PI: Akiyoshi, Donna
Title: A Genome Sequence Survey of Enterocytozoon Bieneusi
Abstract: Enterocytozoon bieneusi is a clinically significant human enteric pathogen associated with immunodeficiencies including AIDS, and against which therapy is not readily available. Given the technical difficulties associated with the lack of in vitro and in vivo propagation methods and limited sources of spores, research on this organism has been limited. Thus, we propose to undertake an E. bieneusi genome sequence survey project which will generate several important genome-related resources (sequence, clones, libraries), which will be available to the research community.

The specific aims are as follows:

1. Construct a small insert plasmid library from which approximately 40,000 clones will be sequenced. The sequence and assembly of reads will obtain 95-98% genome coverage, which should identify the majority of genes. A fosmid library will also be constructed, and a 15-fold coverage will be performed. Master clone banks of both libraries will be created.
2. Perform functional genomic analyses and annotation of the generated sequence data.
   1. Using available computational tools, open reading frames, and non-coding sequences, introns and regulatory elements will be identified. BLAST analyses will be performed to identify genes’ encoding proteins with high similarity to known proteins for assignment of function.
   2. A comparative analysis of the E. bieneusi genomic sequence with that of the Encephalitozoon cuniculi genome sequence will be carried out. Phylogenetic analysis of the cryptic mitochondrion and its function, and the ATP transporter genes will be undertaken.
   3. Establishment of a website to provide access to the sequence data and clones by the scientific community.

PI: Ambady, Nalini
Title: Nonlinguistic Dialects: Behavioral and Neural Correlates
Abstract: The overarching goal of this application is to test a theory regarding nonlinguistic dialects. 6 sets of studies are proposed to test the hypotheses that nonlinguistic emotional dialects are (a) acquired through cultural exposure and (b) provide cues as to cultural identity. Further, (c) emotional dialects of majority group members are understood more accurately by minority group members than vice-versa; (d) emotional dialects are associated with distinct neural patterns of activation; (e) emotional dialect acquisition shows a similar early developmental trajectory to linguistic dialect acquisition, and, (f) bicultural individual is fluent in different emotional dialects will code-switch between dialects in response to subtle primes. The 12 proposed studies are designed to examine emotional communication by members of diverse cultural and ethnic groups, from Japan, India, and the United States. Within these groups, both majority as well as minority group members will be examined. Both the results and the stimuli collected for these studies involving emotions expressed in 3 different channels (the face, body, and voice) from diverse cultural and ethnic groups will be shared and disseminated to other scientists to enhance research and knowledge in this area. The proposed research should have important implications for mental health and inter-group relations. Being less able to understand the emotions being communicated by members of other ethnic or cultural groups probably contributes heavily to a lack of empathy and understanding of less familiar others as well as to inter-group prejudice and discrimination. This work has implications for diverse interactions such as in the client-provider relationships, in which members of diverse ethnic and cultural groups have to interact and sustain relationships.

PI: Anwer, Sawkat
Title: Mechanism of Canalicular Bile Formation and Cholestasis
Abstract: The long-term goal of this proposal is to understand the mechanism of canalicular bile formation and cholestasis. Our present understanding of the pathogenesis of cholestasis is based on studies to define the physiological regulation of transporters involved in bile formation and their deregulation in cholestasis. Cyclic AMP stimulates bile formation by translocating solute transporters to the plasma membrane and reverses acute cholestasis associated transporter dislocation. The focus of the present proposal will be to further characterize the signaling pathways involved in cAMP-mediated transporter translocation and reversal of transporter dislocation in acute cholestasis. The following hypotheses are proposed:

• cAMP stimulates Ntcp translocation by dephosphorylating Ntcp at S226 via activation of PP2B and/or inhibition of ERK1/2.
• PKC-zeta mediates cAMP-induced Ntcp translocation by facilitating PKB activation in hepatocytes.
• cAMP stimulates translocation of Ntcp, Bsep and Mrp2 by activating PKC-delta and/or p38 MAPK.
• cAMP reverses TLC-induced retrieval of Bsep and Mrp2 by reversing the effect of TLC on PKC-alpha and/or PKCE.
• cAMP stimulates net translocation of Ntcp by stimulating Rab4-mediated exocytotic insertion and/or by inhibiting Rab5-mediated endocytic internalization.

Proposed studies will be conducted in perfused rat livers, rat hepatocytes and HUH-7 cell lines. Hepatocytes and HUH-7 cells transfected with wild-type and mutant Ntcp will be used to determine the role of phosphorylation in Ntcp translocation. Role of various kinases will be evaluated by manipulating their activity using chemical inhibitors, wild-type and dominant negative plasmids and siRNA. Immunofluorescence and co-immunoprecipiation studies will be used to determine colocalization of Rab proteins with Ntcp. Collectively, proposed studies should provide further insights into signaling pathways by which cAMP stimulates bile formation and reverses acute cholestasis. In addition, these studies should help define the role of Rab proteins in cAMP-induced vesicle trafficking.

PI: Anwer, Sawkat
Title: Short-Term Training in Health Professional School
Abstract: The overall long term objective of this proposal is to stimulate research interests in talented veterinary students, and to expose them to opportunities inherent in a research career.

The program will provide research training opportunities for veterinary students in the following biomedical research areas:

• Digestive diseases
• Infectious diseases
• Biotechnology/reproductive biology
• Neuroscience/behavior
• International/wild life medicine

This program is specifically designed to introduce students to active biomedical research environments and to train them in following specific areas:

• Critical evaluation of published data
• Development of hypothesis
• Preparation of research proposals
• Experimental designs
• Research ethics
• Analysis and organization of data
• Oral and written presentations of results

The proposed program will accept 15 students per year, and the students will be selected on a competitive basis. All first and second year veterinary students will be eligible to submit a research proposal using a standard format with input from a program faculty of his/her choice. The submitted proposals will be evaluated and ranked in order of merit by a faculty committee using predefined criteria. Apart from the scientific merit of the proposal, preference will be given to minority students, to be actively recruited, and students who exhibit willingness to continue to conduct research beyond this training period.

Training during the months of June, July and August will take place in established laboratories with ongoing projects and funding. Each trainee will attend a seminar series conducted by the program faculty, submit a written report, and give an oral presentation at the annual veterinary student research day. The success and impact of the program will be evaluated by using a survey method to determine the number of trainees actively involved in biomedical research following their initial training period.

PI: Anwer, Sakwat
Title: Veterinary Research Manpower Development for Defense
Abstract: Recognizing that 75% of bioterrorism agents are animal diseases, there is an increased demand for veterinarians to help defend our country. Specially trained veterinarians are needed to support the national biodefense effort. These areas of training and the support that they provide are:

1. Veterinarians trained in research to support the military's research into identification and treatment of weaponized animal diseases, foreign animal diseases, and chemical/biological threats.

2. Veterinarians trained in public health to plan and implement our response to bioterrorisim outbreaks, to ensure a safe food supply, and to assess environmental threats for the nation as well as the military.

Tufts' Cummings School of Veterinary Medicine is uniquely able to train a supply of veterinarians with these important research and biodefense skills. Cummings Veterinary School operates one of the country's leading research programs in biodefense related to identification, prevention, and treatment of food and waterborne and zoonotic diseases, including the National Botulinum Center, the Microbiology Unit of the National Food and Waterborne Diseases Integrated Research Network, and an NIH-funded Regional Biocontainment Laboratory.

The relevance of this proposed program is that it will develop a pipeline of research-ready veterinarians to solve biodefense and public health problems facing our country, in general, and our armed forces in particular. This particular phase of the program (Years 5 and 6) is an extension of this project that will enable students enrolled later in the program to continue, as well as, to enroll additional new students over the continuing two-year period.

PI: Baise, Laurie
Title: CAREER: Integrated Research and Education in Regional Evaluation of Seismic Hazards
Abstract: The proposed work includes an integrated research and education plan that incorporates local soil information into the regional evaluation of seismic hazards, and expands visualization and analysis tools for the evaluation of three-dimensional geotechnical data.

The project will focus on soil amplification and liquefaction. The proposed research aim is to improve visualization and analysis tools that incorporate the overall variability as well as the spatial correlation of geotechnical data to improve regional evaluations of seismic hazards. More accurate, detailed maps of liquefaction and soil amplification susceptibility that account for inherent geologic variability will allow for an evaluation of the effect of this variability and therefore considerably improve the  assessment of these seismic hazards over a regional scale. This in turn will allow communities to better plan and mitigate their effects on the built environment. The proposed educational aim is both to encourage the next generation of engineers and scientists to work on seismic hazards and to educate all students about the cause of geologic hazards and associated risk so that they may be informed citizens.

The PI has developed a long-term research plan that connects geotechnical and geological information over multiple scales to better evaluate the effects of seismic hazards. The proposed research plan grows directly from PI’s research experience in site response, regional wave propagation, and liquefaction hazard. Her long-term career goal is to be a recognized expert in spatial statistics and regional geohazards. The proposed work builds on the PI’s on-going and prior work by focusing on the central theme of regional seismic hazards, and it extends her work through an extensive study of the effect of soil spatial variability on regional seismic hazards.

The PI is committed to undergraduate research and education, and she recently received the UNITE (Undergraduate Initiatives in Teaching Excellence) Award for her mentoring of undergraduates in research and web-based initiatives in undergraduate curriculum. The proposed work will continue her initiatives in undergraduate education and extend them to middle school education. The proposed study will focus on the integration of local geotechnical data into regional seismic hazard evaluations through visualization and spatial analysis tools for research and education. The integrated research and education plan consists of five specific objectives:

1. Develop visualization and analysis tools for multidimensional subsurface data
2. Spatially characterize regional geo-datasets
3. Evaluate spatial simulation methods and address data sampling issues
4. Develop methods for regional mapping of seismic hazards
5. Build middle school seismic hazard curriculum for the Seismic Hazards web-based GIS

Intellectual Merit and Broader Impacts.
The proposed research has significant intellectual merit as it advances the assessment tools for regional seismic hazards. In the 2004 strategic plan for the National Earthquake Hazards Reductions Program (NEHRP) entitled “Expanding and Using Knowledge to Reduce Earthquake Losses,” Goal C in the report was to improve seismic hazard identification and risk assessment methods. Within this goal, the strategy is to “work with cooperators to develop a standard methodology for production of large-scale seismic hazard maps for urban areas” and “[to incorporate] uncertainty into hazard estimates.” The proposed work responds directly to this research need by evaluating the effect of soil spatial variability on regional seismic hazard maps.

The proposed research and education activities have broader impacts in:

1. Multidisciplinary education
2. Exposure to middle school teachers and students to science, math, and technology through seismic hazards
3. Undergraduate research
4. Societal impacts (reduced losses) resulting from improved regional hazard maps

PI: Balbach, Edith
Title: Organized Labor and Tobacco Industry
Abstract: While the shared interests that unions and public health have in worker health has led to cooperation on many health issues, it has often not led to such cooperation on tobacco control. While some unions have been supportive of tobacco control efforts, many have not, and a number have adopted pro-tobacco industry positions, especially protesting tax increases and assisting in the defeat of smoke free air legislation. We will build on our previous study of the relationship between organized labor and public health and continue to explore ways to improve that relationship, which has the potential to improve the health of blue collar and service sector workers, a group with higher than average smoking rates. Our overall research goal is to continue to build an understanding of the relationships among the tobacco industry, organized labor, and public health, focusing on state level activity in 9 states. To achieve this goal, we will pursue three specific research aims:

Specific Aim #1: Continue our comprehensive search of the tobacco industry databases for materials related to the political interactions among public health, organized labor and the tobacco industry, focusing in this renewal on the state level issues related to smoke free worksites and excise tax increases.

Specific Aim #2: Develop 9 case studies based on research conducted under Specific Aim #1, using additional information found through the labor press, local newspapers, and other written sources, and through interviews with key labor leaders and public health activists.

Specific Aim #3: Using comparative case analysis, synthesize the 9 case studies conducted under Specific Aim #2 with three state level case studies completed under parent grant (2002-2005) to build a comprehensive study of public health's efforts to work with organized labor on excise taxes and smoke free worksites.

To lower the high number of deaths caused by smoking, public health must lower smoking rates. Two of the leading interventions to achieve this reduction are worksite restrictions and excise tax increases. To implement these interventions in states with strong unions, public health must work with organized labor. This project will study the factors that facilitate and impede that relationship with a goal of improving it.

PI: Baleja, James
Title: Molecular Interactions in Signaling and Vesicle Sorting
Abstract: Proteins containing EH domains and proteins containing Asn-Pro-Phe (NPF) sequences are ubiquitously involved in fundamental molecular sorting mechanisms. Reps1, an EH domain-containing protein, and FIP2, an NPF-containing protein, are downstream targets of Ras and Ral GTPases and the EGF receptor, suggesting that they are involved in both signal transduction and vesicle-mediated trafficking. Despite the importance of the EH domain-NPF interaction in building the protein networks responsible for vesicle-mediated transport and signal transduction, the factors that regulate their association are largely unknown.

Knowing the basic principles that govern their regulation will lead to strategies to control these proteins and a better understanding of their biological roles. The EH domain of Reps1 binds directly to the NPF sequence of FIP2. Using Reps1-FIP2 as model system, the objective of this proposal is to evaluate the factors that modulate EH domain-NPF interaction. EH domains bind Ca2+ using the EF-hand fold.

Aim 1:  EH domain affinity for Ca2+ and Mg2+ will be measured to test a new regulatory hypothesis operating in vesicle sorting. To examine the conformational changes that accompany metal ion binding, the structures of the apo and Mg2+-bound domains will be determined using NMR and compared to the Ca2+-bound structure. FIP2 and Reps1 both oligomerize through a coiled-coil domain.

Aim 2:  We will determine the structure of the FIP2 coiled-coil domain, and observe how oligomerization governs the strength of the interaction between these proteins.

Aim 3:  To understand the mechanism of binding, the structures from a series of unbound peptides as well as an EH domain-NPF complex will be determined. An inhibitory peptide, designed to be stable in cell-based assays, will also be optimized and tested as a tool to understand the roles of EH domains in vesicle-mediated internalization and secretion.

Ultimately, the results of these experiments will provide insight into the vesicle sorting mechanisms important for diverse processes such as fertilization, neurotransmission, and cell growth. These investigations on the EH domain-NPF interaction are also expected to advance the application of NMR in understanding molecular recognition.

