PI: Rich, Stephen
Title: Genetics and 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: Rogers, Chris
Title: Multi-threaded Instruction: Forming Multi-disciplinary Research Groups To Improve Undergraduate Education
Abstract: The primary goal of the creation of the Robotics Academy is to form a new multi-disciplinary, project-based teaching and learning environment for undergraduate students, involving students from different disciplines in the discovery process and exposing them to the joy of research and design through handson experience with real world problems. A program that provides opportunities for learning in the context of multi-disciplinary problem solving is expected to improve the education of the student and produce engineers that are highly motivated and technically capable. Further, the Academy will include students (and teachers) from multiple universities (Tufts University and University of Nevada, Reno), giving the students and teachers the opportunity to benefit from specialties at other universities, as well as to collaborate with others remotely over the Internet. Under the proposed system, junior students from multiple disciplines (i.e., mechanical engineering, electrical engineering/computer science, human factors, biochemistry/chemistry, and child development/ education) will join the Robotics Academy, completing their major program of studies through a predefined robotics thread. The robotics thread in each discipline consists of a set of courses that introduces the students to robotics and how it applies to their chosen fields. During their two years in the Academy, they will work in a team, composed of four students from different disciplines, on a common robotics project. This project will constitute their undergraduate honors thesis. Each multi-disciplinary Academy team will work on a robotic project in one of 4 areas: Medical Robotics, Tele-robotics, Nanorobotics, and Robotic Toys. Medical robotic projects will be led by the responsible faculty in the Academy, in conjunction with surgeons (Tufts School of Medicine and the Tufts-New England Medical Center), veterinarians (with Tufts Veterinary School), or occupational therapists (Tufts University Boston School of Occupational Therapy). Through their robotics solution, students in these projects will aim to improve the quality of life of the patient, or improve the safety and productivity for a particular medical application. Tele-robotics projects will introduce students to concepts and applications of remote control and manipulation. This work will be done in conjunction with NASA scientists and will teach students about remote exploration. The Nanorobotics project will put students at the forefront of modern robotics, involving them in the cutting edge of nano-fabrication and control techniques. The final project area will be in the design of educational robotic toys. This project will build on the highly successful collaboration with the LEGO Corporation and the development of ROBOLAB at Tufts. As a team, each team member will learn from and contribute to the process in the life cycle of the robotic solution. For instance, the mechanical engineering student will work on robot design and fabrication; the computer science/electrical engineering student will design the circuitry and program the intelligence of the robot. The human factors student will design the user interface to ensure safe and effective use, while the pre-service teacher on the team will implement the engineering education outreach efforts. In addition, senior students in the Academy will act as mentors to junior students from another discipline. This arrangement allows for the students to receive individual attention, while encouraging communication across the disciplines. Each senior will also be responsible for teaching one Academy Hands-On seminar, an afternoon class in some practical aspect of robotics that will be useful to all students in the academy. For instance, a mechanical engineering senior might offer a course in ProEngineer and CAD, whereas a child development student might offer a class in cognition and learning strategies. Five robotics projects are planned for the first year, with two new projects being introduced in each subsequent year. Progress of the proposed program will be assessed throughout the three years of proposed funding. Results of the robotics projects will be documented on the Academy website and disseminated at conferences. The Academy is expected to be self-sustaining after three years, through industrial support for individual projects.
PI: Rogers, Chris
Title: Teaching Through Touching: Using Research to Motivate Education
Abstract: Engineering education can be made exciting and attractive to students of all backgrounds, races, and gender if it includes the excitement of open-ended research. That is, if neither student nor teacher knows the “right answer” to the question. Unfortunately, this kind of engineering or scientific inquiry rarely happens before graduate school. Over the past 15 years, Professor Rogers has been working to change that. During this time, Professor Rogers has built up an internationally recognized program in particle-laden turbulence, in chemical-mechanical planarization (CMP), and in engineering education. His work in education has led to multiple international alliances aimed at bringing engineering into the liberal arts curriculum in universities, into the K-12 classroom, and in improving engineering education for the college engineering student tas well. At the core of these efforts is the desire to teach through research, giving students of all ages the tools and self-confidence to answer their curiosity and to understand math and science. Over the past 5 years, the PI has developed the ROBOLAB™ toolset, a combination of LEGO™ hardware and LabVIEW ™ software that allows students to predict, investigate, test, and understand math, science, and engineering. ROBOLAB is designed to have a very low entry (youngest user was a 3 year old) and high ceiling (it still poses challenges for graduate students).
This proposal has three specific goals: to investigate how students learn engineering both
the kindergartner and the university engineer), to push the ceiling of the PI’s toolset higher by combining the robotics toolset with the CMP research, and to increase the collaboration of all institutions (K-12 and university) dedicated toward bringing openended research problems into the classroom as a teaching method. This work would be the primary focus of a graduate student team, one in education and one in engineering
and a faculty team, some in engineering, some in education. We would involve universities, industry volunteers, government institutions, and science centers around the
world, supplying a support and dissemination structure for these efforts. We would try new directions in both research and education, for instance integrating the virtual world
with the real world of ROBOLAB. We would test new informal education efforts, including integrating math, science, and engineering education with movie cinemas and
the Internet. Professor Rogers’ long-term goal is to increase the engineering literacy of the average graduate, reducing the phobia associated with engineering and breaking down the walls between engineering and the liberal arts courses. To do this effectively, one must start in the kindergarten and continue the effort through college. This proposed
work would provide a big step toward that goal by helping in the dissemination of the engineering material to that K-college body, by helping understand the cognitive
processes behind the learning, and by ensuring that the toolset is flexible enough that all students are challenged.
PI: Rogers, Chris
Title: Tufts Engineering the Next Steps (TENS) GK 12 Project
Abstract: The TENS project focuses on engineering. We adopt a particular emphasis on engineering design, mathematical foundations of computer science, and on helping students to situate engineering in its larger societal context. The primary goals of the Tufts Engineering the Next Steps project are:
(1) To raise teachers. knowledge of, comfort with, and capability to teach engineering and algorithm design,
(2) To increase all students. engineering knowledge and skills
(3) To increase fellows. appreciation for and knowledge about K-12 formal education, teaching, and outreach and
(4) To develop a grade 5-12 community in four schools that is supportive of innovations that integrate engineering and algorithm design.
The TENS proposal builds upon an existing GK12 project that worked with a suburban district to explore whether and how teachers might integrate engineering into their classes. The new challenge that TENS undertakes is to help to integrate engineering into an urban rim. School district that has a population that diverse racially, ethnically, and socioeconomically very diverse. The Tufts Engineering the Next Steps (TENS) GK12 project will work intensively with four schools in the Malden school district. Malden is in the process of revising the K-12 math and science curriculum and intends, with the help of GK12 fellows, to build an engineering strand into the new district curricular frameworks. Eight graduate fellows and seven
undergraduate fellows will guide teachers and provide support for integration of engineering content into grades 5-12. TENS consists of 4 principal and overlapping components: the preparation and education of fellows, professional development of teachers, school and classroom-based efforts, and curriculum enhancement.
The fellows and the teachers work as partners that recognize, capitalize upon, learn about, and slowly develop some of the expertise that the other party possesses. By working with a number of teachers in the same school and district, we aim to develop a cadre of innovative teachers who can support each other's engineering efforts. Intensive contact with one district, its schools, and its teachers over three years is designed to foster curricular and teacher knowledge changes that will enable the continual inclusion of engineering concepts and activities in the district beyond the life of this grant.
