The goal of this project is to establish a mouse model for identification and analysis of genes involved in the spread of tumor cells to hone, liver, lungs and brain, key sites to which metastasis occurs in breast cancer patients with advanced disease.

Biophotonic imaging, a recently developed imaging technique that allows visualization of light emitting cells in living animals, will be used to follow growth and metastasis of genetically engineered 4T1 tumor cells following implantation in the mouse mammary gland.

The genome of these cells has been engineered by incorporation of a light-emitting firefly luciferase gene, drug resistance genes, and a specialized site into which DNA can be inserted for gene silencing experiments. The engineered cells will be introduced in mice and those that spread to bone, liver, lungs and brain will be isolated from the organs. These cells will have undergone genetic changes that facilitate metastasis to the organs from which they were isolated.

Expression of genes in these cells will be compared to the original cells using microarray analysis, a new high throughput genomic technique which allows the investigator to determine the expression of thousands of gene simultaneously. The analysis will reveal differences in gene expression that correlate with observed differences in metastatic properties of the cells. The involvement of specific genes in organ-selective metastasis will then be verified by inhibiting their expression in the tumor cells and studying the effect of the modification on metastasis.

The newly established cell lines will provide valuable tools for further analyzing the roles that identitied organ-selective metastasis genes play in cancer progression. The project will produce new target genes for diagnosis, prognosis and treatment of breast cancer and a novel model for studying metastasis to organs affected in the disease.

PI: Sahagian, Garabed
Title: Genetics of Brain Metastasis in Breast Cancer
Abstract: While breast cancer initially develops in the breast, it is the spread of tumor cells to other organs that is responsible for the disease. A particularly devastating site of breast cancer spread is the brain. While approximately one-third of breast cancer patients are affected, little is known about how tumor cells invade and grow in the brain, and consequently there is little that can be done to prevent or reverse the spread to this organ.

The limiting factor needed to devise effective therapies to counteract brain metastasis is the availability of suitable models for studying the process. Our laboratory has recently developed a mouse model, based on the metastatic mammary tumor cell line 4T1, for studying the spread of breast tumor cells from the mammary gland to all organs affected in breast cancer including brain. The proposed project will use the model to identify genes that are involved in brain metastasis.

Biophotonic imaging, a recently developed imaging technique that allows visualization of light emitting cells in live animals, will be used to follow growth and metastasis of genetically engineered 4T1 cells following implantation in the mouse mammary gland.

The genome of these cells has been engineered by incorporation of a light-emitting firefly luciferase gene, drug resistance genes, and a specialized site into which DNA can be inserted for gene silencing experiments. The engineered cells will be introduced in mice and those that spread to the brain will be isolated and cloned. These cells will have incorporated genetic changes that facilitate metastatic spread to the brain.

Expression of genes in these cells will be compared to the original 4T1 cells using microarray analysis, a genomic technique that allows the investigator to determine the expression of thousands of genes simultaneously. The analysis will reveal differences in gene expression that are likely to be involved in brain metastasis. The involvement of specific genes in brain metastasis will then be demonstrated by inhibiting their expression in the tumor cells and studying the effect of the modification on metastasis.

The newly established cell lines will provide valuable tools for further analyzing the roles of identified brain metastasis genes in breast cancer progression. The project will produce new target genes for diagnosis, prognosis and treatment of breast cancer and a novel model for studying metastasis to brain and other organs affected in the disease.

PI: Saperstein, George
Title: Rare Breeds Preservation Program
Abstract: The objectives of the program are twofold:

• First, to produce and preserve germplasm of rare breeds of domestic animals.

• 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: Saperstein, George
Title: Distribution of Gastrointestinal Parasites of Grevy’s Zebra’s, Plains Zebras, Other Wildlife Species, and Livestock in the Samburu Landscape, Kenya
Abstract: Grevy's zebras (Equus grevyi) are an endangered species and have suffered an 88% decline in their population since the 1970s. As common grazers, Grevy's are critical in the maintenance of ecological health, and therefore conservation of the Grevy's zebras is vital, not only for the Grevy's but for the preservation of overall biodiversity in their environment.

The Samburu landscape in northern Kenya, which includes private land as well as national parks, reserves, and wildlife conservancies, is now a principal range of the Grevy's zebras. Pastoralists, who share the land with the Grevy's zebras, graze livestock across the landscape, thus placing competitive pressure on the Grevy's for water and other resources. This close association with livestock may also promote the transmission of diseases including gastrointestinal parasites.

This study will take place in the Earthwatch Institute Samburu Conservation Initiative site under the supervision of Dr. Paul Muoria of the Earthwatch Institute and the Institute of Primate Research. The main objective of this study is to describe the distribution of gastrointestinal parasites in Gevy's zebras, plains zebras, other wildlife and livestock in the Samburu landscape. The total prevalence, as well as the prevalence of potentially clinically significant parasite burdens in wildlife and livestock will be estimated. The spatial distribution of Grevy's zebras, plains zebras, other wildlife species, and livestock will be related to the parasite species found and the rates of parasitism in these ungulate species.

In addition, parasite loads in the soil of areas grazed by Grevy's zebras, plains zebras, other wildlife species, and livestock will be compared to gastrointestinal parasite loads of these species and examined for overlap of geographic ranges. Fecal and soil samples will be collected and laboratory analysis will occur at the Institute of Primate Research, National Museums of Kenya.

All samples will be analyzed qualitatively by direct smear, flotation, and sedimentation. Additionally, all samples will be analyzed quantitatively by either the Comell-McMaster dilution egg counting technique or the concentration egg counting technique. If further identification of nematode eggs is necessary, fecal culture will be performed.

The data obtained from fecal sample analysis, along with the spatial distribution of these species and data from soil samples, will provide insight to the potential transmission of parasites between livestock and wildlife. This may be useful in better understanding and addressing the human wildlife interface, promoting development of future management strategies, and supporting the conservation of the Grevy's zebras in the Samburu landscape.

PI: Schaefer, Ernst
Title: Molecular Basis of High Density Lipoprotein Deficiency
Abstract: A low level of high density lipoprotein cholesterol (HDL-C) is an independent risk factor for coronary heart disease (CHD). It is estimated that more than 50% of the variation in HDL-C levels in humans is genetically determined. There is great interest in the concept that common genetic variants contribute to inherited differences in the susceptibility to common diseases. One promising approach to address this issue is to relate variation in DNA sequence with patient characteristics in population-based association studies.

We are proposing to identify allelic variants associated with the low HDL trait, using samples from the Veterans Affairs HDL Intervention Trial (VA-HIT, cases), a study designed to explore the benefits of HDL-raising with gemfibrozil in men having low HDL-C (<40 mg/dL), normal LDL-C (<140 mg/dL) and known CHD, and the Framingham Offspring Study (FOS, controls).

Our primary aims are to identify:

1. Susceptibility loci for the low HDL trait
2. Allelic variants associated with levels of apolipoproteinA-l-containing HDL subspecies
3. Allelic variants associated with response to gemfibrozil in VA-HIT

For Aim 1, we will examine biological (n=38) and positional (n=3) candidates. The former will include genes involved in HDL metabolism, insulin resistance and inflammation, while the latter will be selected on the basis of results from genome-wide linkage scans for quantitative trait loci associated with HDL-C levels in FOS.

For each candidate, we will use HapMap data in order to select a maximally informative set of SNPs (tagSNPs), which will allow us to resolve >80% of all haplotypes. Based on this algorithm, we will genotype 1 SNP per 2500 bp across each candidate gene/region. To address the issue of population stratification, we will employ a structured association approach, using a set of 250 markers that has the ability to detect modest amounts of stratification.

The results of this work will provide important insight into the contribution of allelic variation in the pathways of HDL metabolism, inflammation, and insulin resistance to the complex phenotype of low HDL-C.

PI: Schaefer, Ernst
Title: Pharmacogenetics of the Statin Response
Abstract: Coronary heart disease (CHD) is the leading cause of death and disability in our society. Most CHD deaths occur in subjects over 70 years of age. Significant independent CHD risk factors are age, gender, elevated low density lipoprotein (LDL) cholesterol (C), decreased high density lipoprotein (HDL) C, hypertension, smoking, diabetes, elevated lipoprotein (a) or Lp(a) (LDL C> 50% reduction), and elevated C-reactive protein. In this response to RFA HL-03-001 (ancillary pharmacogenetic studies), we propose to study 2804 male and 3000 female participants in the Prospective Study of Pravastatin in the Elderly at Risk (PROSPER), who were selected for age 70-82 years, having vascular disease coronary, cerebral or peripheral) or increased CHD risk due to smoking, hypertension or diabetes and total cholesterol levels between 4.0 and 9.0 mml/L or 151 and 340 mg/dl. In this randomized controlled trial pravastatin decreased LDL C 34% and triglyceride 12% and raised HDL C 5%. C-reactive protein and Lp(a) values have already been measured. Fatal and nonfatal myocardial infarction (MI) were decreased by 19%, and fatal MI 24%, but increased risk of new cancer were noted in the pravastatin group over 3.2 years as compared to the placebo group (all p<0.01) (Lancet 360: 1623-30, 2002). Benefit was greatest in subjects with low HDL C (<1.11 mml/L or 43 mg/dl). No benefit of pravastatin versus placebo on cognitive function or stroke was noted. We and others have shown that statins increase large alpha 1 migrating apolipoprotein A-I containing HDL, decrease plasma lathosterol, a marker of cholesterol synthesis, and increase plasma betasitosterol, a marker of cholesterol absorption as well as decrease cholesterol ester transfer protein (CETP) mass. We propose to measure HDL subspecies, CETP mass, lathosterol, and beta-sitosterol in the 292 subjects who developed CHD while on pravastatin and in a control group (n=292) who did not develop CHD on pravastatin. We propose to isolate DNA in all subjects, carry out sequencing for single nucleotide polymorphism detection in 5 male and 5 female hyper-responders and the same number of hypo-responders (LDL C <10% reduction) and then genotyping at all SNPs on the two 292 patients groups, and the informative SNP detection on the entire 5804 cohort at the following gene loci: ATP binding cassette transporters G5 and G8 (ABCG5, ABCG8), CETP; HMG CoA reductase, apolipoprotein E, lipoprotein and hepatic lipase, microsomal transfer protein, C-reactive protein, connexin, plasminogen activator type I inhibitor and stromelysin I. These genes have been selected because of our own preliminary studies, and their known key role in cholesterol absorption and lipoprotein metabolism or CHD. We hypothesize that response to pravastatin in terms of lowering of LDL C, triglycerides and C-reactive protein, and HDL C raising will be related to specific genotypes and haplotypes. We also hypothesize that subjects with the greatest LDL C- and C-reactive protein-lowering, the greatest increase in large alpha HDL particles, the greatest reduction in lathosterol and the least increase in beta-sitosterol will have the greatest benefit in CHD risk reduction, and that these changes will be related to specific genotypes and haplotypes of the candidate genes being examined. These results can be used to formulate guidelines for identifying elderly subjects for statin treatment to prevent future CHD.

PI: Schaffhausen, Brian
Title: Products of the Transforming Genes of Polyomavirus
Abstract: Murine polyomavirus provides an important model for neoplastic transformation. The virus causes a broad spectrum of tumors. Conclusions reached in the laboratory can be readily tested in animals. Studies on polyoma have repeatedly provided insight into basic mechanisms of cellular growth regulation.

Tyrosine kinase and phosphatidylinositol 3-kinase (PI3K) signaling are two fundamental mechanisms uncovered from studies on polyoma. Transformation results from the action of three viral gene products: large T (LT), middle T (MT) and small T (ST) antigens.

There are four specific aims that will provide new insight into how they work:

Specific Aim 1: Large T. Patterns of host cellular RNA expression that result from different large T pathways will be determined. The mechanism will be established by which LT regulates genes containing CREB/ATF sites. Because acetylation has been connected to transcriptional regulation, the sites of acetylation on LT will be established and their function tested by site-directed mutagenesis.

Recent results show that LT causes G2/M arrest. Experiments will be carried out to determine the mechanism by which LT causes this arrest. We have identified two new LT-binding partners, Pinl and Bubl, which are known regulators of G2/M. Their role in LT function will be probed.

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

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

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

PI: Schaffhausen, Brian
Title: Interdisciplinary Program in Cancer Genetics
Abstract: This is a renewal application for a training program designed to produce postdoctoral and predoctoral trainees in Cancer Genetics. A faculty of 15 mentors are providing intensive training in cancer genetics, with concentrations in viral carcinogenesis, signal transduction, basic mechanisms of gene expression, cell cycle control, and DNA rearrangement. Particular focus is placed on models relevant to breast cancer and leukemia/lymphoma. Since its inception five years ago, our program has grown to include 10 current students and has already produced one PhD To expand the training opportunities available to researchers interested in careers dedicated to the cancer problem, we have developed a postdoctoral component. These two components are well integrated; trainees in both the predoctoral and postdoctoral programs work on research topics directly related to cancer, receive didactic instruction relevant to the cancer problem and are exposed to issues related to cancer as a disease through interactions with New England Medical Center. The postdoctoral program is built on strong research training, but also contains a didactic component focused on cancer genetics, presentation experiences designed to sharpen communication skills, and direct interaction with clinicians treating cancer patients. Career counseling is built into the program. An Executive Committee of the Cancer Genetics Training Program selects postdoctoral trainees based on graduate research productivity, grades and letters of reference. We expect that trainees who have completed our program will be highly competitive for faculty level positions at academic centers or within the biotechnology industry where they will continue to address the cancer problem. Predoctoral students enrolled in the Genetics Graduate Program may apply to enter the Cancer Genetics Program. To encourage application, all Genetics students are required to take Cancer Genetics. Selection into the Cancer Genetics Program is based on successful completion of the first year of the Genetics Graduate Program, a strong interest in cancer, and selection of a Cancer Genetics training laboratory for dissertation research. Predoctoral training includes didactic courses, laboratory rotations, seminars and workshops, but the core of the training is the dissertation research. Upon graduation, we expect our PhD students to be prepared for postdoctoral research in areas relevant to cancer.

PI: Schnitzler, Gavin
Title: Role of Chromatin Remodeling in Transcriptional Repression by Rb
Abstract:

The aims of this proposal are:

1. To measure the effect of hSWI/SNF on higher order chromatin folding, and compare the ability of Rb and HSF to target these effects to specific sites.

2. To measure the location and abundance of stably-altered nucleosomes on these same templates, and determine how these altered nucleosomes relate to folding changes. Also, to establish the importance of stably-altered nucleosomes, we will measure their formation on the cell cycle regulated hSWI/SNF target genes c-myc and cyclin A, and correlate this with gene activity.

3. To determine the extent to which octamer repositioning by hSWI/SNF depends on sequence-based features of the DNA of on the linker histone H1, using mono and polynucleosomes formed from c-myc and cyclin A promoter sequences.

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

PI: Schwob, James
Title: Regulation of Neurogenesis in Olfactory Epithelium
Abstract: The peripheral olfactory system has a remarkable capacity for repair after injury, and the maintenance of that capacity depends on the persistence of neurocompetent stem cells. If the stem cells are destroyed, the injured tissue undergoes respiratory metaplasia, which is a major cause of sensory dysfunction in humans. Despite the importance attached to understanding the regulation of olfactory stem cells, there is much that is not yet known, starting with their identity.

Work during the previous period of support demonstrated that among the population of globose basal cells (GBCs) are cells that have many of the characteristics of stem cells, including the potency to differentiate into all the constituent cell types and the existence of slowly cycling, label-retaining cells (a hallmark of stem cells in other tissues).

The 3 Specific Aims proposed in this application will provide a more in depth understanding of olfactory stem cells and how they are regulated.

Aim 1 will use a transplantation/colony forming unit assay to determine the differentiate and generative capability of different marker-defined subsets of GBCs, including label-retaining ones.

Aim 2 will test whether expression of members of the bHLH transcription factor family signify (and might drive) irreversible commitment to a single lineage.

Aim 3 will test whether the Notch-Hes signal transduction pathway controls the choice point between neurons vs. non-neuronal cells that is made by the multipotent GBCs as they differentiate.

Successful completion of the Aims will advance our understanding of olfactory stem cells and hasten their potential use as a therapeutic modality.