PI: Baleja, James
Title: Molecular Interactions in B Cell Development
Abstract: Maintenance of circulating antibodies requires proper B cell development. Gene rearrangement and editing provide for antibody diversity during development, but can also result in improperly formed antibody chains. There is a proofing mechanism at the pre-B cell stage of development in which the nascent µ heavy chain is probed for proper folding by a light-chain like molecule called the surrogate light chain. The folding and assembly of the surrogate light chain is complex and comprises two polypeptides, VpreB and 14.1. Successful complex formation between the heavy chain and the surrogate light chain results in expression of the pre-B cell receptor on the cell surface. Assembly of the pre-B cell receptor ensures survival and proliferation of that particular B cell. We do not know the mechanism by which the one and only surrogate light chain assembles and is able to pair with each member of the large and diverse group of heavy chains. Such knowledge is key to understanding B cell development.

Together we have the reagents and expertise to test the model that the two polypeptides of the surrogate light chain assemble to form a structure that resembles the structure of a mature light chain. We will provide direct experimental evidence for this hypothesis and extend the model to include the conformation of the non-immunoglobulin regions. The Baleja laboratory uses NMR to determine three-dimensional structures that guide study of protein function and the Stollar laboratory focuses on the molecular mechanisms by which antibodies, principally those associated with autoimmune disease, recognize their antigen. While the Baleja laboratory is well versed in NMR methodology applied to protein-DNA interactions and protein-lipid interactions, this is an exploratory (R21) application in a new scientific area-the protein-protein interactions that underlie B cell development. The specific aims are as follows:

1. Structure Determination of the Variable Domain of the Surrogate Light Chain.
   We will determine the three-dimensional structure for the immunoglobulin-like variable-domain of the surrogate light chain using NMR methods. We will compare the structure with known structures for immunoglobulins. We will also identify the residues of the variable domain of the surrogate light chain that respond to heavy chain binding using NMR methods.

2. Identification of the Role of Non-irnmunoglobulin Domains in Surrogate Light Chain Function.
   We will test the hypothesis that the unique, non-immunoglobulin domains of the surrogate light chain participate in surrogate light chain structure by measuring affinities of a series of surrogate light chain constructs with and without the presence of the unique regions for the VpreB<->14.1 interaction and for the pseudo-ternary complex of VpreB, 14.1, and heavy chain. We will also determine the extent to which the unique, non-immunoglobulin domains of VpreB and 14.1 proteins have three-dimensional structure, either as isolated peptides, or with the adjacent immunoglobulin like domain.

This work is innovative in that no one has obtained a three-dimensional structure of a portion of the surrogate light chain that can be compared to the lambda light chain of mature immunoglobulin and that we couple structural information with other biophysical techniques to measure interactions between the surrogate light chain components and VH. The work is also exploratory because we ask the question whether the unique regions have defined structure, either alone, or in complex with each other. Determining the structures will provide the necessary background for future mutagenesis experiments that probe the role of individual amino acid residues. Finally, these studies use a heavy chain with nucleic acid-binding reactivity that represents a potential precursor of a pathogenic autoantibody associated with systemic lupus erythematosus.

PI: Bedenice, Daniela
Title: Humoral Response to EEE Vaccination in Alpacas
Abstract: The purpose of this study is to establish the serological (humoral) response to an Eastern Equine Encephalitis (EEE) vaccine (Encevacä, Intervet) in 45 healthy alpacas.

PI: Bedenice, Daniela
Title: BVD Viral Infection and Immunity in Alpacas
Abstract: Bovine Viral Diarrhea Virus (BVDV) has been recently recognized as an important and potentially prevalent cause of serious illness, including diarrhea, abortion, wastage and death, in South American Camelids (SAC), posing a significant threat to herd health. The major sources of BVDV transmission are persistently infected (PI) herd members, which shed large amounts of virus throughout their entire life. Based on extensive experience with cattle, PI crias are suspected to be born if their dam was infected by the BVDV in early to mid gestation. At this time, the immune system of the cria is still underdeveloped and may become “tolerant'' of the virus due to its inability and develop an appropriate immune response (“immunotolerance”).

Currently, no known treatment is available for PI patients. Although the virus is generally readily eliminated by adult, acutely infected animals, immunotolerance in PI patients allows for persistent viral shedding. Acute and persistent BVD infection may also result in immune suppression, lading to low white cell counts and secondary "opportunistic" infections.

BVDV is a member of virus family called "Faviviridae", to which the human ''Hepatitis C" virus also belongs. Extensive research continues to be performed in humans to establish an effective treatment to cure Hepatitis C. Commercially available anti-viral medications which are currently considered standard care in human Hepatitis C patients include Pegylated-Interferon (to stimulate the immune system) and Ribavirin (an anti-viral). Additionally, laboratory evaluations have also considered Acyclovir (used in horses against Equine Herpes Virus), BPIP and Ribavirin (anti-virals) to be effective against BVD in cell culture.

The goals of the proposed clinical trial are therefore two-fold:

1. Evaluate the immune function of crias persistently infected with BVDV due to natural exposure(i.e. establish cellular as well as antibody immune responses in PI crias).

2. Evaluate the clinical efficacy of parental administration of Interferon, Acyclovir and BPIP in persistently infected crias.

The first part of the study may establish insight into the cause (pathogenesis) of immune suppression in PI crias by evaluating the presence of specific subsets of lymphcytes (immune cells) called CD 4, CD8 T-lymphocytes as well as B lymphocytes and neutrophils. The second part of our proposed clinical trial is considered experimental, since all dosages and pharmacokinetics of drugs will have to be extrapolated from other species (as is commonly performed for the use of most medications in camelids). For this reason, animals will remain under close veterinary supervision, and only parentera1 medication will be used, limiting suspected species-specific variations in the absorption of medication. All animals (naturally infected PI crias) used in this trial will remain client-owned and treated after written consent by the legal owner.

PI: Bers, Marina
Title: CAREER: Virtual Communities of Learning and Care: Multi-User Virtual Environments that Promote Positive Youth Development
Abstract: Tufts University hopes to sponsor a pilot project on Broadening Participation in Computing. Our project, Computing Undergraduate Scholars Program (CUSP), will take place the during 2006-2007 involving 12 undergraduate students, with 2 students funded on each of six collaboratively linked supplements to existing NSF CISE research grants.

Each of these students will have demonstrated financial need and will come from a group that is underrepresented in computing. It has been widely documented that students with demonstrated financial need face more than just the constraints of time imposed by economic necessity. Many of these students lack role models, such as older siblings, friends, or parents, who have experienced academic success. For women and underrepresented minorities within this cohort, such problems may require even more of an adjustment in order to persist successfully. However, the benefits accruing to students from not having to work for financial support are clearly established in science and engineering majors.

The primary goal of the full instantiation of CUSP planned for the future is to improve the recruitment, retention and graduation rates of women, underrepresented minorities, and first-generation college students who have financial need and interest and ability in computing fields. These students will be welcomed into a collegial and collaborative environment including regular interaction with faculty and graduate students, immediately exposing them to the possibilities afforded in the field of computer science. Most importantly, the full CUSP program will create an academic and social foundation that assists these students, first during their transitions from high school to college as they become exposed to computing as a discipline, and then by engaging them actively in computing research. In addition, each CUSP student will be provided with a stipend for taking on increasing amounts of responsibility within the model: in exchange for actively engaging as mentors, role models, and scholars, students will be relieved of the necessity to work in an unrelated work-study position during the academic year.

The pilot version of CUSP, funded through BPC supplements to active research grants, will focus on the transition from foundational course work to active engagement in research, rather than on the transition from high school to college. The CUSP pilot will create a community of talented Computer Science students comprising roughly 5 sophomores and 5 juniors and 2 seniors at Tufts who have already taken foundational courses in computer science, have expressed some interest in computing, and will benefit from the financial, social, and academic support together with the excitement of conducting computation-based research.

Students will be identified and recruited during the late spring of 2006. To qualify for support under this program, students must:

1. Be pursuing a course of study consistent with a major in Computer Science, Computer Engineering, or Computational Science

2. Enroll full time during the period of their award

3. Maintain a 3.0 GPA in their program or have developed a written plan with their advisor for academic improvement and utilization of support resources

CUSP support will cover the sophomore and junior years of selected students. The CUSP program will include the following components:

1. Intense Advising
2. Weekly Seminars
3. Engaging Students in Undergraduate Research

PI: Blumsack, Marilyn
Title: Osher Institute for Lifelong Learning
Abstract: Founded in fall 2000, The Tufts Institute for Lifelong Learning (TILL) is now a mature, proven center of life-long learning for the retired and near-retired population in the greater Boston area. During calendar year 2004, it served 230 participants who accounted for 435 course enrollments. It offers study groups of varying length, a Lunch and Learn series, and regular off-campus special event. It maintains a collaborative relationship with Lexington's Brookhaven retirement community.

TILL seeks support from The Bernard Osher Foundation for two purposes: to increase its membership and to broaden the scope and number of its core eight-session and four-session study group offerings. These objectives are not mutually exclusive. Both are essential to achieving the financial strength that will ensure the Institute's future. To that end, a first-year grant of $99,000 and any subsequent yearly grants of similar amounts from the Foundation will be used to invest in membership and program development by:

• Hiring a professional-level fulltime associate director with primary responsibility for developing and implementing an enhanced and expanded marketing program.
• Supporting this larger marketing effort with adequate resources and focusing it on attracting members from TILL's "home town" communities of Medford and Somerville and on the suburbs north and west of Boston.
• Supplementing TILL's cadre of volunteer study group leaders each academic year with from four to six faculty or graduate students who will receive stipends of $2,000 to $3,000 per study group.

These faculty members will teach subjects of known interest to TILL's members but in which no member possesses the expertise to serve as a volunteer study group leader. They will be drawn only from Tufts faculty, faculty emeriti, and graduate students and will be used sparingly so as not to undermine TILL's ongoing, fundamental commitment to peer learning. Our goal will be an active membership of 300 in no more than three years.

PI: Bohm, Andrew
Title: Mechanism of Poly(A) Polymerase Processivity
Abstract: Template-independent elongation of the 3' end of mRNA by poly(A) polymerase (Pap) occurs in virtually all organisms. In eukaryotes, the non-coding mRNA extensions added by Pap serve as molecular handles, which interact with nuclear export, translation and mRNA degradation machinery, and strongly effect mRNA stability and translational efficiency. Our recent crystal structure of yeast poly(A) polymerase (Pap1) in complex with the non-extendable ATP analog 3-dATP revealed the catalytic mechanism for base addition, but not the basis for adenosine specificity, the path of the poly(A) tail through the large cleft surrounding the active site, or the structural basis for the high degree of processivity exhibited in vitro by purified Pap1. Pap1 is an excellent model for understanding processivity, since unlike most other polymerases, including mammalian Pap, it does not require additional proteins to achieve processivity. We propose to use x-ray crystallography as well as fluorescence resonance energy transfer, cross-linking, and activity measurements to study the relationship between the structure of Pap1 and its ability to specifically and processively elongate mRNA. We also propose work aimed at a structural understanding of how Pap1 is regulated by two other components, Fip1 and Yth1, of the mRNA cleavage/polyadenylation complex. Our preliminary crystallographic results suggest a basis for nucleotide specificity. Also, we have very recently identified a unexpected RNase activity in highly purified Pap1. Polyadenylation is a major regulator of protein expression. The work we propose will provide a dynamic model of the molecular events associated with this important regulatory process.

PI: Booth, Sarah
Title: Vitamin K and Inflammation
Abstract: Chronic inflammation is characteristic of osteoporosis and coronary heart disease, and markers of inflammation are associated with increased risk of bone loss and cardiovascular events. Vitamin K supplementation may have a protective effect against the progression of osteoporosis and vascular calcification by decreasing production of inflammatory mediators, potentially through its role in the regulation of ceramide. We are currently conducting a three-year randomized, double blind, controlled randomized trial to determine whether supplementation with vitamin K, in an amount that is expected to be both nutritionally optimal and safe, will reduce bone loss at the hip and progression in coronary calcification in 452 older men and women who are calcium and vitamin D-replete. This grant will explore potential mechanisms by which vitamin K may be linked to these outcomes. To the best of our knowledge, this proposed novel mechanism linking vitamin K to inflammation, through its role in the regulation of ceramide levels, has not been examined in humans. The following proposal presents a unique opportunity to examine the potential role of vitamin K supplementation on changes in inflammation among older men and women already being measured for progression of bone loss and vascular calcification. It will also provide the foundation for future studies on vitamin K, inflammation and aging.

Hypothesis 1: Supplementation with vitamin K will lower circulating levels of markers of inflammation in elderly men and women, and the decrease in inflammation in response to vitamin K will be greater in individuals with higher levels of inflammation at baseline.

Specific Aim 1a: To compare the 3-year change in serum IL-6, and other markers of inflammation (IL-1, TNFα, CRP, and PGE₂) in older men and women receiving vitamin K supplementation of 500µg/d to those not receiving vitamin K supplementation.

Specific Aim1b: To determine the association between the baseline levels of IL-6 and other markers of inflammation (IL-1, TNFα, CRP, and PGE₂) and change in bone mineral density (BMD) at the femoral neck in older men and women receiving vitamin K supplementation, and in those not receiving vitamin K supplementation.

Specific Aim 1c: To determine the association between the baseline levels of IL-6 and other markers of inflammation (IL-1, TNFα, CRP, and PGE₂) and change in coronary calcification in older men and women receiving vitamin K supplementation, and in those not receiving vitamin K supplementation.

Hyothesis 2: Vitamin K supplementation modulates IL-6 production through its role in the regulation of ceramide levels.

Specific Aim 2a: To compare the 3-year change in serum ceramide in older men and women receiving vitamin K supplementation of 500µg/d to those not receiving vitamin K supplementation.

Specific Aim 2b: To determine the association between 3-year change in IL-6, and other measures of inflammation, and change in serum ceramide in older men and women receiving vitamin K supplementation, and in those not receiving vitamin K supplementation.

Hypothesis 3: Vitamin K supplementation modulates OPG and RANKL production through its influence on IL-6 levels, and other measures of inflammation.

Specific Aim 3a: To compare the 3-year change in serum OPG and RANKL levels in older men and women receiving vitamin K supplementation of 500µg/d to those not receiving vitamin K supplementation.

Specific Aim 3b: To determine the association between 3-year change in OPG and RANKL, and change in serum IL-6, and other measures of inflammation in older men and women receiving vitamin K supplementation, and in those not receiving vitamin K supplementation.

