We propose to examine the association between aspects of carbohydrate nutrition including dietary intakes of total carbohydrate, dietary fiber and fiber sources, whole and refined grains, glycemic index and load with 1) changes over a 7 year period in body weight and body fat distribution, 2) changes over a 7 year period in several CVD risk factors including blood pressure and concentrations of fasting lipids, insulin, glucose, and 3) the incidence of IRS over approximately seven years of follow-up in the Framingham Heart Study Offspring of men and women. All data, which is derived from physical examinations, questionnaires and blood samples obtained at the 5th, 6th and 7th Framingham Offspring Study examinations is available for testing hypothesis in this proposed application.

The Framingham Heart Study has identified a number of predisposing risk factors for the development of CVD risk or type 2 diabetes, including obesity, cholesterol lipoproteins, and hypertension, among others. This rich database is an important reason why this proposed project has such potential. This is an extremely cost-effective study that will yield important insights into the role of dietary carbohydrates in the development of the IRS and may subsequently lead to simple, low-cost CVD prevention strategies.

PI: Moomaw, William
Title: Tufts Climate Initiative: Taking Action to Slow Climate Change
Abstract: Tufts Climate Initiative is the pioneer in the field of climate change mitigation at institutions of higher learning. In 1999, former Tufts University President, John DiBiaggio committed Tufts to meeting or exceeding the Kyoto target for reducing greenhouse gas emissions. In 2003, President Lawrence Bacow renewed the University's dedication to climate protection by adopting the emissions reduction goals of the New England Governors and Eastern Canadian Premiers (NEG/ECP). Last year, Tufts became the first university to join the Chicago Climate Exchange.

Tufts University was among the very first universities to make sustainability a high priority in its teaching, planning, and operations. TCI is continuing this tradition of being a forerunner; whereas many universities have jumped on the bandwagon and can proudly present a green building on their campus, TCI has chosen the more ambitious approach of fostering change with a comprehensive university-wide program.

PI: Moore, Claire
Title: Biomedical Research Experiences for Engineering Majors
Abstract: Tufts University seeks NIGMS funding for a Summer Internship Program designed to provide biomedical research experiences to Tufts undergraduate students majoring in engineering and computer science. The long-range goal is to encourage cross-disciplinary training for the next generation of biomedical scientists and thus promote an interdisciplinary approach to solving problems related to human health. The specific goal is to increase the number of students who pursue careers in biomedical research. The objectives are: 1. To increase the understanding of students about what a career in biomedical research would entail through distinct, innovative summer research internships on the Tufts Health Sciences Campus; 2. To increase the students' awareness of the benefits of biomedical research and potential careers, collaborations and post-baccalaureate training opportunities through workshops and seminars; 3. To increase students' laboratory skills and confidence by their planning, completing, and presenting a mentored, independent hands-on biomedical research project; 4. To increase the number of students who choose Biomedical Engineering as a major or minor; and 5. To increase the number of graduating engineering seniors who pursue post-baccalaureate training in bioengineering or biomedical research or enter industry careers in these areas. This program consists of a ten-week summer research internship for ten students in which the students interact with and work alongside outstanding research scientists. It builds upon existing strengths at Tufts in engineering, biomedical research, and undergraduate education, a proven commitment of Tufts to undergraduate research as a teaching tool, and a thriving culture of inter-departmental and inter-school collaborations. By exposing the students to potential opportunities and benefits of collaborations with biomedical researchers, it will help the students decide if biomedical research is an area in which they would like to apply their engineering and computer science training.

PI: Moore, Claire
Title: Molecular mechanism of mRNA 3'end formation in yeast
Abstract: The post-transcriptional acquisition of a poly(A) tail onto the 3’ends of eukaryotic mRNAs is an essential process that promotes transcription termination and transport of mRNA from the nucleus and serves as an additional point at which the cell can regulate the type and amount of mRNA derived from a particular gene. The poly(A) tail is also important for optimal translation and for determining mRNA stability. Polyadenylation requires site-specific endonucleolytic cleavage of the primary transcript followed by poly(A) addition to the upstream product. These steps are closely coupled in vivo, but can be experimentally uncoupled in vitro and assayed separately. While many of the protein components required for each step have been identified, much less is known about the biochemical nature of the process. Using the yeast S. cerevisiae as a model eukaryote, we will address the following specific aims: 1. Analysis of the molecular mechanism by which yeast cleavage factors recognize and cleave the mRNA precursor in the 3' untranslated region. These experiments will determine what constitutes the core cleavage complex, and how these factors interact with each other and with the RNA to mediate this step of the reaction. 2. Investigation of the molecular linkage between the mRNA 3'-end processing machinery and the mRNA transport apparatus. This aim seeks to identify connections between disassembly of the polyadenylation complex and assembly of the transport complex. We will ask when the cleavage/polyadenylation factors leave the polyadenylated product, and whether this recycling is facilitated by transport factors. A closely related issue is when transport factors join the mRNA, and whether their recruitment is assisted by the polyadenylation factors. With the research proposed here, we hope to derive a dynamic model of the complex that cleaves mRNA precursor. This work should provide insight into how this complex sets the stage for subsequent polyadenylation and transport of the mRNA. A better understanding of the basic mechanism will also make it easier to determine how polyadenylation is coordinated with other nuclear events and how the cellular environment modulates the activity or levels of the factors involved in this essential process.

