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Funded Research Abstracts

Mentor: Jackson, Rob
Fellow: Huang, Yanmei
Title: Genome-Wide Translational Analysis of Circadian Cycles and Sleep States
Abstract:
There are strong links between circadian timing and several forms of depression including major depressive disorder (MDD), seasonal affective disorder (SAD), and bipolar disorder (BPD). Patients with depression often display abnormal circadian cycle of sleep and wake, body temperature, mood, and hormone release. Chronotherapies such as sleep deprivation dramatically reduces depressive symptoms within hours in many patients tested. As a first step towards understanding of the relations between circadian rhythms, sleep and mood regulation, I propose to systematically identify genes and biological pathways that are regulated by the circadian clock and/or affected by sleep deprivation. The studies will be conducted in fruit fly (Drosophila), which is known to be an outstanding model organism for circadian and sleep research and extremely convenient for large scale genomics studies. I will develop novel methods for directly monitoring genome-wide protein synthesis at any given moment and apply them to identify Drosophila genes that display changes in protein translation in response to circadian events and sleep deprivation. Bioinformatics analyses will be conducted to find mammalian homologs of the identified Drosophila genes for further investigation in the mammalian model systems. The proposed studies will uncover novel genes and biological pathways that are regulated by the circadian clock, including those that are relevant to mood regulation, thus provide insight into the links between biological clock functions and depressive disorders. In addition, identifying genes and pathways that are affected by sleep deprivation might shed light on the molecular basis of the rapid antidepressant effect, thus open new avenues for drugs development and treatment strategies.

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, 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: Jacobsen, Karen
Title: Refugee Livelihoods in Urban Areas: Identifying Program Opportunities
Abstract:
We are proposing a one-year research project that will analyze the livelihoods context of refugees and identify programming opportunities in urban settings. The proposed research builds on earlier studies by the principal investigator and utilizes the experience of local partners and in-country researchers to identify programming opportunities and examples of successful refugee-driven program initiatives in urban areas.

Despite a growing body of research about the livelihood problems of refugees in urban areas in countries of first asylum, there is little evidence about which humanitarian programs work, what livelihoods initiatives refugees undertake themselves, and where opportunities for programming interventions lie. Our research seeks to address this knowledge gap. We will do this by conducting a global desk review of urban livelihoods and program initiatives, and then focus on two urban case studies – Cairo and Quito. These cases were chosen because they represent contrasting refugee policy contexts and livelihoods experience, and offer lessons that could be extrapolated to other host settings. In both Egypt and Ecuador, we will extend our studies to other towns with large refugee populations.

The project will work closely with the following local partners:

  • Cairo – UNHCR, American University of Cairo (CMRS), Africa and Middle East Refugee Assistance (AMERA)
  • Ecuador – UNHCR, Fundación Ambiente y Sociedad (FAS)
The overall goals of the proposed study are to:
  1. Generate ideas from related fields of inquiry, including urban planning, low-income urban (slum) development, youth employment and entrepreneurial initiatives, that could be adapted for refugees in countries of first asylum;
  2. Research and analyze urban livelihoods in two countries of first asylum (Egypt and Ecuador), comparing the experience of refugees and other migrants with non-migrants, and exploring how livelihood initiatives identified in #1 could be applied;
  3. Compare findings from #1 and #2, and present these findings at stakeholder workshops so as to draw lessons and learn from the experience of different cities/regions; and,
  4. Generate a final report that includes a set of programming recommendations for donors, host governments and aid agencies.
To accomplish these goals we will conduct the following activities:
  1. Conduct a global review of the literature on urban development (see #1 above);
  2. Develop a livelihoods analysis methodology for our case studies, based on an appropriate conceptual framework and qualitative methods;
  3. Adapt the methodology for each case study and conduct data collection in two cases, analyze and write up the data into two full case studies. Each case study is expected to take six months.
  4. In each city (Cairo and Quito), hold a stakeholder workshop to compare and discuss findings and generate programming ideas;
  5. Develop explicit programming and practical recommendations for operational agencies based on the findings and the workshop discussion; and,
  6. Disseminate findings via FIC and stakeholders' websites and through practitioner-oriented and academic publications.

The principal investigator (PI) will retain overall supervision and control of the study working in close collaboration with local partners and researchers. The overall study will span one year, including the workshop and final report-writing period.

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.

PI: Jacques, Paul
Title: Project Agreement #3: Strategic Collaboration on a Healthy Aging Platform
Abstract:
The objective of the strategic collaboration on a healthy aging platform is to investigate opportunities to develop/leverage emerging technologies to deliver delicious products with naturally occurring foods/ingredients that can help aging individuals proactively manage their health, with a focus on mobility, mental acuity and metabolic balance.

Tufts University researcher(s) will participate as part of a cross functional Kraft Foods and Innosight LLC project team evaluating approaches for using foods/food ingredients to optimize health of consumers 45+ yrs. old across three strategic focus areas – mobility, mental acuity and metabolic balance. The project will involve participation with consumer research focus and ideation sessions based on the consumer research.

PI: Jacques, Paul
Title: Yogurt Consumption in Americans
Abstract:
The overall goal of the proposed project is to better understand the impact of increased yogurt consumption and develop strategies to increase yogurt consumption. To achieve this goal, it is vital to examine current consumption patterns and trends in yogurt consumption, identify determinants of yogurt consumption, and model potential effects of increasing yogurt consumption on diet quality and health markers in population-based samples of Americans. To this end, we will use available high quality epidemiological Database such as the National Health and Nutrition Examination Survey (NHANES), the corresponding USDA Pyramid Servings Database, and Framingham Heart Study Offspring cohort to address the following specific aims:

  1. Examine the dietary patterns associated with yogurt consumption, the relation between diet quality and yogurt consumption with a focus on the "shortfall" nutrients, and the determinants of its consumption in the US with a focus on personal, lifestyle and dietary characteristics.
  2. Explore the relation between yogurt consumption and biomarkers of health status
  3. Examine the long-term yogurt consumption patterns and model the impact of increasing yogurt consumption in the US diet on diet quality and health status.
  4. Identify potential biomarkers of dairy and yogurt consumption based on a review of existing literature.

Yogurt is an important source of dairy in diets in many countries, but not in the US. Yogurt could be a valuable food for increasing intake of dairy, and consequently increasing the diet quality, in Americans. This study will help document changes in yogurt consumption in the US, identify characteristics of yogurt consumers, identify possible health benefits associated with yogurt consumption and demonstrate the potential benefits of increased dairy consumption through the addition of yogurt to diets of Americans who currently do not consume yogurt.

Understanding the influences of greater dairy intake through increased yogurt consumption on diet quality and markers of age-related chronic disease is clearly within the mission of the HNRCA.

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: Alcon Ltd. Collaborative Research Agreement: Ocular Vitamins
Abstract:
This Collaborative Agreement (CA) will provide Alcon access to HNRCA scientists for questions and input into issues relevant to nutrition, the aging eye, and visual function. Topics to be covered under this CA will be pertinent to all issues in this field, including emerging research information in the area of micronutrients and phytochemicals and the design, conduct, and analyses of in vitro, animal model, and human studies associated with the maintenance of vision during the aging process. Given the role of oxidative stress, inflammation, and angiogenesis in age-related eye disease, and the preventive role of nutrients like vitamins C and E, carotenoids, and polyphenols, issues in which Alcon has interest, overlap substantially with the objectives of several CRIS units including those in the Antioxidants Research Laboratory (to investigate the role of antioxidants on oxidative stress during aging); Carotenoids and Health Laboratory (to investigate the role of carotenoids in the prevention of cataracts and age-related macular degeneration); Laboratory for Nutrition and Vision Research (to determine the primary cause of cataract and age-related macular degeneration); Vascular Biology Laboratory (to determine the mechanism by which antioxidants modulate angiogenesis); and Nutrition and Cancer Biology (to elucidate the mechanisms of action of carotenoids in carcinogenesis).

PI: Johnson, Elizabeth
Title: Fatty Acid Distribution in the Human Brain: Relationship to Cognition
Abstract:
The possibility that the carotenoids lutein (L) and zeaxanthin (Z) could prevent or slow the progression of age-related eye diseases (namely, cataract and macular degeneration) has been carefully studied. Reports on L and Z status, as measured by diet serum, and retina (also known as macular pigment (MP)) have found that increased levels of these carotenoids can be protective against these diseases. Recently, evidence is accumulating that L and Z may also have a role in cognitive health. Data from our laboratory indicates that MP, obtained by non-invasive methods, is related to cognitive function in older adults. Of interest is our research in non-human primates to find a significant relationship between L and Z concentrations in the retina, and frontal and occipital cortices.

Furthermore, our supplementation studies in older adults have found lutein to improve cognitive function. Our laboratory is currently evaluating the relationship between brain and serum concentrations of carotenoids in adult decedents for which cognitive function measures prior to death were obtained. In the evaluation of the role of carotenoids in age-related cognitive decline, it is important to determine if carotenoid concentrations in brain change with age, as has been shown for macular pigment (carotenoids embedded in the eye) with decreases being associated with advancing age.

Therefore, the purpose of this study is to determine the carotenoid concentrations in brain tissue of infant decedents and compare these levels with tissue from adult decedents. Serum will be measured as an indicator of dietary intakes. Our intent is to also analyze brain tissue from younger adults and middle-age decedents using other funding.

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: Joyner, 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 field of research on the development 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.

PI: Joyner, Valencia
Title: GRDS: Collaborative Research: 3D Integrated Imaging Receivers for 10-Gb/s Free Space Optical MIMO
Abstract:
The objective of this research is to create a new class of power-efficient imaging photoreceivers to reach an aggregated data rate of 50 gigabits per second and beyond for broadband wireless optical communication. The approach is to develop a large array of planar tessellated photodetectors with a rate of 10 gigabits per second per pixel. The array includes diversity selection circuits, which are implemented using an optical multi-input/multi-output configuration.

With respect to intellectual merit, this project addresses two well-known challenges in optical wireless communication, channel scintillation and optical beam alignment. The research explores power-efficient InGaAs metal-semiconductor-metal photodetectors that are integrated with silicon integrated circuits for multifunctional operation (decision, amplification, and computing), and the implementation of multi-input/multi-output architectures for signal processing of massive amounts of data. This research is expected to augment the body of knowledge on carrier drift characteristics in the photodetectors at low voltage bias and the physics of complex three-dimensional optoelectronic device structures. This research also explores heterogeneous integration of compound semiconductor device on silicon circuits using three-dimensional wafer bonding for photoreceiver applications.

With respect to broader impacts, the research has the potential to benefit a variety of applications, including wireless networks with reduced security vulnerabilities, networks able to transfer high-resolution digital images in the midst of equipment sensitive to electromagnetic interference in healthcare settings; and optical interconnects in large-scale data centers. A cross-university curriculum is addressed, covering multi-disciplinary topics in information theory, system-level design, analog circuits, device physics, fabrication and integration. Various programs are used to recruit students from underrepresented groups. K-12 outreach efforts are coordinated through the Tufts Center for Engineering Educational Outreach.

PI: Juo, Peter
Title: Molecular and Genetic Analysis of CDK-5 Function in Synaptic Transmission
Abstract:
The long-term goal of this research is to identify and understand the genes and mechanisms that regulate CDK-5 function in synaptic transmission. The cyclin-dependent kinase CDK-5 has diverse cellular functions during development, contributes to several neurodegenerative disorders, and has recently emerged as an important regulator of synapse function and plasticity. The focus of this proposal is to investigate the mechanisms by which CDK-5 regulates glutamate receptor (GluR) trafficking and to identify upstream regulatory signals that control CDK-5 function at the synapse. Activity-dependent regulation of the localization and abundance of synaptic GluRs directly affects synaptic strength and is thought to underlie information storage and processing in the brain. Aberrant regulation of GluRs may contribute to excitotoxicity in ischemia (lack of blood flow), stroke and neurodegenerative disorders. Thus, it is important to define the basic cell biological mechanisms that regulate GluR transport. We use C. elegans as a genetic model to study the genes and mechanisms that regulate synaptic transmission and GluR trafficking in vivo. Advantages of C. elegans include the compact genome (i.e. less gene redundancy), powerful genetic tools and ability of the animal to tolerate severe reductions in nervous system function.

Our preliminary studies indicate that CDK-5 regulates the abundance of the scaffolding protein LIN-10/Mint-1 and the glutamate receptor GLR-1 at synapses in vivo. LIN-10/Mint-1 is a PTB and PDZ domain-containing protein that has been localized to the golgi and synapses and has a conserved role in polarized transport in neurons and epithelia.

In this proposal, we will:

  1. Determine which step of GLR-1 trafficking is regulated by CDK-5,
  2. Define the mechanisms by which CDK-5 regulates the abundance of LIN-10/Mint-1,
  3. Characterize the upstream regulatory signals that control CDK-5 function.

This research may reveal novel targets for therapeutic intervention to control GluR-mediated excitotoxicity after stroke and ischemic (lack of blood flow) brain injury. In addition, since CDK-5 regulates neuronal development and function, and contributes to Alzheimer’s disease and amyotrophic lateral sclerosis (ALS), understanding the mechanisms that regulate CDK-5 activity and how it controls synaptic transmission in healthy neurons will help reveal the pathogenesis underlying the role of CDK-5 in neurodegeneration.