PI: Booth, Sarah
Title: Dietary and Non-Dietary Components of Vitamin K Metabolism
Abstract: Vitamin K has an emerging role in bone health. However, little is known about the vitamin K metabolism, particularly in regards to aging, and maintenance of bone mass. Such limited understanding about vitamin K metabolism currently impedes the ability to establish dietary recommendations for vitamin K, and interpret the results from recent clinical trials on vitamin K supplementation and bone health currently being conducted in primarily women of a narrow age group. The proposed study will be the first to assess the role of dietary and non-dietary factors that influence the response to measures of vitamin K status and bone turnover to vitamin K depletion and repletion in younger and older men and women. This study will also compare the absorption efficiency of vitamin K, relative to current vitamin K status. Men and women [21 younger (21-40y) and 21 older (60-80y)] will participate in a 65-d metabolic study, with a 5-d run-in period, followed by a 30 d dietary vitamin K restriction period (10 µ/g/d), and ending with a 30 d dietary vitamin K supplementation period (500 µ/g/d). Serial measurements of markers of vitamin K status (plasma phylloquinone, urinary Gla, serum % uc and plasma PIVKA-II) and bone turnover (serum osteocalcin and NTx) will provide information on their response to dietary manipulation of vitamin K for both age groups under identical controlled dietary conditions. A stable isotope tracer (deuterium-labeled vitamin K in collards) will be used to compare the absorption of vitamin K during a vitamin K-deplete state (21 d of dietary vitamin K depletion) to that of a vitamin K-replete state (21 d of dietary vitamin K repletion). Because vitamin K is transported in triglyceride-rich lipoproteins, which may vary among individuals due to differences in adiposity, measurement of body composition by DXA and plasma lipids (total and individual lipoproteins) will provide insight into the role of lipids in absorption and transport of vitamin K. The findings of the proposed study are critical for the interpretation of the epidemiologic and clinical data used to determine optimal intakes at which vitamin K may have a protective role in bone health.

PI: Borgers, Christoph
Title: Modeling and Analysis of Persistent Gamma Rhythms and Their Role in Sustained Attention
Abstract: In a variety of animal species and experimental tasks, states of attention depend critically on the cortical neuromodulatory effects of acetylcholine, and are associated with cortical oscillations at gamma frequencies (30-80 Hz). Moreover, cholinergic agonists are known to elicit persistent gamma oscillations in cortical slice preparations. Taken together, these lines of evidence suggest a functional link between gamma rhythms and attentional processes. The nature of this link, however, is not well understood.

The proposed project has two aims. The first is to construct and analyze minimal mathematical models of persistent gamma rhythms, abstracting from non-essential biophysical details in order to clarify essential dynamical mechanisms. The second aim, which depends on the first, is to explore the possible functions of gamma oscillations in attentional processes, particularly the hypothetical role of persistent gamma as a correlate of sustained attention. The proposed work is to be carried out in collaboration and consultation with experimentalists and theorists at Boston University's Center for Memory and Brain.

Broader impacts of proposed activity Oscillatory electrical activity in the brain has been studied for about a century. Much is now known about both the mechanisms of brain rhythms and their correlations with behavioral states. What is still lacking, however, is a bridge between the biophysics of cortical oscillations, most conveniently studied in vitra, and their functional significance, which can only be studied in viva. By modeling the link between gamma oscillations and attention, we hope to design such a bridge.

Graduate and undergraduate students at Tufts University will participate in this work. Strengthening mathematical biology will have significant impact at Tufts, since it has not been strongly represented in the past, although biology, psychology, and the medical sciences are particular strengths of the University.

PI: Bridges, Robert
Title: Endocrine Regulation of Maternal Behavior
Abstract: The long-term goal of this project is to identify the neural and neurochemical mechanisms underlying the regulation of maternal behavior and to elucidate potentially novel mechanisms of neural plasticity associated with the expression of maternal behavior in the adult mammal. The specific hypothesis that will be tested is that the induction, maintenance, and retention of maternal care are under endocrine regulation by a neural lactogenic system, a system that displays significant plasticity as a function of reproductive experience. The first series of studies will examine the role of the neural prolactin (PRL) receptor in the initiation of maternal behavior. Using a rat model, we will determine whether placental lactogens, like PRL, act via the PRL receptor to stimulate the onset of behavior. A second study will use the novel PRL receptor antagonist, S179D-PRL, which will be administered centrally to test the hypothesis that activation of the PRL receptor by lactogenic hormones around the time of birth is essential f or the normal onset of maternal care. The third study using ISHH will measure how pregnancy concentrations of progesterone (P) and estradiol affect expression of mRNA for neural PRL receptors. Then, central sites of P action will be examined to see how P affects the onset of maternal care and to delineate a mechanism of P's action in initiation of maternal behavior. A second series of experiments will determine the involvement of the endocrine system in ongoing maternal care. First, the effects of exposure to PRL-secreting ectopic pituitary grafts on pup-directed maternal care and maternal aggression will be measured. Then, the effects of central administration of the PRL receptor antagonist, S179D-PRL, will be examined in lactating rats. The third set of experiments will delineate the role of the endocrine system in the retention of maternal behavior. The involvement of PRL in the opioid-mediated establishment of the retention of maternal behavior will be assessed. Finally, the effects of prior maternal experience on activation of the neural lactogenic system will be measured to see whether prior maternal experience up-regulates the brain PRL system and makes the female more sensitive to her own neural hormones. The results of these investigations will delineate common endocrine and neurochemical regulators of maternal care in mammals, present a new model for examining neuroplasticity in the adult female, and provide a basis for evaluating the effects of endocrine and neurochemical imbalances on mother-young interactions.

PI: Bridges, Robert
Title: NCRR Research Training for Tufts Veterinary Students
Abstract: There is a need for veterinarians with research training to participate in academic as well as corporate based research in the fields of animal and human health. It is crucial for a set of 21st century veterinarians to acquire the scientific skills and technical training together with the conceptual framework to participate both as independent researchers and collaborators to meet the projected research needs in biomedical sciences. Through the training set forth in the present proposal, the objective is to provide bright, and highly motivated veterinary students with a one-year, in-depth research experience in a productive and active research setting.

Training will involve the use of animal models to develop skills at hypothesis-based, biomedical research. Over a three-year period, the aim is to train a minimum of nine students (three per year). Applicants will be actively recruited from the incoming and existing veterinary classes, with an effort made to insure participation by our minority students. Students will receive research training during a consecutive 12-month period after their first year of veterinary school, but prior to graduation. A program faculty that consists of 20 faculty from the three departments with expertise in numerous research areas that use animal models will offer both intensive laboratory training as well as complementary seminars and course work.

Key research areas include:

• Infectious diseases
• Reproductive biology
• Biotechnology
• Digestive diseases
• Neuroscience and behavior
• Oncology
• Nutrition
• Respiratory physiology

A wide variety of animal models are available for trainee projects, including pigs, horses, goats, sheep, cats, dogs, rats, mice, and shrimp.

Highly successful projects can be submitted to meet the requirements for a Masters Degree in Comparative Biomedical Sciences. All participating students will receive a certificate at commencement acknowledging their training experience. The program itself will contribute funds that can be used to support additional qualified applicants and/or supplement existing stipends.

It is a long-term objective to make this training experience a launching pad for career involvement in biomedical research for the veterinary students. The success of the program will be evaluated by determining the number of trainees that integrate health science research into their professional careers.

PI: Bridges, Robert
Title: Neuropeptidergic Control of Maternal Behavior
Abstract: The following three specific aims are proposed to address the overall hypothesis that AVP has significant roles in the retention of maternal behavior, the expression maternal aggression, and that there are potential connections between central arginine vasopressin (AVP) levels and the expression of maternal behavior and aggression. In addition, the potential connection between AVP’s effects on maternal behavior and its established role in social recognition will be explored.

1. The first specific aim is to investigate the effects of AVP on the initiation of maternal behavior, social recognition, and the retention of maternal behavior in primiparous rats. In contrast to the paucity of research focusing on AVP and maternal behavior or aggression in the rat, there is a wealth of data supporting AVP’s role in social recognition and parental behavior in other rodent species. Since the brain regions implicated in rodent social recognition, such as lateral septum, are also implicated in parental behavior, it is possible that a similar neural pathway controls these two social behaviors. The use of the primiparous model allows for the investigation of interactions between AVP and the experience of pregnancy and parturition. Based upon AVP’s effects on social recognition and social behavior in numerous species, it is hypothesized that the administration of an AVP V1a antagonist during parturition will interfere with the retention of maternal behavior and social recognition.

2. The second specific aim is to investigate the effects if AVP on the expression and decline of maternal aggression. It has been shown that AVP stimulates aggression in non-lactating rodents, but its role in maternal aggression has not been investigated. It is hypothesized that exogenous AVP will increase, and a V1a antagonist will decrease maternal aggression.

3. The third specific aim will characterize AVP and AVP mRNA levels in lactating postpartum primiparous rats and evaluate potential correlations between AVP, AVP mRNA and maternal aggression during lactation. If AVP is involved in postpartum changes in maternal aggression, these changes may be reflected in alterations in central AVP activity. It is hypothesized that relative AVP and AVP mRNA in selected neural regions and postpartum maternal aggression will be positively correlated.

PI: Brodeur, Peter
Title: Organization and Expression of V Genes
Abstract: Antigen specific receptors of B and T lineage cells require the somatic assembly of V, D, and J gene segments during lymphocyte development. Immunoglobulin and T cell receptor loci are targeted by a common V(D)J recombinase in a cell type and developmental stage-specific manner. The fundamental control of this process occurs at the level of alterations in chromatin structure that confer locus "accessibility" to the RAG1/2 complex. The immunoglobulin heavy chain locus (Igh) is the first to rearrange during B cell differentiation and accessibility at different stages is limited to discrete domains. The cis-acting elements and signaling pathways that regulate accessibility throughout the lgh locus are yet to be fully elucidated. Since V(D)J recombination is required for normal lymphocyte development, defects in the process result in both subtle and catastrophic immune deficiencies. Thus, understanding the underlying mechanisms has obvious implications to health and disease.

Our long term goals are focused on the control of Igh locus accessibility within the nearly 3 megabase region containing approximately 200 Vh gene segments. In this application, we propose to extend our studies of a candidate control region in the 5' boundary of the lgh locus that exhibits early B cell specific chromatin remodeling. This novel region interacts, both in vitro and in vivo, with factors that have been implicated in Igh accessibility and rearrangement. We propose to examine sorted early B cell subsets from bone marrow to determine the precise stage of B cell development at which this element is active.

Gel shift and chromatin immunoprecipitation approaches will be used to define the factors that are recruited to the multiple sites mapped within this region. The function of this putative regulatory element will be tested by both genomic deletion and studies of BAC transgenes containing putative control regions and Vh segments of the 5' region of the locus. We will use a novel cell line model as well as primary bone marrow cells to determine the mechanism through which IL-7 receptor signaling selectively mediates D-distal (5') Vh gene accessibility.

PI: Brugge, Douglas
Title: Language, Literacy, Culture: Communication of Health Concepts
Abstract: Spoken communication of key health concepts for many diseases is critical to prevention, screening and clinical management. This communication is made more difficult when words get in the way. Health literacy is affected by general literacy, language and cultural interpretation. Very little is known about maximizing spoken communication of health concepts across these potential barriers. Because of the lack of knowledge, there is a need for first stage studies that lay the basis for future research. We propose a study that will develop methodological approaches to the study of effective spoken communication of three key health concepts: headache, wheezing, and depression.

Our primary hypothesis is that different language groups and groups with different levels of general literacy will have different levels of health literacy which will, in turn, be reflected in their understanding of spoken key health concepts. Further, we hypothesize that we will be able to develop from focus group data a conceptual framework that can be applied to future intervention studies designed to overcome spoken health communication barriers.

The first phase of the study will consist of key informant interviews with medical experts, some of whom are bilingual in Spanish or Cantonese. In the second phase, we will hold 12 focus groups of 8-10 persons in English, Spanish and Cantonese, each at low and high general literacy. We will use highly skilled focus group moderators from Erlich Transcultural Consultants who have many years of experience leading focus groups in each language. The focus groups will be held in a new state-of-the-art focus group facility with extensive technical capability, including an observation room in which the project leaders will observe the focus groups in the presence of a simultaneous translator. Focus groups will be video recorded and the resulting record transcribed and translated. We will use data immersion to analyze the focus groups. This will include a systematic content analysis, a thematic analysis and a logical analysis. Our goal is to develop sufficient understanding of how persons with different language, culture and general literacy understand key health concepts so that an intervention study (R01) aimed at improving spoken health communication can be developed.

PI: Brunken, William
Title: Extracellular Matrix in Synapse Formation in the CNS
Abstract: Laminins are biologically active molecules that function as cell adhesion molecules, regulate various aspects of development, and serve to stabilize complex anatomical structures. They are large extracellular matrix molecules which are composed of three subunit chains, designated alpha, beta and gamma. Five alpha, three beta and three gamma chains have been identified. Laminins are widely expressed in the CNS; as are their receptors. Several disorders of the nervous system are linked to laminin genes: some congenital muscular dystrophies involve the alpha2 chain (merosin); the beta2 chain is reduced in Walker-Warburg syndrome; and a complex group of CNS developmental disorders (muscle-brain-eye disease; retinitis pigmentosa with deafness (RP21 with deafness); Walker-Warburg syndrome) maps to the site of the gamma3 gene. Genetic disruptions in some laminin-related genes also result in human disease and in dysmorphogenesis in animal models. We have identified two novel CNS laminins, alpha4beta2gamma3 and alpha5beta2gamma3 (LN 14 & LN 15, respectively); these are found in the interphotoreceptor matrix and in the matrix of the outer plexiform layer (OPL). These laminins appear to play important roles in the morphogenesis of photoreceptors. First, these chains are expressed prior to the onset of rod genesis and persist into adulthood. Second, ablation of the gene encoding one of the beta2 chains results in the production of dysmorphic photoreceptors; specifically, photoreceptor outer segments are reduced in length and the photoreceptor terminals in the OPL are disrupted. Finally the amplitude of the ERG b-wave is drastically diminished suggesting that transmission from photoreceptors to second order cells is disrupted by loss of beta2-containing laminins. We hypothesize that LN 14 and 15 are critical mediators of synapse assembly and stabilization. Furthermore, we hypothesize that LN14 and 15 form unique substrates with which photoreceptor terminals interact. Specifically, we hypothesize that the molecular assembly and structure of the photoreceptor synapse is dependent on the interactions between these laminins and their receptors. We propose to test several aspects of this hypothesis. We will ask two specific questions:

1. What is the functional composition of the laminin complex in the OPL?
2. How does disruption of the laminin complex alter the functional organization of the OPL?

With these studies, we will: gain insight into the molecular mechanisms of synaptic assembly in the outer retina; define the role of the ECM in this process; and shed light on the basis of a series of genetic disorders in humans.

