ultimate cessation of ferroelectric properties. Despite this, functional ferroelectric films with thicknesses in the nm range have been successfully synthesized. Perovskite films as thin as 1.2 nm have been produced and shown to be ferroelectric. Also, copolymer films have displayed ferroelectricity in 1 nm thick layers. There is, however, a current lack of understanding of the finite domain size required for ordering to occur, the mechanism of ordering and switching, and the effect of the supporting surface on the ordering. Understanding the fundamental nanoscale mechanisms of ferroelectric ordering and transitions is crucial for the continued development of smaller and faster ferroelectric devices. 

The impacts of this research will be broad. Ferroelectrics is an important field that shows great promise for the production of very high density non-volatile computer memory, FeRAM (Ferroelectric Random Access Memory). Information is stored by using electrodes to polarize small volumes of ferroelectric material. These devices are not as fast as current DRAM devices, but they still find application, for example, in the Sony Playstation 2. However, in order for further miniaturization of ferroelectric devices, several fundamental questions must be answered, such as:

1. what is the finite domain size required for ordering to occur? and
2. what is the mechanism of ordering and switching?

By studying simple systems in which variables such as molecular dipole and surface coverage can be altered systematically, our work will shed light on these questions. 

PI: Sykes, Charles
Title: A Single-Molecule Approach for Understanding and Controlling Ferroelectrics
Abstract: I am in a somewhat unique position as the visual appeal and accessibility of my own field of research, scanning tunneling microscopy (STM), allows many difficult concepts in chemical kinetics, quantum mechanics and electronics to be broached and explained in simple terms. As such I have been able to attract minorities like women to my research group who did not originally intend on studying physical chemistry upon entering graduate school. I will continue to make the most of this advantage by using scanning probe data and instruments to teach aspects of physics, chemistry, nanoscience and nanotechnology to a variety of people including the general public browsing the web, high school students thinking about what to pursue at college, and undergraduates performing laboratory experiments. 

I have already begun addressing some of the problems outlined in the introduction and I will describe plans to further this work and impact several different communities:

1. At the undergraduate level we have begun to revamp the physical chemistry lab, removing outdated experiments and replacing them with modem scanning probe and nanoscience related experiments.

2. Female graduate students in the physical sciences are somewhat of a rarity and three are currently supported in my research group. These students will participate in outreach activities that will help attract others to the area. These students are also actively involved in the design and implementation of new teaching experiments based on their own research.

3. At a broader level we have fostered a collaboration with the Center for Engineering Education Outreach (CEEO) at Tufts University that will allow us to travel to local high schools. This will enable me and my graduate students to travel to high schools and give presentations. We will introduce important concepts of physical chemistry, nanoscience and even give live scanning tunneling microscope demonstrations in which students will see atoms for the first time.

4. At the broadest level we are presently working on a website for the general public interested in nanoscience and specifically in scanning probe microscopy. This site is being written and developed by undergraduates and will explain the history behind nanoscience and the term itself.

PI: Teichman, Sherman
Title: Civil Military Relations for a Better World
Abstract: Recent history has shown the need for civil-military cooperation between service agencies and between military and civilian agencies (e.g., between USAID and the Navy; FEMA and the Army). This civil-military gap has led to the growing conviction in the government that security and development are not only linked, but mutually reinforcing. 

For example:

• In 2005, the Department of Defense issued Directive 3000.05 “Military Support to Security, Stability, Transition, and Reconstruction Operations,” which instructs DoD to accord stability operations priority comparable to major combat operations. It supports National Security Presidential Directive-44, which instructs the State Department to develop civilian capabilities and integrate with military capabilities to plan, prepare for, and conduct stabilization and reconstruction missions.

• The new US-Africa Command (AFRICOM) is being established with a unique structure that emphasizes the importance of both civil and military officials to regional issues.

• US military experience in Iraq has highlighted the important role of the “strategic corporal,” in which the actions of enlisted, lower-level soldiers have strategic consequences, both negative and positive. 

These important developments demonstrate the imperative for greater civil-military cooperation, but they do not address a more fundamental issue: the need to cultivate and institutionally establish a sustained commitment to comprehensive civil-military cooperation that embodies a “360-degree” vision necessary to meeting military, stability, and development missions of the twenty-first century. The lack of this vision begins from the earliest stages of service training and academic education. Indeed, on October 30, 2007, Celeste Ward, Deputy Assistant Secretary of Defense for Stability Operations Capabilities, testified to Congress that “strategic success in [civil-military] operations ... will only be possible with a complete architecture for unified civil-military planning, deployment, and action—from the earliest time possible—and dedication of the resources necessary to create and expand the expeditionary capabilities of civilian agencies.” Ms. Ward declared: “We need a strong civilian partner to deal with conflict and instability not only alongside the military but before they become military requirements.” 

The Institute considers the disconnect between the civilian and military sectors to be detrimental to US foreign policy decision making, policy execution, and support for and critique of military initiatives. As the US moves into an increasingly complex world, a comprehensive and sustained understanding between these sectors will continue to be crucial, so that US diplomacy, development, and defense can reinforce rather than inhibit one another. Unless students, cadets, and midshipmen are introduced to each others’ respective perspectives, structures, protocols, and professional jargon during their formative stages of education, conversations between civilian and military personnel will be increasingly disjointed and incoherent. 

In spring 2006, Tufts University’s Institute for Global Leadership (IGL) founded the Alliance Linking Leaders in Education and the Services (ALLIES) to expand and integrate the ongoing relationship between IGL and the military’s educational institutions. The aim is to foster dialogue, encourage joint research opportunities, create activities between students of private universities and future military officers and servicemen, and educate students about the role of the US military at home and abroad. By developing a new relationship between emerging leaders in the civilian and military sectors, ALLIES is helping to build partnerships that promote cooperation that is not only capable of addressing dilemmas central to our common future, but that lasts throughout careers and lifetimes. To date, ALLIES has sponsored the participation of students, cadets, and midshipmen in lectures, roundtable discussions, simulations and conferences at Tufts and the US Military Academy, Naval Academy, and Air Force Academy, on a broad range of issues, including Guantanamo and the tensions between civil liberties and security. ALLIES has also begun working with the United States Institute of Peace and the US. 

The Joint Research Project (JRP) is a major component of ALLIES each year. It brings together future US civilian and military leaders during the month of June in an international research project that (1) enables students, cadets, and midshipmen to collaborate; (2) teaches them about other perspectives through direct contact with international officials; and (3) prepares them to convey their findings at ALLIES exchanges in the subsequent academic year. 

In June 2008, six Tufts undergraduate students, one graduate student from Tufts’ Fletcher School of Law and Diplomacy, two cadets, and two midshipmen will spend approximately three weeks in Jordan, building on the work of the first JRP to Jordan in June 2007. This Second Annual JRP represents a crucial transition. First, it will leverage and expand an already established network of Jordanian contacts from 2007. Second, with Compton Foundation support, it will (1) enhance last year’s post-research dissemination so that the JRP delegation’s findings are shared more widely and have greater resonance in the growing inter-institution ALLIES community, and (2) set a logistical and institutional framework for the 2009 JRP trip, projected to take place in Asia. 

Jordan is important because:

1. it plays an important role in the Israeli-Palestinian peace process;
2. its government is a traditional US ally, often despite popular opinion;
3. its internal political and social environment is marked by a complex political reform process, a constant struggle over national identity, and a rising Islamist movement within its traditionally secularist society;
4. 70% of the Jordanian population is under the age of 30, and the opinions of the youth will have a major impact upon the way these issues are resolved in the future;
5. students can gain insights into Arab attitudes toward, and the consequences of, the US invasion of Iraq. 