PI: Moore, Claire
Title: The coupling of mRNA transcription and 3' end formation
Abstract: This is a supplemental application for our ongoing NIH grant GM068887. The goal of this grant is to characterize the molecular mechanisms involved in the coordination of RNA polymerase II transcription and mRNA 3' end processing. We have identified several new points of interaction between transcription and cleavage/polyadenylation factors which suggest that the presence of processing factors at the promoter might affect the efficiency and/or specificity of transcription initiation. This may serve as a mechanism to insure the proper loading of processing factors onto the transcriptional complex, and in turn, the subsequent polyadenylation of the transcript, which is essential for optimal export, translation, and turnover of mRNA. To investigate this issue, we propose the following specific aims: 1. What is the role ofSsu72 in early transcription events? Ssu72 is directly involved in 3' end cleavage of mRNA as subunit of Cleavage/Polyadenylation Factor (CPF), but also affects the activity of TFIIB in initiation. Our recent data indicates that depletion of Ssu72 causes a severe defect on transcription in vitro, and that Ssu72 is an RNAP II CTD phosphatase with preference for serine-5 of the CTD repeat. We will use assays that discriminate early events in transcription to define the precise role of Ssu72 in transcription and seek additional targets of the phosphatase activity of Ssu72. 2. How is the activity ofSsu72 regulated? Ssu72 interacts in vitro with TFIIB, RNAP II, and the Pta1 subunit of CPF. Our hypothesis is that these interactions regulate the activity of Ssu72 in transcription or 3' end processing. We will determine whether these interactions occur in vivo, and then investigate how disrupting these interactions affects Ssu72 function. 3. How does Swd2 function in RNAP II termination? Depletion of the CPF subunit Swd2 has no effect on 3' end processing, but causes a defect in termination. To examine how Swd2 is involved in termination, we will use chromatin-immmunoprecipitation experiments to identify factors that work with or are influenced by Swd2, use genetic analysis to demonstrate the functional significance of these potential Swd2 interactors and identify novel interactors, and use mutagenesis to verify the importance of these interactions for transcription termination.

PI: Must, Aviva
Title: Obesity and Psychopathology: Longitudinal Study of Youth
Abstract: Obesity and mental illness are two of the largest health issues the United States is currently facing, and their burden to society in human suffering, health care costs, and lost productivity is immense. The known psychosocial consequences of obesity include reduced educational attainment and lower rates of marriage. In adults presenting for the treatment of obesity, co-morbid psychopathology is increased. Widespread stigmatization of obese children is manifested through teasing and increased social stress. Nonetheless, the epidemiology of psychological outcomes subsequent to obesity has not been well characterized, and associations between obesity and psychopathology have frequently been assessed in clinical samples, or have used cross-sectional study designs. The proposed analyses make use of comprehensive data, from a longitudinal community study of determinants of psychological health from childhood to early adulthood, to characterize the specific psychological consequences of obesity and associations with relative weight during adolescence, and to assess the role of psychopathology on the development of obesity. Psychopathology is assessed based on standard Diagnostic and Statistical Manual of Mental Disorders (DSM) diagnostic criteria as well as by symptom scales. With longitudinal statistical modeling, the bi-directional associations of adolescent relative weight with anxiety disorders, mood disorders, and overall psychopathology will be assessed. The extent to which mediating and moderating factors contribute to the development of adolescent obesity and influence subsequent psychopathology will be examined. Relative weight will be assessed based on body mass index (BMI) calculated from self-reported height and weight. The Centers for Disease Control's BMI growth reference will be used to determine age- and sex-specific BMI z-scores. Obesity will be defined as a BMI score above the 85th percentile from the CDC growth reference. Understanding the psychological consequences of obesity will allow for increasingly focused research in prevention and treatment efforts aimed at reducing psychological and physical suffering from obesity and from mental illness.

PI: Ortiz, Daniel
Title: ABC-transporter binding proteins and trafficking
Abstract: The ABC (ATP-Binding-Cassette)-type proteins SPGP and MDR3 are essential for bile formation. SPGP mediates ATP-dependent transport of conjugated bile acids across the canalicular membrane and MDR3 is a phospholipid flippase that mediates transfer of phosphatidylcholine to bile. The transporters, which reside primarily in the canalicular membrane, can be recruited or removed from the apical domain in response to signals such as bile acids, cAMP or changes in osmolarity. Mobilization and targeting of the transporters to and from the canalicular membrane is probably mediated by association with proteins that link SPGP and MDR3 to sorting and trafficking networks. However, outside of the interaction between CFTR and NHERF, which is essential for polarized sorting of the chloride channel, little is known about proteins that bind and regulate trafficking of ABC-transporters. We have identified two proteins that specifically bind MDR3 and SPGP. GST-pulldowns from liver homogenates, FRET analyses and co-immunoprecipitation of transporters with associated proteins, confirmed the validity of these interactions. The goal of the proposed research is to study the role of these binding proteins in regulation of SPGP trafficking in polarized cells. Experiments in Aim 1 will use immunofluorescence microscopy and FRET to establish the sites in hepatocytes where SPGP interacts with binding partners, and the effect of stimuli which induce SPGP recruitment to, or retrieval from, the canalicular membrane. Aim 2 focuses on determining the function of the interacting proteins vis-a-vis SPGP trafficking. The specific amino acid motifs in SPGP which mediate its association with interacting proteins will be identified using yeast two hybrid assays. These moieties will be mutated with the objective of generating mutant transporters that do not bind interacting proteins. Trafficking of the SPGP mutants will be studied in polarized cell model systems to determine the function of the association with the interacting proteins. Mutations which cause abnormal trafficking of canalicular ABC-transporters have been associated with cholestasis of pregnancy and Dubin-Johnson syndrome. Therefore, elucidating the pathways that govern transfer and recruitment of ABC-transporters to the apical membrane, and identifying proteins which control these processes, will provide critical insight into mechanisms underlying cholestasis and suggest targets for therapeutic drug design.