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).

Mentor: Kanarek, Robin
Fellow: Allen, Patricia
Title: Mechanisms Underlying Sex-Specific Effects of Creatine Supplementation on Depression
Abstract:
Preliminary research in this laboratory has established that chronic supplementation with creatine produces reliable sex-specific alterations in the rodent forced swim test, an animal model that is selectively sensitive to agents known to alter depressive symptoms in humans. Specifically, creatine produced antidepressant-like effects in females and pro-depressant effects in males, but the mechanisms underlying these behavioral alterations are presently unknown. These sexually dimorphic effects of creatine are intriguing particularly in light of sex differences in the incidence of unipolar depression. The significance of these findings would be greatly enhanced by additional experiments that address the potential mechanisms underlying these effects. A logical consideration is whether these observed sex differences are attributable to the activational effects of gonadal steroids at the time of testing. For instance, do changing levels of estrogens or progestins mediate the antidepressant-like effects of dietary creatine in females? Does testosterone mediate the prodepressant-like effects of dietary creatine in males? Additionally, brain derived neurotrophic factor (BDNF) – an essential mediator of synaptic plasticity, neural survival, and late phase long-term potentiation associated with depression – interacts with gonadal steroids and may be one molecular mechanism involved in the creatine-induced alterations in depressive behavior. Does creatine have neurotrophic-related neuronal activity? Does creatine influence protein levels or expression of BDNF in a sex-specific manner?

The primary objective of the present proposal is to address these critical gaps in knowledge by evaluating the effects of chronic creatine supplementation on depression-like behavior in the presence or absence of gonadal steroids in male and female rats. Additionally, a goal of this research is to determine the nature of potential creatine-induced alterations in synaptic plasticity.

Specific Aim 1 will determine whether the sex-specific effects of creatine supplementation on depression-like behavior are attributable to the actions of gonadal steroids at the time of behavioral testing. This work will pair classic hormone manipulation paradigms, more specifically bilateral castration or ovariectomy and hormone-maintenance, with behavioral assays to evaluate potential hormone-creatine interactions.

Specific Aim 2 will determine whether creatine-induced alterations in depressive behavior are associated with synaptic plasticity. Specifically, brain-derived neurotrophic factor (BDNF) expression will be quantified in the frontal cortex, hippocampus, and neostriatum to evaluate if there are sex-specific alterations in neurotrophic-related neuronal activity in the presence of creatine. Blood and brain concentrations of creatine will also be assessed in the frontal cortex, hippocampus, and neostriatum to establish if sex or regional differences exist.

Given the mounting functional and clinical significance of creatine in the brain, it is essential to more fully characterize the role of this agent in the pathophysiology of mood disorders. Moreover, addressing the issue of sex differences in response to treatment will profoundly affect our understanding of creatine, its relationship with depressive behavior, and may lead to sex-specific therapeutic strategies.

PI: Kanarek, Robin
Title: Nutritional Modulation of Gait and Cognition in Aging
Abstract:
Establish the effects of nutritional modulation (e.g., berry fruit) on behavioral (e.g., cognition, gait, force and balance) and neuronal deficits in aging to assess the mechanisms involved and the most effective dietary supplements in animal and human models.

Approach:
Conduct studies using a specialized Noraxon treadmill in order to measure parameters of gait including balance, stepping up and down, and walking patterns in humans. These new studies will allow us to focus on more subtle variations in gait that include stride to stride fluctuations (e.g., symmetry) and associated alterations in electromyography (EMG). The Noraxon measurement system allows an objective kinematic analysis of the human gait by means of analyzing the tracks of body surface markers. Additionally, cognition will be measured with a battery of tests that measure memory and vigilance.

PI: Kaplan, David
Title: BioComponent Robot System
Abstract:

TASK 1 — Insect Cells and Tissues: Actuator system components from insect cells and tissue systems will be studied due to the energetics and hardiness. We will focus on the tobacco hornworm, Manduca sexta, due to its environmental robustness (the muscles can even survive freezing), metabolic adaptability and well characterized contractile properties, however, in Task 1 we will also explore other insect cell sources for possible additions to the project.

TASK 2 — Biofuels: Fuels in the form of fats will be the focus. The goal will be to identify and explore multiuse approaches for these fuel sources, as both structural materials for the robots as well as the fuel sources, examining integration, efficiency and time of function. As a backup plan, we also plan to explore polymeric batteries. These bio-ionomers could form the basis of a new generation of battery based on chemical modifications of robust structural proteins.

TASK 3 — Growing Robots and Tissue Engineering: Another concept to be explored in parallel with the above tasks, would be to grow a robot. This task would be based on approaches from the field of tissue engineering, involving complex scaffolds, control of architecture, multigrowth factor delivery to regulate cell outcomes in a regional fashion on the scaffolds, and bioreactor designs to control the environment during robot growth.

PI: Kaplan, David
Title: Bioengineered Living Membranes
Abstract:
While significant advances have been made in the field of pathogen and chemical detection, these methods often require sophisticated equipment and dedicated laboratory space that is not available in forward operating theaters. The U.S. Armed Forces are in need of simple, durable, and low cost detection technologies that will be stable and functional at extremes of temperature, humidity, and in the absence of elaborate support equipment. To that end, we propose the development of a "Living Membrane" system based on recombinant bacterial strains entrapped in cellulosic membranes, making them resistant to environmental degradation. The cellulose-producing bacterium, Glucoacetobacter xylinus is a well-studied organism which produces an extracellular mesh or pellicle of cellulose that entraps itself and any co-cultured organisms as it grows. The pellicle is permeable to small molecules and proteins, providing a natural filter for processing of test samples. It is our intention to couple the power of recombinant DNA with the novel material properties of bacterial cellulose to produce a platform technology for point-of-use diagnostics. Given the wide array of reporter genes available to molecular biologists, we will couple the natural detection capabilities of various signaling pathways with the expression of specific fluorescent proteins to generate a suite of living detectors for the rapid and specific identification of relevant environmental contaminants. The proposed system will be durable and easy to use, requiring no additional power or sophisticated support equipment.

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).

PI: Kaplan, David
Title: Dynamic Camouflage Materials Based on Silk-Reflectin Chimeras
Abstract:
Reflectins are a unique group of structural proteins involved in dynamic camouflage systems in marine organisms. Initial cloning of reflectins, followed by thin film displays of the bioengineered protein, suggests interesting optical features when the recombinant protein is appropriately organized. These useful features include self-assembly and coloration patterns associated with material interference patterns. The goal of this new project is to examine in detail the fundamental relationships between reflectin chemistry, assembly, organization and functional dynamic optical properties. Specifically, we will build on recent efforts to bioengineer silk reflectin chimeric proteins, with the silk component serving as one of the organizing elements for material functions and key structural components for mechanically robust and versatile material formats. The reflectin component will serve as the dynamic optical element. Further contributions may also come from the silk as well, due to its novel light guiding properties and ability to form diffractive optical structures such as planar photonic crystals. Variants in silk block sizes, in reflectin domains obtained from the native reflectin repeats, and different materials assembly approaches will be studied, including thin film/coating and fiber formats. Optical properties will be determined from the recombinant proteins in solution and in the solid state, using a variety of imaging and related spectroscopic tools. Additional plans include modes to bioengineer these novel chimeric silk-reflectin systems with 'triggers', such that can be modulated to impact coloration patterns in a more dynamic way. These approaches may include environmental or external stimuli, such as specific light pulses, enzymatic reactions, or relative humidity. The outcome of this effort will be a new family of dynamic optical materials with potential utility in a range of camouflage and related material needs.

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: Silica-Protein Nanocomposites for Dental Repair
Abstract:
Inorganic materials such as bioactive glasses and composites are used in dental applications but suffer from a number of drawbacks including brittleness, mismatch in mechanical properties with surrounding tissues and poor interfacial stability. In the present proposal we describe a novel biomimetic nanocomposite approach to address these limitations. Importantly, we exploit two critical lessons in materials science and engineering from nature, nanoscale protein structures and control of organic-inorganic interfaces, to optimize material features. We describe new biomaterial nanocomposites formed from bioengineered fusion proteins that consist of two components: (a) a protein self-assembling domain based on mimicking the consensus repeat in spider dragline silk – due to the formation of highly stable (beta-sheet) secondary structures with impressive mechanical properties, and (b) a silica-forming domain derived from the silicatin protein of a diatom that offers versatility in control of the reactions that generate silica and different morphologies. These fusion proteins provide a novel approach to nanoscale materials assembly and control, leading to well-organized composite material structures that can be formed either in vitro (prior to implantation) or in vivo (conformal fill ins, interfacial bonding, avoid shrinkage due to the composite features) in biocompatible approaches.

The hypothesis for the proposed study is that nanocomposite material features (structure, morphology, mechanical) can be optimized and controlled (at different length scales) through appropriate design of chimeric (fusion) proteins in which the self-assembling structural domains and functional (silica forming) domains are linked at the molecular level.

Our goal is to elucidate how alterations in the chemistry of the two domains will lead to predictable changes in composite material properties (structure, morphology, mechanics) (Aim #1), to optimize features for in situ materials formation based on biocompatible reaction conditions (Aim #2), and to assess the new materials for dentinogenic restoration in vitro and in vivo (Aim #3).

Our preliminary data demonstrate the feasibility of the proposed approach and offers a new platform for in situ silica formation with unprecedented control of materials design and functional properties. The silica-forming domain can also be further modified to form other inorganic phases (e.g., hydroxyapatite, titanium dioxide), thus the versatility and opportunities that can be explored with this chimeric biomimetic protein design strategy are expansive.

PI: Kaplan, David
Title: Silk Coatings for Biomedical Devices
Abstract:
Silk-based constructs have led to significant advances in the field of tissue engineering and regenerative medicine. The implantation of various medical devices is a growing medical field, and presents significant challenges in terms of modulating the inflammatory and immune response to decrease device rejection and failure, as well as to minimize secondary infection. The silk fibroin isolated from the silkworm, Bombyx mori represents a unique biomaterial to address these concerns. Once properly purified, the silk fibroin has been shown to be largely inert with relation to the inflammatory response of the host tissue. In addition, the flexibility of its formulation with regards to form (film, gel, sponge, etc.) and inclusion of bio-active compounds (antibiotics, growth factors, immune modulators, etc.) cannot be replicated with other biomaterial systems. Silk-based materials and devices can offer mechanically robust and long-lasting systems, while still fully degradable.

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: Khardon, Roni
Title: RI: Medium: Collaborative Research: Optimizing Policies for Service Organizations in Complex Structured Domains
Abstract:
This proposal defines an important class of highly structured stochastic planning domains we call service domains, and develops theoretical foundations and algorithmic solutions for them. Examples of service domains include optimizing emergency response in a typical city, scheduling doctors and nurses in a hospital to handle a variety of routine and emergency procedures, administering tasks in a typical office, and 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 proposed research will make fundamental advances in automated planning that are required to address these challenges. The result will be scalable and principled planning algorithms for solving service domains, which will significantly broaden the scope and real-world impact potential of AT planning.

To help keep our basic research relevant to real world applications, we will use the domain of municipal fire and emergency management as one of the test beds for evaluation. We have an ongoing collaboration with the fire department of Corvallis, Oregon, which gives us access to domain experts and real world data to robustly evaluate our algorithms. In addition, to help drive basic research on service domains, we will develop a set of synthetic benchmarks with varying complexity that are inspired by real world applications and can be used for evaluation.

PI: Kilmer, Misha
Title: Collaborative Research: Multilinear Algebra Computations with Higher-Order Tensors
Abstract:
Many real world applications need to compress, sort, and otherwise manipulate large volumes of multidimensional data arrays (i.e., higher-order tensors), so there is an increasing need for theoretical and computational tools to deal with multiway data. The subject of multilinear algebra and tensors has gained increasing prominence in the past decade due to a surge in applications such as approximation of Newton potentials and stochastic PDEs, image compression and deblurring, network traffic analysis, biological assay interpretation, unmixing of signals, and many more. Such applications with higher-order tensors involve factorizations of the tensor. There are multiple possible extensions of matrix factorizations to higher-order tensors (e.g. extensions of the matrix SVD), with some more amenable to certain applications. The investigators are advancing the state-of-the-art in both theoretical and computational multilinear algebra via several newly developed tensor constructions based on new notions of tensor multiplication, orthogonality and diagonalizability. Algorithms with compression schemes based on these new constructions are being implemented by the investigators and tested on several datasets from various applications including handwritten digit identification, genomics, the spectral unmixing problem, video compression, and computer image recognition.