The ALLIES delegation will explore Jordanian politics and society from multiple perspectives, meeting with government officials, NGOs, academics, journalists, UN agencies, and think-tanks via formal and informal interviews, organizational site visits, and dialogue sessions. The delegation will expand on the 2007 JRL by making new contacts, in particular with Jordanian university students, and by exploring collaboration on a civil-military dialogue concept with the United Nations University’s International Leadership Institute in Amman. 

PI: Theoharides, Theoharis
Title: Stress Induced Skin Mast Cell Activation and Vasodilation
Abstract: There is a fundamental gap in our understanding of why skin diseases, such as chronic urticaria and psoriasis, are aggravated by stress. The long-term goal of this research is to define the role of mast cells in inflammatory diseases. The objective of this application is to identify the contribution of the corticotropin-releasing hormone (CRH) receptors and neurotensin (NT) receptor-1 in stress-induced skin mast cell activation and increased vascular permeability, using both a mouse model and human skin biopsies. Restraint stress increases skin mast cell degranulation, CRH content and vascular permeability; these effects are unaffected in SP-/- and CRH-/- mice, but absent in W/W^V mast cell deficient mice. CRH increases vascular permeability when injected intradermally in mice, but this effect is blocked by the NTR-1 antagonist SR48692 and is absent in NT-/- mice, demonstrating the critical role of NT. Stress may elicit release of CRH and NT from dorsal root ganglia (DRG) skin terminals and activate mast cells expressing functional CRHR and NTR-1. The clinical relevance of these findings is evidenced by increased expression of CRHR-1 and histidine decarboxylase (HDC) in affected skin from patients with chronic urticaria, indicating the involvement of CRH and mast cells.

The central hypothesis is that CRH released in the skin by acute stress, alone or together with NT, activates mast cells leading to increased vascular permeability and neurogenic inflammation. Guided by strong preliminary data, this hypothesis will be tested by pursuing four specific aims: 

• Determine the importance of CRHR and NTR-1 on stress and intradermal CRH-induced skin mast cell activation and vascular permeability using CRHR-1-/-, CRHR-2-/-, double CRHR-/-, or NTR-1-/- mice.  

• Determine if CRHR or NTR-1 need to be expressed on skin mast cells by reconstituting W/W^V mast cell deficient mice with bone marrow progenitors from the appropriate CRHR-/- or NTR-1-/- mice.  

• Investigate the expression of CRHR and NTR-1 in human skin biopsies from atopic dermatitis, chronic urticaria and psoriasis patients, and correlate findings with serum CRH levels and extent of stress by using the State-Trait Anxiety Inventory (STAI).  

• Investigate the effect of CRH and NT on release from human skin biopsy explants and human cultured mast cells of proinflammatory cytokines.  

Our approach is innovative because it utilizes knockout mice and reconstitution techniques to understand how stress-derived neuropeptides contribute to skin inflammation, and employs novel methods of assaying mediator release. The proposed research is significant because it is expected to advance understanding of how acute stress increases skin vascular permeability and neurogenic inflammation. This is an important and under investigated area of skin pathophysiology that has potential applicability to understanding the pathogenesis of skin diseases and screening compounds that may develop into novel and effective treatments.

PI: Tucker, Katherine
Title: Delta Obesity Prevention Research Unit: Tufts University (ARS HEALTH Project)
Abstract: An objective of the Delta Obesity Prevention Research Unit (Delta OPRU) being conducted in part under this cooperative agreement is to identify barriers and facilitators to adherence to the Dietary Guidelines for Americans (DG) and examine how differential profiles of adherence relate to obesity in children and adults of the Delta region. The specific objective of this cooperative agreement is to establish and support a cooperative partnership between the United States Department of Agriculture (USDA), Agricultural Research Service (ARS) and Tufts University (Tufts) which will permit scientific work amongst ARS/Tufts Nutrition Scientists to construct conceptually and culturally appropriate survey tools to assess DC adherence barriers and facilitators for African-American and Caucasian children and their caregivers of the Lower Mississippi Delta (LMD) region and elsewhere in the U.S. 

PI: Tzipori, Saul
Title: Development and Evaluation of an Innovative System for the Concentration and Quantitative Detection of CCL Pathogens in Drinking Water
Abstract: In a previous EPASTAR award we had developed optimized and validated at Tufts University a novel method, the continuous flow centrifugation or CFC, for the concentration of three protozoa (Cryptosporidium spp. Giardia, Microsporidia) from large volumes of water (l000L). In this application we propose methods which will make it possible to expand this approach to simultaneously concentrate bacteria, algae and viruses (Objective 1) as well. To do this, we will use representative pathogens which include E. colt for bacteria (Objective 1.1), Microcystis aeruginosa for algae (Objective 1.2) and MS2 bacteriophages for viruses (Objective 1.3). We will then integrate the concentration of protozoa, bacteria, algae and viruses from water into a single concentration procedure (Objective 2). The PCFC will then be fine tuned for its ability to simultaneously concentrate representatives from each group of the CCL list (i.e., Coxsackievirus A9, Microcystis aeruginosa, Aeromonas hydrophila, and Enchephalytozoon intestinalis), (Objective 2.1). Objective 3 will focus on detection and quantitative identification of CCL pathogens in water, using multiplex miniaturized fiber optic bead microarrays coupled with a compact confocal-type imaging system and comparing it with EPA approved methods (Objective 3.1). This novel technology, also developed at Tufts University originally for the detection of biodefense related pathogens, requires no nucleic acid amplification step. We propose to apply this powerful technology to detect waterbome pathogens. It is expected that the entire procedure (concentration + detection) will be performed in less than 4 hours. The inclusion of several virulence factors for each pathogen will confirm the integrity and pathogenicity of the organism(s) present in water samples. Once optimized and validated with spiked samples, the concentration/detection procedure will be evaluated with environmental samples, and operational protocols will be formulated. 

PI: Tzipori, Saul
Title: Innate Immunity and Dendritic Cells in Cryptosporidiosis
Abstract: Two species, C. hominis and C. parvum, are linked with human cryptosporidiosis, a serious cause of morbidity and mortality worldwide, and against which there is no effective therapy of prevention. Our goal is to elucidate the mechanisms by which immune cells initiate resistance against cryptosporidiosis with a view that a better understanding of the various components of the immune response will lead to development of effective vaccines and adjuvants to combat the infection in humans. Our central hypothesis is that dendritic cells recognize Cryptosporidium through Toll-like receptor(s) which initiate the process of resistance against parasite invasion. We base this on the observations that:

• Dendritic cells sense pathogens through Toll-like receptors which lead to the initiation of adoptive immune responses
• Proinflammatory cytokine IL-12, predominately produced by dendritic cells, is critical in controlling Cryptosporidium infection
• Bone marrow-derived CD40-positive cells are required for mice to clear C. parvum infection 

Based on these observations, the specific aims are designed to investigate the innate immune response to cryptosporidiosis with a view to determining the mechanisms of Toll-like receptor signaling that lead to such responses, using both mice and human dendritic cells. The specific aims are to:

• Characterize the role of mouse dendritic cells in the innate immune response against C. parvum infection
• Determine the nature of activation of human dendritic cells upon infection with C. parvum or C. hominis
• Identify the Toll-like receptor used by Cryptosporidium and isolate TLR activating ligand(s) expressed by the parasite  

The proposed studies will provide the basis for understanding the mechanisms of innate immune recognition and response to parasite invasion necessary for future design of vaccines and other methods of interventions. 

PI: Utz, Arthur
Title: Understanding and Controlling Gas-Surface Reactivity with State-Selected Reagents
Abstract: In this award funded by the Experimental Physical Chemistry Program of the Division of Chemistry, Prof. Arthur Utz of Tufts University will conduct detailed studies of the chemical reaction dynamics for gas-surface reactions using laser preparation to perform quantum-state resolved reactivity measurements on metal surfaces. These studies will be carried out by combining laser excitation methods with ultra-high vacuum techniques.

