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Funded Research Abstracts

PI: Jackson, Rob
Title: Genetic Analysis of Glia-to-Neuron Communication
Abstract:
There is controversy about the importance of glia-to-neuron communication in the adult brain, notwithstanding studies of “gliotransmission” and indications that astrocytes can modulate neuronal excitability and behavior in mammalian models. For example, some have questioned the importance of glial Ca2+-dependent mechanisms in neuronal modulation. In addition, there is still limited knowledge of the mechanisms regulating gliotransmitter release, although certain studies suggest it involves vesicular exocytosis. In this application, we propose unbiased molecular genetic approaches to identify glial and neuronal factors essential for glia-to-neuron communication. We previously showed that elimination of a Drosophila glial-specific factor called Ebony genetically suppressed the hyperactivity phenotype of a Dopamine Transporter (DAT) mutant, indicating a role for glia-to-neuron communication in the regulation of activity level. In more recent published studies, we have demonstrated that adult Drosophila glial cells can physiologically modulate locomotor activity and circadian rhythmicity. Our studies also demonstrate a critical role for Ca2+-dependent mechanisms and Drosophila astrocyte-like glia in the regulation of locomotor activity.

In this application, we propose studies to identify cell signaling mechanisms and intracellular pathways that are important for glia-to-neuron communication in adult animals. Drosophila is an excellent model for such studies as the glial classes of the adult brain have been well characterized and one class has developmental, morphological and molecular similarities to mammalian astrocytes. Our studies will take advantage of multiple conditional perturbation methods that are cell type-specific and reversible to identify novel, conserved factors and intracellular pathways that mediate the glial modulation of neurons and behavior. Aim 1 will utilize Drosophila strains we recently developed and translational profiling methods to identify neuronal factors and molecular pathways that are modulated by glial signaling. Aim 2 will use behavioral genetic strategies to define glial signaling mechanisms that are important for neuronal modulation. This proposal represents the first broad study of neuronal proteomic changes that occur as a consequence of glial signaling. It also represents the first genetic analysis of glial mechanisms that mediate the modulation of adult neurons. As such, the results will have a major impact on glial biology – they will define molecular pathways within neurons and glial cells that mediate glia-to-neuron signaling, and set the stage for studies of conserved factors in mammalian models to understand their functions in human health and neurological disease.

PI: Jackson, Rob
Title: Glial Cell Regulation of Circadian Behavior
Abstract:
The human brain contains more than 100 billion cells, the majority being non-excitable glial cells. Recent studies, including those from the applicant’s lab, demonstrate that glial cells of vertebrate and invertebrate nervous systems have remarkably dynamic roles in the regulation of physiological and behavioral processes. Studies in mammals have demonstrated that neurons and glia communicate with one another and this has given rise to a model of the “tripartite synapse” wherein a glial cell (an astrocyte) cooperates with presynaptic and postsynaptic neuronal elements to regulate communication events and behavioral processes.

Recent studies from the applicant’s lab describe a role for a defined population of Drosophila astrocytes in the regulation of circadian behavior. Other studies have documented additional functions for fly glia in the regulation of neurotransmission and behavior (reviewed in Jackson and Haydon, 2008). In the present application, we propose experiments to elucidate the functions of glia in circadian timing. Our studies will employ Drosophila so as to be able to utilize sophisticated genetic techniques to study neuron-glia interactions in the circadian system. The work will utilize innovative genetic, behavioral, imaging and electrophysiological approaches and, importantly, the PI and co-I have complementary strengths in these areas.

We propose three specific aims that will test explicit hypotheses about neuron-glia interactions in the circadian system:

  1. Test the hypothesis that gliotransmission or other glial processes are essential for circadian behavior;
  2. Test the hypothesis that glia regulate pacemaker neurons; and
  3. Test the hypothesis that clock neurons regulate glial rhythms.

We expect that the results of these studies will highlight general mechanisms by which neurons and glia cooperate to influence circadian rhythmicity and other behaviors.

In most neurological disorders and psychiatric states, glial cell gene expression profiles are altered, and it is likely that this initiates dramatic structural/functional changes in the brain that lead to these disorders. Alterations of glial cell biology have been implicated in mental and neurodegenerative diseases including multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS), schizophrenia, epilepsy, and Alzheimer’s.

Our proposed studies of glia and circadian control mechanisms have considerable significance for an understanding of pathophysiological conditions such as jetlag and sleep/wake disorders resulting from environmental or genetic perturbations of the circadian system. Molecular components of the circadian system are conserved between insects and mammals, including humans, and Drosophila is an outstanding model for conducting genetic investigations of circadian behavior. It is anticipated that the results of our proposed studies will provide important and general insights about the interaction of glia with the neuronal circuitry controlling behavior, insights which are critical for understanding the roles of glial cells in health and disease.

PI: Jackson, Rob
Title: TUSM/T–NEMC Center Core for Neuroscience Research
Abstract:
Our community consists of 40 neuroscience research laboratories in 14 departments of Tufts University and its affiliated hospitals. Interests among these faculty span a wide range of medically-relevant neuroscience research areas, including synapse biology, ion channel function, sensory & behavioral neurobiology, neural signaling, neural plasticity, and neurological disease. Presently, there are 9 NINDS-funded projects among Tufts neuroscientists and a total of 44 NIH grants. The majority of these neuroscience projects depend heavily on core services that are provided by the NINDS-funded CNR.

The CNR Cores have had significant impact on neuroscience research at Tufts. More than half of the Tufts neuroscience research labs use CNR core facilities, and research conducted in the cores during the last 3.5 years has resulted in at least 90 publications, many of them describing collaborative studies by Tufts investigators. The following four CNR cores are now wholly or partially supported by NINDS funds:

  1. Imaging & Cell Analysis (fluorescence microscopy, confocal & 2-photon microscopy, laser capture microdissection, electron microscopy)

  2. Computational Genomics (gene microarrays, Q-PCR, bioinformatics and computational biology services)

  3. Animal Behavior (rodent behavior testing services)

  4. Electrophysiology & Biophysics (single cell recording, tissue-slice field recording and tissue-slice single-cell recording)

The primary continuing goal of our cores is to provide essential research services to NINDS investigators, other neuroscientists and other investigators of the Tufts community. Secondarily, the CNR continues to try to foster collaborative research enterprises among Tufts neuroscientists and to provide training and educational experiences that benefit the entire Tufts neuroscience community.

Neuroscience is a major component of the recently completed Tufts Medical School strategic plan and thus we are poised to recruit new neuroscientists in the near future. During the coming expansion of neuroscience at the University, we believe that the affordable research services provided by CNR cores will greatly augment the start-up packages provided to newly-hired Tufts neuroscientists and speed their integration into the local neuroscience community.

PI: Jacob, Michele
Title: Molecular Mechanisms of Auditory nAChR Synapse Assembly
Abstract:
Sensorineural hearing loss affects 30 million Americans. Aging, noise-overexposure, infection and ototoxic antibiotics all lead to sensory hair cell degeneration and permanent hearing loss. Recently, hair cell regeneration and partial function restoration were induced in the mature mammalian inner ear. However, little is known about molecular mechanisms that direct functional synapse assembly in either normal developing or regenerated hair cells. Our planned studies will define these mechanisms in vivo and identify molecules required for synapse assembly and hearing restoration in the deafened inner ear. This multi-investigator collaborative project draws on the complementary expertise of Michele Jacob (molecular mechanisms of neuronal synapse assembly), Yehoash Raphael (sensory hair cell regeneration), and Keith Duncan (ion channel function in sensory hair cells). We will focus on efferent olivocochlear (OC) cholinergic inputs from the brain onto sensory hair cells. OC cholinergic activity regulates the sensitivity and frequency selectivity of hearing. α9/10-containing nicotinic acetylcholine receptors (nAChRs) mediate synaptic transmission in hair cells. Further, normal activity requires the functional coupling and close positioning of α9/10-nAChRs to small conductance calcium activated potassium channels (SK2). Early SK2 expression is also required for inner hair cell functional maturation and normal exocytotic activity at the afferent presynaptic inputs onto primary auditory neurons that signal sound reception to the brain. Mechanisms that direct the synaptic localization of α9/10-nAChRs and SK2 channels are undefined. We predict the molecular organization of the OC synapse based on our identification of key components at neuronal α3-nAChR synapses and preliminary findings of shared components at hair cell α9/10-nAChR synapses.

In Aim 1, we will define the core postsynaptic complex of scaffold and cytoskeletal regulatory proteins at avian OC synapses. Aim 2 will define the specific adapter proteins that link α9/10-nAChRs and SK2 channels to postsynaptic complex components. Aim 3 will test the in vivo roles of the adapter proteins in directing α9/10-nAChR and SK2 synaptic localization and functional coupling that are essential for normal hearing. We will test the model in avian developing and regenerated hair cells. We will use loss-of-function and gain-of-function strategies and exploit the spontaneous regeneration and reverse genetic, molecular, morphological, biochemical and functional advantages of chick sensory hair cells.

Our findings will provide new insights into the molecular organization of nicotinic synapses in both developing and regenerated hair cells. We will identify novel binding partners for α9/10-nAChRs and SK2 channels. Further, the studies will provide the first identification of molecular interactions, in vivo, that are essential for synapse assembly and hearing recovery in the deafened inner ear.

PI: Jacob, Michele
Title: Synapse Neurobiology Training Program
Abstract:
Multidisciplinary approaches drive progress in the neurosciences perhaps more than in most other areas of biomedical research. The next generation of research neuroscientists, therefore, must be equipped with a multi-faceted skill set if they are to transition to successful independent careers and contribute meaningfully to the field. To meet this challenge, we propose a new predoctoral Synapse Neurobiology Training Program (SNTP) at Tufts University School of Medicine that will provide in-depth, multidisciplinary research education of 4 trainees in the area of synaptic function-a particular strength of the Tufts neuroscience faculty. The synapse forms the foundation of nervous system function, and research on synapses is, arguably, one of the most interdisciplinary areas in modern neurobiology. The SNTP training plan, thus, includes several mechanisms that position trainees to become leading neuroscientists, working at the interface between traditional disciplines:

  • Each trainee will be co-mentored by two SNTP faculty members who provide training in distinct yet complementary areas.

  • Through subsidized, one-on-one training in imaging, bioinformatics, electrophysiology, and animal behavior methods (provided via the core facilities and PhD-level Managers in the NINDS-funded Center for Neuroscience Research at Tufts), SNTP trainees will acquire the state-of-the-art tools and training required for an effective and influential multidisciplinary approach.

  • Innovative quantitative skills and techniques courses and individualized training plans will further aid each SNTP trainee in mastering the necessary skills to accomplish his/her research career goals.

  • Frequent opportunities for individual interactions with visiting speakers will provide SNTP trainees with further exposure to new methodologies and ideas as well as advice and guidance.

PI: Jacob, Robert
Title: HCC: Medium: Bringing Brain-Computer Interfaces into Mainstream HCI
Abstract:
Brain-computer interfaces (BCI) have made dramatic progress in recent years. Their main application to date has been for the physically disabled population, where they typically serve as the sole input means. Recent results on the real-time measurement and machine learning classification of functional near infrared spectroscopy (fNIRS) brain data lead to this project, in which the PI and his team will develop and evaluate brain measurement technology as input to adaptable user interfaces for the larger population. In this case, brain input is used as a way to obtain more information about the user and their context in an effortless and direct way from their brain activity, which is then used to adapt the user interface in real time. To accomplish this, a multi-modal dual task interface between humans and robots will be introduced, which will serve as a particularly sensitive test bed for evaluating the efficacy of these new interfaces.

The project will create and study these new user interfaces in domains where the effect on task performance of introducing the brain input to the interface can be measured objectively. They are most useful in demanding, high-performance, multitasking situations. Carefully calibrated multitasking applications scenarios from the team's research in Human-Robot Interaction will be employed.

The project will also advance the range of fNIRS brain measurements that can be applied to user interfaces. It will study a recently identified fNIRS signal obtained from the phase relationships among different regions of the scalp at low frequencies (0.1 Hz), as well as a wider range of sensor placement locations than previously examined. As these are developed into usable measurements for real-time signals with machine learning and other analysis approaches, they will be incorporated into new user interfaces.

PI: Jacques, Paul
Title: Flavonoid Intake and Health Outcomes
Abstract:
There is accumulating evidence that foods high in flavonoids (such as tea, berries, red wine, etc) are associated with lower chronic disease risk. However, there is a great deal of uncertainty about the potential effects of flavonoids on chronic disease risk, such as CHD, stroke, diabetes and dementia. This is, in part, a consequence of 1) the absence, until recently, of a relatively complete food flavonoid database and 2) the possibility that distinct classes of flavonoids may have different effects on disease risk. For example, existing evidence suggests that intakes of flavanols and perhaps anthocyanins, but not the intakes of flavones, flavonols, and isoflavones, are associated with a decreased cardiovascular disease risk profile. Flavonoids are ubiquitous in plant-based foods, and given the levels of flavonoids available from foods, they have the potential to impact significantly on disease risk. Given the growing body of evidence relating flavonoids and health, the possible establishment for Dietary Reference Intakes (DRI) for flavonoids is starting to receive some consideration. However much uncertainty remains and the questions regarding the role of flavonoid intake, and in particular the individual classes of flavonoids, in preventing cardiovascular disease, diabetes, metabolic syndrome and age-related cognitive decline still need to be addressed. To address these unanswered questions, we propose to use data from approximately 4,000 Framingham Offspring Cohort. Specifically, we propose to examine the relations between intakes of different flavonoid classes and the 15 year incident diabetes, metabolic syndrome and cardiovascular disease. We also propose to examine the relationship between flavonoid intake and change in cognitive function with age.

Successful implementation of this project should help refine our understanding of the role of flavonoids in age-related health and disease, and provide valuable information for dietary recommendations regarding flavonoid intakes.

Mentor: Jacques, Paul
Fellow: Hruby, Adela
Title: Magnesium, Genetic Risk, and Calcification
Abstract:
Arterial calcification—the "hardening" of the arteries—more than doubles the risk of developing cardiovascular disease (CVD) in as little as 5 years. Severe coronary artery calcification (CAC) is associated with a nearly four-fold higher risk of heart attack and CVD-related death, independent of the Framingham Risk Score. Insulin resistance and diabetes are significant risk factors for CAC, doubling and tripling, respectively, the risk of having more CAC than in those without these disorders. In addition, recent findings point to genetic variants that elevate risk of calcification and these disorders. Relatively little is known about the influence of diet on arterial calcification. Magnesium is a chronically under-consumed but vitally important nutrient, linked observationally to poor outcomes in CVD and diabetes, but so far linked only experimentally to calcification.

To date, no prospective study has examined magnesium intake in relation to calcification, despite in vitro and in vivo evidence and limited clinical data suggesting protective effects. Meanwhile, several studies have shown that higher magnesium intake is associated with beneficial outcomes in glucose and insulin metabolism, but these outcomes have not been examined in relation to genetic variation. Therefore, the overall hypotheses of this project are that higher magnesium intake is associated with lower risk of major arterial calcification and slower rates of its progression, and lower risk of impaired glucose and insulin metabolism in the face of differential genetic risk.

The overall aims of this project are to examine the relationships between

  1. dietary magnesium and CAC and abdominal aortic calcification (AAC), and
  2. magnesium's interactions with genetic risk variants on glucose and insulin metabolism, risk factors for calcification.

The proposed approach is an epidemiologic investigation of magnesium intake through secondary data analyses: CAC/AAC will be investigated in the Framingham Heart Study Offspring and Generation 3 cohorts, and genetic variation in glucose and insulin metabolism will be investigated in a meta-analysis of data from 16 European and US cohorts. This project may provide epidemiologic evidence that increasing intake of magnesium—a low-cost, widely available nutrient—increases longevity and ultimately saves lives by reducing the risk of arterial calcification and factors that accelerate its progression.

PI: Jacques, Paul
Title: Role of Dietary Beef in Optimizing the Mental and Physical Functioning of Older Americans
Abstract:
We recently showed that lower usual beef intakes were associated with less muscle mass in older Americans. Low muscle mass is related to decreases in muscle strength and function that, in turn, lead to falls and poor quality of life. We hypothesize that higher beef intakes by older Americans can be of value in preventing these adverse health outcomes, as well as cognitive impairment. The iron, zinc, and B-group vitamins in beef have previously been linked to good cognitive function. Our proposed project is clearly consistent with the HNRCA's mission and its key scientific priority of preventing age-associated neurocognitive defects through nutrition.

Hypothesis:

  1. Usual beef intake is positively related to gait speed in older Americans

  2. Usual beef intake is positively related to muscle strength in older Americans

  3. Usual beef intake is positively related to cognitive function in older Americans

Objectives:

  1. To show that older Americans with high usual beef intakes take less time to walk 20 feet than older Americans with lower usual beef intakes

  2. To show that older Americans with high usual beef intakes have greater knee extensor force than older Americans with lower usual beef intakes

  3. To show that older Americans with high usual beef intakes perform better on a cognitive function test than older Americans with lower usual beef intakes

Approach:

We propose to use data collected in the 1999-2000 and 2001-2002 National Health and Nutrition Examination Surveys (NHANES) to evaluate cross-sectional associations between usual beef intakes and habitual gait speed, maximal knee extensor force, and cognitive test performance. Our approach will be to create a database (n=1,844) containing the seniors' values for beef intake, gait speed, muscle strength, and Digit-Symbol Substitution Test performance, along with data on potentially confounding factors (e.g., age, sex, race-ethnicity, education, smoking status), and to perform multivariate statistical analyses using beef intakes and potential confounders as independent variables in the models, and the three study outcomes as dependent variables.

PI: James, Thomas
Title: Deflection of Cancellous Bone Screws under a Cantilever Bending Load
Abstract:
High tibial osteotomy (HTO) is a common surgical procedure used to realign the varum or valgus knee, or to reduce stress to the afflicted knee compartment of unilateral osteoarthritis. Following this procedure at Tufts Medical Center, a patient experienced a broken cancellous screw. A research project was initiated to determine the biomechanics of the screw-bone construct. The proposed research is based on the hypothesis that cantilever bending stiffness of cancellous bone screws is primarily dependent on three factors and their interactions:

  1. ratio of major to minor screw diameter,
  2. screw length, and
  3. ratio of cancellous bone engagement length to cortical bone thickness.

The primary goal of this project is to test the hypothesis by elucidating the factors contributing to cancellous screw failure from cantilever bending loads. Synthetic composite bone will be used to test cyclic bending loads on stainless steel cancellous screws representative of loading post HTO. The screws will have varied pitch and length, and the bone samples will have varied cortical thickness.

PI: Jay, Daniel
Title: KillerRed Assisted Mutagenesis to Discover Cancer Drug Resistance Genes
Abstract:
Genetic approaches have contributed greatly to our understanding of biology, but they are limited in cancer cells because genetic loss-of-function in diploid cells is obscured by expression from the wild type allele. To address this limitation, we propose to develop KillerRed Assisted Mutagenesis (KRAM) to generate an unbiased forward genetic screen for somatic cells to identify proteins required for cancer-relevant processes. There are several components to KRAM: first, enhanced retroviral mutagenesis (ERM) will be used to introduce a regulated promoter/guest exon fusion encoding the photosensitizer gene, KillerRed, randomly throughout the genome. The promoter segment provides overexpression of the fused gene, leading to gain of function; while the KillerRed fusion permits Chromophore-Assisted Light Inactivation (CALI), a light-mediated inactivation technology, to destroy the protein fusion, leading to loss of function. As will be discussed later, in contrast to genetic deletion, we expect photo damage by CALI to exert dominant effects regardless of wild type allele expression. Thus, KRAM will provide for the first time a low cost high throughput approach to address the consequences of loss-of-function and gain-of-function globally in diploid cells. KRAM selection would cost markedly less than RNAi and cDNA expression library screens, as it does not require synthesis of specific reagents for every gene. To develop and test KRAM, we will use it to identify genes that act in methotrexate resistance of chronic myelogenous leukemia (CML), a well-characterized process whose genes are known. We will then use KRAM to study imatinib resistance, an important clinical problem in CML, where we expect to identify new genes in this process.

We have assembled a research team whose combined expertise is optimized for the success of the proposed work. The PI pioneered CALI and is the leading authority on this approach and its application to cancer. Dr. Cochran developed the cell lines that will be used to develop KRAM, Dr. Songyang invented ERM screening and Dr. Van Etten is an expert in CML and abl-oncogenes. To establish KRAM, we propose three Aims: 1) optimize KillerRed CALI using β-galactosidase and endogenous proteins implicated in drug resistance; 2) develop KRAM and test its ability to select for genes required for methotrexate resistance; and 3) conduct a full-scale KRAM selection to identify new genes important for imatinib resistance and validate them by overexpression and siRNA. Successful completion of these Aims will provide a proof-of-principle for selections that are generally applicable for other cancers and also for other cancer relevant processes such as proliferation, invasiveness and apoptosis. In addition, it will identify and validate new targets to develop drugs that prevent imatinib resistance in CML, which has potentially high clinical significance. As a generalized low cost approach for gain-of-function/loss-of-function selection in somatic cells, KRAM will have wide application across biomedicine and be a transformative technology.

PI: Jefferson, Douglas
Title: Hepatobiliary Pathophysiology in Cystic Fibrosis
Abstract:
Liver disease affects ~20-30% of cystic fibrosis patients, and is the second leading cause of death in this population. The only cells in the liver that express CFTR are the bile duct cells. Therefore, it is thought that the liver disease seen in CF patients is due primarily to dysfunction of the bile duct cells. Bile duct cells make up less than 5% of the cells in the liver. Bile duct cells form tubes in the liver that act as conduits draining bile from the liver. While the lack of functional CFTR on the surface of bile duct cells has been demonstrated, the mechanism(s) responsible for liver disease remain to be elucidated. It is speculated that CF reduces bile flow by some unknown mechanism causing the initial injury to the bile duct cells. We have isolated bile duct cells from normal individuals and CF patients and studied their function. We have found that another protein (AE2) that transports molecule that regulates how acidic or basic the bile becomes is dysfunctional in CF patients. The affected protein is termed the anion exchanger and our studies have looked at how CFTR and the anion exchanger interact normally and how this interaction is changed in CF bile duct cells. We will investigate if CFTR interacts directly with the anion exchanger on the surface of bile duct cells or if there are other protein(s) that form a complex that might regulate function. We will use a number of techniques to examine the interaction between CFTR and the anion exchanger.

PI: Johnson, Elizabeth
Title: Nutrition and Eye Health
Abstract:
The research to date suggests that certain nutrients may play a role in the prevention or delay in progression of age-related eye diseases, e.g. cataracts, age-related macular degeneration. However, the strength of the evidence for each of these nutrients and diseases needs to be determined with a thorough review of the scientific research that evaluates these relationships.

The intent of this research grant is to conduct a literature search to determine the scientific evidence that supports a role for nutrition in eye health. A systematic review of the literature will be conducted using a Medline search for keywords in the area of nutrition and eye health, e.g. vitamins C and E, zinc, lutein, zeaxanthin, antioxidants, cataracts, age-related macular degeneration, vision. Findings on the relationships between nutrients and eye disease will be summarized in a review organized by nutrient/eye disease. The review article that will result from this literature search will be submitted to a peer-reviewed scientific journal in the area of nutrition and/or visual sciences. The intended readers for this review article are clinicians, health professionals, and visual and nutritional scientists. If the evidence supports a role for nutrition in eye health and disease prevention, dietary guidelines will be developed and communicated in an icon that will be intended for use by the general public.

Hypothesis: Scientific evidence to date supports a role for certain nutrients in the prevention or delay in progression of age-related eye diseases. The strength of the evidence allows for establishing guidelines or recommendations for nutrient/food choices that will be beneficial for eye health.

Objectives: The objectives of this proposal are two-fold:

  • Objective A: To review the scientific research that has evaluated the role of nutrients/foods in the prevention or delay in progression of age-related eye diseases. These diseases include age-related macular degeneration, cataracts and glaucoma.

  • Objective B: If the evidence supports a role, the second objective of this proposal is to define nutritional recommendations for eye health based on the literature review (see Objective A). These recommendations will be communicated in an icon intended for use by the general public. The nutrition/eye icon ("m'eyediet") will be developed using the expertise of various Tufts faculty including a nutritional biochemist with a background in vision research.

PI: Johnson, Elizabeth
Title: The Effect of Consumption of 2 Eggs/D for 6 Months on Cognitive Function in Older Adults
Abstract:
Cognitive impairment affects nearly one in four community-dwelling elders and is a major risk factor for development of dementia later in life. Cognitive impairment in the elderly is receiving increased attention because of the possibility that early intervention may prevent or delay progression to dementia. Findings from our studies suggest that the xanthophylls, lutein (L) and zeaxanthin (Z), which benefit individuals with early stage age-related macular degeneration, may also be important in cognitive function in the elderly. We have previously reported eggs to be a highly bioavailable source of L and Z. Our study proposes to evaluate long-term egg intervention as a treatment strategy for age-related cognitive impairment which could possibly prevent the onset of dementia.

L and Z cross the blood brain barrier and exclusively accumulate in the macular region of the retina, where they are referred to as macular pigment (MP). In a study of healthy older adults, MP was found to be significantly related to cognitive tests that assessed processing speed, accuracy and completion ability. Our work in primates showed that retinal L and Z were significantly related to brain L and Z concentrations. MP is thus a biomarker for brain L and Z concentrations. In the Georgia Centenarian Study population we found that among the serum carotenoids, L had the strongest relationships with global cognitive function, memory, recall, retention, verbal fluency, and dementia severity. In decedents of the same study we found that brain Z concentration was significantly related to ante-mortem measures of global cognitive function, memory, verbal fluency and dementia severity after adjusting for age, education, sex and self-reported diabetes or hypertension. Brain L concentration was related to recall and verbal fluency, but the associations were attenuated after adjustment for covariates. We have also shown that L supplementation significantly improved verbal fluency scores in healthy older women. Our studies have shown that egg interventions can significantly increase MP in older adults. Based on the sum our findings, the next logical step will be to investigate the ability of L and Z in an egg intervention to influence cognitive function.

Specific AIMS:

AIM 1: To determine the effect of consuming 2 eggs/d for 6 months on cognition in adults (>50 y) with age-related cognitive decline and low MP. Tests will include measures of attention, memory, language and executive function.

AIM 2: To determine whether change in MP is predictive of the relative effectiveness (improvement in cognitive function measures) of egg consumption.

PI: Johnson, Elizabeth
Title: The Effect of Daily Consumption of Avocadoes for 6 Months on Cognitive Function in Older Adults
Abstract:
Cognitive impairment affects nearly 1 in 4 community-dwelling elders. It is a major risk factor for dementia in late life. Oxidative stress and inflammation are thought to be risk factors in age-related cognitive decline. Our studies suggest that increased lutein status as measured by serum lutein and macular pigment (MP) may be important in cognitive function in the elderly. Lutein is an antioxidant and anti-inflammatory agent. Avocados are a significant source of lutein. Our study proposes to evaluate long-term avocado intervention as a dietary strategy for age-related cognitive impairment which could prevent the onset of dementia by increasing lutein status.

Hypothesis:

Consumption of one avocado a day for 6 months will result in improved cognitive function in older adults (>50 yrs.) compared to consumption of an isocaloric amount of potatoes or chick peas. The improved measures of attention, memory, language and executive function will be related to increased measures or lutein status (concentrations in serum and macula) and anti-oxidant status and decreased markers of inflammation.

Objectives:

  1. To determine the effect of consuming 1 avocado/d for 6 months on cognition in adults (>50 y) with age-related cognitive decline and low MP density. Tests will include measures of attention, memory, language and executive function.

  2. To determine whether change MP density is predictive of the relative effectiveness (improvement in cognitive function measures) of avocado consumption.

Approach:

The proposed study is a randomized, placebo controlled trial that tests the effects of 6 mo. supplementation with 140g avocados/d on cognition in older adults. Secondary analyses will determine if baseline MP density predicts relative effectiveness of the intervention on cognition. Secondary outcomes include biomarkers of oxidative stress and inflammation. Subjects will be recruited from community-dwelling men and women aged >50 y, and potential subjects will be screened to meet cognitive and functional criteria. Participants will be pre-screened by telephone; those who seem to meet criteria will undergo further screening. The study will include baseline, 3 and 6 month visits.

PI: Joyner Koomson, Valencia
Title: 3D Integrated 80Gb/s SiGe Heterojunction Bipolar Electroabsorption Modulator
Abstract:
The objective of this research is to create a new class of Si-based electroabsorption modulators (EAM) that operate at an unprecedented bandwidth of 80Gb/s. The approach is to use 3D wafer bonding technology to vertically couple light into a nanowire high-index contrast silicon waveguide. A graded base SiGe heterojunction transistor is the baseline electric structure of the modulator, while the free carrier plasma effect is used to generate optical absorption modulation. 

For high capacity fiber-optic systems, a Si-based, monolithically integrated fast EAM is an appealing alternative technology to commercial III-V compound semiconductor based modulators. The proposed research will explore the carrier transport phenomena and its corresponding optical attenuation effect in a SiGe heterojuction transistor, as well as various design trade-offs among modulation depth, signal dispersion, operation speed, power consumption, and local heating effects. The proposed work will design a fully integrated modulator driver circuit using unmodified, silicon fabrication processes. 

PI: Joyner Koomson, Valencia
Title: CAREER: Wireless Optical Sensors for High Resolution Imaging of Biological Structures
Abstract:
Near infrared (NIR) spectroscopy is emerging as a promising non-invasive imaging tool for fundamental studies of biological processes and structures, offering greater biochemical specificity, high temporal resolution, potential for concurrent intracellular and intravascular event measurement, and portability. Time-resolved NIR techniques allow explicit separation of optical absorption and scattering parameters related to biological structures, such as tissue, and (in theory) provide functional and metabolic information based on spectral and spatial imaging information. However, the visibility of superficial and deep structures remains fairly poor due to the lack of imaging sensor technology combining high-resolution spatial mapping, fast pixel response time, and broad spectral response.