PI: Bunnell, Stephen
Title: Unravelling Integrin-Mediated Inflammatory Signaling Pathways Using Dynamic Imaging Techniques
Abstract: Mounting evidence suggests that inflammatory T cell responses play a major role in atherosclerosis, and therefore increase the risks of cardiovascular diseases such as heart attack and stroke. Integrin ligands are upregulated in atherosclerotic lesions and influence the progression of atherosclerosis. Although integrins govern the recruitment of immune cells to sites of inflammation, integrins also transmit costimulatory signals that sensitize T cells to antigen and promote the production of inflammatory T helper 1 (Th1) cytokines. In this manner, integrins may contribute to the development of atherosclerosis by sensitizing T cells to antigens that pose little intrinsic threat. Our specific hypothesis is that integrins enhance the production of inflammatory T helper 1 (Th1) cytokines by directly stabilizing antigen-induced signaling complexes.

To test how integrins influence the development of inflammatory responses, we developed novel methods of visualizing T cell receptor (TCR)-induced signaling complexes in real time. Here, we will determine how the Th1-promoting integrin VLA-4 impacts the formation of these TCR-induced microclusters, and test whether VLA-4 drives Th1-biased signals by altering microcluster formation, persistence, or movement.

AIM 1: How are the TCR and VLA-4 induced signaling pathways coupled? 
We propose that the signaling proteins associated with the TCR and VLA-4 form molecular bridges that integrate the signals from these receptors. We will determine which proteins co-localize with the TCR and VLA- 4 in model systems and in physiological immune synapses. Nanometer-scale interactions among these proteins will be assessed using FRET.

AIM 2: Which signaling molecules are essential for VLA-4 mediated co-stimulation?
We propose that VLA-4 associated signaling molecules promote inflammatory signaling by stabilizing TCR signaling complexes. We will identify these proteins by pairing cytokine production assays with dominant-negative and RNAi-mediated knock-down strategies.

AIM 3: How do essential signaling molecules potentiate inflammatory signaling?
Specific molecular interactions involved in VLA-4 mediated complex stabilization and inflammatory signaling will be identified. Functional and imaging assays will be performed to evaluate mutant variants of the proteins implicated in these processes.

PI: Bunnell, Stephen
Title: Visualizing the Mechanisms of Integrin – Mediated Lympocyte Co-stimulation
Abstract: Autoimmune diseases and immunodeficiency’s result when the immune system incorrectly perceives the “threat” posed by self-antigens or by pathogens. Co-stimulatory molecules play an essential, but poorly understood, role in this process. Integrins are co-stimulatory molecules that respond to ligands upregulated by the inflammatory processes associated with infection and autoimmune disease. The loss of proper integrin function leads to profound immunodeficiencies, and aberrant integrin activation is associated with common autoimmune diseases. To understand how immune responses are controlled, we developed a novel method to visualize the signaling complexes induced by the T cell receptor (TCR) in real time. How integrins modulate the signals transmitted by the TCR remains unresolved. Here we have adapted these assays to reveal how integrins impinge on the signaling complexes induced by the TCR. Our specific hypothesis is that integrins influence the course of immune responses and autoimmune disorders by directly interacting with and enhancing the signaling complexes induced by the TCR. In this proposal we will employ the integrin VLA-4 as a model co-stimulatory protein. VLA-4 is a critical lymphocyte integrin with well-characterized functions in cell adhesion and signaling. VLA-4 has also been implicated in the etiology of autoimmune disorders, such as multiple sclerosis. The mechanisms by which VLA-4 co-stimulates T cell activation are not well understood. These studies will provide insights into general mechanisms of integrin signaling and co-stimulation that are relevant to normal immune function and autoimmune diseases.

Our imaging system, capable of revealing molecular interactions at the TCR in real time, in living cells, provides an ideal basis for these studies. Using this system, we have, for the first time, presented direct evidence of a qualitative effect of integrin engagement on the signaling molecules associated with the TCR; namely, we have shown that VLA-4 immobilizes and increases the persistence of these signaling complexes, an effect that is perfectly correlated with effective co-stimulation. This breakthrough was possible because of our unique ability to track the persistence and movement of individual signaling complexes over time, using our dynamic imaging techniques. Here, we will extend our imaging techniques to explore protein localization and proximity in the immune synapses of living T cells. To do so, we will dynamically visualize multiple proteins in three dimensions, perform FRET experiments in live cells, and automate the reconstruction of immune synapses over time. Our immediate goal, which is to characterize the signals that link VLA-4 to the TCR, will be pursued in the following three aims:

Aim 1: What molecules link VLA-4 to the TCR, as assessed by FRET microscopy?
We propose that TCR and VLA-4-associated signaling proteins form a molecular bridge that integrates the signals from these receptors. We will determine which proteins co-localize with the TCR and VLA-4 in the immune synapse. Direct, nanometer-scale interactions will be confirmed by assessing fluorescence resonance energy transfer (FRET) between these proteins.

Aim 2: Which signaling molecules are essential for VLA-4 mediated co-stimulation?
We propose that TCR and VLA-4-associated signaling molecules play critical roles in the integrin-induced stabilization of the TCR signaling complex. We will identify these proteins by using dominant-negative and RNAi-mediated knock-down strategies.

Aim 3: How do essential signaling molecules exert their co-stimulatory function?
We propose to identify the specific molecular interactions involved in co-stimulation and complex stabilization by performing mutational analyses of proteins implicated in these processes.

By revealing the mechanisms of co-stimulation by integrins, these studies will identify targets for the development of small molecule therapeutics for autoimmune diseases.

PI: Camilli, Andrew
Title: Short-Term Training for Minority Students Program
Abstract: The Sackler program has been in operation for twelve years and has trained 160 minority students during this period. The objectives of this program are:

1. To increase participation of minority undergraduates in biomedical research through summer research internships at the Tufts Health Sciences Campus.

2. To enrich this internship experience with training in communication skills, career counseling, and networking activities.

3. To involve more faculty, postdoctoral fellows and graduate students on the Health Sciences Campus in the mentoring of minority students.

4. To increase the awareness of minority students as to the benefits of biomedical research and potential careers in this area, particularly in the areas of cardiovascular, pulmonary, hematologic and sleep disorder research.

This program, consisting of a ten-week summer research internship for 18 trainees, will give the students an opportunity to interact with, and work alongside outstanding research scientists. It will help them decide if biomedical research is a career they wish to pursue. For those who have already decided on biomedical-oriented careers, the training will give them the confidence and additional qualifications to apply to, and succeed in, the best programs. Our long-range goal is for these students to become independent scientists in academia or industry, and in this capacity, serve as mentors for other minority students.

PI: Camilli, Andrew
Title: Study of Transmissible Forms of Vibrio Cholerae
Abstract: Vibrio cholerae, which lives in association with plankton in brackish, temperate waters the world over, is the causative agent of endemic and epidemic cholera. Hallmarks of the disease include prodigious watery diarrhea resulting from the action of secreted cholera toxin (CT), and infrequent but deadly explosive epidemics. The strong link between explosive epidemics and human crowding accompanied with untreated drinking water suggests a very efficient mode of fecal-oral transmission.

We have discovered a heightened state of transmissibility of stool V. cholerae (referred to simply as "hyperinfectivity"), which persists even after shedding into water reservoirs. Knowledge of the molecular basis for this phenotype, and a general characterization of this transmissible form of V. cholerae, would contribute to the design of vaccines to prevent cholera at the initial stage of infection.

Aim 1 of this proposal will use transcriptional profiling and proteomics to help define this transmissible form. Spotted DNA microarrays will be used to determine the transcriptome of stool V. cholerae incubated in pond water. This will be compared to that of fresh stool V. cholerae to identify potential differences. The results will be validated by quantitatively assaying the steady state mRNA and protein levels from select genes. Microscopy and transcriptome data on stool V. cholerae predict a bacterial state of motility working in the absence of chemotactic signaling. This counterintuitive state is hypothesized to be responsible, at least in part, for the hyperinfective phenotype.

In Aim 2 of this proposal, quantitative immunodetection using paralog-specific antisera will be used to test for reduced expression of all three CheW linker proteins and all three CheR methytransferases in fresh and pond water-incubated stool V. cholerae, as is predicted by current transcriptome data. In addition, capillary tube chemotaxis assays will be performed directly on V. cholerae from these samples to substantiate this hypothesis.

Aim 2 will also test a second hypothesis, that ToxR regulated factors, which are essential for pathogenesis, are not playing a role in the hyperinfectious state.

Finally, Aim 3 will use mutation and infectivity analyses to determine if other metabolic, physiologic or phenotypic properties of the bacteria contribute to the hyperinfective phenotype or, alternatively, to colonization of an environmental planktonic host, Anabaena variabilis. These studies will establish a basis for understanding the hyperinfective phenotype, and the properties in general, that are exhibited by fresh and pond water-incubated stool V. cholerae. In turn, this knowledge will enhance our understanding of transmission of this and perhaps other water-borne pathogens, it will aid in the development of new cholera vaccines that target the antigens of 'incoming' vibrios, and it may suggest new approaches for the prevention of the dissemination of this lethal organism.

PI: Camilli, Andrew
Title: Role of c-diGMP Signaling in Vibrio Cholerae Virulence
Abstract: Knowledge of how facultative pathogens adapt to changing conditions when passing from an environmental reservoir into a human host is limited. Advances in our understanding of the basic mechanisms of adaptation to the host and induction of virulence genes will provide new molecular targets for therapy to reduce the large medical burden imposed by this diverse group of pathogens.

In the water-borne intestinal pathogen Vibrio cholerae we have demonstrated that signaling by external amino acids modulates the cytoplasmic concentration of the secondary messenger cyclic diguanylate (c-diGMP), leading to reciprocal regulation of genes important for biofilm formation and virulence. We hypothesize that c-diGMP also mediates the transition from environmental to virulence gene expression upon entry into the host.

In this project we will investigate the mechanism and general importance of
c-diGMP control of virulence gene regulation. We will characterize the sensory, regulatory and enzymatic properties of a phosphorelay system that lowers the cellular c-diGMP concentration by hydrolysis of c-diGMP and is required for virulence. We will also characterize other c-diGMP hydrolytic proteins that exert control over the cellular c-diGMP concentration during the transition from environment to host. Finally, we will investigate the mechanism by which c-diGMP influences virulence gene transcription.

We anticipate that this work will provide a working model of the central regulatory pathway in V. cholerae that initiates virulence gene expression upon entry into the host. We also anticipate that these studies will have application to a broad range of facultative prokaryotic pathogens since most appear to contain multiple
c-diGMP synthetic and hydrolytic proteins.

PI: Cao, Caroline
Title: CAREER: Adapting to Technology in Minimally Invasive Surgery
Abstract: Advances in robotics and computer-based technology have enabled much complex work to be performed via remote control.  One example of this development is minimally invasive surgery (MIS). Successful minimally invasive surgery (MIS) relies on innovative technology that allows surgery to be performed remotely, and effective integration of this technology into the surgical process.  Technology can improve the performance of the system with increased reliability and precision. However, it can also create unexpected interactions and new forms of errors (Reason, 1990; Cook & Woods, 1994). High error rates in surgery, in particular those associated with technology in minimally invasive surgery, have been documented (Leape et al, 1991; Shea et al, 1996; Wu et al, 2000). There is a great need to improve system performance and patient safety (Bogner, 1994; Kohn et al, 1999), but relatively little systematic research conducted to this end.

Our long-term goal is to enhance human performance with enabling technology in minimally invasive surgery through effective human-machine interface design and training systems. This requires that we understand the system requirements, constraints, and the interactions between various system components upon the introduction of new technology.

This proposal aims to combine research activity and education in an integrated multi-disciplinary program. It fits well with the goals of Tufts University in providing cross-disciplinary education and research opportunities for students in Arts & Science, Engineering, and Health Sciences.  

The specific objectives of this application are the following: 

Research:

1. Study the effects of evolving endoscopic technology on the surgical process in minimally invasive surgery, and how novel technology (e.g., robotics) can be used to better augment surgical performance.

2. Develop a methodology for analyzing complex surgical processes in MIS, and a metric for assessing technology and surgeon performance in MIS.

3. Reduce the possibility for errors by facilitating the integration of technology in MIS and the design of effective interfaces and informational aids to enhance surgeons’ performance.

4. Support the training of surgeons in MIS with modular computer-based training tools.

5. Contribute to the theoretical understanding of human performance and error remediation in dynamic remote environments. 

Education:

6. Develop and promote the interdisciplinary approach to complex systems in graduate and undergraduate curriculum, using the proposed research activities as course material for realistic case-based problem-solving exercises.

7. Develop and promote hands-on learning through laboratory projects and research experiences in undergraduate education, working on small projects related to the proposed research activities.

8. Develop and train successful research scientists and human factors engineers through teaching and mentoring activities.

PI: Castaneda Sceppa, Carmen
Title: Survey of Participants and Leaders to Determine Prevalence of Arthritis and Arthritis-Related Diseases (Strong Living Program: Community-Based Exercise Program)
Abstract: The purpose of this project is to demonstrate the effectiveness of the Strong Living Program in improving health-related quality of life (HRQL) in older adults with arthritis-related disease. This furthers our pilot randomized controlled efficacy study which demonstrated statistically significant improvements in physical performance measures including upper and lower body muscle strength, functional mobility, balance, and flexibility in adults with joint pain due to arthritis as well as multiple chronic medical conditions. We also saw significant improvement in overall arthritis symptoms rheumatologist examination and participant self-report after just 12 weeks of participating in the Strong Living Program. The results of this evaluation will provide evidence supporting the appropriateness of the Strong Living program to reduce the burden of arthritis among older adults in Vermont.

PI: Castellot, John
Title: Training Program in Developmental Biology
Abstract: Continuing support is requested for five predoctoral graduate students per annum to participate in a Training Program in Developmental Biology. Graduate students in this Program become expert in the concepts of modern cell, molecular and developmental biology and the application of molecular, cellular, morphological, and organismic methodologies to the mechanisms underlying embryonic development, reproduction, tissue remodeling, and disease processes involving alterations in cell behavior.