PI: Romero, Michael
Title: Physiology of Stress in Wild Animals
Abstract: All organisms are faced with noxious stimuli (such as predators or storms) from the environment, and somehow these stimuli must be ameliorated in order for the organism to survive. One important system (among many) activated in vertebrates during the response to stress (the physiological mechanisms involved in protecting an organism from an external noxious stimulus) is the hypothalamicpituitary-adrenal (HPA) axis. After several minutes, activation of the HPA axis culminates in glucocorticoid release. Although the stress response allows an animal to respond to and survive noxious environmental stimuli, even after more than 60 years of studying stress we still have only a rudimentary idea of the role glucocorticoids play in wild animals. This project addresses this gap in our knowledge. Results from prior support indicate that several free-living bird species seasonally regulate glucocorticoid release. In other words, both initial and stress-induced corticosterone (the avian glucocorticoid) concentrations depend upon the time of year. The regulatory mechanism involves changes in sensitivity at various regulatory points of the HPA axis. Furthermore, one physiological effect of corticosterone, mobilizing glucose to help combat the stressor, may be less important for free-living animals than in laboratory-housed animals. This proposal builds upon these results and begins to explore some physiological consequences of having different corticosterone responses at different times of the year. Specifically, this proposal asks the question: why do animals seasonally regulate glucocorticoid levels? The following three hypotheses will be tested:
(1) Physiological effects of glucocorticoids are also modulated seasonally (there is a physiological consequence to seasonal modulation of orticosterone release).
(2) Corticosterone’s physiological effects (e.g. mobilizing glucose) are important to coping with stressful events under natural conditions.
(3) Changes in HPA axis function resulting from chronic stress can be distinguished from ormal seasonal modulation of HPA axis function.
Testing these hypotheses will require an integrated approach of both laboratory and field studies. Hypothesis 1 will be tested with field studies by capturing wild house sparrows (Passer domesticus) from their natural environments. Seasonal changes in baseline and stress-induced glucose and triglyceride levels will be monitored by taking blood samples. Injecting exogenous corticosterone and monitoring changes in glucose levels will further connect glucose to corticosterone release. Testing Hypothesis 2 will require field studies to distinguish between glucocorticoid and epinephrine’s effects on glucose, and to determine whether the glucose and triglyceride responses to stress differ when stress is accompanied by access to food (such as when birds are foraging). Hypotheses 3 will be tested in the laboratory by determining whether a chronic stress paradigm will alter the function of HPA axis regulatory points. Together, testing these three hypotheses will help define the physiological consequences of seasonal glucocorticoid release during stress in wild animals. This research will thus have a far-reaching impact on our basic knowledge of the ecological validity of stress responses. The help of students will be vital to completing the above research. Both graduate and undergraduate students will learn how to trap animals, take blood samples, perform assays, write manuscripts, and many other important skills. This research will thus provide important training for these students. My research program also provides breadth to these students since few laboratories currently combine both laboratory and field studies. Answering Hypothesis 3 will also be valuable to researchers in other fields. Conservation biologists especially should benefit from being able to determine whether animals are chronically stressed.
PI: Romero, Michael
Title: Quantifying Physiological Stress in Threatened and Endangered Species Due to Military Activities
Abstract: 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, Irwin
Title: Renal Transplantation, Homocysteine Lowering and Cognition
Abstract: This application is for an ancillary study to determine the cognitive effects of homocysteine Iowering, as an additional outcome in an ongoing, randomized, controlled, double-blind clinical trial in renal transplant recipients (RTRs). Elevated plasma homocysteine levels are associated with diminished cognitive function in the general population, and significantly increase the risk of vascular disease, cerebrovascular disease, stroke and dementia.
Hyperhomocysteinemia is a pervasive feature of chronic renal insufficiency, even after a successful transplant; however, unlike in other types of chronic renal insufficiency, homocysteine levels can be lowered in RTRs by high doses of B-vitamins. The parent trial (FAVORIT - NIH NIDDK UO1 DK61700-01) is designed to determine the effect of lowering plasma total homocysteine levels on atherosclerotic cardiovascular disease outcomes in chronic, stable RTRs. The FAVORIT trial aims to randomize 4000 RTRs with a stable functioning renal graft of > six months to a treatment or placebo group, and to follow them for 4 years or until the occurrence of a cardiovascular event or death. Treatment consists of a standard multivitamin with additional high dose folic acid, vitamin B12 and vitamin B6 and placebo consists of a multivitamin devoid of these vitamins. This application specifically aims to: (1) determine the cognitive effect of homocysteine lowering under this treatment regime; and (2), characterize cognitive function in relation to homocysteine and other risk factors for vascular disease in this high-risk, non-demented population. To do so we will measure cognitive outcomes in 1000 participants in the FAVORIT trial, at randomization and after a 3-4 year follow up. The outcome of this application may be highly significant in improving health care for RTRs and other groups with chronic renal insufficiency. Our long-term goal is to identify risk factors for cognitive impairment that can be modified through nutritional intervention or dietary supplementation in order to reduce the incidence of cognitive decline and dementia in elderly and other vulnerable populations. Demonstrating the cognitive benefits of lowering homocysteine may pave the way to nutritional modification of cognitive decline in RTRs and other high-risk groups and even in the general population.
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: Rosenberg, Naomi
Title: RNA - Tumor Virus Hematopoietic Cell Interaction
Abstract: Abelson murine leukemia virus (Ab-MLV) transformation has long been a valuable model to understand the dynamics by which retroviruses containing v-onc genes induce tumors and transform cells. The virus provides a simple genetic tool to examine the way in which signals that stimulate growth, suppress apoptosis and alter differentiation interface with signals from tumor suppressor genes. Ab-MLV infection of B lineage cells invariably leads to transformation and tumor induction. This is a rapid process: cells infected in vitro begin to divide soon after viral integration and expression of the v-Abl protein, and infected mice succumb 20 to 30 days post infection. Despite this, the road to transformation is not a smooth one. Viral signals that stimulate cell growth and suppress differentiation and apoptosis are countered by a cellular response that promotes apoptosis and erratic growth. Understanding how the virus counters the cellular response and uncovering the pathways that tip the balance in favor of malignant growth will have broad implications for our understanding of malignant disease and illuminate the features that confer oncogenic properties to Ab-MLV and other v-onc gene-containing retroviruses. In the Ab-MLV system, the p53 pathway is inactivated in most transformants, in some instances, the cells acquire p53 mutations and in others, the p53 regulatory protein, p19Arf is down modulated. We are using the virus to understand how signals from the virus counter anti-tumor effects mediated by the host; We will investigate four aims:
1. How does p53 influence the outcome of Ab-MLV infection?
2. How do the products of the Ink4a/Arf locus influence Ab-MLV transformation?
3. How do the p53 and Ink4a/Arf loci affect tumor induction in vivo?
4. How is the target cell specificity of Ab-MLV controlled?
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: 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 PI 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 PI 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 PI 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 PI also plans to examine the feasibility of a proposed new approach to efficient solution of hard problems called adiabatic quantum computation. Finally, the PI 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: Molecular Basis of Bone Metastasis in Breast Cancer
Abstract: The long-term goal of the project is to identify genes involved in organ-selective metastasis that can serve as new targets for breast cancer therapy and to understand how these genes function in metastatic progression of the disease. The specific goals of the project are to isolate, clone and characterize a unique set of variant metastatic cell lines with organ-selective properties and use them to identify genes associated with metastasis to lungs, liver, bone and brain, the primary sites of metastasis in human breast cancer. The specific aims are (1) To obtain and characterize variant tumor cell lines with altered ability to metastasize to bone, liver, lungs and brain. The lines will be generated by repeated rounds of in vivo metastasis and isolation from each of these organs; (2) To identify differences in gene expression associated with phenotypic differences that characterize these variant cell lines. From these experiments, putative organ-selective metastasis gene will be identified (3) To verify the involvement of identified genes of interest in organ-selective metastasis by appropriately altering expression of the genes in the parental and variant lines and determining the effects on in vivo metastasis.