One set of experiments will investigate methane reactivity at surfaces, while another group will test the size limit for mode specific effects by using larger gas reactants for which intramolecular vibrational energy redistribution (IVR) is observed for the isolated molecule in the gas phase. These experiments are designed to understand the role of internal energy in gas-surface reactions and test the limits of bond-selective surface chemistry.

The ultimate goal of this work is to develop a better understanding of the important variables that influence gas-surface reactivity, with an aim towards designing better theories and improved practical applications. The results of this study will provide theorists with much useful data to test the latest theories. The results will also provide important guidance in designing practical catalytic reaction schemes, which are important to industry. Students and postdoctoral research associates who participate in this research acquire new knowledge and skills in preparation for advanced studies or entrance into the scientific/technological job market. 

PI: Van Etten, Richard
Title: Investigating the Homing and Engraftment of CML Stem Cells in the Bone Marrow
Abstract: Chronic Myeloid Leukemia (CML) is a hematopoietic stem cell (HSC) malignancy that is induced upon the balanced translocation of chromosomes 9 and 22 and the resulting formation of a Philadelphia (Ph) chromosome. The product of the Ph chromosome is the BCR-ABL tyrosine kinase. The constitutively active tyrosine kinase activity of BCR-ABL is the crux of the malignancy and causes defective integrin signaling and adhesion in HSCs. Recent studies in our laboratory indicate an abnormal and increased dependency on selectins and their ligands for homing and engraftment of these leukemic stem cells within the bone marrow (BM) niche.

The main objectives of this proposal are to:

1. investigate and further define the nature of the adhesive molecules that contribute to engraftment of malignant stem cells in CML patients; and
2. develop methods that will block engraftment of CML stem cells without affecting the engraftment of normal stem cells. 

To induce CML in donor stem cells we will employ our BCR-ABL transduction/transplantation model system. To delineate the critical adhesion molecules that mediate malignant stem cell engraftment compound donor and/or recipient mutant mice will express genetic mutations for adhesion molecules expressed in HSCs or BM endothelium, respectively. These BM transplantation studies will collectively determine the critical adhesion factors for engraftment of leukemic stem cells. Neuraminidase, anti-selectin reagents, monoclonal antibodies and small molecule selectin antagonists will be tested for their efficacy to block CML stem cell engraftment first in a murine model for CML and then using xenografts of human CML stem cells transplanted into NOD/SCID/γc-deficient mice. The observations from these studies will engender novel clinically applicable methods for blocking CML stem cell engraftment in autographed patients.  

Taken together, these studies will support the NIH mission by contributing to a deeper understanding of the cause of homing and engraftment of leukemic stem cells in CML patients. The development of clinically relevant methods for blocking the engraftment of CML stem cells will improve current techniques of treating CML patients, thereby leading to better management of the disease as well as an increase in patient longevity. 

PI: Vilenkin, Alexander
Title: Fundamental Physics and Cosmology
Abstract: The proposed research focuses on three central topics related to the inflationary universe.

• First, the PI will focus on the large scale structure of the eternally inflating universe and consider models where the dark energy is a stochastic parameter which varies from one place to another. The observational consequences of such models will be investigated and compared to data.

• Secondly the PI will continue a systematic investigation of the formation, evolution and observational signatures of various topological defects including those postulated in "braneworld" scenarios.

• Thirdly the PI will investigate the consequences of the new cosmological paradigm connected with eternal inflation and publish a popular book on the subject.
  

PI: Vilenkin, Alexander
Title: The Measure Problem in the Inflationary Multiverse
Abstract: One of the most intriguing problems of inflationary cosmology is that of calculating probabilities in models of eternal inflation. The crux of the problem is that the numbers of all possible observations performed in the course of eternal inflation are infinite. We have to figure out how to compare these infinities, since otherwise we will not be able to make any predictions at all. We shall explore different proposals for the probability measure, making sure they are not riddled with inconsistencies or obvious conflict with the data. We shall also explore what constraints on the measure are imposed by the global structure of the universe, having in mind in particular the “persistence of memory” effect. 

The most spectacular observational test of the multiverse scenario would be a direct observation of a collision of our bubble with another one. We shall study observational signatures of such a collision and the probability for us to be close enough to detect one. We shall also explore some conceptual issues related to the measure problem. These include the choice of reference class, the choice of prior probabilities for different models, and the possibility of deriving the measure from quantum gravity.

PI: Wachman, Alan
Title: Why Not Mongolia? Chinese Irredentism, Geostrategic Rivalries, and Asian Stability
Abstract: How is Mongolia viewed in the People’s Republic of China (PRC)? Following the disintegration of the Soviet Union, Mongolia—an erstwhile satellite of Moscow— has been utterly and genuinely independent. Since then, Sino-Mongolian relations have been formally proper and substantively cooperative. Yet, there is uneasiness in Ulaanbaatar that the PRC harbors irredentist ambitions that may lead it, first, to insinuate itself economically and then incrementally subordinate Mongolia as Beijing’s power and influence expands. To what degree is Mongolia considered as Chinese irredenta? Evidently, some Chinese today view Mongolia as rightfully part of China. 

More troubling, one finds in PRC publications evidence that some analysts characterize Mongolia as having immense geostrategic significance, or warn that Beijing must be vigilant because Washington views Mongolia in that way. Apparently, some Chinese analysts hold the view that even now Mongolia can serve as a protective screen (pingzhang) to shield Chinese territory. Efforts by some states—the United States chief among them—to exert influence in Mongolia are viewed suspiciously by Chinese authors who regard international competition as essentially geopolitical. To them, Beijing should be wary of conditions under which Mongolia may be used by the U.S. as a bridgehead from which to effect the “containment” of China. 

At present, the PRC seems determined to project an air of unthreatening preoccupation with internal development and the cultivation of harmonious foreign relations with its neighbors. It labors to assuage anxieties abroad about its rapidly expanding economic, political, and military power. Yet, where territoriality is concerned, Beijing has revealed a capacity for stridency, bellicosity, and unyielding assertions of sovereignty.

The PRC makes clear its belief that it will defend every sacred inch of the motherland. It has railed against those it dubs “separatists” in Tibet, Xinjiang and Taiwan—adopting an Anti-secession Law in 2005 to make manifest its determination to “employ non-peaceful means and other necessary measures to protect China’s sovereignty and territorial integrity” if Taipei does not succumb to Beijing’s entreaties to establish “one China.” By contrast, the PRC seems to have accommodated to the “loss” of Mongolia in the early 20th century and to its current status as an independent state. 

Nevertheless, the history of China’s relationship to Mongolia impels one to read with concern expressions of latent irredentism. Considering the assertions that Beijing makes about its sovereignty over Xinjiang, Tibet, and Taiwan, one is inclined to ask: Why not Mongolia? Has Beijing really “written off’ the loss of Mongolia, or should one worry that as PRC power expands Beijing may be more capable and more willing to act on geostrategic impulses to exclude rivals from its periphery. If that happens, Mongolia may be compelled to accept a vassal relationship with the PRC, an eventuality with significant implications for stability in Asia.

To establish the historical background of the contemporary attitudes of PRC elites toward Mongolia, I wish to do archival research the Cold War History Project at the Smithsonian Institution. In addition, I shall review publications from the PRC media, academic presses, and scholarly journals of public and international affairs that touch on the geostrategic role of Mongolia in the PRC’s worldview and/or inferences about objectives of the U.S. in its relations with Mongolia. To supplement what I glean from reading, I shall interview foreign and security affairs specialists in the PRC, Russia, Mongolia, and the U.S. On the basis of what I learn from these various sources, I intend to write a single- authored book suitable for publication by an academic press. Also, I wish to generate at least one article for a policy journal aimed at readers in the world of policy analysis and policymaking. 