PI: Panjwani, Noorjahan
Title: Corneal Epithelial Cell Surface Glycoconjugates
Abstract: The failure of the epithelium to migrate over the wound or of the migrated epithelium to remain adherent to the substratum may lead to the development of a number of debilitating clinical conditions of the cornea including recurrent erosions and persistent epithelial defects. We established during the previous funding period that two carbohydrate binding proteins, galectins-3 and -7, are among the key molecules which mediate corneal epithelial cell migration. For an understanding of the mechanism by which galectins-3 and -7 mediate corneal epithelial cell migration, in Aim 1, we shall identify and characterize the corneal epithelial cell surface and extracellular matrix (ECM) glycoproteins which serve as counterreceptors of galectins-3 and -7, and will establish whether the lectins modulate corneal epithelial cell migration by binding to well-known integrins, growth factor receptors, and/or ECM molecules. In Aim 2, using small interfering RNA (siRNA) and/or antisense adenoviral constructs, cDNA microarrays and glycogene arrays, we shall determine whether galectin-3 mediates corneal epithelial cell migration indirectly by modulating the expression of key adhesion and/or signal transduction molecules. In Aim 3, by determining whether galectins-3 and/or -7 modulate the activation of specific kinases (focal adhesion kinase, protein kinase B, MAP kinases) that are well known for their role in cell migration, we shall establish whether the lectins mediate corneal epithelial cell migration by modulating specific signal transduction pathways. The proposed studies will contribute to a better understanding of the molecular basis of corneal epithelial cell migration and should ultimately help find novel therapeutic strategies for treating nonhealing corneal wounds. In addition, this study will contribute to the basic understanding of the general disorders of impaired or delayed re-epithelialization including chronic wounds in the elderly, decubitus ulcers, and venous stasis ulcer of the skin, conditions that together affect millions of individuals worldwide.

PI: Poltorak, Alexander
Title: Genetic Dissection of Lipopolysaccharide Response
Abstract: The lipopolysaccharide (LPS) signaling pathway has been analyzed using combination of biochemical and genetic methods. However, not all of the proteins that participate in LPS recognition have been identified. In order to find more of them, several inbred strains of mice were evaluated on their LPS response and genetic basis of the differences in this response was examined. Among them, BALB/C and C3H/HeN strains differ significantly in their response to LPS. To further determine the relationship between TLR4 LPS response, (BALB/C x C3H/HeN) F2 intercross mice were analyzed on their LPS response and genotyped for TLR4 locus. These data show that although there is functional polymorphism in TLR4 observed between BALB/C and C3H/HeN mice, additional genes are clearly involved in LPS response. In order to map these genes, the panel of F2 mice was expanded and subjected to genome wide screening with all progeny genotyped for TLR4 locus. Genetic analysis of 80 meioses revealed that hyporesponse of BALB/C mice to LPS is linked to loci on chromosomes 6 and 17. To further characterize candidates and to improve resolution of genetic mapping, two congenic strains were constructed: one with BALB/C allele of TLR4 on C3H/HeN background and another - with HeN allele of TLR4 on BALB/C background. These strains were further analyzed to determine the level of their response to LPS. Two new loci, termed Lpml and Lpm2, may be assumed to contribute to the LPS response. The elucidation of Lpml and Lpm2 will represent an important advance, in that many genes that are known to contribute to complex traits can be identified by such approach. Similar strategy was employed for analysis of intercross response to peptidoglycan in F2 intercross animals. Elucidation of a precise mechanism of anti-bacterial response in a mouse model will undoubtedly contribute to the studies of host resistance to infectious diseases in humans.

PI: Prevelakis, George
Title: Balkans Conference in Hellenic and Southeastern European Studies
Abstract: The Balkan question has been more or less "forgotten" in Western Europe and even more in the United States. However most of the problems of the I990s remain unresolved. A new crisis is more than probable, although it is difficult to predict its timing. Even without an open crisis, the Balkan situation is extremely grave. Security is guaranteed only through the existence of a series of quasi-protectorates, the economic conditions remain disappointing and the steps towards Democratization are uncertain. The economic, political and moral burden of the Balkans remains very heavy. In addition, the Balkans function as a dangerous hearth of organized crime and corruption, threatening neighboring countries, one of which is Greece. Attracting attention and promoting understanding of the Balkans is therefore as important today as it was in the early I990s.

To create interest again, it is necessary to associate the Balkan questions with themes which, for one reason or another, appear more relevant. For the American public, the question of Iraq comes first (and will probably continue to attract attention in the foreseeable future) while the Atlantic relationship (especially in its most acute form, i.e. the French-American relationship) is also a focus of interest. The proposed conference will combine those three elements (Balkans, Middle East and French-American relations).

PI: Rich, Stephen
Title: Genetics & Population Structure of Plasmodium falciparum
Abstract: The current global population of the most deadly human malaria parasite, P. falciparum, is thought to have undergone a bottleneck sometime within the past several thousand years. Despite a recent common origin, P. falciparum populations are characterized by a high degree of protein polymorphism, particularly among certain immunodominant surface proteins. This extensive genetic variation provides the great adaptive potential by which the parasite evades the host immune response. The major objective of this proposal is to determine not only the degree of antigenic diversity in P. falciparum populations, but also to determine the principle mechanisms by which this variation is generated and maintained. We will examine the genetic polymorphism among natural isolates of P. falciparum from several endemic African sites. From these isolates, we will quantify the polymorphism among several molecular markers, which are dispersed on three completely sequenced chromosomes (chr2, chr3, and chr10). The genetic loci to be examined are three encoded surface protein genes--circumsporozoite protein (Csp, on Chr2) and merozoite surface protein-2 (Msp-2, on Chr3), and the 25kd P. falciparum sexual-stage antigen (pfs25, on Chr10). Each of these loci is life cycle-stage-specific in its expression and is the target of one or more vaccines currently in development. In addition to these protein-encoding genes, we will type several highly polymorphic microsatellite loci arrayed on the same three chromosomes. The resulting multi-locus genotype of each isolate will be used to test directly; (1) the level of meiotic recombination among genomes of natural P. falciparum isolates, (2) the extent of diversifying selection among the crucial protein-encoding genes, (3) the role of slipped-strand (mitotic) mutation in maintaining variability in immunogenic, nucleotide-repeat loci (Csp and Msp-2). This work will increase our understanding of the genomic evolution of this parasite will yield valuable information for evaluating appropriate strategies for intervening in disease transmission.