Current applications in the sciences can involve analysis, classification, searching and compression of large volumes of data that is "multidimensional" in nature. Consider, for example, the problem of facial recognition, used to identify a terrorist from within a database of images of known terrorists. The database can be considered multidimensional data in the sense that for each individual there corresponds a specific viewpoint, illumination, and facial expression. It is critical in this scenario to have an accurate and fast algorithm to match an unknown image against a database of images of known terrorists. Another example where a multidimensional representation is useful is genomic data. Here, DNA microarray two-dimensional tabular data from different experiments is concatenated into a multidimensional array. Recently published results have indicated that so-called 'factorizations' of this multidimensional data can be used to discover new molecular-level interactions. Hence, along with advances in computer architecture to store large datasets must come mathematically sound models for the compression and/or analysis of such data. Development of new concepts and ideas is therefore required to deal with the different geometries that arise in the multidimensional case. The investigators are contributing directly to this effort by developing innovative mathematical theory for multidimensional objects that is consistent with two-dimensional proven ideas. With theoretical constructs in place, the investigators are able to create computational tools and algorithms to analyze and compress multidimensional data. A significant component of the proposal is the involvement of undergraduates, graduate students, and researchers. The investigators are leveraging the strengths of both universities in a novel, inter-institutional vertical integrative experience for all students (undergraduate and graduate) involved in the research. This arrangement allows students at all levels, mentored by leading researchers in the field, to advance the state-of-the-art in the analysis and compression of multidimensional data.

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: 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: Targeting Hsp90 with Small Cyclic Peptides
Abstract:
This project aims to discover and characterize small cyclic peptides that modulate the function of the validated cancer target Hsp90. These molecules would provide drug leads, and would also help reveal the roles of Hsp90 in cancer and how we might more effectively target it for cancer therapies.

Heat-shock protein 90, or Hsp90, is a highly conserved chaperone protein that is absolutely required for proper folding of many of the major drivers of oncogenesis, tumor maintenance, invasion and metastasis. Inhibitors of Hsp90 have shown promise in clinical trials but broad application of this anticancer strategy has been limited by side effects and toxicity. There remains a need for molecules that more subtly alter Hsp90 function without completely inhibiting it, in order to minimize these problems. The discovery and characterization of such "Hsp90 modulators" will help us further understand the role of Hsp90 in cancer, and will point the way toward more potent and selective Hsp90-based therapies.

During my postdoctoral work, I developed a novel method of screening large libraries of cyclic peptides, an underexplored class of molecules that can modulate protein function. The first two specific aims of the project employ two independent screening strategies, both performed in living cells: one will uncover inhibitors of specific protein-protein interactions of Hsp90, and another will uncover molecules that bind Hsp90 and alter its structure. The third specific aim uses site-directed mutagenesis, binding assays, and application to cell culture to characterize cyclic peptides uncovered in the first two aims. These experiments will provide immediate insight into how Hsp90 can be targeted to kill cancer cells without general toxicity.

Overall, the cyclic peptide strategy is uniquely poised to deliver new drug leads as well as to provide answers to critical questions about targeting Hsp90 as a broad therapeutic strategy for cancer.

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.

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, Krishna
Title: Fluorinated Cell Surfaces to Modulate Biological Function
Abstract:
The goal of the proposed study is to explore the biological and biophysical properties of living cells that are decorated with unnatural fluorinated carbohydrates on their surfaces. It has been found that this cell-surface modification reduces cell adhesion, and therefore may provide a means to treat human disease processes that are mediated by cell adhesion such as tumor metastasis and inflammation. Part of this project is designed to determine the effect of cell-surface fluorine modification on biological functions related to cellular adhesion. Assays for cell static adhesion, dynamic adhesion, migration, and invasion will be performed. The ability of fluorinated sialic acids to reduce the rate or extent of tumor metastasis will be studied by bioluminescent imaging in a murine model.

The attachment of fluorinated molecules to the cell surface is accomplished by incubating the cells with chemically synthesized fluorinated sialic acid analogues that are taken up and processed into fluorinated glycoproteins and glycolipids expressed on the cell glycocalyx. Part of this project is to determine what the structural requirements are for a fluorinated sialic acid analogue to be expressed on the cell surface. Also, the distribution and nature of modified molecules on the cell surface will be determined by ion mass spectrometry, by colorimetric assay of selectively cleaved cell-surface molecules, and by a chemical genetics approach.

Since fluorinated molecules are virtually absent in unmodified cells, the incorporation of fluorine atoms provides an opportunity for low-background imaging by non-invasive techniques. Tumor bearing mice treated with fluorinated sialic acids will be imaged by 19F MRI. Since many tumor cells are hypersialylated, this technique may be useful for locating tumors before they are visible by other means.

The Specific Aims for this study are to:

  1. Determine the scope of fluorinated sialic acid analogues tolerated for expression on cultured cells;
  2. Evaluate the static adhesion characteristics of cells expressing fluorinated sialic acids;
  3. Study the mechanism of altered adhesion with synthetic models in vitro;
  4. Characterize the surfaces of cells expressing fluorinated carbohydrates;
  5. Evaluate the effect of cell-surface fluorination on tumor metastasis in mice; and
  6. Evaluate 19F MRI of tumor-containing mice expressing fluorinated carbohydrates.

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.

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: Kuperwasser, Charlotte
Title: Breast Cancer and Cellular Reprogramming
Abstract:
Much attention has focused on the promise of using human embryonic stem (ES) cells and their progeny for regenerative therapies in the treatment of various diseases including spinal cord injury, severe burns, or other pathological conditions. However, there are various limitations to using cells derived from fertilized embryos which led to the recent breakthrough of creating ES cells from adult cells using a process called cellular reprogramming. By introducing specific genes or treating cells with small molecules, cells from skin, liver, brain and other organs have been reprogrammed back into ES cells. Unfortunately, reprogrammed cells are often cancerous limiting the potential utility of these cells for therapy.

The Kuperwasser laboratory has recently made a serendipitous discovery connecting mechanisms of cancer progression and cellular reprogramming. Specifically, we have found that a population of human breast epithelial cells can spontaneously and naturally reprogram back into skin 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 goal of this work is to gain a deeper understanding of reprogrammed breast cells and assess whether they can form other skin-derived structures such as hair follicles and other tissues. We will also examine the genetic and molecular changes within these cells. 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.

Mentor: Kuperwasser, Charlotte
Fellow: Rudnick, Jennifer
Title: Mechanisms by Which Stromal Fibroblasts Promote or Suppress Aggressive Breast Cancer Cell States
Abstract:
The tumor microenvironinent is pro-inflammatory. The tumor microenvironment is composed of several cell types including lymphocytes, endothelial cells, pericytes, macrophages, mast cells and myofibroblasts, all of which work collectively produce inflammatory signals and growth factors necessary to maintain and fuel breast cancer growth and progression. However, the myofibroblasts are a particularly interesting cell type constituting a significant fraction of cells within the tumor associated stroma. These myofibroblasts (referred to as cancer associated fibroblasts, or “CAFs” in the context of the tumor microenvironment) express smooth muscle alpha actin (SMA) and fibroblast activated protein (FAP) REF. They are apparent during both wound healing and tumorigenesis, although their cell of origin is largely unknown REF. Several reports have shown that CAFs promote tumor growth in mice when coinjected with breast cancer cells compared to normal reduction mammary fibroblasts (RMFs). One reported mechanism by which these CAFs promote tumor growth is through secretion of SDF 1, which facilitates the recruitment of endothelial progenitor cells from the bone marrow, thereby promoting tumor angiogenesis REF. Unlike in acute inflammation and normal wound healing where myofibroblasts are present only transiently, myofibroblasts are chronically apparent in tumor associated stroma REF. It remains unknown why these cells linger at the tumor site and if they are epigenetically programmed to support tumor growth. Signals from the tumor microenvironment may serve to maintain the activated state of these myofibroblasts, whereas these signals may be short lived or nonexistent during acute inflammation.

CAFs, unlike normal fibroblasts, co-evolve with tumor cells and are chronically exposed to high levels of cytokines and prostaglandin E2 (PGE2). Thus, CAFs have the potential to be influenced by other surrounding cell types and by high levels of pro-inflammatory molecules within the tumor microenvironment. Moreover, CAFs themselves are a source of pro-inflammatory cytokines. PGE2, a major pro-inflammatory lipid based hormone, has been shown to influence IL-6 secretion by human breast fibroblasts. IL-6 is known to be secreted by CAFs, and high IL-6 levels can be detected in the sera of patients with breast cancers correlating with poor clinical outcome.

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: Lee, Kyongbum
Title: Phenotype-Targeted Inference of Flux-Enzyme Correlations in Adipocyte Metabolism
Abstract:
Obesity has become one of the most pressing health concerns in the United States. With very few (3 as of 2007) drugs approved for long-term treatment, there is a critical need to develop additional therapeutics. Currently available drugs affect metabolism throughout the body and suffer from efficacy limitations and numerous side effects. The goals of this project are:

  1. To identify potential enzyme targets through a global analysis of adipocyte intermediary metabolism; and
  2. To achieve a better understanding of the relationship between metabolism and lipid accumulation.

The approach is to perturb metabolic enzymes specifically (at the level of protein translation) via RNA interference (RNAi), perform a metabolomic assessment of the local and global effects of these perturbations, and integrate the measured data into a predictive model that quantitatively relates metabolic reaction fluxes to cellular lipid accumulation. The overall hypothesis is that we can limit the hypertrophic growth of adipocytes, which is principally driven by lipid accumulation, by modulating the activities of a small number of metabolic enzymes.

A key driver for this work is the notion that the state of cellular metabolism – as expressed by the engagements of a network of metabolic reactions – influences the outcomes of specific biochemical functions, in this case lipid synthesis, breakdown, and storage, in a predictable manner. In prior work, several enzymes of adipocyte intermediary metabolism have been identified as promising initial targets for knockdown. This project will assess the local and global impacts of these knockdowns by collecting metabolite and protein expression data. The collected data will be used to establish a metabolic control analysis (MCA) model and determine parameters that characterize the metabolic system’s sensitivity to perturbations in specific enzyme activities. The metabolic model’s predictions will be tested through additional, multiple knockdown experiments.

The specific aims are:

  1. Achieve specific, sustained knockdown of identified target enzymes using sequence optimized small, interfering RNAs (siRNAs);
  2. Quantify the local and global metabolic impacts of the knockdowns;
  3. Perform a proteomic assessment of the knockdowns’ effects on the expression levels of metabolic enzymes; and
  4. Develop a model to characterize and predict the effects of specific enzyme activity changes on adipocyte lipid accumulation and fuel utilization.

The project’s final outcomes will be a comprehensive evaluation of selected metabolic enzymes as prospective intervention targets for limiting adipocyte lipid accumulation; and a first, predictive metabolic model of adipocyte hypertrophy. These results should lead to a clearer understanding of the metabolic regulation underlying hypertrophic growth of adipocytes. In addition, the expected technological advances should broadly support other systems’ biological approaches for rational selection of targets for obesity drug development.

PI: Leekeenan, Debbie
Title: Teaching and Learning and Critical Friendship in Early Childhood: The Early Childhood Professional Development Consortium
Abstract:

Tensions and Challenges: There are tensions in early childhood education that bear heavily on how professional learning communities develop. Early childhood teachers in the United States are likely to work in programs that do not have a well conceptualized philosophy and organization; they are expected to make due without one or to make it up as they go along (Phillips & Bredekamp, 1998). Early childhood educators also tend to be a disparate group and convening around a particular theory can be difficult.

Early childhood teachers often have little training, and depend upon on-the-job professional development to gain the knowledge and skills necessary to do their work (Cassidy & Lawrence, 2000; Einasdottir, 2003; Wilcox-Herzog & Ward, 2004). Teacher meetings are centrally used as a vehicle for professional development but can quickly become a venue for school-wide issues and matters that do not directly affect teacher practice and children's opportunities to learn (Horn, 2005; Kuh, 2008; Little, 1999). Thus, conversations over-focused on scheduling, staffing, paperwork, and school-wide issues create barriers to professional activity that focuses on teaching and learning, jeopardizing experiences for young children.

Our Vision: If schools are to produce more powerful learning on the part of students, then teachers must be afforded more powerful learning opportunities that are woven into the ongoing work of teaching (Feiman-Nemser, 2001). When, as is too often the case, early childhood teachers do not look critically or have intellectual control over their work, they cannot fully understand, reflect upon, or examine their practice with children (Lubeck, 1998). There are few efforts to promote transformational learning communities specifically among early childhood educators, yet the School Reform Initiative's philosophy and process holds promise for acculturating teachers of young children to innovative ways of approaching their craft.

Our goal is to create an early childhood critical friends movement within our own institution and among our community partners in order to train key change agents in the work of critical friendship and instill the goals of transformational learning communities in diverse settings, increasing the equity within and among sites. In addition, we want to document this process to assess and disseminate our findings in presentations and journals.

PI: Lerner, Richard
Title: Constructing a Positive Youth Development Short Form Measure
Abstract:
Interests in the strengths of youth, the plasticity of human development, and the concept of resilience coalesced in the 1990s to foster the development of the concept of positive youth development (PYD). All current theoretical conceptions of the PYD developmental process have been framed within the relational, developmental systems meta-theoretical perspective. The 4-H Study of Positive Youth Development exemplifies the use of this theoretical orientation in PYD research. In the 4-H Study PYD, or thriving, is conceptualized as the growth of the "Five Cs" of PYD - Competence, Confidence, Character, Connection, and Caring.