PI: Selhub, Jacob
Title: Folic Acid Fortification: Effects on DNA Methylation, Global Gene Expression, and Interaction with Common MTHFR Mutations
Abstract: Folic acid fortification of grain products in the United States was mandated by the FDA and implemented in 1998 as a preventive measure to reduce birth defects caused by nutritional deficiencies in early pregnancy. This policy is deemed efficacious since the incidence of neural tube defects decreased concomitant with folic acid fortification. However, little is known about the effect of this policy on the long-term health of the general population.

Folic acid is an essential dietary co-factor in the pathway termed one-carbon metabolism, which provides a biochemical input to the methylation of genomic DNA, a known gene silencing mechanism linked to cancer and age-related pathologies. Our group has shown that the folate-de pendent enzyme, methylene tetrahydrofolate reductase (MTHFR), in this pathway has a common variant form encoded by the C677T mutation, which interacts with a low-folate diet in homozygotes to cause a 50% reduction in global DNA methylation.

The population frequency of MTHFR C677T homozygotes in the US is 12%, but varies among different ethnic groups.

This polymorphism is therefore likely a major genetic determinant that influences dietary requirements among subsets of the population.

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 Sµ 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 Sµ 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 Sµ tandem repeats or the Msh2 protein show shifts in switch targeting. These shifts indicate that a 4-5 kb domain downstream of the lµ promoter is accessible for switching even if the Sµ 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 Sµ 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 Sµ and lµ 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 µ 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 µ 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: Shang, Fu
Title: Role of Ubiquitin in Quality Control of Lens Proteins
Abstract: Ubiquitin is a 76 amino acid peptide which is involved in various cellular processes including selective degradation of damaged or regulatory proteins (c-jun, c-fos, p53, cyclin, G-proteins, etc.), cell cycle control and DNA repair. One of the best understood functions of the ubiquitin dependent pathway (UDP) is its role in selective protein degradation. The activity of the UDP has been identified in lens cells. Like many other enzymes in the lens, the activity of the UDP is highest in the lens epithelial cells. Our recent data indicate that the activity of the UDP in bovine lens epithelial cells (BLEC) is limited by ubiquitin activating enzyme (E1) (see PRELIMINARY STUDIES), and that UDP activity increases during recovery from oxidative stress. Several stresses elicit similar responses of the UDP in many types of cells. Together with literature regarding the function of the UDP in other types of cells, our data allow us to hypothesize that the increased activity of the UDP plays a role in the restoration of the normal function of lens epithelial cells after stress and that ubiquitin activating enzyme (E1) and ubiquitin onjugating enzymes (E2s) are primarily responsible for the increased UDP activity. The specific aims of this project are to elucidate the function of UDP in lens epithelial cells, particularly in response to stress. In this study we chose oxidation as a model of stress to lenses since oxidative damage is observed upon aging and cataractogenesis.

PI: Sheoran, Abhineet
Title: Pathogenic and Immunogenic Studies on Cryptosporidium Genotypes
Abstract: C. parvum is an enteric protozoan that infects the gastrointestinal tract of most mammalian species and is the major cause of cryptosporidiosis in humans. C. Parvum comprises two known genotypes, type 1 and type 2. Type 1 (anthroponotic) is found exclusively in humans, while type 2 (zoonotic) is found in all mammals including humans. C. meleagridis (which infects birds and mammals) has also been associated with human cryptosporidiosis recently. C. Parvum type 1 is responsible for the majority of human infections and differs from type 2 in many aspects. Evidence from animal studies in this laboratory and in Ugandan children indicates that types 1 and 2 display several reproducibly distinct phenotypic traits including sporozoite proteins. Type 2 infects rodents but not type 1. Co-infections of animals with both types show no evidence of genetic recombination, and, therefore, it is likely that they maintain independent reproductive cycles. In contrast, recombination among type 2 isolates has been demonstrated experimentally. In addition, significant differences in DNA and amino acid sequences of types 1 and 2 surface protein gp40/IS exist. These observations suggest that the two types exhibit prominent genotypic and phenotypic differences and consequently, may belong to separate species of Cryptosporidium. While C. parvum types 1 and 2 and C. meleagridis display differences in host range, they share a common denominator in that all three appear to infect humans. Several type 1 isolates have recently been successfully adapted to continuously propagate in gnotobiotic piglets, enabling for the first time to begin studies on type 1 isolates and compare them with what is already know about type 2 isolates. In this application, the investigators aim to exploit the piglet model to define differences, if any, between C. parvum types 1 and 2. C. meleagridis (the only known species to cross the vertebrate class barrier) has also been successfully adapted to propagate continuously in piglets and will be included in this comparative study, largely because of its major epidemiologic and public health significance. The long-term goal of the candidate's research is to characterize molecular, functional, and immunologic parasite antigens associated with virulence and protection. The proposed study is the first step in that direction and the outcomes will provide basis for the future studies.

The following are several hypotheses that will be tested in this study: 1) as observed in human volunteer studies, there will be differences in virulence among isolates of type 1, 2, and C. meleagridis, in terms of clinical manifestation, infectivity, and the extent and distribution of the mucosal damage (Aim 1); and 2) while the nature of systemic and mucosal immune responses are likely to be almost identical among the three types/species of Cryptosporidium, immunodominant antigenic differences will be identified (Aims 2 and 4) which will result in greater cross-protection within homologous isolates than heterologous, and that C. meleagridis will be considerably different than either type (Aim 3).

The following specific aims are designed to address these hypotheses:

1. Compare the nature of host parasite interaction of C. parvum types 1 and 2, and C. meleagridis in the gnotobiotic piglet model with respect to disease manifestation and pathogenesis.

2. Characterize systemic as well as local cellular and humoral immune responses against C. parvum types 1 and 2 and C. meleagridis in the gnotobiotic piglet model.

3. Determine the extent of cross-protection between C. parvum types 1 and 2 and C. meleagridis in the gnotobiotic piglet model.

4. Identify immunodominant molecules that are unique to each C. parvum type and C. meleagridis, and also that are common among C. parvum types and C. meleagridis, which will help in identification of key specific- and cross-protective parasite antigens.

PI: Shirihai, Orian
Title: Erythroid Transporter Function in Hemoglobin Synthesis
Abstract: A number of disorders due to abnormalities in heme biosynthesis influence erythropoiesis. During normal erythroid maturation, heme precursors are generated in the mitochondria, modified in the cytosol and then returned to the mitochondria for final assembly with iron. Remarkably, accumulation of toxic heme intermediates or substrates, and secondary mitochondrial damage, are key elements in the pathophysiology of all heme biosynthesis disorders.

However, the mechanisms of export or import of heme products, by-products and intermediates to or from the mitochondrial matrix are as yet poorly understood. This proposal will focus on such mechanisms taking advantage of our discovery of ABC-me, a novel mitochondrial erythroid transporter. ABC-me expression is controlled by the erythroid transcription factor GATA-1 and is down regulated by heme. In differentiating erythroleukemic cells, ABC-me is rate limiting for heme biosynthesis. As such, ABC-me is the only mitochondrial inner membrane transporter implicated in heme biosynthesis.

To explore its function in heme biosynthesis, we have developed ABC-me deficient cell culture models and functional assays of reconstituted transporter in proteoliposomes. We have established that differentiating erythroid cells with reduced ABC-me activity produce less heme and exhibit signs of mitochondrial stress. We postulate that transporters involved in the heme pathway serve as gatekeepers for reactive substrates, intermediates and byproducts and thus couple production of essential end products to protection from toxic intermediates.

We propose a combined biochemical and biophysical approach to the study of the function of ABC-me in heme biosynthesis and the consequences of its malfunction. We will address the following questions:

1. Which steps in the heme biosynthetic pathway are facilitated by ABC-me?
2. Does ABC-me deficiency result in the accumulation of heme precursors in the cytosol and mitochondria?
3. What is the source of mitochondrial stress associated with ABC-me deficiency and what are the morphological and functional consequences?
4. Does ABC-me transport a heme biosynthesis intermediate, product or co-factor, and in which direction?
5. Does ABC-me facilitate heme biosynthesis indirectly by opposing mitochondrial stress?

PI: 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: Sommers, Samuel
Title: Evaluating Diversity: The Influence of Racial and Gender Composition on Information Processing and Group Decision Making
Abstract: In an increasingly multicultural society, “diversity” has become a ubiquitous word. It is nearly impossible in contemporary America to find a corporate CEO, university administrator, political candidate, or human resources manager who does not publicly claim to champion diversity, particularly with respect to race and gender. Despite its status as a buzzword, however, we know little about the actual effects of diversity.

The rationales for diversity initiatives often center on righting historical wrongs and ensuring equal access for all citizens. These are important and noble considerations, but what of the effects of diversity on group performance? Does a group’s composition predict its level of interpersonal conflict? On a more positive note, are there additional, perhaps even more persuasive justifications for diversity than concerns about fairness and adherence to the precepts of political correctness? The present proposal addresses these questions by examining the influence of heterogeneity on group decision-making, and more specifically, by considering the psychological processes through which racial and gender diversity affect group outcomes and individual cognitive tendencies.

These are issues of theoretical and practical significance in the continuing investigation of group processes and intergroup relations. Examining them empirically will lead to objective conclusions regarding the observable effects of group heterogeneity, and has the potential to identify specific ways in which diversity can truly “work” in the real world.

Research has demonstrated that there are both benefits and disadvantages to group diversity. However, there exist multiple definitions of “diversity,” and few investigations have examined the specific cases of race and gender. Moreover, most analyses of group composition rely exclusively on the informational prediction that “group diversity leads to diversity of ideas.” That is, the traditional informational assumption has been that any benefits of diversity are generated by the novel perspectives and opinions contributed by minority group members.

Previous research conducted by the present investigator, however, offers a broader consideration of the informational, motivational, and cognitive processes through which a group’s composition is influential. This work suggests that the benefits of diversity are not solely informational, and they can be enjoyed by majority as well as minority group members. Specifically, this previous research indicates that diversity can lead group members to scrutinize information more carefully, think in more complex ways, and be more amenable to the discussion of controversial topics.

The present proposal builds on this research and was designed with the following objectives:

1. To examine how membership in a racially diverse group influences individuals’ cognitive tendencies (e.g., how carefully they scrutinize information, how complex their thoughts are) and social motivations (e.g., the desire to avoid the appearance of prejudice)

2. To identify boundary conditions for these effects, namely whether they emerge across different types of groups and decisions

3. To further generalize these results by assessing the effects of gender heterogeneity, both in terms of information exchange and non-informational routes through which a group’s gender diversity is influential

4. To consider the question of decision quality—specifically, the possibility that diverse groups make objectively better decisions than do homogeneous groups in some contexts

PI: Sonenshein, Abraham
Title: Molecular Genetics of Basic Cell Functions
Abstract: Continued support is requested for an interdepartmental training program in genetics of basic cell functions. The emphasis is on rigorous training focused on studies of basic cell processes, such as chromosome replication and segregation, cell growth and division, regulation of gene expression, cellular differentiation, and host-parasite interactions. This training program has been the beneficiary of continuous support from NIGMS since 1975. In the present application, support is requested for six predoctoral trainees per year for a period of five years. The 19 members of the training faculty are from the Departments of Molecular Biology and Microbiology, Biochemistry, Pathology, Dermatology, and Medicine. They are highly interactive and dedicated to close, joint supervision and mentoring of graduate students. Their laboratories are fully equipped for modern molecular genetics research and have direct access to more sophisticated equipment in shared facilities. Past trainees include leading researchers in academia and industry. The training program is administered by the Graduate Program in Molecular Microbiology of the Sackler School of Graduate Biomedical Sciences. Starting with a pool of about 140 applicants, the graduate program annually admits 7-8 new students, of whom 70-80% are typically eligible for training grant support. The program has been successful in attracting an unusual number of students from underrepresented minority groups, all of whom have been supported by this training grant. Of the 167 students admitted since the inception of the graduate program in 1964, 108 have graduated with the PhD degree and 9 with the MS degree. Forty-one students are currently in training. Seven of the current students are from underrepresented minority groups. Nearly all graduates of the program have obtained high-quality postdoctoral appointments and are still active as researchers, as teachers, or in allied fields. Entering students take required courses in Genetic Analysis and Biochemistry and pursue 9-week rotation projects in four different laboratories. At the end of the first academic year, they choose a thesis supervisor and begin thesis research. Student progress during the first year is monitored closely by the faculty as a whole and thereafter by the thesis advisory committee. In the second and third years, the students complete required and elective coursework and prepare a research proposal (unrelated to their thesis topic) as a PhD qualifying examination. All students are required to complete a seminar course in scientific ethics.

PI: Sonenshein, Abraham
Title: Regulation of Glutamate Synthesis in Bacillus Subtilis
Abstract: The biosynthesis of glutamate lies at the intersection of carbon and nitrogen metabolism, linking the Krebs citric acid cycle to nitrogen assimilation through glutamine synthetase. In Bacillus subtilis, the genes for glutamate synthesis and for the pathways leading to the precursors of glutamate are tightly regulated by a host of proteins that respond to a variety of metabolic signals. The long-term goal of this project is to unravel and understand the network of genes, enzymes, and regulatory proteins that allow the cell to maintain tight control over glutamate accumulation. Building on knowledge gained from previous work, this proposal aims to focus on the roles of two of these regulatory proteins, CcpC and GItC. Two aspects of CcpC function will be investigated: interaction with the inducer, citrate, and the role of multimerization in repression. For GItC, the metabolite or protein that regulates its activity will be identified, in addition, the broad role of GltC in gene regulation and its functional interaction with other regulatory proteins will be explored. One of the Krebs cycle enzymes, aconitase, may have a second, non-enzymatic activity, perhaps as an RNA binding protein. The putative secondary activity of aconitase will be tested by seeking targets of such a function and by creating mutants that retain enzymatic activity but have lost the non-enzymatic activity. The implications of this second activity for sporulation in B. subtilis will receive particular attention. Since B. subtilis is a model organism for the gram-positive branch of the bacterial world, the knowledge gained here will be applied to a related, pathogenic species, Listeria monocytogenes. Thus, this proposal seeks to take advantage of the apparent conservation of regulatory proteins, gene organization and regulatory sites between B. subtilis and L. monocytogenes and thereby make rapid progress in an unexplored aspect of the life of an important pathogen.

PI: Sonenshein, Abraham
Title: Isolation of Early Sporulation Genes
Abstract: Bacillus subtilis, an endospore-forming bacterium, is, after Escherichia coli, nature's best studied organism and has proved to be a useful model for other gram-positive bacteria, pathogens and non-pathogens alike. Endosporulation in Bacillus and Clostridium spp. is a response to severe nutrient limitation. Thus, the metabolic signals that the cell perceives are a crucial component of the regulation of this differentiation process. In recent prior work, the B. subtilis CodY protein has been identified as a metabolite-sensing repressor that controls a number of genes that are turned on when cells experience nutrient deprivation. Some of these genes must be critical for sporulation, because a codY mutant sporulates under conditions of nutrient excess. It has long been recognized that the intracellular concentration of GTP drops when cells make the transition from rapid exponential growth to stationary phase. CodY has now been shown to respond to the intracellular level of GTP, losing activity as a repressor as the GTP concentration drops. CodY also responds to isoleucine and valine, two of the branched chain amino acids. These novel findings suggest that CodY plays a surprisingly general role in cellular metabolism and differentiation. In fact, microarray analysis has shown that hundreds of genes respond to the absence of CodY and are thus either direct or indirect targets of the regulatory protein.

This proposal seeks to address several fundamental questions about the role of CodY and the mechanism by which it regulates transcription. The specific aims of the proposal are:

• To determine the role of CodY in initiation of sporulation
• To determine the global role of CodY in metabolism
• To determine the molecular architecture of CodY and its interaction with effectors
• To characterize the interaction between CodY and its DNA targets
• To identify a hypothetical second signaling pathway that mediates the switch from stationary phase to sporulation.

The results of this research have the potential to resolve longstanding mysteries, such as why the onset of sporulation is accompanied by a transient drop in the GTP pool, and to provide new insight into the metabolic regulation of stationary phase and early sporulation events. Given the existence in other gram-positive bacteria of proteins that are remarkably similar to CodY, it is likely that the findings of this project will have general application to a broad group of important prokaryotes.