PI: Saperstein, George
Title: Rare Breeds Preservation Program
Abstract: Under the direction of Dr. George Saperstein (Principal Investigator), TUSVM will conduct studies to produce and ciyopreserve germplasm of rare and endangered breeds of domestic animals. The purpose is to continue to sustain a paramount rare breeds germplasm repository. Breeds will be identified based on their respective genetic trait(s), value, extent of threat to the breed, and the programmatic interests of Sponsor. Efforts will focus on sheep, goats, and cattle. TUSVM will provide the expertise in germplasm preservation and herd health management, and advise on animal husbandry and biosecurity at Sponsor facilities in Newport, RI (“SVF”; “On Site”) and at TUSVM facilities, located in N. Grafton, MA. Tufts veterinary students will actively participate in the program in support of the school’s educational mission. The objectives of the program are twofold: first, to produce and preserve germplasm of rare breeds of domestic animals; and, second, to establish and implement a comprehensive herd health and biosafety plan for Sponsor’s animals. Student education and training activities will be integral aspects of the program. The study will be conducted predominately on site with some laboratory activities carried out at TUSVM in support of program requirements. In addition, the off-site germplasm collection and cryopreservation program will continue, largely focused on cattle embryos, semen, and cells as well as small ruminant semen.
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: Selsing, Erik
Title: Immunity in Transgenic Mice
Abstract: Class switch DNA recombinations are important for isotype switching in B cells and also appear to be involved in chromosomal translocations of some oncogenes. However, the mechanisms of switching and the processes that target these mechanisms to certain DNA regions are still not known. Switch (S) regions containing tandemly repeated sequences are located upstream of all H-chain antibody constant region genes and are the target of the switch recombinational machinery. However, the roles of S regions in targeting are unclear. We have found that the S? tandem repeat region is not required for switching but does play a role in providing highly efficient switching. In mice lacking the DNA mismatch repair protein, Msh2, we have also found that the S? tandem repeats are critical for switching, indicating that different DNA regions need different proteins to complete switch recombination. Finally, measurements of switch site distributions in mice that lack either the S? tandem repeats or the Msh2 protein show shifts in switch targeting. These shifts indicate that a 4-5 kb domain downstream of the l? promoter is accessible for switching even if the S? tandem repeats are not within this domain. In addition, in the absence of Msh2, switching is focused to the tandem repeat region within the domain. The current proposal seeks to further analyze the targeting of switching by S regions using a variety of mutant mouse strains that affect the isotype switching process. First, the roles of S? region sequences in targeting switch recombination through possible formation of R-loop structures, or by promoting specific chromatin structures will be analyzed in wild-type and mutant mice. Second, the roles of the Mlh1 and Exo1 mismatch repair proteins in switching will be compared to the role of Msh2 to determine whether these proteins affect the same or different pathways in the switching mechanism. Third, the abilities of S? and l? regions in regulating switch targeting will be analyzed by relocating these sequences and assessing whether switch targeting is also relocated. Finally, we will assess the importance of the AID protein, which is critical in initiating switch recombination, for the u transgene chromosomal translocations that were discovered in our laboratory. Although aberrant targeting of oncogenes by the switch mechanism has been suggested to be involved in some oncogene lgH translocations, recent studies by another laboratory have indicated that these translocations do not involve AID. If transgene translocations also do not involve AID then this would provide a convenient model system for genetic analyses of the sequences and proteins important for the IgH translocation process.
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 play a 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.
The method consists of probing single crystal ice surfaces with a combination of surface sensitive vibrational spectroscopy, specifically sum frequency generation (SFG), and chemical probes (CH3OH, CO, NH3, HCl, and CF3Cl). Determining the accessibility of the hydrogen atoms is all-important for a mechanistic understanding of reactions at the ice surface. For example, accessible and mobile hydrogen atoms are believed to promote ionization of ionic molecules and easily rearranged hydrogen bonds likely lead to a liquid-like layer on the surface. Comparison of single-crystal ice surface measurements with previous low-temperature polycrystalline ice results is expected to determine whether the surface or edges dominate icecatalyzed reactions under atmospheric conditions. The results will not only directly contribute to understanding of interactions at this important surface, they will also form the basis for extending investigations to lower and higher temperatures. Lower temperatures are relevant to interstellar cloud chemistry where the recent discovery of ice and small molecules (H2CO, CO, H2O2, and NH3) may account for formation of numerous precursors of amino acids, the building blocks of life. At higher temperatures, mobility in the liquid-like layer may hold the answer to questions about the mechanism for the efficient activation of chlorine reservoir species by HCl, formation of cloud condensation nuclei by efflorescence of salts, and adhesion of ice to a variety of surfaces. The broader impacts of the proposed work include training of students at all levels: graduate, undergraduate, and postdoctoral, in methods for generating a molecular-level picture of reactions at surfaces. There is an increasing trend to incorporation of these pictures into secondary and college texts and it is important that these pictures be accurate. As students think on the molecular level, their interest in and fascination with chemistry specifically, and science in general increases significantly.
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: Sloan, Nancy
Title: Community-Based Kangaroo Mother Care to Prevent Neonatal and Infant Mortality
Abstract: The overall objective of the Community-Based Kangaroo Mother Care to Prevent Neonatal Mortality study is to conducting a single, rigorous evaluation of CKMC as the most efficient way to determine if CKMC, implemented by piggy-backing CKMC instruction into the existing community-based National Nutrition Programme (NNP), reduces newborn and infant mortality. The study tests whether CKMC will increase skin-to-skin contact, delay newborn bathing, increase the duration of breast feeding (by averting cessation associated with infant death), including predominant breast feeding and will consequently reduce the reported incidence of diarrhea and respiratory diseases and neonatal and infant mortality by 27.5% and 25%, respectively. The study will also assess whether, in addition to averting infant death, family planning and other birth spacing information and services made available during the postpartum period help women avoid or delay subsequent pregnancies. This activity will advance and promote an effective practice to prevent newborn deaths and thus contribute to the mission strategic and global health objective to reduce infant mortality in Bangladesh. The activity is intended to contribute to the SO intermediate results 1.1: Expanded and improved package of health and family planning services and 1.2: Changed behaviors related to high priority health problems. The objective of the activity is to prevent neonatal modality. The study assumes the following intermediate results to achieve the objective in the project area: (1) Increased birth spacing as consequence of improved neonatal and infant survival; (2) Increased proportion of women with surviving infants and continued breastfeeding; (3) Reduced fertility and maternal mortality.
PI: Soto, Ana
Title: The Stroma as a Gatekeeper of Tumor Development
Abstract: The majority of human cancers are of epithelial origin (carcinomas). Alterations in the stroma (the connective tissue) have been thought to occur as a consequence of the malignant transformation of epithelial cells. However, using a tissue recombination rat mammary gland model, we recently observed that epithelial cells are transformed into malignant cells when only the stroma was exposed to the chemical carcinogen nitrosomethylurea (NMU); epithelial tumors appeared regardless of whether or not the epithelial cells were exposed to the carcinogen. These data suggest that the stroma, rather than the epithelium, is the crucial target of NMU. In a separate experiment, we surgically removed the epithelium from the mammary glands leaving a cleared mammary stroma or cleared fat pad (CFP), and injected epithelial cancer cells (ECCs) isolated from NMU-induced rat mammary tumors. Those ECCs formed tumors when injected in CFPs of young hosts. In contrast, tumor formation was substantially decreased or absent when tumor cells were injected into CFPs of mature hosts or CFPs of hosts that have had multiple pregnancies. Instead normal ducts formed. The objective is to further investigate the role of pregnancy on the normalization of ECCs. We aim
i) to discern whether treatment with ovarian hormones, estrogen (B) and progesterone (P), at concentrations found during pregnancy allows the stroma to normalize the ECCs and
ii) to identify the molecular components mediating this phenomenon. Study design: We will compare the ability of CFPs to normalize ECCs and to inhibit tumor formation by injecting ECCs into CFPs of 1) young virgin animals (high tumor incidence expected), 2) animals that have undergone pregnancy (lowest tumor incidence expected), 3) virgin animals of similar age to those that have undergone pregnancy (intermediate incidence expected), and 4) virgin animals treated with E+P at concentrations found during pregnancy (incidence expected to be similar to Group 2).