The Five Cs are a means to operationalize the developmental characteristics that a youth needs to become a successful and contributing member of society. These Five Cs were linked to the positive outcomes of youth development programs reported by Roth and Brooks-Gunn. In addition, these "Cs" are prominent terms used by practitioners, adolescents involved in youth development programs, and the parents of these adolescents in describing the characteristics of a "thriving youth". Heck and Subramaniam note that the Five Cs model of PYD is the most empirically supported framework to date. Empirical evidence indicates that this construct has good psychometric properties.

However, despite its strong psychometric characteristics (i.e., factorial, concurrent, and predictive validity and, as well, high internal consistency reliability), many practitioners find it difficult to use the existing PYD measure as a gauge of the status of, or changes in, thriving among program participants or as a tool in program evaluation. The obstacle preventing such use is the length of the PYD measure. In Grades 5 to 7, 77 items are used to index the Five Cs; in Grades 8 to 12, 78 items are used.

Accordingly, the purpose of the proposed work is to develop a Positive Youth Development Short Form (PYD-SF) measure. Our goal is to develop a measure that will be useful for practitioners and program evaluators and, as well, that will have strong psychometric characteristics (indexed through assessments of factorial, concurrent, and predictive validity, and by internal consistency reliability).

PI: Lerner, Richard
Title: Promoting the Thriving Journey for America's Adolescents: Igniting the Sparks for Positive Youth Development Through Enhancing Selection, Optimization and Compensation
Abstract:
We propose to create a set of new tools for mentors to guide youth in a thriving journey. Our work will capitalize on the past work of the Thrive Foundation – by framing our tool development efforts within the Foundation’s “Theory of Change” and employing a version of the Thrive Conversation Aid – we will provide tools to mentors that will introduce youth to a discussion of his/her sparks; the importance of a commitment to positive growth (i.e., a “growth mindset”); the identification of positive purposes/goals; and the importance of goal management, through selection, optimization, and compensation (SOC) actions and strategies; in order to successfully undertake a thriving journey that attains the development of the “C”s of positive youth development (PYD). We will develop and evaluate the effectiveness of a mentor guide for launching and maintaining, across time, this discussion. This guide constitutes the first deliverable of the proposed work.

Within the context of this mentor guide, the mentor helps the youth identify his/her sparks and discusses the importance of committing oneself to actions that increase one’s abilities to activate and actualize one’s sparks (this commitment reflects a growth mindset). Within this discussion the mentor will introduce rubrics for SOC and PYD. The form and substance of these rubrics will need to be developmentally (and culturally) appropriate, to fit the particular characteristics of the young person (most notably his/her sparks) and his family and community contexts. Moreover, these rubrics will need to be constructed to possess age equivalence, so that scores can be compared across points in development. The development of these rubrics will be the second deliverable of our work.

The tools we will develop will enable the youth and mentor to make baseline assessments of the status of the young person in regard to his/her spark(s), PYD, and SOC skills. Through using (the actual or a revision of) the Thriving Conversation Aid, the youth and mentor will set the immediate, intermediate, and long-term goals (the growth) that they will pursue. At this point we will introduce another tool, the videotaped exemplars of youth who have used SOC to ignite and attain their spark(s) to traverse successfully a thriving journey, that is, to develop the Cs of PYD. These materials constitute the third deliverable we will produce.

We hope to use this third product as a basis for a simulated SOC (SIM-SOC) video (electronic) game, but the creation of this game is not a focus of the proposed work. Nevertheless, the prototype for SIM-SOC will be the fourth deliverable we will produce (in the second year of the proposed work).

The evaluation of (a) the overall tool kit, that is, of the first three deliverables, and (b) whether the use of these tools within different youth-serving programs enhances SOC, PYD, and spark-related achievements, constitutes our fifth deliverable. The outcome of this two-pronged evaluation will provide the evidence needed to refine, scale, and disseminate our work.

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: Biophysical Controls of Vertebrate Organ Regeneration
Abstract:
The regeneration of tissues and organs lost to injury or disease is a key goal of biomedicine. Induction of regeneration in clinical contexts will require a molecular dissection of the relevant patterning signals operating in animals that are able to regenerate. This field has been dominated by a focus on chemical signals and is ready for fresh approaches to the problem.

Our lab merges functional physiology with molecular genetics to understand novel biophysical controls of patterning and use them to control tissue growth. When amputated, the Xenopus tail forms a regeneration bud that rapidly produces a perfect duplicate of the original tail, including nerves, blood vessels, and muscle. Using this powerful vertebrate system, we discovered that endogenous ion fluxes and membrane voltage gradients play a crucial role in regeneration. Our drug screen implicated a H+ pump, a K+ channel, and a Na+ channel as required for regeneration but not for wound healing or primary tail growth; the activity of these transporters establishes a moderate zone of depolarization in the bud that is crucial for regeneration. We used mutant transporter constructs to inhibit or rescue regeneration, demonstrating that H+ flux is necessary and sufficient for inducing regeneration. These biophysical events function upstream of and control: known regeneration marker expression, up-regulation of cell proliferation in the bud, and axon patterning.

We propose to begin to understand the role of ion flux in regeneration by characterizing: (1) the time-course and properties of blastema currents, (2) the expression of implicated electrogenic genes, (3) the downstream steps linking membrane voltage to molecular and morphogenetic events during regeneration. Our data provide the first induction of regeneration by molecular modulation of ion flows, and the proposed work will answer the most important open questions in this new field. This proposal incorporates a high degree of novelty because it is focused on a paradigm that has not been previously addressed using molecular genetic tools: electrical controls of regeneration. It is high-reward because it would lay bare a new set of control parameters for the regeneration of a complex vertebrate structure (including spinal cord).

This will have important implications for understanding basic morphogenetic mechanisms as well as establishing a foundation for promising medical approaches to augment or induce regeneration in non-regenerating tissues. The ability to regenerate tissues and organs is crucial to the medical management of injury, aging, infection, or surgical removal of cancer. Our work will provide an entirely new modality that may, one day, allow human beings to regenerate important tissues and organs (including muscle and spinal cord).

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: 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 (funded significantly by the Mathers Foundation) 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]

Our work is unique because 1) we are asking fundamentally new questions (not identifying additional mechanisms for existing questions in the field), 2) we will be generating necessary datasets that will not only serve as a crucial base for this proposal, but will also be mined for years to come as the field identifies additional aspects of this new direction; 3) we are using a concordance of state-of-the-art techniques to transcend the artificial boundaries which obscure fundamental insight in several fields; and 4) we seek a deep synthesis of theory to guide future reductive experiments.

PI: Levine, Peter
Title: Civic Health Index
Abstract:
NCoC will recruit an expected 8 "teams" (composed of local institutions and their partners) in various states and localities. CIRCLE (Center for Information & Research on Civic Learning & Engagement) at Tufts University, under the direction of Dr. Peter Levine, will provide each team with the following:

  1. A memorandum depicting the condition of its local civic health, based on CIRCLE's analysis of the Census Current Population Supplement data and other information collected at the discretion of CIRCLE staff.
  2. Up to 10 hours of time for additional data queries and explanations.
  3. Fact-checking the team's final report
CIRCLE staff will also provide general guidance to NCoC and all the state teams about methodology and interpretation.

PI: Levy, Stuart
Title: Genetics of Soil Survival and Persistence of Pseudomonas fluorescens
Abstract:
This fundamental research proposal seeks support to expand upon previously funded (USDA/CSREES) work accomplished within the area of Plant Health and Production and Plant Products. It focuses on identification of genes and gene networks involved in survival of Pseudomonas fluorescens in soils, specifically addressing the FY2009 priority of “characterization of mechanisms used by microorganisms to survive or respond to environmental changes”. The studies fit within the overall goals of the USDA to improve health and growth of crops.

We propose three major aims:

  1. Continue to identify, and characterize genes associated with soil survival and persistence using IVET technology and transcriptome sequencing;

  2. Determine the function of genes, controlled by transcription factor AdnA, identified by microarray studies; evaluate strains deleted or over-expressing the AdnA-regulated genes; determine role of AdnA-regulated transcriptional regulators;

  3. Identification and analyses of cryptic genes involved in growth and survival of P. fluorescens in soils including characterizing overlapping genes for production of a protein or a regulatory non-coding RNA and expansion of proteomic studies under different environmental conditions.

In all studies, where appropriate, the mutant strains will be examined for survival alone and in competition with parental Pf0-1 in both sterile and live soil. The prior grant’s annotation of Pf0-1 will greatly facilitate this work. The data can be used to develop strains of P. fluorescens Pf0-1 improved for soil survival, a characteristic sought for a bio-control and/or growth promotion agent which must remain in soil long enough to perform a desired activity.

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: 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: Evaluation of Glycemic Index to Assess Diet-Associated Chronic Disease Risk
Abstract:
The objective of this proposal is to investigate the intra-individual reproducibility (within the same individual, when repeatedly measured) and inter-individual variability (among individuals) of glycemic index (GI) and glycemic load (GL) value determinations for individual foods and food combinations. The specific aims to accomplish this objective are to evaluate reproducibility and variability of GI value determinations in volunteers differing in biologic characteristics – body mass index (BMI), age and gender; assess the effect of macronutrient amounts and combinations, and fiber on variability of GI and GL value determinations; assess the effect of prior meal macronutrient composition (‘second meal’ effect) on GI value determinations; and relate these data to chronic disease risk factors monitored prior to and during the intervention period.

These aims will be accomplished by assessing intra-individual reproducibility and inter-individual variability of repeated GI value determinations for white bread, commonly used as a reference food, relative to glucose, volunteers selected to represent a range of BMI’s (18.5-24.9, 25-29.9, ≥30) and ages (18-49.9, 50-85 y), and on the basis of gender, and relate these data to body composition and insulin sensitivity (Phase I). This work will then be extended to address issues related to variability potentially introduced by differences in macronutrient and fiber combinations and loads (Phase II), and finally by ‘second meal’ effects (Phase III). Prior to each set of food challenges (glucose and test food[s] in random order) volunteers will be characterized on the basis of fasting HbA1c; fructosamine; lipids and lipoproteins; insulin, glucose and C-reactive protein.

During the 5-hour challenge (sampling at 0, 15, 30, 45, 60, and every 30 minutes thereafter) volunteers will be monitored for changes in blood glucose, insulin, triglycerides, total and high density lipoprotein cholesterol and non-esterified fatty acid levels. The concepts of both GI and GL are in the public domain and it has been suggested that the concepts be incorporated into U.S. federal dietary guidance (U.S. Dietary Guidelines and Dietary References Intakes [DRI]) formulated to promote health and reduce chronic disease risk.

This proposal addresses some of the understudied areas for which additional information would be useful in order to determine whether GI and GL should be used to classify foods on an individual basis, as has been suggested, and when formulating dietary guidance for the general population.

PI: Lichtenstein, Alice
Title: Nutrition and Cardiovascular Disease Predoctoral Research 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 rationale 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: 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: 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.

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.

PI: Malicki, Jarema
Title: Cilia in Eye Development and Disease
Abstract:
This research proposal focuses on the role of cilia in photoreceptor morphogenesis. Cilia are essential for development, differentiation, and function of many tissues. In the vertebrate eye, the photosensitive part of the photoreceptor cell, the so-called outer segment, forms as a highly differentiated cilium. In the absence of ciliary axoneme, the outer segment does not form, the photoreceptor is not functional, and it degenerates. Milder cilia defects frequently cause the visual pigment mislocalization in the photoreceptor cell. This is a serious defect, known to cause photoreceptor death. Many forms of human blindness involve cilia malfunction.

Nephronophthisis (NPHP) and Meckel-Gruber syndrome (MKS) are ciliary disorders that in addition to other abnormalities involve photoreceptor degeneration and blindness. Although several NPHP and MKS genes have been identified, the function of their protein products in the cell is poorly understood, if at all. We hypothesize that NPHP and MKS proteins contribute to the transport of the visual pigment to the photoreceptor outer segment. Accordingly, their defects lead to visual pigment mislocalization and photoreceptor death. Using biochemical and genetic approaches, we identified binding interactions between MKS as well as NPHP proteins and molecular complexes involved in ciliary protein transport. Here we propose to study these interactions further, and to test how MKS and NPHP proteins contribute to opsin transport in the photoreceptor outer segment.

The studies of human carriers of NPHP and MKS defects identified many molecular lesions that cause photoreceptor death. How do these lesions affect protein function remains, however, unknown. We will test how human mutations impact the ability of NPHP and MKS proteins to localize to cilia and to bind their partners. Together with experiments outlined above, these studies will reveal fundamental mechanisms, necessary for photoreceptor morphogenesis, function, and survival. They will also offer a way to test the impact of human mutations on specific aspects of protein function in the photoreceptor cell.

PI: Malicki, Jarema
Title: Genetic Analysis of Photoreceptor Differentiation
Abstract:
Photoreceptors are highly specialized light-sensitive cells of the retina, essential for visual perception. The loss of photoreceptors in the human eye due to genetic causes is a frequent cause of blindness. Although a lot of effort has been devoted to the studies of the photoreceptor cell, some essential aspects of its differentiation and function remain poorly understood. In particular, molecular mechanisms that lead to the assembly of the sophisticated features of photoreceptor morphology, such as the photosensitive outer segment, remain to a large extent unknown. Defects of these mechanisms frequently cause photoreceptor death and lead to blindness in humans.