The goal of this CAREER program is to develop a new class of highly integrated wireless imaging sensors, combining photonic devices, broadband analog/RF circuits, and free-space optical communication to improve the spatial resolution of time-resolved NIR images; and establish an interdisciplinary educational environment for engineers. The long-term goal is to further expand the field of biological imaging by developing true mixed-mode integrated systems combining microwave, acoustic, photonic, and nanoscale electronic circuits for concurrent measurement of multiple imaging modalities to increase the visibility of sub-millimeter structures.

Mentor: Juo, Peter
Fellow: McGehee, Annette
Title: Regulation of Glutamate Receptor Trafficking by TGF-Beta Signaling in C. elegans
Abstract:
The goal of this proposal is to understand the mechanism by which the DAF-7/TGF-beta signaling pathway regulates the abundance of glutamate receptors (GluRs) at synapses in C. elegans. The regulation of GluR abundance and localization can be controlled by activity, and has been implicated in learning and memory. Excessive transmission through GluRs is linked to excitotoxic nerve death following stroke and trauma. Thus, understanding the cell biological mechanisms that regulate GluRs is of significant interest. I have preliminary data indicating that multiple mutants in the DAF-7/TGF-beta signaling pathway have increased abundance of the C. elegans GluR GLR-1 at synapses in the ventral nerve cord (VNC). TGF-beta signaling regulates multiple cell biological and developmental processes in many organisms, including several aspects of neuronal development. My preliminary data identify a novel function for the DAF-7/TGF-beta pathway in regulating GluRs, and suggest that an extracellular factor (TGF-beta) is required to maintain GluR levels in the mature nervous system. This proposal will test the hypothesis that the DAF-7/TGF-beta signaling pathway acts cell autonomously in interneurons of adult animals to specifically regulate the abundance of GLR-1 in the VNC (Aim 1). I will determine the mechanism involved by testing whether the DAF-7/TGF-beta pathway affects GLR-1 transcription and/or various receptor trafficking steps (Aim 2), and whether the increased abundance of GLR-1 in the VNC has functional consequences for behavior (Aim 3).

PI: Kaplan, David
Title: BioComponent Robot System
Abstract:
This proposed work is to develop BioComponent systems for use in mobile robots and manipulators. The overall goal is to exploit the powerful capabilities of biological systems to assemble and operate new types of moving devices. Biological materials and living cells have extraordinary potential for self-assembly (from molecular structures to integrated devices), they are powered by energy-dense, safe, hydrocarbons such as lipids and sugars and are both biocompatible and biodegradable making them a potentially "green" technology.

The primary components that are needed are:

  1. actuators (synthetic or living muscles),
  2. a mobile body structure (built from biopolymers in any desired configuration),
  3. a supply of biofuel (the appropriate mobilization of glucose or lipid reserves),
  4. sensors and controls.

These new technologies should also provide additional capabilities that are difficult to achieve with traditional robotics including silent operation and the possibility of extreme compliance and conformability in different environments. Because such robots could be built (or grown) on a massive scale from simple, inexpensive raw materials using parallel fabrication and replication methods, they also have potential in tasks that require disposable devices or swarm-like interactions.

PI: Kaplan, David
Title: Bioinspired Composites of Nano Crystalline Cellulose (NCC) and Polymeric Proteins
Abstract:
Bioinspired material systems are derived from different living organisms such as plants, arthropods, mammals and marine organisms. These biomaterial systems from nature are always present in the form of composites, with molecular-scale interactions optimized to direct functional features. These interfacial features are critical to the development of advanced biomimetic composites with diverse mechanical and other functions.

Herein, we propose a novel approach that employs sustainable raw materials to fabricate nano-structured composite biomaterials made of NCC high porous foams with polymeric proteins such as silks and resilin with elite physical properties.

The general significance of this proposal is on development of bioinspired composite materials with molecular-scale control of properties, as replacements for synthetic materials. Its particular significance to the field of regenerative medicine is in the development of durable composite foams from NCC, silks and resilin for tissue engineering of physically stressed tissues which currently lack proper solutions. Development of these composite foams will be beneficial for improved health of many patients due to the relatively low cost and minimally invasive procedures that will save hospitalization time, post surgery morbidity and wide distribution across hospitals and medical care centers.

PI: Kaplan, David
Title: Biomaterial Applications of Recombinant Bacterial Collagens
Abstract:
There is a need for well-defined standardized collagen proteins amenable to easy sequence modifications and large scale production levels for biomedical and biomaterial applications. This proposal aims to express recombinant bacterial collagens in high yield in E. coli to use both as a basic science tool for characterizing biologically active collagen domains and as a scaffold to promote bone generation by human stem cells. Bacterial collagen proteins expressed from S. pyogenes and four other bacteria in high yield have been shown to form triple-helical molecules of high stability despite the absence of hydroxyproline. Higher order structural aggregates formed by bacterial collagens will be characterized and cross-linked for stability, and the mechanical properties of materials formed will be characterized. Collagen variants will also be designed to contain a trimmer coiled coil adjacent to the bacterial collagen-like domain to expand the opportunities for increased molecular stability, formation of heterotrimeric molecules to foster protein association, and the inclusion of specific cell binding and matrix metalloproteinase (MMP) cleavage sites in order to better define collagen cell receptors and control turnover of the protein. The ability of tailored bacterial collagen proteins to support growth and differentiation of human bone marrow derived mesenchymal stem cells as potential biomaterials for bone replacement will be investigated. Bacterial collagens with and without inserted biological signals will be compared with extracted mammalian collagens using a range of osteogenic phenotypic and genotypic markers. In addition, inflammatory responses to the various collagens will be compared in vitro and in vivo to animal collagens using macrophage and dendritic cell screens, and subcutaneous implants in mice. These comparisons will provide baseline data on biological responses at the cell, tissue and animal level to the collagen variants. High yields and straightforward genetic manipulations in this system will allow combinations of different biologically active sites and permit interactive optimization to design biomaterials of appropriate structural and biological properties to fine tune stem cell responses related to basic cell biology and applied biomaterials and tissue engineering needs.

The interdisciplinary team of collaborators brings important complementary expertise to make this project successful, with Dr. Brodsky's extensive work on collagen structure, Dr. Kaplan's track record with stem cells and biomaterials, Dr. Ramshaw's experience with collagen, biomaterials and product development, and our consultants’ expertise in matrix metalloproteinases (Dr. Nagase) and DDR receptors (Dr. Leitinger).

Mentor: Kaplan, David
Fellow: Amy Thurber
Title: Craniofacial Tissue Regeneration Using Membrane Potential to Control Stem Cell Differentiation
Abstract:
Regeneration of craniofacial tissue provides one means to restore function to a disease- and injury-free state. Biochemical factors have successfully been used to differentiate stem cells to many cell types. However, little progress has been made in being able to spatially pattern tissue. Recent evidence indicates the ability to use bioelectric signaling as an alternative method to direct stem cell differentiation. Due to advances in specialized tools, it is now possible to spatially and temporally control bioelectric stimulation. The hypothesis of this project is that membrane potential (Vmem) is an instructive differentiation signal capable of inducing specific molecular pathways depending on the precise Vmem. Inducing tissue regeneration with Vmem stimulation could profoundly impact oral and maxillofacial defect treatment by for the first time enabling spatial patterning of hard and soft tissues.

The goal of this project is to develop a system to screen modulation of Vmem for the ability to induce stem cell differentiation to a specific cell type and to study the mechanism behind Vmem regulation. The focus will be on the differentiation of human dental pulp stem cells (hDPSCs) to osteoblasts and adipocytes, two important craniofacial cell types.

The first aim of this project is to develop a screen to identify beneficial Vmem for differentiation to particular cll types. The screen will require reproducible induction of Vmem and analysis of differentiation. Electrophysiological recordings from hDPSCs will be used to correlate media ion concentrations with induced membrane potentials. Currently available differentiation marker fluorescent protein (FP) reporter constructs will be validated as a means to monitor hDPSC differentiation. As a proof of principle, FP expression in hDPSCs cultured in decreased biochemical induction factors known to affect differentiation will be observed. It is expected that changes in FP expression will correlate with the effects on differentiation and provided similar resolution of monitoring differentiation as other forms of analysis, such as qPCR, but with the added benefit of being nondestructive.

In aim 2, the screen will be used to identify membrane potentials beneficial and instructive to osteogenic and adipogenic differentiation. Hyperpolarizing and depolarizing membrane potentials will be induced and changes in FP expression will be used to identify effects on differentiation in media with or without biochemical induction factors. Changes in differentiation molecular pathways will be investigated to identify Vmem signal transduction mechanisms. The outcome of this aim will provide evidence of the utility of using bioelectric signaling to induce differentiation of specific cell types. This work will enable future development of patterned tissue regeneration for the purpose of craniofacial reconstruction and other clinical applications.

PI: Kaplan, David
Title: Electrogelation for Reversible Silk Adhesives
Abstract:
Novel silk-based reversible adhesive systems have been identified. These systems are aqueous-based, function in a range of environments, require activation by low voltage electric fields but are stable in the subsequent absence of the field, and can then be deactivated and returned to a liquid state if the electric field is reversed. The system is robust on many surfaces, can carry significant loads and has many technological and medical implications. In the seedling, we will focus on exploiting this new adhesive system to meet the requirements for Zman (i.e. human climbing on glass and painted aluminum). Thus, we will interact with the Draper team to optimize features of the silk electro gelation process to determine compatibility with the needs of Zman, including reaction conditions, time, surfaces, shear effects, compatibility with Zman-developed micro- and nanostructures, and related needs. Samples will also be provided to continuously garner feedback on needs and fits to the program. Tufts will coat Zman structures provided by Draper with e-gel and both test them in our laboratory and provide samples to Draper for testing.

PI: Kaplan, David
Title: Electrogelation of Biopolymers for New Functional Materials
Abstract:
In recent studies we have gained extensive insight into both the fundamental mechanistic behavior of silk protein self-assembly into organized structures, as well as various applications for these assembled materials. We have extended our understanding of this novel protein-based polymer system into new material formats, such as ultrathin coatings, as well as into control of gel states. In this renewal application to AFOSR, we plan to build upon our prior studies, and in particular our recent observations on electrogelation (e-gel formation) with silk proteins as a novel way to control surface gelation and reversible material features, in order to generate a new family of functional biopolymer-based high performance materials. We anticipate that silk initially will serve as a useful material toward this goal, while in the longer term it can provide a model system upon which to design and implement new synthetic strategies that exploit the self-assembly and functional features of these systems.

To address the overall plans we will exploit our knowledge base on silk protein polymers as a model polymeric system that offers precise and extensive control over polymer chemistry, polymer block design and assembly, all leading to new paradigms in polymer science and windows into a new generation of functional high performance materials. Thus, our model system will initially consist of native silkworm fibroin to provide the polymer building blocks and components for the planned studies. We will exploit these proteins as outlined below towards the formation of e-gel based hydrogel surfaces and materials with new functions, and develop a fundamental understanding for the basis for e-gel as a route to a broader materials base. For the latter, we will use a larger base of biopolymers towards the goals of the study. A variety of characterization tools will be employed to assess the mechanical, thermal, and structural features of these new materials.

Aim #1: E-gel System for New Functional Materials – The goal of this aim is to exploit e-gel controlled assembly of biopolymer systems (silk, silk-elastin block copolymers, others) to study material features. The goal will be to elucidate the role of environmental factors (e.g., pH, salts, second polymers, voltage, time, molecular weight) on the dynamic nature of e-gel properties – both during e-gel formation and related to reversibility. We will initially use silk as the model system as the Preliminary Studies demonstrate suitable function. We will then expand the biopolymer base towards other systems – silk-elastin block copolymers, then collagens and other biopolymers. Options for entrapment of second polymers, small molecules, enzymes and dyes will be pursued as part of this aim. The material features will be assessed using a variety of analytical tools including FTIR, TEM, polarized optical microscopy for structure and DMA for properties and adhesion. The outcome of this Aim will be a series of phase diagrams that describe the nature of environmental and experimental variables on e-gel formation, properties and reversibility features.

Aim #2: Dynamic System to Evaluate E-gel Properties – The goal of this aim will be to develop and utilize the dynamic nature of e-gels as a route to forming and studying dynamic surfaces. Towards this goal, we plan to design and implement a novel materials assembly platform with which to study these properties. The experimental platform will allow us to track and characterize e-gel formation in a dynamic fashion during formation, growth and then reversal with a range of variables. The outcome of this dynamic interface and assembly platform will provide insight into the potential utility of e-gel in a range of dynamic material surfaces for potential applications in future Air Force needs – as in self-repairing materials, shedding surfaces, dynamic sensors, remote adhesion, underwater adhesion and related needs.

Aim #3: E-gel Mechanisms – The goal of this aim is to develop a solid understanding of the mechanisms related to e-gel formation and reversibility as a route to better control and utilization of this material assembly technique – related both to the hydrogelation mechanism and the adhesion function. Towards this goal, we will start with our rudimentary understanding from the Preliminary Data, including slight increases in helical structure, a decrease in pH, and likely charge-interactions, towards a more robust understanding of the role of polymer chain dynamics, organization and adhesion mechanisms on different surfaces. The outcome of this Aim will be a solid foundation of understanding of the mechanisms involved in e-gel formation, leading to improved control and new applications for the process. Further, we will expand on the utility of the e-gel systems, based on the mechanistic understanding, towards new material processing routes – e.g., novel spinning techniques and other types of materials formation.

PI: Kaplan, David
Title: Electrotherapeutic Strategies for Connective Tissue Repair
Abstract:
The role that biophysical forces play in regenerative medicine is expanding, with increased interest in the use of intrinsic electrical forces (via regulation of cell membrane channels) and externally applied electric fields (via bioreactor environments) as important control points. Despite significant potential of electrical signals for regenerative medicine, they have not yet been integrated into the design of tissue engineering systems. We propose a radically new strategy to improve connective tissue regeneration by electrotherapeutic control of cell function, through the integrated use of molecular and electrical control of cell function and tissue formation. Our hypothesis is that the synergistic application of molecular control of transmembrane ion flux and externally applied electric fields will improve the quality of cartilage and bone regeneration and accelerate their integration in vivo. We will rigorously test this hypothesis by studying the regeneration of composite bone/cartilage grafts. The regulation of cell function and tissue regeneration will be first studied in vitro using controlled bioreactor environments, and then in vivo in an orthotropic animal model of cartilage and bone regeneration. Three specific aims will be pursued: (a) Biophysical regulation of chondrogenesis and osteogenesis in adult human stem cells, (b) Electrotherapeutic bioreactor models for regeneration of cartilage/bone tissues, and (c) Animal studies of cartilage/bone regeneration. The anticipated scientific impact will be in significant new insights into the biophysical control of connective tissue repair by modulation of electrical regulatory signals. The main technological impact will be in improved regeneration of cartilage/bone tissues, and the new generation of electrotherapeutic medical devices termed BioDomes.

PI: Kaplan, David
Title: In vitro Bioreactor System for Platelet Formation
Abstract:
Millions of platelet transfusions are conducted each year, yet the supply of this blood component is limited, thus patient access to treat disorders is problematic. There are also many diseases where platelet production or function are impaired, resulting in severe consequences and where there are limited clinical options available. To address these current limitations, new modes to generate functional platelets in vitro would provide a major benefit to many patients, as well as provide an approach to permit the systematic investigation of mechanisms involved in functional platelet formation. Our goal in this project is to build upon our recent successful studies where a novel bioreactor system was engineered to house megakaryocytes (Mks) and to generate functional platelets in vitro. We will exploit this system to address our hypothesis; engineered microenvironments in vitro can be tailored to optimize the formation of functional platelets. To address the needs in the program we will: (Aim 1) establish a bioreactor-based 3D tissue system to study the mechanisms of Mk development and platelet release, with functional outcomes in terms of functional platelets generated and recovered from the system, and (Aim 2) to use this 3D tissue system to investigate mechanisms of platelet production related to biochemical signalling and environmental components (i.e. matrices, oxygen tension) as well as pathological megakaryopoiesis by including Mks from patients with platelet related diseases. With insight from this system we will be positioned to interrogate the maturation of Mks from both normal vs. diseased sources in order to begin to establish differences in Mk outcomes (adhesion, migration, proplatelet formation, platelet production and function).

The outcome for the proposed study would be twofold: (1) a new laboratory model for Mk development, proplatelet formation and platelet release in normal and abnormal (disease) states, and (2) mechanistic insight into these processes. In the long run, building upon this new in vitro tissue system would allow for the more systematic understanding of the processes involved in Mk development, as well as insight into modes to intervene in disease states associated with these cells.

Mentor: Kaplan, David
Fellow: Burke, Kelly
Title: In vitro Type Two Diabetes Mellitus Tissue Model to Investigate Insulin Resistance
Abstract:
The goal in the project is to develop a physiologically-relevant 3D tissue system for type two diabetes mellitus (T2DM), a disease state hallmarked by insulin resistance. While adipose tissue is not the primary site for glucose disposal in humans, glucose uptake, secretion of adipokines, and lipolysis are all altered in T2DM. Increased lipolysis in insulin resistant adipose tissue can directly contribute to insulin resistance in liver and skeletal muscle through secretion of free fatty acids (FFAs) that activate pathways known to disrupt insulin signaling. Animal models have given great insight to both obesity and T2DM, but there are several important differences between human and rodent adipose tissue function that necessitate the development of a relevant in vitro model derived from human cells. 3D tissues are proposed to be essential for in vitro disease models, but these studies have not focused on adipose tissue. It is not known if a 3D culture will better represent in vivo adipose tissue than 2D, but it is hypothesized that endothelial cells will polarize better in 3D than 2D, and that 3D is necessary for subsequent organization into lumens. The 3D tissue is also hypothesized have enhanced cell-extracellular matrix (ECM) interactions from a greater amount of ECM that may be deposited around the cells.

Aim 1 will investigate 2D and 3D co-cultures of human adipocytes with endothelial cells to establish baseline differences in structure and function of the co-cultures in the different geometries.

Aim 2 will add complexity to the model by incorporating human fibroblasts and monocytes, which may activate to M1 macrophages to create a pro-inflammatory environment like that found in obese adipose tissue. The cultures will be characterized by quantifying DNA, relative gene expression by qRT-PCR, secreted proteins by ELISAs, triglyceride accumulation and lipolysis using colorimetric assays, relative protein expression by Western blot, and energy use by glucose and lactate assays. Microscopy (light, fluorescence, confocal) will be used to examine cellular organization and adipocyte size and lipid accumulation. Functional responses to hormones will assess physiological responses.

Aim 3 will expose the 2D and 3D cultures developed in Aim 2 to insulin and FFAs, two stimuli elevated in obese type 2 diabetics, and proteins that alter ECM remodeling. The tissue responses to the T2DM stimuli and the altered remodeling conditions will be compared separately first and then together. Activation of insulin signaling pathways known to be affected by FFAs and inflammatory cytokines will also be examined using Western blot to determine how pathway activation may change in response to these stimuli and with geometry.

The proposed work will thus advance understanding of adipose tissue engineering by developing a new 3D in vitro model for inflamed adipose tissue, directly comparing structure and function of 2D and 3D cultures, and by understanding how insulin signaling is impaired in response to stimuli found in obese type 2 diabetics and may change in 2D and 3D models and with altered matrix remodeling.

PI: Kaplan, David
Title: Living Membranes as Environmental Detectors
Abstract:
The principle goal of this proposed research is to exploit the capability of cellulose producing bacteria to provide a novel living membrane for biologically-mediated rapid and specific detection of chemical and biological contaminants. We will accomplish these goals by completing the following two specific aims:

  1. Coupled synthesis and processing of living membranes: To examine the co-cultivation of the cellulose producing organism Gluconacetobacter xylinus with recombinant Escherichia coli strains to determine the growth kinetics and viability of both organisms under a variety of environmental conditions (oxygen tension, carbon sources, agitation) and to elucidate the three dimensional structure and composition of resulting polymeric/cellular material. Of specific interest will be the secretion of fusion reporter protein constructs recovered on the membrane directly. This goal is significant because it will establish the fundamental concept of co-cultivation with these novel living membranes leading to an expanded range of membrane functions. Thus, an ability to combine membrane formation (G. xylinus) with inducible fusion reporter protein expression will be studied. With this concept established, future efforts can address the direct incorporation of organisms to target specific chemical and biological contaminants.

  2. Development of recombinant detection suite: To develop a platform E. coli strain which can be modified to recognize specific chemical and biological contaminants. Most pathogens and chemical agents have well-defined cellular activities that can be utilized to detect their presence. By generating fusion receptors based on these cellular actions, we will generate a chimeric signaling pathway by which the recombinant E. coli strain responds to the specific stimulus with rapid and high levels of reporter gene expression.

The living membrane systems described are envisioned for application in a forward operating area by members of the armed forces with limited or no scientific training. The reading of these devices will be a simple binary yes or no as determined by the presence or absence of a fluorescent signal. The use of bacteria in a lyophilized membrane will allow long-term storage in a variety of conditions, with a minimal time required to rehydrate the device prior to field use.

PI: Kaplan, David
Title: Multifunctional Tropoelastin-Silk Biomaterial Systems
Abstract:
New multifunctional, degradable, polymeric biomaterial systems are needed that can be tailored to specific cell and tissue needs in vitro and in vivo. While protein-protein composites dominate tissue structure and function in our bodies, we have been unable to recapitulate the complexity and control of such systems in vitro for new biomaterials in order to direct cell and tissue functions. For example, material systems that can form mechanically robust and durable biomaterials to give highly flexible and dynamic biomaterials, remains a challenge.

Our goal is to construct a panel of composite protein biomaterials that can cover a range of physical properties, to mimic the elasticity of diverse tissue structures and the consequential ability to control biological function – such as to direct stem cell responses. The hypothesis is that combinations of a highly elastic and dynamic structural protein (tropoelastin) with tough, durable proteins (silk) will generate new multifunctional protein composite systems that can offer a broad platform of utility to the biomaterials field. We propose to generate a new family of highly controllable composite fibrous protein systems, based on combinations of these two well-established structural proteins, tropoelastin and silk, both biodegradable protein polymers with good biocompatibility. These proteins encompass a range of biomaterial needs; tropoelastin provides highly flexible and dynamic structural features, silk provides mechanical toughness and slow degradation. Our findings of molecular-scale interactions between these two structural proteins, forms the basis of the present proposal.

The experimental plans are focused on: (a) further elucidation of the mechanistic interactions between tropoelastin and silk to optimize control of material structure and function, (b) assessment of cell interactions for the range of materials generated to understand relationships between the protein composites, material compliance and cell outcomes, using hMSCs and cortical neurons, and in vivo screens of material degradation profiles and inflammatory responses, and (c) exploitation of the dynamic material properties achievable with these systems towards support of cell functions in vitro. The experimental plans are supported by extensive preliminary data that demonstrate our ability to generate the required materials (tropoelastin, silks), to functionalize the materials (e.g., surface chemistry) to probe the interactions among the two components with mechanistic insight, to process the proteins into new material formats, and to direct cell outcomes on these materials with outcomes dependent on the composition.

The overall outcome from the plans would be a new protein composite biomaterials platform that would fill an important need in the field of biomaterials, with direct relevance to tough but flexible systems and strong, durable systems.

PI: Kaplan, David
Title: Tissue Engineering Cornea Replacements
Abstract:
Corneal diseases are responsible for extensive vision loss throughout the world, second only to cataracts. A number of sources are currently available for cornea replacement and include allogenic and synthetic materials. However, each of these options has problems, including disease transmission, inflammatory responses post implantation and poor material performance that may result in tissue rejection or device failure. These concerns are compounded by the growing demand for corrective surgery which renders would-be donor corneas unsuitable for grafting. Thus, a crucial need exists to develop new cornea replacement devices that provide the required material and biological properties to address the above limitations, while also offering an integration strategy that allows the device to be replaced by the patients' native tissues.

To address these critical shortcomings, we propose a tissue engineering approach in which a novel combination of biomaterials and cells will be utilized. The system for study consists of silk protein micropatterned films organized and engineered to match the morphology, mechanical properties and biological needs of the cornea. The proposed system will exploit the novel material features of silk (e.g., biocompatibility, slow in vivo degradation, transparency and durability). In combination with human corneal cells, a 3D stacked lamellar cornea tissue system will be constructed that will match all of the functional performance requirements of a human cornea. The hypothesis is that thin film micropatterned silk protein lamellar systems coupled with human corneal cells can be bioengineered to match all cornea functional requirements and thus provide a novel, highly compatible source of urgently needed human cornea replacements. The outcome of the proposed program will be the in vitro optimization of human cornea material-cell systems followed by in vivo transplantation and assessment.

PI: Kaplan, David
Title: Tissue Engineering Resource Center
Abstract:
The P41 Tissue Engineering Resource Center (TERC) has established innovative biomaterials, bioreactors, and tissue engineering models since its inception in August 2004. The core themes for TERC continue to evolve along with the scientific and technological progress. Initially the focus was on functional tissue engineering achieved through the integration of the key elements in the field via a systems approach — cells, scaffolds and bioreactors. This renewal continues this path, with an expanded impact for the Center themes in areas of clinical relevance, disease models, and research tools applicable to biological inquiry. In the renewal plans, we maintain a focus in two critical areas: (a) skeletal systems and (b) cardiovascular systems, while progressing toward new impact for the underlying fundamentals and translational strategies. The common aspects for both areas (skeletal tissues, cardiovascular systems) include: cell sources, genetic tools, imaging (molecular, cellular, tissue levels), biomechanics (from cells to tissues), modeling (computational biology, transport, electrical and mechanical signal transduction) and the use of animal models, with strong focus on advancing biological research and translation into medical applications. The Center will continue to be led by two long-time collaborators, David Kaplan at Tufts University and Gordana Vunjak-Novakovic at Columbia University, who also head the two cores in the Center (biomaterials, bioreactors). A large group of faculty and collaborators will contribute the activities in the Center, and a stellar Scientific Advisory Board will continue to provide critical guidance to the Center.

PI: Kaplan, David
Title: Tissue Regeneration by Biophysical Signaling
Abstract:
Traditional approaches to tissue engineering have focused on biochemical cocktails to direct cells toward tissue-specific outcomes; in some cases mechanical forces have also been utilized. However, there is also a significant literature that details the role of biophysical signaling during tissue development and tissue regeneration, which has not yet been incorporated into the field of tissue engineering to date. The field of developmental biology has tracked the role of biophysical factors, such as membrane voltage potential and ion fluxes, during tissue regeneration, in wound healing, in embryonic patterning, and in many other critical tissue-related events. These data provide a clear link between membrane potential and cell behavior that determine tissue-specific outcomes. However, many molecular details are still unclear and this novel cell control modality has not been capitalized upon to advance tissue regeneration. The focus of the present proposal is to fill this void by specifically studying biophysical regulation of bone and adipose tissue regeneration, development and patterning. We will utilize 3D human tissue systems for bone and adipose tissue. The goal is to determine the utility of biophysical factors, such as membrane potential, on tissue-specific outcomes in the context of tissue regeneration in Aim #1, tissue development in Aim #2, and tissue patterning in Aim #3. We will compare the role of membrane potential during tissue regeneration and formation to the use of traditional biochemical cocktails as the controls. In the last aim, we will focus on spatial control of tissue outcomes via light-activated regulation of ion transport, mediated via a 3D optically-addressable scaffold system, to generate tissue patterns in vitro, analogous to morphological control during limb development. The outcome of the proposed study will be an entirely new approach to the regulation of tissue formation in vitro, with implications in many areas of regenerative medicine. Understanding and exploiting the role of bioelectrical signals on tissue outcomes in non-excitable cells will provide new insight into fundamental control of tissue regeneration, as well as novel approaches toward generating complex pattern development in tissues both in vitro and in vivo.

PI: Kaufman, Gretchen
Title: Determining Disease Risk from Elephant Endotheliotropic Herpes Virus in Breeding Elephants in Nepal
Abstract:
Elephant endotheliotropic herpesvirus (EEHV) is a growing threat to the health and viability of the Asian elephant population worldwide. The disease is characterized by sudden onset of lethargy, edema of the head, proboscis and limbs, oral ulcers, and internal hemorrhaging. If left untreated, the infection will result in death within one week of the onset of symptoms. There is currently no knowledge of the prevalence of EEHV in Nepal, a country within the natural range of wild Asian elephants. However, in recent years, several calves have succumbed to unconfirmed but presumed herpesvirus.

The objective of this project is to determine whether latent EEHV infections are present in the captive breeding herd at the elephant breeding center in Chitwan National Park, Nepal. Conjunctival swabs will be collected twice per week for 4 weeks from 15 elephants. Serial trunk wash samples will also be collected when possible from selected animals and compared with conjunctival samples from the same animal. Polymerase chain reaction (PCR) analysis will be performed to determine whether or not EEHV DNA is present in the samples. If EEHV DNA is detected, positive samples will undergo DNA sequencing to determine which strains of EEHV are present in the breeding herd.

This project is the first of its kind in Nepal. It will assist in understanding the prevalence of EEHV in the captive breeding herd and will lead to recommendations for management of this disease to minimize morbidity and mortality in captive animals in Nepal. It is expected that this work will put in place the capacity for local EEHV diagnosis and will lead to future investigations into disease occurrence and dynamics in captive and wild Asian elephants in Nepal.