PI: Rios, Maribel
Title: BDNF Mutants: Genetic Models for Depressive Disorders
Abstract: Depressive disorders are debilitating conditions that affect millions of individuals and create an enormous burden on society. Close to 100 billion dollars per year are spent treating patients with severe and mild forms of depression in the United States alone. However, the underlying molecular mechanisms that trigger depression remain to be elucidated so that treatment alternatives for patients that are unresponsive to the current forms of therapy can be created. A potential target for the design of novel treatment strategies is brain derived neurotrophic factor (BDNF). Compelling evidence shows that BDNF modulates affective behavior but the specific role and the mechanism of action of this neurotrophin remain elusive. We recently generated conditional mutations of BDNF using the cre recombinase/IoxP system. These mice have a pre or postnatal depletion of BDNF in the central nervous system that does not compromise their viability as the global depletion of BDNF does. These mutants display dramatic changes in behavior including hyperaggression and hypersensitivity to stress, both of which are often symptoms of depression. We propose using these mutants, and others that we are currently generating, as genetic models of depressive disorders to dissect the role of BDNF in the regulation of behavior. Different lines of mutants that through genetic manipulation have depletion or over expression of BDNF in different regions of the brain associated with mood disorders will be tested using standard behavioral models for depression and aggression.

PI: Roberts, Susan
Title: Dietary Energy Restriction and Metabolic Aging in Humans
Abstract: Reducing morbidity and delaying mortality are recognized as major goals of aging research, and are addressed by this proposal to conduct a 2-year human caloric restriction (CR) intervention. A 1-year pilot study will be conducted in 32 overweight men and women to develop an effective CR regimen when fed at 70% of energy requirements determined at baseline. As part of this pilot we will refine all aspects of a CR intervention, including exercise and behavioral counseling, and will obtain necessary information on outcome variability with which to perform power calculations for the main study. Subjects will be randomized to two dietary regimens with different levels of dietary fat and glycemic index (GI) (20% fat and moderate GI vs. 35% fat and low GI) and dietary compliance and key outcome measurements will be determined at 5 periods throughout the year. Dietary factors such as dietary variety, liquid sources of energy, and dietary fiber will then be taken into account in the design of the interventions. Following identification of an effective CR regimen, a randomized 2- year intervention will be conducted in 117 overweight men and women fed 70%, 80% or 100% of energy requirements determined at baseline. The hypothesis will be tested that, compared to control subjects fed 100% of baseline energy requirements. The parameters to be evaluated will include immune function, oxidative stress, fasting insulin, hemoglobin Alc, and cardiopulmonary function. W further hypothesize that, compared control subjects, individuals randomized to 70% or 80% of baseline energy requirements will not experience adverse change sin thyroid and reproductive hormones, bone mineral density, disease incidence, mood or cognitive function. Dose-response relationships between the extent of CR and changes in outcome variables are anticipated. As part of the study, changes in total energy expenditure and resting metabolic rate, body composition and body temperature will be quantified to document the effects of CR on energy metabolism. We anticipate that the results of this study will have a major impact on our understanding of the relevance of CR to human health. In addition, this study will contribute to the development of new avenues for long- term treatment of overweight and obesity.

PI: Roffler-Tarlov, Suzanne
Title: Albinism: Defects in Tyrosinase, Amines, or Melanin
Abstract: This proposal requests funding for examination of the hypotheses that (1) tyrosinase activity in the embryonic eye results in the formation of developmental signals, that (2) these signals, perhaps amines, direct the generation of ganglion cells in the embryonic eye and that (3) the signals made through the activity of tyrosinase are transient and are made before tyrosinase switches to the synthesis of melanin in the pigment epithelium. The effects of amines on spatiotemporal features of neurogenesis would in turn lead to designation of the crossed and uncrossed projection of retinal ganglion cells. Our studies will focus on two lines of mice that are genetically identical except for a mutation at the tyrosinase locus. One line is pigmented (C57B16 Tyr+), the other, the albino (C57B16 Tyrc2j) carries a point mutation in the gene that codes for tyrosinase. The albino is known to have aberrant ganglion cell projections with the ipsilateral pathway reduced by half. Lack of functional tyrosinase in the albino is also correlated with changes in the timing of generation of retinal ganglion cells. Using histological and biochemical techniques, we will determine whether catechol or other amines are formed transiently in the developing eye, as is the case in peripheral tissues. We will test whether these substances are formed as a consequence of tyrosinase activity, again, as occurs in peripheral tissues. We will examine developing eye to see if there is a switch from the formation of developmental signals by tyrosinase to the formation of melanin, as also may be the case in peripheral tissues. We will correlate these events with the birth and differentiation of retinal ganglion cells. If we do find candidates for developmental signals for generation of retinal ganglion cells, we will test their effectiveness as signals in retina in vitro, and in vivo using tissues from albino mice including the OA1 knockout in which a G protein coupled receptor within melanosomes is mutated. Ultimately, if such signals deriving from tyrosinase-driven pathway unrelated to melanin production, are found, this research will open doors to therapeutic intervention in individuals carrying the OA1 gene.

PI: Romero, Michael
Title: Quantifying Physiological Stress in Threatened and Endangered Species Due to Military Activities
Abstract: Organisms must respond to unpredictable, novel, and/or dangerous
conditions in their environment to maintain homeostasis and optimize fitness (survival and reproduction). Response of wildlife populations to human disturbance has long been of concern to ecologists, which is reflected in strong federal regulations (e.g. the Endangered Species Act of 1973, as amended) requiring federal agencies to assess the effects of their actions on listed species. Physiological indicators of stress have been suggested as surrogate measures of human- caused stress in species of concern, notably measures of energy expenditure and the adrenocortical response. Human-related stressors characteristic of military operations that are episodic, unpredictable and varying in intensity have received relatively little attention in terms of physiological response. Mission critical military training and testing have been impacted by known, unknown, or potential impacts on endangered species. This study will evaluate physiological indicators of stress in two endangered avian species, the black-capped vireo and golden-cheeked warbler, to determine sensitivity and plasticity of response to military training disturbance.