A very productive way to gain insight into the genetic causes of photoreceptor degeneration in the human eye is to study animal models of photoreceptor loss. The zebrafish is one of the leading animal models used to study the genetic causes of retinal disease. Using a mutagenesis approach in zebrafish, we have identified and characterized several mutations that lead to photoreceptor death. A newly discovered mutation, jj203, causes a loss of rod photoreceptors and a delay in the differentiation of cone outer segments. We characterized the molecular nature of the jj203 locus and found that it encodes a molecular motor, kinesin. This analysis demonstrated for the first time that kinesins function differently in rods and cones, raising important questions regarding their cargo specificity and the interaction with ciliary transport machinery.

We are planning to characterize the role of kinesins in photoreceptor differentiation and survival using chemically-induced mutant alleles recovered both via forward and reverse genetic approaches. We will ask what aspects of photoreceptor phenotype are affected in different kinesin mutants and whether different kinesins can substitute each other. We will also study how kinesins interact with the intraflagellar transport, a process essential for outer segment formation. These studies will provide fundamental insights into the molecular mechanisms that drive photoreceptor differentiation.

PI: Mann, Anthony
Title: High Energy Physics at Tufts University
Abstract:
The proposed grant will enable our continued research in three frontier areas of elementary particle physics: (i) Neutrino Physics, with focus on the fundamental parameters governing neutrino state oscillations and neutrino interactions; (ii) 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 (iii) 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: Margalit, Dan
Title: CAREER: Group-Theoretical, Dynamical and Combinatorial Aspects of Mapping Class Groups
Abstract:
This project concerns the mapping class group, which is the group of isotopy classes of homeomorphisms of a surface. There is particular emphasis on the Torelli group, the subgroup consisting of elements that act trivially on the homology of the surface. We take a three-pronged approach, on the homological, dynamical, and group-theoretical properties of mapping class groups. The techniques include methods from combinatorial Morse theory, Teichmüller theory, and geometric group theory. In joint work with Bestvina and Bux, we aim to determine the homological finiteness properties of the Torelli groups; in particular to answer the famous question of whether or not they are finitely presented. With Farb and Leininger, we work on understanding the structure of all low entropy pseudo-Anosovs; in particular to address the Symmetry Conjecture, which asserts that all such pseudo-Anosovs are obtained as the product of a rotation with a map supported on a subsurface of uniformly bounded genus. With Brendle, we study the group structure of the hyperelliptic Torelli group, in particular focused on the conjecture of Hain, which states that the group is generated by Dehn twists.

The focus of this project is the mapping class group, which is the collection of symmetries of a surface, such as a sphere or a torus. This is a central object in mathematics, with connections to algebraic geometry, hyperbolic geometry, and dynamics. Discoveries in this area have had influence on fields outside of mathematics, such as string theory and cryptography. With my collaborators, I have made progress on decades-old questions about the global structure of mapping class group, and this project is dedicated to answering some of the most important remaining questions. The techniques involved in this study are wide-ranging, from combinatorial to algebraic to topological to dynamical, and the proposed work has applications to algebraic geometry, 3-manifolds, and group theory. I will work on the research aspects of the proposal with several collaborators and with students at various levels. In addition, I will host a workshop for young researchers in topology, with the aim of training and cultivating future researchers and educators.

PI: Marrow, Helen
Title: Social Status and Qualities of Intergroup Contact Among Native and Immigrant Groups
Abstract:
Given the notable increase in immigration over the past half century, coupled with its recent geographic dispersion into new communities nationwide, there is a pressing need to understand the nature and consequences of immigrant-native contact in 21st century America. Surprisingly, we know little about contact experiences among natives and immigrants in shared settings. Separate strands of research have developed to examine immigrant adaptation/assimilation on the one hand, and natives' responses to immigrants on the other hand, but few studies have combined these approaches to examine contact experiences among multiple groups in the same context. Additionally, existing research on intergroup relations still rests largely on a black-white paradigm, and needs to be broadened to consider: (a) how multiracial contexts, especially those with rapidly changing demographics and little history of immigration, may reveal distinct patterns of contact across multiple immigrant and native groups; and (b) how status characteristics in addition to socioeconomic and race/ethnicity may inform immigrant-native relations. Exploring these issues can help us to identify mechanisms that promote positive intergroup attitudes, as compared to enhancing feelings of intergroup threat, and (c) explain how and why relations between immigrant and native groups may play out differently across varied economic, demographic, political, and institutional contexts. Our project proposes to address these lacunae, by studying the contact experiences of multiple immigrant and native groups together; by exploring how these contact experiences might vary by various status characteristics across groups and in different receiving contexts; and by exploring how these contact experiences in turn shape individuals' attitudes toward other groups, their opinions about policies, and their own civic engagement.

To accomplish our goals, we propose to conduct an initial pilot survey in metropolitan Philadelphia among two different groups of immigrants (Mexicans and South Asian Indians – hereafter referred to simply as "Indians") and two different groups of native-born citizens (whites and blacks). We have selected these four target groups to explore the intersection of socio-economic status and race/ethnicity among new immigrants to the United States, and to expand the research agenda beyond these standard indicators of status to consider how a broader array of status dimensions may inform immigrant-native contact. We plan to examine the roles that immigration-related status characteristics such as legal status and citizenship, as well as skin color, language and religion play in shaping immigrant and native perceptions of intergroup contact and threat, alongside and in interaction with the status dimensions of socioeconomic status and race/ethnicity.

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: Maxwell, Daniel
Title: Linking Food Security Analysis to Improved Crisis Response
Abstract:
Mortality in recent food security crises has declined compared to earlier time periods, but as recently as the mid 2000s, research showed that the response to crisis has not improved as might have been expected. Responses were limited to a narrow range of programs, mostly not based on sound analysis, and many programs ignored information even when it was available. Food security responses consisted mostly of general food distribution, supplementary feeding, or seeds-and-tools support. Food aid was mostly provided in kind by donors. Program choices were made on the basis of institutional inertia or donor resources, not analysis. Crisis response generally was not funded on the basis of need.

Since the L'Aquila G-8 summit in 2009, a growing consensus has emerged to significantly ramp up international assistance to address food security issues globally. The main thrust of these initiatives is rightfully on agricultural technology and marketing to address long-term trends in production and food availability. But a comprehensive strategy for addressing food insecurity and malnutrition also has to take into account the problem of short-term — and increasingly, longer-term — crises of food access. Roughly 925 million people were food-insecure in 2010. Of these, about half lived in crisis-affected countries, and over 160 million were in countries in protracted crises (FAO 2010).

Much has changed with regard to the nature of food security response in crisis since the time the studies noted above were released. Significant effort and funding has been devoted to improved analysis. The range of responses has grown beyond the limited choices noted by Levine and Chastre. New tools and processes have been developed to help program designers and donors choose among these responses. And significant effort has gone into assessing impact — at least at the level of individual projects and programs. But beyond lowered mortality in crises, it is not entirely clear how the overall enterprise of food security response in crisis has progressed. A number of old questions remain, and several new ones have emerged. This concept notes briefly reviews the changing nature of food security and nutrition crises and suggests several major areas for research and assessment of the impact of the changing nature of response. More specific proposals address each of the major research topics in greater detail.

PI: Mazurana, Dyan
Title: People First! Phase III
Abstract:
People First!'s overall objective is to document and define remedy and reparation based on the lives, experiences and perspectives of people who had suffered and survived grave violations and mass atrocities. People First!'s overall goal is to empower and amplify citizens' voices so that they will be prepared to advance their own agendas for accountability, remedy and reparation. The project prioritizes women and youth's perspectives and priorities. Compton Foundation support in 2009 and 2010 set this research into motion, enabling victims and their communities to mobilize around and express their shared concerns. The goal of Phase III is to use culture and art to address key issues concerning violence and inequality the villagers themselves identified that have been exacerbated during the conflict and which pose serious challenges to the wellbeing of individuals and the harmony of communities during the post conflict situation. The project will culminate in a music and drama festival.

PI: McCanta, Molly
Title: Lunar Volatiles and Magma Ocean Differentiation: Reconciling New Results with Old Ideas
Abstract:
The proposed research seeks to integrate new data with older models of lunar igneous evolution. A combination of experiments and modeling will be used to investigate lunar magma ocean differentiation processes, the effects of lunar volatiles on melt crystallization and eruptive mechanisms, and the timing of lunar solidification. The research proposed on volatile addition to lunar magmas would provide new data on lunar petrogenesis and enhance the understanding of water distribution throughout the solar system. The experimental data generated will provide insight into the range of water contents that may be applicable to lunar magmas and the effects of water addition on lunar magma compositions, mineral liquidus temperatures, and magma physical properties (i.e., density and viscosity), specifically in regard to eruptability, magma transport, and plagioclase floatation. In addition, these experiments will produce a dataset that can be compared with natural lunar samples to study the potential effects of water on lunar igneous processes and that can be used as input in future modeling efforts.

The proposed work on coupled magma ocean solidification, including both chemistry and physics, will form an important link between theoretical modeling of early planetary formation and mission data from the planet today. These models make predications about the age and composition of the oldest lunar crust, about the compositions and ages of mantle reservoirs, and about the volumes and locations of water on the planet today. The results will provide insight into magma ocean crystallization and the timing of lunar evolution that are applicable to early geologic processes on other bodies.

The research tasks proposed are in direct fulfillment of the NASA strategic goal to advance scientific knowledge of the origin and history of the solar system and of many goals specific to the LASER program.

PI: McClellan, Andrew
Title: Mellon Postdoctoral Fellowship in the Humanities
Abstract:
Tufts University requests renewal funding from the Andrew W. Mellon Foundation to support the Mellon Postdoctoral Fellowship in the Humanities program at Tufts University. When we applied for initial funding in 2004, we were hopeful but uncertain about its benefits. Four years later we are in a position to say the program has been an unqualified success, and we are keen to see it continued into the future. Because the program was new to our campus, we adhered closely to the Foundation's proposal guidelines, following the model of overlapping fellowships, selecting fellows based on promising research and teaching synergies with senior faculty, and encouraging course development in directions that stood to enhance the teaching portfolios of our fellows while adding new dimensions to our curriculum. Fellows worked closely with their faculty mentors and remained grounded in departments; at the same time they were encouraged to explore interdisciplinary stimulation across campus and the extraordinary scholarly and cultural resources of the greater Boston area. Supervised by the Deans of Academic Affairs for Arts & Sciences, the program was directed by Professor Bruce Hitchner (Classics), who was given responsibility for coordinating regular events (e.g., lunches and research presentations) and logistical details (e.g., advertising, budgets, and travel) for the fellows. Hitchner also chaired the committee that selected fellows and solicited faculty participation.

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: 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: McVey, Mitch
Title: CAREER: Interactions between Error-Prone and Error-Free DNA Double-Strand Break Repair Pathways in Drosophila melanogaster
Abstract:
Precise and controlled repair of DNA double-strand breaks is crucial for cell survival and genomic integrity. Double-strand breaks can be repaired by a number of both accurate and inaccurate mechanisms. Although accurate repair has been characterized in substantial detail, error-prone double-strand break repair is not well understood. The importance of these error-prone pathways is highlighted by evidence of their involvement in genome destabilizing processes such as chromosome rearrangement and telomere fusion. As outlined in this proposal, we will employ and optimize powerful double-strand break repair monitoring assays to elucidate the molecular mechanisms of error-prone DNA end-joining repair. Our central hypothesis is that error-prone end-joining repair is linked, at both the mechanistic and regulatory levels, to a failure to complete repair by homology-driven or error-free end-joining pathways.

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: Mecsas, Joan
Title: Building Diversity in Biomedical Sciences
Abstract:
In the past ten years, Short Term Training for Minority Students Program (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 Building Diversity in Biomedical Sciences Program (BDBSP) will provide:

  1. An intensive 10-week mentored summer research experience with a focus on cardiovascular and pulmonary diseases;
  2. Training in oral and written communication through experiences that build skills and enhance self-confidence;
  3. Exposure to the breadth of biomedical science careers through workshops;
  4. Seminars, field trips and career counseling;
  5. Career models through interactions with graduate students as well as trainees in our post baccalaureate and postdoctoral training programs that are centered on outreach to underserved individuals; and
  6. 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.

Mentor: Mecsas, Joan
Fellow: Alison, Davis
Title: Role of the Y. pseudotuberculosis YscF Protein in Toxin Delivery into Host Cells
Abstract:
Gram-negative enteropathogenic bacteria, including Yersinia, Salmonella, Shigella and both enteropathogenic and enterohemorrhagic E. coli, cause a wide variety of gastero intestinal diseases in humans. All of these pathogens harbor an essential virulence factor called the Type III Secretion System which delivers bacterial toxins directly into host cells. The Type III Secretion System produces a structure in the bacterial membrane with a needle-like appendage extending out from the bacterial surface through which toxins are secreted. The secretion system also produces a pore in the host plasma membrane, termed the translocon, which allows access of the toxins to the host cell cytosol. It is not known how the needle on the bacterial surface and the translocon in the host cell membrane cooperate to enable toxin delivery. In an effort to determine the requirements for a productive needle-translocon interaction during infection I have isolated mutations in the needle protein of Yersinia pseudotuberculosis (YscF) that are incapable of delivering toxins into the host cell, but retain the ability to secrete toxins out of the bacteria into the extracellular space. In this proposal, I aim to use both WT Yersinia and the YscF mutants to:

  1. Determine if the needle is in direct contact with the translocon during infection
  2. Determine if the needle-translocon interaction is required for pore formation in the host cell
  3. Map the regions in YscF and the partner protein that are required for needle-translocon interactions

Characterization of the bacteria-host cell interactions using both WT and mutant Yersinia will provide insight into the mechanism of toxin delivery into host cells used by Y. pseudotuberculosis. This virulence mechanism is conserved amongst other enteric pathogens, and thus this work will be directly applicable to the study of disease caused by Salmonella, Shigella and E. coli, and will uncover candidates for targets of anti-microbial agents.