PI: Sonenshein, Abraham
Title: Clostridium Difficile Toxin Gene Regulation
Abstract: Clostridium difficile is the principal causative agent of antibiotic-associated colitis and the only known cause of pseudomembranous colitis, a potentially lethal disease. Two large toxin proteins, encoded by the toxA and toxB genes, which lie within a 19 kb pathogenicity islet, are the primary virulence factors. Previous work from the applicants' laboratories has shown that transcription of the tox genes depends entirely on TxeR, a novel RNA polymerase sigma factor encoded within the same pathogenicity locus.

The level of expression is strongly modulated, however, by the growth state of the cells and by environmental conditions. For instance, for cells growing in broth medium, tox gene transcription is restricted to stationary phase cells and is repressed by rapidly metabolizable carbon sources, such as glucose. Other factors that influence toxin synthesis are the availability of biotin and certain amino acids and the temperature of cultivation.

The present proposal seeks to determine the molecular mechanisms that control toxin synthesis in response to environmental signals. Candidate regulatory proteins, based on comparative genomics, will be specifically tested for their participation in such regulation. These proteins include CodY, CcpA and VirR, whose homologs in Bacillus subtilis or Clostridium perfringens have been shown to mediate the types of regulatory effects in question.

In addition, general, unbiased searches for the relevant regulatory proteins will be carried out based on affinity chromatography. A fourth protein, TcdC, will be tested for its potential activity as an antagonist of TxeR.

The project makes use of the expertise in C. difficile biochemistry, genetics and physiology of the three collaborating research groups and recent advances made by each of the groups in making this experimental system amenable to detailed molecular genetic analysis.

PI: Sonenshein, Abraham
Title: Bacillus Spores as Vaccine Delivery Systems (GC#2)
Abstract: We will construct spore-forming bacterial strains that prevent vaccine antigens to the mammalian immune system in a way that will induce immunity to important childhood diseases (diphtheria-pertussis-tetanus and rotaviral diarrhea) Spores of each constructed strain will be fed to mice to assess safety and to measure the extent to which an appropriate immune response is induced. Promising candidate vaccine strains will then be tested in humans to assess safety. The advantages of using spores as the vaccine delivery system are 4-fold:

1. Bacterial spores are among the most resistant biological entities known with respect to extreme variations in temperature and hydration
2. Spores of Bacillus subtilis, the bacterium of choice, are known to be harmless when ingested
3. Industrial-scale production of spores is a well-characterized, inexpensive and safe method that can be easily applied in the developing world
4. A spore-based vaccine can be used for oral immunization

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

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

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

PI: Soto, Ana
Title: Prenatal Xenoestrogen Exposure and Mammary Cancer
Abstract: Increased breast cancer incidence has prompted scientists to consider the possible role of hormonally active environmental chemicals. However, epidemiological studies searching for correlations between adult environmental exposures and breast cancer incidence have been mainly inconclusive.

Yet, epidemiological studies do suggest that fetal estrogen level fluctuations have long-term consequences on the risk of developing breast cancer as an adult. Studies in mice reveal that prenatal exposure to low doses of the xenoestrogen bisphenol A (BPA) alters the development of these mammary glands. These effects manifested long after exposure ceased. The increased number of terminal end buds and terminal ducts in these mammary glands is particularly relevant since carcinomas originate in these structures.

We hypothesize that prenatal exposure to low doses of BPA may increase the risk of mammary cancer and that the mechanism responsible for the neoplastic outcome may include the extemporaneous expression of genes that mediate mammary gland development. Xenoestrogens administered prenatally may alter the expression of estrogen-responsive genes that would then affect downstream genes in the mammary development program and predispose these animals to cancer.

To test this hypothesis we will use a rat model whereby exposure to the carcinogen nitroso-methyl-urea (NMU) at puberty induces mammary cancer. A pilot study performed using Wistar rats showed that this model could be modified to assess the effect of fetal exposure to environmentally relevant xenoestrogen levels. It produced data consistent with the proposed hypothesis.

Aim 1: To determine the levels at which prenatal BPA exposure results in an increased incidence of mammary cancer. Tumor incidence and latency will be measured in Wistar rats exposed to BPA from gestational day 9 to birth. At 50 days of age, animals will be injected with a sub-carcinogenic dose of NMU.

Aim 2: To test the hypothesis that prenatal BPA exposure alters gene expression in the mammary gland during both the period of BPA exposure and throughout life. Total RNA will be isolated from mammary glands and analyzed by DNA microarray technology. Where appropriate, laser-capture microdissection and/or RNA linear amplification techniques will also be used to obtain samples. Up- and down-regulated genes will be confirmed by QRTPCR. Cellular distribution will be assessed by in situ hybridization.

Developmental points to be analyzed are:

• During gestational BPA exposure
• 5 days postnatal
• Peripubertal
• After ductal development is completed

Aim 3: To test the hypothesis that in utero exposure to BPA alters the histo-architecture of the rat mammary gland and results in the expression of pre-neoplastic phenotypes. This Aim will assess whether exposure to low doses of BPA results in specific morphological alterations in the rat mammary gland that may confer a propensity to carcinogenesis, such as an increase in the number of terminal end buds (TEBs) and terminal ducts (TDs) at the time of NMU administration), the persistence of TEBs beyond puberty, and the appearance of pre-neoplastic lesions at several age points. In addition, the histo-architecture of the mammary gland at the time-points studied in Aim 2 will be examined.

Aims 1, 2 and 3 are intimately linked, because they seek to study the same phenomenon at different levels of biological organization. Gene expression alterations may suggest histo-architectural consequences and vice-versa.

This exploratory research is central to the generation of testable hypotheses about cause-effect relationships linking prenatal hormonal exposure and mammary gland carcinogenesis and may finally provide the direct link between environmental exposures and incidence of breast cancer researchers have been looking for over the past 20 years. Moreover, if, as suspected, low doses of BPA increase the incidence of mammary cancer, this work may have a great impact on the way we study risk factors and conduct epidemiological studies. It may even influence public policy about prevention.

PI: Soto, Ana
Title: The Stroma as a Gatekeeper of Tumor Development
Abstract: Rat mammary gland neoplasia is the surrogate model that most closely resembles the human disease. The main assumption guiding research in mammary gland carcinogenesis has been that carcinogens induce neoplasia by causing mutations in the DNA of epithelial cells (somatic mutation theory, SMT).

However, using a tissue recombination model, we recently observed that neoplastic transformation of epithelial cells occurred when only the stroma was exposed to the chemical carcinogen N-nitrosomethylurea (NMU). Neoplastic transformation occurred regardless of whether or not the epithelial cells were exposed to the carcinogen.

These data suggest that the stroma is a crucial target of NMU and favor the concept that carcinogenesis and neoplasia would be tissue-based, emergent (supracellular) phenomenon akin to development gone awry (tissue organization field theory, TOFT).

Unlike the SMT that is based on the cellular level of organization, the TOFT posits that neoplastic cells may be reprogrammed to behave like "normal" cells when placed within normal tissues. Evidence favoring this idea has been published regarding teratocarcinomas and hepatocellular carcinomas. Regarding breast neoplasms, Bissell and collaborators have shown reversion of the malignant phenotype of breast cells in a three-dimensional culture model by modifying the composition of the extracellular matrix.

PI: Soto, Ana
Title: Mechanisms of Developmental Toxicity of Bisphenol-A
Abstract: The increase of the incidence of malformation of the genital tract and hormone-related cancers is a main health problem in the industrialized world. Environmental estrogens are suspected to be the casual agent. The long-term goal of this proposal is to understand the mechanisms underlying the toxicity of the environmental estrogen Bisphenol-A (BPA) on the female reproductive system and mammary gland. During the first period of funding, we found evidence that perinatal exposure to low- environmentally relevant doses of BPA produces irreversible alterations in the structure and function of estrogen-target tissues in female rodents. Increased body weight, changes in morphology of the ovary, uterus and mammary gland, and alterations in pattern of estrus cyclicity suggested that perinatal exposure to BPA may decrease reproductive success, and increase the risk of mammary gland cancer. Given the similarity of toxic effects of the synthetic estrogen diethylstilbestrol in rodents and humans, our results raise concern the exposure to BPA may put human health at risk.

The overall hypothesis to be tested is that perinatal exposure to BPA results in morphological and functional alterations of the hypothalamus and/or pituitary as well as estrogen sensitive peripheral organs (i.e. mammary gland) by an ER-mediated process. These effects of BPA in development would in turn result in an altered response to ovarian and pituitary hormones in adulthood. The following complementary Specific Aims are proposed:

Aim1: To assess the hypothesis that perinatal exposure to BPA alters a) sexual differential of the brain and b) the functionality of the circuitry involved in the luteinizing hormone and prolactin surges thus altering permanently the hormonal milieu of peripheral organs (i.e. mammary gland).

Aim 2: To assess the hypothesis that in utero exposure to BPA directly affects the prenatal development of the mammary gland anlage by altering the expression of ER and downstream homeobox genes Msx2, Wnt10b).

Aim 3: To assess the hypothesis that the morphological changes observed in the mammary glands are due to an altered response to gonadal and pituitary hormones. The responsiveness to various sex hormones will be assessed in ovariectomized mice following in utero exposure to BPA.

The following techniques will be used: immunocytochemistry, in sit hybridization, quantitative PCR, morphometry, radioimmunoassays. The information obtained from these studies will considerably advance our understanding of the mechanisms underlying the developmental and reproductive toxicity of BPA. These data are needed to develop biomarkers and research strategies to apply these data to assess the toxic impact of xenoestrogens on human development and reproduction.

PI: Squires, Catherine
Title: Fusion of the Ribosomal RNA Promoters to Other Operons
Abstract: The long-term goal of our research is to understand fundamental aspects of ribosome synthesis and function. We propose experiments to study factors and conditions influencing rrn gene expression and specific aspects of ribosome function. Expression will be studied from two different perspectives, the rrn transcription antitermination system, and how the transcription apparatus responds to several conditions that lead to down-regulation of rRNA synthesis. Functional studies will highlight our new rrn deletion strains and are aimed at addressing questions uniquely suited to the use of these strains. In fast growing bacteria how transcription of rRNA genes occurs at increased elongation rates and bypasses terminators is very poorly understood. We will examine antitermination and map out the RNA/protein and protein/protein interactions involved. We propose to examine, using electron microscopy, how rRNA operon transcription responds to conditions requiring drastically reduced levels of operon expression. We will also ask what features of the ribosome guide its response to two specific problems in translation - sticky peptides in the exit channel and switching to tmRNA when stalled. Our ability to produce strains encoding homogenous populations of mutated ribosomes allows us to use these strains to ask where sticky peptides and their effecters interact with the ribosome and what specific features of the ribosome enable it to switch to a tmRNA molecule when a block in further translation is encountered. We have developed an rrn D strain in which we can shut off all synthesis of rRNA. Using this special strain we propose to questions such as:

• What rules govern the efficiency of mRNA translation when ribosomes are limiting?
• Are newly synthesized ribosomes required for cells to respond to a cold shock?
• How stable are ribosomes?

The current renaissance of information and studies of the ribosome and translation process put our work into the context of contributing to fundamental information about the cellular translation machinery that apply to all living systems.

PI: Stadecker, Miguel
Title: Immunoregulation of Schistosomiasis
Abstract: Shistosomiasis continues to be a major helminthic disease suffered by millions of people throughout the world. Morbidity and mortality are largely due to the consequences of a host CD4 T cell-mediated immune response against parasite egg antigens, yet the magnitude of the resulting granulomatous and fibrosing inflammation varies greatly from individual to individual, and also among inbred mouse strains in an experimental model of the disease.

In the majority of individuals, in whom the egg antigen-specific CD4 T cell response readily attains effective Th2 polarization, there is relatively limited immunopathology with improved survival; conversely, an unrelenting pro-inflammatory Th1 milieu is conducive to severe disease and death.

To date, the failure of some to turn off the life-threatening Th1 response is still not clear. The studies proposed in this application will examine two plausible mechanisms that drive Th1 responses with the hypothesis that both contribute to the development of the pronounced immunopathology typically seen in the C3H and CBA mouse strains.

The first is the production of IL-12 by dendritic cells derived from normal (CBA) mice and the second is an oligoclonal expansion of a CD4 T cell population reacting against the immunodominant epitope 234-246 (Sm-p40234-246) of the major Sm-p40 schistosome egg antigen with a Th1-biased response.

Aim 1 of this proposal examines the pathogenicity of IL-12 and related cytokines.

Aim 2 examines the pathogenic role of CD4 T cells that recognize Sm-p40234-246 with focus on a transgenic mouse expressing a T cell receptor specific for this epitope.

Aim 3 assesses the relative contribution of these two mechanisms with the use of MHC congenic mice.

These studies will provide a better understanding of the major pathways leading to severe immunopathology in murine schistosomiasis. They should lead to the design of highly focused, realistic and practical strategies for amelioration of disease, which could be amenable for consideration and possible implementation in the human patient population.

PI: Stadecker, Miguel
Title: Immunoregulation in Schistosomiais: Practical Applications
Abstract: Schistosomiasis continues to be a major parasitic disease suffered by millions of people throughout the world. The underlying potentially lethal immunopathology in schistosomiasis is a granulomatous inflammation around parasite eggs, which is mediated by the host's T cells sensitized to parasite egg antigens. The proposed studies are based on the concept that such pathogenic T cell responses are amenable to down-regulation by immunotherapy, thereby resulting in the prevention or amelioration of disease. This approach has been referred to as "anti-pathology" vaccine.

Specific T cell hybridomas will be used as probes to identify and isolate the major sensitizing egg antigens. Antigens will be cloned and their dominant T cell epitope peptide(s) will be synthesized. The type of elicited T cell response will be characterized and the genetic restriction of the T cell response will be determined.

The major schistosomal egg antigen Sm-p4O will be the subject of close analysis by investigating the interaction of its dominant epitope 13mer peptide with the MHC class II molecule I-A^k, and by assessing its intrinsic pathogenicity. A broad range of experiments will test selected strategies involving specific homologous or altered peptides for the purpose of down-regulating the pathogenic T cell response.

The long-term goal of this project is the achievement of effective and lasting specific T cell tolerance by way of a suitable biological vector capable of delivering the peptides into the host suffering from, or susceptible to, disease.

PI: Stadecker, Miguel
Title: Glycan Residues on the Sm-P40 Schistosome Egg Antigen
Abstract: Schistosomiasis is a major infectious disease caused by trematode helminths of the genus schistosoma. It is now well established that morbidity and mortality in schistosomiasis occur as a consequence of the immune system's hypersensitivity to the parasite's egg ags. In the case of infection with Schistosoma mansoni, there is typically perioval granuloma formation in liver and intestines, which in a smaller but significant proportion of patients leads to severe fibrosis, portal hypertension, portal-systemic shunting, hemorrhage and death. Despite great progress and success in socio-economic, geographic, and therapeutic measures, as well as major efforts in developing a vaccine, schistosomiasis is still prevalent in many tropical areas of the world.

The purpose of this project is to develop a novel immunological approach conducive to preventing and/or ameliorating irnmunopathological tissue damage associated with this disease. The proposal is based on strong initial evidence from the murine model which demonstrates that the oral administration of the major egg ag Sm-p40, in recombinant form, leads to significant reduction of granuloma formation. The mechanism underlying such an "anti-pathology" vaccine is the down-regulation of pathogenic CD4+ T helper lymphocytes responsible of mediating the granulomatous inflammation. Additional major schistosomal egg ags are in the process of being identified with the aid of available T cell probes and will be obtained in purified and recombinant form. The strategy of preventing and/or ameliorating pathology by suitable administration of a critical combination of specific ags/peptides is proposed as an important alternate/additional immunological approach which should make a significant contribution in curtailing the adverse consequences of infection with this major neglected disease of mankind.

PI: Stephanopoulos, Maria
Title: A New Class of Oxidation Catalysts: The Role of Atomically Dispersed Metals in Nanostructured Oxides
Abstract: Structures with nanoscale features have been in use for centuries and in heterogeneous catalysts for more than one hundred years. Empirical studies suggest relevant variables, but the lack of synthetic approaches, which have proved so successful for homogeneous catalysts, limits the level of structure- property understanding. The complexity introduced by the typical nanoscale "size" of the catalyst and, in some cases, the support to which it is attached, makes separation of possible contributing factors to the structure- property relationship a difficult task.