The objectives of this proposal are to: (1) develop “dose/response” models for physiological response of selected priority endangered species to military stressors, (2) determine capacity of species of concern to habituate to “non-threat” disturbances, and (3) test predictive models for physiological stress response based on life history characteristics and taxonomic affiliation of endangered species. Meeting these objectives will fill knowledge gaps on the threshold level of disturbance necessary to elicit a physiological response and modulation of this response to repeated disturbance, and will provide a predictive framework for physiological response to disturbance based on life history characteristics and taxonomic affiliation.

PI: Rosenberg, Naomi
Title: Abelson Leukemia Virus Transformation
Abstract: Abelson murine leukemia virus is a rapidly transforming retrovirus that induces pre-B cell lymphoma and transforms pre-B lymphocytes and NIH 3T3 fibroblasts in vitro. The v-Abl protein tyrosine kinase mediates these responses and transmits signals that stimulate growth and suppress apoptosis and differentiation. All of these signals must be appropriately integrated for transformation to occur. A combination of genetic approaches will be used to determine how v-AbI accomplishes this. We will focus on three areas. A combination of genetic and biochemical approaches will be used to study the role of the v-Abl SH2 domain in mediating signals from v-AbI to downstream mediators. Two mutants will be used, a conditional mutant that is compromised for transformation at 39 degrees celcius, and a mutant that encodes a chimeric v-Abl/v-Src protein that fails to transform cells. In a second aim, we will examine the mechanism by which the COOH terminus of v-Abl affects lymphoid cell transformation. The role of the Ras pathway in this response will be investigated using a genetic complementation strategy and sequences at the extreme COOH terminal end of the protein will be identified using a series of deletion and truncation mutants. The mechanism by which these sequences influence the transformation process will be studied by identifying the cellular targets that are affected by these sequences. In the last aim, we will use a unique weakly oncogenic AbMLV mutant that generates highly oncogenic variants in vivo to understand the selective pressures that affect c-onc gene evolution. The dynamics and complexity of the viral population during the selection process and the role of target cell growth stimulation will be tested to uncover features that control this process. These events are likely to mimic those that occur during the v-onc gene capture and should advance our understanding of the way in which v-onc gene containing viruses arise and induce tumors.

PI: Rosenblatt, Michael
Title: Nature of PTH/PTHRP--Receptor Bimolecular Interactions
Abstract: This research proposal is focused on elucidating the interactions of parathyroid hormone (PTH) with its receptor (Rc, the hPTH1-Rc). Formation of the complex between PTH and the hPTH1-Rc leads to a sequence of events, namely hormone binding, Rc activation, and signal transduction, which culminate in expression of hormonal bioactivity. The impetus for this research program comes from a desire to understand: (1) the fundamental nature of molecular recognition between a peptide hormone and its G protein-coupled Rc; (2) the mechanism of action of the hormone (PTH) responsible for minute-to-minute regulation of calcium levels in blood; and (3) the differences in Rc states (conformations) which translate into hormone agonism, antagonism, inverse agonism, etc. The introduction of PTH as a major new agent for treatment of osteoporosis also focuses attention on the mechanism of anabolic action of this hormone. By gaining insight into the nature of the hormone-Rc complex, the discovery of small molecule PTH-mimetics may be facilitated by structure-guided design in the future. During the previous grant award period, by integrating photoaffinity scanning, molecular biology, pharmacology, and structural biology (conformational studies of hormone and Rc, and molecular modeling), we succeeded in generating an advanced experimentally derived model of the PTH-hPTH1-Rc bimolecular complex that provides structural detail and reveals some of the dynamics of hormone-Rc interaction. We are now positioned to take the next major step in mapping the interface of PTH and its Rc, and to extend our studies to identify the shifts in Rc conformation associated with activation. Specifically, we plan to: (1) improve the resolution of the map of the hormone--Rc interface; (2) study the interaction of PTH ligands covalently tethered to the Rc; (3) investigate the ability of dual-reactive analogs to simultaneously make contact with two sites in Rc; (4) perform "reverse" crosslinking from Rc to PTH; (5) prepare Rc and constitutively active Rc mutants on a large scale for structural studies of antagonist and inverse agonist interaction; (6) use disulfide bridge formation as a probe of Rc states; and (7) integrate all the above efforts in a molecular modeling initiative.

PI: Roy, Ananda
Title: Molecular Mechanisms of Acute Promyelocytic Leukemia
Abstract: The promyelocytic leukemia (PML) gene codes for a tumor suppressor protein that is associated with distinct subnuclear macromolecular structures called the PML bodies. The PML gene is frequently involved in the t (15;17) chromosomal translocation of acute promyelocytic leukemia (APL). The translocation results in a fusion gene product, PML-RARalpha, in which the PML gene fuses to the retinoic acid receptor alpha (RARalpha) gene. PML-RARa behaves as a potent transcriptional repressor for apoptotic genes and disrupts the architecture of PML bodies, a phenotype reversed by treatment with all trans retinoic acid (ATRA). Besides its role in APL, PML bodies have also been linked to viral infection. A variety of viruses targets the nuclear bodies and often causes their disruption. Moreover, upon interferon treatment of normal cells, PML is induced and the number of PML bodies increases dramatically, suggesting that PML may function as a mediator of interferon function and behaves as an immune surveillance factor. Although the activities of several transcription factors are modulated by virtue of physical association with PML bodies, the molecular mechanisms of PML or PML-RARalpha-mediated gene regulation remain elusive. Given the biological importance of PML and PML-RARalpha proteins, it is critical to ascertain their functional role and mechanisms of action. The TFII-I family of multifunctional transcription factors is activated in response to growth factor and antigenic signals to regulate growth-controlling genes, thus linking signal transduction events to transcription. We show a novel association of TFII-I family of factors with PML bodies. We further show a previously unknown function for PML-RARalpha it hyper-activates c-fos promoter in response to growth factor signaling. Based on these and other results, we hypothesize that the growth-regulatory and antigenic signals are processed through PML bodies in normal and APL cells to activate genes that control cellular growth or death via TFII-I family proteins. We propose to elucidate this novel pathway that will lead to a better understanding of PML and PML-RARalpha function.