PI: Walker, Peter
Title: Ambiguity and Change
Abstract: The 2004 Ambiguity and Change report, produced by Tufts University’s Feinstein International Center, spelled out some of the predicted big drivers of the humanitarian environment over a ten year period. It focused on environmental changes, urbanization, migration and HIV/AIDS as well as changes within humanitarian agencies. It was underwritten by a grouping of operational international humanitarian agencies, the International Working Group (IWG). 

While agencies, their senior management and boards found the report a useful tool for thinking and planning for the near future, it was neither sufficiently comprehensive nor explicit when it came to the practical “so what” for operational agencies. Agencies concerned with the challenges of the future need analyses that extend further than those offered in the original report. Greater attention has to be given to the use of the analyses. A futures-oriented time frame, to be useful and realistic, should be at least fifteen years. The practical utility of such analyses is based upon a highly dynamic and interactive dialogue between agencies and analysts at the strategic and operational levels. 

It is with this in mind that Tufts University’s Feinstein International Center [FIC] and the Humanitarian Futures Programme [HFP] at King’s College, London, have joined forces to propose a two year action-oriented research project to help agencies prepare for the future and ensure that they continue to focus on the livelihoods of communities in crisis. As the originator of the Ambiguity and Change concept and approach, FIC offers a unique, tried and tested perspective, with an internationally renowned research base. With the HFP’s extensive research programme on humanitarian organisations’ anticipatory and adaptive capacities, the collaborative effort will hopefully meet the needs of the IWG and the wider humanitarian community. 

Some aspects of the future are better known than others. Technological innovation may be more predictable than socio-political or economic forecasts; demographic trends may be more certain than climatic trends. In most instances, however, prediction is a hazardous art, and one that does not necessarily serve organisations well. Far more important is an organisation’s ability to be sensitive to what might be, and to ensure that sensitivity is matched by a capacity to adjust to changes in its operating environment whilst staying on track with its mission. 

Therefore, the proposed project is underpinned by two broad concerns. The first deals with compelling future drivers and their consequences for marginalised communities. The second concerns organisational capacities for dealing with such drivers and continuing to serve the aforementioned communities. In identifying those drivers that may directly or indirectly impact humanitarian crises in a fifteen year perspective, one needs to be clear about the best ways to identify compelling possibilities, to determine their degrees of certainty and to establish ways to monitor their probability. When it comes to organisational capacities for dealing with such possible futures, the ways that organisations formulate strategies, link such strategies to operational programmes and projects, and innovate and seek knowledge and information are all critical determinants for dealing with future drivers. 

It is the amalgam of these two concerns that forms the basis of the project. These are reflected in the project’s abiding objective, its key components, methodology and its administration, staffing and budget, as noted below:

The project has one abiding objective — to help the humanitarian community prepare for the complexities and uncertainties of the future by enhancing their anticipatory and adaptive capacities.

The project has four key components to achieve this objective:

1. Analysis of external drivers affecting the humanitarian environment in fifteen years with a particular emphasis on impact on livelihoods of marginalised communities;
2. Analysis of internal drivers influencing the capacities of humanitarian organisations in the future;
3. Institutional anticipatory and adaptive capacity assessments, both at the HQ and field level for dealing with the future; and
4. Exploring possible futures through scenario development again, both at the HQ and field level. 

PI: Walker, Peter
Title: Climate Change and Humanitarian Spending
Abstract: This proposal outlines research to be carried out and presented by November 15th 2008 to:

1. Provide a succinct, explicit, understandable account of the humanitarian consequences of increasing/changing extreme weather events and climate trends with quantitative estimates based on disaster data and trends analysis; and
2. Initial cost estimates of additional humanitarian action in response to increasing extremes due to climate change. 

The work will be carried out in collaboration between the three institutions mentioned above, with the FIC providing overall management and administration to the project. 

The study will be a desk top and literature study, it will not involve direct field work. The study will model from historic data the relationship between extreme hydrometeorological events, recorded disasters, international humanitarian response and international humanitarian spending. This modeling will be based primarily on data from the CRED EMDAT database of disasters and the UN FTS data base of humanitarian funding, along with data from the OECD-DAC humanitarian contribution database. We would also expect to work closely with the Red Cross Climate Change Center and climate change experts accessed through them. 

The model will then be projected into the future for the time period for the next two decades using assumptions on extreme hydrometeorological events and trends from the recent IPCC reports and other studies on climate change. The data we are particularly interested in are those predicting national and regional trends. This will allow for sample projections which can then be extrapolated to global projection by reference back to the global historic record. 

 Wherever data is available, projections will be done for three models: high, medium and low climate change. The projected models will be used to describe the likely extent of humanitarian crisis, the potential needs they will generate and the possible cost of response. 

PI: Walsh, Genevieve
Title: Symmetry and Commensurability
Abstract: Two n-manifolds are commensurable if they have homeomorphic finite-sheeted covers. This notion is particularly useful for 3-manifolds. For 3-manifolds which admit a geometry, commensurability respects this geometry. In light of the recent proof of geometrization for 3-manifolds, classification into commensurability classes is a refinement of the classification of 3-manifolds by which geometry they admit. This applies to 3-manifolds that have been decomposed along 2-spheres and 2-tori. Questions concerning virtual properties of 3-manifolds, such as if a 3-manifold is virtually Haken or virtually fibered, are also questions about its commensurability class. Commensurability also respects certain number-theoretic properties of 3-manifolds. A classification of hyperbolic 3-manifolds up to commensurability would be very useful for the field. A first step towards understanding commensurability classes of hyperbolic 3-manifolds is understanding commensurability classes of hyperbolic knot complements. The PI proposes to work on the conjecture that there are at most 3 hyperbolic knot complements in a given commensurability class, and to understand how these commensurabilities can occur. Commensurability is also a useful equivalence relation on hyperbolic surfaces, with the definition that two hyperbolic 2-manifolds are commensurable if they have isometric finite-sheeted covers. The PI proposes to further develop this theory, in particular to understand commensurability classes of surfaces with large symmetry groups. 3-manifolds are spaces which locally look like a 3-dimensional ball. In particular, our universe is a 3-manifold and we do not know which one it is. A deeper understanding of commensurabilities amongst 3-manifolds would significantly refine geometrization for 3-manifolds. Knot complements are a naturally occurring class of 3-manifolds, and thus good candidates for study. 2-manifolds, or surfaces, are spaces which locally look like a 2-dimensional disc. Hyperbolic surfaces are the most common type of surface and are used in an extremely wide variety of scientific contexts. The study of symmetry and commensurability is very useful for these surfaces. The PI regularly speaks on her work to diverse audiences. Any results from this research will be described in research seminars, posted on the arXiv, and broadly distributed.