Several major themes of research are available to the students:

1. Early vertebrate development and reproductive biology
2. Tissue and organ development and remodeling
3. Disease processes, such as cancer, aging and vascular disease, resulting from disturbances in cell behavior and tissue remodeling
4. Basic molecular and cellular mechanisms relevant to development

The unique strengths of the Program are derived from:

1. The established strength of the faculty
2. The sustained retention of a strong pool of matriculants into the Program over the past decade or more
3. The intense concentration and collaboration on mechanisms of dynamic cell behavior in the above systems
4. The broad range of technical expertise of the faculty
5. The availability of top-grade facilities that allow sophisticated technology to be used
6. The location of the Program in a very strong Health Science research environment that facilitates collaborations
7. The interactive nature of the Program members
8. The close attention given to trainees in the small to moderate-size laboratories of the Program

On graduation, trainees will receive a Ph.D. in Cell, Molecular and Developmental Biology from the Tufts University Sackler School of Graduate Biomedical Science.

PI: Castellot, John
Title: Smooth Muscle Cell Growth Control by CCN Protein
Abstract: The long-term goal of this project is to elucidate the cellular, molecular, and biochemical mechanisms regulating the proliferation and motility of vascular smooth muscle cells (VSMC). VSMC hyperproliferation is a key early event in the pathogenesis of arteriosclerosis, and is the major cause of the high failure rate (restenosis) of many vascular surgical procedures. A hallmark of restenosis is intimal SMC hyperplasia during the first few weeks following surgery. Clearly, a detailed understanding of the mechanisms and molecules that regulate VSMC mitogenesis and migration will provide a therapeutic rationale for controlling VSMC hyperplasia following vascular surgery, and may provide important insights into the pathophysiologic basis for atherogenesis. Our laboratory has provided strong evidence that CCN5, a heparin-induced growth arrest-specific gene, inhibits proliferation and motility in cultured VSMC. Based on this evidence the following hypothesis will be tested: CCN5 is an autocrine regulator of VSMC proliferation and motility in culture and in vivo, and exerts it anti-proliferative and anti-motility effects, at least in part, through regulation of extracellular matrix synthesis and composition. To test this hypothesis, we will:

1. Continue our functional analysis of CCN5 on proliferation, motility, and extracellular matrix in SMC cultured from normal and injured arteries, using adenovirus vectors, recombinant CCN5, small inhibitory RNAs and anti-sense mRNA approaches.

2. Examine the physiologic and developmental functions of CCN5 in knock-out and transgenic mice, with particular attention to cardiovascular defects. In situ hybridization and immunohistochemistry will be employed to determine the spatial and temporal expression pattern of CCN5 mRNA and protein in developing embryos of wild-type, CCN5 heterozygotes and homozygotes. Functional analysis of VSMC cultured from the arteries of genetically altered mice that over or under-express CCN5 will be carried out.

3. Characterize the role of CCN5 in both mouse and rat models for vascular injury. We will determine if CCN5 gene or protein therapy might be a useful approach for suppressing restenosis in both the mouse wire-injury and rat balloon angioplasty model systems. The experiments proposed in this application should provide novel insights into VSMC pathophysiology.

PI: Castellot, John
Title: Regulation of Uterine Fibroids by CCN5
Abstract: The long-term goal of this project is to elucidate the cellular, molecular, and biochemical mechanisms regulating the proliferation and motility of human uterine smooth muscle cells (UtSMC). UtSMC hyperproliferation is the cause of fibroids, a condition that afflicts 20-25% of all women and 75% of African-American women. Fibroids cause severe pain and bleeding, impair fertility, and result in >200,000 hysterectomies annually in the U.S. There is no known treatment-medical or surgical─that permanently reduces or eliminates fibroids, other than hysterectomy.

Clearly, a detailed understanding of the mechanisms and molecules that regulate UtSMC mitogenesis and migration will provide a therapeutic rationale for controlling fibroids, and may provide important insights into the pathophysiologic basis for fibroid formation. Our laboratory has provided strong evidence that CCN5, an estrogen-induced growth-arrest specific gene, inhibits proliferation and motility in cultured UtSMC. Furthermore, we have demonstrated that human leiomyomas have greatly reduced levels of CCN5 mRNA and protein compared to normal myometrium from the same uterus. Based on this evidence the following hypothesis will be tested: CCN5 is an autocrine regulator of UtSMC proliferation and motility in culture and in vivo, and exerts it anti-proliferative and anti-motility effects, at least in part, through regulation of extracellular matrix synthesis and composition.

To test this hypothesis, we will:

1. Continue our functional analysis of CCN5 and its regulation by estrogen on proliferation, motility, and extracellular matrix in SMC cultured from matched pairs of normal and fibroid human uterine tissue. To do this we will use adenovirus vectors, recombinant CCN5, and small inhibitory RNA approaches.

2. Examine the physiologic functions and estrogen regulation of CCN5 in animal models, including normal cycling rats, ovariectomized rats, pregnant rats, wild-type mice, and genetically manipulated mice that either under or over-express CCN5. Quantitative PCR, Western blot analysis, and immunohistochemistry will be used to determine the spatial and temporal expression pattern and estrogen regulation of CCN5 in each of these animal models.

We will also explore the possibility that CCN5 gene or protein therapy might be a useful approach for suppressing human fibroids in a novel nude mouse model system. The experiments proposed in this application should provide new and important insights into UtSMC pathophysiology in humans.

PI: Catley, Andy
Title: Case Study on Emergency, Pastoralist and Food Security in South Sudan
Abstract: There is an increasing recognition that complex emergencies and man-made disasters have become the most important causes of food insecurity and famine. Responses to such emergency situations are generally of a humanitarian/emergency nature that, while contributing to saving lives and (sometimes) to protect livelihoods, are generally inadequate in addressing in a sustainable manner the complex root causes of the problem. This may be partly attributable to a dearth of long-term policies and strategies for addressing food security problems in a sustainable manner in protracted emergency contexts. Such policy gap was highlighted during the 'International Workshop on Food Security in Complex Emergencies: building policies frameworks to address longer-term programming in complex emergencies' organized by FAO-ESA (Tivoli 23-25 September 2003).

A series of case studies were thus launched by FAO-ESA to contribute to such on-going research process in three countries under complex emergencies conditions: RDC, Somalia and Sudan. In the cases of Sudan a preliminary review was undertaken during the months of June and July 2004 that:

1. Defined and tested a framework for analysis of food security in complex emergencies context
2. Provided an overview of the food security situation in Sudan (with a focus on south Sudan) and related responses and of the links of the responses with stakeholders strategies and information flows
3. Extracted some preliminary hypothesis and lessons with respect to the existence and the limitations of the responses by donors' agencies, local institutions and local people that though undertaken in an emergency context may have long-term potential effects on food security
4. Identified research areas that may deserve further attention and define the related frameworks for analysis

The objective of the project is to provide empirical evidence, based on hands on experience to the research process through a case study on emergency, pastoralists and food security in south Sudan. The case study will focus on livestock as a key component of the livelihoods of south Sudan people and the lessons that could be learned from an experience that although undertaken in an emergency context presents a number of "developmental" elements.

PI: Cebe, Peggy
Title: Polymer-Based Nanocomposites: An Opportunity for Deaf and Hearing-Impaired Students
Abstract: A small group of college-age students will perform an internship at Tufts University for six weeks during the summer. The classroom and laboratory components address the materials, chemistry, and physics of polymers and polymer-based nanocomposites, as well as crystallization and melting of polymers. The interaction of X-rays and light with polymers, mechanical properties of polymers, and the connection between thermal processing, structure, and ultimate properties of polymers are also addressed.

To prepare the students for participation in the scientific community, a strong component of pre-professional training is incorporated, including discussion of ethical issues in the performance of research and in scholarship. The goals of the laboratory research component are to:

1. Expose the students to the laboratory environment
2. Introduce them to the concepts of formulating and testing hypotheses
3. Assist students to conduct systematic studies while controlling variables
4. Illustrate the use of modern analytical equipment
5. Demonstrate the connection between processing variables, structure, and properties

The students will make and characterize polymer-based nanocomposite films comprising a semicrystalline polymer matrix and an additive, which will be either organically modified silicates (clay) or glass nanospheres. This research will contribute to a fundamental understanding of the effects of nanoparticles on the structure and properties of semicrystalline polymers. In this renewal, we will emphasize mechanical property measurements, using newly available instrumentation.

The program is aimed at a disadvantaged minority population, of deaf and hard of hearing college age students. The unique attributes of this program are its emphasis on:

1. Teamwork
2. Performance of a start-to-finish research project
3. Materials science-based approach
4. Diversity

Students of all disability levels are candidates for this program, including those students who neither hear nor voice. The broader impact will be to bring deaf and hard of hearing students into the larger scientific community as professionals, by providing positive scientific experiences at a formative time in their educational lives. The long-range goal of the proposed program is to increase participation of deaf and hard of hearing students in science and engineering, and provide enrichment and mentoring for these students.

PI: Cebe, Peggy
Title: Constraints in Semicrystalline Polymers During the Transition from the Liquid to the Solid State
Abstract: The ultimate mechanical, electrical, and thermal properties of crystallizable polymers depend upon the relationship between the crystals and the non-crystalline amorphous chains. During solidification, the growing crystals act as thermo-reversible cross links that constrain the mobility of the amorphous phase. The solidification process of semicrystalline polymers and its impact on the amorphous phase, from early stages of crystal growth to the completion of crystallization, forms the subject of this proposal.

We will investigate the confining effect of crystals during the transformation from liquid to semicrystalline solid of semicrystalline polymers, which are modeled as comprising the following nanophases: lamellar crystals, mobile amorphous, and immobile amorphous. Our research will answer the following specific questions about structure and relaxation dynamics vis-à-vis confinement:

1. Does the length of cooperatively rearranging regions, CRR’s, at the glass transition correlate with the development of the solid fraction, the crystal fraction, or both?
2. Do the alpha relaxation (glass transition) dynamics vary in a manner correlated with the development of these same fractions?
3. What is the structure of the resultant nanophases during solidification?

The answers to these questions will provide a fundamental level of understanding about the relationship between the crystals and the non-crystalline portions of the semicrystalline polymer as confinement occurs during the transition from the liquid to the solid state. An important part of these studies will be to test specific predictions of recent models of the early stages of crystal growth [Strobl 2000], as well as two prevalent lamellar structural models, the Heterogeneous, and the Homogeneous, Stack Models for the later stages of crystal growth.

Specifically, we will investigate the relaxation behavior, and the thermal and structural properties, of isotactic polystyrene, poly(butylene terephthalate), and poly(ethyleneterephthalate). A major, and novel, aspect of this research will be the use of quasi-isothermal measurements of the reversing heat capacity to study the time development of formation of the crystal constraints. The time development of the solid fraction, and the crystalline fraction, respectively, will be assessed from the earliest stages of crystallization, through spherulite impingement, and finally to completion of the crystallization process.

The alpha relaxation (glass transition process) will be studied using dielectric relaxation spectroscopy (DRS), and lamellar structure will be determined using small angle X-ray scattering (SAXS) and atomic force microscopy. DRS and SAXS will be performed simultaneously so that structural and relaxation dynamics information are obtained together.

Experience gained in the prior NSF grant (for which this proposal is a renewal) has positioned our group uniquely to be able to perform the proposed thermal, structural, and relaxation studies.

Broader Impacts: The PI will continue her unique outreach to the deaf and hard of hearing, by visiting Gallaudet University, Rochester Institute of Technology’s National Technical Institute for the Deaf, Connecticut School for the Deaf, and the Clark School (elementary through ninth grade). Prof. Cebe will present a popular lecture and discuss careers in science and engineering with deaf students. The PI will also participate in the Tufts University “Windows on Research” freshman advising option which will allow a diverse group of freshmen to join the advanced students in performing materials-related research. Dissemination of research results will be through the Cebe group’s website: http://www.tufts.edu/~pcebe.

PI: Chaisson, Eric
Title: Educational Tools for Chandra Mission
Abstract: The Chandra X-Ray Observatory was designed to be the next great leap forward in x-ray astronomy. Mainly a NASA mission, this Hubble-class vehicle was launched into high-Earth orbit on July 23, 1999. Its resolution and sensitivity makes this telescope the premier device for high-energy astronomy into the twenty-first century.

The Chandra X-Ray Observatory Center, operated by the Smithsonian Astrophysical Observatory, the Chandra X-Ray Observatory Center control science and flight operations of Chandra for NASA from Cambridge, MA. The operation center consists of two facilities and has a staff of over two hundred people. The operation and control facility in Kendall Square is electronically linked to the science support facility at the Harvard-Smithsonian Center for Astrophysics. Scientists from the CFA and MIT have played key roles in designing the mirrors and scientific instruments for the telescope.

The Wright Center has partnered with the Chandra X-Ray Observatory Center (1) to create broadcast-quality video animations of the spacecraft in orbit and of the main science results resulting from the spacecraft's observations of a variety of cosmic objects, and (2) to plan and organize a series of teacher workshops, including the construction of a suite of educational modules for use by pre-college teachers, in order to disseminate the main science findings of this x-ray mission.

PI: Chen, Jinkun
Title: Cell Differentiation in Periodontal Regeneration
Abstract: Traumas as well as congenital disorders cause bone and soft tissue defects in oral and maxillofacial region. Periodontal disease results in destruction of alveolar bone and tooth loss. The ultimate therapeutic goal for these diseases is tissue regeneration, which requires (1) differentiation of reparative cells and (2) production of extracellular matrix components. Our long-range goal is to expand current understanding of the cellular and molecular mechanisms involved in formation and regeneration of periodontal tissues and to provide further insight into the development of products for skeletal regeneration and tissue engineering. The objective of this application is to determine differentiation potential of cells involved in the processes of tissue repair and regeneration. The central hypothesis to be tested is that, in periodontal regeneration, the transcription factor Cbfa1 selectively induces osteogenic and cementogenic differentiation and the subsequent expression of BSP which enhances tissue formation and mineralization. The rationale is based on evidence that the conversion of non-differentiated mesenchymal cells and pre-osteoblastic cells into mature and functional osteoblasts is a crucial step in bone formation, as well as in bone regeneration.

Aim 1: To determine in vivo the regulating and promoting effects of Cbfa1 in cell differentiation occurring during periodontal regeneration. Using a gene-therapy approach, for the first time, the "master gene" Cbfa1, will be introduced into an artificially created periodontal defect and its inductive and modulating effects documented.

Aim 2: To determine the role of BSP expressed by differentiated cells in enhancing mineralization in the process of bone tissue formation and regeneration in rive. Using a cell-based method, BSP overexpressing cells will be transplanted into periodontal defects and for the first time the effect of BSP in enhancing biomineralization and bone maturation in vivo determined. Results derived from these in rive studies should provide novel and important insights into osteogenic gene therapy and molecular control of differentiation of cells, the most important component and the most powerful engine in tissue formation and periodontal regeneration.