The basis of modern studies of nanostructured systems lies in the ability to design, control, and fabricate relevant structures with an unprecedented degree of precision. A general goal of this proposal is to bring to bear on these problems of heterogeneous catalysis an array of techniques, which encompass preparation and characterization and reflect the interdisciplinary requirements for the solution of a real problem. More specifically, we wish to examine in depth a recently discovered atomic-scale Interaction between gold and cerium oxide, which led us to the identification of the active site for the water-gas shift reaction and the prediction of the reaction light-off temperature.

This proposal is based on very new data that indicate that for a large class of active oxidation catalysts based on nanostructured metal/cerium oxide, the metal nanoparticles are not participating in certain low-temperature oxidation reactions, such as the water-gas shift reaction. It appears that the catalytic sites involve non-metallic charged clusters and atomically dispersed species. The practical impact of this finding is both economic and intellectual. An active catalyst now can be prepared using only 10% of the gold or noble metal initially thought to be required (for gold, < 0.5 wt% in ceria). This cost reduction has economic impact on the production of hydrogen as a fuel for fuel cells, currently an important national problem. In addition, the extensive literature on size effects in supported catalysts, and the attempts to explain these effects in heterogeneous catalysis, may not have always identified the proper source of catalytic activity. It is vital that these intellectual issues be resolved.

Our objective in this proposal is to investigate the metal ion or cluster interaction with ceria and other oxide supports, and to study the metal ion interaction in the absence of underlying lattice oxygen, by imbedding patterns of metal ions in thermally stable biopolymers. We will investigate systems with one nanoscale dimension as film edges, prepared with n0 high temperature fabrication step; and isolated metal ions in nano-layered peptide structures; and we will study their catalytic behavior and make comparisons with traditional nanocatalysts. Theoretical calculations will complement and guide the experimental effort.

Broader Impacts

An interdisciplinary team of experts from Chemical Engineering, Chemistry, and Materials Science at Tufts University, and from Chemistry at Brookhaven National Laboratory, has been assembled for this project. At the end of the interdisciplinary effort, we hope to have answered key questions regarding the activity and selectivity of atomically dispersed metals in nanostructured catalytic supports, and be in a position to provide rational designs for practical catalyst preparation. These materials are used in automotive catalytic converters, and as electrocatalysts and anode films for fuel cell applications. Thus, the impact of these findings could lead to better power systems design. There are several other tangible benefits for each of the disciplines involved here: new methods for catalyst synthesis, new materials properties specific to the nanoscale of importance to sensors, fuel cell components, as well as to catalysts; new catalyst design; new applications of biomaterials to catalysis. An overall benefit will be a template for rational design of catalysts derived from the interdisciplinary activities of the project. In what has become a tradition in our laboratories, we regularly exchange information with industrial colleagues. In this project, we plan to involve industrial colleagues as technical advisors, both for science and possibly for technology transfer.

The interdisciplinary nature of the proposal clearly impacts the education of graduate students and postgraduate fellows. Extensive training of young researchers at Brookhaven National Laboratory is also planned in the project. To bring this material to a wider audience, we plan the establishment of a Nanotechnology Seminar series to be given four times per year on a University wide basis. Another effort deals with pre-college education. The ability to reach high school-level science teachers in this regard is made much more feasible as Tufts is the home of the Wright Center for Science Education. The Center sponsors high school science teachers as fellows on a year- long basis as well as short- term fellows. We plan to develop an appropriate Nanotechnology workshop for these high school teachers designed to develop an appreciation for this technology.

PI: Strissel, Katherine
Title: Do infiltrating macrophages have a role in adipose tissue remodeling in obesity?
Abstract: This proposal seeks support for a novel line of investigation into obesity and the specific obesity-associated complications of adipose tissue inflammation and insulin resistance. It is now established that bone-marrow derived macrophages infiltrate adipose tissue of obese individuals and promote adipose tissue inflammation and systemic insulin resistance. The functional significance of adipose tissue macrophages is poorly understood. Recent work from our laboratory has further demonstrated that human and murine obesity are associated with dramatically elevated rates of adipocyte turnover, and that adipose tissue macrophages are almost exclusively associated with moribund adipocytes. I propose that the function of macrophages in adipose tissue of obese individuals is to remodel the adipose tissue to accommodate increased demand for lipid storage under conditions of chronic over-nutrition. The thesis that macrophages actively participate in this transition is supported by demonstrated roles for macrophages in wound healing and developmental tissue remodeling. This remodeling entails macrophage participation in both adipocyte mover (clearance) and in proliferation and differentiation of new adipocytes from precursors. I will test this hypothesis in proposed experiments. Importantly, in preliminary data, I demonstrate that at least one aspect of this remodeling (clearance of moribund adipocytes) is highly pro-inflammatory (i.e., TNF-alpha-inducing) and is coincident with the onset of insulin resistance.

This research stems directly from recent work of our laboratory of the identification of macrophage "crown-like-structures" surrounding dead adipocytes of obese mice and humans (Cinti S, et al, 2005). Those observations are being extended by studies that address the underlying proximate cause(s) and cell biological mechanisms of adipocyte death in obesity. By addressing macrophage function as opposed to adipocyte biology, the proposed pilot studies fundamentally differ from, but fully complement the other initiatives in our laboratory. Importantly, by providing the first assessment of the relative importance of macrophages to adipose tissue function, the proposed pilot studies have the potential to alter our current view of adipose tissue macrophages as inherently pathologic. This alternative view may impact the development of obesity therapeutics directed at adipose tissue macrophages.

The proposed studies address an underlying cause of obesity complications (inflammation, insulin resistance) that are significant public health concerns.

PI: Tang, Guangwen
Title: Formation and/or Degradation of Golden Rice Cartetenoids
Abstract: In 2004, the first batch of field Golden (GR1) was harvested from a protected field in Crowley, LA. Analysis of GR1 found that the rice which was held for a 3-month period showed a 3-5 fold lower amount of pro-vitamin A β-carotene than the rice that was newly harvested.

It is known that β-carotene can be bleached through either auto-oxidation and/or enzymatic oxidation. However, β-carotene in vegetables (spinach, carrots), which is associated with chloroplasts or chromoplasts, is relatively stable when the vegetables are kept at low temperature for freshness. However, rice is usually kept at room temperature. Further, the β-carotene in rice is associated with rice endosperm, and little is known of its stability. By common storage practice, the rice is kept either as milled rice or whole rice grain. What will happen to the high levels of β-carotene in the rice grain during the storage period? In addition, the grain development during the ripening process to the levels of rice β-carotene over time has not been studied. That is, the formation of β-carotene during the rice ripens needs to be followed to determine the optimal harvest period to ensure the optimal nutrient content.

The objective for this proposal is to investigate Golden Rice:

1. Carotenoid formation during grain development
2. Carotenoid stability in grains at different time points of harvest and storage in both milled and un-milled forms with/without deactivation enzyme activity

We expect to determine the optimal harvest time and to detect and identify a few cleavage metabolites during the storage period.

PI: Taylor, Allen
Title: Ubiquitination Lens Proliferation/Differentiation
Abstract: Lens formation requires a chronologically and spatially executed program of cell division and proliferation, as well as exit from the cell cycle and differentiation into lens fibers. These processes are controlled in most cell types by the timely degradation of cell cycle regulators by Ubiquitin Proteasome Pathways (UPPs). Aberrations in these processes frequently result in microphthalmia or cataract.

Yet there are few published papers that address either the lens cell cycle or its control by UPPs. During the ongoing grant, we demonstrated that Ub and Ubc3 (a Ub ligase) are required for proliferation and differentiation. However, regulation of the cell cycle by these moieties occurs at the G2/M transition and not, as predicted, at G1/S. We now seek to determine if the same controls are observed in vivo by directing expression of mutant Ub to the epithelial and differentiating lens cells in transgenic animals.

Our recent data beget two new overall hypotheses:

1. Proteolysis involving Ub and Ubc3 is required for proliferation, differentiation, and lens formation in vivo.
2. UPP which involves an undescribed Ubc3-E3 interaction is involved in control of G2/M events in lens.

These overall hypotheses are separated into 4 specific aims to test the hypotheses that:

1. An active UPP is required for proliferation, differentiation and lens formation in vivo.
2. Ubiquitination is required for the lens cell cycle, particularly in G2/M.
3. Lens Ubc3 cooperates with an E3, which we will identify, to control the G2/M transition.
4. Control of the cell cycle at G2/M requires ubiquitination of the APC regulators in a Ubc3-dependent process.

These studies will address a major objective of the NEI lens and cataract program: to characterize controls of lens cell division and differentiation, and their roles in formation of secondary cataract. The long-term objective is to prolong function of:

1. The natural lens by gaining a better understanding of processes involved in control of lens cell proliferation and differentiation, as well as in lens formation.
2. Implanted lenses by limiting secondary cataract due to overgrowth.

Our recent papers show that these results will also lead to a better understanding of corneal wound healing and retina responses to stress. This information, and our novel "reagents", will also find use in new ways to limit secondary cataract, prolong function of glaucoma medication, limit cancer and in fighting other proliferative maladies. We are joined in this effort by excellent collaborators, each of whom is a leader in his field.

PI: Taylor, Allen
Title: Exploiting Nutrition to Delay Cataract
Abstract: Cataract, or opacification of the lens, is a major cause of visual disability. Cataract extraction is the most frequently performed surgery, and costs for cataract-related problems account for the largest line item in the Medicare budget. Delaying cataract by 10 years would halve the number of cataract extractions and save over $1 billion annually.

Studies suggest that nutrition may be exploited to diminish the prevalence of or retard the progress of age-related cataract. However, there is little epidemiologic research regarding relations between major components of diet (other than for antioxidants) and risk for prevalence or progress of cataract. This knowledge is important in order to know if people at high risk or with early lens opacities might benefit from proper dietary management.

This NEI R03 proposal from the Nutrition and Vision Project (NVP) - a collaboration between Tufts University and the Nurses' Health Study (NHS) at Harvard University and Harvard Medical School - exploits unique existing ophthalmologic and nutritional databases to assess three previously untested hypotheses about the extent to which nutrition affects risk for prevalence and progress of opacification.

The NVP ophthalmologic data set includes two sets of graded lens images, gathered five years apart, from 451 women (ages 53-73 y) who are a subset of the NHS. The NHS data set includes long-term nutritional data from 5 food frequency questionnaires and a plethora of data on other personal and environmental factors, all gathered during a 15-year period before the baseline eye exam.

Combined, these data will be used to test three hypotheses: that risk for cataract is lower in persons who:

1. consume higher levels of specific fats (monounsaturated, polyunsaturated, and saturated dietary fats, n-3 fatty acids, etc.)
2. consume lower glycemic index foods
3. eat according to particular dietary patterns

PI: Telford III, Sam
Title: Perpetuation of Francisella Tularensis
Abstract: Multi-locus variable number tandem repeat analysis (MLVA) demonstrates that F. tularensis in dog ticks from Martha's Vineyard comprises diverse haplotypes; using 2 informative loci, we calculated a Simpson's index of 0.86. It may be that between epizootics F. tularensis is maintained in small isolated natural foci. Epizootics occur when local amplification causes natural foci to coalesce. In such a scenario, the greatest diversity would be evident during the initial stages of the epizootic as previously isolated foci of transmission merge together. To test this hypothesis, we examined the diversity of F. tularensis in dog ticks from a single site on MV over 4 years using MLVA. Over the course of the study, the diversity index fell linearly from 0.87 in 2002 to 0.36 in 2005. During this time the prevalence of infection in host-seeking ticks rose from 0.6% in 2002 to greater than 2.5% in all subsequent years. The decrease in diversity, despite the increase in prevalence, appears to be due to the emergence of a few dominant haplotypes. These findings suggest that certain haplotypes are more readily transmitted, and are consistent with the hypothesis of epizootic development as a result of the expansion of natural foci.

PI: Theoharides, Theoharis
Title: Acute Stress-Induced Neurogenic Bladder Inflammation
Abstract: Corticotropin-releasing hormone (CRH) is typically released from the hypothalamus, but has proinflammatory effects outside the brain, possibly through activation of mast cells that express CRH receptors (CRH-R) with selective secretion of vascular endothelial growth factor (VEGF), which may be involved in the pathogenesis of the painful bladder syndrome/interstitial cystitis (PBS/IC) that is characterized by bladder inflammation and is worsened by stress. Here we investigated the effect of intravesical CRH and acute restraint stress on VEGF release from mouse bladder explants and the role of mast cells.

PI: Theoharides, Theoharis
Title: Mast Cells, Antidepressants and Chronic Fatigues Syndrome
Abstract: Chronic fatigue syndrome (CFS) is characterized by fatigue, malaise, sleep and autonomic disturbances; it is considered a neuroimmune disorder with dysregulation of the hypothalamic-pituitary-adrenal (HPA) axis, precipitated by stress and associated with high disability. CFS often occurs with comorbid diseases such as fibromyalgia, irritable bowel syndrome (IBS), interstitial cystitis (1C) and migraines, all of which also worsen by stress. There are no reliable animal models for CFS. Mast cells have emerged as a major regulator of neuroimmune endocrine processes affected by stress and have been implicated in all comorbid diseases associated with CFS. We have shown that:

1. Mast cells have functional associations with nerve endings.

2. Acute stress activates mast cells, an action blocked by pretreatment with corticotropin-releasing hormone (CRH) neutralizing antiserum

3. Stress increases blood-brain-barrier (BBB) permeability, which is inhibited by the CRH-receptor-1 (CRH-R1) antagonist Antalarmin and does not develop in mast cell deficient W/W mice

4. Human mast cells express CRH receptors, activation of which leads to selective release of vascular endothelial growth factor (VEGF)

5. Some of the stimulatory effects of CRH on mast cells are mediated by neurotensin (NT), which has been shown to regulate the HPA axis

Tricyclic antidepressants are helpful in CFS and in the other comorbid diseases, but this mechanism of action is unknown. Our preliminary results show that the tricyclic antidepressant amitriptyline can inhibit rat mast cell secretion and intracellular calcium ion levels. Hypothesis: CRH, or the structurally related urocortin (Ucn), secreted by stress activates diencephalic mast cells, either alone or together with other neuropeptides such as NT leading to release of molecules that contribute to the central pathogenesis of CFS, and secretion of which can be inhibited by tricyclic antidepressants.

We will investigate:

Aim 1. The dose-response (0.1-100 uM) and time-course (0.5,1,6, 24 h) effects of three different classes of antidepressants, (a) the tricyclic (amitriptyline, imipramine), (b) the selective serotonin uptake inhibitors (fluoxetine, sertraline) and (c) bupropion on secretion of histamine, IL-1, IL-6, IL-8, IL-13, TNF, tryptase and VEGF from normal human umbilical cord-derived cultured mast cells (hCBMCs) derived from CD34+ progenitors triggered by IL-1, CRH or Ucn (100 nM).

Aim 2. The effect of those antidepressants shown to be effective in Aim 1 for their ability to inhibit "brain mast cells" developed by culturing human umbilical cord matrix stem cells (hCMSCs) that are CD34- in the presence of 10 nM IL-4 and nerve growth factor (NGF), stimulated as in Aim 1 q NT (0.1-100 mM).

Results from these studies will further our understanding of molecules released in response to stress hormones and which antidepressants may be useful in inhibiting these effects. Future studies will build on these findings to develop in vitro and in vivo models of CFS and lead to clinical trials with select antidepressants or other molecules that inhibit brain mast cells.

PI: Thorley-Lawson, David
Title: Epstein-Barr Virus Latency
Abstract: Epstein-Barr virus is a herpesvirus 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.

• 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?

• Does EBV, like other herpes viruses, 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?

• 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?

• 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: Thorley-Lawson, David
Title: Computer Simulation of Epstein Barr Virus Infection
Abstract: Epstein-Barr vires persistently infects >95% of the adult human population. It causes infectious mononucleosis and is associated with several important human cancers. Considerable progress has been made recently in understanding how EBV establishes and maintains persistent infection. It is apparent that EBV uses various combinations of latent gene expression patterns to manipulate the biology of normal B lymphocytes. This allows EBV to establish latent persistent infection in resting memory B cells in the blood and replicate in plasma cells in the lymphoepithelium of Waldeyer's ring (tonsils and adenoids).