PI: Ruskai, Mary Beth
Title: Noisy Quantum Communication and Computation
Abstract: This project is concerned with a number of mathematical problems in quantum information theory .The theory of quantum particles has the potential to provide the basis for vastly more powerful computers, and new methods of secure communication. Although building quantum computers remains a formidable experimental challenge, the feasibility of several methods of quantum communication and encryption have already been convincingly demonstrated. The main focus in this proposal is on problems related to quantum communication.

As with conventional classical communication, one must be prepared to deal with noisy channels when transmitting information via quantum particles. The P.I. plans to study a number of questions associated with the capacity (i.e., the maximum rate of reliable transmission of information per bit) of noisy channels. The resulting knowledge of how to encode message to minimize the effects of noise is clearly important. Some of this work also has other implications for experimental design. The P.I. has identified some situations in which noise can affect the phenomena which make quantum communication advantageous. When designing channels this information can used to decide how to best allocate resources to minimize certain types of noise. Conversely, the same information can be used to learn how to most effectively jam transmission through quantum channels.

Another important issue in both noisy communication and fault-tolerant computation is error correction. The leading proposal for dealing with errors in quantum computation is based on the use of large, concatenated codes which have many practical drawbacks. One alternative is to build computers which are resilient to some types of errors and focus on correcting the dominant, possibly correlated, errors. The P.I. proposes to construct a new code to deal with permutation errors, and to investigate whether it may lead to new methods for constructing other codes for adaptive error correction.

The P.I. also plans to examine the feasibility of a proposed new approach to efficient solution of hard problems called adiabatic quantum computation. Finally, the P.I. plans to study some mathematical problems about relative entropy and information geometry , upon which much of the theory of quantum information is based.

PI: Sahagian, Garabed
Title: Xenogen IVIS 200 Imager
Abstract: Biophotonic imaging of small animals using bioluminescent and fluorescent probes has become a state-of-the-art technology for studying normal and disease processes in vivo and is providing powerful new approaches for studying tumor biology, infectious disease, and gene expression. This application requests funds for a Xenogen MS 200 biophotonic imaging system to support a wide range of research being carried out at Tufts University Schools of Medicine and Dental Medicine and the Tufts-New England Medical Center in Boston. A core group of 12 well-funded NIH investigators will be the primary users of the instrument and will account for approximately 79% of its use. A small animal imaging facility will be established to house the imager. Establishment of the facility is strongly supported by the Chairman of the Department of Physiology, who has offered laboratory space for the facility and support for administration of the grant, by the Dean of the Medical School, who will provide funds for renovation of the laboratory space, and by the Vice President of Research at Tufts-New England Medical Center who has offered funds for maintenance of the instrument. The facility will be directed by the PI and the instrument will be operated and managed on a day-to-day basis by a full time research associate who will also train new users. An advisory committee consisting of the PI, and representatives from Tufts-New England Medical Center, the Division of Laboratory Animal Medicine and the Department of Physiology will oversee use of the MS 200 system and develop policies for use and equitable sharing of the instrument. At present, the PI has an MS imager on loan from Xenogen that is being shared by several of the major users. However, this instrument will have to be returned to Xenogen and no other suitable imager is available for use at Tufts University or the Tufts-New England Medical Center. The presence of the new MS 200 imaging system and imaging facility will greatly enhance the research carried out on the Tufts University Health Sciences Campus in Boston.

PI: Schlegel, Amy
Title: Ilya and Emilia Kabakov: The Center for Cosmic Energy
Abstract: The Center for Cosmic Energy is a “total” art installation by the former Soviet artist Ilya Kabakov (b. 1933) and his collaborator/wife Emilia Kabakov (b. 1945), a conceptual monumental “maquette” for a series of hypothetical architectural structures collectively devoted to the study of harnessing “cosmic energy.” The Tufts University Art Gallery's installation will focus on one building in particular, the Building "Communication with the Cosmos," a human-size (not actual size) structure containing a SO-seat auditorium set on a 30-degree angle inside a cylindrical shaft which itself is set on a 60-degree angle, the incline considered by the Kabakovs to be perfect for receiving cosmic energy from an "ancient reservoir" at a central axis. The visitor's attention inside the auditorium is captured by a 2O-minute audio lecture/performance on "communication with the cosmos." The Building for Communication with the Cosmos also includes a "subterranean" component installed in the gallery below --an archeological excavation of the aforementioned "ancient reservoir." The Center for Cosmic Energy therefore focuses on connecting individual, human consciousness with physical sites imbued with cosmic energy across the Earth, regardless of national boundaries and geography. The Center is, ultimately, a fictional research facility and a humanist, utopian model of an abstracted, psychologically charged space.

PI: Schwob, James
Title: Development of the Primary Olfactory Projection
Abstract: The fundamental event during the differentiation of a peripheral olfactory neuron is the selective expression of one allele of one olfactory receptor (OR) gene from among the 1296 members of the superfamily of OR genes. The choice of OR will determine the stimulus sensitivity of the neuron and will determine where that neuron should send its axon, although the means by which the OR directs axon targeting is still poorly understood. This grant proposes to answer 3 fundamental questions about OR choice. 1) Precisely how does OR choice reflect spatial location in the olfactory epithelium? Preliminary evidence suggests that the conventional 4-zone model relating OR expression to location is insufficient. We will take advantage of the explosion of genomic information to test specific hypotheses relating OR family membership and chromosomal location to the pattern of expression. 2) Where does the memory of spatial position reside that allows reconstitution of OR expression after injury? We will transplant progenitor cells into foreign parts of the epithelium to see whether the progenitor cells encode a memory for place or whether cues that derive from the local environment direct expression. 3) How does OR choice govern axonal connectivity during the course of epithelial reconstitution and neuronal regeneration? Data gathered during the prior period of support suggests that recapitulation of the precise one OR-one glomerulus organization occurs during the recovery from extensive peripheral lesion only when a substantial population of pre-existing axons are spared. We will take advantage of strains of mutant mice in which expression of an OR is coincident with expression of a marker protein to extend our analysis of the consequences of mild, moderate, and severe lesions of the periphery. The data obtained will allow us to determine whether fasciculation of growing axons with spared like-axons occurs when the projection is restored to its pre-lesion precision, and fails when it is not. We will use both confocal microscopy and immuno-EM analysis to test our hypothesis. Successful completion of the aims has implications for our understanding and treatment of human olfactory disease. Attempts to foster recovery in the clinical population require an understanding in depth of the inherent limits and capacities for regeneration.