PI: Khardon, Roni
Title: RI: Medium: Collaborative Research: Optimizing Policies for Service Organizations in Complex Structured Domains
Abstract:
This project studies an important class of complex structured planning domains called “service domains” using simulators and probabilistic action models. Examples of service domains include optimizing emergency response in a typical city, scheduling doctors and nurses in a hospital, administering tasks in a typical office, optimally delivering products to shops from distribution centers. These domains share many characteristics such as relational structure, parallel actions, multi-time-scale decision making, exogenous events, and the need for human interpretable solutions that make them highly challenging. The project develops scalable and principled planning algorithms for service domains through a variety of techniques including a novel hierarchical framework of multi-time-scale optimization, new model-free simulation-based planning algorithms, and model-based planning via composition of first-order decision diagrams. These techniques are applied to the real-world problem of optimizing the fire and emergency response in cities through a collaboration with the fire department of Corvallis, Oregon.

The results of the project include new algorithms and frameworks to solve service domains, prototype implementations of the algorithms in the emergency response domain, and new testbeds of service domains for research. The broader impact of the work includes more cost-effective emergency response systems, and development of new research-oriented courses, tutorials and special workshops on the next generation decision support systems for service domains.

PI: Kilmer, Misha
Title: Collaborative Research: Innovative Integrated Strategies for Nonlinear Parametric Inversion
Abstract:
The investigators aim to reduce drastically the costs of numerical inversion (as occurs, for example, in medical imaging) by blending new parametric level-set approaches and nonlinear least squares methods together with innovations in linear solvers, preconditioning, and model reduction of parameterized systems. Four strategies are combined to reduce the computation necessary while not degrading accuracy of the solution. First, the dimension of the inverse problem is drastically reduced by developing low-order parametric inversion methods replacing the usual voxel-based inversion. Second, the optimization underlying parametric inversion incorporates novel nonlinear least-squares solvers specifically designed to deal with ill-conditioned Jacobians. Third, the high cost of solving many large forward problems is reduced through new model reduction techniques that are particularly well-suited to the structure of the inverse problems under consideration. Fourth, the high costs of computing reduced models and solving forward problems is reduced by innovations in Krylov subspace recycling and efficient reuse of preconditioners for parameterized linear systems.

The inverse problems studied here involve recovery of images describing how unknown quantities of diagnostic interest (such as electrical conductivity) are distributed throughout a given medium (such as human tissue or soil). These images can reveal the presence or absence of anomalies, such as tumors in human tissue or contaminant plumes in soil. The computational extraction of high quality images from noisy surface measurements in reasonable time is a very difficult task. As rapid advances in technology make it possible to take vastly more measurements, computational bottlenecks become ever more acute, impeding innovation in medical and other areas of imaging.

This project aims to combine innovations in diverse fields within computational linear algebra, systems theory, and optimization to create dramatically improved strategies for image extraction.

PI: Kirker-Head, Carl
Title: Tendon Coating Scaffold in a Rabbit Tendon-Bone Model
Abstract:
The objective of this study is to determine if a calcium delivery device consisting primarily of calcium sulfate (CaSO4) and collagen with two different CaSO4 concentrations affects the biomechanical healing properties of a tendon to bone healing in a bone tunnel.

PI: Kounaves, Samuel
Title: Collaborative Research: Development of a Sensor Array for In-Situ Real-Time Measurement of Deep Ocean and Hydrothermal Vent Chemistry
Abstract:
The study of dynamic biogeochemical processes in the ocean, especially in the deep ocean or near hydrothermal vents, is a daunting challenge and requires the ability to monitor a variety of chemical species in-situ in real time under extreme conditions. Understanding these dynamic processes is critical for our ability to predict and/or mitigate the long term effects of natural and human impacts on the oceans. I propose to develop and demonstrate a prototype ion selective electrode (ISE) sensor array capable of providing simultaneous in-situ real-time measurements of a variety of ionic chemical species at extreme depths and near vent temperatures in the ocean. The proposed sensor array will be based on the ISE sensor array that was designed, built, tested, and successfully used on the surface of Mars as part of the 2007 Phoenix Mars Mission, to analyze for ionic species in an aqueous solution containing a soil sample.

The longer term objective of this initial effort is to enable follow-on research that, with improved detection limits and selectivity, will provide the ocean sciences community with a new tool for in-situ real-time mapping of a broad range of chemical species in seawater. The resulting sensor array device will also be of use to researchers in a variety of related disciplines for studies or monitoring of other bodies of water such as lakes, estuaries, ground water, drinking water, and rivers.

PI: Kounaves, Samuel
Title: In situ Wet Chemical Analysis Laboratory and Sensor Array
Abstract:
The recent success of the Phoenix MECA Wet Chemistry Lab (WCL) emphasizes the need for additional chemical analyses to assess the soluble species in the soil which cannot be attained solely through in situ elemental analysis or orbital measurements. The WCL on Phoenix evaluated a limited set of analytes and occupied more than 80cc of volume per sample analyzed. Future rover missions will need to provide for the analysis of many more samples and the addition of reagents without a corresponding size increase.

This proposal leverages the heritage and previous NASA funded work on the WCL, RCAL, and other instruments, to create a novel design for high density arrays of ISE sensors, and a new low volume and low mass wet chemistry lab for planetary focused chemical analyses and science. The objective of this research is to develop and demonstrate at TRLG an instrument that will provide the aqueous chemistry of surface materials on Mars or the inorganic ionic species in melted ice on Mars, Europa, Ganymede, Enceladus or other planetary bodies.

The system will significantly improve the current instrumentation by adding enhanced sensor technology and an advanced multi-sample handling system. Major goals for this project are: the development of an improved hyper-redundant sensor array with advanced algorithms for rapid and accurate chemical analysis; the integration and reduced scaling of the WCL system so it is capable of analyzing more samples and more analytes per sample to improve the richness of the data returned on a given mission; to demonstrate that it can be integrated with an automated sample preparation system to provide a "robotic" chemistry laboratory system with a capability to obtain reliable and accurate real-time chemical analyses under realistic conditions.

PI: Krautz-Peterson, Greice
Title: RNAi-Based Functional Analysis of Schistosome CD59-Like as Complement Regulators
Abstract:
This application proposes to functionally characterize a group of putative complement regulatory proteins (CRPs) in schistosomes to assess their potential as therapeutic targets by suppressing their gene expression through RNA interference (RNAi). Schistosomes are flatworm parasites that cause a chronic and debilitating disease afflicting more than 200 million people worldwide and causing more than 250,000 deaths/per year. These worms are well adapted to live in the vasculature of their vertebrate hosts for decades, and apparently have developed strategies to escape from the host's immune surveillance, including the potent complement system. We hypothesize that schistosomes express CRPs in their tegument (a syncytial membrane that covers the entire worm surface in direct contact with the host) as a mechanism of complement evasion.

Using genome and transcriptome analysis, seven putative schistosome CRPs with significant sequence homology to human CD59 were identified. These homologues are designated here as SmCD59-1, 2, 3, 4, 5, 6, 7. CD59 has broad distribution in human tissues. It is a potent inhibitor of the pore-forming membrane attack complex (MAC or C5b-9) through interaction with the final complement components, C8 and C9. The hypothesis to be tested here is that one or more of these SmCD59 genes protects schistosomes from complement killing just as CD59 protects the host's cells. First, studies will be conducted to determine which of the SmCD59 proteins are complement inhibitors by expressing each homologue individually in mammalian cells and testing for their ability to prevent complement damage to transfected cells. Then, the expression of selected pools of SmCD59 genes will be suppressed in schistosomula by RNAi to assess whether these parasites become more vulnerable to complement killing in vitro. In preliminary results, gene expression was detected for all SmCD59 homologues in schistosomula and adult worms (both life stages adapted to survive in the bloodstream) and the gene expression of each SmCD59 target individually, or in pools of up to 4 gene targets have effectively been suppressed in schistosomula.

If the RNAi studies are successful, this will be strong evidence that SmCD59 expression in the tegument is a major parasite defense mechanism against the host complement. This project could be then readily expanded into a larger project to explore the SmCD59 complement inhibition mechanism and to pursue strategies that exploit this knowledge in the development of new therapies or vaccines aiming to reduce the incidence and/or morbidity of schistosomiasis.

PI: Kritzer, Joshua
Title: Drugging the Undruggable: Targeting Transcription Factors with Small Cyclic Peptides
Abstract:
Transcription factors such as Myc, STAT3 and HSF1 represent key targets for novel cancer therapies. Because they lack the structural features of traditional drug targets, these proteins are often classified as "undruggable." This assumption is perpetuated by the fact that, despite intensive screening efforts using traditional methods, there are few known compounds that target transcription factors. However, potent natural products such as cyclosporine and rapamycin can bind diverse, non-traditional targets that would otherwise be overlooked. Cyclic peptides resemble these natural product macrocycles, and thus have the potential to target promising proteins, including transcription factors, previously dismissed as "undruggable."

Novel screening technologies will be required to discover bioactive cyclic peptides (CPs). We recently described a method of genetically encoding CP libraries for phenotypic selections in the yeast Saccharomyces cerevisiae. Since transcription is such a fundamental process, it is critical to perform selections in live organisms to ensure activity and selectivity for the target protein in the context of the full eukaryotic transcriptional machinery. This technique allows tens of millions of CPs to be screened in live cells in a single day without expensive robotics, and provides a rapid route for secondary testing and optimization of hits.

This proposal describes application of this method to discover CPs that inhibit diverse human transcription factors implicated in cancer. This will be accomplished by: (1) engineering yeast selection strains that report on protein-protein and protein-DNA interactions of Myc, STAT3 and HSF1, (2) applying CP libraries to the selection strains to isolate molecules that selectively disrupt these interactions, and (3) elucidating the mechanisms of action of the most promising CPs at the molecular and cellular level. These experiments will provide first-in-class drug leads suitable for further development, and will also demonstrate a rapid, inexpensive approach to drug discovery for previously overlooked targets.

PI: Kritzer, Joshua
Title: Rapid Generation of Isoform-Selective Histone Deacetylase Inhibitors
Abstract:
The therapeutic potential of histone deacetylase (HDAC) inhibitors has been demonstrated for several cancers. More recently, HDAC involvement has been demonstrated in polyglutamine repeat disorders, motor neuron disorders, and recovery from post-stroke ischemic injury. Application of broad-spectrum HDAC inhibitors has shown activity in animal models of these and other nervous system disorders. These data have led to speculation that selective inhibition of HDACs in neurons could be a viable therapeutic strategy for nervous system disorders. Despite over a decade of drug discovery research, our ability to address whether HDACs are practical therapeutic targets for nervous system disorders is still hampered by a shortage of isoform-selective inhibitors. Development of isoform-selective HDAC inhibitors is a key unsolved problem in the pursuit of novel therapeutic strategies for nervous system disorders.

The objective of this project is to apply a novel drug discovery strategy to generate isoform-selective HDAC inhibitors. The generation of isoform-selective HDAC inhibitors is significant because such compounds are necessary to address whether HDACs are practical therapeutic targets for nervous system disorders. The proposed approach takes advantage of a novel technology developed by the Principal Investigator to rapidly screen millions of cyclic peptides in the yeast Saccharomyces cerevisiae for inhibitors of enzyme activity or protein-protein interactions. Cyclic peptides are an underexplored class of compounds with proven potential for selective HDAC inhibition, and the proposed approach represents the first high-throughput screening method for cyclic peptide inhibitors of HDACs.

Several selection strains will be constructed and employed to isolate inhibitors of specific HDACs and inhibitors of protein-protein interactions involving HDACs. Each inhibitor will be analyzed using in vitro protein and cell-based assays to quantitatively measure HDAC inhibitory potency, HDAC selectivity profiles, and ability to inhibit protein-protein interactions involving HDACs. This approach represents a rapid, inexpensive alternative to traditional drug discovery that is uniquely well-suited for the development of novel HDAC inhibitors. Once developed, cyclic peptide HDAC inhibitors will be valuable tools for the neuroscience community as well as starting points for drug discovery.

Mentor: Kuliopulos, Athan
Fellow: Foley, Caitlin
Title: MMP1-PAR1 Signaling in Vascular Integrity During Sepsis
Abstract:
Protease-activated receptor 1 (PAR1) is a G-protein coupled receptor that is activated by extracellular proteolytic cleavage at the N terminus. PAR1 is activated by a diverse array of serine proteases, including thrombin and activated protein C (APC), and by the zinc-dependent collagenase, matrix metalloprotease 1 (MMP1). PAR1 is expressed highly on endothelial cells and acts as a major regulator of vascular integrity and coagulation.

Sepsis is a deadly complication of infection and is characterized by an excessive inflammatory response and aberrant activation of the coagulation cascade. A key early event in sepsis is loss of vascular integrity, causing leakage of vascular fluid and increased tissue factor generation. PAR1 plays a key role in this endothelial retraction and pharmacologic blockade of PAR1 early in sepsis reduces vascular leakage and improves survival in mouse models. However, the upstream PAR1 activation cascade in early sepsis is poorly understood.

Lipopolysaccharide (LPS), a component of the Gram negative outer cell membrane, is an important initiator of the inflammatory response in Gram negative sepsis. Macrophages are known to secrete MMP1, a PAR1 agonist, in response to LPS. This led to the hypothesis that MMP1 signaling through PAR1 in early sepsis leads to loss of vascular integrity and activation of the coagulation cascade. Preliminary results indicate that endothelial cells secrete MMP1 in response to LPS stimulation and that pharmacologic blockade of MMP1 in vivo leads to improved survival in mouse models of sepsis.

The purpose of this work is to determine the effect of MMP1-PAR1 signaling on vascular integrity and coagulation during sepsis. MMP1-PAR1 signaling in response to LPS will be studied on endothelial cells using in vitro assays of vascular leakage, actin retraction, and RhoGTP generation. The role of MMP1-PAR1 signaling in vivo will also be studied by examining the effects of pharmacologic MMP1 blockade on clinical markers of vascular function and coagulation in a mouse cecal ligation and puncture (CLP) model of sepsis. Additionally, the enzymatic activity and expression of the putative mouse homologue of MMP1 and the hypothesized PAR1 agonist in vivo, mColA, has not been fully characterized. The collagenolytic and PAR1 agonist activity profile of mColA and other secreted mouse collagenases will be determined in vitro and plasma expression levels and activity of these collagenases during sepsis progression will be quantified in vivo. Ideally, this work will delineate a novel mechanism endothelial function regulation and will identify novel approaches to sepsis treatment.

Mentor: Kuliopulos, Athan
Fellow: Austin, Karyn
Title: The Role of Metalloprotease-PAR1 Signaling in Vascular Injury and Repair
Abstract:
Cardiovascular disease (CVD) is the single leading cause of death in America. The major underlying pathology contributing to CVD is atherosclerosis. The dynamic nature of atherosclerosis involves remodeling of the blood vessel wall to accommodate for changes in vessel compliance. Initially, this process is adaptive, later it becomes injurious. An important component of vascular remodeling includes the phenotypic modulation of smooth muscle cells (SMCs) in and around the atherosclerotic plaque. SMCs undergo dramatic changes following blood vessel injury; cells proliferate, become invasive, and modify their metabolic properties. The molecular mechanisms underlying SMC behavior are poorly understood.

An important characteristic of the pathologic SMC phenotype is the production of proteases that allow for SMC invasion and vessel remodeling. Matrix metalloproteases (MMPs) are a family of endopeptidases that are largely responsible for the degradation of extracellular matrix, and play important roles in vascular remodeling and modulation of SMC behavior. MMP1 is a key player in the process of vascular repair, and is often over-expressed in atherosclerotic plaques. Published data show that MMP1 is an important activator of protease-activated receptor 1 (PAR1). SMCs express high levels of PAR1, and expression is increased in atherosclerotic plaques. The role of PAR1 signaling in SMCs represents an interesting paradox in vascular physiology; the cells only contact the natural activator, thrombin, during times of injury, which raises numerous questions about the role of PAR1 on SMCs. Furthermore, MMP1 and thrombin cleave and activate PAR1 at differing sites, leading to the hypothesis that thrombin and MMP1 differentially activate PAR1 to modulate SMC activity following vascular damage.

The long-term goal of this project is to better understand the pathophysiology of maladaptive vascular remodeling. The immediate objective is to investigate the mechanism of a thrombin-MMP1-PAR1 signaling axis in vascular repair. To accomplish these goals, two specific aims have been identified. First, to characterize the role of thrombin- vs. MMP1-activated PAR1 signaling in vascular repair, and second, explore the contribution of PAR1 signaling in mouse models of vascular injury. To accomplish aim 1, in vitro assays for proliferation, migration, and invasion will be performed on SMCs utilizing PAR1 and MMP1 agonists/antagonists. Furthermore, we will interrogate the mechanism of thrombin- vs. MMP1-activated PAR1 in SMCs. Additionally, the role of a thrombin-MMP1-PAR1 autocrine axis will also be explored. Mouse models of vascular repair will be used to investigate aim 2. Carotid artery wire injury will be performed on PAR1 and MMP1 knock-out mice, and vessels will be analyzed for neointimal lesion formation. Finally, the efficacy of PAR1 targeted pepducins will be tested as therapies for the treatment and prevention of vascular neointimal formation.

PI: Kumamoto, Carol
Title: Contact Sensing and C. albicans-Host Interaction
Abstract:
This research project investigates the mechanisms used by the opportunistic fungal pathogen, Candida albicans to produce disease. The long term goal of this research is to understand how C. albicans cells cause infection.

Environmental sensing is likely to be important for regulation of activities that promote virulence, such as the ability of C. albicans cells to convert into invasive filamentous hyphae, which penetrate into host tissue. Studies of invasiveness under laboratory conditions have shown that C. albicans cells sense contact with a semi-solid matrix, causing them to produce invasive hyphae. A plasma membrane protein that is needed for invasive growth in response to contact with agar medium and may be a sensor of contact has been characterized.

To evaluate the importance of contact sensing for invasion during infection, a mutant lacking the sensor will be studied in animal models of infection (Aim 1). Preliminary studies suggest that other plasma membrane proteins also participate in contact sensing. Aim 2 will focus on analyzing double or triple mutants lacking several components that are important for invasiveness in animal models of infection. In Aim 3, signaling pathways that are activated by the plasma membrane proteins of interest will be investigated. To understand how the contact sensing process takes place, Aim 4 will seek to identify and characterize proteins that function with the contact sensing protein. These studies will increase the understanding of regulatory mechanisms that are important in C. albicans pathogenesis.

PI: Kumar-Singh, Rajendra
Title: Age-Related Macular Degeneration
Abstract:
Age-Related Macular Degeneration (AMD) affects approximately 1 in 3 individuals over the age of 65 and is hence the most frequent cause of blindness in the elderly. There are currently no efficacious therapies available for this disease. Recently, genetic and biochemical evidence has emerged that AMD is caused by or is associated with local chronic inflammation of the retina with hallmarks similar to other diseases of aging such as Alzheimer's and atherosclerosis. There currently exists no animal model that accurately resembles human AMD (i.e. one that is initiated by inflammation). We propose developing such a model and using this model to develop genetic therapies that would be potentially used to alleviate symptoms or reverse AMD in human patients. Progress in these studies will have impact not only on the treatment of AMD, but potentially also in other diseases of aging that have a similar genetic and biochemical origin.

PI: Kumar-Singh, Rajendra
Title: Gene Therapy for Retinitis Pigmentosa
Abstract:
The long-term objective of this study is to develop a therapy for Retinitis Pigmentosa (RP), a disease that causes photoreceptor degeneration and blindness. There is currently no therapy available for this disease. RP is caused by mutations in genes expressed exclusively in the rod photoreceptors. The most common causes of recessive RP are mutations in the gene encoding the β subunit of cGMP Phosphodiesterase (βPDE). Despite the availability of a variety of animal models with mutations in βPDE, e.g. rd1, rd10, rcd etc., no evidence yet exists for long-term rescue of rod photoreceptors in these models, a prerequisite to clinical trials. This is in stark contrast to the success gene therapy approaches have had in other ocular tissues such as the retinal pigment epithelium (RPE) and specifically in animal models of Leber's congenital amaurosis. The lag in progress for rescue of rod photoreceptor diseases is due to the absence of efficacious gene transfer vectors for photoreceptors. The most commonly used vector in ocular gene therapy to date is adeno-associated virus (AAV), most serotypes of which have a limited cloning capacity of 4.8 Kb. This capacity is insufficient for the inclusion of large gene regulatory elements needed to achieve rod-specific and regulated transgene expression. Furthermore, AAV has been recently shown to cause hepatocellular carcinoma at the alarmingly high rate of 56% in animals by insertional mutagenesis. Hence, there is a need for gene therapy vectors that persist episomally. These significant deficiencies with AAV vectors and other deficiencies discussed in the proposal can be overcome through the use of helper-dependent adenovirus vectors (Hd-Ad) that have a 36K cloning capacity and persist episomally for years in non-human primates. However, Hd-Ad vectors do not have a tropism for photoreceptors but instead, they target only the RPE. Recently, we have shown that adenovirus (Ad) vectors with deletions in the RGD domain of penton base or Ad vectors that display transferrin on their capsid can very efficiently transduce photoreceptors. These modifications should readily be transferable to Hd-Ads. Hence, this proposal has 3 specific aims:

Specific Aim 1: Determine whether in the context of first generation Ad vectors. Do Ad capsids containing an RGD deletion in penton base combined with transferrin on their capsid, transduce photoreceptors more efficiently than either modification alone?

Specific Aim 2: To develop a 36Kb-capacity Hd-Ad that has been modified to contain an RGD-deletion in its penton base and transferrin on its capsid. Examine rod specific transgene expression using very large (>10Kb) 5' upstream and downstream gene regulatory elements, including a 10Kb βPDE promoter.

Specific Aim 3: To examine whether the photoreceptor targeted Hd-Ad system can express βPDE in the rd1 and rd10 murine retina specifically in rod photoreceptor cells using native (10Kb) βPDE 5' and 3' gene regulatory elements and potentiate long term rescue of photoreceptor degeneration.

PI: Kumar-Singh, Rajendra
Title: Non-Viral Gene Therapy for Retinal Degeneration
Abstract:
Retinal degeneration is one of the most genetically heterogeneous groups of disorders known, involving over 184 loci. Several ocular gene therapy clinical trials have remarkably demonstrated that gene therapy is a valid approach to treat retinal diseases. Each of these clinical trials and almost every preclinical gene therapy study thus far have utilized viruses as the gene transfer vector. Viruses have significant advantages as gene transfer vectors — primarily their ability to efficiently deliver genes to post-mitotic retinal cells in vivo. However, viruses also have some disadvantages, including induction of host immune responses, a limited transgene capacity, insertional mutagenesis and difficulty in production. Despite these disadvantages, viruses are the current vector of choice in almost all ocular gene therapy studies because of a lack of alternatives. If the above disadvantages could be resolved by the development of non-viral gene transfer vectors that could deliver genes to post-mitotic tissues such as adult retina, it would have substantial impact on the field of preclinical and clinical ocular gene therapy. Unfortunately, non-viral vectors only work efficiently in cell culture or in neonatal retina where mitosis is ongoing. Hence, unlike viruses, non-viral vectors generally fail to rescue animal models of retinal degeneration unless applied in neonatal murine retina – results from which cannot be directly translated to post-mitotic human retina.

Recently, we developed a 3.5 Kd peptide (POD) that can form nanoparticles resembling viruses in size (136nm) when complexed with DNA and enable transgene expression in post-mitotic retina. Although gene transfer with POD nanoparticles was not as efficient as with viruses, it was sufficient to enable a short-term delay in retinal degeneration in vivo. This is only one of two studies thus far demonstrating a delay in retinal degeneration in an adult mouse using a non-viral vector. The major limitation of our study was that of short-term transgene expression from POD nanoparticles. The primary objective of this study is to prolong transgene expression from POD nanoparticles by use of nuclear DNA integration or DNA retention elements. The second objective of this study is to improve the efficiency of gene transfer of POD such that it could be more potent and the third objective is to validate the improvements in POD in two relevant animal models of retinal degeneration. The high level of genetic heterogeneity observed in retinal degeneration hampers the timely availability of therapies for patients as each gene and virus combination needs to be developed through a lengthy process. Such approaches are not economically feasible for the >184 loci. Hence, we propose to use POD nanoparticles not to deliver individual genes but instead, genes encoding neurotrophic factors such as to develop a non-viral, non gene-specific approach to treat retinal degeneration. Upon completion of these studies we will have a novel non-viral vector ready for use in clinical trials pending toxicology studies. If successful, these studies would be a paradigm shift in ocular gene therapy.

Mentor: Kumar-Singh, Rajendra
Fellow: Binder, Christina
Title: Optimization of Peptide Nanoparticles for the Treatment of AMD
Abstract:
Age-Related Macular Degeneration (AMD) is the most common cause of blindness in individuals 50 years of age or older in western cultures. More than 8 million Americans suffer from AMD, and this number is predicted to increase by more than 50% in the next 10 years. Evidence indicates that dysregulation of the complement system plays a role in AMD pathogenesis. Additionally, complement dysregulation has been indicated in other chronic diseases such as atherosclerosis, Alzheimer’s, diabetic angiopathy, and rheumatoid arthritis. Some studies have found a correlation between cognitive impairment and incidence of AMD. Currently, dry AMD (90% of AMD patients) is untreatable, and wet AMD can be ameliorated by repeated injections of anti-Vascular Endothelial Growth Factor therapeutics. Ocular injections are not free from complication, however, and methods to decrease the frequency of injections are under research. One approach is to deliver DNA that can alter the disease course. Delivery of a complement inhibitor to the cells of the retinal pigment epithelium (RPE) has potential to benefit both wet and dry AMD patients.

No preferred method exists among DNA delivery strategies because each method has restrictions. Non-viral methods avoid the dangerous and limiting complications of viral delivery, but are historically less efficient and of shorter duration. Thus, a need exists for the development of an efficient and safe delivery system that enhances longevity of transgene expression. This study proposes to significantly upgrade upon a non-viral delivery system termed POD (Peptide for Ocular Delivery). When pegylated (PEG) and injected subretinally, POD can deliver transgene DNA to RPE cells in vivo. When injected intravenously, PEG-POD delivers transgene DNA systemically to other tissues, especially the lung. However, transgene delivery by PEG-POD is subject to a short duration and entrapment in the lysosomal-endosomal pathway. Thus, the primary goal of this proposal is to enhance the PEG-POD technology such that it can be used to deliver CD59 (a complement inhibitor) expressing plasmids to the RPE for treatment of AMD. The improved POD delivery agent will be validated in an in vivo model of wet AMD. Should POD-delivered complement inhibitors demonstrate efficacy in the wet AMD model, they will have a high therapeutic potential for dry AMD as well.

These results will present a novel solution to a common, debilitating disease. The use of non-viral methods in ocular gene-therapy opens many possibilities for long-term protection from not only AMD but other aging diseases of complement dysfunction.

PI: Kumar-Singh, Rajendra
Title: Platform for Rapid Delivery of Biologics and Drugs to Ocular Cells and Tissues Following Combat Associated Trauma
Abstract:
Multiple cell death pathways are triggered in neurons following exposure to blast waves. A large group of biologics are currently available that can block cell death. None of these biologics are currently used in the combat theater. Following blast pressure wave associated trauma, there is a limited window of opportunity within which to intervene before cell death programs such as apoptosis are irreversible. The barrier to the rapid delivery of biologics into neurons is the cell membrane. We have developed a small peptide called peptide for ocular delivery (POD) that can enter neurons without toxicity. POD can be conjugated to cargo and rapidly ferry the cargo into the cell. We envisage that in the combat theater, members of the Armed Forces will be equipped with lyophilized POD conjugated to anti-apoptotic proteins in the medical kit and this therapeutic will be suspended and applied directly to the eye or injected into the vitreous subsequent to exposure to blast.

Hypothesis: POD may enable delivery of functional anti-apoptotic proteins across the plasma membrane of cells/neurons in the retina end cornea and may attenuate apoptosis and loss of cells.

Specific Aims and Study Design: We will design and synthesize several anti-apoptotic proteins fused to POD and measure the ability of these proteins to cross the plasma membrane and inhibit ultraviolet radiation induced apoptosis in vitro and in vivo.

PI: Kuo, Catherine
Title: Identification of Muscle-Derived Soluble and Mechanical Cues to Direct Differentiation toward Tendon Lineage
Abstract:
Tendons have poor healing ability and require surgical repair with grafts when ruptured. The grafts are fraught with problems ranging from failure to donor site morbidity, motivating stem cell-based tissue engineering strategies for tissue replacement. However, differentiation of cells toward the tendon lineage (tenogenesis) has been challenging due in part to a poor understanding of tendon development. Developmental biology studies have demonstrated that muscle plays a significant role in tendon embryogenesis, though the mechanisms of its contributions are not well understood because the respective physical (mechanical) and soluble signaling factor influences of muscle tissue have been difficult to study in a complex in vivo environment.

Our objective is to identify and characterize muscle cell-produced soluble and mechanical tenogenic cues to direct tenogenesis. We hypothesize that soluble factors secreted by muscle cells, as a function of their developmental stage, will regulate tenogenesis of TPCs in vitro and that this process will be enhanced by dynamic mechanical stimulation. Using a unique in vitro co-culture system we will characterize muscle cell secretion of putative soluble factors and their potential tenogenic roles (Aim 1), and investigate the potential for mechanical loading to enhance chemoregulation by studying TGFβ2 as a model soluble factor (Aim 2). The outcomes of this study will subsequently be translated in a future study to develop a mesenchymal stem cell-based regeneration strategy through controlled application of these influences.

Our long-range goal is to use developmental biology as motivation and a guide in developing novel mesenchymal stem cell-based strategies in regenerating new tissue to replace injured or diseased tendons and ligaments. The outcome of this research effort would significantly advance knowledge of tendon developmental biology, help define rational soluble factor dosing and mechanical loading parameters for progenitor cell differentiation, and lead to advanced strategies to engineer tendons with mesenchymal stem cells.