PI: Chisholm, Karen
Title: MACC AmeriCorps*VISTA Program 2006-2007
Abstract: Colleges and universities across the Commonwealth of Massachusetts face the challenge of responsibly and successfully addressing the needs of low-income communities. Lack of proper training, resources, and quality service providers contribute to the inability of higher education institutions to meet the needs of their communities. Massachusetts Campus Compact (MACC) AmeriCorps*VISTA members act as transformative agents between institutions of higher education and their surrounding communities by building the necessary infrastructure and partnerships for campuses to develop high quality, effective, community-based service programs. Because this is a statewide program and the projects and programs developed and sustained by these 27 VISTA members are specific to the needs of the campus and community in which they serve, the social issues addressed and community outcomes generated vary from site to site. VISTA members build mutually beneficial relationships with community-based organizations; generate, assess and refine non-curricular service opportunities; educate on, evaluate, and create service-learning courses and partnerships; and improve and increase student leadership and participation in service programming. MACC VISTA members assist campuses in the implementation of programs that meet identified community needs with deliberate community-based outcomes in mind. Their goal is to create programs with long-term lasting results. They build, support, and develop the capacity for MACC member institutions and their students to responsibly address the issues identified by surrounding communities and make a greater number of appropriate campus resources available to impact a greater number of people in low-income communities. MACC VISTA members revolutionize higher education by building thoughtful and successful service programs which make students more active, responsible, engaged citizens.

PI: Choi, Sang Woon
Title: Epigenetic Effects of Chronic Alcohol Consumption on Colonic Mucosa
Abstract: Epidemiologic studies have demonstrated that alcohol consumption enhances colorectal carcinogenesis especially in individuals with folate depletion and/or methylenetetrahydrofolate reductase (MTHFR) homozygous variant genotype, indicating that the co-carcinogenic effect of alcohol is conveyed through the folate mediated one-carbon metabolism, as well as advocating an interaction between alcohol and MTHFR gene, which maintains a balance between biological methylation and nucleotide synthesis. Recently, we identified the fact that chronic alcohol consumption induces hyperhomocysteinemia and genomic DNA hypomethylation in the rodent colon, indicating the potent effects that alcohol exerts on one carbon metabolism and epigenetic phenomena, and subsequently lending that chronic alcohol consumption modifies critical gene expression through epigenetic changes and provides a co-carcinogenic milieu in the colonic mucosa.

Our long-term goal is to find effective strategies for the chemoprevention of alcohol-associated cancer. The studies outlined in this proposal are aimed at defining epigenetic mechanisms by which alcohol consumption affects colorectal carcinogenesis. We therefore hypothesize that chronic alcohol consumption disturbs one-carbon metabolism in the colon and thereby alters epigenetic phenomena including DNA methylation and histone methylation as well as critical gene expression. We further hypothesized that conditions which alter the balance of one-carbon metabolism, such as folate depletion, aging and MTHFR polymorphism, aggravate these epigenetic phenomena induced by chronic alcohol consumption.

This application is innovative because two proposed epigenetic phenomena, histone methylation and promoter DNA methylation, have never been explored for the study regarding the alcohol-associated carcinogenesis and the proposed epigenetic interaction between alcohol and MTHFR gene is a new concept that enables individually tailored chemoprevention by genotypes and nutrition status. Based on the results of this application we will apply for a research project grant to finalize the epigenetic effect of chronic alcohol consumption on colorectal carcinogenesis. If those studies can precisely define the epigenetic mechanism for alcohol-associated carcinogenesis, we can develop a new chemopreventive strategy for those cancers.

PI: Cochran, Brent
Title: Stat3 as a Therapeutic Target for Glioblastroma
Abstract: The STAT (signal transducers and activators of transcription) family of transcription factors was initially described in my lab as regulators of the c-fos proto-oncogene and is key mediators of cytokine and growth factor responses. They have been shown to mediate cell growth, differentiation, and survival in a variety of different cell types. The STATs are unusual among transcription factors in that they have characteristics of cytoplasmic signaling molecules such as a Src homology (SH2) domain and a tyrosine phosphorylation site. Upon tyrosine phosphorylation in response to receptor activation, the STATs dimerize through interaction of the SH2 domain with a phosphorylated tyrosine residue and translocate to the nucleus where they directly bind DNA. Growth factor receptor kinases, JAK family kinases, and Src family kinases have been implicated in STAT activations.

Among the seven mammalian STAT genes, STAT3 has been most strongly implicated in the transformation of both mouse and human cells. A constitutively activated STAT3 is an oncogene for fibroblasts. This constitutive STAT3 has been shown to cooperate in the transformation of human cells as well. Dominant negative STAT3 inhibits transformation by v-src. Remarkably, knockout of STAT3 in the mouse epidermis completely prevents induction of skin tumors by tumor and STAT3 activation appears to be sufficient to generate gastric tumors in mice. Moreover, STAT3 is essential for the growth and survival of several human cancers including breast, prostate, and head and neck cancer. Recent studies have indicated that inhibition of STAT3 function can inhibit the growth of some of these tumors both in vitro and in vivo. Strikingly, STAT3 activation has been shown to be required for the differentiation of astrocytes in response to CNTF. STAT3 has also been found to be activated in >90% of primary human glioblastoma multiforme tumors.

Previous work has shown that STAT3 can regulate genes involved in several of the "hallmarks of cancer" including growth promoting genes (cyclin D and c-myc), angiogenic genes (VEGF), anti-apoptotic genes (survivin and bcl-xl), and genes associated with tumor invasion (LIV-1) 21-25. The catalytic subunit of telomerase (hTERT) is overexpressed in many tumors and is believed to play an important role in cancer development. 70% of glioblastoma multiforme (GBM) tumors have hTERT expression, and it has been suggested to be a promising target for therapy in GBM. We have now found that STAT3 regulates this critical hallmark of cancer, i.e. the expression hTERT in some glioblastomas and that RNAi knockdown of hTERT, as well as STAT3, causes cell death in GBM cell lines. We have shown that knockdown of STAT3 by RNAi induces apoptosis in several glioblastoma cell lines. Since the expression of activated STAT3 is low in uninjured brain and in normal human astrocytes, STAT3 would seem to be a promising target for glioblastoma therapy. Surprisingly, however, very little work has been done to evaluate the efficacy of STAT3 as a target for therapy of GBM despite the fact that such therapy has shown promise for other tumor types in animal models. Here we propose to rectify this situation.

PI: Coffin, John
Title: Retrovirus Evolution
Abstract: Retroviruses exhibit a wealth of evolutionary phenomena, including the ability to undergo rapid genetic change in response to varying selective pressure, the ability to vary in the use of host cell receptors, and the ability to become integrated in the genome of their host species and passed down through the generations as endogenous proviruses. In the prior project period, we have studied all these aspects of retrovirus evolution:

• We have looked at the mechanism of evolution of env genes by analyzing an unusual mutant that extends the host range of ALV beyond chicken to quail, dog, and even human cells.
• We have isolated and studied an unusual mouse endogenous provirus that appears to occupy a special phylogenetic location between mouse and non mouse species.
• We have extensively analyzed the coevolution of humans and their endogenous proviruses, particulary the relatively recently inserted HERV-K family.
• We have developed sophisticated mathematical models for the evolution of replicating virus populations, describing the effects of mutation, selection, drift, linkage, and recombination on the accumulation (or loss) of deleterious mutations.

Future work will address the following aims.

1. How do retroviral envelope genes evolve to use new receptors and to alter other important properties? We will use our extended host range mutants to test specific hypotheses for this process.

2. What are the functional and pathogenic properties of human endogenous retroviruses, particularly HERV-K? Can we isolate infectious virus? Is their expression or reintegration associated with malignancy?

3. How do important the forces of mutation, selection, recombination, and drift combine to direct retrovirus evolution?  We will combine mathematical and in vitro modeling of virus replication to test specific evolutionary models.

Relevance: In addition to its inestimable value as a tool of basic discovery, retrovirus evolution has important consequences for public health. In recent history, several retroviruses have evolved to use humans as hosts, with devastating consequences. Furthermore, evolution of viruses within a given host can have important consequences, such as use of different receptors, increased virulence, or resistance to drugs. Understanding of both how this evolution has occurred in our past and how it occurs in simple models will leave us better prepared to deal with such events as they happen.

PI: Court, Michael
Title: Mechanisms of Adverse Host Responses to Antibiotics
Abstract: Recent events of bio-terrorism and the rapid global spread of emerging infectious diseases has revealed a compelling need to develop effective and safe therapeutics that can be readily applied to large and genetically diverse populations, typical of the United States. In a comprehensive review of the 2001 anthrax bio-terrorist attack, investigation of "adherence, barriers to adherence, and adverse events associated with long-term use of antimicrobial agents" was considered to be a high priority research area. The objective of this research is to elucidate molecular mechanisms underlying adverse host responses to antimicrobial agents used to treat NIAID Priority Pathogens.

This proposal focuses on the fluoroquinolones, particularly ciprofloxacin, which was the primary antibiotic used for anthrax post-exposure prophylaxis, and is an option for treatment of other NIAID Priority Pathogens. Side effects of the fluoroquinolones leading to non-adherence most commonly involve the gastrointestinal (GI) tract. The novel hypothesis to be explored in this proposal is that many of these adverse side effects result from the formation of reactive fluoroquinolone metabolites in the liver and/or GI tract.

Major metabolites of the fluoroquinolones in humans are acyl glucuronides, which have a high potential for covalent adduct formation with biomolecules. In preliminary studies, we show evidence for spontaneous degradation and acyl migration of ciprofloxacin and trovafloxacin glucuronides similar to that observed with glucuronides of the nonsteroidal anti-inflammatory drugs. Consistent with the exploratory nature of an R21 Research Project, the specific aims of this proposal are as follows.

1. Firstly, we will use primary human hepatocytes and human intestinal cell lines to quantify the Cytotoxicity of 3 fluoroquinolones frequently associated with adverse GI side effects in people (trovafloxacin, grepafloxacin and sparfloxacin) compared with 3 fluoroquinolones less commonly associated with these side effects (ciprofloxacin, ofloxacin, and norfloxacin).

2. Secondly, we will determine whether inhibition of enzymes potentially responsible for reactive metabolite formation (UGT, CYP and acyl CoA ligase) minimizes the in vitro toxicity of these drugs.

By the completion of these studies we will have developed an understanding of the biochemical pathways potentially responsible for clinically important adverse side effects of the fluoroquinolones. In future work we will verify these findings through in vivo studies and identify host genetic factors that may account for individual variability in side effect susceptibility.

Our ultimate goal is to optimize the broader clinical application of these drugs through a pharmacogenetic approach in situations where long-term treatment of large and genetically diverse populations is necessitated.

PI: Crane, Gregory
Title: A Reading Environment for Arabic and for Islamic Culture
Abstract: Students learning Arabic or reading English texts about Islamic Culture are confronted with a dizzying set of references as well as quotations from religious texts and classical poems that are part of the vocabulary of even ordinary, and apparently, unrelated discourse, understood by native speakers but unintelligible to outsiders. This project develops and evaluates a reading environment, based on automatic linking within a digital library framework that automatically provides lexical and/or encyclopedic background information adapted to individual users.

The work proposed here transfers digital library research to a pedagogical setting. Heavy and growing usage of a reading environment for Greco-Roman culture suggests that a similar set of tools may attract substantial use for Arabic and Islamic studies. This project goes beyond implementation, studying the impact of these technologies on two core groups of learners: Arabic language students moving beyond annotated texts to real world Arabic, and students reading texts about Islamic culture, especially non-specialists from other disciplines.

We focus upon two broad areas. First, to what extent does this reading environment enhance existing practice? Can readers move more quickly through new text? Can readers begin working with culturally complex documents at an earlier stage? Second, to what extent does this reading environment change the goals of learners? Can they address more challenging topics? Do they make broader use of more complex source materials?

We will examine three core user groups:

1. Students enrolled in Arabic courses at Tufts University
2. Students working with Islamic cultural materials as part of courses taught as International Relations
3. Individuals making use of the reading environment online from outside Tufts

Instruments include reading comprehension tests for both Arabic and English texts on Islamic culture, field observations of practice, structured interviews with students, instructors and focus groups, and analysis of lookup patterns. By encoding our data in standard formats, we can represent that information in a range of forms, not only as text on text, but printed as braille and as sound generated by speech synthesizers.

The materials produced under this grant will be made available under the open access terms defined for the Tufts University Open Courseware Initiative (http://ocw.tufts.edu), providing a public commitment, backed by the university leadership, to make these results freely available.

PI: Crivello, Natalia
Title: Vitamin K and Sulfatides in Brain Aging
Abstract: Neurological disorders such as Alzheimer's disease (AD), and to a lesser extent normal aging, are associated with declines in motor and cognitive behavior. There is a strong need to identify those modifiable factors that regulate the proper maintenance of those brain regions involved in controlling behavior. Dysregulation of sulfatides, a subclass of spingolipids in the brain, is a risk factor for cognitive dysfunction observed in AD and normal aging.

We propose to initiate a series of novel animal studies that examine the role of dietary interventions, which are potentially modifiable, on sulfatide metabolism. Vitamin K is present in high concentrations in the brain, and has been implicated in a positive regulation of sulfatide metabolism. The primary objectives of the proposed study are to develop a novel animal model for the study of the interplay between vitamin K and sulfatides in brain regions controlling motor and cognitive behavior, and to determine if age and sex-specific differences in vitamin K status influence sulfatide metabolism. Vitamin K and sulfatides status will be assessed in myelin (axon insulator) and synaptosomes (nerve terminals), isolated from the cortex, hippocampus and striatum of Fischer 344 rats (n=80), following 28 days of intake of 500 fg/kg of diet of one of two forms of vitamin K [phylloquinone (K1) or dihydrophylloquinone (dK)]. There is a tissue-specific conversion of K1 to menaquinone-4 (MK-4), which is the major form of vitamin K in the brain. Conversely, there is no conversion of dK to MK-4 in the brain, nor is there accumulation of dK in the brain.

The proposed study will exploit this inability of dK to convert to MK-4 to develop a novel animal model that can manipulate vitamin K status in the brain, while conserving vitamin K-dependent coagulation. We will apply this model to examine the role of age (2m, 12m, and 24m) and gender on vitamin K and sulfatide metabolism. These studies will provide the foundation for future studies on the role of dietary vitamin K in sphingolipid metabolism as it relates to healthy aging, and ultimately its role in motor and cognitive behavior.