PI: Schwob, James
Title: Regulation of Neurogenesis in Olfactory Epithelium
Abstract: The peripheral olfactory system has a remarkable capacity for repair after injury, and the maintenance of that capacity depends on the persistence of neurocompetent stem cells. If the stem cells are destroyed, the injured tissue undergoes respiratory metaplasia, which is a major cause of sensory dysfunction in humans. Despite the importance attached to understanding the regulation of olfactory stem cells, there is much that is not yet known, starting with their identity. Work during the previous period of support demonstrated that among the population of globose basal cells (GBCs) are cells that have many of the characteristics of stem cells, including the potency to differentiate into all the constituent cell types and the existence of slowly cycling, label-retaining cells (a hallmark of stem cells in other tissues). The 3 Specific Aims proposed in this application will provide a more in depth understanding of olfactory stem cells and how they are regulated. Aim 1 will use a transplantation/colony forming unit assay to determine the differentiate and generative capability of different marker-defined subsets of GBCs, including label-retaining ones. Aim 2 will test whether expression of members of the bHLH transcription factor family signify (and might drive) irreversible commitment to a single lineage. Aim 3 will test whether the Notch-Hes signal transduction pathway controls the choice point between neurons vs. non-neuronal cells that is made by the multipotent GBCs as they differentiate. Successful completion of the Aims will advance our understanding of olfactory stem cells and hasten their potential use as a therapeutic modality.

PI: Shirihai, Orian
Title: Erythroid transporter function in hemoglobin synthesis
Abstract: A number of disorders due to abnormalities in heme biosynthesis influence erythropoiesis. During normal erythroid maturation, heme precursors are generated in the mitochondria, modified in the cytosol and then returned to the mitochondria for final assembly with iron. Remarkably, accumulation of toxic heme intermediates or substrates, and secondary mitochondrial damage, are key elements in the pathophysiology of all heme biosynthesis disorders. However, the mechanisms of export or import of heme products, by-products and intermediates to or from the mitochondrial matrix are as yet poorly understood. This proposal will focus on such mechanisms taking advantage of our discovery of ABC-me, a novel mitochondrial erythroid transporter. ABC-me expression is controlled by the erythroid transcription factor GATA-1 and is down regulated by heme. In differentiating erythroleukemic cells, ABC-me is rate limiting for heme biosynthesis. As such, ABC-me is the only mitochondrial inner membrane transporter implicated in heme biosynthesis. To explore its function in heme biosynthesis, we have developed ABC-me deficient cell culture models and functional assays of reconstituted transporter in proteoliposomes. We have established that differentiating erythroid cells with reduced ABC-me activity produce less heme and exhibit signs of mitochondrial stress. We postulate that transporters involved in the heme pathway serve as gatekeepers for reactive substrates, intermediates and byproducts and thus couple production of essential end products to protection from toxic intermediates. We propose a combined biochemical and biophysical approach to the study of the function of ABC-me in heme biosynthesis and the consequences of its malfunction. We will address the following questions: 1) Which steps in the heme biosynthetic pathway are facilitated by ABC-me? 2) Does ABC-me deficiency result in the accumulation of heme precursors in the cytosol and mitochondria? 3) What is the source of mitochondrial stress associated with ABC-me deficiency and what are the morphological and functional consequences? 4) Does ABC-me transport a heme biosynthesis intermediate, product or co-factor, and in which direction? 5) Does ABC-me facilitate heme biosynthesis indirectly by opposing mitochondrial stress.

PI: Shultz, Mary Jane
Title: The Ice Surface: Chemical Probes of the Native Surface, Interactions, and Reconstruction
Abstract: Ice is one of the most important solids in the Universe. In interstellar space, the surface of ice, a veritable chemical factory, has been recognized as a significant constituent of dense interstellar clouds and is well recognized as a major component of comets that spread materials throughout the Universe. Closer to terra firma, generation and interparticle transfer of charges on ice particles are responsible for most lightning storms. In the stratosphere ice surfaces playa key role in enabling and catalyzing reactions that are responsible for the now-famous ozone hole. Throughout the troposphere, ice and aqueous particles transport and cycle countless species as well as providing a staging site for reactions that are either forbidden or very slow in the gas phase. Despite its importance, until recently, the surface of ice was a mysterious material at the molecular level. The emerging picture includes reasonable agreement about the configuration of the oxygen atoms at low temperatures «130 K). The hydrogen atoms are more elusive and their location is controversial with some measurements indicating a very low density of dangling-OH groups and others finding evidence for plentiful dangling-OH groups and ordering. The location of the hydrogen atoms, the density of dangling-OH groups, however, profoundly affects the reactivity of ice. The objective of the proposed work is to generate an atomic-level picture of the hydrogen atoms and characterize their rearrangement due to changing temperature or interaction with incoming molecules. Due to the development of surface-sensitive spectroscopy, the tools are now available and the time is ripe not only to locate the hydrogen atoms, reconciling these seemingly contradictory measurements, but also to determine their mobility, producing a molecular-level understanding of reactions on this important surface.