PI: Kuo, Catherine
Title: Regulation of Embryonic Tissue Differentiation by Matrix Elasticity and Mechanical Forces
Abstract:
Increasing evidence indicates biomechanical forces are critical regulators (mechanoregulators) of embryonic development. An altered biomechanical environment radically affects normal skeletal tissue development, as evidenced by human infant disorders resulting from fetal akinesia, and animal studies demonstrating skeletal defect formation due to muscle paralysis. Internal (via tissue matrix) and external (via environment, e.g., muscle) mechanical influences on embryogenesis have been modeled using theoretical simulations. Recent stem cell differentiation studies demonstrate that long-term lineage commitment can be specified by matrix elasticity. Collectively, these studies indicate mechanics play a significant role in development. However, mechanoregulatory mechanisms by physical factors have not been elucidated in isolation of confounding soluble factors in vivo, and embryonic tissue mechanical properties are not well characterized due to difficulty of applying traditional test methods to tiny, delicate embryonic tissue. Thus mechanoregulation of skeletal tissue development remains poorly understood. We have recently demonstrated feasibility of force-volume atomic force microscopy to measure mechanical properties of delicate embryonic tissue, and the use of dynamic loading to mechanoregulate stem cell differentiation. Using tendon as our model skeletal tissue, the objective of this study is to understand how these forces may modulate tissue embryogenesis.

We hypothesize that matrix elasticity and dynamic mechanical forces couple synergistically to mechanoregulate differentiation of embryonic tendon cells toward the tendon lineage. Employing a unique interdisciplinary approach, we will apply engineering tools to interrogate the effects of individual and coupled internal and dynamic external forces on differentiation of embryonic tendon cells. An understanding of the mechanoregulatory cues required for normal tissue embryogenesis would significantly enhance our knowledge of developmental biology and enable development of preventative and treatment strategies for mechanically influenced debilitating disorders.

PI: Kuperwasser, Charlotte
Title: Analysis of the Cellular and Molecular Determinants of the Human Breast Hierarchy
Abstract:
The identification of human mammary progenitor cells and understanding their regulation is critical to enumerating the origins and events that control pathologic conditions. Determining the identity and activity of normal primitive cells could lead to a better understanding to the connection between stem cell biology and cancer. Our study is directed towards understanding the mechanisms that underlie human breast development and the regulation of primitive progenitor cells by hormone-growth factor signaling.

The limited understanding of human breast development and stem cell biology has largely been due to the lack of appropriate model systems and assays to detect, analyze, and characterize stem cell properties. In recent years, our laboratory has developed and optimized various in vivo and in vitro tools to study the biology and mechanisms governing human breast development. We have pioneered and implemented the use of an innovative in vivo model to study human mammary development by exploiting the mouse mammary fat pad of immunocompromised mice as a source of important endocrine signaling events and using grafted human stroma to support the growth and differentiation of the human mammary epithelium.

In recent work, we have identified:

  1. four distinguishable epithelial differentiation states (two luminal phenotypes and two basal phenotypes) within human breast tissues that differ on the basis of CD24, EpCAM and CD49f expression,
  2. the existence of bipotent progenitors that contribute to structurally distinct elements (ducts and lobule/alveoli),
  3. the hormonal combinations that enhance stem/progenitor cell activity within human epithelial cells, and
  4. a hormone growth factor mechanism through TBX3 expression can regulate breast stem-like cells.

In Aim 1 of this project, we will delineate the epithelial hierarchy in adult human breast tissue. In Aim 2, we will determine how hormone-growth factor signaling regulates human breast progenitor cell activity. Our studies provide innovative insight into the identity of human breast progenitor cells and provide critical molecular underpinnings by which hormones drive human breast progenitor cell morphogenesis that could serve as focal points during the development of pathological conditions.

PI: Kuperwasser, Charlotte
Title: Breast Cancer and Cellular Reprogramming
Abstract:
Breast cancer is the most frequently diagnosed cancer among women and accounts for more than 40,000 deaths annually. Pre-malignant benign lesions, which include fibrocystic disease, metaplasia, and epithelial hyperplasias, are also common conditions of the breast that affect more than 50 percent of all women. Over the years, one of the major issues has been to determine to what extent these lesions are a risk factor for the subsequent development of breast cancer. As the diagnosis of benign breast diseases becomes more common, it is crucial to identify those women who are at an increased risk for breast cancer.

Both benign and malignant breast neoplasms can be regarded as diseases of inappropriate cellular proliferation and altered differentiation state, in which both genetic and epigenetic abnormalities play a causative role. Metaplastic breast carcinomas (MBCs) are a distinct aggressive form of estrogen receptor (ER)-negative invasive breast cancers that histologically resemble cells and tissues such as those found in skin, bone, and cartilage. These cancers have a high rate of systemic metastasis and few treatment options; therefore, an understanding of the pathobiology of metaplastic carcinomas is critical to develop new avenues of therapy for this disease.

The Kuperwasser laboratory has recently made a serendipitous discovery connecting the origins of this aggressive form of cancer with cellular reprogramming. Specifically, we have found that a population of human breast epithelial cells can spontaneously and naturally reprogram back into skin (and possibly ectodermal) stem cells in the absence of genetic or chemical manipulation. These cells may have the capacity to regenerate adult mature skin and possibly other skin-derived structures (hair follicle, sweat glands etc.). The ability of breast cells to lose their identity and revert back to a more embryonic state reminded us of the process called induced pluripotent stem (iPS) in which cells can be reprogrammed back into embryonic stem cells.

The goal of this work is to gain a deeper understanding of reprogrammed breast cells and assess the genetic and molecular changes within these cells. Specifically, we will determine if and how epigenetic reprogramming allows these cells to access embryonic-like multipotent states normally restricted in adult tissues. In addition, we will examine the epigenetic process of DNA methylation and post-translational modification of histones in the requirement of reprogramming and cancer.

Because this is the first study of its kind, it has the potential to provide for the first time new information regarding the link between cellular reprogramming and the process of tumor progression. In addition, information from these studies may lead to a new and suitable alternative to ES cells for the treatment of severe burn that require autologous grafts. Finally, as the specific mechanisms governing cell differentiation state (or de-differentiation) in cancer remain almost completely undefined, this study will have broad implications for the identification of novel epigenetic targets in breast cancer therapy.

PI: Kuperwasser, Charlotte
Title: Mechanisms of Breast Cancer Associated with Obesity
Abstract: In the United States obesity rates are increasing, and understanding the molecular and cellular basis for breast cancer associated with obesity is of significant clinical relevance and impact. Obese women are more likely to be diagnosed with non-familial estrogen receptor negative tumors than lean women, and these tumors are more likely to be associated with nodal metastases. Since breast stromal tissue is a reservoir of subcutaneous fat the stromal tissue microenvironment is known to have a profound effects on breast cancer development and progression, the changes that take place within the fat depots of obese individuals may play a significant role in the pathogenesis of breast cancer. Therefore, elucidating the mechanistic role of adipocytes and adipose tissue biology in breast cancer is critical for prevention and treatment of obesity-related cancer.
Fat cells within the breast tissue of obese women secrete a monocyte chemo-attractant protein-1 (MCP-1) that promotes the recruitment of macrophages into breast adipose tissue. These cells when recruited induce local tissue inflammation, but role of macrophages in obesity-associated inflammation during is currently unknown. Here, we aim to test from bench to human clinical trials, the hypothesis that breast tissue in obese women exhibits increased neoangiogenesis and inflammation prior to overt tumor formation due to the recruitment of macrophages by MCP-1 producing adipocytes, thereby leading to increased vascularity and malignancy.

In Aim 1 of this project, we will investigate the molecular mechanism by which bone marrow-derived macrophages promote obesity-induced angiogenesis and cancer initiation. Aim 2 will test whether bone marrow-derived macrophages are necessary for obesity-induced tumor progression, and Aim 3 will determine whether obesity-induced preneoplastic changes can be reversed. Our studies provide innovative insight as to why obesity is associated with increased cancer incidence and aggressiveness. In addition, this work will provide a novel paradigm for the prophylactic treatment of high risk obese patients.  

This investigator initiated research grant seeks to investigate from pre-clinical models to community partnered trials, the relationship between obesity and the initiation and aggressive behavior of ER- breast cancer in Caucasian and African American women. This is in response to Provocative Question (PQ1) "How does obesity contribute to cancer risk?"

PI: Kuperwasser, Charlotte
Title: Mechanisms of Breast Cancer Progression Associated with Obesity
Abstract:
Since obesity has become an epidemic in the United States it has recently been identified as the most important risk factor associated with breast cancer in postmenopausal women. In these women, obesity is also significantly correlated with an overall poorer prognosis. Breast stromal tissue is a reservoir of subcutaneous fat, a fact that has been largely ignored in the study of breast cancer. Obese adipose tissue is associated with chronic inflammation and recruitment of pro inflammatory macrophages, due to elevated localized expression of monocyte chemoattractant protein-1 (MCP-1) produced by activated adipocytes. This inflammatory microenvironment may foster breast cancer development and progression.

In order to study the mechanisms of obesity in human breast cancer progression, we have created a model derived from human breast reduction mammoplasty tissues in which primary breast preadipocytes were enforced to express MCP-1 mimicking preadipocytes in obese adipose tissue. These obesity-like preadipocytes were found to recruit inflammatory macrophages, promote cancer development in a humanized model of breast cancer, and significantly enhance neoangiogenesis compared to control preadipocytes. Macrophages stimulated with conditioned media from obesity-like preadipocytes significantly induced stromal-derived factor (SDF)-1α secretion which has been shown to enhance angiogenesis.

Based on these and other findings, we hypothesize that preadipocytes in obese individuals enhance neoangiogenesis through the recruitment of inflammatory bone marrow-derived macrophages that increase localized SDF-1α concentrations, thereby leading to increased vascularity and malignancy. Accordingly, we propose to 1) determine whether bone marrow-derived macrophages are necessary for obesity-induced angiogenesis, and 2) determine the effect of inhibiting macrophages and signaling pathways by using small molecule inhibitors on obesity-induced tumor progression and angiogenesis.

PI: Kuperwasser, Charlotte
Title: The Role of Breast Cancer Stem Cells in Metastasis
Abstract:
It is reported that 4%-45% of patients with early stage breast cancer already have disseminated breast cancer cells that do not appear to be growing in the bone marrow by the time the primary tumors are surgically removed. Despite the removal of the primary tumor, relapse in these patients and metastases at distant sites such as bone, lung, and brain is still the major obstacle in the clinic. Recently, our laboratory developed several models of human breast cancer metastasis to lung, brain and human bone. We have also shown that a population of breast cancer-initiating cells (BCICs) can be found within human breast cancer cell lines and these cells correlates with the ability of the cell line to form a tumor and metastasize in animal models. Furthermore, we observed that these are resistant to Paclitaxel (Taxol) and 5-Fluorouracil (5-FU), both commonly used chemotherapies for the treatment of breast cancer. We now wish to extend this work by identifying the cellular and molecular mechanisms that permit cancer initiating cells to survive in the bone marrow. This work if successful will identify important novel targets aimed specifically at preventing the expansion of breast cancer cells in the bone marrow and possibly even seeding other tumors at distant sites.

PI: Labato, Mary
Title: Cat Pimobendan Study
Abstract: Chronic kidney disease (CKD) is one of the most common reasons geriatric cats present to the veterinarian. CKD is considered irreversible and progressive, and effective treatments are limited. A common comorbid condition often appreciated in feline CKD patients is heart disease. Two of the investigators in this study (ML, JR) have administered pimobendan to cats with combined kidney and heart disease after these patients developed congestive heart failure (CHF) secondary to intravenous fluid administration, a typical standard of care for kidney disease. In some of these patients, addition of pimobendan resulted in a greater improvement in kidney values and clinical response (appetite and energy level) than has been usually noted in comparable historical cases. Pimobendan is a benzimadole-pyridazinone that increases contractility of heart muscle and relaxes the body's blood vessels. It is an effective drug for the management of CHF in dogs and may support kidney function through improved cardiac output and kidney perfusion. Tolerability and safety of this drug has already been established in cats with heart disease. This will be a pilot study to assess the tolerability of pimobendan in cats with chronic kidney disease and search for benefits in comparison to the current standard of care. Investigating these observations in a larger study will help establish whether pimobendan could be a novel treatment for cats with kidney disease. Benefits could include decreased hospitalization time, improved appetite, improved kidney values, and the ability to treat kidney disease while simultaneously protecting against CHF in patients with concurrent heart disease.

PI: Labato, Mary
Title: Evaluation of Clopidogrel-Induced Platelet Inhibition in Dogs with Protein-Losing Nephropathy
Abstract:
Diseases frequently complicated by clot formation are becoming well defined in veterinary medicine, and the use of blood thinners is on the rise. Guidelines for use of blood thinners in sick dogs have not been well defined. Protein-losing nephropathy is a condition frequently complicated by clot formation, and these dogs are routinely treated with blood thinners such as heparin, clopidogrel, or aspirin. Since heparins require a protein called antithrombin that is often depleted in dogs with protein losing nephropathy to be reliably effective, clopidogrel is often used as an alternative. Clopidogrel is a platelet inhibitor has shown to have platelet blocking effects in healthy dogs at the traditional dose of 2mg/kg. Studies in healthy dogs also suggest that these effects are also seen at lower doses. The goal of this study is to document the efficacy of 1 mg/kg clopidogrel on platelet function over a 24 hour period using thromboelastography Platelet Mapping and aggregometry in dogs with protein-losing nephropathy and to compare this effect to normal dogs that do not receive clopidogrel.

This study will be a valuable addition to the veterinary literature as the first study to confirm the antiplatelet effect of clopidogrel in dogs with a naturally occurring disease commonly complicated by clot formation. Results of this study will serve as the foundation to providing evidence-based guidelines for the administration of anti-platelet drugs to dogs with hypercoagulable states for the prevention of potentially devastating clot formation.

PI: Leong, John
Title: Actin Pedestal Formation by EHEC O157:H7
Abstract: Enterohemorrhagic E. coli (EHEC) serotype O157:H7, an important agent of diarrheal disease, triggers the formation of filamentous actin pedestals on intestinal epithelial cells beneath sites of bacterial attachment. The ability to generate actin pedestals promotes late stage intestinal colonization and permits the formation of large aggregates on the epithelial surface. To generate pedestals, EHEC injects two effectors, Tir and EspFU, into mammalian cells via a type III secretion system. Tir is inserted into the host cell membrane and acts as a receptor for the bacterial outer membrane protein intimin. The C-terminal cytoplasmic domain of Tir is recognized by IRTKS (Insulin Receptor Tyrosine Kinase Substrate), a mammalian adaptor/effector that promotes the formation of F-actin and protrusive membrane structures at the plasma membrane. IRTKS also binds PI(4,5)P2 and deforms membranes, and binds the GTPase Rac, which is also known to stimulate actin assembly. Importantly, a C-terminal IRTKS SH3 domain binds to EspFU,, potentially linking it to Tir. EspFU contains multiple 47-residue proline-rich repeats and activates the actin nucleation promoting factor (NPF) N-WASP by mimicking and displacing an autoinhibitory N-WASP peptide. N-WASP is required for efficient translocation of Tir and EspFU, but if this block is overcome, EspFU can trigger an N-WASP-independent pathway for actin assembly, presumably by interacting with an alternative mammalian actin NPF. These findings suggest a model in which host actin assembly initially promotes translocation of Tir and EspFU, both of which bind IRTKS to assemble a complex at the plasma membrane, clustered by interaction with bacterial intimin, that potently stimulates two pathways of actin assembly. Tir/EspFU-mediated actin assembly may in turn promote more efficient type III translocation, and, by unknown means, epithelial colonization in vivo. IRTKS may play a role in pedestal formation in addition to linking Tir to EspFU, since pilot experiments suggest that the IRTKS binding sequence of EspFU enhances pedestal formation even when EspFU is directly fused to Tir.

The following aims will be pursued to investigate both N-WASP-dependent and independent mechanisms of actin pedestal formation, and to examine potential roles of pedestal formation during mammalian infection:

  1. Determine whether Tir-, EspFU-, PI(4,5)P2-, and/or Rac-binding activity is important for IRTKS to promote actin pedestal formation;
  2. Identify mutants of EspFU that are defective for the N-WASP-dependent and/or -independent pathways of pedestal formation;
  3. Determine the relative importance of the N-WASP-dependent and N-WASP- independent pathways in pedestal formation on polarized intestinal epithelial cells, and
  4. Investigate whether pedestal formation promotes stable bacterial attachment, disruption of tight junctions and/or translocation in vitro, and the clonal expansion of microcolonies on intestinal epithelium during infection. 

PI: Leong, John
Title: EHEC-induced Actin Rearrangement and Stx2 Translocation Across Epithelium
Abstract: To test the hypothesis that EHEC mediated actin rearrangement contributes to the disruption of intestinal epithelial barrier function and enhancement of Stx-mediated disease, we outlined the following aims:

  1. Assess the ability of EHEC-mediated actin rearrangement to enhance Stx2 translocation across polarized epithelial cells in vitro.

  2. Assess the ability of EspFu to enhance Stx2-mediated disease in a mouse model for EHEC.

Mentor: Leong, John
Fellow: Bhowmick, Rudra
Title: Role of Hepoxilin A3 in Lung Inflammation Caused by Streptococcus Pneumoniae Infection
Abstract:
The overall objective of the proposed project is to understand the molecular mechanisms that lead to the hallmark of bacterial pneumonia: inflammation. A clear understanding of these mechanisms is crucial for understanding the robust neutrophil influx, the key event of inflammation, which if uncontrolled, cause major damage to the lung itself. From the preliminary data generated by us, we hypothesize that the inflammatory eicosanoid, hepoxilin A3 (HXA3), is required for the transepithelial migration of neutrophils during pneumococcal pneumonia. Therefore, understanding the key molecular mechanisms that culminate in the synthesis and subsequent release of HXA3 is of utmost importance for devising therapies that could lead to simultaneous control of the bacterial insult and attenuating the resulting inflammation, both of which are responsible for the pathogenesis of bacterial pneumonia.

PI: Lerner, Richard
Title: Evaluating the Williamson Model: Promoting Character Development and Productive and Engaged Citizenship among Young American Men
Abstract: Most educational programs focus separatelyon either character, moral, or civic education. The Williamson School (WS) is one prominent exception. Across its more than 120 year history, the WS, a three-year junior college located in Media, PA, fosters among deserving but socioeconomically disadvantaged young men the values of faith, integrity, diligence, excellence, and service, character attributes that correspond with many of those included by Sir John in the Foundation's charter (e.g., diligence, entrepreneurship, future mindedness, generosity, honesty, purpose, and reliability). Thus, the model has the potential to have a positive enduring impact on socioeconomic sectors of our society often bereft of hope and positive purpose.

Accordingly, we propose to conduct a cohort sequential longitudinal evaluation of the integrative WS model. Across three years, we will measure existing and entering class cohorts of WS students, and operationalize and evaluate the implementation and impact of the WS theory of change, thereby providing a sample that includes newly-enrolled students and follows current students post-graduation. Using a life narrative interview method, we will also assess a representative sample of WS graduates 5 to 50 years post-graduation. The quantitative data from the WS will be contrasted with data derived from matched groups of male students enrolled in other vocational education or junior college programs in the greater Philadelphia area.

The results of this research will address two Big Questions (about the role of character development in academic and life achievement, and about how to enhance the educational achievements of American men), and potentially have enduring impacts on enhancing the education of young men, on promoting the role of character education in enhancing life success, and on formulating a potentially powerful means to break the cycle of social dependency among lower income American families.

PI: Lerner, Richard
Title: The Development of Entrepreneurship in Adolescents and Young Adults: A Longitudinal Study of the Individual Basis for American Free Enterprise
Abstract:
Entrepreneurship is the foundation of American freedom and democracy, and understanding how individuals develop the capacity for successful entrepreneurship is essential to preserve and enhance our way of life. Yet there is little strong scientific knowledge about the development of entrepreneurship during adolescence and early adulthood — periods when developmental theory and prior Templeton Foundation-supported research suggest that such development is likely to occur. Without knowledge of the bases of the development of entrepreneurship, efforts to create effective educational programs to foster entrepreneurial skills in young people will be limited. Only through a rigorous longitudinal study will we be able to identify the capacities that we must cultivate in youth to develop successful entrepreneurship.

Both developmental theory and the present investigators' prior review of the entrepreneurship literature suggest that the characteristics of youth expressing entrepreneurial purpose correspond in large part to the attributes of character noted by Sir John Templeton. For example, in addition to the quality of entrepreneurship, among the defining features of youth with entrepreneurial purpose are the character attributes of creativity, curiosity, diligence, future mindedness, and reliability. As such, we propose a cutting-edge longitudinal study that will examine the development of entrepreneurial purpose, achievements, and character attributes from ages 18 through 26 years across diverse U.S. youth.

The results of the study will offer entrepreneurship educators and policymakers invaluable, scientifically validated information about the bases of entrepreneurship and therefore knowledge about how to promote entrepreneurship purpose and achievement among young people. This information will provide a model for future entrepreneurship research and a knowledge base for educational practice in this essential but under-studied area of American life.

PI: Leventhal, Tama
Title: Neighborhood Context and Immigrant Young Children's Development
Abstract:
Neighborhoods historically have played a central role in the lives of immigrant families who typically settle in communities comprised of other immigrant families with a shared language, values, practices, and social ties. However, efforts to explore neighborhood effects on immigrant children's development remain largely theoretical. The primary goal of the proposed study is to expand and apply to children from immigrant families a conceptual framework that I developed to explore how neighborhood context contributes to child development in ways that both promote and inhibit well-being.

This study will use data from the Project on Human Development in Chicago Neighborhoods, a multilevel, longitudinal study; children were sampled from 80 diverse neighborhoods and followed every 2 years over a 6-year period. I will use data for 3 cohorts first seen at 6 months and 3 and 6 years of age (N= 3,248; 50% male and 44% immigrant families). At each assessment, mothers reported on children's behavioral functioning and child care use and family processes (maternal depression, warmth/harshness, and developmental stimulation), and children's English verbal/reading abilities were evaluated. Neighborhood structure (concentrated poverty, high SES, immigrant concentration, racial/ethnic composition, and residential stability) and processes (norms and collective efficacy, relationships and ties, and institutional resources) were measured through independent sources. Hierarchical linear modeling will be used to test if neighborhood conditions have unique associations with immigrant children's development compared with non-immigrant children (generally or for specific racial/ethnic groups).

My project is aligned with several research areas of interest to FCD including: (1) factors contributing to positive and negative outcomes of immigrant children in their communities, (2) early education, and (3) language development. It will provide much needed information on young immigrant children and their neighborhoods, which cannot be ascertained from other studies. I plan to disseminate my results to both academic and policy/practice audiences. In addition, I will collaborate on a comprehensive, integrated, child-oriented community-based intervention targeting immigrant and low income families in Somerville, MA modeled on a well-known initiative in Harlem, NY.

PI: Levin, Michael
Title: Collaborative Research: CDI Type-1: A Computer Framework for Modeling Complex Pattern Formation
Abstract:
The mechanisms living systems use to establish and maintain complex 3-dimensional shapes during embryonic development are poorly understood even though molecular and cell biologists have generated mountains of data about genes and their effects on organisms. Fundamental advances in controlling biological form are stymied by the difficulty of obtaining shape information through the analysis of gene networks such that it is currently difficult or impossible for scientists to generate testable models of shape based on experimental results from current biological research. These investigators will apply state-of-the-art computational science and artificial intelligence to create a novel suite of computational tools that will fundamentally integrate numerous areas of biology and engineering to promote research into the mechanisms used by organisms for establishing and maintaining their 3-dimensional shape. This "Bioinformatics of Shape" project will integrate experimental data, a new mathematical language, a system for storing and mining data, a modeling environment within which rule sets for regulatory mechanisms can be simulated on computers, and an artificial intelligence module that will help scientists discover and test novel ideas about how shape is generated through genetics. The benefits to society of this new kind of collaboration between computer scientists and biologists include the translation of molecular and cell biological data into a new level of understanding that could have implications for regenerative medicine, adaptive and self-repairing devices for robotics and other engineering applications.

The work will provide unique training opportunities for students, establish a proof-of-principle for new educational tools at the boundary between artificial intelligence and biology, and facilitate data to knowledge production in a number of fields, such as developmental biology, evolutionary biology, and the engineering of complex adaptive systems.

PI: Levin, Michael
Title: IDBR: A Workstation for Optogenetics in Embryogenesis and Regeneration
Abstract: The orchestration of cell behavior into multicellular pattern formation is a central issue for developmental, regenerative, cancer, evolutionary, and synthetic biology. Modern genetics has made remarkable strides because tools were developed for the experimental control of biochemical signals.
Bioelectric signals are a fascinating and important (epigenetic) layer of control. Steady-stale (long-term, not action potential) endogenous flows of ions, voltage gradients, and electric fields in most cells are crucial, well-conserved, instructive determinants of cell behavior and tissue patterning. The biophysical signals, produced by ion channel and pump proteins, regulate gene expression and are now known to be involved in the control of cell migration, proliferation, differentiation, with important roles in morphogenesis and cancer suppression, alongside biochemical gradients and gene regulatory networks.

Our lab pioneered the development of molecular-genetic tools for the investigation of bioelectric signals in vivo. We developed protocols for non-invasive dynamic monitoring of bioelectrical gradients in entire embryos, and used misexpression of ion channel and pump proteins to make targeted, quantitative, molecular-level changes in ion fluxes in a variety of vertebrate and invertebrate model systems. This implicated specific bioelectric events in development (left-right asymmetry, craniofacial patterning, eye induction), regeneration (tail and limb regeneration in vertebrates, and anterior-posterior polarity in planaria), and is now being applied to detection and suppression of neoplasia in situ. By developing 1) tools that allow molecular-level changes in physiological properties in vivo, 2) building quantitative computer models synthesizing physiological and molecular data, 3) fleshing out pathways that show in detail how biophysical signals are produced, propagated, and transduced into downstream canonical biochemical/transcriptional responses, and 4) disseminating protocols and reagents to many labs in related fields, bioelectricity has been brought into a new age. Crucially, the field is held back by a lack of tools: transformative impact requires the ability for labs in related fields to be able to exert tight spatio-temporal control over ion flux and transmembrane voltage in vivo.

Current technology (pharmacology and misexpression of channels) is very limited in where the modulating reagents are delivered and how tightly the ion flows can be controlled. Optogenetics (using light-sensitive ion channels) is an exciting advance, but has never been applied outside of excitable cells (nerve and muscle) because available devices do not allow flexible control of sufficiently-bright light delivered over large areas – a necessity when working with developing or regenerating systems (organs, whole animals, or bioengineered constructs). Our system is an automated, highly versatile, optogenetics research station that expands past existing technology by more than an order of magnitude along several criteria. Built around a computer controlled microscope that is suitable for working with anything from small animals to individual cells, our platform will allow any lab that has access to molecular biology and microscopy to perform screens and targeted experiments on the role of physiology and bioelectricity In any context. We will significantly impact several basic fields by transforming the state of the art in how functional in vivo physiology experiments are planned, executed, and analyzed. In Aim 1, we capitalize on our commercial partnership and local collaborators in engineering and optics to build the platform. In Aim 2, we validate it by proof-of-principle applications in 3 very different systems.

PI: Levin, Michael
Title: Probing the Fundamental Nature of Bioelectric Signals that Mediate Information Processing in Cells and Tissues
Abstract:
The fundamental property that defines life is the ability to process information and utilize it to maintain highly adaptive structure and function of the organism. Biological information comes in two forms:

  • Spatial information – which determines the 3-dimensional structure of the organism and is used to guide pattern formation during embryonic development, regeneration, and cancer suppression
  • Temporal information – which arises from stored patterns inferred from environmental signals and used to guide behavior by memories and intelligence

The central question is how do living systems process these two kinds of information — through what mechanisms do cells organize exquisitely complex tissues and organs into an organism that can show such remarkable robustness and flexibility in attaining its goals?

Significant progress has been made in understanding patterning cues (developmental biology) and neuronal information processing (neurobiology). However, we are still very far from unlocking the fundamental theory that will explain this unique property of life (and thus illuminate every field of endeavor, including such seemingly disparate fields as biomedicine, astrobiology, engineering, and artificial intelligence). The community is currently pursuing those areas that are tractable given today's technology, but there are huge areas of this field of science in which we don't even know how to pose the correct questions (experimentally or theoretically). The major focus of funding has been on biochemical signals (to the significant exclusion of physics), gene regulatory networks (with a lack of corresponding advances in understanding how transcriptional activity is translated into actual anatomical pattern), and on the computational properties of neural networks (with very little attention paid to the information processing capabilities of non-neuronal cells). Bioinformatics is in its infancy, dealing with primary gene/protein sequences but having no formalism or computer-aided tools to formulate or test models of pattern formation based on the enormous glut of molecular and functional data pouring out of popular high-throughput techniques. Reductive approaches have blossomed, but the necessary corresponding syntheses of the data into actual understanding of how shape and function are controlled have not been developed. Specifically, largely missing (a significant brake to fundamental progress) are:

  • An attempt to integrate the two kinds of information and find out how the structure of complex tissue underlies its computational (information storage) capacity
  • An understanding of how bioelectrical gradients store the morphogenetic templates guiding pattern formation
  • Mathematical and computer-aided tools to help derive insights about large-scale spatial and temporal functions of complex systems from genetic pathway data; moving beyond the avalanche of expression and interaction data to patterning properties would revolutionize many fields
  • A realization that proteomic and genomic profiling is not sufficient to capture the functional state of a cell, because physiological signals work above the protein level – dedicated efforts in "physiomics" are needed to understand cellular behaviors
  • A concerted effort to fund work at the edges of the known, where the best questions need additional data in order to be defined

This project addresses the above and strikes out in a new direction. Our work over the last few years has revealed the outlines of the radically new approach that is needed, and enabled us to produce the tools (reagents and techniques) that render this work technically feasible. Our goals will be to understand bioelectric (physiological) networks as prepatterns for gene expression domains and anatomy, and develop conceptual tools for deriving emergent properties from functional data. Using a highly interdisciplinary combination of biophysics, molecular genetics, and computer science, we will produce the following specific deliverables over the next 3 years of funding:

  • Reveal the code that maps membrane voltage gradients to organ identity and shape;

    • Uncover the molecular details of how physiological networks encode the target morphology of organisms [planarian regeneration]

    • Characterize long-range bioelectrical signaling systems to understand how non-local controls of cell behavior are utilized by a host organism [planaria, Xenopus tadpoles]

  • Understand the feedback loops and crucial temporal aspects of large-scale pattern formation and its control by bioelectric gradients

    • Develop light-based controls of physiological state [Xenopus tadpoles]

    • Develop computer algorithms and formalized descriptive systems within which patterning properties of complex systems (described by molecular-genetic, biochemical, and bioelectrical data) can be simulated and understood [planarian regeneration]

  • Understand the plasticity of non-neural tissues

    • Develop quantitative models showing how brain and body tissue structure underlies their ability to communicate, exhibit sensory and behavioral plasticity, and store memories [Xenopus tadpoles with ectopic eyes and limbs]

    • Establish models for the investigation of information transfer between somatic tissues and brains [planarian memory outside the brain]

PI: Levine, Peter
Title: Civic Education, Youth Electoral Engagement and Public Policy
Abstract:
The purpose of this project is to generate research relevant to practice and policy that enhances the equitable and informed political participation of young Americans.