The pattern of gene expression in the CFPs of high and low susceptibility groups will be compared to identify candidate genes expressed in the stroma that may mediate the normalization of cancer cells. Potential outcomes and benefits of the research: We anticipate that these experiments will result in a better understanding.
PI: Tang, Guangwen
Title: Retinol Equivalency of Plant Carotenoids in Women and Children
Abstract: This project is to determine the vitamin A value (equivalence) of dietary provitamin A carotenes from spinach, Golden Rice, and pure Beta-carotene (B-C) in oil. These experiments will be conducted in children (ages 6-8) with/without adequate vitamin A nutrition. As plant provitamin A carotenoids are a major and safe vitamin A source for a vast population in the world, it is essential to determine the efficiency of provitamin A carotenoid (mainly B-C) conversion to vitamin A. By introducing B-C into rice endosperm, Golden Rice may directly benefit consumers by providing vitamin A nutrition. Our investigation uses hydroponically grown, decadeuterium labeled spinach and Golden Rice, synthetic Beta-C-d10 and a vitamin A isotope reference, decadeuterated retinyl acetate (RAc-d10), to evaluate the bioavailability and the bioconversion of plant provitamin A carotenes to retinol as compared with B-C in oil capsules in vivo. Our objectives will be to test the following hypotheses and to make the following determinations:
(1) The absorption and bio-conversion of provitamin A carotenes taken by children are different between spinach, Golden Rice, and B-C in oil capsules.
(2) The absorption of provitamin A carotenes and their bioconversion to vitamin A are different in children with or without adequate vitamin A nutrition.
(3) To define the vitamin A equivalence(s) of dietary spinach, Golden Rice, and a B-C in oil dose by using an isotope reference method in children with or without adequate vitamin A nutrition and to compare those values with values derived from model based compartmental analysis.
(4) To determine the number and time of blood samples needed for future studies in various field settings on the retinol equivalence of a large number of plant sources.
Seventy-two children each will take two meals, lunch and supper, containing equal amounts of B-C in labeled spinach (along with white rice), or Golden Rice (along with light colored vegetables), or B-C oil capsules (along with white rice and light colored vegetables), every day for 7 days. Before the two meals, the volunteers will take a breakfast with a RAc-d10 dose as a reference for 7 days. The enrichment of labeled B-C and labeled retinol in human circulation will be determined using advanced liquid chromatography / mass spectrometry and gas chromatography / mass spectrometry. Through the applications of these novel technologies, we will be able to determine the relative biological activities of endogenous carotenoids; that is, the vitamin A value of spinach, Golden Rice, and B-C in oil capsules for children with/without vitamin A malnutrition. This study will be of importance in planning vitamin A deficiency prevention strategies and also will provide useful information regarding the potential efficacy of a bioengineered crop to provide vitamin A nutrition.
PI: Tang, Guangwen
Title: Vitamin A Value of Spirulina Carotenoids in Humans
Abstract: The goal of this project is to determine the vitamin A value (equivalence) of spirulina. The investigation will use intrinsically deuterium labeled spirulina and an isotope vitamin A reference dose, octodeuterated retinyl acetate (RAc-dg), in males (n = 16). Up to 45 blood samples (10 ml sample) will be collected from each subject over a two-month period to evaluate the bioavailability and bioconversion of spirulina B-carotene to vitamin A. We will produce deuterium labeled spirulina with octodeuterated B-carotene (-cia) by culturing cells in a nutrient solution with heavy water (deuterium oxide). Serum response kinetics of B- carotene-tie and retinol-d from a deuterated spirulina dose, and serum response kinetics of retinol-dg following a RAc-d dose will be determined using advanced mass spectrometries. By using RAe-c as a reference, the equivalence of spirulina provitamin A carotenoids to vitamin A will be quantitatively determined. The conversion factor of spirulina n-carotene to vitamin A will be compared with those of spinach, carrot, and pure n-carotene in oil capsules (through similar approaches, funded by a USDA National Research Initiative Competitive Grant) in normal adult subjects. Two doses will be studied (10 g, n = 4 and 5 g, n = 4) in the US. To test the effect of vitamin A status on the bioavailability of spirulina, Chinese adults (10 g, n = 8) with marginal vitamin A status will also be studied. Further data analysis will use Model-Based Compartmental Analysis to determine fractional absorption of provitamin A carotenoids (intact or converted to vitamin A), vitamin A absorption efficiency, vitamin A disposal rate, and total traced mass of vitamin A (i.e., vitamin A stores). The research will be conducted in the US and China. Because the following advanced technologies are required: 1) growing isotopically labeled spirulina and 2) tracer analysis to determine the enrichment of the blood samples, the samples collected from the volunteers will be analyzed in the US only. However, a graduate student from China will be trained to carry out the sample analysis in the U.S. The results from this study will be of importance for planning the use of spirulina in rational vitamin A deficiency prevention programs.
PI: Taylor, Holly
Title: Collaborative Research: Development and Evaluation of a Natural Hazard Interactive Laboratory for Improving Decision Making
Abstract: Mitigating the increasing costs associated with extreme natural hazards will require the best-available science, effective strategies for communicating risks to the public, and a well-trained, environmentally-literate society capable of making risk-benefit decisions. Schools must provide students with both the basic knowledge needed to understand these hazards and the critical thinking skills required to make informed decisions regarding their risks in the presence of uncertainty. Designing effective learning exercises (i.e., those that succeed in integrating basic knowledge with critical thinking skills) requires the delineation of specific instructional methodologies that are proven to influence higher-order cognitive skills. Integral to these efforts is the identification of assessment techniques that can measure changes in higher-order cognitive skills. We propose to fully develop, implement, and assess the cognitive impact of an educational tool designed to improve decision-making and risk analyses; a computer-based interactive laboratory involving the volcanic hazard assessment (Eruption). Our pilot studies evaluated a prototype version of Eruption using an innovative evaluation strategy that incorporates cognitive science methodologies. We found that students who used Eruption demonstrated a significant (p < 0.005) improvement in decision-making skill. A similar improvement was not found with students using an informationally-equivalent paperand-pencil exercise.
Three additional pilot studies, described in detail in peer-reviewed publications, involving the design, development and assessment of three other computer-based earth science laboratories allowed us to identify specific design principles that impact higher-order thinking and increase the effectiveness of CAI laboratories. These design principles will be incorporated into the fully developed Eruption laboratory to increase the potential of Eruption to positively impact student populations with weaker math and science backgrounds and/or those traditionally underrepresented in the sciences. The existence of a proven prototype along with well-defined refinements known to promote decision-making skills are compelling evidence in support of the full development of Eruption. The proposed project will result in a unique, broadly available, resource that promotes the integration of knowledge and thinking skills to make effective decisions regarding risks. This objective is central to the HSD emphasis area on Decision Making and Risk. In addition, the project will directly impact the training of the undergraduate and graduate students involved in the project. Further, the proposed project is a collaboration between PIs at two different institutions (Dartmouth and Tufts) and from two different disciplines (Earth Sciences and Psychology). As such, this project brings a unique perspective for development of risk assessment content and the evaluation of educational impact as well as broad opportunity for dissemination. The unique interaction between two institutions guarantees twice the immediate impact of the project (in terms of number of students using the software) and, by forcing us to accommodate the different institutions, will undoubtedly improve the robustness of the final product, thereby increasing its potential for further dissemination.