PI: Meiri, Karina
Title: A Collaborative Approach to Real World Science in the Classroom
Abstract:
Engaging students in the biomedical sciences while they are still in high school is a critical first step toward educating a scientifically literate citizenry as well as initiating the pipeline that will eventually result in increased numbers of biomedical and health related professionals. This proposal is a collaboration between biomedical scientists at Tufts University School of Medicine, biology teachers at the Madison Park Technical and Vocational High School, the Boston Latin Academy and the Boston Latin School, all inner city Boston public high schools, and members of the Wright Center for Innovation in Science Education, also at Tufts. Its goal is to engage the imagination of 11th and 12th grade students who do not see the science of their real world experiences mirrored in the classroom. It accomplishes this goal by developing and disseminating a novel inquiry-based high school biology curriculum that focuses on biomedical research in the context of five great diseases that challenge global health – infectious, neurological, cardiovascular, cancer and diabetes. A key element of the proposal is addressing the challenge of aligning content and process in the classroom to enable teachers to create the knowledge-centered classrooms that are critical for learning transfer.

Accordingly the first aim is to first develop a learning community that will interactively guide the in-depth professional development underlying each of the 5 curricular modules. The learning community will then collaborate on the second aim; to develop curricular content and to generate deliverables for implementation of the curriculum in the classroom. This material, which will include a syllabus and web-based virtual and real interactive inquiry-base laboratory exercises, will be disseminated into the Boston Public School system and the broader educational community as part of the third aim. The fourth aim is to design and implement an evaluation strategy that will allow for ongoing revision during the project to ensure optimization of outcomes. The involvement of Tufts medical and biomedical graduate schools ensures that the program is highly sustainable and our final goal is to establish it as a model of how medical schools and school districts can interact to disseminate understanding of the biomedical research underlying disease at the high school level.

PI: Meiri, Karina
Title: Mechanosensitive Regulation of Axon Guidance
Abstract:
During development of the embryonic nervous system, growing axons navigate to their synaptic targets by following guidance cues located in the environment. The precise spatial and temporal regulation of guidance cue expression elicits orchestrated responses from the highly motile tip of the growing axon, its growth cone. These responses enable the growth cone cytoskeleton to remodel in a spatially specific manner that enables directional outgrowth. It is well understood that the cytoskeleton is responsive to the mechanical as well as the chemical environment, but the extent to which mechanotransduction impacts guidance cue responses is poorly understood.

This proposal takes biochemical and cell biological approaches using mechanosensitive cortical neurons in culture to address the hypothesis that the interactions between the membrane and cortical cytoskeleton that are critical for regulating directional guidance of axons require activation of signaling mechanisms responsive to a highly elastic embryo-like mechanical environment in concert with those activated by guidance cues. We have developed an in vitro model that reproduces the elastic properties of the embryonic cortex and identified a growth cone cytoskeletal modulator responsive to both mechanical stimuli and guidance cues. This protein, GAP-43, is a major determinant of central nervous system cytoarchitecture in vivo, regulating how the tubulin and actin cytoskeletons interact with the growth cone membrane in response to directional guidance cues. GAP-43 is also highly mechanosensitive: We have shown that it recapitulates its in vivo responses to guidance cues like Netrin-1, only under in vitro mechanical conditions that reproduce the embryonic environment.

The results suggest that understanding how the outside in signaling initiated by Netrin-1 coordinates with mechanotransduction initiated by the environment coordinates to regulate GAP-43 interactions with the growth cone cytoskeleton, will provide information broadly relevant to other neuronal and non-neuronal cells.

The experiments proposed here have two main goals: the first is to investigate how substrate elasticity impacts the outside-in signaling mechanisms elicited at the growth cone by Netrin-1. The second is to understand how these signaling mechanisms are transduced to potentiate microtubule-regulated neurite elongation at the expense of the actin-regulated growth cone stalling seen under non-elastic in vitro conditions.

The intellectual merit of these studies is that for the first time they will directly address how mechanotransduction coordinates with directional guidance using a molecule, GAP-43 known to be a key determinant of cytoskeletal organization in vivo.

PI: Meydani, Simin
Title: Calorie Restriction and Immune Response in Humans
Abstract:
Aging is associated with impaired regulation of the immune system, which contributes to the increased incidence of infectious, inflammatory and neoplastic diseases observed in elderly subjects as well as their prolonged post-illness recovery periods. In addition, these changes were shown to be predictive of morbidity and mortality in animal models and humans. While all cells of the immune system contribute to the impaired immunity of old age, T cells are the main contributors, with age related changes reported in both in vivo and in vitro measures of T cell function across all species including humans. Calorie restriction (CR) has been shown to affect many age sensitive immunological responses in animal models, but information related to the effects of CR on immune response of humans is lacking. Preliminary results from the pilot phase of the two-step NIA-supported multi-center clinical trial to determine the biological effects of CR in humans [Comprehensive Assessment of Long-Term Effects of Reducing Intake of Energy (CALERIE) Phase 1], showed that CR significantly improved delayed type hypersensitivity skin response, as well as T cell proliferation, in humans while decreasing production of T cell suppressive factor, PGE2.

Thus, we hypothesize that long term CR intervention in adult subjects will enhance the immune response, as indicated by improved T cell-mediated function and reduced production of inflammatory mediators. Furthermore, we hypothesize that these CR-mediated effects on T cells are due to decrease in PGE2 production and/or intrinsic changes in T cells.

We propose to test this hypothesis utilizing subjects enrolled in the second phase of the NIA supported multi-center randomized controlled clinical trial, CALERIE Phase 2. The overall aim of CALERIE Phase 2 is to test the effects of 2 year, 25% CR from baseline energy intake, in adult subjects, on physiology, metabolism, body composition, risk factors for age-related pathologies, and its potential adverse effects. The specific aims of this project are to determine the effect of 2 y, 25% CR on T cell-mediated functions of adult subjects, as well as to explore its underlying mechanisms. Specifically the effect of CR on immune cell profile, proliferate ability of specific T cell subsets, intracellular and extracellular IL-2, IFNγ, as well as PGE2 production, will be evaluated before, and following 1 and 2 years of 25% CR.

The results from this study will be the first documentation of the impact of CR on immune response in humans, a biologically meaningful and clinically relevant marker shown to be sensitive to CR in various animal models. The proposed studies will also explore the underlying mechanisms of CR-induced modulation of the immune response and will add valuable information to the parent CALERIE Phase 2 studies by providing insight into the cellular and molecular mechanisms of CR induced health effects.

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.

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: Miller, Eric
Title: Collaborative Research: CI-P: Computationally-Enhanced Optical Imaging Infrastructure
Abstract:
The objective of this CI-P proposal is to plan for the enhancement of a computationally enhanced optical imaging infrastructure for biology research. Optical imaging is playing an increasingly important role in today's biology research. Images acquired at microscopic and macroscopic levels can reveal important molecular and cellular processes. Advancements in optics, engineering, physics, and biochemistry have propelled the development of optical imaging techniques at a fast pace over the last ten to twenty years. Modern biological research often applies not only one but multiple optical imaging techniques in experiment, including microscopy, high-throughput imaging, and in vivo small animal imaging, all of them generate a large amount of image data. As a result, there is a growing need to use advanced computational technique to process and analyze optical images efficiently.

The CI will feature upgraded hardware, computational techniques, and a versatile data structure and software toolbox. The principal investigators in this proposal have been working to develop computational analysis capability to help investigators use optical imaging to address biological questions. First, the enhanced infrastructure will provide biologists with advanced imaging techniques as well as corresponding computational tools to automatically process, analyze, and quantify the images. Second, the infrastructure will bring new data types and challenges to computational scientists by connecting them to biologists to synergistically address biological questions from the perspective of computations. The multi-disciplinary environment will provide students abundant training opportunities in computer science, applied mathematics, biology, engineering, and optical imaging.

Our planned CI is based at the three institutions in the Boston area, Brigham and Women's Hospital, Northeastern University, and Tufts University, to take advantage of each institution's strength and expertise. We plan to design the CI to serve both local and national user communities. The versatile data structure and software interface will allow virtual access via the internet. In addition to biologists, the other two user communities that may benefit from the infrastructure are computational scientists who can use the CI as a platform to develop novel techniques and collaborate with biologists and researchers working on optical imaging who can take advantage of the hardware and software capabilities of the CI to model imaging process and improve instrument performance. During the planning phase, we will coordinate our efforts to design the CI and identify consensus need of a broad community. We will hold a workshop and follow-up meetings at Northeastern University to invite representative users for inputs on the CI. The planning effort will identify the consensus needs of the broad user communities in terms of technical requirements on hardware, software, computational techniques, and data structure. We will also use interaction with the user community to identify training and educational opportunities that the CI can provide to postdoctoral fellow, college students, and high-school students.

PI: Miller, Eric
Title: Harvard IIC Support for Amelio Vasquez
Abstract:
These funds will support the work of Mr. Amelio Vazquez-Reina, a Tufts Computer Science graduate student, who will work under the supervision of Prof. Miller at Tufts and Profs. Hanspeter Pfister (Harvard SEAS/IIC), Jeff Lichtman (Harvard Center for Brain Sciences), and Clay Reid (Harvard Center for Brain Sciences) developing image processing methods for the identification of axonal and dendritic processes in large volume nanoscale imagery. More specifically, Mr. Vazquez-Reina will work with the Harvard Center for Brain Sciences (CBS) on the Connectome Project to develop algorithms for the visualization, segmentation and tracking of neuronal processes in electron and optical microscopy imagery. He will develop software tools for the labeling and identification of neuronal connections in large image volumes of brain tissue. These tools will include algorithms for volumetric color and grayscale segmentation, methods for shape analysis and tracking, and computer programs and libraries for the recognition of synaptic connections and neuronal circuit extraction.

Mr. Vazquez-Reina will develop algorithms employing a wide range of state-of-the-art techniques including

  • Constrained deformable models for the estimation and segmentation of 3D neuronal structures such as mitochondria, synaptic vesicles and cellular membranes
  • Probabilistic graphical model techniques and graph-theoretic segmentation ideas for tracking these structures across slices in a stack of electron microscopy data.

Mr. Vazquez-Reina will be expected to publish his accomplished results in the journals and conferences most suitable for his work.

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.

PI: Moore, Claire
Title: Training in Education and Critical Research Skills
Abstract:
Our program is based on the need for university faculty that is prepared to meet the multiple challenges faced by young assistant professors pursuing their first independent position. We will provide our postdoctoral trainees with the career skills they will need to succeed in an academic research environment by integrating three training components: rigorous bench research; mentored teaching experience; and career development skills. By partnering with two local minority serving institutions, Roxbury Community College and the University of Massachusetts, Boston, our trainees will also acquire a greater knowledge and sensitivity to diversity issues and provide enriched course material to institutions serving a minority population. Our faculty of 42 is committed to research excellence and has a strong training record. Research opportunities focused on multiple biomedical problems with special emphasis on basic biological problems, cancer, neuroscience and infectious disease will be available to trainees. Research committees and research advisors will monitor progress and career development with respect to research. The teaching component uses workshops, discussion groups and tutorials to prepare trainees to develop and present a course of their own design at one of the minority serving institutions. A series of career building workshops, along with checkpoints that provide mentoring and constructive feedback round out the program.

Our goal is to produce the academic scientific leaders of the future. University faculty must develop independent research programs, an activity that requires creative thought, high intellectual capacity, outstanding facility with oral and written communication and considerable experimental skill. These activities must be sustained while faculty participate in classroom teaching, serve on a variety of committees and mentor trainees. Success also requires additional skills in interpersonal relations, budgeting and time management. The TEACRS program will provide trainees with the research portfolio needed to compete for academic positions and the skill set that postdoctoral fellows need to meet the challenges they will face as they enter academic careers.