A primary purpose of education in the United States has always been to produce capable, informed, and engaged citizens; that is why public schools were founded. Other educative institutions, such as families, civic associations, news media, political parties, and elections, also influence young people's civic development. Engagement should be assessed along dimensions of quantity (how many people participate in each aspect of politics?), quality (are they informed and responsible?), and equality (are people of different background equally likely to engage effectively?). We focus on young people because patterns of engagement in young adult years tend to persist.

Of all forms of civic engagement, voting is by no means the only important one, but it is fundamental; it is a universal right of citizens; and it has been found to correlate with other forms of engagement. Thus, this project focuses on young adults' voting, with a concern for quantity (measured by turnout rates), quality (measured by levels of political knowledge and understanding), and equality (with special focus on race/ethnicity and social class). In short, this is a project about informed and equal voting.

For the past 40 years, voting by youth (18-24) has remained fairly flat and significantly lower than that of older adults. Young people who do vote are more likely to be college students and come from more well-off families. Political knowledge is generally considered poor. For example, only one quarter of high school seniors reach the "proficient'' level on the National Assessment of Educational Progress (NAEP) Civics Assessment. Factors that may influence informed voting include civic education (in and out of schools), the electoral process, and the interactions between those two influences and students' social circumstances. Our research will investigate those relationships and produce evidence suitable for guiding policy decisions and practices to increase informed voting.

The project as a whole will produce a research-based, high-profile national report, supported by a set of other materials that include a literature review, a state policy scan, reanalysis of public data, and new surveys of youth and teachers. The national report, written by a diverse and distinguished group of scholars (the ''commission"), will make recommendations for policy and practice.

PI: Levine, Peter
Title: Evaluation of Democracy Projects
Abstract:
To reduce polarization, misinformation, and the corrupting Influence of money in US politics, ON's Democracy Fund has provided support for several experiments during the 2012 cycle. CIRCLE will coordinate the evaluation of six of these projects in order to help assess their outcomes and impacts – complimenting self-reported data provided by the grantees, other evaluative activities that are already planned, and observations made by ON. The findings of the evaluation will be disseminated to other funders through a funder affinity group, Philanthropy for Active Civic Engagement, as well as other informal funder networks. They will also be presented to a group of media reform and media leaders that are being convened by the New America Foundation as part of a separate grant that has already been approved by ON (which is conducting several media related experiments).

CIRCLE will undertake the following activities:

  1. Conduct formal evaluations of the six projects as briefly summarized below. CIRCLE would be responsible for IRB, data-collection, analysis, write-up, etc. for all projects.
  2. Produce draft reports for ON's private use.
  3. Prepare a shorter and more strategic report for external audiences.
  4. Plan and staff meeting of leading experts to discuss the draft evaluation summaries.
  5. Support dissemination to external audiences, including other funders.

PI: Levy, Stuart
Title: Microbial Community Activity: Communication among Soil Microbes
Abstract:
In natural environments, bacteria generally live in complex communities consisting of multiple bacterial species. In addition to signaling other members of the same species by quorum sensing, other forms of interspecies communication are important for the survival of members of these polymicrobial communities. Preliminary experiments have demonstrated a signaling between Pseudomonas fluorescens and Pedobacter. In response to a diffusible signal from Pedobacter, P. fluorescens produces an agent which inhibits growth of fungi. This project focuses on understanding how the interspecies communication works in terms of the type of signal, the genes involved in its synthesis, and the genetic response of P. fluorescens to the signal.

The studies will extend understanding of the response of P. fluorescens to signals from Pedobacter through identification of the genes expressed in response to the signal. Studies will also identify the genes required for the synthesis of the anti-microbial agent as well as genes for the synthesis of the signal molecule. Through biochemical, chemical and molecular work, we shall identify the signal compound and the anti-microbial agent. We shall determine the spectrum of activity of the antimicrobial agent by testing it against other fungi and bacteria. These studies will shed light on the molecular basis by which two different kinds of bacteria communicate with each other in the production of an agent which helps them survive in soils. The knowledge can be used in the development of agents to control microbial threats to agricultural crops.

PI: Levy, Stuart
Title: The Multiple Antibiotic Resistance (MAR) Regulon
Abstract:
Multidrug resistance (MDR) is an increasingly more common presentation of antibiotic resistance in hospitals and communities worldwide. Resistance may occur through acquisition of resistance determinants from other organisms, or through chromosomal mutations in the drug target genes or in transcriptional regulators such as MarA and its homologs, or by mutations in, or duplications of, genes for multidrug efflux pumps. MarA of the marRAB operon in E. coli controls the expression of 80 or more genes in the mar regulon. Initially discovered in E. coli, MarA homologs are found in all bacterial species examined. MarA has its own functional homologs (SoxS and Rob) in E. coli. Recent studies in this laboratory and others have demonstrated that MarA and its homologs can affect not only MDR but also influence the infectivity of the organism. Thus this single regulatory protein contributes to both MDR and virulence.

This proposal asks for funding to continue work, ongoing for two decades since the discovery of the mar locus in this laboratory, on the molecular basis for the functions of MarA and its marRAB operon. The new project period will examine:

  1. The control of expression of the marRAB operon, both by its repressor, MarR, which has recently been shown to interact directly with other cell proteins which modify its activity, as well as by other proteins that regulate marRAB; the crystal structure of MarR with DNA and other ligands

  2. The molecular requirements for MarA-mediated repression of gene expression, the interaction of MarA with RNA polymerase/DNA, the interplay of MarA with other regulators of the hdeAB operon involved in pH stress response

  3. A gene newly-found to affect MDR by controlling the amount of OmpF porin

  4. MarA-regulated products which are associated with the infectious process in an E. coli pyelonephritis mouse model

  5. MarA homologs in Serratia marcescens, an opportunistic pathogen of increasing threat to immunocompromised patients

With the E. coli marRAB operon as the focus, the proposed studies seek to define the role of the proteins regulated by MarA, including the effect of interactions of other cellular proteins with MarR and MarA on drug resistance and virulence. Work in this area should serve as a paradigm for similar regulatory proteins in other bacteria and suggest novel approaches towards preventing and curing bacterial infectious diseases.

PI: Lichtenstein, Alice
Title: Diet Quality and CVD Risk Factors in a Family Based Weight Management Study
Abstract:
The Family Weight Management Study (FamWtStudy) is designed to evaluate the effects of a 12-month, family-based intervention designed to improve BMI z-scores and CVD risk factors in children ages 7-12 years living in a community with a high minority representation, who have a BMI z-score >85th percentile. The families recruited will be randomized to either the Control or Experimental Intervention group. The Control Intervention group will receive a booklet with targets for healthy eating behaviors, a handout on how to increase physical activity, and quarterly visits that will include a brief review of lifestyle recommendations. The Experimental Intervention group will receive weekly sessions for 3 months that include a core diet and physical activity curriculum followed by monthly post-core sessions for 9 months promoting adherence to the curriculum. Targeted diet strategies in the core curriculum include increasing highly pigmented fruit and vegetable, and fish intake; substituting non-fat and low-fat for full-fat milk and dairy products; and reducing meat, fried food and savory snack intakes. During the one-year intervention period social support and tailored individualized counseling will be provided to the families, augmented with telephone calls and home visits addressing barriers to adherence. The goal of this ancillary study application is to expand the scope of the FamWtStudy to include:

  1. Measures of diet quality biomarkers in child-mother/female guardian pairs,
  2. Measures of CVD risk factors in children and mothers/female guardians, and
  3. Assessment of associations within child-mother/female guardian pairs for diet quality biomarkers and CVD risk factors pre-intervention and post-intervention change.

Each biomarker for diet quality was chosen because it is an indicator of a different component of the diet targeted by the parent study and can provide an indication of adherence – red blood cell (RBC) 18:3n-3, 20:4n-6, 20:5n-3 and 22:6n-3 for vegetable oil, meat and fish; plasma carotenoids and phylloquinone for highly pigmented fruits and vegetables; RBC 15:0 for full-fat milk and dairy products; and RBC trans fatty acids and plasma dihydrophylloquinone for fried food and savory snacks. Measuring the CVD risk factors (total, HDL and LDL cholesterol, triglyceride, hsCRP, glucose and insulin concentrations, blood pressure and waist circumference) in the children and mothers/female guardians, will allow for an assessment of ‘spill-over’ effect within families and concordance within child-mother/female guardian pairs. Regardless of the outcomes, the results will provide critical data with which to design subsequent interventions in minority communities to increase the impact of family-based obesity prevention programs and public health importance because the data will provide information in an area that continues to challenge current efforts to stem the tide of childhood obesity in a high-risk subgroup and an area that threatens to overwhelm our healthcare system. The response is time-sensitive because the FamWtStudy intervention started in August, 2009 and it is critical to have resources to collect, process, archive, transfer and analyze plasma and RBC samples.

PI: Lichtenstein, Alice
Title: Effect of Diets Enriched in Stearate Relative to Palmitate and Oleate on Inflammatory Response and Cardiovascular Disease Risk Indicators
Abstract: Although the relationship between inflammation and lipoprotein concentrations, and cardiovascular disease (CVD) risk is well established, critical questions remain unanswered about the relative effects of stearic acid (18:0), oleic acid (18:1) and linoleic acid (18:2). The comparison of 18:0 and 18:1 is of importance due to the potential conversion of the former to the later and the comparison of 18:1 to 18:2 is of importance due to the controversy about their biological similarities. Lack of data with regard to these issues impedes efforts to refine current dietary guidance and revise nutrient labels, and undermines public health efforts to promote and improve diet quality. Previously, addressing these questions was hampered by our inability to design experimental diets enriched in the fatty acids of interest. Fortunately, legislative mandates to remove partially-hydrogenated fat (trans fatty acids) from the food supply has spurred development of a wide range of plant oils selectively high in the fatty acids of interest. Consistent with our long term goal to identify optimal dietary parameters to reduce CVD risk, the overarching goal of this proposal is to simultaneously address two separate research gaps:

  1. metabolic comparability of 18:0 to its metabolic product 18: 1 and a shorter chain saturated fatty acids (using palmitic acid 16:0] as representative fatty acid) and

  2. comparability of 18:2 (polyunsaturated fatty acid) to 18: 1 (monounsaturated fatty acid) on the basis of immune status, innate immune activation and plasma lipoprotein profiles.

The experimental diets will be enriched in the fatty acids of interest by taking advantage of bioengineered safflower, cottonseed, sunflower and palm oils and will be made available to us by the supplier. The experimental diets will be consistent with the 2010 Dietary Guidelines for Americans recommendations.

Aim 1: Determine the effect of habituation to diets enriched in 16:0, 18:0, 18:1 and 18:2 on systematic inflammatory makers (TNF-α, IL-6, CRP, ICAM, VCAM, E-selectin, P-selectin and MCPI).

Aim 2: Determine the effect of habituation to diets enriched in 16:0, 18:0, 18:1 and 18:2 on the ability of T cells to proliferate and of immune cells to produce cytokines and PGE2 using whole blood culture method, and also proliferation of subset T cells (such as CD4 and CD8, naive and memory) using peripheral blood mononuclear cells (PBMC).

Aim 3: Determine the effect of habituation to diets enriched in 16:0, 18:0, 18:1 and 18:2 on CVD risk indicators (plasma lipids, lipoproteins, apolipoproteins, glucose homeostasis, endothelial function, and biomarkers of coagulation and oxidative stress).

Results from randomized controlled human dietary intervention trials weigh heavily in ongoing activities to update clinical treatment guidelines and dietary guidance for CVD risk reduction. Regardless of the actual findings, the data generated from this dietary fat trial will expand our knowledge about CVD risk indicators, elucidate mechanisms underlying the observations and yield data on innate immune response, which in turn will provide critical information for updating evidence-based nutrition recommendations and translating these recommendations into population-based guidance for CVD risk reduction. Collectively, these data will contribute to ongoing public health efforts for primary prevention at a passive level (better food options at point of purchase) and active level (improved individual food choices) to reduce population-wide CVD risk burden.

PI: Lichtenstein, Alice
Title: Nutrition and Cardiovascular Disease Predoctoral Training Program
Abstract: The objective of this proposal is to obtain funds for the training of highly qualified PhD scientists committed to a research career in the area of nutrition and cardiovascular disease (CVD) at a basic, clinical, epidemiological, and/or translational level. Despite advances in the past two decades, CVD is still the leading cause of disability and death in the U.S. It costs the nation approximately $432 billion annually, a figure in excess of 20% of the total national healthcare expenditures. Given the continued demographic shift in the U.S. towards the older age groups, this burden is predicted to increase. Lifetime risk for CVD and median survival rate is strongly associated with risk factor burden at age 50 years. Understanding the underlying mechanism(s) of this association and refining optimal approaches to minimize CVD risk factor burden is critical to advancing approaches to decreasing CVD incidence and improving prognosis. The rational for this proposal is based on the firm belief that nutrition is the most significant modifiable lifestyle behavior that can alter CVD risk. This proposal seeks funds to train the next generation of researchers to address CVD genesis, prevention, and treatment at a molecular, cellular, whole organism, and population level. Continued support is requested for the four predoctoral training slots for each of five years.

All trainees are first admitted to the PhD program at the Friedman School of Nutrition Science and Policy. After one or two years of coursework (depending on prior training) students are eligible to be admitted to the Training Program. Acceptance is predicated upon outstanding academic and research achievement, and establishing a doctoral research project in the area of nutrition and CVD. Training Program faculty have a proven record in their ability to provide exemplary training to predoctoral students. Critical components of the Training Program include didactic training in nutrition, basic sciences and epidemiology; independent mentored research in the area of biochemical and molecular nutrition or nutritional epidemiology (doctoral thesis); preparation and submission and/or publication of research in peer-reviewed journals; oral and poster presentation of research in public forums; NIH-style research proposal preparation and defense; and training in scientific ethics and research responsibility. Training Program administration and trainee supervision will be the responsibility of the program director and Steering Committee, which will meet semi-annually to review and discuss trainee selection and progress, adequacy of mentoring, and transition of trainees to independent researchers. We believe the training environment at the Friedman School and Tufts Univeristy provides an outstanding opportunity for future researchers in the area of nutrition and CVD and continued support in both the scientific future of the trainees and the public health of the nation is a good investment. 

PI: Linsenmayer, Thomas
Title: Corneal-Epithelial Nuclear Ferritin and UV Protection
Abstract:
Data suggests that avian corneal epithelial (CE) cells have evolved a novel mechanism for preventing UV-induced oxidative damage. This involves having the iron-sequestering molecule ferritin in a nuclear localization rather than the cytoplasmic location it has in other cell types. This nuclear localization of ferritin involves a tissue-specific nuclear transport molecule termed ferritoid. Recently it has been shown that ferritoid not only serves as the nuclear transporter for ferritin, but once within the nucleus, ferritoid retains its association with ferritin, where together they form a stable ferritin-ferritoid complex(es). This complex has structural and functional properties that make it unique among eukaryotic ferritins, including:

  1. Its size – which is approximately half that of a "typical", cytoplasmic ferritin,
  2. Its intrinsic low content of iron – which may make it exceptionally effective in sequestering iron and thus preventing iron-mediated oxidative damage, and
  3. Its ability to bind to DNA – where it could be most effective in preventing damage by sequestering DNA-associated iron and through physical association with the DNA.

One aim of the proposed studies is to examine further the structural and functional properties of the nuclear ferritin-ferritoid complex(es) of CE cells. These analyses will involve determining whether the complex(es) are a singular molecular type or whether there are multiple types of complexes. These analyses will utilize column chromatography, analytical ultracentrifugation, and electron microscopy. Also, as one proposed mechanism of protection afforded by the complex(es) involves abrogating the deleterious effects of free iron, analyses will also be performed on their uptake of iron.

Another aim of the studies will be to examine UV protection by ferritin-ferritoid complexes. Certain of these studies will employ a whole corneal organ culture system in which the CE cells maintain their normal, stratified arrangement and recapitulate the events observed for ferritin and ferritoid production during normal development. This organ culture system also allows the manipulation of synthesis of ferritin and ferritoid in CE cells by using the iron chelator deferoxamine – which reversibly blocks the synthesis of both components. Other studies will be performed using human CE cells.

The studies will evaluate UV protection by the endogenous heteropolymeric nuclear ferritin-ferritoid complexes and by homopolymeric complexes of ferritin and ferritoid. Lastly, the possibility will be examined that nuclear ferritin may have an additional function(s) in protecting cells from UV damage and death – which involves affecting cell signaling. The proposed experiments include analysis of developmental changes in signaling activity (before and after the developmental acquisition of nuclear ferritin) and gain- and loss-of-function experiments. These manipulations will involve UV-B irradiation followed by evaluation of cell damage and death.

PI: Liscum, Laura
Title: Building Diversity in Biomedical Sciences
Abstract: In the past ten years, SMRP has trained over 155 individuals from groups underrepresented in biomedical science. All of our trainees have completed college or are currently enrolled in college. Over 50% of those who have graduated are enrolled or have completed advanced biomedical sciences degrees. To increase diversity in biomedical sciences, the BDBSP will provide: an intensive 10-week mentored summer research experience with a focus on cardiovascular and pulmonary diseases; training in oral and written communication through experiences that build skills and enhance self-confidence; exposure to the breadth of biomedical science careers through workshops; seminars, field trips and career counseling; career models through interactions with graduate students as well as trainees in our postbaccalaureate and postdoctoral training programs that are centered on outreach to underserved individuals; and awareness of the ways in which biomedical research can address health issues, including health disparities, through seminar presentations. A group of 51 faculty, with a strong focus on NHLBI-funded research, will train 18 undergraduates each summer. A formative and summative evaluation plan will support the program and contribute to a better understanding of the ways in which programs designed to increase diversity in biomedical sciences can be most effective.

PI: Liscum, Laura
Title: Investigation of the Mechanism by Which NPC1 Dysfunction Leads to Liver Disease
Abstract:
Niemann Pick disease type C (NPC) is an autosomal recessive lipidosis that is characterized by lysosomal storage of cholesterol and glycosphingolipids. NPC patients suffer from cholestasis, prolonged jaundice, and hepatosplenomegaly. The mechanism by which NPC1 protein dysfunction leads to liver disease is unknown. We have developed a unique mouse model with which to study NPC1 liver disease. Treatment of mice with a NPC1 specific antisense oligonucleotide leads to liver specific and reversible knockdown of NPC1 protein expression. Our goal is to determine the mechanism by which lysosomal lipid storage leads to liver disease.
Our hypothesis is that NPC liver disease is initiated by the massive lipid storage, which leads to lysosome destabilization and release of pro-apoptotic proteases and lipids. Hepatocyte apoptosis is then propagated through the TNFα pathway. We expect that if NPC1 were re-expressed in the knockdown mouse, then the fibrotic liver would recover. Because our model of NPC1 knockdown is reversible, we have the ability to study disease regression.

Specific Aim #1 – To determine if the lysosomal lipid storage in NPC mouse hepatocytes leads to rupture of lysosomes and release of their contents to activate apoptosis. Our preliminary results suggest that NPC1 knockdown leads to release of cathepsins from lysosomes. We will determine if cathepsin knockdown reduces NPC hepatocyte apoptosis.

Specific Aim #2 – To determine if NPC liver disease is propagated through the TNFα pathway. Our preliminary results indicate that NPC1 knockdown in TNFα-deficient mice leads to a less severe disease phenotype. We will test this hypothesis using TNFα knockout mice, liver-specific knockdown of the TNFα-receptor, TNF-RI, and an inhibitor of the TNFα pathway.

Specific Aim #3 – To determine the course of reversal of NPC liver disease upon re-expression of the NPC1 protein in the NPC1 knockdown mouse model. Our preliminary results indicate that NPC1 re-expression leads to reduced liver injury and inflammation. We will determine the extent to which NPC1 re-expression fully reverses the hepatic disease phenotype.

PI: Loo, Cheen
Title: Post-Doctoral Training in General, Pediatric and Public Health Dentistry
Abstract:
The objectives and activities of this project include enhancement of the current program at the School of Dental Medicine and community partnerships with Tufts Medical Center to increase access to comprehensive dental services for the underserved and unserved populations of Boston. Pediatric Dentistry graduates will expand the current network of providers that can meet the oral needs of the pediatric underserved populations, including individuals with special health care needs. The recruitment and retention of minority and disadvantaged students and faculty to mirror the diversity of our local/ national pediatric population will play an integral role. In addition, this program will educate community medical providers and organizations about oral health, cultural competency, ethnic disparities and access to care issues.

PI: MacLachlan, Scott
Title: Fast Multigrid Solvers for Transport with Forward-Peaked Scattering
Abstract:
The primary goal of this proposal is to develop fast, efficient, and robust multigrid-based solvers for the solution of the linear Boltzmann-transport equation in the regime of highly forward-peaked scattering. Recently, the project team have developed an angular multigrid algorithm for a model problem that captures the essential features of the Boltzmann-transport equation for scattering in a two-dimensional "Flatland" model. The research goals of this project are, thus, to extend this approach to an efficient and effective method for true three-dimensional scattering, in realistic media, with accurate discretizations. Among the challenges of extending the already developed technique to three dimensions are improving the discretization to allow discontinuous coefficients, and local grid refinement needed in regions of interest. Additionally, the project team will investigate theoretical analysis of the convergence of these algorithms, in both two and three spatial dimensions, providing critical insight into the design of these algorithms for realistic scattering kernels.

Accurate and efficient models of forward-peaked scattering are of significant interest in both biomedical and nuclear engineering applications. This regime describes the scattering of electron beams, used in radiation therapy for the treatment of certain cancerous tumors, as well as the transport of charged particles in reactor physics and astrophysics. While there is a long history of interest in efficient and accurate algorithms for modeling charged-particle transport, the problem still poses formidable challenges. The approach considered here offers a new direction for research in this area. The proposed work leads directly to simulation tools for biomedical and nuclear engineers and scientists. The active roles of the PI and co-PI in the computational science and mathematical biology communities ensure timely and widespread dissemination of the resulting algorithms. Furthermore, the project directly involves a doctoral student, who is actively mentored by the PI and co-PI, contributing to the training of an early career scientist in an important field of research.

Mentor: Maddox, Keith
Fellow: Schultz, Jennifer
Title: I-Sharing is Caring: Using I-Sharing to Improve Interracial Interactions
Abstract:
Interracial interactions, especially those focused on race-related issues, can be fraught with anxiety and discomfort, yet people from different groups need to be able to interact comfortably to work together in general as well as to tackle issues related to prejudice and discrimination. The current research tests an intervention to promote more positive interracial interactions. Specifically, this work investigates whether shared subjective experiences with an in-group or out-group member transfer to influence cognitive, affective, and behavioral outcomes in subsequent cross-group interactions, and it examines these effects for discussions in which race-related issues are or are not salient.

PI: Maguire, Jamie
Title: Investigating GABAergic Control of the HPA Axis in the Co-Morbidity of Depression
Abstract:
It has been known for decades that there is a co-morbidity of depression in epilepsy and recently, depression has been identified as a risk factor for epilepsy, highlighting the overlap in the pathophysiology of these diseases. However, very few studies have addressed the mechanisms mediating the co-morbidity of depression and epilepsy. Stress is a trigger for both of these disorders, and we hypothesize that dysfunction in the body's stress response, mediated by the hypothalamic-pituitary-adrenal (HPA) axis, and may play a role in the co-morbidity of depression and epilepsy. A hallmark characteristic of depression is hyperexcitability of the HPA axis and seizure activity activates the HPA axis. The output of the HPA axis is mediated by corticotrophin-releasing hormone (CRH) neurons in the paraventricular nucleus (PVN), the activity of which are under robust GABAergic control. This proposal will test the hypothesis that dysfunction in GABAergic control of the HPA axis results in hyperexcitability of the HPA axis, leading to increased seizure susceptibility.

We have developed a sophisticated set of tools to test this hypothesis, including a novel, conditional knockout of one of the principal GABAARs regulating the HPA axis, the Gabrd gene. We intend to cross these mice with CRH-Cre mice to generate mice with GABAergic deficits specifically in the CRH neurons regulating the output of the HPA axis. Further, we will investigate whether an initial seizure insult alters GABAAR subunit expression in the PVN, as it does in other brain regions, thereby leading to HPA axis hyperexcitability and future seizures. Insight into the role of GABAergic control of the HPA axis in the co-morbidity of epilepsy and depression may identify novel therapeutic targets for both epilepsy and depression as well as the co-morbidity of the two, which complements the mission of the NINDS to reduce the burden of neurological diseases through research and the new strategic plan to identify new potential therapies for neurological diseases.

Mentor: Maguire, Jamie
Fellow: Lee, Vallent
Title: Role of Tonic Inhibition in Hippocampal Function and Epileptogenesis
Abstract: Epilepsy is a devastating condition with no cure as yet, and many patients suffer from seizures that are resistant to current treatment options. Many changes in the brain occur during epileptogenesis, but it has been difficult to distinguish those that are directly pathological from those that are merely compensatory. This complexity presents a major obstacle in identifying novel therapeutic targets. One potential target is the GABAA receptor d subunit, which mediates tonic inhibition in many brain regions. The expression of this subunit decreases in principal neurons in the hippocampus during epileptogenesis, but the interpretation of this change is uncertain because it is not understood why the brain utilizes two forms of inhibition, tonic and phasic. This project utilizes a novel conditional-knockout mouse line to selectively examine the function of tonic versus phasic inhibition. By dissociating the two types of inhibition, we will uncover the role of tonic inhibition in normal hippocampal function as well as under pathological conditions, such as epileptogenesis. This proposal employs a multifaceted approach, from the examining the role of tonic inhibition in individual neurons to an entire network of neurons, to animal behavior and in vivo models of epileptogenesis. The results will provide information as to the role of tonic inhibition in the CNS as well as to evaluate the potential of these receptors in the development of new treatments for epilepsy.

PI: Mann, Anthony
Title: High Energy Physics at Tufts University
Abstract: This project is part of our continued research in three frontier areas of elementary particle physics:

  • Neutrino Physics, with focus on the fundamental parameters governing neutrino state oscillations and neutrino interactions;
  • Collider Physics, studying hadronic interactions at the highest accessible energies and searching for particles heretofore unobserved whose existence may point the way to unification of the nuclear force with the electroweak interaction; and
  • Theory investigations of the spin structure and quark-gluon dynamics of protons and neutrons which may lead to more quantitative methods of calculation for systems governed by quantum chromodynamics.

Our research tasks share two overarching objectives:

  1. We seek to test the predictions of the Standard Model of elementary particle physics, and

  2. We seek to discover phenomena which lie outside the purview of the Standard Model and which present new challenges to theories of fundamental particles and their interactions.

PI: Marchesini, Danilo
Title: Size Growth at the Top: WFC3 Imaging of Ultra-Massive Galaxies at 1.5<z<3
Abstract:
The science goal of the accepted HST GO proposal is to obtain WFC3 F160W imaging of a sample of 30 ultra-massive galaxies at 1.5<z<3.0 with log(M/Msun)>11.6 to accurately measure their sizes, morphologies, and light profiles. Our objective is to confirm if size-downsizing exists, and if so, which redshift the most massive galaxies reach their final sizes. We will extend the mass range over which the stellar mass-size relation has been measured at high-redshift by a factor of ~5. Finally, we will use ACS imaging available in the archive for 60% of the sample (COSMOS) to study the occurrence of color gradients and the distribution of the stellar mass-weighted sizes in these ultra-massive galaxies.

Most of our targets are located within 30 arcminutes of each other and therefore we can obtain imaging of 2 targets within a single orbit, for a total observing time of 15 orbits for 30 targets. For each object we will take four dithered exposures. The native pixel scale of WFC3 is somewhat large (0.13") compared to the expected sizes of our galaxies (0 .1" – 0.5"). Dithers will be made with half-pixel offsets so that the images can be drizzled to improve the spatial resolution, allowing to robustly measure sizes as small as 0.1".

PI: Mason, Joel
Title: Defining the Promoting Effect of Folate on Colorectal Cancer in a Novel Animal Model
Abstract: Of great contemporary concern in the US, as well as in other countries, is the question of what constitutes a healthful dietary intake of the B-vitamin, folate. Although adequate amounts of folate intake protect against colorectal cancer, accumulating evidence indicates that overly abundant quantities among those who harbor pre-cancerous or cancerous lesions may produce a paradoxical acceleration of tumorigenesis. There are many sources of folic acid in the American diet that supplement the quantities naturally present in foods: voluntarily-fortified foods, vitamin supplements and mandatory fortification. The seminal question is whether these sources are collectively sufficient to produce the cancer-promoting effect of folate; a grave concern for which there is already supportive evidence from several pre-clinical and clinical studies.