PI: Crott, Jimmy
Title: Effect of Folate Status on Genetic/Epigenetic Stability and Tumorigenicity
Abstract: The proposed project requires the development of a novel in vivo mouse model that will test whether moderate extended folate depletion can induced genetic, epigenetic and proteomic aberrations in mouse embryonic fibroblasts (MEF) and whether folate depletion alone is sufficient to promote tumorigenesis after injection into immunodeficient nude mice. We will harvest MEFs from mice that ubiquitously express luciferase. These cells will then be cultured in conditions of high and low folate for up to 10 weeks. At weekly intervals, aliquots of cells will be injected into nude mice to study their tumorigenic capacity. Since these MEFs express luciferase, we will be able to accurately monitor tumor growth in vivo using fluorescent imaging. At the same intervals, cell aliquots will be studied for genetic stability (DNA uracil content, p53 strand breaks, micronucleus formation, karyotype, mitochondria1 DNA deletions), epigenetic aberrations (global and promoter methylation), global gene expression patterns and the abundance of specific proteins. In this fashion we will be able to thoroughly characterize the cellular response and damage induced by folate depletion and also determine which molecular and genetic events are required (and those that are not) for folate depletion-induced tumorigenesis. In addition to the utility of this novel model in studying folate depletion-induced carcinogenesis, we propose that it may find broad application in the study of other nutrients, chemicals, radiation and putative tumor suppressor genes in tumorigenesis.

PI: Dawson-Hughes, Bess
Title: Effect of Potassium Bicarbonate on Bone and Muscle
Abstract: Metabolic acidosis causes bone loss and muscle wasting. On a daily basis, adults on normal Western diets generate approximately 75-100 meq/d of acid. As individuals age, their renal function and ability to excrete acid decline and they become more acidotic. Alkalinizing the diet with supplemental potassium bicarbonate (KHCO3) reduces urinary calcium excretion, lowers bone turnover rates (biochemical markers of bone resorption) and reduces urinary nitrogen excretion (an indicator of muscle catabolism) acutely in subjects on acidogenic metabolic diets.

Currently there is no concensus on the relative contributions of potassium (which may increase renal calcium retention) and bicarbonate (shown to reduce net acid excretion) on decreasing bone turnover. We postulate that HCO3 may also reduce N2 (muscle) wasting in older persons. There is no evidence at this time that KHCO3 or its components will have a sustained impact on bone turnover or nitrogen wasting under field conditions. 

The proposed study will determine the effect of potassium and bicarbonate, alone and in combination, on serum PTH, biochemical markers of bone turnover, calcium excretion, calcium absorption and urinary nitrogen. 192 men and women will be randomly assigned to treatment with 67.5 mmol of KHC03, KCI, or NaHCOS or with placebo daily for 12 weeks. Subjects will visit the Center at baseline, 3, 7, and 12 weeks for assessment of their diets, pill compliance, and weight. They will have the following measurements on selected study visits: muscle function tests, serum calcium, potassium parathyroid hormone, osteocalcin, 24-hr urinary calcium, N-telopeptide, calcium absorption, potassium, sodium, creatinine, titratable acid, renal net acid excretion, and nitrogen.

In the analysis, the final value will be the response and the baseline value will be a covariate. If effective in reducing calcium excretion, bone turnover, and/or nitrogen wasting, then treatment with potassium, bicarbonate, or the two combined, would warrant further evaluation as a potentially inexpensive and safe means to reduce bone loss and muscle wasting in the elderly.

PI: Degterev, Alexei
Title: Caspase-Independent Cell Death and Neurodegeneration
Abstract: The overall goal of this project is to characterize a novel cell death pathway, termed "aponecrosis", and determine its role in neurodegeneration.

Extensive evidence was generated in recent years suggesting that two "classic" cell death pathways, apoptosis and necrosis, do not explain the variety of physiological and pathological cell death mechanisms. Existence of the third pathway, termed "aponecrosis", is proposed.

This pathway is activated in cells that are induced to undergo apoptosis, yet prevented from its completion. It is proposed to represent a novel safety mechanism aimed at elimination of damaged and potentially dangerous cells. Aponecrosis shares programmed cell death nature with apoptosis and execution subroutines and phenotypic features with necrosis. Using high throughput screening of small molecule library, several chemical inhibitors of aponecrosis were isolated. These compounds were found to selectively inhibit aponecrosis, but not apoptosis, underscoring distinct mechanism of the aponecrotic cell death process.

Using selected inhibitors aponecrosis was shown to represent a major cell death process in a variety of caspase-independent cell death paradigms in vitro. Aponecrosis was also implicated in amyloid-beta toxicity in PC12 cells and ischemic brain damage in vivo. Analysis of the effects of selected inhibitors in various systems suggests that two of them target cell type specific signaling pathways, whereas another compound blocks uniform downstream execution step, providing the opportunity to characterize various steps in aponecrosis with the help of individual inhibitors. Two aponecrotic regulators (p38 kinase and cathepsin B) were discovered.

Specific aims of this study are as follows:

1. To evaluate the role of aponecrosis in neuronal cell death in vitro and in vivo
2. To characterize molecular events involved in aponecrosis
3. To identify novel genes involved in aponecrosis using chemical, cell and molecular biology approaches

PI: Dunlap, Kathleen
Title: Presynaptic Receptors on Embryonic Sensory Neurons
Abstract: The influx of calcium (Ca) through voltage-gated ion channels and the consequent rise in intracellular Ca concentration influence many cellular activities (secretion, contraction, regulation of membrane permeability). Recently, multiple types of Ca channel have been described in several tissues, leading to speculation as to the involvement of Ca channel subtypes in the control of separate cell functions. Modulation, by drugs and neurotransmitters, of the various classes of voltage-dependent Ca channel may offer the flexibility necessary to differentially control these multiple cellular responses. Experiments outlined in this proposal will use electrophysiological and biochemical techniques to study three types of Ca channel in embryonic chick dorsal root ganglion neurons in vitro.

First, using patch clamp recording, we will determine if current flow through transient (N- and T-type) Ca channels is modulated by norepinephrine and amino butyric acid, two transmitters already known to inhibit the long-lasting, L-type Ca current in these neurons. Further experiments will address the mechanism(s) underlying any observed modulation; in these studies we will investigate the biochemical pathways mediating the action of the transmitters on Ca channel function. Finally, transmitters and ligands selective for different classes of dorsal root ganglion neuron Ca channel will be tested for their effects on 1) neuropeptide secretion and 2) synaptic transmission between dorsal root ganglion neurons and spinal cord cells. These experiments will determine the type of Ca channel(s) involved in neurosecretion and the biological ramifications of its modulation. Thus, we will acquire new insight into the mechanisms fundamental to neuronal exocytosis and the means by which intercellular communication can be modulated by endogenous transmitters.

PI: Dwyer, Johanna

Title: Optimizing Food and Nutrition Services in Assisted Living Facilities: The FANCI (Food and Nutrition Care Indicators) Survey

Abstract: As the number of elders needing supportive services and assisted living facilities (ALF) grow, policies related to them are evolving, but as yet they have not included attention to nutrition related supportive services. Our project on optimizing food and nutrition supportive services in ALF for elders, the Food and Nutrition Care Indicators (FANCI) Study conducted by Johanna Dwyer DSc, RD (Principal Investigator), and Shirley Chao MS, RD (Co-Investigator) will explore the views of national experts as well as stakeholders in one state.

PI: Easterbrooks, Anne
Title: Touchpoints Early Child Care & Education Evaluation
Abstract: Since its inception, the Brazelton Center’s Touchpoints Program (TP) has garnered much public and professional acclaim. The Touchpoints Program, built on Dr. Brazelton’s vast clinical experiences helping parents manage their young children’s often bewildering and stressful developmental transitions, trains a wide variety of child-serving professionals. Its primary goals are to increase professionals’ knowledge of child, parent, and family development, and to expand professionals’ repertoire of respectful, collaborative, and strengths-oriented strategies for working with parents. Improved relationships between professionals and parents, and between parents and children, should result, and these in turn should yield enhanced child well-being. 

The conceptual model used to frame this evaluation is Jacobs’ Five-Tiered Approach to Evaluation (FTA). The FTA is explicitly incremental, developmental, and systemic in nature. It organizes evaluation activities at five levels — moving from generating descriptive and process-oriented information at the earlier stages:

To determining the effects of programs later on:

The tiers are structured for consecutive use, at least on the first “go through”; a program would need the information gathered at an earlier tier to facilitate a competent evaluation at the next. 

Participants will be child care providers and the children and families for whom they care. Site selection awaits further exploratory research, but our communications with the Boston Touchpoints staff suggest that the city of Springfield, Massachusetts may contain appropriate sites for the evaluation. Our aim is to recruit four sites providing center-based childcare, with the availability of approximately 30 teachers at each site. Two sites would receive Touchpoints ECCE training at the beginning of the intervention, while two other sites would serve as comparison groups, and would receive Touchpoints ECCE training at the close of the evaluation. This would eliminate differences among centers in motivation to receive Touchpoints intervention. 

Measures of certain key constructs will be reviewed for their applicability and feasibility for this project; large-scale evaluations of Head Start, Early Head Start, and the NICHD Early Child Care Research Network will serve as important reference points for assessment tools. The constructs include:

1. Child development knowledge (of provider)
2. Provider-parent relationships
3. Provider-child relationships
4. Parent-child relationships

PI: Economos, Christina
Title: Strategies for Overcoming Obesity through Nutrition, Physical Activity and Education
Abstract: Tufts University requests the support of the PepsiCo Foundation in strengthening the capacity of our John Hancock Center for Physical Activity and Nutrition to develop replicable community-based solutions to the growing problem of childhood obesity, with an emphasis on behavior change. Building upon our ongoing and successful intervention program, "Shape Up Somerville," in the schools and wider community of Somerville, Massachusetts, the Center will engage in evaluation of our current work, expansion of our intervention, revision and improvement in the curriculum, technical assistance to other interested communities nationwide, and extensive publication of our results in both the scholarly and popular literature. A key component of our work will be to deepen the understanding of ethnic groups and people of color as it relates to fashioning workable tools for behavioral change around diet and exercise. Further, we will expand our association and affiliation with a wide variety of organizations, both locally and nationally, to even more broadly disseminate our efforts.

PI: Economos, Christina
Title: Children in Balance Online HEAT Club
Abstract: The Children in Balance initiative of the Friedman School of Nutrition Science and Policy proposes to develop a pilot on-line course of the HEAT Club, an after school nutrition and physical activity curriculum and training designed for programs working with elementary school aged children.

"The Course": This pilot program has the potential to increase healthy eating and active time for participating children, and to increase nutrition awareness in the children's families and wider community. After school program leaders who are trained in the HEAT Club will begin developing the tools they need to work towards improved nutrition and physical activity in their schools and communities.

PI: Economos, Christina
Title: Better Breakfast Initiative Nutritional Guidelines
Abstract: Since 2004, Project Bread and the Child Nutrition Outreach Program (CNOP) have been working with food service directors in several Massachusetts communities on the Better Breakfast Initiative (BBI), a program to improve the nutritional content of school breakfast. Participating food service directors are required to meet Better Breakfast nutritional guidelines, a standard that exceeds the current USDA guidelines for school breakfast. As the project expands beyond the pilot phase, Project Bread hopes to contract with The Friedman School of Nutrition Science and Policy, Tufts University to create a comprehensive toolkit for distribution to other communities interested in participating in the program.

PI: Economos, Christina
Title: PEP Grant Somerville School Department
Abstract: Tufts University, Friedman School of Nutrition, will collect all height and weight data on 4th–8th graders during the 2006-2007 school year, train Somerville School personnel to conduct the data collection, and provide 3 Nutrition education trainings for Somerville after-school providers, physical education teachers, and food service workers.

PI: Edwards, Aurelie
Title: Model of Transport in Renal Medullary Microvasculature
Abstract: The microcirculation in the renal medulla plays an essential role in the regulation of fluid and electrolyte excretion and in the long-term maintenance of arterial blood pressure. The overall goal of this research is to extend our mathematical model of the renal medullary microcirculation in order to understand the effects of specific transporters, osmolytes and hormones on medullary blood flow and its distribution. Our specific aims are as follows:

Aim 1: To determine the importance of UTB urea transporters in providing a route for volume efflux that shunts blood flow from descending vasa recta (DVR) to ascending vasa recta (AVR), secondarily reducing blood flow to the inner medulla. We will incorporate new in vitro findings (including permeability and reflection coefficient measurements in UTB knockout mice) into our mathematical model in order to examine the role of water channels and urea transporters in regulating blood flow to the inner medulla.

Aim 2: To examine the effects of the dependence of solute permeability on blood flow rate. Permeability of DVR to sodium and raffinose varies with perfusion rate; this dependence may be mediated by NO generation. We will investigate whether flow dependent increases in permeability reduce transmural concentration gradients and therefore water efflux from DVR, thereby enhancing blood flow to the inner medulla and decreasing concentrating ability.

Aim 3: To determine whether lactate plays a significant role in the medullary microcirculation. It has been suggested that the accumulation of lactate could augment water removal from the thin descending limb and enable mathematical models to accurately predict cortico-medullary osmolality gradients. We will examine whether the presence of lactate can enhance volume efflux from DVR across transcellular pathways, thereby affecting blood flow to the papilla and axial small solute concentration gradients.

Aim 4: To examine the effect of nitric oxide on overall medullary blood flow as well as on the distribution of blood flow between the inner medulla and the interbundle region of the outer medulla. We will investigate the extent to which NO generation may abrogate medullary hypoxia, and whether the production of nitric oxide by the thick ascending limb could constitute a feedback system aimed at specifically protecting the mTAL from ischemic injury I to the detriment of the inner medulla.

PI: Feig, Larry
Title: Genetic Analysis of Ras and G Protein Function
Abstract: The overall goal of this proposal is to gain a better understanding of how Ras proteins couple extracellular signals to intracellular signaling pathways in normal and disease states. The Ras superfamily of GTPases participate in the regulation of a wide variety of cellular processes by functioning as molecular switches that cycle between the active GTP-bound and inactive GTP-bound states. A key step in how Ras protein function is modulated in cells is by their activation by a family of guanine nucleotide exchange factors (GEFs), whose role is to respond to a specific extracellular signals by promoting the exchange of GTP for GDP bound to Ras proteins.

This proposal will investigate how the Ras-GRF family of GEFs contribute to the control signaling pathways mediated by the Ras family of GTPases. Ras-GRF1 and Ras-GRF2 each have two GEF domains, one used to activate the Ras proto-oncogenes and another used to activate the Rac GTPase. Calcium/calmodulin binding regulates the ability of Ras-GRFs to activate Ras and Rac. Ras-GRFs can also be stimulated by phosphorylation. Both Ras-GRF proteins are expressed predominantly, but not exclusively in neurons.