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: 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: Moss, Stephen
Title: The Role of Dynamic-1 in the Trafficking of GABAB Receptors
Abstract:
GABA (gamma-aminobutyric acid) is the main inhibitory neurotransmitter in the mammalian nervous system. The slow action of GABA is mediate by GABAB receptors (GABABRs), which belong to the group C G protein-coupled receptors. They function as heterodimers composed of GABABR1 and GABABR2 and couple to adenylyl cyclase, presynaptic voltage-gated Ca2+ channels and postsynaptic inwardly rectifying K+ channels. They have been implicated in epilepsy, nociception, depression and cognition. They also represent attractive targets for the treatment of withdrawal symptoms from addictive drugs such as cocaine. We suggest that GABABRs mediate their plethora of functions as part of multi-protein complexes that are subject to exquisite modulation. Thus we have proposed our general hypothesis: "GABABRs modulate the efficacy of inhibitory synaptic transmission as part of multi-protein complexes." Previous attempts at identifying these complexes have been only partially successful. We have now employed a unique approach to identify GABABR associated proteins in neurons using immunopurification and mass spectrometry. As a result, we have obtained several candidates involved in protein turnover. To maximize the impact of our screen and open a new line of research we have prioritized the study of one candidate, namely dynamin-1, a protein with an established role in protein trafficking. Here we propose to investigate its role in GABABR turnover in the context of the following hypothesis: "Dynamin-1 defines the trafficking properties and membrane availability of GABABRs." We will develop the following specific aims:

  1. Specific Aim 1: Characterize the endocytosis of GABABRs in neurons.
  2. Specific Aim 2: Evaluate the specificity of the interaction to dynamin-1.
  3. Specific Aim 3: Test the functional consequences of the interaction to dynamin-1.

Together with the parent grant, our proposed studies are likely to contribute to uncover novel mechanisms of trafficking and assembly of GABABRs into inhibitory sites and the identification of signaling events with potential therapeutic value.

PI: Nelson, Miriam
Title: StrongWomen – Healthy Hearts: A Community-Based Program for Midlife and Older Women
Abstract:
Heart disease is the leading cause of death of midlife and older women in the United States. Key lifestyle factors that put women at elevated risk for heart disease are overweight and obesity, sedentary lifestyle and poor nutrition. Approximately, two-thirds of adults in the United States are overweight or obese, over 75% of women are sedentary, and less than half of women consume enough fruits and vegetables to meet the Dietary Guidelines. All of these risk factors lead to increased risk of heart disease in addition to other chronic diseases such as type 2 diabetes, as well as decreased longevity. This project aims to reduce obesity rates, increase physical activity, and improve nutrition through a targeted community-based program and by increasing skills-based capacity of extension educators. This project is a partnership with Tufts University, Penn State Cooperative Extension and the National Extension Association of Family and Consumer Sciences (NEAFCS).

Objectives include:

  • To train extension educators in the StrongWomen – Healthy Hearts curriculum.

  • To monitor Reach, Effectiveness, Adoption, Implementation, and Maintenance of the StrongWomen – Healthy Hearts curriculum throughout Pennsylvania.

  • Engage an estimated 18 Penn State Cooperative Extension educators who will each recruit 15 participants and assess them at baseline and following the 12 week extension educational program. Underserved populations (low-income, minority and rural women) will be specifically targeted.

  • To work in partnership with Tufts University and NEAFCS to implement and monitor the StrongWomen – Healthy Hearts curriculum nationally.

The overall goal is to improve body weight, physical activity patterns and nutrition of midlife and older women. This will be accomplished by enhancing extension educators' knowledge, skills and leadership competence related to implementing a community-based healthy hearts curriculum. Objectives will be achieved by conducting a leadership training workshop for Pennsylvania extension educators and a special training for extension educators attending the NEAFCS annual conference. This training and program delivery approach offers significant potential to provide effective, inexpensive, and accessible programming to midlife and older women in community settings throughout the United States.

PI: Nelson, Miriam
Title: Tufts – USDA Doctoral Fellowship Program in Obesity
Abstract:
Obesity is a major health concern in the United States. More than 65% of adults are either overweight or obese, and one-third of children are at risk for being overweight or overweight. Progress at abating this crisis has been unsuccessful and the prevalence is still increasing. The goal of this proposal is to reverse this trend by obtaining funds for the education and training of three PhD scientists committed to research careers in obesity. The John Hancock Center for Physical Activity and Nutrition at the Friedman School of Nutrition at Tufts University is uniquely positioned to fulfill the needs of the CSREES TESA for Human Nutrition — specifically obesity, diet, and exercise. The eight core faculty and ten associate faculty engaged in this program has national and international research reputations in relevant fields: energy balance, nutrition, exercise, sociology, epidemiology, communications, behavior change, endocrinology, and food policy. The broad range of expertise represented at Tufts will prepare the fellows to critically examine the problem of obesity at many levels. Faculty will mentor and guide fellows throughout their doctoral training, which includes appropriate coursework, a qualifying examination, and original, high-quality research. The training that the fellows receive will prepare them to become future leaders in the field. Their collective work will increase knowledge and understanding of obesity, and ultimately, will contribute to reducing obesity rates among Americans.

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 our hypothesis 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: Obin, Martin
Title: Metabolic Health in Obese TWEAK KO Mice
Abstract:
Obesity is associated with adipose tissue inflammation caused in large part by infiltrating immune cells (macrophages) that are recruited to scavenge dying adipocytes. One candidate mediator of adipocyte death is tumor necrosis-like weak inducer of apoptosis (TWEAK/TNFSF12). TWEAK is reported to be an important potentiator of cytokine-induced cell death in rodent models of chronic inflammation. We and others have shown that TWEAK and its receptor are upregulated in adipose tissue of obese humans and mice and that TWEAK gene expression is upregulated in adipose tissue by a subset of macrophages surrounding dead adipocytes.

Hypothesis:
We hypothesize that the absence of TWEAK protects mice from adipocyte death in response to a diet high in saturated fat.

Objectives:
In the proposed experiments we will use archived blood and tissues from TWEAK and WT mice fed a high fat diet to elucidate mechanism(s) by which TWEAK promotes adipocyte death, inflammation and insulin.

PI: Olum, Ken
Title: Does General Relativity Permit Time Travel?
Abstract:
Does semiclassical gravity permit the construction of time machines or other exotic phenomena? Given any spacetime, Einstein's equations tell you what source is necessary to produce it. Exotic spacetimes require sources that violate the averaged null energy condition (ANEC), which requires that the total energy seen by an observer traveling on a null geodesic be nonnegative. In free field theory in flat space, ANEC is obeyed, but quantum fields in curved backgrounds can easily violate it. The goal of this project is to determine whether such violations can be self consistent, i.e., whether their curvature can be generated by a stress-energy tensor produced with that very same spacetime as the background. If not, exotic phenomena would be impossible.

The best possibility seems to be to start with an ANEC-obeying curved background and produce ANEC violation by a quantum field on that background. Since we must be far from the Planck scale to be in the regime of semiclassical gravity, the quantum field effects are small compared to the background, so corrections at the next order can be ignored. But if the background gives zero contribution to ANEC, even a small correction could produce a violation.

PI: Olum, Ken
Title: Large Parallel Cosmic String Simulation
Abstract:
Cosmic strings connect fundamental theories of basic physics with observation of the universe on the largest scales. Cosmic strings are infinitesimally thin or even fundamental objects of cosmological length. They can arise from symmetry breaking in field theory or they can be the fundamental strings of string theory or 1-dimensional "D-branes". 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. To interpret a cosmic string observation or to set limits on cosmic string properties from non-observation requires a quantitative understanding of the cosmic string network. In particular, since most cosmic string signatures arise from string loops, it is vital to know the density, sizes, and shapes of the loops whose effects might be detected. Because of the highly nonlinear nature of cosmic string processes, accurate understanding of the string network requires numerical simulation.

This project will develop a new parallel simulation code, using algorithms adapted to parallel computing, and with more flexibility in generating initial conditions and analyzing the simulation data than previous codes. Parallel computing will provide for a simulation volume hundreds, perhaps a thousand times larger than has been simulated before, and an increase in the simulated time interval of up to a factor of 10. This will allow much more time for initial condition artifacts to be damped out and for the string network to assume its final scaling form, from which the important network parameters can be extracted.

The project will have a broader impact through the involvement of graduate and undergraduate students and through opportunities for students from under-represented groups to participate in research. Its results will be broadly disseminated through publication in journals and made available to the public on the World Wide Web. It will benefit society by further connecting studies of the universe with studies of the fundamental laws of nature.

PI: Ordovas, Jose
Title: Aragon Workers Cardiovascular Health Study (AWCHS)
Abstract:
This research program will evaluate the impact of environmental factors (i.e., nutrition, smoking, physical activaty) in the modulation of cardiovascular risk.

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 herein 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: Peattie, Robert
Title: A Combined Experimental-Computational Method to Evaluate Abdominal Aortic Aneurysm Wall Stress
Abstract:
Abdominal aortic aneurysms (AAAs) represent permanent, localized swellings of the aorta that occur where the diseased vessel is weakened. AAA rupture accounts for 14,000 deaths in the U.S. annually. A growing body of literature has developed in recent years concerned with methods for quantitatively evaluating the distribution and magnitude of stress in patient-specific models of the aneurysm wall, since mechanical stress is thought to be the cause of rupture. However, the role of blood flow in creating wall stress has not been addressed to date. Accordingly, the research objective of this award is to develop a combined experimental-computational physiologic wall stress analysis (PWSA) procedure that evaluates wall stress distributions and maxima in models accurately replicating the shape, non-uniform thickness and mechanical properties of individual patient AAAs. To accomplish this aim, AAA wall tissue samples will be collected from patients undergoing surgical repair. The mechanical properties of those samples will be measured, and an elastic material replicating the diseased wall stiffness will be used to fabricate a model replicating the shape of the patient lesion. Wall pressure in this model will be measured under flow conditions emulating the patient aorta. The measured wall pressure data will then be used as loading in a corresponding computational evaluation of stress levels within the wall.

Establishment of a protocol for accurate assessment of AAA wall stress using patient-specific information has the potential to significantly impact clinical AAA management. The proposed studies will provide the first quantitative measurements 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.

PI: Peattie, Robert
Title: Collaborative Research: Engineering an Angiogenic Cardiac Patch
Abstract:
The goal of the proposed project is to develop and evaluate a biocompatible implant capable of preserving cardiac function following ischemic injury. By facilitating restoration of functional myocardium, this implant will significantly reduce morbidity and mortality associated with ischemia and the resulting infarction. The implant will be fabricated as a reinforced hydrogel (the cardiac patch), easily securable to the heart and capable of storing and releasing into the diseased tissue exogenous factors of interest. Functional tissue restoration will be achieved through a mature, perfusable neovascularization response elicited by delivery of those factors.

Currently, 920,000 cases of myocardial infarction occur annually in the US. Inadequate revascularization and the resulting insufficient respiratory gas transport are critical limitations to infarcted myocardial healing. Development of mature microvessel networks in the damaged tissue can therefore provide a major advance towards restoring myocardial function. The cardiac patch will consist of a novel composite biomaterial composed of electrospun silk fibers dispersed through a covalently crosslinked, thiol-modified hyaluronic acid (HA) and heparin (Hp)-based gel, creating a biomimetic synthetic extracellular matrix (sECM). Mature microvessel networks will be induced by controlled, sequential release of multiple growth factors (GFs) from the gel and by synergistic interactions of those GFs with HA.

In the proposed project, we will test the hypothesis that Hp-regulated GF release can dramatically improve the revascularization of damaged myocardium, eliciting functional, perfused vessels that restore cardiac function after ischemic injury. Physicochemical 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 GFs then sutured onto ischemic hearts will be quantified by measurement of microvessel proliferation and maturity in retrieved myocardial tissue samples post-implantation. The functionality of elicited vessels will then be assessed by longitudinal non-invasive MRI monitoring of perfusion changes in the ischemic border region. Simultaneously, the efficiency of treated hearts will be evaluated. By supporting the expansion and reparative activity of resident stem and progenitor cells in the ischemic border, the patch will serve as the foundation for tissue repair. The development of this superior sECM can ultimately significantly impact the therapeutic options available for treatment of ischemic and/or congestive patients.

PI: Peattie, Robert
Title: MRI: Acquisition of a High-Resolution Multi-Material Printing System
Abstract:
The objective of this proposal is to obtain support for the purchase of a state-of-the-art, ultra-high-resolution, 3-dimensional printing system for rapid prototyping and rapid manufacturing of parts with complex shapes and complicated material properties. The printing system will find its primary use in the activities of the Tufts University biomechanics, biology and biomedical engineering groups. The term "rapid prototyping" refers to the automated construction of physical objects directly from computer aided design or animation software. A variety of competing rapid prototyping technologies are now commercially available. However, for our purposes, the least costly, easiest to use, fastest and most flexible technology in terms of the variety of different materials that can be processed to fabricate parts is 3-dimensional printing. In this approach, photopolymer liquids are forced through inkjet print heads to create the layers of the part, which then are cured and fused together by ultraviolet light. The printing system will be the centerpiece of a biomimetic biomechanics facility, in which it will be used to fabricate essential parts that are the basis of the research activities of the Tufts University biomechanics group, and that cannot be produced by any other method. Use of this printing system in our interdisciplinary approach to biomechanics will lead to (1) major breakthroughs in the understanding of biologic mechanical systems and (2) novel applications of biologically inspired principles to engineer mechanical devices and systems. A particularly novel aspect of this work is that we seek to understand the role of tissue material properties in living movements and to exploit materials to show similar properties to design, build and control devices emulating biologic mechanical systems. Thus acquisition of the printing system will significantly advance the field of biomechanics, by providing a crucial part of the key instrumentation used in a novel and innovative, biologically inspired, closed loop research and design facility.