However, these studies have all been static and an understanding of the dynamics of the infection is lacking. In the absence of a suitable animal model, we propose to develop a new generation of computer simulation. The application of supercomputing on distributed clusters of processors will allow us to develop an unprecedented level of sophistication and complexity.

We will employ an agent-based approach, which makes it possible to represent the actual anatomy of the relevant tissues and organs and the dynamic changes that occur over time and space. These features are not possible with traditional mathematical models based on ordinary differential equations. A simulation involving approximately 108 agents is within reach and should produce realistic results, based on previous experience with similar simulations of traffic and wireless communication systems.

We will use sensitive PCR and immunological techniques to accurately measure levels of virus shedding, virus infected cells, EBV specific CD4 and CD8 cells and neutralizing antibody as acute infection resolves into persistent infection. The data will then be used to inform the computer simulation. This approach will allow us to define the infection parameters necessary to predict the observed kinetics of infection.

Ultimately, the simulation will be used to test the effects of perturbations such as lowering the numbers of T cells (immunosuppression) eliminating infectious virus (anti-viral and/or vaccines) and to ask if complete clearance (i.e. cure) of the virus is realistic or even possible.

PI: Thorley-Lawson, David
Title: LSRII Four Laser Analytical Flow Cytometer
Abstract: We are applying for funds to support the purchase of instrumentation necessary to serve the flow cytometry needs of the research community at Tufts University School of Medicine (TUSM) and Tufts-New England Medical Center (T-NEMC). The facility has been in existence for 17 years and has been run for the last 6 years by Mr. Allen Parmelee, a skilled and respected operator, under the direct supervision of the P.I. and the Core Advisory Committee.

Under their direction the facility has grown, become highly successful and maintained financial stability through competitive pricing and Departmental support. 85% of facility time is dedicated to the NIH funded work of the Core User Group. We are now asking for an LSR II (BD Biosciences) analytical machine. This instrument will double our analytical capacity, while providing access to a powerful range of techniques not currently available to us, including:

1. The measurement of intracellular calcium in cells expressing GFP variants, using Indo-1 (355nm laser)
2. The tracking of cell cycle, viability, and proliferation, using Hoechst or DAPI (355nm laser)
3. The analysis of protein expression, using CFP (405nm laser)
4. The measurement of molecular association at the nanometer scale, using CFP-to-YFP FRET (405nm laser)
5. Any analysis requiring 5 or more colors

The cost efficiency and high standards of our facility have encouraged use to the point where the facility (one FACSCalibur and one, soon to be two, MoFLos) is now over-extended. In addition, new faculty hiring, combined with growing interest in flow applications throughout TUSM and, recently, T-NEMC has increased the size of our NIH funded Core User Group from 6 to 17. This renders the acquisition of a new LSR II essential, if we are to meet the flow cytometry needs, both in time and available technology, for the community of NIH funded researchers at TUSM and T-NEMC.

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. 

Intellectual merits of the proposed activity: 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.

Broader impact of the proposed activity:  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: Tseng, Florina
Title: Investigation of Secondary Anticoagulant Rodenticide Toxicosis in Birds of Prey in Urban and Suburban Areas of MA
Abstract: Anticoagulant rodenticides kill target species by interfering with the animal's blood-clotting system, causing the animal to bleed to death. Secondary poisoning of non-target species that ingest the dead or dying rodent has been documented in a variety of wild birds and mammals. Birds of prey that feed on rodents are particularly at risk of being poisoned and dying from severe, acute blood loss. At Tufts Wildlife Clinic (TWC), we began selectively testing suspected cases of secondary rodenticide toxicosis in April 2005. To date, we have identified the rodenticide brodifacoum from five out of five blood or liver samples analyzed from red-tailed hawks (Buteo jarnaicensis) with signs of severe hemorrhage and delayed blood coagulation. These hawks were all recovered from the city of Boston, MA or urban areas immediately surrounding Boston.

The most commonly used anticoagulant rodenticides, including brodifacoum, accumulate in the liver, and sublethal residues in the liver persist over many weeks. The persistence of these compounds allows the possibility that an animal will build up a lethal level of the rodenticide by consuming multiple sublethal doses. The presence of sublethal levels of these compounds has been suggested to have direct toxic effects on the liver, potentially causing a more chronic illness and eventual death in animals that do not die from acute poisoning. The purpose of this proposed study is to screen birds of prey presented to the Tufts Wildlife Clinic for exposure to anticoagulant rodenticides and investigate potential sublethal effects of these compounds on the liver. Thus far, we have tested only those birds showing clinical signs of severe hemorrhage or anemia. It is likely that a larger percentage of birds received by TWC are exposed to these rodenticides at sublethal levels. Our goal is to document the occurrence of sublethal exposure in birds of prey presented to TWC and to investigate the potential health effects of sublethal doses in order to raise awareness of the impact of these rodenticides on wildlife.

PI: Tucker, Katherine
Title: Lower Mississippi Delta Human Nutrition Intervention Research Initiative
Abstract: The objectives of this project are:

1. Review the existing data describing the socioeconomic, nutritional and health status of Lower Mississippi Delta residents.

2. Review the existing nutrition and community assessment research methodologies in order to identify potential assessment methods for the Lower Delta Research Initiative.

3. Conduct an assessment to determine the nutritional status of a population of Lower Delta residents (including dietary intake, food consumption and biochemical, physiologic, and anthropometric measurements) and conduct follow-on assessments so that the nutritional status of the population can be monitored over time.

4. Assess community factors impacting nutritional status.

5. Identify nutritionally responsive health problems and their determinants/causes in the Lower Mississippi River Delta populations so that interventions may be designed for testing.

6. Identify aspects of the community that are amenable to interventions that impact nutritional health of community members.

7. Review intervention research methodologies in order to identify potential interventions aimed at remediating the nutritional problems identified in the Lower Delta.

8. Evaluate, in a research setting, potential remediations for the targeted nutritional problems so that successful interventions can be implemented on a larger scale.

PI: Tucker, Katherine
Title: Center for Research on Nutrition and Health Among Older Puerto Ricans in Boston, MA
Abstract: Puerto Rican older adults living in the U.S. mainland have been identified as a group highly at risk of excess chronic conditions, particularly diabetes, depression, and physical impairment. Few studies have been conducted on this rapidly growing and generally low-income ethnic group. To reduce health disparities, it is necessary to understand the factors that combine to progress to poor health outcomes. The overall aim of this Center is to perform a series of inter-related studies involving a cohort of older adults of Puerto Rican origin to evaluate specific stressors affecting the Puerto Rican community, and to determine the effect of these stressors on allostatic load and, in turn, on disease-specific outcomes. The Center will include four research projects.

Project 1 is a prospective 2-year cohort study that will investigate both baseline and 2-year prospective associations between psychosocial stressors and allostatic load; and in turn, allostatic load and functional decline, specifically depression, cognitive decline and physical disability; along with the role of support, and vitamin intake and status in modifying these associations.

Project 2 is a sociological investigation of psychosocial stressors and their measurement using both qualitative and quantitative methodology to gain contextual understanding of the sources of stress in this population that relate to allostatic load, and adapt instruments for its measurement.

Project 3 consists of intervention studies. Using subsets of the baseline study, researchers will investigate the effectiveness of three different 2-year interventions in reducing indicators of allostatic load. Each is designed to be feasible for expansion by community agencies if effective. These include: 1) vitamin supplementation; 2) food coupons and nutrition education; and 3) social support and participation.

Project 4 will investigate genetic contributions of allostatic load. Investigators will explore the relationship between selected gene variants and allostatic load at baseline and with change over time, and will investigate the interaction between gene variants and responses to the differing nutrition and social interventions.

Three cores will work with all projects, including administrative, statistical, and laboratory cores. A pilot grants program during years 2 through 4 will encourage additional investigations relevant to the Center theme.

PI: Tucker, Katherine
Title: Psychosocial and Nutritional Associations with Depression
Abstract: Relative to Non-Hispanic whites, Puerto Rican elders living in the US mainland are at particularly higher risk for several conditions, among them depression. Differences between these two groups in the prevalence of depression persist despite controlling for socio-demographic variables, which suggests the contribution of socio-cultural and/or biological factors in the existing health disparity among Puerto Rican elders. Research with B vitamins suggests that folate, cobalamin and pyridoxine status correlate with depressive symptomatology. Puerto Rican elders have been identified as having lower plasma and dietary status of these vitamins compared to Non-Hispanic white elders. This population faces more stressors than other groups, which is thought to contribute to health disparities. Social support has also been shown to mediate stressors that lead to disease outcomes.

The specific aims of this research are to

1. Examine the associations of folate, cobalamin, and pyridoxine on depressive symptomatology using dietary intake and plasma vitamin measures.

2. Assess cross-sectional and two year relationships of stress on nutritional status and depressive symptomatology.

3. Assess how social support mediates the above associations. These data will be gathered from Puerto Rican adults between the ages of 50-70 in the greater Boston area. Understanding these relationship in this at-risk population is necessary to reduce health disparities.

PI: Tzipori, Saul
Title: Specific Human Monoclonal Antibodies for HUS Prophylaxis 
Abstract: Our objective is to develop an immunotherapeutic formulation for the treatment and prevention of complications associated with Stx-producing Escherichia coli (STEC) infections.

Clinical isolates of STEC are known to predominantly produce Stxl, Stx2, and/or Stx2c. Children are particularly susceptible to development of Stx-mediated HUS. Our hypothesis is that administration of Stx-specific antibodies will prevent or modify the outcome of infection for individuals at risk of developing HUS. In the earlier awards, we have generated a panel of human monoclonal antibodies (Hu-mAbs) specific for Stx 1 or Stx2. Using the gnotobiotic piglet model, we have shown that Stx-specific Hu-mAbs neutralize Stx and prevent development of Stx-mediated complications.

We now wish to determine which Hu-mAbs should be included in a formulation suitable for clinical evaluation. Based on superior efficacy, four Hu-mAbs specific for Stx2 (3 against the A subunit and 1 against the B subunit), and 2 for Stxl (both against B subunit) have been selected as candidates. The next step is to determine which combination of Hu-mAbs, is both compatible and highly effective.

In this proposal we plan to define the structural and functional characteristics, which facilitate protective efficacy of Stx-1 and Stx2-specific Hu-mAbs (Specific Aim 1). Affinity and efficacy of each HumAb will then be studied against their respective toxin (Specific Aim 2). The efficacy of protection of a given antibody dose will then be determined in terms of time after bacterial infection (Specific Aim 3). Finally, combinations of Hu-mAbs specific for B subunit of Stx 1 and A or B subunits of Stx2, will be examined for relative efficacy and compatibility to determine which is the most effective and thus suitable for clinical evaluation (Specific Aim 4).

At the conclusion of these experiments we will have determined the components, and optimized the formulation of Hu-mAbs which will be recommended for testing in human patients. The Hu-mAbs will first be characterized and ranked according to their efficacy, affinity and compatibility with each other. The optimal amount of each Hu-mAb in the formulation required to provide the longest protection after bacterial infection will also be established.

This is not a hypothesis-driven proposal, but an essential segment for the characterization of a promising therapy for HUS, against which currently there is no effective treatment.

PI: Tzipori, Saul
Title: Studies on 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 two-fold:

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

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:

Aim 1:  Conduct a comprehensive analysis of ten type 1 C. parvum isolates.

Aim 2: The host-parasite interaction of type 1 and type 2 isolates will be compared using the pig model and cell culture models.

Aim 3:  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: Tzipori, Saul
Title: Food and Waterborne Diseases Integrated Research Network
Abstract: The purpose of this contract is to establish research units who will collaborate and participate in the Food and Waterborne Diseases (FWD) Integrated Research Network (IRN). The FWD IRN will facilitate the integration of research programs to develop products to rapidly identify, prevent, and treat food and waterborne diseases that threaten public health. The FWD IRN will: 1) evaluate vaccines, therapeutics, and rapid detection methods; 2) integrate human mucosal immunity with clinical research; 3) increase research and product development activities, and 4) include the ecology and microbiology of food- and water-borne zoonoses as well as drug-resistant pathogens.

PI: Tzipori, Saul
Title: Development and Evaluation of an Innovative System for the Concentration and Quantitive Detection of CCL Pathogens in Drinking Water
Abstract: In a previous EPASTAR award, we had developed, optimized, and validated at Tufts University a novel method, the continuous flow centrifugation or CFC, for the concentration of three protozoa (Cryptosporidium spp. Giardial, Microsporidia) from large volumes of water (1000L). In this application we propose methods which will make it possible to expand this approach to simultaneously concentrate bacteria, algae and viruses (Objective 1) as well.

To do this, we will use representative pathogens which include E. coli for bacteria, Microcystis aeruginosa for algae and MS2 bacteriophages for viruses. We will then integrate the concentration of protozoa, bacteria, algae and viruses from water into a single concentration procedure (Objective 2). The PCFC will then be fine tuned for its ability to simultaneously concentrate representatives from each group of the CCL list (i.e., Coxsackievirus A9, Microcystis aeruginosa, Aeromonas hydrophila, and Enchephalytozoon intestinalis).

Objective 3 will focus on detection and quantitative identification of CCL pathogens in water, using multiplex miniaturized fiber optic bead microarrays coupled with a compact confocal-type imaging system, and comparing it with EPA approved methods. This novel technology, also developed at Tufts University originally for the detection of biodefense related pathogens, requires no nucleic acid amplification step.

We propose to apply this powerful technology to detect waterborne pathogens. It is expected that the entire procedure (concentration + detection) will be performed in less than 4 hours. The inclusion of several virulence factors for each pathogen will confirm the integrity and pathogenicity of the organism(s) present in water samples. Once optimized and validated with spiked samples, the concentration/detection procedure will be evaluated with environmental samples, and operational protocols will be formulated.

PI: Tzipori, Saul
Title: Regulation of Innate Immunity to Enterocytozoon Bieneusi Infection
Abstract: Of the 14 species affecting human health, E. bieneusi is clinically the most significant emerging enteric pathogen that infects the gastrointestinal tract of most mammalian species, and is the major cause of chronic diarrhea, wasting and cholangitis in patients with HIV/AIDS, malnourished children, and those receiving immunosuppressive therapy.

The technical difficulties that were associated with the lack of in vitro laboratory propagation methods as well as limited sources of spores, has contributed to the very slow progress on understanding the biology, pathogenesis and protective immune responses against this emerging pathogen. These have, to a large extent, been overcome recently by our group, which provides the impetus to this application.

The long term goal of this application is to enhance our understanding of the mechanisms involved in E. bieneusi protective immunity. This information is critical for the development of effective immunotherapeutic approaches that may help resolve otherwise fatal infection in immunodeficient individuals.

Our preliminary data indicate that IFN-gamma is an important component in providing initial resistance to E. bieneusi infection. An investigation into the molecular basis of cellular activation by E bieneusi, including a characterization of the innate immune receptors that initiate this initial resistance is unknown.

The goals of this application are to identify the specific cellular receptors and adaptor proteins that are responsible for initiating innate immunity during E. bieneusi infection, and to determine which IFN-gamma-dependent components are regulated by the host immune system during infection of epithelial cells. The role of TLRs in innate immunity and the IFN-gamma regulated genes that are essential in the context of this infection will be investigated.

The specific aims are:

1. To examine the expression of E. bieneusi-specific IFN-gamma regulated genes that may be involved in innate immunity to infection.

2. To determine the role of Toll-like receptors (TLR)/MyD88 signaling pathway, in the induction of IFN-gamma, in response to E. bieneusi infection.

Elucidation of the mechanistic basis of regulation of the innate immunity will lead to a better understanding of resistance to E. bieneusi infection. Moreover, innate immunity significantly affects the generation of acquired immunity to many infections. Thus, the proposed studies will form a foundation on which to build further studies to examine how regulation of innate immunity impacts acquired immunity to this emerging infection.