This proposal will use both genetic and biochemical approaches to focus on three major areas of study. The first is to reveal how the Ras-GRF family contributes to receptor/calcium channel signaling in neurons by exploiting our recently derived mouse strains that lack Ras-GRF1, Ras-GRF2 or both Ras-GRFs. The second is to reveal how two classes of newly identified Ras-GRF binding proteins contribute to Ras GTPase regulation. These binding proteins include IB1 and IB2 (scaffolds for components of the Jnk and p38 MAP kinase cascades, respectively) and spinophilin (a scaffold for the p70 S6 kinase and protein phosphatase 1). Finally, the third goal is to investigate the biological function of the newly identified Ras-GRF1 target, R-Ras, in oncogenesis. These studies will exploit our recent finding that constitutively activated R-Ras is a potent activator of estrogen independent proliferation in MCF-7 breast cancer cell lines.

PI: Feig, Larry
Title: The Function of the Ras-Related Ral Proteins
Abstract: The overall goal of this proposal is to gain a better understanding of the functions of the Ras related Ral-GTPase family.

RalA and RalB have been implicated in diverse cell functions including the control of vesicle sorting, gene expression and cell proliferation. As GTPases, Ral proteins function as molecular switches to transmit extra-cellular signals to specific intracellular signaling cascades. Ral proteins reach the active GTP bound state in cells by interacting with one of a family of Ral-specific guanine nucleotide exchange factors (Ral-GEFs). One class of Ral-GEFs binds to and is activated by GTP-bound Ras, and a growing body of evidence supports the idea that elevated Ral-GEF/Ral signaling has the potential to contribute to human oncogenesis.

This proposal will attempt to reveal the mechanisms underlying two newly identified processes by which the Ral-GEF, Ral-GDS, is regulated. One process positively regulates Ral-GEF activity through interaction with the PDK1 protein kinase, and the other negatively regulates Ral-GEF activity through protein kinase D-mediated phosphorylation. Active GTP-bound Ral proteins bind to and alter the activity of a set of downstream "effector" proteins to influence cellular processes.

Studies in this proposal will expand upon our recent finding that RalA, but not RalB, functions in the maintenance of cellular polarity by enhancing the rate of delivery of membrane proteins to the basolateral surface of epithelial cells through its newly identified effector, the exocyst, and possibly through an exocyst-independent mechanism. Thus, one set of goals is to define the biochemical basis for the difference in activities of these two closely related Ral family members. Another goal is to identify the additional RalA "effector" that participates in basolateral membrane delivery. We also plan to define how RalA binding to the exocyst or other Ral effectors promotes membrane delivery in MDCK epithelial cells. Understanding how Ral functions in this process is important because faulty delivery and polarization of membrane proteins can lead to serious diseases including cystic fibrosis, I cell disease, familial, polycystic kidney disease and possibly cancer.

Finally, there is a growing appreciation that Ral-GEFs contribute to downstream signaling from GTPases by mechanisms that are independent from their ability to activate GTPases. Therefore, another aim of this proposal is to evaluate the contribution of Ral-GEF binding proteins for their ability to contribute functions that complement those of active Ral in cell processes mediated by the Ral-GEF/Ral signaling cascade.

PI: Fielding, Roger
Title: Lower Extremity Muscle Power and Function in the Elderly
Abstract: The focus of this research has been to examine the physiologic and functional effects of a muscle power training intervention in comparison to traditional progressive resistance training in a community-based group of elderly men and women with moderate mobility limitations.

During the current project period, we have successfully demonstrated that peak lower extremity power is closely associated with functional limitations and self-reported disability in older community dwelling men and women. We propose to extend our previous findings by examining the physiological mechanisms that contribute to the age and gender-related declines in peak muscle power. To compliment our existing data on skeletal muscle fiber contractile properties, we will assess the neural contributions to the generation of muscle power with advancing age.

We will test the hypothesis that skeletal muscle single fiber contractile properties (i.e. single fiber power, shortening velocity, specific force), neural factors (i.e. intermuscular coordination patterns, central activation ratios/and motor unit control properties) and lower extremity power will be reduced with advancing age and degree of risk for mobility disability, and these differences will be attenuated in women compared to men. Middle-aged healthy (40-55 yrs), older healthy (70-85 yrs), and older (70-85 yrs) men and women at risk for mobility disability will be studied under controlled conditions (Study 1).

In addition, in light of our recent observations that differences in lower extremity maximal velocity occur at relatively low external forces (eg: 40% 1 RM) and are most closely associated with gait velocity in older individuals, we propose conducting a randomized controlled trial of high velocity low resistance exercise training in individuals at risk for mobility disability. This to test the hypothesis that a short-term resistance training intervention performed against a low external force and at maximum voluntary velocity will induce significant reductions in mobility disability as evidenced by improvements in gait velocity during performance of a 400 M walk in older individuals at risk for mobility disability (SPPB score ≤9, 70-85 yrs) (Study 2).

The results of these studies will have important implications in understanding the proximal determinants of physical disability in the frail elderly and in designing intervention strategies targeted at improving mobility.

PI: Fielding, Roger
Title: Activity-Dependent Signaling in Aging Skeletal Muscle
Abstract: The age-related loss of skeletal muscle mass is associated with well-characterized functional limitations and physical disability. Although resistance training attenuates age-related muscle loss, the cellular processes that initiate muscle hypertrophy and the extent to which they are preserved with age are not well understood. The 70-kDa S6 protein kinase (p70S6K) is a downstream target of the protein kinase B/mammalian target of rapamycin (Akt/mTOR) pathway that has been implicated in the regulation of muscle size during overload and disuse atrophy. This and other kinases of the Akt/mTOR pathway affect protein translation by regulating translational inhibitors such as glycogen synthase kinase 3 (GSK-3), phosphorylating key ribosomal proteins, and influencing the availability of eukaryotic initiation factors (elF's).

We propose to test the hypotheses that

1. Aging is associated with a reduced activation of the Akt/mTOR pathway
2. The reduced phosphorylation of p70S6K and mTOR results in a decreased number of elF4E-elF4G complexes and a reduction in muscle protein synthesis
3. Chronic contractile activity results in blunted muscle hypertrophy in older animals due to a reduced activation of the Akt/mTOR pathway

We propose to use electrical stimulation to simulate acute resistance exercise in young and old rat hindlimbs, and surgical ablation of synergistic muscles to model the effects of chronic contractile activity.

Specifically, we will

1. Characterize Akt/mTOR signaling after a single bout of resistance exercise at young age
2. Assess the effects of resistance exercise on the number of capped mRNA binding sites (elF4E-elF4G) available for protein translation at young age
3. Compare the activation of Akt/mTOR, formation of elF4E-elF4G complexes, and protein synthesis of young, middle aged, and old rats in response to acute contractile activity
4. Compare the activation of Akt/mTOR, formation of elF4E-elF4G, and protein synthesis of young, middle aged, and old rats in response to chronic ablation of synergistic muscles

Muscle samples at several time points will be analyzed for p70S6K, Akt, mTOR, 4EBP1, and GSK-3 phosphorylation, elF4E-elF4G complexes, and skeletal muscle protein synthesis. We believe that the identification of molecular dysregulation in the Akt/mTOR pathway associated with age-related muscle atrophy will establish the groundwork for studies aimed at correcting these deficiencies and improving the efficacy of resistance training and other therapeutic interventions in the elderly.

PI: Folstein, Marshall
Title: Micronutrients, Stroke and Cognition in Aging
Abstract: A major determinant of the quality of life for elderly individuals is the efficiency of their mental processes. Loss of neurocognitive functioning has been noted in elderly individuals; the severity of this loss ranges from simple memory deficits to profound dementia of the Alzheimer's variety. It is important to discover to what extent such functional decline is preventable and reversible. Specific to this project is the possibility that nutritional factors are important. Since estimates of the incidence of micronutrient deficiency in the elderly is quite large, intervention could significantly reduce the public health burden.

The aim of this multidisciplinary study is to determine the prevalence of elevated homocysteine, brain disease, and cognitive decline in the homebound elderly. From 7500 homebound elderly, representative of all registered cases from a specified geographical area, a cohort of 1600 elders will be assessed. The assessment will include dietary history and serum nutrient levels, as well as comprehensive neuropsychological testing. Clinical examination and MRI scans will be performed on a subset of 400 subjects. Known risks for cognitive impairment such as diagnosed depression, anxiety, hypertension, diabetes, atrial fibrillation, medications, APOE, saturated fat intake, and mutations of MTHFR will be measured and included in analyses as covariates and effect modifiers.

PI: Forgac, Michael
Title: Structure, Mechanism and Regulation of the V-ATPases
Abstract: The long term objectives of this proposal are to determine the structure, mechanism and regulation of the vacuolar (H+)-ATPases (or V-ATPases). The V-ATPases are responsible for acidification of intracellular compartments in eukaryotic cells and serve an important function in a variety of cellular processes, including receptor-mediated endocytosis, intracellular membrane traffic, protein processing and degradation and coupled transport of small molecules. V-ATPases in the plasma membrane of specialized cells also function in renal acidification, pH homeostasis, bone resorption and tumor metastasis. Understanding how V-ATPases are regulated is thus crucial to understanding many disease processes, including viral entry, osteoporosis and metastasis. The V-ATPases are organized into two functional domains: a peripheral V1 domain responsible for ATP hydrolysis and a integral V0 domain responsible for proton translocation. Electron microscopic images of the V-ATPase complex reveal multiple connections between the V1 and V0 domains. To determine the arrangement of subunits within the V-ATPase complex, unique cysteine residues will be introduced into the B subunit and used as sites of attachment of a photoactivated crosslinker. In addition, electron microscopy of complexes decorated with subunit-specific antibodies will be performed. The function of a unique domain of the catalytic A subunit will be addressed by deletion and random mutagenesis. The structure of the 100 kDa a subunit and its interactions with the proteolipid subunits of the V0 domain will be determined using cysteine mutagenesis, chemical labeling and disulfide bond formation. Finally, the in vivo dissociation of the V-ATPase complex, which has been proposed to be an important regulatory mechanism, will be investigated. Dissociation in response to glucose depletion will be compared in V-ATPases located in different intracellular compartments and mutants defective in dissociation will be selected and analyzed. These studies should provide further insight into the structure and regulation of this important family of (H+)-ATPases.

PI: Forrester, Janet
Title: Nutritional Status in HIV-Positive Hispanic Drug Abusers
Abstract: The BIENESTAR study is a longitudinal cohort study of the effect of drug abuse on nutritional status and outcomes among Hispanics with HIV infection. The cohort is comprised of 3 groups: HIV+ drug abusers, HIV- drug abusers, and HIV+ non-drug abusers. Efforts will focus on expanding and maintaining this unique cohort.

New hypotheses addressed in this competitive renewal reflect current trends, as HIV becomes a chronic disease. Liver disease is an increasingly common complication in patients with HIV. While it is generally accepted that HCV is largely responsible for liver disease in HIV infection, studies suggest that drug abuse itself is a risk factor for liver disease among persons with HCV. Animal studies have shown that cocaine can cause liver toxicity by the induction of oxidative stress with tissue damage. Hispanics of the NE USA preferentially inject cocaine more than other races, and therefore may be at increased risk of oxidative stress and liver dysfunction. Low serum antioxidant micronutrient levels are common in drug abusers, further increasing the risk of oxidative tissue damage.

This study proposes to examine antioxidant status, oxidative stress, and liver dysfunction in Hispanic drug abusers with HIV infection. There are 4 specific aims:

1. To examine the association between the type (cocaine versus heroin) and frequency of drug abuse with plasma antioxidant capacity and oxidative stress. The specific interest is to know if cocaine differs from heroin in its ability to alter antioxidant capacity and cause oxidative stress.

2. To examine the association between the type and frequency of drug abuse and liver dysfunction. If cocaine or heroin cause oxidative stress to hepatic tissues this may result in detectable alterations in liver function.

3. To examine the association between oxidative damage and liver dysfunction. An association between oxidative stress and liver dysfunction will imply that drug abuse alters liver function through a mechanism involving oxidative stress.

4. To examine the influence of infection with HIV, infection with HCV, and alcohol consumption on the associations described in specific aims 1 to 3. This will determine if drug abuse is an independent risk factor for liver dysfunction or whether, like alcohol, it is an accelerator of liver dysfunction in persons with HIV and/or HCV infection.

PI: Freeman, Lisa
Title: Interdisciplinary Research Training for Veterinarians
Abstract: Well-trained comparative medical scientists are needed to meet the research needs of the 21st Century. Veterinarians currently are underrepresented in biomedical research but can make a unique contribution because of their expertise in clinical practice and fundamental biology, as well as their knowledge of spontaneous animal models of human disease.

The goal of the proposed education program is to attract veterinarians to NIDDK-relevant research. This program is focused primarily on veterinary residents, a highly motivated group for which research training usually is not provided. This program is designed to first create the desire to pursue research and then to nurture these candidates with strong mentoring and programs, as well as by providing readily accessible research opportunities. Finally, common barriers to research will be addressed.

A major aspect of the program will be biannual symposia on spontaneous animal models of human disease to disseminate information to the scientific community on the vast array of models available and to create new opportunities for collaboration. This program will utilize the strengths and resources of Tufts University to develop an untapped resource for research scientists.

This will be achieved under the mentoring of a program faculty that has been recruited specifically to provide an interdisciplinary team of collaborative scientists in a variety of disciplines and that provides experienced and positive role models. The program faculty consists of 17 faculty from 9 departments on three different campuses, providing a network of research training in nutrition and endocrine, digestive, kidney, urologic, and hematologic diseases.

Veterinary residents will be actively recruited, with particular attention paid to minority candidates. The program will consist of six parts:

1. Biannual symposia on spontaneous animal models of human disease to provide greater interaction with researchers from other disciplines and to increase opportunities for collaborative research
2. A multi-function website to facilitate research, including two web-based courses on laboratory techniques and applied statistical methods
3. A resident research and development (R&D) seminar series that will include topics to foster an interest in research and to facilitate research training
4. Short-term introductory research electives
5. Intensive research training electives
6. An active mentoring program

The inclusion of a specialist in outcomes assessment on the program faculty will ensure timely and accurate assessment of the program. Short-term evaluations will be used to guide development in the early stages of the program. Mid-range impact of the program will be evaluated by the number of participants, research presentations, grants, and papers. Long-term outcomes will be assessed by the number of Tufts-trained residents that do postdoctoral training and pursue research careers, and by using existing and novel outcomes assessment instruments. These evaluations, continued refinement, and the commitment of the program faculty will help to ensure that this becomes a self-sustaining program.