By allowing the fabrication of complex parts with advanced, non-uniform material properties, acquisition of the 3D printing system will permit us to undertake a large set of projects that are presently not possible due to part fabrication limitations. These projects will be central to the biomimetic biomechanics facility, allowing us to design and fabricate novel biomimetic devices and systems. Development of such devices and systems can in due course significantly impact understanding of biomechanical systems and thereby underlie new biologically inspired research and design approaches. Furthermore, the proposed project will permit the PIs to continue to engage undergraduate as well as graduate students in our ongoing research efforts, expanding the research training, educational experience and opportunities available to the students involved. Moreover, formal courses in which the printing system will be used have been identified from the Tufts Departments of Biology, Biomedical and Mechanical Engineering, and the Tufts Gordon Institute. In addition, as part of the process of recruitment of students into our projects, full attention will be given to the infrastructure in place at Tufts for recruiting and retaining women and underrepresented minorities in research. These include the New England Board of Higher Education Excellence through Diversity Program, a major forum for recruitment of underrepresented minority students, and formal ongoing outreach programs in Computer Science, Biomedical Engineering and robotics, as well as the Tufts School of Medicine NIH-funded minority outreach program, which has been highly successful in attracting underrepresented undergraduate students to summer internships for the last ten years.

PI: Pennell, Kurt
Title: Design and Application of Microbial Uranium Reduction Monitoring Tools (MURMoT)
Abstract:
For this aspect of the MURMoT project, Dr. Pennell will train graduate students from Dr. Löffler's research group in the proper experimental techniques to be used for the column studies described in Objective 4. The students will intern at Tufts University during the first two summer sessions of the project. Dr. Pennell will provide hands-on training in the following areas: soil characterization (e.g., measurement of specific surface area, soil bulk density, particle density), column packing (wet and dry packing methods with Ottawa sand and field soils), hydraulic conductivity and permeability (constant head method), hydrodynamic dispersion (non-reactive tracer tests), innoculum injection, electron donor addition, side-port and effluent sampling, column sectioning and solid-phase extraction at conclusion of each experimental study. In addition, Dr. Pennell will train the graduate students in the use of mathematical models (e.g., CXTFIT) that will be used to simulate solute transport in one-dimensional soil columns and obtain transport parameters (e.g., dispersion coefficient, mass transfer coefficient). Throughout the duration of the project Dr. Pennell will be involved in the design, implementation, and mathematical modeling of the column experiments described in Objective 4.

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: Laboratory Revitalization for Environmental Sustainability Research and Training at Tufts University
Abstract:
Over the past 40 years, the field of environmental engineering has evolved from a discipline focused primarily on "sanitary engineering" to one that brings a multidisciplinary approach to solve environmental problems in natural and engineered systems. This multidisciplinary approach is essential for addressing the growing need for sustainable approaches to using, managing and conserving natural resources. Water is a critical resource requiring sustainable management of both quantity and quality. One of the most critical threats to current and future clean water supplies is emerging contaminants, specifically engineered nanomaterials, pharmaceuticals and personal care products (PPCPs), and pathogens. To address this need, this award will renovate the existing environmental engineering laboratories to create a centralized, high-quality laboratory space to enhance ongoing and future research programs in environmental sustainability. The outcome of the renovation will be the new Environmental Sustainability Laboratory (ESL) that will be used extensively to support the research activities of eight researchers and their collaborators. The renovated space will also allow for integrated research and research training of undergraduate students, graduate students and postdoctoral associates from a range of disciplines and backgrounds. Within this renovated space, Tufts University will be able to pursue the following research goals: (1) acquire fundamental knowledge for enhanced mathematical modeling of engineered nanomaterial transport, distribution and persistence in multi-media environmental systems, (2) understand the influence of wastewater treatment operations and reactive transport processes on the environmental fate of PPCPs in water reuse systems, (3) develop real-time monitoring devices and modeling tools to assess the prevalence and fate of waterborne pathogens in urban areas, and (4) create and implement multi-disciplinary undergraduate and graduate student research training in environmental sustainability.

PI: Pennell, Kurt
Title: Secondary Impacts of In Situ Remediation on Groundwater Quality and Post-Treatment Management Strategies
Abstract:
Although substantial progress has been achieved in the remediation and management of hazardous waste sites at Department of Defense (DoD) installations, many sites contain recalcitrant contaminants, such as chlorinated solvents, often in complex hydrogeologic settings. As a consequence, these sites are characterized by failed or protracted remediation efforts, escalating costs with minimal progress toward remedial objectives, and an inability to achieve site closure with reasonable institutional or engineered controls. The unfortunate reality is that for these problematic sites significant amounts of the contaminant mass (>10%) are likely to remain even after aggressive source zone treatment. As a result, the subsurface remediation paradigm has shifted from the development of stand-alone, "silver-bullet" technologies toward the integration of complementary remediation technologies that can be deployed in parallel (simultaneous) or in series (sequential) to more efficiently address difficult remediation scenarios and meet cleanup goals. However, the potential impacts of in situ remediation technologies on long-term groundwater quality and the impacts of an aggressive primary treatment on microbial reductive dechlorination processes are not well understood. For example, physical-chemical treatments may alter aquifer geochemistry and the microbial ecology, both of which can be either detrimental (e.g., aquifer clogging, reduced microbial diversity, undesired by-product formation) or beneficial (e.g., enhanced electron donor availability) to subsequent bioremediation and/or natural attenuation processes.

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 (a) identify secondary impacts of two prominent in situ remediation technologies, thermal treatment and anaerobic bioremediation, on long-term groundwater quality; (b) establish methods to predict the extent of both positive and negative post-remediation impacts on groundwater quality; and (c) 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: Pfeifer, Blaine
Title: GOALI: E. coli Metabolic Engineering to Produce the Marine Anticancer Agent Lomaiviticin A
Abstract:
Intellectual merit: This project originates from a complementary partnership between Tufts University and Wyeth Research. The Wyeth Natural Products Discovery Group is recognized as a leader in industrial natural products-based drug discovery and development and has recently sequenced the gene cluster responsible for a potent anticancer compound called lomaiviticin A. The gene sequence was isolated from the marine bacterium Salinispora pacifica naturally responsible for lomaiviticin A production. Unfortunately, lomaiviticin A production from S. pacifica is time-consuming (7-14 days), inconsistent, and low-yielding (0.5 mg/L). In this project, E. coli will be used as a heterologous host for lomaiviticin A biosynthesis with the logic that E. coli will overcome the current S. pacifica production limitations. Here, the Principal Investigator's (PI's) group at Tufts University will lead efforts to transfer the lomaiviticin A gene cluster to E. coli for subsequent heterologous production. The Tufts team specializes in genetic transfer strategies to facilitate complex natural product production through convenient heterologous hosts. Together, the industrial-academic collaboration is well-suited for the production of this important marine anticancer agent through perhaps the ideal biological host.

Broader impacts: The synergistic and complementary academic-industrial partnership between Tufts and Wyeth will promote unique broader impact opportunities. Through Tufts, the project will use research as an educational and outreach mechanism. The research itself crosses numerous disciplines that include: molecular biology, microbiology, chemistry, genetics, and chemical and biological engineering. Students at two educational levels (graduate and undergraduate) will have the opportunity to participate in and learn from this multi-disciplinary approach. In addition, individual research responsibilities have been designed to re-enforce the academic level of the student. The graduate student will oversee and manage the experimental efforts in Aims 1 and 2 while simultaneously guiding undergraduate assistants. Hence, the graduate student will develop as an independent scientist and will also gain mentoring experience as he/she trains younger students. The undergraduate student will learn process culturing and analysis skills that coincide with their chemical and biological engineering curriculum. As part of the outreach initiatives, the undergraduate will be selected from the Tufts NSF-sponsored Computer Science, Engineering, and Mathematics Scholars program that encourages underrepresented groups to pursue science and engineering and for whom the PI serves as an academic advisor. Finally, the students will be able to compare academic and industrial research opportunities and gain first-hand access to both climates through summer internships hosted by Wyeth.

PI: Pokras, Mark
Title: Development of a Digital Image Database for Identifying Individual Common Loons
Abstract:
The status of populations of Common Loons (Gavia immer) is of great interest to New England and northern states from New York to Washington. Loon populations in the region have been unstable and in 1970 loons were listed as a Threatened Species in at least one New England state. Banding plays a crucial role in monitoring loon abundance. However, there are several problems that arise with the use of banding to census and monitor loons. Banding can incur unnecessary stress upon individuals and chicks, who must be captured and removed from the water with their parents to prevent predation. After being banded, loons can become wary and recapture can become much more difficult. Additionally, banding requires many person hours, can be very costly and may have unidentified deleterious affects on survival and reproduction.

Recently, various image analysis programs have been introduced and successfully used in several marine species including whale sharks, manta rays, right whales, and dolphins. These programs allow researchers to annotate spot patterns or fin shapes in photographs of individuals and subsequently search a database of individuals for matches. The use of this software allows researchers to acquire the same information as tagging, without the cost, person hours, and invasiveness. Additionally, the use of this software is capable of involving citizen scientists, as anyone is able to photograph individuals and submit their work to the database, which is then analyzed by researchers. This aspect greatly increases the number of possible samples and/or tracking capabilities, since data may be collected by many people, at any time, rather than a small group of researchers only periodically.

It is possible that this technique could be applied successfully to Common Loons, as they have extremely complex spotting and striping patterns. The goal of this project is to develop an approach for identifying Common Loons in this manner. To do this, we will need to first determine which of the many program options may be most successful in loons, or if a new program will need to be developed. Secondly and most importantly, we will need to determine if the patterns loons possess are discrete across individuals and consistent in the breeding plumage over the lifetime of the bird, and if so, what specific parts of the pattern will lead to the most effective identification. If successful, the implementation of this technique will not only cut costs of species monitoring and management activities and spur the involvement of citizens of New England in loon conservation efforts, but would also alleviate the stress induced by capturing and banding on individuals. Ultimately, the successful application of these methods in Common Loons could lead to more studies involving other avian species, such as penguins, that are negatively affected by capturing and banding.

PI: Pokras, Mark
Title: Shaping the Future: Education Resources for Conservation Medicine
Abstract:
The Cummings School of Veterinary Medicine at Tufts University has been at the forefront of the development of the field of conservation medicine. This emerging area of study explores the relationships between animal, human and environmental health and seeks to develop and apply health management practices and programs that sustain biodiversity and protect environmental systems essential to animal and human health.

It is the goal of Tufts Center for Conservation Medicine (TCCM) to train interdisciplinary leaders for today's and tomorrow's conservation challenges. We do this by inspiring veterinary students and giving them the tools they need to solve complex problems and become leaders in the U.S. and around the world. An important part of this strategy is to build collaborative educational, policy and research linkages among disciplines and with a wide variety of institutions.

To help achieve these goals, we are requesting support to pursue and expand the following efforts:

  1. Lecture series in wildlife and conservation biology
  2. Wildlife & Conservation Biology Symposium
  3. Short course on wildlife capture & immobilization techniques
  4. Acquisition of specialized conservation-related library resources
  5. Travel subsidies to support student involvement in conferences and field research projects

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 components, 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 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: Poltorak, Alexander
Title: Understanding Genetic Basis for Resistance to TNF-induced Lethal Shock Using New MSM/Ms Mouse Model
Abstract:
Mortality from toxic shock, caused by infective agents that trigger excessive synthesis of tumor necrosis factor (TNF) and other inflammatory molecules by macrophages, remains a major health problem. Here we describe for the first time resistance to lipopolysaccharide (LPS) and TNF-induced death in vivo of wild-derived mice of MSM strain, representing a new genetic model of resistance to toxic shock. The remarkable resistance of these mice to toxic shock is further emphasized by their lack of sensitivity to acute liver injury caused by the co-administration of TNF and D-galactosamine, a well-established in vivo model of endotoxic liver damage. In support of the in vivo resistance data, we show that primary MSM macrophages exhibit another phenotype in that they are resistant to TNF- or TLR-agonists-induced regulated necrosis (“necroptosis”) but not apoptosis cell death in vitro. Based on these results, we hypothesize that the two traits are mechanistically linked and propose to examine the connection between necroptosis and toxic shock traits by means of genetic linkage analysis aimed at identification of the loci, which confer both traits.

In further extension of the in vitro results, we discovered that TNF-mediated in vivo liver injury can be prevented by a specific inhibitor of a key necroptosis regulator, RIP1 kinase. In addition to cell death phenotype, we demonstrate that TNF synthesis by MSM macrophages is reduced and may also be influenced by RIP1 signaling in vivo. Based on our preliminary data, we suggest that RIP1 kinase is a mediator of acute toxic shock and may represent an important new target for therapeutic intervention. We propose to further characterize the scope of changes in in vitro MSM hepatocyte and macrophage responses to TLR agonists and TNF in order to better determine the cellular basis for MSM resistance to toxic shock.

Overall, these studies may provide important new insights into mechanisms of both toxic shock in vivo and necroptotic RIP1 kinase signaling, which has also been implicated in other acute pathologies, such as stroke, myocardial infarction and brain trauma.

 

 

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