Due to ethical and chronological constraints, this dilemma cannot be resolved by human studies alone.
Consistent with this reality are the conclusions of a recent international conference on the topic, which included among the high priority recommendations the need to pursue the issue in appropriate animal models. The overall goal of this proposal, therefore, is to answer these questions within the context of a highly innovative and powerful animal model. This novel animal model of colorectal carcinogenesis, GEM-1, allows one to monitor development of colonic neoplasms in vivo over a period of several months. This will enable us to determine critical parameters such as the threshold of dose at which dietary folate has a promotional effect and to develop a mathematical model that defines the relationship. Our proposed studies will:

  1. characterize the GEM-1 model's response to supraphysiological quantities of dietary folic acid in regard to cancer promotion,

  2. define the quantitative nature of the dose-response relationship between supra physiological folic acid intake and acceleration of tumorigenesis,

  3. determine whether delivery of supraphysiological quantities of a natural form of the vitamin, 5-methyltetrahydrofolate, lacks the cancer-promoting effects observed with the synthetic form, folic acid, and

  4. provide evidence that the molecular pathway by which excess folic acid is driving carcinogenesis is by providing abundant quantities of the rate-limiting substrate for DNA synthesis, thymidine.

PI: Matson, Douglas
Title: Electromagnetic Levitation Flight Support for Transient Observation of Nucleation Events (ELFSTONE)
Abstract:
The purpose of this project is to understand phase selection in structural alloys and to investigate the effects of convection on time-dependent nucleation phenomena during rapid solidification from the melt. This research addresses fundamental issues relating to rapid solidification behavior, metastable phase selection and analysis of the processes governing microstructural evolution.

PI: Mazzotti, Jose
Title: Documenting Isconahua (isc) in Peru: An Interdisciplinary Project
Abstract:
This project will document and revitalize the Iskonawa language, one of the 30 existing Pano languages from the Amazon. Iskonawa is almost extinct, but still fluently spoken by a few elders and partially by some of their descendants, totaling around 14 known speakers. There is also an Iskonawa community of around 80 members living in voluntary isolation on the border between Peru and Brazil. 

First, the project intends to develop a high-quality audiovisual database including lexical and textual material, testimonies, and ethnographic information. Second, the project will train the younger members of the Iskonawa community in re-learning the language, compiling material, documenting it and teaching it to future generations. The knowledge gained through the study of the Iskonawa language and culture will help to preserve a vast legacy of cultural production in the form of songs, rituals, dances and, mainly, oral narratives. Iskonawa represents a critical case of an indigenous language in a rapid process of disappearing. It has not been completely assimilated by the Shipibo-Konibo language, as linguists usually thought. 

Recent field research (summer of 2011) has demonstrated that Iskonawa still has an independent life of its own. Specialists in different disciplines form the team of researchers. Their goals transcend just language documentation and involve ethnographic compilations, community development, language revitalization and cultural empowerment.
An important feature of this project is the training and participation of both students and native speakers in language documentation, linguistic analysis and development of materials. The project will produce a comprehensive database in a website that will organize the basic information about grammar, vocabulary and the oral tradition of the Iskonawa language and people. Part of this database will be included in a trilingual DVD (Iskonawa, Spanish, English). The project will also contribute to the better education of international public and Peruvian people about the richness of their linguistic and cultural diversity.

PI: McCanta, Molly
Title: Collaborative Research: Effects of Composition and Cooling Rate on Fe XANES Glass Calibrations
Abstract:
Magmas are probes of the physical conditions and chemistry of the Earth’s interior. Exploring the mineralogy of these erupted melts provides insight into the continuing geologic evolution of the planet. In addition to basic geochemistry, parameters such as temperature and pressure are important to quantify to constrain the conditions present within the Earth. Of equal importance to the above physical parameters is oxygen fugacity (fO2), a measure of the oxidative environment a magma crystallized under. Of all the elements in magmas, iron is the only major rock-forming element that commonly exists in multiple valence states. Thus, accurate knowledge of the valence state (Fe3+/Fe2+) provides the best proxy for the amount of oxygen that was present when the minerals crystallized, and allows for estimation of magmatic oxygen fugacity. The oxygen fugacity of a magma is one of the principal parameters, along with temperature, pressure, and water content, that determines a magma’s crystallization path, as well as the composition of the resulting minerals. In order to decipher the origin of any igneous rock, it is essential to first understand the compositional differences, phase changes, and crystallization sequence variations that can be caused by magmatic processes in a closed system at a given fO2

Oxybarometers have been developed to quantify the fO2 of a magmatic system using the chemistry or partitioning behavior of multivalent elements such as iron within a mineral (e.g., FeTi oxides, pyroxene) or glass. The best-calibrated methods for evaluating Fe3+/Fe2+ are wet chemistry and Mössbauer spectroscopy, both of which use utilize bulk samples that obfuscate small-scale variations. Fe XANES allows for in situ Fe3+ measurements in glass, but there are significant analytical issues primarily related to changes in melt structure arising from composition and cooling history. Therefore composition-specific calibrations and sophisticated multivariate analysis techniques are necessary for robust microanalyses of Fe3+/Fe2+ in glasses.

The goal of this proposal is thus to provide a broad range of independently-analyzed Fe3+/ΣFe standards for glasses spanning a broad range of geologically-feasible conditions. The investigators propose a wide ranging series of experiments under carefully controlled fO2 conditions with the resulting products subjected to multiple analytical techniques to quantify Fe3+. The results of these experiments will provide a robust calibration of the Fe XANES technique for a wide range of geologically-relevant compositions and cooling histories.

PI: McCobb, Emily
Title: Tufts Shelter Medicine Program
Abstract:
The Tufts Shelter Medicine Program was formally launched in September of 2008. Our mission is to improve the health and quality of life for homeless, displaced, abused and neglected animals through the education of veterinary students, veterinarians and veterinary paraprofessionals in the principles, core competencies and best practices of shelter medicine. We accomplish this mission by working to develop a community that is more capable of meeting the needs of at risk animal populations: shelter animals, animals displaced by natural and manmade disasters, and animals belonging to underserved populations such as the low income pet owners. Specific program goals include creating a pool of educators and students who are well versed in animal welfare issues and instilling in our veterinary students a sense of urgency and obligation, driven by compassion, to give back to their communities. Our program also serves as an expert community resource on animal policy issues for vulnerable populations and conducts humane research in the field of shelter medicine with the goal of advancing the health and welfare of all companion animals.

PI: McCobb, Emily
Title: Tufts Shelter Medicine/Sterling Animal Shelter High Volume Spay Neuter Rotation
Abstract:
The goal of the proposal is to increase the number of spay and neuter surgeries performed by veterinary students at the Cummings School of Veterinary Medicine (TCSVM) and to expose veterinary students to pediatric surgical sterilization techniques. This goal will be accomplished through the establishment of a new rotation in partnership with Sterling Animal Shelter. Students at TCSVM who participate in all of our current offerings (core junior surgery course and 3 electives) through the Shelter Medicine Program have the potential to perform approximately 4 dogs spays, 4-5 cats spays, 4-5 dog neuters and 5-10 cat neuters only and they do not get the opportunity to perform surgery on pediatric animals. While spay/neuter exposure has been vastly improved from the days before the Shelter Medicine Program when students would each perform only 2 dog spays, students would benefit from a concentrated opportunity to improve their surgical skills. This rotation would allow them to perform a higher number of surgeries (15-20 or more) in a single week and would expose them to pediatric surgery in a shelter setting. The structure of the rotation would be that the students would report to the shelter Monday through Thursday and then perform surgery in the Cummings School's Lerner Spay/Neuter Clinic on campus on Fridays. Currently students who are enrolled in the Friday high quality/high volume clinic rotation only get to perform surgery on Fridays, so this experience would represent a substantial increase in their surgical exposure.

PI: McKeown, Nicola
Title: Relationship between Plasma Alkylresorcinols and Progression of Coronary-Artery Atherosclerosis in Postmenopausal Women with Coronary Artery Disease
Abstract:
Validation of plasma Alkylresorcinols (ARs) as biomarkers of whole grain intake in both postmenopausal women and free-living adults in the U.S. is lacking. Controlled intervention studies have demonstrated that plasma ARs are responsive to whole grain wheat and rye intake in small bioavailability trials. However, the use of plasma ARs as surrogate biomarkers of whole grain intake has yet to be examined in a larger study with free-living adults. This study will be the first to examine the association between a whole-grain biomarker and coronary artery disease (CAD) over a three year period.

PI: McKeown, Nicola
Title: The Role of Whole and Refined Grains in Changes in Body Weight and Abdominal Adiposity
Abstract:
The proposed project aim is to examine the relationship between whole and refined grains on the development of overall obesity and abdominal adiposity over a maximum of 13 years of follow up. We will accomplish our objective by utilizing existing data collected in the Framingham Heart Study offspring (FOS) cohort. We propose to use longitudinal measures of dietary intake and anthropometric measures collected at or between the 5th (1991-1995) and 8th (2005 -2008) examination cycles and between the 1st and 2nd exam of the Gen III Cohort. Thus, we will have a maximum of 13 years of follow-up in this study. Weight-change categories will be defined to examine the role of whole and refined grains in weight gain and weight loss, separately. Individuals will be classified into 3 categories (1) weight stable or minor weight gain/weight loss; (2) major weight gain or (3) major weight loss.

To date, few prospective studies have considered the role of whole and refined grains in the progression of abdominal obesity. Abdominal obesity is often identified by using waist circumference (WC) as a surrogate marker, since WC correlates with abdominal fat mass (subcutaneous and intra-abdominal). In adults, higher intake of whole grains have been associated with less weight gain, while higher intake of refined grains have been associated with greater weight gain. Of these prospective studies examining the relationship between weight gain and fat accumulation, few have considered the impact of underlying insulin resistance.

In a cross-sectional analysis of data collected at the 7th examination of the Framingham Heart Study Offspring cohort (FOS), a differential association was observed between whole- and refined-grain intake and visceral adipose tissue (VAT). That is, increasing whole-grain intake was associated with lower VAT in adults, whereas higher intake of refined grains was associated with higher VAT. Furthermore, after considering the joint relation between whole- and refined grain intake on VAT volume, the association between high whole-grain intake and VAT was diminished in people consuming ≥4 servings refined grains per day, indicating that refined grains may offset any potential benefits of whole grains on abdominal adiposity. To date, no prospective study has considered the joint effect of whole- and refined-grain intake on progression of weight gain/abdominal adiposity and whether this relationship differs according to underlying insulin resistance.

The strength of the Framingham Heart Study data is that all anthropometric measurements were taken by trained research staff in the Framingham Heart Study clinic according to standardized protocols, over repeat examination cycles. WC has been consistently measured at the level of the umbilicus with the participant standing. Fasting insulin concentrations have been measured in plasma as total immunoreactive insulin. Changes in body weight and WC can be defined as the difference between measurements taken at FOS exam 5 and exam 8. Approximately 2,812 of the FOS participants have anthropometric measures collected at both exam 5 and exam 8, providing us with approximately 13 years of follow-up. In addition to FOS data, dietary data collected in the third generation of the Framingham Heart Study (Gen 3) is available at exam 1 (2002-2005; mean age of 40 years). Gen 3 is a younger cohort as compared to the FOS cohort at exam 5, which had a mean age of 54 years. Gen 3 data at exam 2 (2008-2010), provides 4.5 years of follow-up in approximately 4,000 adults. As such, we propose to extend these analyses to examine the above hypotheses in a younger cohort, taking into account family structure.

Whole-grain intake will be estimated as grams per day, expressed as % of whole-grains, in addition, estimates of whole grain will be calculated with the inclusion and exclusion of added bran. This study will contribute to the literature on whole-grains and disease risk, as there is a lack of data to support the whole-grain health claim.

Specific Aims:

  1. To evaluate the influence of whole and refined grain intake on the development of overall obesity and abdominal adiposity over 3-4 y (Gen III) and 13 y (Offspring) follow-up.

    Hypothesis tested: Consuming a diet high in whole-grain foods and dietary fiber is associated with less gain in abdominal adiposity, while consuming a diet high in refined grain foods, or with a high GI, is associated with greater gain in abdominal adiposity.

  2. To examine insulin resistance as an effect modifier of the relationship between whole and refined grain intake and development of abdominal adiposity over 3-4 y (Gen III) and 13 y (Offspring) follow-up.

    Hypothesis tested: In the presence of underlying insulin resistance, higher intake of refined grains will lead to greater gain in abdominal adiposity than in those without underlying insulin resistance while a higher intake of whole grains will lead to a lower development of abdominal adiposity, mediate in part by lower insulin resistance.

PI: McLaughlin, Kelly
Title: Controlling Cardiogenesis via the Notch Signaling Pathway
Abstract:
This research is directed at discovering and characterizing molecules that mediate the differentiation of heart progenitor cells, as well as understanding the mechanism by which these molecules influence cardiogenesis. Specifically, this proposal focuses on investigating the role of the Notch signaling pathway during discrete periods of the early stages of heart development: the specification and prepatterning of the cardiac mesoderm. Although numerous studies have demonstrated roles for Notch signaling during later stages of cardiogenesis, its role during early heart development, in particular the formation of the mesoderm that is fated to become heart tissue, is not well understood and is the focus of this application. Using a newly-developed, innovative approach to manipulate Notch signaling during discrete windows of development, we are able for the first time to investigate:

  1. When does the Notch signaling pathway play a role in creating the cardiac precursor cell population that gives rise to the vertebrate heart? and
  2. What is the underlying molecular basis of how Notch signaling mediates specific stages of cardiogenesis?

For the research described in this application, we will utilize a newly developed molecular approach designed to enable the examination of discrete periods of cardiogenesis, and identify windows of development when Notch signaling functions to mediate the specification and patterning of cardiac cell fates. Subsequently, we aim to elucidate the underlying molecular mechanisms by which Notch signaling mediates early cardiac mesoderm development by examining the function of downstream mediators of Notch signaling.

Notch signaling mediates multiple, distinct developmental events during discrete windows of cardiogenesis. Moreover, although extensive studies have examined the regulation of basic helix-loop-helix (bHLH) proteins by Notch signaling, the mechanism by which specific Notch responsive-bHLH genes function during discrete windows of early cardiac development, as well as the identity of their bona fide targets, remains elusive and will be examined during the specification and prepatterning of the cardiac mesoderm.

Due to the technical challenges involved with studying the embryonic circulatory system in mammals, both the identity and function of downstream components of Notch signaling during heart development remain enigmatic. To overcome these obstacles, our lab uses a versatile amphibian model system that is both ideal for examining embryonic organ development and is amenable to working with undergraduate researchers, making it an ideal model system to integrate research and education objectives.

PI: McNinch, George
Title: Reductive Algebraic Groups in Positive Characteristic
Abstract: This project will study reductive algebraic groups G. The project will investigate the rational nilpotent classes in the Lie algebra in the case of a “local” ground field. A related issue concerns the structure and adjoint representation of the special fiber of a parahoric group scheme attached to G. Another related issue concerns optimal rank 1 subgroups of a reductive group: does any such subgroup determine a Donkin pair?

The conjugacy classes – and adjoint orbits – of a reductive group G over a field K represent an important aspect of its structure. For instance, the study of rational adjoint nilpotent orbits for p-adic groups plays an important role in the theory of smooth representations of the group of K-points of such a group. The methods to be used for the investigations of these classes continue McNinch's investigations of nilpotent orbits in the Lie algebra of G, where results from Geometric Invariant Theory have seen successful use in positive characteristic in place of the Jacobson-Morozov theorem. The main goal is to remove restrictions concerning the validity of a description – due to DeBacker – of the nilpotent orbits of the group
G(K) using the affine building associated to G by F. Bruhat and J. Tits. The project will investigate DeBacker's description from a more Galois-cohomological viewpoint. Investigations so far have led to some questions about good-filtration subgroups which have independent interest and will be investigated jointly with C. Hague (presently at Tufts).

A second component of the project concerns the action of certain non-smooth diagonalizable subgroups on a reductive group G. There remain some awkward structural omissions in the literature, which should be cleared up by investigations of the project. Moreover, in the case of the multiplicative-type group M = µp, the project will investigate – jointly with Paul Levy (U. Lancaster, UK) – the polynomial invariants on a representation of the centralizer of M on an M-weight space in the Lie algebra of G.

The most important examples of multiplicative-type subgroups of G are the maximal tori. Several results in the literature concerning the harmonic analysis of G(K) for a local field K of characteristic 0 depend on the finiteness of the number of conjugacy classes of such tori. For a local field having positive characteristic, this finiteness is no longer valid, and the project will investigate this difficulty.

A complete understanding of the possible mappings between reductive groups is out of reach in positive characteristic; however, a description of all completely reducible mappings should be tractable. A few years ago, McNinch-Testerman described all completely reducible homomorphisms from SL2 to a reductive group. The techniques are free of case-considerations for the image group, and the results hold over an arbitrary field. This project will investigate jointly with Donna Testerman (Lausanne) a substantial obstacle to the extension of these techniques to the case of a group of rank > 1 – namely, the lack of a suitable, general notion of “restricted” mapping.

The mathematical importance of the structure of linear algebraic group – and the substantial tools available to the investigator for their investigation – demonstrate the intellectual merit of the present project. Moreover, the project will support collaborative mathematics, it will foster the growth of an algebraic group’s community in the Tufts University math department, and it will facilitate the role of McNinch as a graduate mathematical educator.

PI: McVey, Mitch
Title: CAREER: Interactions between Error-Prone and Error-Free DNA Double-Strand Break Repair Pathways in Drosophila melanogaster
Abstract: Our ultimate goal is to characterize the dynamic interactions between various double-strand break repair pathways in the context of developmental, cell cycle, and chromatin-based cues. We have chosen Drosophila melanogaster as an optimal model system in which to initially pursue these studies for several reasons:

  1. Our preliminary data suggest that error-prone end joining is a robust repair mechanism in flies and that it may share components with other repair pathways,

  2. Innovative reporter systems have been successfully used in Drosophila to study the relative contribution of different pathways to the repair of a single, well-defined double-strand break, and

  3. An unparalleled collection of sophisticated genetic, genomic, and molecular tools has been developed in this multicellular model organism.

We expect that the knowledge gained during our investigations will have direct relevance to the study of genomic stability and its relationship to human disease. In addition, it will provide insight into the mechanisms by which error-prone DNA repair pathways can promote genetic diversity.

To accomplish our goals, we will use a multi-pronged approach that employs both in vivo and in vitro methods. Our first aim is to further develop and refine assays that monitor the relative use of multiple pathways involved in the repair of a single double-strand break. These assays will provide quantitative data concerning repair pathway choice in various genomic contexts and will allow recovery of individual repair events for subsequent analysis. Our second aim is to identify and characterize the genes involved in the process and regulation of error-prone end joining. We will employ a candidate gene approach, based on knowledge gleaned from other organisms and from the Drosophila genome sequence, to assess the involvement of likely genes. In addition, we will screen for other mutations that alter the normal balance between error-free and error-prone end joining. We will synthesize data from these first two aims to formulate a model describing the mechanism and regulation of error-prone end joining. Our third aim is to determine how this basic model is impacted by cellular cues specific to different tissue types and developmental stages. Together, these three objectives represent crucial initial steps that will lay the groundwork for a comprehensive understanding of the regulation of DNA double-strand break repair in a multicellular eukaryote.

PI: McWayne, Christine
Title: Partnerships for Early Childhood Curriculum Development: RISE
Abstract:
RISE curricular materials and approaches will build on the research team's work in early STE teaching and learning (Betty Zan, Kimberly Brenneman, & Daryl Greenfield), approaches to learning (Christine McWayne & Daryl Greenfield), and effective home-school collaborations (Christine McWayne & Jayanthi Mistry). The design team will include educators and parent leaders, alongside the research team. Over the course of three years, an iterative design and evaluation process will be used to develop curricular approaches and materials. Initial curricular materials and resources that both integrate content/activities across the STE domains and incorporate information from families will be developed in Year 1. We expect that five Mentor Teachers (MTs) will do some initial piloting of activities and approaches as these are drafted throughout Year 1 and that fidelity measures will be developed and piloted simultaneously. Formative assessments will inform redesign for Year 2 when the materials will be implemented and further refined by these same five teachers.

The Year 2 implementation study will involve both qualitative and quantitative data sources, collected to assess changes in teaching practice, classroom quality, teachers' efforts to engage families in discussion groups, and child outcomes. Fidelity of implementation data will be collected via teacher survey and live and video observation. In Year 2, pre-post data will be collected from teachers (beliefs, attitudes, instructional quality) and for 10 randomly selected children in each of the five classrooms using standard measures. Project-specific measures of learning for STE concepts and skills will assess learning from specific units and activities. Thus, Year 2 data will allow for: the assessment of fidelity with which MTs implement project activities; continued modification of the RISE curriculum based on this information; and codifying procedures for fidelity monitoring in the full curricular intervention year – Year 3. In the summer between Years 2 & 3, 5 five additional expansion teachers (ETs) will be trained in the RISE approach, including classroom-based STE experiences and HSC methods.

In Year 3, the curriculum will be implemented and evaluated in all 18 participating classrooms across the two programs (10 intervention; 8 control), with the continual support of the Research Team. Pre-post data will be collected from teachers (beliefs, attitudes, instructional quality) and for 10 randomly selected children in each classroom as in Year 2 and fidelity of implementation data will also be collected. Across all years, formative assessment data will be collected and an external evaluator will assess the extent to which each phase of the project is accomplished as outlined (as per NSF guidelines).

PI: Meiri, Karina
Title: Mechanosensitive Regulation of Axon Guidance
Abstract:
The usefulness of any model experimental system depends on the extent to which it is able to mimic normal in vivo responses. A case in point is models that use controlled tissue culture environments to investigate how neurons grow. Most researchers fail to account for all key influences that neurons encounter in the embryo, particularly the mechanical behavior of embryonic tissue. This omission turns out to be critical. Evidence from the PI's laboratory shows that unless the tissue culture environment mimics the mechanical status of the embryo, certain neurons are unable to respond normally to critical guidance cues. Very little is known about how mechanical signals impact neuronal responses to molecular guidance cues. These experiments address that deficit using a tissue culture model developed by the PI to account for the mechanical status of the embryo. They focus on a protein that plays a key regulatory role in how neurons respond to both mechanical and molecular signals. With the aim of unraveling mechanisms that coordinate neuronal responses to molecular guidance cues in the embryo, these investigators will use biochemical and molecular approaches to manipulate the mechanical environment. Their ultimate goal is to construct a tissue culture model with maximum in vivo relevance to both neurons and other mechanosensitive cells.

PI: Miczek, Klaus
Title: Behavioral Neurobiology of Aggression: Alcohol, GABA, and 5-HT
Abstract: The proposed research will increase our understanding of the neural mechanisms via which alcohol escalates aggressive behavior in some individuals but not in others. Violent outbursts are one of the most costly, horrifying and damaging consequences of alcohol consumption, representing one of the most significant problems for the public health and criminal justice systems. The overarching hypothesis is to assess how escalated aggression, particularly under the influence of alcohol, is a function of dysregulation of serotonergic activity in the raphe cells by feedback via somatodendritic autoreceptors and by GABAergic and glutamatergic influences, especially by feedback from the prefrontal cortex, and by CRF input.

We propose that the dysregulation of feedback control on serotonergic neurons projecting to prelimbic, infralimbic and orbitoventral regions of the prefrontal cortex characterizes those individuals who engage in escalated aggressive behavior after alcohol consumption. Specifically, experiments in mice and rats are designed to answer the following questions:

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

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

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

The experimental work relies on quantitative ethological methodology for the analysis of species-normative and escalated forms of aggression, voluntary alcohol self-administration, real time PCR, in situ hybridization histochemistry, genetic point mutations, in vivo microdialysis and HPLC, and intracerebral microinfusions. The anticipated outcome will identify targets for therapeutic interventions.

Mentor: Miczek, Klaus
Fellow: Burke, Andrew
Title: CRF Type 1 Receptor, Social Defeat Stress and Escalated Cocaine
Abstract: Reports from emergency rooms treating victims of violence and statistics from the criminal justice system on violent crimes committed by drug users as well as epidemiological evidence and neurobiological data all link social stress and drug use. Furthermore, approximately 43% of cocaine addicts also meet the DSM-IV criteria for posttraumatic stress disorder and experience of symptoms associated with this disorder is often concurrent with increased cocaine use. Only specific types of social stress can promote drug abuse and each stressor activates discrete neurobiological mechanisms. However, the precise neural mechanisms responsible for this connection have yet to be determined. Therefore, deciphering this underlying neurocircuitry will contribute to the discovery of pharmacological therapies for the treatment of addiction. 

Corticotrophic releasing factor (CRF) is a neuropeptide that mediates adaptive and maladaptive responses to social stress. We focus on CRF receptor subtype 1 (CRF-R1), based on the growing evidence and our own preliminary data that implicate this system in the mechanisms of social stress escalation in drug abuse-related behavior. We utilize a well-established social defeat method to stress rats because it is an ethologically relevant stressor for the rat. Furthermore, social defeat has been shown to increase several different aspects of cocaine self-administration that are indicative of addiction-like behaviors in the rat. 

This proposal seeks to characterize the link between CRF, social stress and escalated cocaine self-administration at the behavioral level and proceed to the study of critical nodes at the level of neurocircuitry. Initially, we will utilize pharmacological agents to activate or inhibit CRF-R1 action in specific brain regions and test for effects on cocaine-induced behavior. We focus on subregions of the ventral tegmental area (VTA) as a most relevant anatomical site for the modulation of dopamine by CRF. Dopamine cells are located in the VTA and are most often implicated in abused substance abuse behaviors. Prior social defeat experiences cause subsequent increased dopamine release in brain regions specifically implicated in addiction. Therefore we also will determine if CRF-R1 agents acting in the VTA just prior to defeat stress can block or potentiate later cocaine-induced increases of dopamine in specific brain regions using in vivo microdialysis techniques. Social defeat stress also increases later cocaine self-administration behaviors. Therefore, we hypothesize that CRF-R1 antagonists infused directly into the VTA prior to each social defeat stress experience will prevent the escalation of cocaine self-administration. We investigate aspects of self-administration that have, in the past, contributed substantially to the development of pharmacological therapies for drug addiction. Importantly, the current proposal advances our understanding of the stress-CRF-cocaine link through its focus on the social dimension of stress. Furthermore, we focus on stress-escalated cocaine self-administration as a preferential target for CRF-R1 modulation specifically in the VTA dopamine cell body region.  

PI: Miczek, Klaus
Title: Neuropeptides, Social Stress and Drugs of Abuse
Abstract: The close link between social stress and drug use is based on reports from emergency rooms treating victims of violence and statistics from the criminal justice system on violent crimes committed by drug users as well as epidemiological evidence and neurobiological data. Some specific types of social stress can promote drug abuse and trigger relapse, whereas others do not, each stressor activating discrete neurobiological mechanisms. The present application focuses on the neuropeptide CRF, particularly receptor subtype 1 (CRF- R1), based on the growing evidence and our own preliminary data that implicate this system in the mechanisms of social stress leading to escalated drug intake.

Specific Aim One tests the hypothesis that CRF-R1 modulation of discrete dopaminergic neurons is a critical mechanism for stress-escalated behavior, with a focus on different phases of cocaine self-administration (acquisition, maintenance, binge, relapse). The proposed research employs discrete intracerebral microinjections to stimulate and block CRF-R1 in discrete neural regions, in vivo microdialysis for sampling extraneuronal fluid, high performance liquid chromatography for assaying these samples to determine dopamine and other amines and behavioral measures as indices of neuroadaptive changes.

Specific Aim Two tests the hypothesis that blockade of CRF-R1 in the VTA attenuates stress-escalated cocaine self-administration, whereas antagonism of CRF-R2 intensifies cocaine self-administration.

Specific Aim Three tests the hypothesis that antagonists of CRF-R1 in the VTA will not only protect (Aim 1), but more importantly reverse social stress-induced behavioral sensitization and stress-escalated cocaine self-administration by modulating the activity VTA DA cells.

Specific Aim Four tests the hypothesis that conditional CRF-R1 knockout mice will fail to show stress-induced psychomotor and neural sensitization in response to a cocaine challenge. We hypothesize that genetic prevention of CRF 1 receptor expression in forebrain structures alters behavioral, physiological and neurochemical responses to social stress.
The proposed research on CRF-R1 in the ventral tegmental area promises to identify one critical target in the neurocircuitry of social stress for therapeutic intervention, especially in cases of intense "binge"-like cocaine intake. 

PI: Mirkin, Sergei
Title: Replication of Simple DNA Repeats
Abstract:
Expansions of simple DNA repeats are implicated in nearly thirty hereditary disorders in humans. This proposal concentrates on molecular mechanisms responsible for repeat expansions. During the previously funded period we have found that replication forks stalled at expandable repeats in mammalian cells. The length of the repeat, which caused replication inhibition, closely matched the threshold length for its expansion in human pedigrees. We have further studied the control of repeat-mediated replication blockage in yeast to discover that the fork stabilizing proteins, Tof1 and Mrc1, facilitated replication through expandable repeats. Most significantly, we have developed a new experimental system to analyze large-scale repeat expansions in yeast. The unique advantage of this system is that it allowed us to monitor expansions of the carrier-size repeats well into the disease-size range. Expansion rates were strongly elevated upon inactivation of the replication fork stabilizer, Tof1, while significantly decreased in the lack of the DNA helicase Sgs1 or the post-replicative repair regulator Rad6. Altogether these data implicate DNA replication and/or post-replicative repair in repeat instability; we will further assess this hypothesis in yeast and mammalian cells in this proposal.