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

1. Dendritic cells sense pathogens through Toll-like receptors which lead to the initiation of adoptive immune responses

2. Proinflammatory cytokine IL-12, predominately produced by dendritic cells, is critical in controlling Cryptosporidium infection

3. Bone marrow-derived CD40-positive cells are required for mice to clear C. parvum infection

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

1. Characterize the role of mouse dendritic cells in the innate immune response against C. parvum infection

2. Determine the nature of activation of human dendritic cells upon infection with C. parvum or C. hominis

3. Identify the Toll-like receptor used by Cryptosporidium and isolate TLR activating ligand(s) expressed by the parasite

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

PI: Ultz, Arthur
Title: Vibrational and Rotational State Resolved Dynamics of Gas-Surface Reactions
Abstract: Despite the tremendous economic and technological importance of heterogeneous chemistry, mechanistic details of many important gas-surface reactions remain controversial. This situation is particularly true for internally energized gas-phase reagent molecules that may dominate reactivity in highly activated reaction systems. This lack of detailed mechanistic understanding has serious consequences. Systematic efforts to control, manipulate, or promote chemistry on surfaces are hampered by an incomplete understanding of the underlying reaction mechanism. Recent advances in theoretical surface chemistry hold the promise to revolutionize our approach to catalyst design, but stringent experimental tests are needed to test and verify computational results.

Prior NSF support funded development of a powerful new approach for studying gas-surface reaction dynamics. That technique combined state-resolved infrared laser excitation of molecules in a supersonic molecular beam with spectroscopic quantification of the surface-bound reaction products. Those studies revealed directly the dramatic role that internal energy can play in promoting reactivity.

This renewal proposal describes experiments that build on that foundation by addressing central questions in the field of gas-surface reaction dynamics through experimental study of model reaction systems. Results will reveal key molecular-level details of the chemical reactions studied, provide detailed experimental data for testing theoretical predictions of gas-surface reactivity, and establish new mechanistic insights into heterogeneous chemistry. Studies will focus on activated dissociative chemisorption reactions, where internal energy can playa crucial role in promoting chemical transformation. Specific lines of inquiry include:

1. Survey of vibrational mode specificity in gas-surface reactivity: Statistical models of reactivity are remarkably successful at predicting chemical reaction rates. Recent measurements in our laboratory reveal that such models are inadequate for describing CH 4 dissociation on Ni(100). Proposed studies will search for mode-specific dissociation probabilities in gas phase reagents that span a range of internal state density. Results will reveal trends useful for predicting mode-specific behavior in gas-surface reactivity.

2. Studies of orientation, reorientation and stereochemical effects: Polarized infrared laser light excites an anisotropic spatial distribution of vibrational excitations in the reagent molecules. Experiments with this oriented ensemble of molecules will test predictions of "steering" in gas- surface reactivity. Tailoring the nature of the vibrational excitation through isotopic substitution will reveal how localized excitations translate into enhanced reactivity.

3. Trapping-mediated reaction channels for internally excited molecules: Molecular beam reflectivity techniques will quantify the trapping probability of gas phase molecules containing chemically significant amounts of internal energy. High trapping probabilities could allow these energized molecules to react with much higher probabilities than one would expect from a single "direct" encounter and without the need for extensive energy exchange with the surface.

4. Prospects for bond-selective surface chemistry: The observation of vibrational mode selectivity in CH 4 dissociation on Ni(100) establishes a key element necessary for bond selective chemistry. Studies of CHD 3 and HOD dissociation on Ni(I00) will explore the possibility of exercising bond- selective control over gas-surface reactions. Excitation of the localized C-H oscillator in CHD 3 also facilitates a direct comparison with theoretical calculations.

PI: Vetter, Douglas
Title: Investigations into the Mouse Olivocochlear System
Abstract: Corticotropin releasing hormone (CRH) receptors, and urocortin, a member of the CRH family of peptides, has recently been discovered expressed in outer hair cells and olivocochlear terminals, respectively. While analysis of the role played by urocorUn, the only known ligand expressed in the inner ear capable of activating the CRH receptors, has been analyzed using urocortin deficient mice, nothing is known of the developmental expression pattern or role of the CRH receptors in the inner ear. The CRH receptors are G protein coupled receptors, and stimulate the cAMP second messenger-signaling cascade.

We hypothesize that activation of the CRH receptors may represent one phenomenon underlying protection from noise induced hearing loss. The mechanisms of action may include phosphorylation and inactivation of the sK2 calcium-activated apamin-sensitive potassium channel. In order to further analyze the morphological aspects of the CRH system in the cochlea, its functional role in hearing and protection from noise induced hearing loss, and finally, to assess the cellular mechanisms of action associated with activation of the CRH receptors, three specific aims are proposed.

First, we will establish the developmental and adult expression pattern of the CRH receptors in the inner ear. Successful completion of the experiments of this specific aim will establish the precise identity of the cells within the inner ear that express the CRH receptors, and identify the postsynaptic elements of the urocortin immunopositive fibers at the ultra structural level.

Second, we will establish the functional roles CRH plays in hearing, and whether they participate in protection of the inner ear from noise induced hearing loss. This aim will be accomplished using mice that lack the gene for either the type 1 or the type 2 CRH receptor, or that lack both.

Finally, we will use these mice to establish whether there are alterations in cAMP induced phosphorylation of targets in the outer hair cells following CRH receptors gene ablation. Success in this aim will allow us to identify individual proteins phosphorylated due to activation of the CRH receptors, as well as their role in modulating normal olivocochlear synaptic activity.

This will functionally link the urocortin hormone/CRH receptor and classical ACh neurotransmitter systems together in a unified model explaining inner ear based protection from noise induced hearing loss.

PI: Vilenkin, Alexander
Title: Fundamental Physics and Cosmology
Abstract: Inflationary scenarios explain the observed homogeneity of the universe and small deviations from homogeneity originating as quantum fluctuations during inflation. At the same time, these scenarios predict that on very large scales the universe is very inhomogeneous, with vast regions still in the state of exponential inflationary expansion. The cosmological parameters, such as the dark energy density or the amplitude of density fluctuations, may have different values in different parts of such an eternally inflating universe. These variable parameters cannot be predicted with certainty, and one can only hope to determine the corresponding probability distributions. The proposed research will focus on the large-scale structure of the eternally inflating universe and will address the conceptual and technical problems that have been encountered in the calculation of probabilities in such a universe. Of particular interest are models where the dark energy density is a stochastic parameter which varies from one part of the universe to another. Observational signatures of such models will be studied in detail and compared with the data.

Phase transitions in the early universe are likely to produce topological defects, such as domain walls, strings, or monopoles. These remnants of the big bang could still exist in the present universe and could produce a variety of observable effects. It is proposed to continue a systematic study of the formation, evolution, and observational signatures of various defects. Defect formation and properties in currently popular "braneworld" cosmological models will also be investigated. If topological defects are discovered, we would learn a great deal both about particle physics and about the early universe.

Research on eternal inflation, combined with new developments in string theory, points to a new cosmological paradigm, where distant parts of the universe have diverse properties and different particle physics. This paradigm shift has far-reaching philosophical implications and has already inspired some inter-disciplinary research.

Accounts of the new worldview that is emerging from inflationary cosmology will be published in a popular book and presented at conferences, including joint meetings with philosophers. Much of the proposed research will be done with active participation of graduate students.

PI: Waldor, Matthew
Title: Medical Scientist Training Program at Tufts University
Abstract: Medical scientists with dual training in MD and PhD programs provide an important bridge between basic research and its application to human diseases. The MD/PhD program at Tufts University began in 1976. Its goals are to provide the highest quality scientific and clinical training to students with outstanding research and academic potential. We seek to develop a group of young physician-scientists with a creative approach toward the study of human diseases. The MD/PhD program at Tufts University has graduated 26 MD/PhDs who are now in academic or industrial positions. Thirty-five students are currently enrolled in our MD/PhD program. Strengths of the MD/PhD program at Tufts include a dedicated and interactive faculty whose research topics are highly related to human diseases. The MD/PhD program at Tufts is small, so individual attention can be paid to each trainee. Students have easy access to the MD/PhD program co-directors and to members of the MD/PhD Executive Committee. A variety of special meetings for MD/PhD students promotes student interactions with one another as well as with the MD/PhD faculty.

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:

1. The site-specific integration of phage DNA into the bacterial chromosome
2. The repression of most phage gene expression following integration
3. 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: Intestinal Colonization of Enterohemorrhagic E. coli
Abstract: Enterohemorrhagic Escherichia coli (EHEC) are a group of non-invasive intestinal pathogens that cause illnesses ranging from non-bloody diarrhea to the potentially fatal hemolytic uremic syndrome. The O₁₅₇:H₇ serotype accounts for most cases of EHEC-induced disease. The O₁₅₇:H₇ genome contains ~1.4 megabases of DNA not present in the genome of non-pathogenic E. coli K₁₂. This additional DNA is interspersed as 'O-islands' (of >5obp) along the common backbone of sequences that is shared by both organisms. We hypothesize that the O-islands encode the majority of the pathogenic properties of E. coli O₁₅₇:H₇. Relatively little is known about the EHEC factors that are required for intestinal colonization. The lack of a readily available animal model of human EHEC infection has hindered the study of EHEC intestinal colonization. We have recently established that EHEC infection of infant rabbits reproduces some of the key aspects of human E. coli O₁₅₇:H₇ infection. Infant rabbits develop diarrhea and colitis after EHEC ingestion and these manifestations of EHEC infection are dependent on several of the known EHEC virulence factors. In our work, we are using the infant rabbit model of EHEC infection to identify O-island encoded colonization factors. Our Specific Aims, remain to

1. Identify EHEC O-island genes that promote intestinal colonization.
2. Characterize the expression, regulation, function, transmission and distribution of acfO, an EHEC intestinal colonization factor encoded on O-island 57.

PI: Walker, Peter
Title: A Collaborative Partnership
Abstract: This MOU provides a framework to allow Oxfam America HRD and the Feinstein International Famine Center to collaborate around mutual interests in humanitarian, livelihoods and policy based research and its application to programming and institutional change.

This agreement assumes a shared commitment to the highest quality output of research “products,” with applicable and measurable contributions to disaster response and recovery, as well as prevention, mitigation, and preparedness programming. Shared commitment to building upon the joint working relationship, and where appropriate, the building of joint programming and advocacy responses to researched subjects. The Famine Center and Oxfam America HRD will work together to develop collaborative opportunities combining research and programming with the overall goal of enhancing the quality of the Oxfam America’s humanitarian programming.

Examples of such collaboration might include:

• Developing new and innovative studies and applications for programming in the realm of disaster prevention, preparedness, mitigation, response, and recovery.

• Researching policy and practice options for local and national governments confronted with humanitarian crises.

• Working together on providing a rigorous evidence base for advocacy around genocide, crimes against humanity and the impact of global trade arrangements on crisis affected populations.

• The placing of Masters-level interns into OA field programs to both support those programs and provide meaningful field experience for the interns.

• Trainings conducted by Feinstein Center researchers for tsunami team and potentially indigenous research partners.

PI: Walker, Peter
Title: Omidyar Core Funding
Abstract: Our ten-year vision presents a justification for developing the Center and identifies six key areas for strategic growth:

• People to teams, programs to themes: A new approach to research

• Institutional change: An integral component of all our work

• Teaching and education: Consolidating the gains and working with African universities

• Staying close to the action: Securing our day-to-day presence in Africa

• Effective communication: A global network and new technologies

• Networking: Building a global humanitarian coalition

This Strategic Plan describes how we will make substantial progress in each of these six growth areas during the next three years. By the end of 2008, we expect to have a far more effective communications system and network in place for sharing ideas and information, and for achieving impact. Our office in Africa will be fully operational. It will coordinate community-based research, support our local academic partners, and continue with (and expand) a range of in situational change processes that are already in progress.

Our strategy for supporting greater multidisciplinary research will be well advanced with the blending of all existing programs into our three main Center themes. Stronger linkages with other centers and programs in the Friedman School, and other schools at Tufts, will be in place. And we will have consolidated our existing teaching and training courses, and developed specific strategies for providing affordable, quality education in humanitarian studies to users in developing regions. All of these changes will be underpinned by strong administrative and communications support in our Medford and Addis Ababa offices.

Our Strategic Plan recognizes that the Center’s key asset is its people. At present we have a small team of academics, practitioners and support staff who share a deep personal commitment to improving and professionalizing interventions to assist people in crisis and those affected by armed conflict. We recognize our strengths and achievements over the last ten years, but also realize that at times we have been stretched and overworked. This has impaired our ambitions to gel as a team and work collectively on cross-cutting issues. It has also limited our capacity to share our work and make the best use of advances in communication.

Our proposed strategy is to invest in more junior and mid-level researchers and support staff in order to free the time of senior researchers. We are aiming to build a Center within the Friedman School that enables a far more creative and reflective working environment that has strong day-to-day engagement with communities and partners in Africa.

The crucial changes we will bring about relate to a reshaping of programs in order to make better use of our collective experience, and to ensure that we strengthen our administrative and communications systems. We will retain our ability to conduct high-quality analyses to inform policies and interventions as new emergencies appear.

Although we are committed to further developing the Center, we also recognize that some of the Center’s current activities reflect firm commitments to partners and donors and will of course have to be completed. We will be able to adapt some projects more quickly than others to the changing environment in the Center. Similarly, existing teaching commitments will need to be fulfilled while simultaneously exploring new ways to build courses with our African academic partners.

PI: Walker, Peter
Title: Impact Assessment of Innovative Humanitarian Projects in Sub-Saharan Africa
Abstract: The Famine Center will provide support to the operational NGOs funded by the Bill and Melinda Gates Foundation in order to develop baseline project information, monitoring systems and participatory evaluation techniques for their innovative emergency interventions. With the selected agencies, the Center will develop and field-test an impact assessment toolkit. The Center will lead a final evaluation of the selected projects focusing on their true impact on the targeted communities. The Center will produce a final evaluation report and will disseminate the Impact assessment toolkit within the humanitarian aid community.

The goal of this work is to improve the ability of the humanitarian community to carry out impact assessment of its work and thus improve its effectiveness and accountability to the affected communities and the donor community.

The specific objectives of this project are:

1. The development of an impact assessment approach and methodology with participating agencies.

2. The application of this methodology to selected agency projects to produce a single comprehensive impact evaluation report.

PI: Walker, Peter
Title: Humanitarian Policy and Practice Complex Emergencies
Abstract: The first project takes forward recent work at the Center on various aspects of humanitarian practice in complex emergencies, with particular reference to the issues (described below) of universality, terrorism, integration, and security. The second project involves a pilot study in Darfur, which is both an extension of recent research and an element in a larger region-wide examination of remittances, livelihoods, and conflict.

Both projects are collaborative in approach, drawing on the perspectives and insights of southern researchers and institutions. Taken together, the two projects provide distinctive but related perspectives of the challenge of providing assistance and protection in the political and institutional framework post 9/11. 

The research will result in a number of publications for the policy and practitioner communities in the form of reports and articles in peer-reviewed journals. It will also serve as a springboard for discussions with communities of interest in donor capitals but also, and particularly, in countries of the south, using mechanisms and a format already in place. Each of the two component parts is described in the following section of the proposal.

PI: Walker, Peter
Title: WFP Khartoum Workshop
Abstract: WFP has a long history of operations in Sudan. The first WFP development project in the world was launched in 1963 in Sudan. Since that time, WFP has continued to provide development and relief assistance to the people of Sudan. WFP Sudan is most known for Operation Lifeline Sudan, drought response and more recently for its emergency operation in the Darfur region.

In 2006, WFP Sudan is implementing a Country Program, a Protracted Relief and Recovery Operation, and an Emergency Operation to address the immediate and longer-term food needs of some 6.5 million people in the South, East, Three Areas, and the Darfur.

The signature of the Comprehensive Peace Agreement (CPA) - and the expectation that an agreement reconciling the conflict in Darfur will take place in 2006 as well as settlement of the conflict in the east - represent a landmark opportunity to ensure sustainable peace for the people of Sudan.

WFP Sudan needs to ensure its strategy for implementation and priorities are aligned with this post-conflict environment to best help the people of Sudan achieve food security and sustainable peace.