PI: Walt, David
Title: Microsensor Arrays for Saliva Diagnostics
Abstract: This application aims to develop a portable, diagnostic system useable by minimally trained personnel in the field for the diagnosis of pulmonary diseases using saliva as a sample specimen. The ultimate goal of the project is to create a universal platform for performing systemic diagnosis using saliva. The device will be fully integrated and self-contained. A saliva specimen will be loaded onto a disposable microfluidic card containing all the necessary components and reagents to process the sample and measure multiple clinically relevant analytes with an optical fiber microarray. The results will be read optically and reported both via a liquid crystal display and over a wireless network. Pulmonary diseases will be targeted including asthma, chronic obstructive pulmonary disease (COPD)/emphysema, cystic fibrosis, and acute pneumonia. Saliva samples from both diseased and control patients will be obtained and analyzed using a variety of fluorescent-based assays. The initial phase will be carried out in an outpatient clinic setting, where patients will be followed at regular intervals when stable and during exacerbations of their disease. Samples will be collected and analyzed at baseline and during clinically unstable conditions allowing analysis of change from baseline as well as single sample correlations with clinical status. To directly test our overall hypothesis that saliva contains substances that correlate with disease, we will also collect and analyze key representative respiratory secretions and compare these results with those obtained from salivary analysis. Analytes that correlate with disease will be selected and converted into bead-based sensors for use with optical fiber microarrays. Analytes to be examined will include DNA/RNA, proteins such as cytokines, and small molecules and ions. All sample preparation and pre-treatments will be incorporated onto a microfluidic cassette. The disposable cassette will be integrated with the sensor array, enabling many clinical parameters to be measured simultaneously. The array will be interrogated by a portable optical reader that will be designed and integrated with the microfluidic cassette and the optical fiber array. We will demonstrate efficacy of the system at the lab bench and later provide one Alpha and three Beta units to the clinical investigators for pilot studies and expanded field trials. 

PI: Wanke, Christine
Title: Protease Inhibitor Related Dyslipidemia
Abstract: Protease inhibitors are used as therapy in HIV patients and have been reported to cause elevations in plasma triglycerides, cholesterol, and glucose, and rarely to induce severe hypertriglyceridemia, pancreatitis, and diabetes mellitus with insulin resistance, excess fat deposition, and lipodystrophy. Our aims are to measure fasting Serum cholesterol (C), triglyceride (TG), remnant lipoprotein (RLP) C and TG, low density lipoprotein (LDL) C, high density lipoprotein C, lipoprotein(a), apolipoproteins A-I and B, apo E genotype, homocysteine, free fatty acids, glucose, insulin, and blood pressure. We will also assess smoking status, carotid artery wall thickness by ultrasound, and coronary artery calcification by computerized tomography in our prospective cohort of 400 HIV patients whose nutritional status is being evaluated and who are taking a variety of antiviral agents including protease inhibitors. Comparisons will be made on and off inhibitors and also longitudinally, and with controls. Our comparison group are participants in the Framingham Offspring Study who have had all the same parameters measured (n=3250). HIV patients who become hyperlipidemic on protease inhibitors will be treated with either gemfibrozil or atorvastatin. We will also examine the effects of protease inhibition in Hep G2 and CaCo2 cells with or without supplementation with fatty acids and cholesterol on lipoprotein assembly and secretion and apolipoprotein, LDL receptor, and microsomal transfer protein (MTP) gene expression. The effects of protease inhibition on lipoprotein metabolism and aortic foam cell formation will also be assessed in FIB hamsters on chow and on diets high in cholesterol and saturated fat. In addition, using a primed constant infusion in the constantly fed state and deulerated leucine, the secretion and catabolism of apoB-48 and apoB-100 within lipoproteins will be determined by GC/MS analysis and multicompartmental modeling in the presence or absence of protease inhibition with ritonavir in 10 males and 10 female HIV patients. We will test the following hypothesis:

• protease inhibitors increase triglyceride and cholesterol by increasing RLP
• elevated RLP leads to increased carotid wall thickness and coronary calcification
• these increases can be ameliorated with diet, gemfibrozil and/or atorvastatin treatment
• in cell culture these RLP increases are elated to enhanced secretion of apoB-100 due to less intracellular degradation, and excess cellular lipid content
• in hamsters there are increased RIP in serum in animals on the atherogenic diet, especially with protease inhibition, and this leads to increased aortic foam cell formation
• in humans protease inhibition causes increased triglyceride-rich lipoprotein apoB-100 secretion

This research should define the nature of the problem, its mechanism, and methods for treatment with regard to the hyperlipidemia induced by protease inhibitors in HIV patients.

PI: Webb, Patrick
Title: Nutrition in Emergency Funding Appeals
Abstract: By improving our understanding of what is (or is not) included under the rubric of ‘nutrition’ across sectors in CAPs, Flash appeals and Natural Disaster appeals, the intent is to define:  

1. trends since the mid-1990s in emergency nutrition appeals and resultant funding
2. evolution in approaches to appeals (and the related programming)
3. scale of nutrition activity relative to other activities in related emergency sectors (food, health, watsan, multi-sector, etc.)
4. value-added of nutrition cluster to the appeals, changes in how nutrition is characterized and promoted in the process, and insights into how emergency nutrition might be ‘framed’ differently in future appeals 

PI: White, Robert
Title: Modeling and Characterization of Micromachined Ultrasound Elements
Abstract: The goal of this project is to produce, document, and experimentally validate a computational model of a micromachined ultrasonic array element. The testable elements will be provided by Sonetics Ultrasound. Computational and experimental work will be carried out at Tufts University in consultation with Sonetics Ultrasound. This will provide a vehicle for better understanding micromachined ultrasonic transducers, and also for educating a graduate student in the area of MEMS, acoustics, device modeling, and device characterization. 

PI: White, Robert
Title: In Situ CMP Characterization with Patterned Substrates
Abstract: Polishing small, fragile features requires a delicate mechanical polish. This can be achieved using reduced down force, but this results in lower material removal rates. Increased polish time leads to added cost and consumables. In terms of cost and environmental impact, it would be preferable to reduce structural damage while maintaining a high material removal rate. 

Previous research indicates that as little as 0.1% of the wafer surface is in contact with the polishing pad during CMP. These small contact areas, which bear the bulk of the imposed loads, result in large local lateral forces in the small contact regions. If the contact area could be increased by appropriate changes to the pad surface topology, it might be possible to reduce local shear forces at the micro- or nano-scale while maintaining high material removal rates. 

The goal of this research is to experimentally investigate this approach. We will measure the local shear force, estimate the local contact area, and measure the wafer-scale forces and dynamic position during polishing of metal and oxide using in situ experimental methods developed over the last few years at Tufts. We will correlate these in situ measurements to surface damage observed on patterned structures, as well as material removal rate. The work will focus on the effects of advanced conditioning using highly controlled diamond conditioners from Advanced Diamond, Inc., as well as the effects of grooving on pads supplied by Cabot Microelectronics. We will microfabricate micron-scale test structures at Tufts. Intel Corporation will supply patterned wafers with smaller features for additional testing. 

PI: Widmer, Giovanni
Title: Genetics of Cryptosporidium Parvum
Abstract: Cryptosporidium panvum is an opportunistic intestinal protozoan parasite which infects AIDS patients and other immune-compromised individuals. For lack of effective drugs, cryptosporidiosis may become persistent, particularly in immunodefficient individuals. Genes controlling virulence phenotypes in this parasite are unknown, but their identification is needed to guide the development of drugs, to better understand pathogenesis, and to improve the annotation of the genome. The long-term objective of this revised application is to apply genetic methods to map genes controlling specific phenotypes. In contrast to linkage analysis used in the first funding period, we propose to apply Linkage Group Selection, a strategy which does not require the cloning of progeny lines. Instead, genetic loci controlling selectable traits are identified by crossing two phenotypically distinct C. parvumlines, exposing progeny populations to one or multiple rounds of selection, and quantitatively genotyping progeny populations in bulk. Based on well-defined phenotypic differences between the parental lines, we will select for early oocyst differentiation, oocyst resistance to bleach, and parasite infectivity to interferon-γ knock-out mice. During selection, parasites carrying alleles conferring the phenotype selected against will be eliminated from the progeny. As a result of meiotic recombination the proportion of alleles from the sensitive parent will increase with distance from the locus under selection, giving rise to a “selection valley”. Such valleys will be identified by quantitatively genotyping a selected and a control progeny population with a panel of single nucleotide polymorphisms. This study will be performed in collaboration with Dr. Paul Hunt, a malaria geneticist who has developed and successfully applied Linkage Group Selection to the study of malaria drug resistance.