We will study large-scale expansions of various tri-, tetra- and pentanucleotide repeats in our yeast experimental system by analyzing the rates of repeat expansions and visualizing the replication fork progression through various repeats. Expanded repeats inhibited gene expression in yeast as they do in human diseases. We will, therefore, analyze the mechanisms responsible for gene repression in our system by studying the effects of various repeats on transcription, RNA splicing and RNA stability. For repeats that are unstable when transcribed, we will develop a different system for their large-scale expansions, so that they are positioned in a non-transcribed area. To get insight into the genetic control of repeat instability, we will analyze repeat expansions and contractions in our large collection of mutants affecting DNA replication, repair and recombination in yeast. We will further perform genetic screens for yeast mutants that show either an increased rate of large-scale repeat expansions or a decreased rate of repeat contractions using gene disruption with a mutagenized yeast genomic library. In mammalian cells, we will evaluate replication of various expandable repeats in the pSV2neo episome using two-dimensional electrophoresis of replication intermediates.

We will also study whether expandable repeats trigger episomal fragility. Expression of mammalian homologues of the genes, which came up from the yeast screens, will be knocked down by siRNAs to study their role in repeat-mediated replication blockage and fragility. Finally, we will attempt to develop a new system for monitoring large-scale repeat expansions in human cells using a specifically designed HyTK selectable cassette combined with the Flip-In integration approach. The long-term goal of this proposal is to understand molecular mechanisms responsible for repeat expansions and contractions in humans.

PI: Moaveni, Babak
Title: BRIGE: Continuous Structural Health Monitoring Framework for Bridge Structures
Abstract:
This Broadening Participation Research Initiation Grant in Engineering (BRIGE) provides funding for the development of a probabilistic continuous structural health monitoring framework. The novel framework will allow structural damage to be estimated as a loss of stiffness in probabilistic terms. The framework permits estimation of confidence in damage identification results as a function of uncertainty in the identified modal parameters which are directly calculated from continuous measurements. This new framework will be applied to a prototype footbridge (Dowling Hall Footbridge) located at the Tufts University campus. The footbridge is exposed to a wide range of environmental conditions and is large enough to exhibit complex structural behavior, providing an opportunity for a realistic assessment of structural integrity in the presence of varying environmental effects. As part of this framework, modal parameters of the Dowling Hall Footbridge are extracted based on the low-amplitude ambient vibration response measured using accelerometers and strain gages. Separation methods are then used to remove the effects of changing environmental conditions (temperature and relative humidity) from the identified natural frequencies. The continuous stream of identified natural frequencies, mode shapes, and their statistical characteristics are fed into a recursive Bayesian finite element model updating algorithm for probabilistic damage identification.

If successful, the results of this research will improve the accuracy and confidence of damage detection algorithms, thus permitting enhanced monitoring and maintenance of infrastructure by providing a more effective engineering basis for better allocation of limited financial resources. In addition, the prototype continuous monitoring system provides a live, cross-disciplinary laboratory for integrated research and teaching in health monitoring of civil infrastructure systems. During this project, participation of students from underrepresented groups will be promoted through K-12 outreach and undergraduate research.

Mentor: Moore, Claire
Fellow: Nazeer, Fathima
Title: Mechanisms of mRNA 3' End Processing Regulation Following DNA Damage
Abstract:
The DNA damage response (DDR) is an elaborate protective mechanism against the accumulation or detrimental genetic mutations and is involved in various functions including DNA repair and prevention of cell proliferation following DNA damage. Defects in the DDR can lead to cancer. One way of controlling cell proliferation is to control the amount of mRNA made by the cell to reduce the amount of protein synthesized, thereby limiting cell growth when DNA is damaged. The levels of cellular mRNA can be controlled by reducing the levels of mRNA synthesis and/or reducing levels of mRNA maturation. mRNA maturation requires a series of processing events following mRNA synthesis and one of these events is the cleaving of the new mRNA and addition of a ‘tail’ structure at the end to help in its stability. This process requires several dedicated protein factors.

Previous work in mammalian cells show that this cleavage step of mRNA maturation is blocked following certain types of DNA damage and this requires interactions between tumor suppressor proteins and factors involved in the cleavage and ‘tail’ addition event. The significance of this blocking event is underscored by studies showing that certain cancer cell lines carrying mutations in these tumor suppressor proteins cannot block this cleavage step following DNA damage. However, the molecular mechanism involved in this blocking event is not very well understood.

My work so far has shown that both the cleavage and ‘tail’ addition steps are blocked in yeast cells following treatment with certain DNA damaging agents. I have also shown that the levels of protein factors involved in these steps change following DNA damage. Since the mammalian and yeast factors required for cleavage and ‘tail’ addition are relatively similar, I propose to study the molecular mechanism and regulation of this blocking event after DNA damage in the simpler and more cost effective yeast system. I will mostly use well characterized biochemical approaches and various yeast strains carrying mutations in relevant protein factors to carry out these studies.

At the end of this study, I will be able to contribute to the understanding of molecular mechanisms and regulatory pathways involved in controlling mRNA maturation following DNA damage. These studies could be then extended to mammalian cells to identify mechanisms that may lead to carcinogenesis. Furthermore, reducing levels of some of these mRNA cleavage and ‘tail’ addition factors have been shown to increase sensitivity to DNA damaging agents (used in cancer therapeutics) and help reduce tumorigenic properties of cancer cells. Therefore, by studying the regulatory mechanism causing reduced levels of these factors (as I have seen in my preliminary studies), we could potentially find strategies to improve cancer therapeutics.

PI: Moore, Claire
Title: Training in Education and Critical Research Skills
Abstract: The Training in Education and Critical Research Skills (TEACRS) Program serves the national need for university and college faculty trained in biomedical research who are optimally prepared to meet the multiple challenges faced by young Assistant Professors pursuing their first independent position and who understand the value of diversity in the scientific workforce. These individuals must to be fully prepared to meet the demands of setting up and managing a productive research lab, obtaining grant funding, developing and delivering exciting and effective courses and participating in the vibrant life of an institution of higher learning. They need to be able to inspire the next generations of scientists through the teaching and research opportunities they offer. Our goal is to provide talented and qualified postdoctoral scholars with the research portfolio and career skills they will need to succeed in an academic research environment that includes training and mentoring of future biomedical researchers. To achieve this goal, we have partnered with three local minority-serving institutions, the University of Massachusetts, Boston, Pine Manor College and Bunker Hill Community College. Tufts will provide these scholars with rigorous bench research training leading to the development of an independent research program as evidenced by peer-reviewed publications and also provide instruction and activities that build career skills in teaching, written and oral communication, grant writing, ethical conduct of research, laboratory management and mentoring. By working with our partners, we will offer these trainees direct, in-classroom teaching experience with a diverse student body in a mentored setting. TEACRS will also enhance the capacity of our partner institutions to deliver exciting science curriculum and increase accessibility of faculty and students at these institutions to biomedical research. We plan to gradually expand to admitting four trainees each year who will receive receiving 75% support from this grant and 25% support from their research mentor for a maximum of four years.

PI: Moss, Stephen
Title: Constructing Inhibitory Synapses
Abstract:
Gamma-aminobutyric type A receptors (GABAARs) are the principle sites of synaptic inhibition in the brain. It is unclear how neurons regulate the accumulation of synaptic GABAAR subtypes at inhibitory synapses. The impact these processes have on behavior also remains poorly understood. We hypothesize that the synaptic accumulation of GABAARs is dependent on the direct binding of conserved amino acids within the intracellular domains of the α1 and α2 subunits to the inhibitory postsynaptic scaffold protein gephyrin. This interaction is subject to dynamic modulation via the phosphorylation of serine residues within these intracellular domains, a process that determines both receptor number at synaptic sites and the efficacy of synaptic inhibition. Selectively disrupting the binding of GABAARs to gephyrin in the cortex of mice leads to deficits in paired-pulse inhibition, reminiscent of the sensorimotor deficits seen in humans with schizophrenia. To test this central hypothesis we will focus on four specific aims:

  1. To test the hypothesis that selective binding of gephyrin to a conserved motif within the receptor α1 and α2 subunits mediates binding to the inhibitory scaffold gephyrin. We will delineate the residues within the α1 and α2 subunits that are responsible for gephyrin binding and then use isothermal titration calorimetry to determine the affinities of these subunits for gephyrin.

  2. To test the hypothesis that inhibiting the interaction of gephyrin with the GABAAR α subunits modifies both receptor accumulation at synaptic sites and the efficacy of neuronal inhibition. We will compare the properties of miniature inhibitory postsynaptic currents in neurons expressing fluorescent wild-type and mutant α1 subunits, which have reduced capacity to bind gephyrin then go on to examine the effects of adeno-associated viruses that we have created on the synaptic clustering of endogenous GABAARs containing α1/α2 subunits.

  3. To test the hypothesis that phosphorylation of GABAAR α subunits by casein kinase 1 acts as a molecular switch to regulate their interaction with gephyrin. We will assess the role of casein kinase 1-dependent phosphorylation in regulating the membrane trafficking of GABAAR-containing α1/α2 subunits, their accumulation at inhibitory synapses and the efficacy of neuronal inhibition.

  4. To test the hypothesis that inhibiting the binding of gephyrin to GABAAR α subunit in the cortex leads to schizophrenic-like behavior in mice. We will evaluate if deficits in paired pulse inhibition are reversed by antipsychotics and subtype-selective benzodiazepines. We will also assess if blocking GABAAR clustering in the cortex induces other schizophrenia-related phenotypes in the absence and presence of psychostimulants.

PI: Moss, Stephen
Title: Deficits in Tonic Inhibition and the Pathology of Autism Spectrum Disorders
Abstract: Gamma aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the brain and mediates its fast inhibitory action via the activation of a type of receptor to this neurotransmitter, GABA-A receptors. These receptors mediate both phasic and tonic inhibition. It has recently emerged that modified tonic inhibition may be of significance in the pathology of autism spectrum disorders (ASDs). Modified expression of the receptor beta 3 subunit, a critical component of GABA-A receptors that mediate tonic inhibition, is common in ASDs. To directly test the role that the beta 3 subunit plays in ASDs we have created a mouse line in which the phosphorylation of this subunit has been prevented. Phosphorylation of the beta 3 subunit is an accepted endogenous regulatory mechanism to increase tonic inhibition. Our new mouse model has decreased levels of tonic inhibition and modified neuronal morphology. This mouse exhibits enhanced anxiety and decreased social interaction, hallmarks of humans with ASDs. We will use this animal to further determine the link between beta 3 subunit expression and neuronal excitability. We will also test if neurosteroids that preferentially act to increase tonic Inhibition are able to reverse the behavioral deficits in this unique animal model. By doing so will be able to provide novel insights into the pathology of autism spectrum disorders which may lead to the development of new therapies.

Mentor: Moss, Stephen
Fellow: Terunuma, Miho
Title: GABAB Receptor Phosphorylation Regulates Neuroprotective Effects
Abstract:
GABAB receptors are G-protein coupled receptors (GPCRs) that mediate slow and prolonged synaptic inhibition in the brain. Consistent with their roles in mediating neuronal inhibition, deficits in GABAB receptor function play significant roles in both neurological and psychiatric disorders including ischemia, epilepsy, depression, schizophrenia, addiction, and nociception. Previous studies from Dr. Stephen Moss' laboratory have revealed that GABAB receptors are phosphorylated by intimately associated 5'AMP-dependent protein kinase (AMPK) and this process enhances their activity and the strength of neuronal inhibition. Transient anoxia also increases AMPK dependent phosphorylation of GABAB receptors and neuronal survival. The recent finding of Ca2+/calmodulin-dependent protein kinase kinase-beta (CaMKKβ) as an upstream kinase of AMPK, suggests that an increase of intracellular calcium acts as a second pathway to activate AMPK. Given the established role of glutamate receptors in ischemia-induced excitotoxicity and neuroprotection, I will explore the link between the glutamate receptor activation and the regulation of AMPK-dependent phosphorylation of GABAB receptors. The key observation has led me to formulate a central hypothesis driving the experiments in this proposal: Prolonged exposure of neurons to glutamate inhibits AMPK-dependent phosphorylation of GABAB receptors. This deficit in phosphorylation enhances GABAB receptor ubiquitination, endocytosis from the plasma membrane and thereby reducing the efficacy of neuronal inhibition mediated by GABAB receptors and leads to neuronal death. Using cultured neurons and brain tissue from mice in which ischemia has been induced via occlusion of the middle carotid artery. I will use a combination of molecular and cell biological approaches to address the following specific aims. My effort will center on three complementary but distinct experimental goals.

  1. I will test the hypothesis that glutamate receptors modulate GABAB receptor phosphorylation and its effector coupling.
  2. I will test the hypothesis that glutamate receptor activation modulates the endocytosis of GABAB receptors via PP2A and proteasome activity.
  3. I will test the hypothesis that blocking GABAB receptor dephosphorylation promotes neuronal survival after anoxia.

PI: Moss, Stephen
Title: GABAergic Regulation of Glutamine Synthetase and Its Role in Preventing Epilepsy
Abstract: Fast neuronal inhibition in the adult brain is critically dependent on the ability of neurons to synthesize the inhibitory neurotransmitter γ-aminobutyric acid (GABA) that mediates its actions via ionotropic GABAA and metabotropic GABAB receptors. Deficits in GABAergic inhibition are central to epilepsy and a plethora of other neuropsychiatric disorders. The major metabolic precursor for GABA synthesis by neurons is glutamine, which in turn is supplied by astrocytes. The ability of astrocytes to export glutamine is dependent upon the activity of the astrocyte-specific enzyme glutamine synthetase (GS). The significance of GS for brain function has been revealed by the use of specific inhibitors and gene deletion. These manipulations lead to seizures and death that result from decreased synaptic inhibition. Consistent with this, deficits in GS expression are found in the brains of epileptics and animal models of epilepsy. To date, however, there have been no systematic experiments to evaluate how the activity of GS is regulated to meet the demands of neurons for glutamine, and if deficits in these processes contribute to epileptogenesis. These issues will be addressed here. Preliminary results suggest that GS expression is subject to powerful regulation by astrocytic GABABRs. GABABRs are heterodimeric G-protein coupled receptors which couple to Gi/o, to modulate Ca2+ transients, and inhibit the activity of adenylate cyclase. Preliminary studies have revealed that astrocytic GABABRs act to stabilize GS by reducing its ubiquitination and subsequent degradation. To understand the significance of this finding, we have created a mouse in which the expression of astrocytic GABABRs can be specifically ablated. These mice have decreased steady state expression levels of GS, spontaneous seizures, and premature death. Based on these observations we hypothesize that: Astrocytic GABABRs prevent the ubiquitin-dependent degradation of GS and thereby ensure the continued availability of glutamine for neuronal GABA synthesis. This proposal will center on three aims that are detailed below:

Aim 1. To test the hypothesis that astrocytic GABABRs regulate the stability of GS.
Aim 2. To test the hypothesis that ablating the expression of astrocytic GABABRs results in GS degradation, spontaneous seizures and death.
Aim 3. To test the hypothesis that reducing astrocytic GABABR expression compromises synaptic inhibition and neuronal viability.

Together these experiments will provide unique insights into the role that astrocytic GABABRs play in regulating GS expression, fast synaptic inhibition and epileptogenesis. Collectively these studies may lead to the development of novel therapies to increase the activity of GS to alleviate the burdens of epilepsy  
Our studies are intended to provide insights into the role that gamma aminobutyric acid type B receptors play in regulating a specific enzyme found only in astrocytes. These insights have the potential to lead to the development of novel therapies to modulate the activity of this enzyme and ultimately to alleviate the burdens of epilepsy.

Mentor: Moss, Stephen
Fellow:
Comenencia-Ortiz, Eydith
Title: Neurosteroid Modulation of Extrasynaptic GABAA Receptors
Abstract:
Gamma-aminobutyric acid type A receptors (GABAARs) are the principal mediators of inhibitory transmission in the mammalian central nervous system. GABAARs can be localized at postsynaptic inhibitory specializations or at extrasynaptic sites. While synaptic GABAARs are activated transiently following the release of GABA from presynaptic vesicles, extrasynaptic GABAARs are activated continuously by resting concentrations and thus mediate tonic inhibition. These extrasynaptic GABAARs are predominantly composed of α4 β2/3 and δ subunits, exhibit high affinity for GABA and have little or no desensitization. Steroid metabolites of progesterone and deoxycortisone (known as neurosteroids) have been shown to be potent positive allosteric modulators of extrasynaptic GABAA receptors. However the exact mechanisms by which neurosteroids alter extrasynaptic GABAARs function are not well understood. Previous experiments have suggested that Protein Kinase C (PKC) activity is required for neurosteroid-mediated modulation of GABAARs.

We hypothesize that neurosteroids mediate PKC phosphorylation of extrasynaptic GABAARs to alter receptor function. In order to test this hypothesis, our laboratory has developed DNA constructs that were critical serines for PKC phosphorylation have been mutated to alanines (α4S443A and β3408/409A). I have first examined how these point mutations in PKC phosphorylation sites within α4 and β4 subunits alter the neurosteroid-mediated changes in the magnitude of GABAergic currents. I have shown that the rundown of α4β3-mediated GABAergic currents is prevented by phorbol ester stimulation of PKC and by the S443A mutation in the α4 subunit. I will assess the physiological significance of mutations in PKC phosphorylation sites within the α4 and β3 subunits and determine whether PKC-dependent phosphorylation plays a role in neurosteroid-mediated enhancement of tonic inhibition. These studies will utilize knock-in mice in which sites of neurosteroid-induced phosphorylation of GABAARs have been mutated, which will also allow us to examine the behavioral significance of this phenomenon. Neurosteroid levels are known to fluctuate during various physiological states and pathophysiological conditions. The work proposed in this grant will provide insight into neurosteroid-mediated alterations in neuronal excitability and might contribute to the development of novel pharmacological targets for the treatment of postpartum depression, epilepsy and anxiety disorders.  

PI: Moss, Stephen
Title: Phospho-Dependent Regulation of GABAB Receptor Membrane Trafficking and Its Role in Determining Neuronal Survival
Abstract:
γ-aminobutyric acid type B receptors (GABABRs) mediate slow and persistent synaptic inhibition in the brain. GABABRS are the prototypic G-protein coupled receptors composed of GABABR1 and R2 subunits. Recent studies from our laboratory have revealed that activity of post-synaptic GABAB receptors, which are enriched on spine necks, is subject to modulation via the activity of N-methyl-D-aspartic acid (NMDA)-type glutamate receptors. Prolonged activation of NMDA receptors (NMDARs) leads to increased endocytosis and enhanced lysozomal degradation of GABAB receptors, a process that is dependent on protein phosphatase 2A-dependent dephosphorylation of serine S783 with the GABABR2 subunit. To analyze the significance of this process we have created mouse lines in which S783 has been mutated to an alanine residue to block NMDA-dependent dephosphorylation. Our preliminary studies have demonstrated that NMDAR-dependent degradation of GABABRs is ameliorated in these animals. We will thus explore the significance of NMDA-dependent degradation for the efficacy of GABAB-mediated neuronal inhibition in animal behavior and determine its role in cognition and in mediating neuroprotection after ischemic injury.

PI: Murray, Maureen
Title: Rodenticides in Four Species of Birds of Prey: Assessing Results of Recent EPA Action
Abstract:
This study will investigate exposure and risk of toxicosis in four species of birds of prey from several rodenticides (rodent poisons) which have been impacted by regulations enacted by the U.S. EPA in June 2011. These new regulations prohibit the sale to general consumers of one category of rodenticide, the second generation anticoagulants (SGARs), which have been shown to cause illness and death in birds of prey. However, SGAR use will still be allowed by licensed pest professionals and agricultural users, allowing them to potentially remain in the food chain. The restriction of SOARs from the general market has led to their replacement with two other categories of rodenticides, the first generation anticoagulants (FGARs) and the neurotoxic agent bromethalin. The risks of FGARs to birds of prey are not well studied, and there is little information on the potential effects of bromethalin. This project will be the first to investigate the risk of bromethalin to birds of prey. The study population will be four species of free-living birds of prey from an area where widespread exposure to SGARs prior to new EPA regulations has been documented. This study presents a rare opportunity to evaluate whether recent EPA action will have the intended result of decreasing the threat of poisoning from SGARs in wildlife. Additionally, this study will begin evaluating SGAR alternatives, the risks of which to wildlife are not well understood, as their availability to general consumers increases following EPA action.

PI: Nephew, Benjamin
Title: Central Vasopressin and Maternal Behavior
Abstract: The mission of the NICHD is to ensure that every person is born healthy and wanted, that women suffer no harmful effects from reproductive processes, and that all children have the chance to achieve their full potential for healthy and productive lives, free from disease or disability, and to ensure the health, productivity, independence, and well-being of all people through optimal rehabilitation. Postpartum anxiety and depression- associated disorders can have negative effects on the health of both mother and offspring through effects on maternal behavior, and little is known about the etiology of these disorders. Although chronic stress is a significant risk factor for depression, it is unknown how chronic stress during lactation affects maternal behavior. Recent molecular studies indicate that the neurohormones arginine vasopressin (AVP), oxytocin (OXT), and corticosteroid releasing hormone (CRH) may be involved in the modulation of maternal behavior in lactating rats. It is hypothesized that the central AVP system may be a potential target for the treatment of postpartum behavioral disorders, as central manipulations of AVP alter behavior. The overall objective of the current protocol is to investigate the role of AVP in postpartum maternal behavior and aggression, and the effects of social stress on this system. The specific aims of the current proposal are to characterize the roles of central AVP, OXT, and CRH in the control of maternal behavior and maternal aggression, investigate the effects of chronic social stressors on these behaviors and central AVP, and investigate a potential AVP-mediated mechanism for the effects of chronic social stress on maternal behavior. These aims will be pursued using behavioral, endocrine, and molecular methods to quantify the effects of chronic social stressors, molecular and imaging (fMRI) techniques to identify the neural regions implicated in the acute and chronic control of maternal behavior, and stereotaxic surgical techniques to manipulate central AVP activity. The overall goal is to gain valuable training in behavioral neuroscience techniques and study the neuropeptidergic control of behavior. The long term career goal is to develop new targets and treatments for behavioral disorders of mothers.

PI: Ng, Wai-Leung
Title: Signal Transduction Mechanisms in the Major Quorum-Sensing Circuit of Vibrio Cholerae
Abstract: The goal of this research is to understand the molecular mechanisms underlying quorum sensing, a cell-cell communication process that allows bacteria to coordinate population-wide gene expression and function as coordinated groups. Quorum sensing relies on the production, detection, and response to chemical signal molecules called autoinducers. Disruption of any one of these steps renders pathogenic bacteria avirulent. The research proposed here will provide an understanding of the molecular mechanisms underpinning signal production, detection and response in quorum-sensing which will enable the design of interference strategies that can be developed into new therapies to combat infectious diseases.

The human pathogen Vibrio cholerae, the causative agent of the disease cholera, regulates virulence factor production and biofilm formation through quorum sensing via a new class of autoinducer, called CAI-1 ((S)-3-hydroxytridecan-4-one). CAI-1 is produced by the CqsA synthase and detected by the transmembrane histidine kinase receptor CqsS. Histidine kinase receptors are ubiquitous in bacteria and play important roles in bacterial pathogenesis, nonetheless, little is known about their molecular mechanism of signal transduction across the membrane. This is, in part, due to a profound lack of chemically defined ligands for histidine kinases. By determining the biosynthetic pathway for and structure of CAI-1, and examining the CAI-1-CqsS interaction as well interactions between CqsS and a series of related, synthetic molecules, my postdoctoral work demonstrated that the CqsA/CqsS/CAI-1 signaling pathway offers an unprecedented opportunity to study ligand-receptor interactions and transmembrane signaling by histidine kinase receptors. With this work as the foundation, going forward, my first aim is to use CAI-1/CqsS as a model system to define how this important class of receptors recognizes and propagates external signals internally into the cell. My second aim focuses on co-evolution of signal production by the CqsA synthases and signal recognition by the partner CqsS receptors. As a postdoc, I showed that signal production and signal recognition in each vibrio CqsA/CqsS pair are matched. That is, either a particular CqsA synthesizes multiple related molecules and the cognate CqsS receptor detects them all, or the CqsA enzyme produces one specific molecule and the cognate CqsS receptor is exquisitely selective for detection of that ligand. The biological significance of this co-evolution and the molecular mechanism that specifies stringency or lack thereof are unclear and I will study this phenomenon. Our understanding of the role quorum sensing plays in V. cholerae physiology is limited to virulence factor production and biofilm formation. However, the V. cholerae quorum-sensing regulon is composed of more than 100 genes in addition to those known to be involved in virulence and biofilm formation. My recent work showed that V. cholerae mutants unable to activate a quorum-sensing response are exquisitely sensitive to low pH. In my final aim, I will study the molecular mechanisms underpinning quorum-sensing dependent regulation of Acid Tolerance Response. The long term goal is to define the roles of V. cholerae quorum-sensing response in coping with the changes in the environment and in the host. 

It is now well established that quorum sensing is employed by many bacterial species to regulate both harmful and beneficial traits. A long standing goal of the quorum-sensing field is to develop pro-quorum-sensing and/or anti-quorum-sensing small molecules to manipulate these behaviors. This study will make progress toward this important goal.

PI: Ngatia, Muthoni
Title: Social Interactions and Individual Reproductive Decisions
Abstract:
I would like to carry out a follow-up survey to a randomized experiment I carried out in Central Malawi looking at the role that social networks play in individuals’ decision to get tested for HIV. I find that individuals’ probability of getting tested is increasing in their own payment to test. Secondly, I find a significantly negative peer effect of having a social contact that gets tested with a low experimental payoff and that each additional dollar received by a social contact to get tested mitigates this negative effect. These findings are robust to looking separately at male and female contacts and comparing peers and relatives.

I would like to examine the following questions, to help understand the results from the experiment and measure additional outcomes from the study.

  1. Patterns of information dissemination: I will ask respondents to list the people whom they told their IDV status, and whom they encouraged to get tested. One of the hypotheses I am testing is whether there are strategic substitutes in testing. Social networks are characterized by homophily, that is a strong tendency to form links with partners similar to one’s self. In that case, if a node’s links get tested and tell her their results, she might be less likely to get tested herself since her results will be the same as those of her social contacts.

  2. Changes in knowledge, stigmatizing attitudes and self-reported behavior: I will compare knowledge about HIV and stigmatizing attitudes at follow-up with data collected at the baseline to see whether the intervention had long-lasting effects on knowledge, attitudes and behavior, one year later. An ongoing debate among practitioners is whether there’s any value to testing on long-run behavior. Thorton (2008) finds little change in self-reported behavior or condom purchases after individual's learning their HIV status.

  3. Changes in the social network: I will also resurvey the social networks of study respondents to see whether the intervention changed networks in any way. Research reports that people living with HIV/AIDS face social isolation from family, friends and the larger community. Since the experiment provides random variation in who finds out their status, I can provide direct evidence on whether this is true.

PI: Olum, Ken
Title: Cosmic String Network Simulation and Analysis
Abstract:
Cosmic strings are microscopically thin or even fundamental objects of cosmological length. They can arise from symmetry breaking in field theory or from superstring theory. They can produce observational signatures in the cosmic microwave background, in cosmic rays, in ultra-high-energy neutrinos, or in gravity waves. If cosmic strings are detected, they will provide a window onto fundamental physics and energies beyond the reach of any accelerator. Over the past three years, the Tufts cosmology group has developed a large, parallel computer simulation of cosmic string networks. This simulation can handle a much larger volume for a much longer time than any other. This project will make use of this simulation in order to study the shapes of cosmic string loops, the power spectrum of excitations on long cosmic strings, and the specific processes which give rise to the spectrum of cosmic string loops. All these questions are of paramount importance to the observational signatures of a string network.

PI: Omenetto, Fiorenzo
Title: INSPIRE: Resorbable Electronics – Materials, Manufacturing, and Modeling
Abstract:
This project will create an electronic platform with the ability to resorb into the surrounding environment in a benign way, at prescribed times, and with well-defined rates. This behavior is contrary to traditional expectations for conventional electronics, where the goal has typically been to achieve physical forms that display extended lifetimes. The work described will develop a scientific and engineering base of knowledge on materials, manufacturing strategies, and modeling tools for a resorbable electronics technology. This approach will enable important new classes of devices that cannot be achieved with conventional electronics. These could include medical monitors, sensors, or actuators that fully resorb into the human body at the end of their functional life and environmental monitors that eliminate the need for collection or recovery because they decay into their surroundings in a programmed way. Other concepts could include compostable circuits that will facilitate disposal of used electronics or circuits that incorporate strategic segments with timed transience to induce controlled transformation in electronic function.

PI: Omenetto, Fiorenzo
Title: Workshop: Silk – New Threads of Discovery
Abstract:
Silk-New Threads of Discovery will be held at the Tufts European Center in Talloires, France, from July 20th through July 23rd, 2012. This highly interactive workshop, organized by Professors Fiorenzo Omenetto and David Kaplan (Tufts University) and Rajesh Naik (Air Force Research Lab), will tackle the future directions in research and product development around silk biomaterials. The invitees, who will include program managers from funding agencies, industrial partners, and academics, will explore funding opportunities and development paths.

Silk is an active topic of research as evidenced by the increasing number scholarly articles that are expanding its reach into interdisciplinary domains of research that encompass materials science, nanotechnology, sensing, and biomedical devices. Though silk is featured in the media with increasing frequency, challenges remain in harnessing the protein's behavior and capturing the power of its diversity.

This workshop will build on the success of the first silk workshop ("Silk: Is it all spin?") held in 2011 at the New York Academy of Sciences and continue in the goal of bringing together an international group of interdisciplinary academics and industrial practitioners that are actively involved in silk research. The assembled group is highly interdisciplinary and links diverse backgrounds ranging from bioscience, chemistry, physics, materials science and engineering. We hope to establish this conference as a reference workshop to discuss the current state of the art, assess progress, and identify opportunities and key questions for the future.