PI: Walt, David
Title: Optical Tweezer Arrays on Optical Imaging Fibers
Abstract: This project aims to develop a platform for simultaneous and parallel micromanipulation of thousands of particles including both living cells and microspheres. The technology should offer a powerful tool for moving, trapping, assembling, positioning, examining, and sorting single particles and cells. Such capabilities have a broad range of potential applications in the fields of diagnostics, drug discovery, therapeutics, and basic research. An imaging fiber bundle will be coupled to a laser and the laser beam will be divided into an array of thousands of small focused spots, forming an array of optical traps. Initial work will focus on improving the system's capabilities to array particles to enable dense arrays of up to 1000 traps to be created. A microfluidics platform will be integrated into the system to enable solutions to be delivered to the array in a reproducible manner. The optical tweezers will optically trap single cells and microparticles of various sizes, ranging from 1 micron to tens of microns. In addition, the system will be integrated with a device for controlling each individual optical trap in the array, thereby enabling the system to selectively trap and release individual particles or cells. After system improvements have been performed to enhance the existing capabilities of the instrument, several proof-of-concept experiments will be performed to determine the system's capabilities and limitations. Such applications include DNA arrays and cell trapping. The long-term goal of the proposed work is to develop a platform technology for performing screening and selecting many particles or cells simultaneously. The technology has applicability to cell selection, blood and tumor diagnosis, combinatorial screening, nanoassembly, gene expression analysis and a wide variety of screening, selection, and diagnostic applications.

PI: Wang, Xiang-Dong
Title: Lycopene Chemoprevention of Lung Cancer in Ferret
Abstract: Increasing epidemiological evidence supports the role of lycopene as a micronutrient with important health benefits. It appears to provide protection against a broad range of epithelial cancers, including lung, prostate, stomach and colon. However, both the conflicting results of the p-carotene intervention trials in cigarette smokers (which used high doses of p-carotene and increased lung cancer risk) vs. the observational epidemiological studies showing that diets high in fruits and vegetables containing carotenoids (but at much lower concentrations than in the intervention studies) are associated with a decreased risk for lung cancer, and the conflicting results of lycopene effects on lung carcinogenesis in animal studies motivate us to focus our attention on the dosage of lycopene supplementation, biological activities of lycopene, and interaction of lycopene metabolism with cigarette smoke.

The objective of this investigation is to understand the mechanistic basis for the possible chemopreventive efficacy of lycopene (and its metabolites) against lung cancer development and the metabolic pathway of lycopene under well-controlled experimental conditions, using the ferret model, which is highly analogous to humans.

We hypothesize that:

1. There is a dose-dependent association between lycopene (or its metabolites) and the prevention (or promotion) of premalignant and malignant lung lesions in smoke-exposed, carcinogen-treated ferrets

2. Lycopene (or lycopene metabolites) inhibit lung carcinogenesis by up-regulating insulin-like growth factor binding protein-3 (IGFBP-3) as a molecular target and interrupting the signal transduction pathway of IGF-I as a mechanism for the chemopreventive efficacy of lycopene

3. Both oxidative metabolism of lycopene and expression of carotene 9',10'-monooxygenase (a cleavage enzyme for carotenoids) can be altered by lycopene supplementation and smoke-exposure

Our specific aims are:

1. Determine the effectiveness of lycopene (and apo-10'-lycopenoic acid) in both physiologic (low and median) and pharmacological (high) doses on plasma and tissue levels of lycopene (and apo-10'-lycopenoic acid), smoke-induced oxidative stress, DNA damage, and development of lung preneoplastic lesions and tumor formation in the carcinogen treated, cigarette smoke-exposed ferrets

2. Examine whether the induction of IGFBP-3 with lycopene or its metabolites inhibits the signal transduction pathway of IGF-1 and cell proliferation, and promotes apoptosis in both ferret and cell culture models

3. Investigate the metabolic pathway of lycopene by examining both the expression and activity of carotene 9', 10'-monooxygenase and the formation of apo-10'-lycopenoids from lycopene in vivo and in vitro

Our research effort will provide important insights regarding the mechanisms leading to the bioactivity of lycopene and its metabolites. This information is critically needed for future human studies involving lycopene for prevention of lung cancer and cancers at other tissue sites. In addition, the establishment of a lung carcinogenesis model in ferrets provides a valuable tool for both lung cancer and carotenoid research fields.

PI: Wanke, Christine
Title: Feasibility Study of Probiotics for Growth Faltering
Abstract: Diarrheal disease and growth faltering that occur at the time of weaning are significant public health problems. Malnutrition is associated with more than half of the 10 million deaths per year in children under the age of five in the developing world. Exposure to contaminants in the environment is a risk that increases at the time of weaning and is linked to an increased incidence of diarrheal disease when exclusive breast feeding is supplemented by additional foods.

Many of the potential interventions for interrupting this cycle of malnutrition and diarrheal disease are not economically or technically feasible for communities in the developing world. The proposed project consists of a study that will determine the feasibility of conducting a randomized-double blinded controlled study of the probiotic Lactobacillus GG (LGG) given in yogurt to infants at the time of weaning as a culturally and economically appropriate intervention for this cycle of malnutrition and diarrhea.

The proposed project will also obtain preliminary, background data on the incidence and severity of both diarrheal disease and growth faltering by weight for height and weight for age z scores before and after the time of weaning in children in a slum community in Pakistan.

This study will examine the feasibility of enrolling infants at birth, following them weekly at home until the time of weaning and testing the ability to identify the time of weaning. Further, this study proposes to examine the feasibility of following children daily at home after weaning, to monitor health and nutritional status and to administer LGG in yogurt to a group of infants randomized to this intervention.

The primary outcomes for the feasibility study are the ability to collect the required data on nutritional, general health, and neuro-developmental status, and on the rate and duration of diarrheal disease in the community in Pakistan. Additional primary outcomes include the determination of the background rates and severity of diarrheal disease and growth faltering by height for age and weight for age z scores, and the preliminary ability of LGG to ameloriate both diarrheal disease and growth faltering. These data will be used, with the feasibility data obtained, to prepare a proposal for a full clinical trial of LGG as an intervention for growth faltering and diarrheal disease at the time of weaning in these children.

PI: Wanke, Christine
Title: Improve HAART Adherence in a ARV Treatment Expansion Program in Kenya: Operational Evaluation and Cost Analyses 
Abstract: This proposal proposes to investigate the incidence of infectious diarrhea in infants living in an indigent section of Karachi, Pakistan, as well as the effectiveness of preventive treatment with Lactobacillus GG (LGG), a probiotic supplement.

This application is responsive to most of the criticisms offered in the review of its previous version. It exhibits a number of important strengths including the high public health significance of its proposed research, an excellent rationale for this, a reasonable study design, and a well-qualified investigative team.

Aside from some minor procedural issues that seem easily addressable, the notable concern about this application that holds over from the previous submission derives from the practical organization of this project, which involves the administratively responsible institution and lead personnel in Boston being geographically separated from the actual study site in Karachi. Although the PI and the main study-site co-investigator have a record of productive scientific collaboration, the cultural and structural discrepancies between the administrative site and the study site seem such as to threaten the practical do-ability of the proposed research. Nonetheless, this application is recommended for the 2-year support-term requested, and with some enthusiasm.

PI: Widmer, Giovanni
Title: Genetics of Crystosporidium Parvum
Abstract: Cryptosporidium parvum infects AIDS patients and other immunodeficient individuals and is a frequent cause of childhood diarrhea. Studies in human volunteers and animals have revealed pronounced differences in virulence among isolates, but the genetic determinants of virulence are unknown. The recent development in our laboratory of a method to cross C. parvum lines in mice opens new possibilities for studying this pathogen using genetic methods. We have found that C. parvum lines with recombinant genotypes are produced in mixed mouse infections and that recombination between different genotypes can produce highly virulent progeny lines. Since multiple recombinant lines with different virulence properties are obtained in experimental crosses, linkage analysis can be used to identify genetic determinants of virulence, without prior knowledge of virulence determinants. It is our central hypothesis that a majority of clinically or biologically relevant phenotypic properties in C. parvum are quantitative traits controlled by multiple genes which can be identified by linkage analysis.

Specific Aims:

1. Establish a high-density map of polymorphic genetic markers for C. parvum type 2. The almost completed C. parvum genome sequence will facilitate the identification of polymorphic microsatellites, restriction fragment length polymorphisms and single nucleotide polymorphisms.

2. Determine the mating structure and the inheritance of an extrachromosomal element in genotypically mixed C. parvum infections. We will test the working hypothesis that in mixed C. parvum infections fertilization results from the random fusion of identical or dissimilar gametes, and that the ratio of self- to cross-mating conforms to the model of random mating. The inheritance of a viral RNA genome will be studied in the context of this specific aim.

3. Identify virulence markers in type 2 C. parvum using Quantitative Trait Locus (QTL) analysis. This aim is based on preliminary observations that recombination between C. parvum lines generates progeny with distinct capacities to kill mice. We will test the following two working hypotheses:

   1. Virulence is determined by multiple genetic loci.
   2. QTL analysis can be used to identify genomic segments associated with virulence.

PI: Widmer, Giovanni
Title: Functional Genomics of Cryptosporidium Parvum
Abstract: This is a revised grant application to study global gene expression in Cryptosporidium parvum and in host cells infected with this parasite. Cryptosporidium parvum is an opportunistic pathogen in people with AIDS and is a common cause of diarrhea in children worldwide. The completed sequence of the C. parvum genome provides new opportunities for identifying drug targets and for research on this parasite on a genomic and functional level. In this project DNA microarrays will be used to study parasite gene regulation and the response of the host cell to this infection. Analysis of global parasite transcription, primarily during excystation, host cell invasion, and the initial phase of intracellular development will identify parasite biochemical pathways, which are differentially expressed during this phase of the life cycle. Analysis of the transcriptional response of infected host cells in culture and in animal models will identify mechanisms by which the host responds to the infection, and shed light on the molecular pathways leading to the intestinal symptoms of cryptosporidosis.

This project is a collaborative effort between two research groups with complementary expertise in the biology and genomics of C. parvum and extensive collaborative experience. In Specific Aim 1 a comprehensive DNA microarray containing all of the genes of C. parvum will be developed. This task will take advantage of the genomic sequence by converting genomic information into a tool for identifying genetic processes associated with parasite development. In Specific Aim 2 microarrays developed under aim 1 will be used to probe global gene expression during oocyst excystation, host cell invasion, and first generation merogony. Specific Aim 3 will use commercially available human and mouse gene arrays to study the response of the host cell to C. parvum. A comparison of the host cell response to C. parvum type 1 and type 2 will be performed to probe the molecular basis of the observed difference in virulence between types.

PI: Wilde, Parke
Title: Household Tradeoffs in the Food Stamp Budget: A Revitalized Engel Approach
Abstract: This project has two broad goals:

1. To measure the effect of food stamp benefits on food spending at-home and away-from-home. Serving almost one out of 12 U.S. residents, the Food Stamp Program is the nation’s largest food assistance program. The principal vehicle by which the U.S. Food Stamp Program (FSP) is expected to improve food security and reduce hunger for low-income American families is by raising their food spending level through normal channels of trade. From its inception, an important secondary purpose of the Food Stamp Program has been to support food retailers, manufacturers, and farmers, by promoting increased consumer demand for their products and services. The program provides targeted benefits, which generally may be spent only on groceries intended for home consumption (“at-home food spending”), and not on restaurant or cafeteria food (“away-from-home food spending”). Thus, in addition to influencing overall food spending, the Food Stamp Program may affect the tradeoffs some households make between food at-home and away-from-home, which may have households make between food at-home and away-from-home, which may have different implications for nutrition and well-being.

2. To measure the effect of food stamp benefits on household food security. Improving household food security is the Food Stamp Program’s primary purpose. As part of the “Healthy People 2010” objectives, the Federal Government adopted a goal of reducing the national prevalence of household food insecurity by half, from 12 percent to 6 percent, between 1995 and 2010 (Nord and Andrews, 2002). The Food and Nutrition Service’s strategic plan for 2000-2005 also set a quantitative target for reducing the prevalence of food insecurity with hunger among households with income under 130 percent of the Federal poverty standard (Wilde, 2004). For the United States, the world’s most prosperous nation by many measures, reducing hunger and food insecurity is a defining national dilemma.

PI: Willson, Robert
Title: Very Large Array-SOHO Investigations
Abstract: This Small Research Grant Proposal involves radio observations with the Very Large Array (VLA) to support NASA Solar Missions that study eruptive phenomena on the Sun. Coordinated VLA observations will provide images at decimeter and meter wavelengths with a spatial and temporal resolution comparable to the Solar and Heliospheric Observatory (SOHO), the Transition Region and Coronal Explorer (TRACE), and the Ramaty High Energy Solar Spectroscopic Imager (RHESSI) solar satellites.

Our main objective is to use long-wavelength VLA data (20, 92 and 400 cm) to extend the results of the 50110, TRACE, and RHESSI missions concerning non-thermal coronal energy release outside flares. The wavelengths of observation refer to heights that increase with wavelength from the low corona at 20 cm wavelength and into the middle corona at 400 cm wavelength.

VLA images at these wavelengths will provide direct information on the size, location, temporal behavior and magnetic structure in regions of the low and middle corona where stored energy is often released, producing shock waves and non-thermal particle acceleration. The non-thermal radiation of these events will be observed as long-lived Type I noise storms originating in the low corona, as trans-equatorial loops associated with non-thermal activity and low-level Type-ifi bursts from the middle corona.

By comparing these radio data with simultaneous 50110, TRACE, and RHESSI data, we can relate the acceleration sites and mechanisms to evolving coronal loops and other structures at lower levels in the solar atmosphere.

PI: Woods, Margo
Title: Nutrition Intervention for Metabolic Complications of HIV+
Abstract: As the HIV population survives and ages, a new syndrome is being observed that appears to be affected by PI medications but is also seen independent of PI use. This syndrome is characterized by hyperlipidemia, lipodystrophy and insulin resistance. Elevated triglycerides are a common observation with or without hypercholesterolemia. Since statin do not reduce serum triglycerides and may be counter-indicated to lower serum cholesterol because of potential liver damage in the HIV+ population that are on PI, dietary interventions have been getting more attention. Literature suggests that a diet lower in fat with reduced levels of saturated fat relative to polyunsaturated fat, increased omega 3-fatty acids intake, high fiber, and use of carbohydrates lower in glycemic index may be beneficial when they were studied individually. We propose to use a nutrition intervention in a HIV+ population that has elevated triglycerides (>220 mg/dl) to test whether a diet that combines all of these factors can have a significant effect on reducing serum triglycerides. The nutrition intervention will be a low fat diet (25% of calories from fat) with a 1:1:1 ratio of Saturated:Monounsat: Polyunsaturated fat, high in fiber (40 g/day) with carbohydrates of lower glycemic index (< 70 whenever possible). This diet will contain 3 g/day of omega 3-fatty acids which will be supplemented with 3.0 g of omega 3-fatty acids from capsules to give a total of 6 g/day of omega 3-fatty acids and a ratio of n-6/n-3 of 4:1. In addition to measuring triglycerides, serum cholesterol and its sub-fractions will be determined as well as insulin area under the curve (AUC) and body composition using CT scan. HIV+ participants eligible for the study (N=100) would be randomized into a control or nutrition intervention group and be tested for changes after 3 weeks, 13 weeks and 6 months of intervention. During the first 3 weeks the intervention group will be given all their meals at the hospital General Clinical Research Center, followed by an additional 10 weeks in which some food products are supplied to them along with the continued use of omega 3-fatty acids supplements at 3 gms/day (in 10 capsules). After 13 weeks the participants will be asked to continue to take the omega 3-fatty acid capsules but food products high in n-3 fatty acids will not be supplied. A 6-month follow-up will then remeasure all the study parameters to determine if the nutrition intervention group had experienced an improvement of the listed risk factors compared to the control group.

PI: Wortis, Henry
Title: CD22, 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 it 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: 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: Tufts Post-Baccalaureate Research Education Program
Abstract: This is a program designed to increase the number of minority post-baccalaureates who successfully pursue careers as health related research scientists. It is based on the idea that an enriched experience in research is the best preparation for PhD training in the biomedical sciences. The training consists of a two-year academic research laboratory apprenticeship at the interface of basic and clinical science. This will be complemented with interactive training that is designed to increase skills in scientific writing, seminar presentation and the critical reading of scientific literature. Additional elective didactic training will be offered. This will be structured around the needs of the trainee, ranging from remedial work to graduate biomedical science courses. Collegiality, cooperation and a relationship to the community outside the walls of academe will be fostered. The Sackler School of Graduate Biomedical Sciences and Tufts University School of Medicine propose a program that will be built on the foundation of well funded and highly regarded research laboratories; highly successful PhD training in biomedical research and several successful minority-oriented programs already in existence at Tufts. Assessment of the trainees' understanding of their own research, their bench skills, presentation technique and writing skills will be conducted regularly. The ultimate success of this PREP training program will be measured by outcomes: the aim is to have all entering trainees go on to complete MD/PhD or PhD training in a biomedical area.