Specific Aims:

1.Cross phenotypically distinct isolates of Cryptosporidiurn parvumand use Linkage Group Selection to identify chromosomal regions controlling specific phenotypes. Populations of uncloned recombinant parasites derived from crosses between phenotypically distinct parental lines will be subjected to selective pressure and quantitatively genotyped using a set of 100 SNP markers. Regions under selection will be progressively resolved by iterative recombination and selection.

2. Identify genes within a selection valley controlling the phenotype under selection and identify the mutations responsible for the phenotype. A set of closely spaced SNPs located within the selection valley will be proportionally sequenced to identify genes and mutations under selection. Mutations will be mapped to the genome of C. hominis, C. muris and more distantly related apicomplexan to identify those most likely to affect protein function or transcriptional regulation. 

PI: Wilde, Parke
Title: Dynamics of Food Insecurity Over Five Years in the Survey of Program Dynamics (SPD)
Abstract: This project will analyze longitudinal patterns of food security and food insecurity for families and individuals over a five-year period. The project will identify predominant patterns of food security/insecurity over time (never insecure, always insecure, in and out of insecurity, etc.), develop longitudinal measures of food insecurity and describe the population in terms of these patterns and measures. Then determinants of occasional and persistent food insecurity and of changes in food security status will be investigated, including the roles of selected cash and food/nutrition assistance programs. 

ERS publishes annual food security statistics, which provide a cross sectional snapshot of households’ food security. Less is known, however, about how many years households typically remain food insecure and what proportion of households are food insecure at some time during a multi-year period. To date, the relatively few studies that have examined dynamics of food insecurity have analyzed data from just two points in time. These studies have found that there is considerable churning in the food-insecure population, with perhaps as many as half of food- insecure households in one year becoming food secure by the following year and being replaced by as many newly food-insecure households. Little is known about longer term dynamics and, in particular, the extent of long-term persistent food insecurity and the size of the pool of households that is exposed to food insecurity at some time over a period of several years. These longer term patterns of food insecurity have implications for the design of programs intended to improve the food security of U.S. households. 

The Survey of Program Dynamics (SPD) collected data on households’ food security annually from 1998 through 2002. This project will utilize the SPD data to analyze longitudinal patterns of food security and food insecurity for families and individuals over that five-year period.  

PI: Wilder, Andrew
Title: Winning Hearts and Minds? Examining the Relationship between Aid and Security in Afghanistan, Pakistan, and the Horn of Africa
Abstract: There is a widely held assumption in military and foreign policy circles that development assistance is an important “soft power” tool to promote security. Counterinsurgency doctrine in particular emphasizes the importance of humanitarian and reconstruction assistance in “winning hearts and minds,” which in turn helps promote security by undermining support for insurgents and/or terrorist organizations. This assumption is having a major policy impact on how development assistance is apportioned and spent, and provides an important rationale for the growing “securitization” of development assistance. Given how widespread the assumption is, and given its major impact on aid and counterinsurgency policies, there is remarkably little empirical evidence that supports the assumption of a causal link between reconstruction assistance, “winning hearts and minds,” and improved security. The objective of this study is to address this evidence gap by conducting a comparative study in Afghanistan, Pakistan and the Horn of Africa that critically examines the assumption that aid projects contribute to improved security by helping to “win hearts and minds.” By helping to clarify the relationship between aid and security the study hopes to make development assistance more evidence-based and effective in addressing the needs of the poor and marginalized in crisis-affected communities and states around the world. 

PI: Will, Leslie
Title: The AAOF Craniofacial Growth Legacy Collection: Pilot and Planning Application
Abstract: This proposal describes a project in two parts, to be completed in one year:

The first will consist of a pilot study that will include digital scanning the longitudinal cephalometric records from ten subjects from each of the ten collections whose curators met at the Bolton-Brush Study Center on November 15-16, 2007 to discuss collaboration. This meeting was called in response to the Request for Proposals issued by the American Association of Orthodontists Foundation.

The ten orthodontically untreated subjects will be selected by each curator on the basis of the criteria established in the AAOF Awards Materials; i.e., “male subjects with records spanning ages 8 to 18 years, female subjects with records spanning ages 8 to 16 years; at least four readings within the age span including records at (1) age 8, (2) 16 or 18 (as defined above), and (3, 4) two intermediate record sets 2 years apart from any other time point. For subjects that meet the minimum requirements, records from all available time points should be entered (i.e.,all intermediate time points and (or) time points outside the requisite age spans).” Additionally, Class III and Class II patients will be included preferentially, and sex distribution will be equal. The films will be scanned at the agreed-upon specifications and will contain information on scale from fiducials and will be stored in a uniform format, enabling samples from the entire collection to be examined and processed seamlessly as a group.

The primary records to be included in this pilot project will be lateral cephalograms (closed and open). Submissions from the participating collections ultimately may include other materials such as dental casts, other radiographs, photographs, measurements and other information sources. However, only the lateral cephalograms will be uploaded to the two web sites during this pilot project.

The purposes of the pilot project are as follows:

1. To demonstrate the ability of the participating collections to achieve consensus and collaborate on the establishment of a joint “Legacy Collection,” including agreement as to the protocol used for the digitizing process and the configuration of a common data base. It was agreed that Microsoft Access^® would be used as the common indexing software.

2. To examine existing software at two university sites (The University of Iowa and at The University of the Pacific San Francisco), and to establish a pilot AAOF Craniofacial Growth Legacy Collection Websites, using lateral cephalometric images and associated material submitted from each of 10 facial growth collections in North America. The software for these two web sites will be modifications of the University of Iowa and the University of the Pacific craniofacial growth website source codes.

3. To assess and compare the utility of both the Iowa and Pacific web platforms and other relevant software programs that might support the broader, complete AAOF Craniofacial Growth Legacy Collection. Based on this assessment, a platform will be selected for use in support of the broader collection.

4. To make available the radiographs submitted by each collection for orthodontic and craniofacial research.  

The second portion of the proposal is the final planning for the comprehensive archived database, hereafter referred to as the AAOF Legacy Collection. It is anticipated that this collection, also a representative sample from among the ten collections in North America, ultimately will include approximately 1,000 subjects, depending on budgetary constraints. In addition to selecting the platform to be used, procedures for setting priorities for further record archiving, establishing a process for accessing and using records, and establishing a structure to receive three-dimensional DICOM files, quality assurance of records, and assurance of statistical significance will be addressed by a steering committee in conjunction with consultants and co-investigators. 

PI: Wise, Timothy
Title: GDAE's Working Groups on Development and the Environment Expanding Policy Influence in Latin America
Abstract: The Globalization and Sustainable Development Program of Tufts’ Global Development and Environment Institute (GDAE) has been carrying out empirical, policy-relevant research on the effects of “free trade” agreements and policies on developing countries, with a particular focus on Mexico and the rest of Latin America. Since 2003, that work has included collaborations with working groups of Latin American economists to examine the extent to which economic integration has brought social, economic, and environmental improvements to the region. Following a successful pilot project in 2004 on the effects of globalization on the region’s environment, GDAE has organized two further Working Group projects, one on foreign investment, the other on agriculture. Policy reports from the two projects are due out in English, Spanish, and Portuguese in Spring 2008, with launch events planned for the second half of the year. 