PI: Theoharides, Theoharis
Title: Restraint Stress-Induced Neurogenic Bladder Inflammation
Abstract: The urinary bladder is often the site of subacute or chronic inflammation, in the absence of infection, as in interstitial cystitis (IC), a painful bladder disorder occurring mostly in women. Symptoms of urinary frequency and pelvic pain commonly worsen perimenstrually and under stress in IC. Bladder mastocytosis with mast cell activation has been documented in IC. We also showed that acute immobilizationstress in rodents induced bladder mast cell activation, a process that was dependent on the neuropeptides neurotensin (NT) and substance P (SP), as it was absent in rodents treated with capsaicin to deplete sensory nerve fibers of their SP content and was also inhibited by the NT receptor antagonist SR48692. Moreover, pretreatment of bladder with estradiol increased the stimulatory effect of SP, by activating high affinity estrogen receptors that we have identified on bladder mast cells. It was recently shown that bladder inflammation could not occur in mast cell deficient mice infected with the neurotropic pseudorabies virus. Mast cells are located perivascularly close to nerve processes and may secrete many vasoactive, proinflammatory and neurosensitizing molecules in response to allergic triggers, as well as by direct nerve stimulation and by acute immobilization stress. Corticotropin releasing hormone (CRH) is released from the hypothalamus under stress and activates the hypothalamic-pituitary-adrenal (HPA) axis. However, both CRH and its structurally related urocortin (Ucn) are also released in the periphery where they have proinflammatory effects. CRH and Ucn induced rat skin mast cell activation and increased vascular permeability, both of which were inhibited by pretreatment with neutralizing antiserum to CRH or the CRH-receptor (CRH-R) antagonist, antalarmin. CRH or acute stress-induced skin vascular permeability was absent in W/W v mast cell deficient mice, but was present in their +/+ controls indicating it is mast cell dependent. Acute stress also triggered rat bladder mast cell activation that was blocked by a NT-receptor antagonist. The proinflammatory cytokines interleukin-6 (IL-6) and tumor necrosisfactor-alpha (TNF-alpha) were recently shown to be elevated in urine of IC patients. We are hypothesizing that acute stress releases (CRH) and/or (Ucn) in the bladder leading, directly or through SP or NT, to mast cell activation, increased vascular permeability and the expression of proinflammatory molecules. We propose to use normal and genetically deficient female mice to investigate the effect of acute stress and CRH/Ucn on: (1) bladder mast cell and urothelial Nuclear Factor kappa B (NF-kappaB)activation, as well as the levels of histamine,lL-6 and TNF-alpha in the urine collected from an indwelling catheter; (2) bladder vascular permeability quantitated by 99Technetium-gluceptate (99Tc) extravasation; (3) Vascular permeability, urine mediator release, as well as NF-kappaB activation in W/W v mast cell deficient mice, as well as in CRH knock-out mice and their +/+ controls; (4) mouse bladder mast cell and urothelial NF-KB activation, as well as secretion of histamine, IL-6 or TNF-alpha induced by intravesical administration of CRH/Ucn. These studies will help us understand how acute stress triggers bladder mast cell activation leading to increased vascular permeability and proinflammatory molecule release. Our findings may be relevant to the pathophysiology of IC and may suggest new therapeutic approaches.
PI: Thorley-Lawson, David
Title: Epstein-Barr Virus Latency
Abstract: Epstein-Barr virus is a herpes virus that infects >90% of the human population. It has two distinguishing characteristics. First it is able to maintain a life long persistent infection in healthy hosts and second it is associated with several human lymphomas and carcinomas. This proposal will address central issues related to these two properties. Persistence- EBV establishes a persistent latent infection in memory B cells. Much is known about how it does this but less is known about how the latently infected cell produces infectious virus to spread to other hosts. Our preliminary data indicate that the signal for viral replication is the terminal differentiation of the latently infected cell into a plasma cell. We will use standard molecular biological tools to identify the role of plasma cell specific transcription factors in activating the EBV lytic cycle. Since plasma cells replicate the virus only when they are fully differentiated it is likely that they release infectious virus when they migrate to the bone marrow. This would result in early B cell progenitors becoming infected. These cells could also provide a site of life time persistent infection. Therefore, the second aim of this study will be to test the role of the bone marrow as a second site of viral persistence. This will be achieved by fractionating the bone marrow into the known subsets of B cells and testing for the presence of the virus by quantitative DNA and RT PCR techniques that we have developed. Neoplasia: One of the commonest tumors associated with EBV is nasopharyngeal carcinoma. There remains little known about the molecular basis of this tumor and its association with EBV. We will use Affymetrix chip technology to provide a molecular genetic definition of NPC that distinguishes Type II and Type III (poorly and undifferentiated NPC) and tumors negative and positive for the EBV encoded oncogene LMP1. Our preliminary data demonstrates the feasibility of this approach. This analysis will identify candidate marker genes for the different types of NPC and test their roles in in vitro and in vivo models. Specifically this approach will be used to identify genes and signaling pathways activated by LMP1. This will provide a molecular basis for explaining the role of LMP1 in NPC.
PI: Thorley-Lawson, David
Title: Host Immunity to EBV Infection in Vitro and in Vivo
Abstract: The long term objective of this study is to develop a deeper understanding of persistent infection with Epstein-Barr virus (EBV). EBV has the capacity to drive the proliferation of resting B lymphocytes and this makes it a risk factor for human cancers such as Hodgkin's disease, Burkitt's lymphoma, immunoblastic lymphoma and nasopharyngeal carcinoma. However, the virus is able to persist in a quiescent state in vivo where it specifically targets resting memory B cells. By understanding how EBV can persist in most individuals without causing disease we hope to gain insight into what goes wrong when the virus does cause neoplastic disease. This study will employ sophisticated cell fractionation techniques and quantitative RealTime DNA and RT PCR assays to address four unresolved issues around EBV persistence.
1. Does acute EBV infection, infectious mononucleosis (AIM), represent a disordered state of EBV infection or simply an amplified version of the stable, long term carrier state?
2. Does EBV, like other herpesviruses, shut off the expression of all protein coding genes when it reaches its final site of persistence - long lived memory B cells in the peripheral blood?
3. What is the nature and origin of the latently infected memory cells proposed as the site of EBV persistence? Are they bona fide memory cells? Does antigen play a role in the production and/or maintenance of these memory cells or do latent proteins perform these functions? How rapidly do the infected cells turn over?
4. Are epithelial cells of the nasopharyngeal lymphoid system e.g. tonsils infected with EBV in vivo or infectable in vitro?
Previous studies have analyzed EBV infection of epithelial cell lines and tissues from sites other than the site of persistent infection - the nasopharyngeal lymphoid tissue. However, epithelial tissues are biologically diverse so we will focus our studies on the biologically relevant epithelium from the tonsil.
PI: Thornton, Ronald
Title: Web-Delivered Interactive Lecture Demonstrations (WEBILD): Creating an Active Science Learing Environment Over the Internet
Abstract: Educational research has shown that traditional instruction in physics (and other sciences) is rarely effective for teaching conceptual understanding even with academically-talented students and almost totally ineffective for under-prepared students. This project intends to develop further methods and tools that can facilitate effective teaching even in traditional environments. We are proposing a web delivery system for a proven interactive pedagogy, Interactive Lecture Demonstrations (ILDs) which we developed earlier. Prototype WebILDs, like in-class ILDs, have been shown to lead to very significant changes in conceptual understanding and are even easier to implement. Starting with this proven, research-based, pedagogical method and our successful WebILD prototype software we wil:
(1) refine, using student learning as a guide, the Motion, Force, and Energy WebILD prototype by testing it with larger and more diverse introductory course audiences and using it for learning at a distance
(2) create WebILD sequences in additional physics topic areas
(3) create additional WebILD sequences in another science disciplinary area
(4) refine the delivery and administration software and make it and the WebILD sequences nationally available.
This project focuses on the learner and uses well established conceptual evaluations to measure Learning. We will use the flexibility of internet-delivery to adapt ILDs to increase their already considerable efficacy with under-prepared students including women and minorities. This project meets a need for learning at a distance, for pedagogical methods that work for internet delivery, and for methods to teach teachers. Even during Development we will reach thousands of students per year.