PI: Skelly, Patrick
Title: Gene silencing in schistosomes using RNAi
Abstract: Schistosomes are extracellular blood worms that infect over 200 million people globally and cause several thousand deaths annually. Large-scale genome sequencing projects for each of the three major human schistosome species: Schistosoma mansoni, S. haematobium and S. japonicum, are currently underway. Despite the wealth of new data to be generated by all of these undertakings, there is still no routine technique available that allows us to exploit the data through manipulation of the schistosome genome. Because of this, our understanding of the molecular and cellular biology of schistosomes has been severely hampered and lags behind that of most other important human pathogens. In this application, we propose to utilize a relatively new technology in gene manipulation called RNA-mediated interference (RNAi) to examine gene function in schistosomes. In preliminary experiments, we have achieved considerable inhibition of select genes involved in nutrient acquisition by the parasites. We plan to build on this success by first optimizing the protocol for gene suppression using RNAi in different schistosome life cycle stages by varying the amount and nature of the dsRNA employed, the mode of delivery and the duration of exposure. Such a systematic assessment of the relative importance of the factors that impinge on the phenomenon could have wider applicability for researchers using RNAi in other biological systems. Next we will utilize the optimized protocol to test specific hypotheses concerning an important area of schistosome biology: The involvement of proteases in hemoglobin degradation and nutrient acquisition. The work proposed here is designed to provide a simple, powerful and widely applicable protocol for the schistosome research community to facilitate functional schistosome genomics. In addition, our application of the technology is designed to provide significant new information about schistosome metabolism and biology.

PI: Sonenshein, Abraham
Title: Clostridium difficile Toxin Gene Regulation
Abstract: Clostridium difficile is the principal causative agent of antibiotic-associated colitis and the only known cause of pseudomembranous colitis, a potentially lethal disease. Two large toxin proteins, encoded by the toxA and toxB genes, which lie within a 19 kb pathogenicity islet, are the primary virulence factors. Previous work from the applicants' laboratories has shown that transcription of the tox genes depends entirely on TxeR, a novel RNA polymerase sigma factor encoded within the same pathogenicity locus. The level of expression is strongly modulated, however, by the growth state of the cells and by environmental conditions. For instance, for cells growing in broth medium, tox gene transcription is restricted to stationary phase cells and is repressed by rapidly metabolizable carbon sources, such as glucose. Other factors that influence toxin synthesis are the availability of biotin and certain amino acids and the temperature of cultivation. The present proposal seeks to determine the molecular mechanisms that control toxin synthesis in response to environmental signals. Candidate regulatory proteins, based on comparative genomics, will be specifically tested for their participation in such regulation. These proteins include CodY, CcpA and VirR, whose homologs in Bacillus subtilis or Clostridium perfringens have been shown to mediate the types of regulatory effects in question. In addition, general, unbiased searches for the relevant regulatory proteins will be carried out based on affinity chromatography. A fourth protein, TcdC, will be tested for its potential activity as an antagonist of TxeR. The project makes use of the expertise in C. difficile biochemistry, genetics and physiology of the three collaborating research groups and recent advances made by each of the groups in making this experimental system amenable to detailed molecular genetic analysis.

PI: Soto, Ana
Title: Prenatal Xenoestrogen Exposure and Mammary Cancer
Abstract: Increased breast cancer incidence has prompted scientists to consider the possible role of hormonally active environmental chemicals. However, epidemiological studies searching for correlations between adult environmental exposures and breast cancer incidence have been mainly inconclusive. Yet, epidemiological studies do suggest that fetal estrogen level fluctuations have long-term consequences on the risk of developing breast cancer as an adult. Studies in mice reveal that prenatal exposure to low doses of the xenoestrogen bisphenol A (BPA) alters the development of these mammary glands. These effects manifested long after exposure ceased. The increased number of terminal end buds and terminal ducts in these mammary glands is particularly relevant since carcinomas originate in these structures. We hypothesize that prenatal exposure to low doses of BPA may increase the risk of mammary cancer and that the mechanism responsible for the neoplastic outcome may include the extemporaneous expression of genes that mediate mammary gland development. Xenoestrogens administered prenatally may alter the expression of estrogen-responsive genes that would then affect downstream genes in the mammary development program and predispose these animals to cancer. To test this hypothesis we will use a rat model whereby exposure to the carcinogen nitroso-methyl-urea (NMU) at puberty induces mammary cancer. A pilot study performed using Wistar rats showed that this model could be modified to assess the effect of fetal exposure to environmentally relevant xenoestrogen levels. It produced data consistent with the proposed hypothesis. Aim 1: To determine the levels at which prenatal BPA exposure results in an increased incidence of mammary cancer. Tumor incidence and latency will be measured in Wistar rats exposed to BPA from gestational day 9 to birth. At 50 days of age, animals will be injected with a sub-carcinogenic dose of NMU. Aim 2: To test the hypothesis that prenatal BPA exposure alters gene expression in the mammary gland during both the period of BPA exposure and throughout life. Total RNA will be isolated from mammary glands and analyzed by DNA microarray technology. Where appropriate, laser-capture microdissection and/or RNA linear amplification techniques will also be used to obtain samples. Up- and down-regulated genes will be confirmed by QRTPCR. Cellular distribution will be assessed by in situ hybridization. Developmental points to be analyzed are: 1) during gestational BPA exposure; 2) 5 days postnatal; 3) peripubertal; and 4) after ductal development is completed. Aim 3: To test the hypothesis that in utero exposure to BPA alters the histoarchitecture of the rat mammary gland and results in the expression of pre-neoplastic phenotypes. This Aim will assess whether exposure to low doses of BPA results in specific morphological alterations in the rat mammary gland that may confer a propensity to carcinogenesis, such as an increase in the number of terminal end buds (TEBs) and terminal ducts (TDs) at the time of NMU administration), the persistence of TEBs beyond puberty, and the appearance of pre-neoplastic lesions at several age points. In addition, the histoarchitecture of the mammary gland at the time-points studied in Aim 2 will be examined. Aims 1, 2 and 3 are intimately linked, because they seek to study the same phenomenon at different levels of biological organization. Gene expression alterations may suggest histoarchitectural consequences and vice-versa. This exploratory research is central to the generation of testable hypotheses about cause-effect relationships linking prenatal hormonal exposure and mammary gland carcinogenesis and may finally provide the direct link between environmental exposures and incidence of breast cancer researchers have been looking for over the past 20 years. Moreover, if, as suspected, low doses of BPA increase the incidence of mammary cancer, this work may have a great impact on the way we study risk factors and conduct epidemiological studies. It may even influence public policy about prevention.