GDAE is seeking support from the Moriah Foundation to help the Working Groups expand their reach and influence in Latin America. With new governments in the region actively looking for alternative policies, this project can help inform the debates in many countries over the direction of these policies. Funding shortfalls prevented the first Working Group project from reaching as many policy-makers in the region as it could have. GDAE has secured adequate funding for the research and publication of the two policy reports in the three languages, as well as to cover U.S. launches and meetings for the English editions of the reports. We seek additional funding to ensure that each of the two groups is able to carry out at least two substantive presentations of the project, in Brazil and in at least one major capital in Latin America. 

PI: Wood, Richard
Title: Services for Sample Analysis Per Statement of Work
Abstract: The overall purpose of this study is to study the association of serum ferritin and serum transferrin saturation with the risk of gastric adenocarcinoma and the risk of esophageal squamous cell carcinoma, adjusting for the likely confounding variable of inflammation (CRP).  

PI: Wortis, Henry
Title: Immunologic Aspects of Disease
Abstract: This is a renewal application to support a well-established pre-doctoral training program in immunology at the Sackler School of Graduate Biomedical Sciences located on the Tufts University Health Sciences Campus in downtown Boston. Currently, 21 faculty members are training 34 students of whom seven receive direct support from this grant. Highly qualified students are selected from a pool of over 100 applicants based on research experience, recommendations, coursework/grades, interviews, and GRE scores. Training is designed to provide an appreciation of clinical problems together with the technical expertise and scientific insight to successfully perform research on important immunological questions. It aims to stimulate originality, curiosity and the use of rational reasoning to understand biological mechanisms. The didactic component consists of required courses: Biochemistry, Introduction to Immunology, Immunogenetics, Immunochemistry, Immunological Mechanisms in Disease and an elective. These courses provide the foundation for required participatory courses: Journal Club, Advanced Journal Club, Seminars and Student Workshop. Students take four first year laboratory rotations and pass a qualifying examination before entering a thesis laboratory. Thesis research, considered the core of the Ph.D. training, is overseen by individual thesis advisory committees that meet with the student each semester. It is the thesis committee's role to critique the project proposal and to assess the student's progress towards producing an original and substantive contribution to scientific knowledge. While publication is not a formal requirement, graduates are expected to be first authors of peer-reviewed papers. Students are explicitly trained in oral and written presentation of scientific ideas and data and have opportunities to present their research findings at the weekly Student Workshop as well as the annual retreat and mini-symposium. Students also routinely attend and present at national and international meetings. A Program Student Adviser acts as student consultant and advocate. Requirements and expectations are established in writing and the Student Adviser keeps students well informed of their progress. The program successfully graduates a high proportion of all entering students (89%). In the past 10 years there have been 65 graduates, three are in transition, twelve are in clinical training or service and of the remaining 53, a high proportion, 45 (85%), are actively engaged in research, including four in tenure track positions. The program has benefited from a recently increased University commitment of resources to graduate biomedical education, new research facilities and the hiring of new faculty members. 

PI: Wortis, Henry
Title: Babesiosis as a Model of Age-Related Immunosenescence
Abstract: As people age, a greater number becomes susceptible to infections. Because the aged represent an ever-growing segment of the populations in the western world, morbidities due to infection are significant. We are interested in identifying alleles that underlie this age-acquired susceptibility. We developed a mouse model of human babesiosis due Babesia microti, an emerging infection particularly severe in the aged. We observed that young DBA/2 mice experience significant parasitemia but young C57BL/6, B10.D2 and BALB/c mice do not. In DBA/2 mice an early phase characterized by an intense and transient parasitemia is followed by a late phase of low grade, but persistent parasitemia. As age advances, both acute and chronic parasitemia become greater in DBA/2, but not in C57BL/6, B10.D2 or BALB/c mice. Using B10.D2 x DBA/2 (BXD) and BALB/c x DBA/2 (CXD) crosses as well as chromosome substitution mice, we have established that i) resistance is a complex trait in young and old mice, and ii) acute resistance in young mice maps to QTL that differ from those of old mice. We conclude that the greater susceptibility of old DBA/2 mice maps to age-restricted QTL. We now propose to identify resistance and susceptibility alleles and to uncover how they modulate the response to B. microti infection.

• In Aim #1, we will identify the major genetic determinants of acute resistance in old mice of the BXD cross.
• In Aim #2, we will identify the major genetic determinants of acute resistance in old mice of the CXD cross. Both aims combine a positional cloning approach with bioinformatic analyses and gene expression studies.
• In Aim #3, we will identify the cells that contribute to resistance and are affected by the polymorphisms underlying the major QTL. Strain differences in cell function will be studied and used to guide the genetic analysis.

We are hopeful that the identification of age-sensitive polymorphisms that modulate immune resistance of old mice to babesiosis may inform the search of alleles that predispose aged individuals to infection. 

PI: Wortis, Henry
Title: Light Weight Modeling of Epstein-Bar Virus
Abstract: Eipstein-Barr Virus (EBV) infects more than 90% of all humans, usually without symptoms. It can also be responsible for acute infectious mononucleosis (AIM) and is associated with fatal malignancies including immunoblastic lymphoma, Hodgkin's lymphoma, Burkitt's lymphoma, nasopharyngeal carcinoma and X-linked proliferative disorder (XLP). Our long-term goal is to understand these processes in sufficient detail to guide clinical intervention. Our overall model of normal and malignant EBV biology puts us in a good position to build computer models of EBV infection. Our specific aims include the following:

1. Evaluate the relative impacts of various factors known to play a role in EBV biology.
2. Assess the probability of the varying fates of a cell once it has entered a particular infected state.
3. Understand the overall dynamics of these models as dynamical systems. This includes distinguishing possible long-term behaviors and the transitory states that lead to them.

We will pursue these goals by building and analyzing multiple light weight computer models of EBV infection. By "light weight" we mean that these models are easy to write, modify and run. This will allow investigations not possible with larger agent-based computer models. Epstein-Barr Virus is widespread in the human population. While it is usually asymptomatic, it is also associated with fatal malignancies. Computer simulation is a way to study the normal asymptomatic course of this infection and the ways in which this turns malignant. We hope that a better understanding of these processes will show us how they can be controlled. 

PI: Yee, Kathleen
Title: A Role for Pax6 in Cochlear Nucleus Development
Abstract: We are interested in how information-transmitting cells in the brain (neurons) obtain their identity and acquire characteristics that allow them to perform very specific functions. To address these questions, we study a region of the brain that forms the cochlear nucleus, the first and only direct target for cochlear input. While a large body of data exists on features of mature cochlear nucleus neurons, studies are only beginning to examine the role of genes during development. Our preliminary data shows that the molecule, Pax6, a transcription factor, is expressed in the developing cochlear nucleus. The Pax6 gene has long been known to produce deficits in eye development, often manifesting as aniridia, type II (AN2) in humans. Only recently has it been recognized that these patients also have hearing deficits. Studies have implicated higher order brain centers as the sites of aberrant auditory processing. Our data demonstrating Pax6 gene expression in the cochlear nucleus suggests that AN2 patients may hear problems much earlier in the primary auditory pathway. Our r data indicate that there are structural changes in the cochlear nucleus in heterozygous mouse mutants. This proposal will examine the extent of anatomical changes and how these changes affect function. 