PI: Trimmer, Barry
Title: Central and Peripheral Actions of Nitric Oxide
Abstract: Nitric oxide (NO) in the central nervous system (CNS) is usually thought to communicate with a large number of local targets simultaneously. The range of such signaling has not been rigorously defined and may vary between tissues and species. Experiments in the original grant period identified NO producing and responding neurons, established that NO production is coupled to nicotinic acetylcholine receptors and characterized the physiological responses of some identified neurons to NO. In addition, it was shown that NO signaling is less general than first thought and may in fact involve precise and directed cell-cell communication. Another aspect of NO signaling that has not been examined in detail is the interplay between central and peripheral actions. Nitric oxide synthase (NOS) can be detected throughout the cytoplasm of most NO-producing neurons. Because NO release does not depend on specialized synaptic structures it can be produced in the dendrites and cell bodies where it acts as a central neurotransmitter or modulator. NOS is also found in tissues outside the CNS including muscles and axons, but the function of peripherally released NO in insects is largely unknown. The immense differences in size, metabolic function and cellular architecture of NO target tissues raise important questions about the mechanisms of NO signaling at different locations. Experiments in the current proposal will take advantage of the well-characterized NO/cGMP system of the insect Manduca sexta to establish how NO acts at central and peripheral sites to control both patterned neural activity and neuromuscular functions. Detailed characterization of the actions of NO will be made using several semi-intact preparations that allow precise physiological and pharmacological manipulations. In addition, the coordinating functions of NO will be explored using a newly developed RNA interference procedure to down-regulate the expression of NOS in intact, freely moving larvae.
The long-term goal of the proposed activity is to understand the specializations and limitations of NO in carrying out its diverse signaling roles. The identification of prominent NO-dependent motor effects will lead to a better understanding of how NO coordinates neural activity. This information will be combined with our current knowledge of the NO responses of specific neurons, and data from a forthcoming study of reactive oxygen and nitrogen species in the CNS, to formulate a comprehensive description of neural NO signaling. A final step will be to synthesize our understanding of neural signaling with concomitant actions of NO in peripheral tissues. Concepts learned from this tractable model system will be important in understanding how insects function and are expected to apply to NO signaling in all multi-cellular organisms. In addition to its intrinsic scientific importance this research will help to provide training opportunities at all levels of education. Manduca is used as a teaching-aid in grade school biology classes and as an experimental organism in undergraduate physiology classes. In previous NSF funded studies on NO signaling in Manduca undergraduates, graduates and post-doctoral associates received training in molecular biology, physiology, biochemistry and behavior. In addition to the individuals directly sponsored by the proposed grant it is expected that 12-15 undergraduates will carry out their own research and prepare formal reports on topics related to this work. Although it is not the direct goal of these experiments the findings will identify new ways for NO to control insect behavior (e.g., developing novel, pest-specific antifeedants). Furthermore, understanding how NO coordinates changes in central and peripheral tissues will be an important contribution to the field of distributive control systems.
PI: Tzipori, Saul
Title: Studies of Cryptosporidium Parvum Type 1
Abstract: The goal of this project is a comprehensive characterization of Cryptosporidium parvum type 1, the most common agent of human cryptosporidiosis in the general population and in people with AIDS. Because until recently type 1 C. parvum could not be propagated in the laboratory, this subgroup is rarely studied and remains poorly characterized. As a consequence, phenotypic properties of calf-propagated type 2 oocysts are extrapolated to the whole species. In light of the significant public health importance of cryptosporidiosis caused by type 1 C. parvum, the emphasis of this proposal is twofold: 1) to extensively characterize C. parvum type 1 with respect to genotypic and phenotypic properties, as well as its life cycle and interaction with the host, and (2) to compare C. parvum type 1 and type 2 with the aim of gaining a better understanding of the species C. parvum and the disease caused by this parasite. The recent development of a gnotobiotic pig model suitable for propagation of type 1 and type 2 will facilitate the study of several type 1 isolates and the comparison of standardized C. parvum isolates of both types originating from the same host. The interaction of type 1 and type 2 isolates in mixed infections will also be studied in the pig model. Type 1 isolates included in this study will originate from chronic and acute infections and from different geographical areas. The study includes three specific aims: the first aim is a comprehensive analysis of ten type 1 C. parvum isolates. In the second aim the host-parasite interaction of type 1 and type 2 isolates will be compared using the pig model and cell culture models. The third aim investigates the extent of genetic variation and exchange within and between the two types of C. parvum to determine whether they belong to one or two species.
PI: Waldor, Matthew
Title: Molecular Biology and Virulence of CTX Phage
Abstract: CTXphi is a filamentous bacteriophage that encodes cholera toxin. This is the principal virulence factor of Vibrio cholerae, the Gram-negative bacterium that causes the severe diarrheal disease cholera. CTXphi is the first filamentous bacteriophage shown to mediate the horizontal transfer of a virulence gene. CTXphi integrates into the Vibrio cholerae chromosome and, in the lysogenic state, most CTXphi genes are not expressed due to the activity of the CTXphi repressor, RstR. Generally, the integrated form of CTXphi is found as part of tandem arrays of prophage DNA interspersed with the related genetic element RS1. RS1 encodes a protein, RstC, that can counter RstR repression and lead to markedly enhanced expression of CTX prophage genes including ctxAB, the genes encoding cholera toxin. The long-term goal of this work is to understand the molecular events in the life cycle of CTXphi and the role that this phage plays in the pathogenesis of cholera. The proposed studies will explore 3 processes central to the phage life cycle: i) the site-specific integration of phage DNA into the bacterial chromosome; ii) the repression of most phage gene expression following integration; and iii) the activation of phage gene expression and virion production by environmental and genetic stimuli.
Experiments in Aim 1 to identify the mechanism and factors that mediate the integration of CTXphi DNA into the V. cholerae chromosome will reveal how the chromosome encoded recombinases XerC and XerD interact with phage and chromosome sequences to accomplish CTXphi integration. These studies will elucidate a novel mechanism of phage integration and may shed light on the mechanism of ctxAB amplification as well.
Experiments in Aim 2: to characterize the regulation and mode of action of RstR will clarify how CTXphi can be maintained in a quiescent state. rstR autoregulation and modulation of RstR levels by environmental factors will be explored. RstR's binding to its unusual operators will also be studied.
Experiments in Aim 3 to determine the mode of action of RstC-will explore how RstC can inactivate RstR-mediated repression. RstC's ability to bind to either RstR and/or RstR's binding sites will be investigated and the expression of rstC during infection will be measured. All of these studies will yield insights into fundamental aspects of phage biology. In addition, they may reveal ways in which changes in phage gene expression or copy number can contribute to the pathogenicity of V. cholerae.
PI: Waldor, Matthew
Title: Role of HFQ in Vibrio Cholerae Virulence
Abstract: Vibrio cholerae causes the severe diarrheal disease cholera. We found that Hfq, an RNA-binding protein, is essential for Vibrio cholerae virulence. Deletion of hfq abolished V. cholerae colonization of the suckling mouse intestine, but had a minimal effect on growth in vitro and did not influence expression of known colonization factors. Thus, Hfq appears to control previously undescribed pathways essential for cholera pathogenesis. In E. coli, Hfq binds to numerous small untranslated RNAs (sRNAs), modulates their activities, and thereby controls expression of a wide variety of genes. Hfq also binds to some mRNAs in E. coli and alters gene expression directly. Although Hfq in V. cholerae probably acts by similar mechanisms, the distinct phenotypes of hfq V. cholerae and E. coli suggest that the proteins bind different sets of RNAs and control distinct regulons. No RNAs bound by V. cholerae Hfq and no V. cholerae sRNAs have been characterized to date. The goals of this R21 application are to identify pathways controlled by Hfq in V. cholerae, particularly Hfq-regulated genes that contribute to V. cholerae virulence, and to characterize the mechanisms controlling their expression.
Experiments in Aim I - to define the Hfq regulon - will generate the first knowledge of Hfq-regulated effectors in V. cholerae. Experiments in Aim II - cloning of sRNAs and mRNAs that interact with Hfq - will utilize a new, unbiased approach for cloning interacting RNAs. Experiments in both Aims I and II will explore which of Hfq's interaction partners and downstream effectors contribute to V. cholerae virulence and thus illuminate currently unknown mediators of pathogenesis. Experiments in Aim III - to match sRNAs to the genes they regulate and characterize processes of Hfq dependent gene regulation - will create the foundation for detailed analyses of the mechanisms by which Hfq controls gene expression in V. cholerae. These studies will also facilitate disruption of Hfq-mediated pathways. As Hfq contributes to the virulence of several other Class B Priority Pathogens in addition to V. cholerae, Hfq or its downstream effectors may prove to be valuable targets for new antimicrobial agents.