The format of the workshop will include focus discussions around 5-interlinked scientific topics over 2.5 days, and be arranged so that each successively builds upon the knowledge transfer from the previous section. The focus of the sessions will be generated by short presentations; an opening presentation from members of the panel outlining the objectives for discussion, followed by the relevant group from the proposed participants giving a brief overview of the recent progress in this field. Each session will be wrapped-up by a round table discussion as to what objectives have been met in light of the talks and where future effort is best focused.

PI: Ordovás, José
Title: Aragon Workers Cardiovascular Healthy Study (AWCHS)
Abstract: The societal burden of cardiovascular disease in terms of mortality, morbidity, and economic cost is staggering. Preventive strategies, however, have been limited by the modest discrimination ability of available prediction algorithms to identify individuals at high risk of cardiovascular events (e.g., Framingham or Score algorithms). New technologies, including imaging, genomic, and high-throughput metabolomic techniques, hold considerable promise in providing novel information that may help increase the sensitivity and specificity of available risk factor approaches to identify individuals at high risk.

Hypothesis:

This study will provide unique data on novel determinants of the development and progression of atherosclerosis and it will take advantage of imaging, genetic, and metabolomic techniques already used in the HRP-Biolmage Study, a prospective study to determine the value of advanced Imaging, genetic, and metabolomlc techniques for improving prediction of cardiovascular events being conducted in Chicago.

Objectives:

  1. To identify the prevalence of subclinical atherosclerosis & its progression over 5 years in a population of apparently healthy middle age subjects using a combination of traditional & advanced imaging.

  2. To apply high-throughput metabolomic and genomic techniques to identify novel biochemical and genetic determinants of the prevalence & progression of subclinical atherosclerosis in the study population.

  3. To identify novel biochemical and genetic markers associated with the prevalence and progression of established cardiovascular risk factors, including diabetes, metabolic syndrome and its components, hypertension, hyperlipidemia, excess adiposity, and inflammatory and oxidative stress markers.

  4. To develop prediction algorithms.

Approach:

In order to close some of the current knowledge gaps & contribute to a better prevention of cardiovascular diseases in the aging population, we propose to carry out the most sophisticated cardiovascular imaging techniques in combination with genomics & metabolomics in a large cohort of over 8,000 individuals. Moreover, we will use a longitudinal design that has been proven to be invaluable to accomplish these objectives without the bias of case-control studies. All Aragon Workers Cardiovascular Health Study (AWCHS) participants have detailed determinations of cardiovascular risk factors. In addition, a group of individuals potentially at higher risk will undergo carotid and aortic echocardiography, ankle-brachial blood pressure index, and calcium coronary scores. These techniques will be repeated after 5 years to establish atherosclerosis progression.

PI: Panetta, Karen
Title: INDIVIDUAL: Nomination of Dr. Karen Panetta for Innovations in Engineering Education Mentoring for Attracting and Retaining Women in Engineering through Her Nerd Girls Program
Abstract: In this program, Dr. Panetta helps individuals build confidence in their research skills by creating the connection between math and science using real world projects that serve humanity. 98% of all students graduating from Dr. Panetta's program pursue a graduate degree within 3 years of receiving their undergraduate degree in engineering. Her work has contributed substantially to our success in attracting and retaining female students. Indeed, Tufts University is now recognized in the ASEE Prism Magazine as a top 20 university for recruiting women in engineering. In addition, Dr. Panetta has conducted outreach and mentoring activities across the globe for thousands of students and educators to help youth realize their potential to positively affect the world.

Over 25 years ago, Karen Panetta recognized that female students needed to be encouraged to stay in engineering and overcome the negative social and media portrayal of engineers. This, coupled with the lack of public awareness for the role that engineers serve in society, has created barriers discouraging young girls from pursuing STEM careers. Based upon this perceived need, Dr. Panetta developed an innovative program, entitled the "Nerd Girls", which utilized the diverse talents of female and minority engineering student teams and state-of-the-art technologies to solve real world problems. The project learning approach teaches students to use their engineering and team skills to take on challenging interdisciplinary projects that help improve the quality of lives for individuals, communities and the environment. The students interact with professional engineers, government agencies and conduct research, while disseminating their experiences to young girls in K-12. They serve as young role models that dispel negative "geek" stereotypes of female engineers and scientists.

This award will recognize Dr. Panetta's international success in developing programs for attracting and retaining women in engineering. It will also provide funding to support more community based Nerd Girl engineering projects, including designing solar energy systems for a lighthouse, a wireless remote monitoring system and a water filtration system for a New England island that is home to a National Fish and Wildlife preserve. These highly visible public projects allow communities to see first-hand what engineers do and provide students the opportunity to develop their engineering design skills.

PI: Panjwani, Noorjahan
Title: Pathogenesis of Acanthamoeba Keratitis
Abstract: Acanthamoeba keratitis (AK) is a serious, debilitating, and intensely painful infection of the cornea caused by parasites of the genus Acanthamoeba. At present, diagnosis of the disease is not straightforward and treatment is very demanding. During the current funding period, we have characterized a major virulence protein of Acanthamoeba, the mannose-binding protein (MBP) that mediates the adhesion of parasites to host cells. Specifically, we have cloned and characterized the Acanthamoeba MBP and have shown that it is a transmembrane protein with characteristics of a typical cell surface receptor. Recent studies have revealed that normal human tear fluid contains anti-MBP IgA antibodies that inhibit the adhesion of parasites to host cells and that, compared to tear fluid of normal individuals, tear fluid of AK patients contain significantly reduced levels of antibodies against the N-terminal domain of MBP, referred to here in as truncated MBP (T-MBP). Collectively, these data lead us to hypothesize that: (i) anti-MBP IgA in tears of normal individuals provide the first line of defense by inhibiting the adhesion of parasites to host cells and that (ii) the absence of sufficient quantities of antibodies against adherence-inhibiting and/or possibly other protective epitopes within the T-MBP, poses risk of infection. In Aim 1, we will characterize the structure and the function of the T-MBP epitopes, against which, the antibodies are present in normal tears, but are absent or present in reduced amounts in tears of AK patients. Because amoebae bind to host cells via a mannose-based recognition mediated by the MBP, we hypothesize that the presence of antibodies in tear fluid specifically against the carbohydrate recognition domain (CRD) of MBP should be protective. In Aim 2, using deletion mutants of the MBP, we shall identify and characterize the sequence encoding the CRD of MBP and will determine whether the CRD encompasses the protective epitope addressed in Aim 1. In Aim 3, we will test a hypothesis that subsequent to the adhesion of parasites to the host cells via the CRD of the extracellular domain of the lectin, a cascade of signal transduction events begins via the intracellular cytoplasmic (CT) domain of MBP, leading to the expression of cytotoxic proteinases that ultimately lead to the development of cytopathic effect. Specifically, in this Aim, using deletion mutants we will determine the function of the CT domain of MBP in the pathogenesis of AK. Since many of the complement proteins are mannose-containing glycoproteins, in Aim 4, we will determine whether the CRD of MBP modulates the innate immune system by influencing the function of the human complement system. The studies proposed have implications for both: (a) a basic understanding of the fundamental mechanisms of many ocular infections in general, and (b) meaningful translation to studies on patients. We are hopeful that the proposed studies will help develop novel, rationally designed strategies to manage and protect against: (i) AK in the short run, and (ii) keratitis produced by other organisms in the long run.

PI: Panjwani, Noorjahan
Title: The Role of Galectins in Corneal Hem- and Lymphangiogenesis
Abstract:
Corneal neovascularization is a vision-threatening condition affecting ~1.4 million individuals each year in the United States alone. It is associated with a wide range of ocular disorders and is the major cause of corneal graft rejection. Indeed, the frequency of rejection reaches as high as 90% for corneal grafts placed in high-risk vascularized host beds. In addition to blood vessels (BV), lymphatic vessels (LV) also play a key role in graft rejection and other ocular disorders provoked by immune response. The presence of BV and LV essentially abrogates the immune privilege status of the cornea.

The goal of this project is to understand the molecular mechanisms that regulate hemangiogenesis (HA) and lymphangiogenesis (LA) in the cornea and to develop effective strategies to prevent the growth of blood and lymphatic vessels in the cornea. HA and LA are predominantly mediated by a family of vascular endothelial cell growth factor (VEGF) receptors and integrins. In a recent study, we demonstrated that a carbohydrate-binding protein, galectin-3 (Gal3) is a mediator of the VEGF-mediated angiogenic response. Also, in a pilot study, we have shown that Gal3 promotes LA in vitro and in vivo. In the proposed study, we will characterize the mechanism by which galectins modulate HA and LA and will determine whether galectins can be exploited to prevent corneal neovascularization.

In Aim 1, we will determine whether the function of select VEGF receptors (VEGFR-2 and -3) and integrins (αvβ3, αvβ5, α5β1, α4β1, α9β1, α1β1), well-defined for their role in HA and/or LA, is modulated by Gal3. Specifically, we will determine:

  1. which specific VEGFRs and integrins bind to Gal3 and

  2. whether the binding activates the receptor and transmembrane signaling, and, if so, whether Gal3-mediated phosphorylation and activation of one or more VEGFRs is independent of its ligand, VEGF.

In Aim 2, we will determine whether any other members of the galectin family, besides Gal3, influence HA and/or LA. After establishing, which specific galectins are proangiogenic, we will characterize the molecular mechanism by which these galectins influence HA and/or LA, and will determine whether suture-induced corneal neovascularization is reduced in the select galectin(s) knockout mice.

In Aim 3, we will determine whether specific inhibitors of galectins can be used to prevent the growth of BV and LV in the mouse model of suture-induced inflammatory HA and LA. It is our hope that this study will lead to the development of novel, more effective strategies for corneal allograft survival as well as for prevention and regression of BV and LV in numerous other inflammatory disorders of the ocular surface.

The impact of the proposed study extends far beyond diseases of the cornea because HA and LA dysfunctions have been found in numerous diseases including neovascular age-related macular degeneration, diabetes, cancer, AIDS and in all solid organ transplants. 

PI: Panzer, Matthew
Title: Solid Ionogel Electrolytes for Flexible Charge Storage Applications
Abstract:
The objective of this research is to demonstrate that high-performance, safe electrochemical double layer capacitors (supercapacitors) can be effectively fabricated on flexible substrates, transforming the rigid and bulky sealed devices of today into lightweight, solid-state energy storage platforms. The approach is to utilize the in situ immobilization of an ionic liquid in contact with high surface area electrodes to form a flexible ionogel electrolyte.

PI: Panzer, Matthew
Title: Thin Film Supercapacitors Integrated Into Uniforms and Equipment
Abstract:
The technical innovation proposed here is the demonstration of a new type of ultrathin, flexible, and conformable power storage device based on the extraordinary capacitance of the electrode/electrolyte interface in a solid-state electrochemical capacitor (EC), or "supercapacitor." ECs are relatively simple devices, consisting of two oppositely charged, high surface area electrodes in contact with an ion-conducting electrolyte. In order to retain the high ionic conductivity of a liquid electrolyte in a solid-state film near room temperature, we intend to utilize polymer and/or silica network-supported ionic liquids (ionogels) as the electrolyte layer material. Inherent advantages of the ionogel electrolyte material include: nonvolatility, nonflammability, and high electrochemical stability.

PI: Paul, Ligi
Title: Micronutrient Powder Supplementation for the Control of Micronutrient Deficiency and Anemia
Abstract: Supplementation with micronutrient powder that does not alter the taste or color of the foods to which they are added has been tested for efficacy to prevent and control anemia in developing countries including Peru. Efficacy studies have shown important reductions in anemia in periods from 60-90 days, with consumption of 1 sachet every day. This study will be used to generate evidence that can be used to guide program design by providing cost-effectiveness estimates for different durations of supplementation and assist governments in setting reasonable goals for programs, based on the selected duration and frequency of supplementation.

Hypothesis:
Supplementation with micronutrient powder will control micronutrient deficiency and anemia.

Objectives:
To evaluate the status of micronutrients at baseline, midline and endline and presence of anemia and compare by daily or intermittent supplementation for 60 or 90 days. This information will assist in developing similar programs for supplementation in the elderly who are highly susceptible to micronutrient deficiencies and anemia.

Approach:
Four hundred infants 6-11 months of age will be enrolled from Cajamarca, Peru and randomized to one of the intervention groups (daily or intermittent supplementation for 6 or 12 months). Blood samples will be collected and analayzed for hemoglobin and micronutrients at baseline and endline. Micronutrients and hemoglobin will be determined from blood at midline as well as post-endline for the groups consuming supplement for 6 months. Vitamin Metabolism and Aging Laboratory will measure folate and vitamin B12 concentrations of the study samples to determine the effectiveness of the supplementation.

PI: Peattie, Robert
Title: A Combined Experimental-Computational Method to Evaluate Abdominal Aortic Aneurysm Wall Stress
Abstract: The primary aim of this multidisciplinary research collaboration is to develop an integrated approach to analyze abdominal aortic aneurysm (AAA) biomechanics, wall stress development and rupture risk for single patients. The transformative impact is that all steps of the analysis will be fully based on information exclusively derived from that patient, including lesion shape, wall material properties, experimentally measured flow field characteristics and wall pressure, and wall stress computation.

Current methods for analyzing AAA wall stress are limited by the inability to incorporate accurate wall material properties and experimentally verified flow fields, both taken from the patient being studied. Here we propose the development of a physiologic wall stress analysis (PWSA) procedure, that is, a finite element (FE) computational technique that evaluates the distribution, maxima and principal values of wall stress in models accurately replicating the shape, the non-uniform thickness and the mechanical properties of individual patient lesion walls. Wall loading will be derived from experimental pressure measurements in corresponding phantoms fabricated with patient-derived material properties matching lesion wall stiffness, under conditions dynamically simulating in vivo hemodynamic flow fields. To carry out this analysis procedure, wall tissue samples will be obtained from patient aneurysms at the time of surgery, and their material properties quantitatively characterized using uniaxial and biaxial loading-unloading sequences. A synthetic elastic material replicating the diseased wall stiffness at physiologic loading will then be developed from castable elastomers. Using that synthetic polymer, a thin-wall, elastic phantom replicating the bulge shape as determined by non-invasive imaging will be fabricated and installed in a flow loop. Bulge wall pressure distribution will be measured at a series of flow rates dynamically simulating in vivo conditions from rest-state to exercise, and used as applied loading for the finite element model. Wall internal stress distributions will be calculated, and rupture potential characterized by comparing maximum stress values to the tissue sample ultimate strength.

The proposed studies will provide the first quantitative, phenomenologic observations of pressure distributions using aneurysm phantoms with both realistic geometries and patient-derived mechanical properties, as well as the first quantitative evaluation of wall stress based entirely on patient properties. Establishment of a protocol for accurate assessment of AAA wall stress using patient specific information has the potential to significantly impact clinical AAA management.

PI: Peattie, Robert
Title: Collaborative Research: Engineering an Angiogenic Cardiac Patch
Abstract: The goal of the project is to develop and evaluate a biocompatible implant capable of preserving cardiac function following ischemic injury. By facilitating restoration of functional heart tissue, this implant will significantly improve healing and recovery after heart attack. The implant will be fabricated as a reinforced hydrogel (the cardiac patch), easily securable to the heart and capable of storing and releasing growth factors into the injured tissue. Functional tissue restoration will be achieved through the growth of a new capillary network surrounding the injured tissue in response to the implant.

Currently, 920,000 heart attacks (myocardial infarction) occur annually in the US. Inadequate blood supply to the injured heart tissue is a critical limitation to healing. Development of mature microvessel networks in the damaged tissue can therefore provide a major advance towards restoring heart function. The cardiac patch will consist of a novel composite biomaterial composed of electrospun silk fibers dispersed through a covalently cross-linked, thiol-modified hyaluronic acid and heparin-based gel, creating a synthetic extracellular matrix. Mature microvessel networks will be induced by controlled, sequential release of multiple growth factors from the gel and by synergistic interactions of those growth factors with hyaluronic acid.

In this project, we will test the hypothesis that heparin-regulated growth factor release can dramatically improve the revascularization of damaged myocardium, eliciting functional, perfused vessels that restore cardiac function after ischemic injury. Physico-chemical properties of the composite will be evaluated in vitro and its composition optimized with regard to specified design criteria. The angiogenic capacity of patches pre-loaded with selected growth factors then sutured onto ischemic hearts will be quantified by measurement of microvessel proliferation and maturity in heart samples post-implantation. The functionality of elicited vessels will then be determined by non-invasive magnetic resonance imaging in live animals over time. Such imaging will also allow determination of improvements in heart function.

PI: Pennell, Kurt
Title: Fate and Transport of Metal-Based Nanoparticles in the Subsurface
Abstract:
Despite the rapid development of nanotechnologies over the past decade, our current understanding of nanomaterial fate and transport in the environment remains quite limited. For example, it is not known how most engineered nanomaterials will interact with soil matrices, whether or not their transport can be modeled as colloidal particles using classic particle filtration theory, or how unsaturated conditions impact nanoparticle transport, retention and persistence in natural soils. Although some effort has been devoted to investigate the behavior of carbonaceous nanomaterials in the environment, a very limited number of studies have focused on the transport of metal-based nanoparticles, a particularly important class of nanomaterials because of their potential toxicity and widespread use in personal care products. For this reason, the proposed research will focus on three representative metal-based nanomaterials; nano-silver (Ag), nano-titanium dioxide (TiO2), and nano-manganese oxide(s) (MnOx).

The research program is designed to couple detailed laboratory experimentation with mathematical modeling to elucidate mechanisms governing the fate and transport of metal-based nanoparticles in quartz sands and natural soils. To achieve this goal, the research is structured around four specific tasks:

  1. Characterize metal-based nanoparticle suspensions and soil properties,
  2. measure the transport and retention of metal-based nanoparticle under water saturated conditions,
  3. measure the transport of metal-based nanoparticle under unsaturated conditions, and
  4. develop and validate mathematical models for the prediction of metal-based nanoparticle transport and persistence in subsurface systems.

Particularly novel aspects of the research plan include the consideration of surface coatings and emulsifying agents commonly used in sunscreens and cosmetic products containing TiO2 and the use of force-volume microscopy to image nanoparticles deposited on sand grain surfaces. Information gathered from each task will be integrated to advance our fundamental understanding of the mechanisms governing metal-based nanoparticle transport in porous media, with the ultimate goal of developing a numerical simulator that can be used to predict the transport behavior of engineered nanoparticles in the subsurface based on known input parameters.

PI: Pennell, Kurt
Title: Impacts of Surface Coating Aging on Nanomaterial Fate and Transport in Porous Media
Abstract:
Despite rapid advances in nanotechnologies over the past decade, our current understanding of nanomaterial fate and transport in the environment remains limited. Recent studies have demonstrated that new approaches are needed to accurately predict the transport and retention of nanomaterials in soils and aquifers. In addition, nanomaterial mobility is known to depend upon surface coating properties and the presence of stabilizing agents, such as surfactants and organic matter. Although most commercially-available nanomaterials are produced with surface coatings, very little information is available regarding their longevity and impact on nanomaterial fate over time. To address these gaps in our current understanding of nanomaterials, the project combines laboratory experiments with mathematical modeling to quantify and predict the effects of surface coating aging on the fate and transport of several representative engineered nanomaterials (iron and manganese oxides) in sands and natural soils.

The research is structured around three tasks:

  1. Determination of nanoparticle deposition and release rates as a function of surface coating properties using a quartz crystal microbalance (QCM),
  2. Measurement of nanoparticle transport and attachment in soil columns and aquifer cells, and
  3. Development and validation of mathematical models to predict the effects of surface coating properties and aging on nanoparticle mobility and persistence in soils and aquifer materials.

Novel aspects of the research include the assessment of nanoparticle deposition rates, utilization of scanning electron microscopy (SEM), magnetic resonance imaging (MRI) and light transmission (LT) analysis to visualize nanoparticle mobility, and the integration of experimental and mathematical modeling to assess nanomaterial fate in the environment. 

PI: Pennell, Kurt
Title: Secondary Impacts of In situ Remediation on Groundwater Quality and Post-Treatment Management Strategies
Abstract: The overarching goal of this project is to develop a fundamental understanding of the impacts of in situ remediation technologies on groundwater quality and relevant subsurface processes. This information is needed to realize the full potential of combined remedies, more effectively treat difficult hazardous waste sites, and develop ecologically sound long-term site management strategies.

The specific objectives of the project are to

  1. identify secondary impacts of two prominent in situ remediation technologies, thermal treatmentand anaerobic bioremediation, on long-term groundwater quality;

  2. establish methods to predict the extent of both positive and negative post-remediation impacts on groundwater quality; and

  3. develop strategies to overcome, or take advantage, of secondary impacts (e.g., pH reduction, release of electron donor) to achieve both immediate and long-term remedial objectives.

PI: Pirie, Christopher
Title: Indocyanine Green Fluorescence Angiography (ICGFA) of the Normal Canine Eye Using a Camera Adaptor
Abstract:
The purpose of this pilot study is to validate a novel and inexpensive camera adaptor for performing ICGFA. This technique is routinely performed within human based medicine; however, its use is limited within the veterinary profession, simply due to equipment costs. While dogs have been selected for this study, the target population could include any animal demonstrating ocular signs associated with an ocular and/or systemic disease requiring ICGFA imaging.

This study aims to address a significant limitation, pertaining to diagnostic imaging, within the veterinary profession. Validation of this new technology could significantly enhance our diagnostic and therapeutic capabilities, thereby improving patient outcome. This is relevant to the Morris Animal Foundation, as it could prevent, diagnose, and treat numerous sight-threatening ophthalmic conditions including uveitis, chorioretinitis, hypertensive retinopathy, and sequalae of pre-iridal fibrovascular membranes (e.g. glaucoma).

It is our hypothesis that ICGFA of the dog eye will be possible using this adaptor and that ICGFA will be a more informative technique, as compared to sodium fluorescein angiography. The objectives of this study are to determine if ICGFA of the dog anterior (e.g. iris vasculature) and posterior (e.g. retinal/choroidal vasculature) segment(s) is possible using this adaptor system and to establish a standardized protocol for its use. Additionally, we seek to document normal parameters (e.g. vascular patterns, filling times) of ICGFA of the dog eye and to compare these results with a more established imaging technique (e.g. sodium fluorescein angiography).

PI: Poltorak, Alexander
Title: Genetic Analysis of Inflammatory Responses in Wild-Derived Mice
Abstract: A central scientific question of this competing proposal's renewal is finding novel functions of immunologically relevant genes by means of classical genetic analysis in genetically diverse wild-derived mice. We have shown and continue to show that, with respect to regulation of immune responses, wild-derived mice resemble human phenotype better than classical laboratory mice. One line of inquiry continues investigation of TIRAP-dependent activation of IRAK2 followed by specific recruitment of the p38 MAP kinase, which is hyperactivated in wild- derived but not laboratory mice. We have proposed a model of MyD88-independent recruitment of IRAK2 and TIRAP in wild-derived mice, which leads to a specific activation of p38. If confirmed, this model will challenge several well-established paradigms such as simultaneous activation of MAP kinases via TLRs and MyD88-dependent activation of p38 thus broadening existing models of TLR-mediated activation and providing additional mechanistic insight. Another phenotype that we propose to investigate in wild-derived mice is their remarkable resistance to TNF-induced lethality, which is, according to our preliminary data, a genetic trait that is conferred by four loci, which we propose to identify. Given our expertise and track record in mapping and positional cloning, it is likely that we will find novel components of TNF-receptor pathway, which protect mice and presumably humans from TNF-induced lethality. To explain the trait, we generated our central hypothesis in that TNF-resistance in MSM mice is biased towards pro-survival as compared to cytotoxic signaling that leads to necrosis, and we provide feasible scientific plan to prove that. Thus, the scientific impact of this proposal is high because it will identify component, which are capable of defining the outcome of TNF-activation. In addition, the proposed genetic analysis will help identifying genes that otherwise would be difficult to predict in the absence of "strong educated guess". Most importantly, the identification of these genes will be of high relevance to human health given several hundred thousand of patients suffering each year from septic shock. In addition to cloning of the TNF-resistance, which is clearly a priority of this proposal, we provide a research plan aimed at revealing in vivo functions of two genes that we identified in the previous cycle.

PI: Pothos, Emmanuel
Title: The Effects of Magnetic Excitation on Monoamine Neurotransmitter Secretion and Cell Viability in Peripheral and Central Systems
Abstract: The human population worldwide is increasingly exposed to magnetic waves emitted from various sources (antennas, computers, cellular telephones, medical devices etc.) with unknown health consequences. Unfortunately, very little is known or established about the properties of magnetic fields (i.e. amplitude, frequency, energy) that may affect biological systems, and their eventual consequences, which could potentially be either harmful or beneficial.

Our objective is to discover the main cellular mechanism(s) through which exposure to magnetic fields becomes harmful or beneficial to biological systems. In the present study we put forward the theory that cyclotron frequencies of magnetic waves known to selectively resonate ions relevant to neurotransmitter secretion are the ones responsible for significant and possibly harmful effects on biological systems. We have in place a systematic collaboration between engineers and biologists, such as Dr. George Sergiadis of MIT Media Laboratory and Aristoteleion University, Drs. Kathleen Dunlap and Alexei Degterev of Tufts University and other consultants, in order to properly identify and manipulate the parameters involved in magnetic field excitation, and analyze their effects on neurotransmitter release from individual secretory and neural cells and cell death.

Specifically, we propose to take a systematic approach in studying the selective effects of magnetic excitation on the release of biogenic amines and cell death in rat adrenal chromaffin cells and CNS cortical neurons. Adrenal chromaffin cells were chosen as representative of secretory cells in the periphery and as model cells to study exocytosis from dense core vesicles; and prefrontal cortical slices as representative of CNS tissue typically exposed to magnetic excitation in humans. The tools we will use include carbon fiber electrophysiology to monitor real-time neurotransmitter release from individual vesicles, whole cell patch clamp to monitor Ca2+ current kinetics, real-time PCR to measure mRNA differences in regulators of monoamine release and assays to detect apoptotic and necrotic cell death.

All the preparations mentioned above will be exposed to an experimentally designed magnetic field applied through magnetic coils emitting magnetic waves at cyclotron frequencies in the range of 30-60 Hz. Within that range we will test frequencies that selectively donate kinetic energy to ions involved in neurotransmitter release, such as Na+, K+, Mg2+, Cl- and Ca2+. We have new evidence to suggest that the cyclotron resonance frequency specific for Ca2+ ions (44-48 Hz) is the one to significantly increase neurotransmitter quantal size, alter properties of Ca2+ channels, increase Ca2+ entry into the cytosol and, therefore, likely to induce cell death.

We have so far concluded and accordingly hypothesize that magnetic exposure to cyclotron resonance frequencies can affect biological systems through excitation of Ca2+ ions and alteration of voltage-gated Ca2+ channel properties.

Specific Aims:

  1. To study the effects of exposure to the cyclotron resonance frequencies given in Table 1 on monoamine release from single vesicles in real time through carbon fiber amperometry in cultured rat adrenal chromaffin cells and rat acute prefrontal cortical slices.

  2. To study the effects of exposure to the cyclotron resonance frequencies on Ca2+ currents and Ca2+ channel properties through whole cell patch clamp on the same type of preparations described in Aim 1.

  3. To study the effects of exposure to the cyclotron resonance frequencies given in Table 1 on cell death (through appropriate assays) and on mRNA levels (through QPCR) and protein levels (through immunocytochemistry) on protein regulators of monoamine neurotransmitter release in the same type of preparations described in Aim 1.

Table 1: Cyclotron Resonance Frequencies

Cyclotron Resonance Frequency in Hz for the Most Significant Ions in Biology

Ion

Earth Magnetic Induction

30 µT

50 µT

60 µT

70 µT

Li+

66.37

110.61

132.74

154.85

Na+

20.04

33.40

40.08

46.75

Mg2+

37.90

63.16

75.80

88.43

Cl-

13.37

22.29

26.74

31.20

K+

11.78

19.64

23.56

27.49

Sr2+

10.51

17.52

21.02

24.53

Ca2+

22.99

38.31

45.98

53.64

PI: Quinto, Eric Todd
Title: The Urban Math and Science Teacher Collaborative
Abstract:
The Tufts University Urban Mathematics and Science Teacher Collaborative is a collaboration among three Tufts University Arts and Sciences departments (Education, Mathematics, and Physics & Astronomy) and three Boston public schools in the 5-12 grade range (Boston Arts Academy, Fenway High School, and Mission Hill School). A local non-profit organization, the Center for Applied Special Technologies (CAST) is providing professional development around Universal Design for Learning (UDL), a framework for designing curriculum and instruction to address barriers to learning. The Collaborative is preparing 16 new Mathematics and Science Teaching Fellows (TFs), and contributing to the development of 4 Master Teaching Fellows (MTFs) over 6 years. It builds on more than 10 years of experience in the preparation of teachers for work in urban schools through the Urban Teacher Training Collaborative (UTTC), a school-based teacher residency program in which pre-service teachers work in their assigned schools full-time for a full academic year under the guidance of mentor teachers and university professors while earning a Master of Arts in Teaching (MAT) degree. Two new courses integrating science and mathematics content with pedagogy are being developed collaboratively by education and STEM faculty in the physics and mathematics departments. Ongoing professional development is provided through the creation of an online community (through Digital Toolboxes and blogs) to encourage communication and discussion among TFs, MTFs, University Participants, and CAST. The courses, course materials, examples of effective teaching, and teacher discussion forums are being built as free, open-source, cross-platform modules that universities and schools can use and modify under Creative Commons licensing. The project seeks to increase teachers' understanding of the challenges of teaching in urban settings; increase the numbers and retention of teachers of Mathematics and Science in urban schools; transform teachers' practices in Mathematics and Science; increase student understanding and performance in Science and Mathematics for students taught by TF and MTFs; and develop stronger relationships with local urban schools.

 

 

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