PI: Yelick, Pamela Crotty
Title: Dental Stem Cells and Tooth Tissue Engineering
Abstract: Our long-term goal is to define cell populations and methods that can be used for autologous tooth tissue engineering in humans. The ability to identify and manipulate adult dental stem cells (DSCs) would significantly facilitate this process. Although a considerable body of literature supports the existence of epithelial and mesenchymal DSCs, DSCs themselves remain poorly characterized relative to other stem cells including neural, bone marrow stromal, hematopoetic, spermatogonial and keratinocyte stem cells. In particular, tools such as antibodies to DSC surface markers that could be used to generate enriched DSC populations, to identify adult DSC populations, and to examine the induction of odontogenic potential in non-dental tissues, are currently lacking. We hypothesize that the identification and characterization of post-natal dental stem cell (DSCs), combined with state-of the art tissue engineering techniques, will facilitate whole tooth tissue engineering efforts. Our recent tooth tissue engineering results demonstrate our ability to isolate, and maintain in culture, epithelial and mesenchymal post-natal DSCs, and to use these cells to bioengineer small tooth crowns containing dentin and enamel.

Based on these promising initial results, here we propose studies whose successful completion will define methods to bioengineer teeth of predetermined size and shape. We propose:

1. to establish and characterize enriched epithelial and mesenchymal DSC populations using well-characterized stem cell sorting techniques, and to confirm their identity using our functional tooth tissue-engineering assay; and

2. to define scaffold materials and design for optimized bioengineering of tooth tissues of specified size and shape.

The successful completion of the proposed aims will significantly improve our knowledge and understanding of DSCs and their potential utility in tooth tissue engineering, and will provide the foundation for eventual autologous tooth tissue engineering in humans. 

PI: Yelick, Pamela Crotty
Title: Bioengineered Dental Tissues from Human Tooth Bud Cells
Abstract: Aim 1 – Optimize harvesting/culturing conditions for human post-natal tooth bud cells.
We are continuing to optimize methods forth harvest and culture of human post natal tooth bud dental stem cells (DSCs), and to characterize DSCs for dental tissue engineering applications, including: 1) determining the ideal tooth developmental stage for optimal DSC yield; 2) correlating human chronological age, gender, and pre-existing health conditions of the tooth donor with: tooth soft tissue weight; length of time in cell culture to confluency, colony forming units (CFU) assay, and initial harvested cell count, and final dental cell number. In this manner, we are establishing a table for predicting USC yield from harvested human teeth at defined developmental stages. 

Extracted teeth were classified into one of five developmental stages, based on the degree of mineralization of the crown structures, and tooth root length measured from the dentin-enamel junction. Extracted tooth germs were examined using both radiographic and light microscopic analyses. Stage one teeth consisted of only tooth crown structures; Stage 2 teeth contained crowns and 1-2 mm tooth roots (avg. 1.5); Stage 3 teeth contained 3-4 mm length root (avg. 3.5);Stage exhibited 5-6mm tooth roots (avg. 5.5); Stage 5 teeth exhibited 7-8 mm roots (avg. 75); and Stage 6 teeth exhibited fully formed tooth roots with open apex (avg. 9.0). 

Human DSC cultures were prepared from the soft tissues isolated from harvested teeth at developmental Stages 1, 2 and 3 only. Extracted teeth at developmental Stages 4 and 5 were cryopreserved for future studies. Extracted tooth bud cell yields obtained from harvested impacted molar teeth were variable, depending on the developmental stage, and chronological age of the individual. 

Currently, our study sample consists of 39 patients, some of whom donated two teeth, for a total of 49 tooth germs, from 35 female and 14 male patients. Stage 1 teeth were obtained from 15 females and 5 males, Stage 2 teeth were obtained from 9 females and 9 males, and Stage 3 teeth were obtained from 11 females. As stated above, developmental stage was defined clinically through analysis of recent panoramic X-ray.  

Aim 2 — Perform molecular/cellular characterization of human post-natal tooth bud cells.
To investigate cytogenetic integrity of harvested and cultured human DSCs, we are establishing methods to assess chromosomal stability. Chromosomal preparations were made from in vitro expanded hDSC cultures and analyzed by G banding and FISH, using an alpha-centromeric X chromosome probe. Our results showed that ~70% of the cells exhibited abnormalities including polyploidy, aneuploidy and ring chromosome structures. Although initial cell culture protocols were identical, karyotypic changes were observed after clonal selection, and when methods to maintain hDSCs in culture without feeders. These results indicate the need to analyze cytogenetic stability of cultured DSCs prior to their use in clinical therapies.

Aim 3 — Generate and characterize bioengineered human tooth tissues.
We are examining hDSC interactions with nanoscale topography of biopolymer materials, for potential use in clinically relevant human tooth tissue engineering applications. PGA fiber scaffolds, with and without collagen surface treatment, are being characterized by atomic force microscopy (AFM) specifically using the intermittent contact mode, and by scanning electron microscopy (SEM). Our preliminary results show that PGA fibers with collagen exhibit morphological periodicity at the nanoscale level. Similar analysis of naturally formed human teeth revealed the same morphological periodicity observed on PGA with collagen, suggesting that the collagen treatment promotes a morphological mimic of the natural environment.

PGA scaffold fibers with and without collagen treatment were seeded with cultured hDSCs isolated from teeth extracted from patients scheduled for diagnostic third molar extraction. After 12 hours, cell adhesion was evaluated using tapping mode AFM and SEM. Our results revealed that hDSCs did not adhere to PGA scaffolds without collagen treatment. In contrast, hDSCs exhibited excellent adherence on fibers surface of PGA scaffolds with collagen treatment. These results indicate that the nanoscale morphology induced by collagen surface treatment can favor hDSCs adhesion. In addition, evaluations of hDSC/scaffold interfaces at the nanoscale level further emphasize the importance of collagen coating on PGA scaffold surface to facilitate hDSC adhesion for tooth tissue engineering applications.

Adult nude rat hosts (14) were used for construct implants, with each one receiving 2-4 subcutaneous implants. Two nude rats received intact third molar tooth germ implants as positive controls. We will allow the implants to grow for 20 to 30 weeks, prior to harvesting for analysis. Although certain technical difficulties exist during manipulation of nude rats due to the fragility of their health and occasional death prior to implant harvest date, our results to date reveal mineralized tissues formation using X-ray. Histological and immunohistological analysis are now being performed. 

PI: Young, Helen
Title: An Exploratory Study into Pastoralist Perspectives on the Impact of the Darfur Conflict on Livelihoods
Abstract: Tufts/FIC have been leading a series of research studies and related institutional support activities since 2004 in the Darfur Region. The current project “Darfur Research On Livelihoods, Migration And Remittance Flows And Support For The Darfur Research Consortium” started in July 2006 and is funded by DFID. This project has included a range of field based research activities, support to the UN Resident Coordinators Office/UNOCHA and also support of an informal Darfur research network. The wider goal of the Tufts research project on livelihoods, migration and remittances is to: “generate a common and shared understanding of the key causes of the conflict, impact on livelihoods, and wider processes and institutions, and implications for future recovery. This understanding will be used to generate improved livelihood policy and programmatic responses”. 

This livelihoods research includes an exploratory study on pastoralist perspectives, as described in the attached concept note. This proposal is for additional support from UNEP in order to ensure the environmental issues are adequately addressed. The objectives of the pastoralist perspectives study are: 

1. To promote understanding and raise awareness in Sudan and internationally about the livelihood challenges facing specific pastoralist groups in Darfur.
2. To engage a broader group of stakeholders and promote a dialogue between pastoralist groups and local stakeholders in order to promote their inclusion as stakeholders in relevant national and international level processes (peace processes and recovery).
3. To sharpen the focus and effectiveness of strategic humanitarian action aimed at supporting the livelihoods of these groups now and in the future. 

To complement this, an additional objective will be added to be supported by UNEP:

1. To review the major environmental issues affecting pastoralists, including livelihood adaptations to apparent climatic change such as changing rainfall patterns, impact of conflict on access to water, pasture and fodder for livestock, and changes in local systems of natural resource management in order to inform policies and programmes (range, forestry and water programming). 

This work will start in the middle of March 2008 and be completed by the end June 2008.