PI: Walker, Peter
Title: Desk Review of Non-food Programs in Emergency Situations
Abstract: The Feinstein International Famine Center (FIFC) of Tufts University proposes to complete a desk review as part of the WFP/SENAC project on strengthening needs assessment capacity within WFP. The desk review will focus on assessment methods and corresponding non-food programs implemented in emergency situations in response to household food insecurity. The review will examine assessment methods and programs implemented by a range of operational agencies, including U.N. agencies, international and national NGOs, and national governments. The review will document and analyze best practices and lessons learned in these assessments and interventions. The goal of the review will be to assist WFP in increasing its knowledge regarding the range and variations of assessment methods and programs. A discussion of the positive and negative aspects of previous and existing programs will provide guidance on possible models for WFP. The desk review will be conducted by a FIFC team and will adhere to academic standards. WFP and interested stakeholders will have the opportunity to provide comments and feedback on the desk review prior to finalization. The desk review team will make every effort to incorporate comments and feedback as appropriate, but the team is under no obligation to alter its independent analysis based on these comments. The intent is for the desk review to serve as the first phase in a larger process involving the design and testing of survey methods leading to non-food interventions. This next step will be considered following the completion of the desk review and in close consultation with WFP and the review team. If this next step occurs, the review team will make recommendations for possible country case studies and may seek involvement in the field testing. At the FIFC, the project will be a collaborative effort under the management of one Center research member. An estimate 3-5 people will play a role in the review, including the FIFC director and one or two PhD or MA students. The core team will fly to Rome to meet with WFP after the submission of the draft report in late October and will work together with WFP to plan the next phase of the project.
PI: Waller, William
Title: New England Space Science Initiative in Education (NESSIE)
Abstract: New England contains one of the greatest concentrations of space science professionals in the country. The six-state region also presents a diverse set of educational challenges and opportunities. K-i 6 schools cover the gamut from rural schools of Aroostook County, Maine, to urban neighborhoods of Boston, and some of the world’s leading research universities. The region is also home to innovative curriculum development institutions, educational publishers, public television studios, museums, and science centers. The New England Space Science Initiative in Education (NESSIE) will weave a rich tapestry between space science researchers on the one hand, and educational institutions on the other. The goals of the project are to: (a) facilitate a wide range of educational and public outreach (E&PO) activities to enhance awareness of space science throughout the New England region; and (b) contribute to our understanding of how space science education can contribute to scientific and technological literacy.
Activities can be visualized on a grid defined by two dimensions: (1) Levels of interaction. Space science professionals can share their knowledge and passion for science by interacting with people on a variety of levels, ranging from personal contact with small numbers of students, to developing television programs an& school curricula that reach large numbers of people. (2) Educational settings. Educational opportunities for various audiences can be defined along a spectrum from formal education (involving students and teachers in accredited programs) to informal learning (such as visitors to planetariums and science centers).
Our role as a regional clearinghouse of space science research and education will be enabled through a frilly-maintained website with linkages to relevant institutions and programs. Special efforts will be made to reach underserved groups by first identifying the educational needs and interests of these communities.
PI: White, Joel
Title: Characterization of Mechanisms Underlying Vapor Phase Responses of DNA-Based Sensors
Abstract: We have observed that fluorescent-labeled, short, single-stranded DNA oligomers can be used as sensing materials when dried onto the detector substrates used in the Tufts Medical School artificial nose. This proposal is directed at addressing the mechanisms that govern how these molecules react to vapor phase compounds (odors). How does ambient humidity within the sensing chamber affect sensitivity and breadth of response to a define odor set? How do oligomer length, sequence, and the position of the fluorescent dye label affect response? How do different salts, buffers and substrate materials influence response?
PI: Widmer, Giovanni
Title: Genetic Transformation of Cryptosporidium Parvum
Abstract: The goal of this project is to develop a transient genetic transformation system for Cryptosporidiumparvum. Genetic transformation is an essential tool for analyzing gene function, but is not available for C. parvum. Together with the almost completed sequence of the C. parvum genome, genetic transformation is needed to translate genomic sequence into biologically and clinically relevant information by elucidating the function of unknown genes and identifying regulatory sequences. We propose to identify genes which are highly expressed in trophozoites and early meronts and insert upstream and downstream intergenic regions from these genes into plasmids carrying the green fluorescent protein reporter. Electroporation methods for the transient transformation of C. parvum sporozoites will be developed with these plasmids. Transformed C. parvum will be identified directly by fluorescent microscopical analysis of infected cell cultures. Alternatively, GFP specific antibodies or reversetranscription PCR will be used. Consistent with the goals of this Program Announcement, this project is exploratory/developmental and is not hypothesis-driven. The goal is the development of a new technique relevant to the study of a pathogenic microorganism.
The specific aims are: 1. Identify C parvum genes upregulated in trophozoites/early meronts and construct transient transformation vectors incorporating regions regulating the expression of these genes. Approach: rnRNA transcripts expressed at high level in trophozoites and early meronts will be identified by quantitative real-time PCR. Intergenic regions flanking these genes will be inserted into GFP expression plasmids. 2. Establish a transient transformation system for Cryptosporidium parvum.
PI: Wise, Timothy
Title: Policy Space for Development in the WTO
Abstract: In the face of increasing poverty, inequality, and environmental degradation across the developing world, the world community has reasserted the need for development through the Millennium Development Goals and the global commitment to sustainable development signed at the World Summit for Sustainable Development. At the same time, most of the world’s nations have also embarked on a new round of global trade negotiations—the so-called Doha Round under the World Trade Organization (WTO). Although developing countries agreed to enter a new round of trade negotiations only on the condition that development would be the centerpiece, there are growing concerns that this promise will go unfulfilled. Key among those concerns is the notion that a new trade agreement will not give the developing world the “policy space” to use the very instruments and tools that many industrialized nations took advantage of to reach their current levels of development.
The case of Mexico can serve as a laboratory to draw out lessons for the Doha Round. Mexico began an economic integration process in 1985 that culminated with the 1994 North American Free Trade Agreement (NAFTA) and continues to this day. Mexico succeeded in transforming itself from a protected, resource-based economy into one of the most open economies in the world, with strong foreign investment and growing manufacturing exports. Many developing countries are just now considering liberalization measures that Mexico has experimented with for almost twenty years, and they would do well to examine Mexico’s relatively poor track record. To what extent is this poor track record due to the fact that Mexico’s policy space significantly shrank under the NAFTA? Building on its earlier grant from the Rockefeller Foundation that examined the extent to which Mexico’s model of economic development has raised standards of living, the Global Development and Environment Institute (in collaboration with Mexican researchers) proposes to assess the policy options available for Mexico at the national level and in bi-lateral policy with the United States in terms of agriculture and investment policy. To help formulate a pro-active agenda for developing nations committed to preserving policy space under the WTO, the institute will also examine the extent to which such policies in Mexico would be permissible under proposed WTO commitments and draw out recommendations for developing countries with similar development concerns.
This project cuts across several programs at the Rockefeller Foundation. While rooted in the Global Inclusion program, this research project will also continue to identify “push and pull” factors for migratory flows and remittances in the North American community, and dovetail with the Foundation’s emphasis on global commodity chains under the Working Communities program. The institute proposes to develop a series of policy papers, followed by subsequent dialogue with policy-makers in Mexico and the U.S., WTO negotiators, civil society, and the media.