PI: Wortis, Henry
Title: Immunogenetics of Infectious Disease
Abstract: Humans are genetically susceptible to infection. That is, without sanitation, vaccination, antibiotics and other relatively recent innovations, people would survive on average only into their early thirties. In the highly developed world, average lifespan has greatly increased thanks to major advances in diagnosis and treatment of infectious disease over the last 50 years. Nonetheless, infectious diseases are still the leading cause of death worldwide and the second leading cause of death in the United States.

In addition to the need to prevent and treat infectious disease in the developing world, there are four clear warning signs that indicate the worldwide need for accelerated research into infectious disease and immunity:

1. The spread of diseases once confined to specific geographic areas (as a result of global travel)
2. The increased incidence of antibiotic resistance in major pathogens
3. The continuing threat of bioterrorism
4. Increased susceptibility to infection in a rapidly aging population

Tufts University, with its excellence in geographic medicine, its strong research programs in defense against potential bioweapons, the premier voice in antibiotic resistance, and a focus on aging and immunity, is well positioned to respond to this challenge. We have the necessary intellectual capital, highly skilled researchers, and a rare collaborative environment, but lack certain essential equipment. A partnership between The W.M. Keck Foundation and Tufts to erase this deficit will have both immediate and long-range benefits.

PI: Wright, Andrew
Title: Integration of Nucleotide Synthesis and DNA Replication in E. coli
Abstract: Cytological studies in several bacterial species have shown that multiple proteins involved in DNA replication, repair and chromosome condensation are highly enriched at the cell center, forming a replication ‘factory’. Increasing the localized concentration of such proteins is thought to facilitate the processivity and efficiency of DNA replication.

Multiple lines of evidence, from phage infected bacterial cells to mammalian cells, indicate that there is a multi-enzyme complex involved in the synthesis of deoxynucleoside triphosphates (dNTPs), physically associated with components of the DNA replication machinery. Although the coupling of these complexes is non-essential, the implication for coupling is that there would be an increased concentration of dNTPs at active sites of replication, maximizing the efficiency and stability of replication forks. Data presented in this proposal are consistent with a model in which nucleotide synthesis and DNA replication are spatially and temporally coupled to maximize the efficiency of DNA replication.

The intellectual merit of the proposal is its focus on two important questions, using the bacterium E.coli as a model system:

1. How is nucleotide synthesis coordinated with DNA replication?
2. What factors constitute or are associated with the replication factory?

Additionally, a key feature of these studies is that they have the potential to define the protein interaction network associated with DNA replication.

Understanding these processes is of fundamental importance because faithful and accurate transmission of genetic information from mother cell to daughter cell is crucial in all organisms. Additionally, loss of replication fork stability can lead to fork collapse, resulting in mutation or loss of viability.

The broader impact of these studies is that they will not only lead to greater understanding of DNA replication and nucleotide synthesis in bacterial cells, but will provide new insights into these processes in higher cells. The educational component of this project includes the research carried out primarily by students in the Wright laboratory, which forms a major part of their Ph.D. training. Undergraduate students, often from minority groups, conduct summer research in the Wright laboratory, including one currently working on this project. The summer undergraduate program includes attendance at group meetings, oral presentations and a final poster presentation to summarize work accomplished. Mentoring undergraduates provides an excellent teaching experience for the graduate students in the laboratory.

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 β-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-β-catenin 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 HBPl is a suppressor of Wnt-β-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 D1 promoter, which is activated Wnt-β-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 Chr. 7q31–a region that is frequently deleted in breast and other cancers. Deletion in cancer is a hallmark of tumor suppressor genes. Lastly, the impact of HBP1 on breast tumorigenesis in a Wnt mouse model will be addressed. 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.

PI: Yee, Amy
Title: Phytonutrient Suppression of Wnt Signaling in Cancer
Abstract: Breast cancer recurrence is characterized by invasiveness, high proliferation, and distant metastases. Yet, relatively few diagnostic and therapeutic tools are available to predict and prevent recurrence and distant metastases, which is often fatal. Our previous studies have identified the HBP1 transcription factor as a gene that is mutated in clinical specimens of invasive breast cancer, which is a sub-type with high risk of recurrence and of mortality. Importantly, HBP1 mutations were detected before lymph node metastasis, thus representing a critical stage in breast cancer progression. Our previous and ongoing functional studies provide evidence that HBP1 regulates both proliferation and invasiveness, both of which are increased in the transition to invasive breast cancer (Paulson, et al submitted). Using HBP1 as a beginning, the project below attempts to delineate a gene expression program for the molecular mechanisms of early invasiveness and which may predict recurrent disease. The funded grant also investigates the major green tea compound EGCG in suppressing breast cancer, as this compound increases HBP1 levels (Kim et al, submitted). By defining the HBP1-regulated gene expression program for early invasive breast cancer, we can better investigate the possible role of EGCG in suppressing breast cancer transitions through its induction of HBP1.

This project was designed for Vincent (Vinny) Unanue, who is a recent Tufts graduate and will spend up to two post-baccalaureate years in my laboratory (see qualifications below). Aim 3 of this grant seeks to identify gene that determine invasiveness and that are susceptible to regulation by HBP1 and the major green tea compound EGCG. The objective is to identify critical genes and pathways that green tea may regulate to decrease breast cancer relapse. We have obtained microarray data for the first part of Aim 3 (comparing normal an HBP1 knockdown cells) in efforts to identify the relevant candidate HBP1 target genes. Ongoing efforts are directed at the gene expression profile upon EGCG treatment. Vinny's project will be to assess the HBP1 and EGCG regulation on selected target genes using quantitative PCR. Several molecular criteria were outlined in the grant application, e.g. induction upon HBP1 knockdown, the presence of HBP1 sites in the promoter, correlations to proliferation or invasion. Vinny will additionally screen the candidate genes against databases for breast cancer relapse. This strategy selects for genes that may have molecular relationships to invasive breast cancer and a clinical relationship to relapse. Three databases for clinical relapse have recently become available in the NCBI GEO datasets and on oncomine.org, which is a searchable database with normalized gene expression data that allows comparisons from different platforms.

Vinny will then test the candidate genes for HBP1 and for EGCG dependence by real-time PCR in cell models that mimic invasive breast cancer and early transitions. As described in the grant, we have recently implemented cell-based models for invasive breast cancer and have added an additional 3D cell model that mimics the transition from DCIS to invasive disease. RNAi-mediated knockdown will be used to assess the impact of the candidate genes on HBP1 and EGCG-regulated invasiveness and proliferation in these cell-based models of breast cancer transitions, as outlined in the grant. We expect to test about 20 genes that survive our molecular and clinical selection criteria. The final stage will begin testing candidate genes for expression in clinical specimens and attempt to correlate changes in expression with pathology. We have used a similar approach in our recently completed HBP1 study and will use the same access to clinicians and statisticians in configuring the study.

PI: Yee, Kathleen
Title: The Role of Neuregulin 1 Signaling in the Developing Cochlear Nucleus
Abstract: We are interested in how information-transmitting cells in the brain (neurons) obtain their identity and acquire specific characteristics that endow them to perform very specific functions. To address these questions, we study a region of the brain that forms the cochlear nucleus, the first and only direct target for cochlear input. While a large body of data exists on features of mature cochlear nucleus neurons, studies are only beginning to examine the role of genes during development. Our preliminary data shows that a receptor molecule, erbB4, along with a protein that binds to this receptor and activates it, neuregulin1, are both expressed in the developing cochlear nucleus. ErbB4 and neuregulin1 are molecules that can be associated with components of neurons that are important for neuron-to-neuron communication and interestingly, data supports that NRG1 is a schizophrenia susceptibility gene. Impaired auditory processing is a feature of the schizophrenic disorder and we propose to examine how the cochlear nucleus is altered in mutant mice with impaired erbB4/neuregulin1 signaling. Our findings will reveal how early gene expression controls development of the cochlear nucleus and how erbB4/neuregulin1 signaling may be critical for organization and wiring of the cochlear nucleus in schizophrenia.

PI: Yelick, Pamela
Title: Bioengineered Dental Tissues from Human Tooth Bud Cells
Abstract: We have reported the successful bioengineering of dental tissues, using immature pig and rat tooth bud cells seeded onto biodegradable scaffolds, and grown in the omenta of adult rat hosts. Based on this success, we hypothesize that human tooth bud cells can also be used to bioengineer organized tooth tissues. To test this hypothesis, and to bring this methodology closer to clinical relevance, here we propose to generate and analyze bioengineered dental tissues generated from human dental stem cells (DSCs), obtained from impacted and/or supernumerary human teeth. The Specific Aims of this research are:

Aim 1. Optimize harvesting/culturing conditions for human post-natal tooth bud cells.

Aim 2. Perform molecular/cellular characterization of human post-natal dental stem cells.

Aim 3. Generate and characterize bioengineered human tooth tissues.

The significance of the research proposed in this FIRCA is the extension of our previous successful tooth tissue engineering approach in pigs and rats, to the use of human tooth bud cells. Although we anticipate that higher vertebrate human tooth progenitor cells can be used to bioengineer human dental tissues, we also anticipate that human DSCs will exhibit distinct and unique features that distinguish them from those of rat and pig. Based on the anticipated unique properties of human DSCs, the proposed FIRCA research to characterize human DSCs is essential to the eventual use of human cells for autologous tooth tissue engineering applications. The specific aims of the FIRCA relate to the aims of the parent grant in that they will use our previously characterized methods used to analyze pig and rat dental progenitor cells, to now characterize human dental stem cells. The training received by the Duailibi's during their tenure in my laboratory makes them superbly qualified to perform the proposed research. Furthermore, the fact that the Duailibi's have easy accessibility to human tooth buds, extensive trained expertise in human tooth extraction, combined with the expertise of their Brazilian collaborator Dr. Esper Georges Kallas, guarantees the successful completion of the proposed studies.

PI: Yeum, Kyung Jin
Title: Effects of Antioxidants on Human Macular Pigments
Abstract: Long Term Objectives: Age-related macular degeneration (AMD) is the leading cause of blindness in the United States. Low dietary intake or low blood levels of lutein and zeaxanthin, which are the only pigments found in the macular region of the human retina, has been associated with an increased risk for AMD. We have reported that the dietary supplementation of lutein and zeaxanthin can increase the macular pigments (MP) of the eye. MP effectively absorbs blue light as well as quenches reactive oxygen species (ROS). Green tea polyphenols are also effective scavenger of ROS in vitro. However, the bioavailability and biological functions of tea polyphenols in humans are far from being understood.

Our goal is to elucidate how to effectively increase MP by physiologic levels of antioxidant supplementation. We focus our attention on the antioxidant functions of lutein and tea polyphenols in relation to AMD. We hypothesize that lutein and tea polyphenols protect the macula of the eye by increasing MP carotenoids effectively through an antioxidant mechanism.

Specific Aims:
Aim 1: To determine the changes of MP density of the eye and changes of antioxidant capacity of circulation using our newly developed assay when consuming lutein, which is a major MP.

Aim 2: To determine the possible synergistic effect of lutein and green tea extract on MP density and antioxidant capacity of circulation using our sensitive assay.

Research Design and Methods:
Forty-four men & post-menopausal women (all 50-70 yrs) will be randomly assigned to be supplemented with either 1) lutein (12 mg/d), or 2) lutein (12 mg/d) + green tea extract (400 mg/d) for 16 wks. Macular pigment density will be determined monthly using heterochromatic flicker photometry. The spatial distribution curve of MP will be obtained. Blood samples will be collected monthly for analyses of 1) antioxidant capacity in both the lipophilic and hydrophilic compartments of plasma by fluorescence methods; 2) lipid peroxidation (MDA-TBA adducts) by HPLC; 3) epigallocatechin gallate, major component of green tea extract, & its metabolites, and lutein & zeaxanthin levels by HPLC.

This study will allow us to obtain new information regarding biological functions of antioxidants on human MP and to characterize factors that might predict individual differences in response to lutein supplementation. Due to the budgetary limitations, we are proposing to study only two groups (I, Lutein; II, Lutein + green tea extract), with 22 study participants in each group for an 18-wks period of intervention. If we obtain positive results from the current study, we plan to submit a larger (R01) proposal, which would 1) increase the study groups to four (I, Placebo; II, Lutein; III, green tea extract; IV, lutein + green tea extract) and 2) determine how long the increased macular pigments can be sustained once the intervention has been discontinued.

PI: Zeng, Li
Title: Nkx3.2 Nuclear Localization and Cartilage Formation
Abstract: Arthritis is a widespread debilitating disease in which the joint cartilage becomes disintegrated. Regenerating cartilage in vitro may be part of a plausible treatment for these conditions. The understanding of how cartilage is degraded and how to produce new cartilage requires a thorough understanding of the signaling events that take place during cartilage formation.

Our long-range goal is to unveil the mechanisms that govern cartilage formation in the embryo, which will provide the knowledge for treating arthritis and other skeletal diseases. The objective of this proposal is to investigate the mechanism of nuclear localization of the important transcription factor Nkx3.2 during chondrogenesis. Our central hypothesis is that the control of Nkx3.2 nuclear localization is a key mechanism to cartilage differentiation.

We will test our hypothesis by pursuing the following Specific Aims:

1. Investigate the significance of Nkx3.2 nuclear localization in cartilage precursors in the embryo.

2. Investigate the mechanisms of Nkx3.2 nuclear localization controlled by prochondrogenic signals Shh, BMP and Sox9.

3. Investigate if cartilage-inhibiting signals TNF-α and IL-1ß affect Nkx3.2 nuclear localization. 

Nkx3.2 is an important protein because it not only promotes cartilage formation, but also prevents chondrocyte hypertrophy. Thus, the understanding the regulation of this protein will help us in our future research of introducing Nkx3.2 in adult stem cells to direct and maintain differentiated chondrocyte phenotype in our endeavor of cartilage regeneration.

PI: Zoukhri, Driss
Title: Stimulus-Secretion Coupling in Diseased Lacrimal Gland
Abstract: Sjogren's syndrome, a systemic inflammatory autoimmune disease which occurs almost exclusively in females, is the leading cause of aqueous tear deficient dry eye. To date there is no cure for this disease and the precise mechanisms responsible for the decreased tear secretion are largely unknown. Studies from this laboratory point to a potentially pivotal role of the proinflammatory cytokines, interleukin-1alpha (IL-1a), IL-1beta (IL-lb), and tumor necrosis alpha (TNFa), in the impaired function of the lacrimal gland associated with Sjogren's syndrome. Specifically, we found that these cytokines have a dual target in the lacrimal gland: the nerve endings (i.e., inhibition of neurotransmitter release) and the epithelial cells (i.e., inhibition of lacrimal gland protein secretion). However, the mechanism(s) by which these cytokines interfere with lacrimal gland nerve endings and lacrimal gland acinar epithelial cell functions remain to be elucidated. Thus, the long term objective of the present proposal is to define the intracellular signaling pathways activated by proinflammatory stimuli to inhibit lacrimal gland secretion. A second objective is to test the effects of selective inhibitors of these pathways on lacrimal gland secretion using a murine model of Sjogren's syndrome.

To obtain these goals, the following specific aims have been proposed:

1. Define the signaling pathways activated by inflammatory stimuli to inhibit neurotransmitter release and lacrimal gland secretion

2. Determine if JNK, ERK, and iNOS are involved in the impaired lacrimal gland secretion associated with Sjogren's syndrome

3. Determine if ICE activity is necessary for inflammation-induced inhibition of neurotransmitter release and lacrimal gland secretion

Lacrimal gland slices will be prepared from diseased and control female mice. Activation of JNK, ERK, and iNOS will be measured by western blotting. The release of neurotransmitter from lacrimal gland nerve endings and protein secretion will be measured spetrophotometrically. Selective inhibitors of JNK, ERK, and/or iNOS will be given subcutaneously to diseased and control female mice.