PI: Wood, Richard
Title: Functional Role of Cat1 and Calbindin D in Ca Transport
Abstract: Absorbing too little calcium (Ca) in the intestine can be a risk factor for negative Ca balance and the development of osteoporosis. An important gap in our knowledge is that the molecular determinants of Ca influx into the enterocyte and transcellular Ca transport have not been unambiguously identified. This lack of knowledge limits the development of specifically targeted therapeutic agents to manipulate Ca absorption. The long-range goal of our research program is to identify the molecular factors responsible for age-related calcium malabsorption, and to target the development of safe and effective therapeutic agents that can modulate active calcium absorption. The recent discovery of the CaT1 (also known as ECaC2 and TRPV6) gene and the demonstration that its protein product functions in non-intestinal cells as a membrane Ca transporter, and our recent report that the expression of CaT1 in the Caco-2 human intestinal cell line is regulated by 1,25-dihydroxyvitamin D, offers an exciting new opportunity to further knowledge of the molecular mechanisms of vitamin D-mediated intestinal Ca absorption.
We have two specific aims in this proposal (1) to establish the role of CaT1 and calbindin D in vitamin D-mediated calcium transport and (2) to identify to what extent CaT1 expression is a modulator of the rapid non-genomic vitamin D response in the enterocyte. We will determine the functional role of these proteins in the enterocyte by using Caco-2 cells transfected with sense and antisense CaT1 and calbindin D cDNA to alter the level of these proteins independently of vitamin D. The central hypothesis of our first research aim is that CaT1 acts as the primary gatekeeper of intestinal Ca absorption by controlling, under regulation by 1,25-dihydroxyvitamin D, the rate of Ca entry across the apical brush border membrane of the enterocyte. The cytosolic vitamin D-dependent calbindin D acts in concert with CaT1 as both intracellular buffer and transporter, delivering Ca that has crossed the brush border membrane to the basolateral membrane of the enterocyte for Ca exit into the blood. We hypothesize in our second research aim that the CaT1 molecule either is responsible for or modulates the rapid 'non-genomic' response to 1,25-dihydroxyvitamin D in Caco-2 cells by determining the rate of Ca entry into the enterocyte following 1,25-dihydroxyvitamin D treatment and thereby modulating the rise of free intracellular Ca and subsequent downstream activation of protein kinase C and phospholipase D.
PI: Wortis, Henry
Title: Babesiosis as a Model of Age-Related Immunosenescence
Abstract: While the increased susceptibility of the aged to infection by a variety of organisms is widely documented, its mechanistic basis is little understood. We are not aware of any well-developed experimental system for analysis of this phenomenon. We believe that we can exploit a unique animal model for this purpose. The organism we propose to study is the pathogen Babesia microti, a protozoan hemoparasite that most frequently produces severe disease in otherwise healthy individuals aged 50 and above. Data generated in our laboratory demonstrate for the first time that susceptibility of mice to B. microtiincreases with age. Specifically, we have shown that as DBAI2 mice age from two to six, twelve and 18 months there is an increase in the frequency and persistence of parasite infected erythrocytes. We have also demonstrated that there is a marked strain variation in age-associated susceptibility, as two, six, twelve and 18-month-old BALB/c and C57BL/6 mice show marked resistance to infection. Nonetheless, older BALB/c and C57BL/6 mice display a modest increase in early parasitemia but never manifest detectable persistent parasitemia. We have additional data demonstrating that SCID BALB/c mice sustain prolonged high parasitemia. Transfer of naive BALB/c splenocytes to SCID mice prevents such persistent parasitemia, showing that a cell transfer system can be used to identify effector cells. Importantly, there is an age-associated loss of transferable protective immunity by DBA/2 and BALB/c spleen cells. Since the age-associated differences in the ability of DBA/2 spleen cells to transfer resistance is only revealed at a lower parasite load, we conclude that BALB/c spleen cells confer a greater protection than DBAJ2 cells. We now propose to test the hypothesis that there are functional differences in specific populations of lymphocytes and/or antigen presenting cells that are responsible for the age-associated loss of resistance. We will use our adaptive transfer system in SCID mice to define these cell populations. Since SCID mice do not succumb to high numbers of B. microti, innate immunity may contribute to resistance. We will use a genetic approach to test this hypothesis. We further propose to use formal genetic analysis utilizing recombinant inbred and classic matings to map genes critical for this age-associated decline in resistance to B. microti. We will attempt to identify candidate genes by coordinated genetic analysis and transcriptional profiling (microarray) studies.
PI: Wortis, Henry
Title: CD 22, A Regulator of B Cell Activation
Abstract: CD22, a surface molecule expressed on B cells, negatively regulates activation, in part by attenuating calcium signals. Its absence is reported to increase autoantibody formation. CD22 is a member of the sialic acid binding immunoglobulin domain containing lectin (siglec) family of proteins that are expressed by hematopoietic cells. Like CD22, many siglecs have ITIMs and act as negative regulators of cell activation. As CD22 is currently the best studied member of the siglec family, an understanding of its function might be expected to provide insight into mechanisms that are widely used in regulating hematopoietic cell functions. We have evidence that CD22 can augment Ca2+ extrusion following B cell activation. Regulation of Ca2+ extrusion of lymphocytes has not been previously described. We will characterize the mechanism for this regulation and see if impacts B cell function in vivo. We have recently established that CD22 requires its lectin activity for full function. We will ask if lectin function plays a role in the association of CD22 with the B cell receptor. We have discovered that the conserved cytoplasmic juxtamembrane region of CD22 contributes to its function, and we will study the basis for this, including a test of the hypothesis that it contributes to association with the BCR. While the importance of ITIMs for negative regulation is well established, we have discovered that CD22 acts as a negative regulator in the absence of ITIMs. We ask how this ITIM-independent negative regulation pathway functions. Overall, our goal is to understand how the several components of CD22, particularly the lectin binding region (specific for (2,6 sialic acids), the juxtamembrane portion of the cytoplasmic tail and the six cytoplasmic tyrosines (including three ITIMs) serve the molecule to diminish calcium signals and to prevent autoantibody formation.
PI: Yee, Amy
Title: Mechanisms of Transcriptional Repressor HBP1 in Cancer
Abstract: Breast cancer is a major killer of women in whom the etiology of tumor induction is poorly understood. The Wnt pathway has emerged as a major oncogenic pathway with a complex interplay of oncogenes and tumor suppressor genes. A key feature is the stability of f3-catenin, which functions as a transcriptional co-activator with the LEF and TCF family of transcription factors. The oncogenic phenotypes are ultimately established by the regulation of promoters for key growth regulatory genes. For example, Cyclin D1 is activated by, the Wnt-betacatenin pathway. Cyclin D1 mRNA is increased in many breast cancers, and its role in breast cell proliferation is well established. While the components and the activation of the pathway have been an area of intense study, the molecular mechanisms that inactivate the Wnt pathway in normal tissues are not well understood. These suppressive mechanisms are excellent candidates for the identification of new tumor suppressor genes. The working hypothesis of this proposal is that HBP1 is a suppressor of Wnt-beta-catenin signaling through the transcriptional repression of oncogenes and other gene targets. We had previously identified HBP1 as a transcriptional repressor and cell cycle inhibitor. Like LEF and TCF, HBP1 is an HMG box transcription factor. However, HBP1 remains one of few examples of repressors within this important transcription factor family. Recent work indicates that HBP1 is a transcriptional repressor of the Cyclin Dl promoter, which is activated by Wnt-B-catenin signaling. Experiments are specifically designed to test the role of HBP1 and transcriptional repression in breast tumorigenesis. The possible role of HBP1 as a tumor suppressor gene in human breast cancer will be tested directly. HBP1 is located in human Chromosome 7q31--a region that is frequently deleted in breast and other cancers. Deletion in cancer is a hallmark of tumor suppressor genes. The long-term goals are the mechanisms that may govern normal breast cell proliferation and that may become aberrant in tumorigenesis. This is critical to understanding tumor suppression and to how mis-regulation may lead to oncogenesis. Together with other work, the proposed studies may provide insights into new diagnostic and/or therapeutic strategies for breast cancer.
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