PI: Quinto, Eric Todd
Title: Geometric Analysis on Euclidean and Homogeneous Spaces
Abstract: This award provides support for participants in a workshop on Geometric Analysis on Euclidean and Homogeneous Spaces at Tufts University, January 8-9, 2012. The workshop will encompass the related fields of integral geometry, harmonic analysis, differential geometry, representation theory, microlocal analysis and partial differential equations. These subjects find applications in areas as diverse as inverse problems, tomography, and signal and data analysis, all of which will be represented at the workshop. The workshop will be organized so that researchers and graduate students in the different fields will have ample opportunities to interact with the hope of developing collaborations. This range of fields is not typically represented together at workshops, and cross-fertilization between these fields is an important goal of the workshop. The workshop is preceded by the annual Joint Mathematics Meeting in Boston and uses the momentum of this national event to bring together well-known experts, young researchers, and graduate students. Women, graduate students, postdoctoral fellows, and young faculty will have the highest priority for support. The PIs plan to disseminate the research through proceedings that will include works by researchers from both the special session and the workshop. The interface between the topics of this conference has led to fruitful research and applications to medicine, industry, and science in the past. This meeting will provide the opportunity to push this positive development forward by bringing together, engaging, and motivating graduate students and young emerging researchers as well as more established experts. In particular graduate students are involved in the workshop through participation in a poster session. This allows the students to present their work and interact with each other and established experts in an informal setting. Supporting a diverse group of graduate students and beginning researchers is the highest funding priority.
PI: Quinto, Eric Todd
Title: Local Tomography and Microlocal Analysis
Abstract: The main goal of this project is to solve problems in tomography. Professor Quinto will develop and test local reconstruction algorithms and apply them to electron microscopy (ET), single photon emission tomography (SPECT), Sonar, and X-ray tomography. Professor Quinto is developing his fully three-dimensional tomography algorithm, and collaborators at Sidec and the Karolinska Institute, Stockholm, are testing it on electron microscope data of biological specimens. He is adapting and testing the algorithms on new scanning geometries (ways of taking the data) in ET and SPECT. With Andreas Rieder and Thomas Schuster, he is developing local algorithms for three-dimensional Sonar, and he is applying it to more general data acquisition geometries. Professor Quinto is developing X-ray tomography algorithms for novel X-ray source curves and applying them to medical tomography. In each case, he will develop pure mathematics to analyze what the algorithms do to boundaries or singularities, and he will improve the algorithms using this information. The PI will develop the microlocal analysis behind the physical models and his algorithms. Along with Hans Rullgard, he will quantify the strength of singularities of objects and their tomographic data.
The principal investigator will solve specific mathematical problems and apply them to practical problems with scientists in industry. His electron microscopy project is undertaken jointly with biologists and mathematicians, and his algorithm is being used to help scientists better image and understand the structure of individual molecules. The X-ray tomography (CT) project is motivated by a novel X-ray CT scanner for the operating room, and his algorithm can be used to image the inside of the body as doctors operate. His research on measuring strength of singularities from Radon data could help researchers quantify what is visible from tomographic data, much as the principal investigator's previous research on qualitative singularity detection shows where these singularities are located in the body. The work in this proposal also supports the mathematical education of Prof. Quinto's students. He will use some of his NSF support to work with at least two undergraduate students on his proposed research projects.
PI: Quinto, Eric Todd
Title: The Urban Math and Science Teacher Collaborative
Abstract: The Tufts University Urban Mathematics and Science Teacher Collaborative is a collaboration among three Tufts University Arts and Sciences departments (Education, Mathematics, and Physics & Astronomy) and three Boston public schools in the 5-12 grade range (Boston Arts Academy, Fenway High School, and Mission Hill School). A local non-profit organization, the Center for Applied Special Technologies (CAST) is providing professional development around Universal Design for Learning (UDL), a framework for designing curriculum and instruction to address barriers to learning. The Collaborative is preparing 16 new Mathematics and Science Teaching Fellows (TFs), and contributing to the development of 4 Master Teaching Fellows (MTFs) over 6 years. It builds on more than 10 years of experience in the preparation of teachers for work in urban schools through the Urban Teacher Training Collaborative (UTTC), a school-based teacher residency program in which pre-service teachers work in their assigned schools full-time for a full academic year under the guidance of mentor teachers and university professors while earning a Master of Arts in Teaching (MAT) degree. Two new courses integrating science and mathematics content with pedagogy are being developed collaboratively by education and STEM faculty in the physics and mathematics departments. Ongoing professional development is provided through the creation of an online community (through Digital Toolboxes and blogs) to encourage communication and discussion among TFs, MTFs, University Participants, and CAST. The courses, course materials, examples of effective teaching, and teacher discussion forums are being built as free, open-source, cross-platform modules that universities and schools can use and modify under Creative Commons licensing. The project seeks to increase teachers' understanding of the challenges of teaching in urban settings; increase the numbers and retention of teachers of Mathematics and Science in urban schools; transform teachers' practices in Mathematics and Science; increase student understanding and performance in Science and Mathematics for students taught by TF and MTFs; and develop stronger relationships with local urban schools.
PI: Ramsburg, Andrew
Title: Exploration of the Mechanisms Controlling Emulsion-Based Alkalinity Release During Subsurface Remediation
Abstract: At present, there is considerable interest within the geoenvironmental community to harness coupled physical, chemical and biological processes for remediation and stewardship of contaminated sites. Many of these processes require control of pH during treatment and, in some cases, long after. The central hypothesis of this proposal is that carefully designed oil-in-water emulsions can be effective tools for modulating alkalinity release during remediation. Emulsions have been employed to sequester contaminants and deliver remedial amendments, but controlling amendment release, both the extent and rate, remains highly empirical. This empiricism restricts our ability to realize the full potential that oil-in-water emulsions hold for controlling release of amendments within complex systems of porous media. The proposed project integrates experiments and modeling to elucidate the mechanisms controlling alkalinity release from oil-in-water emulsions during subsurface remediation.
Project objectives are:
- assess mechanisms controlling the stability of CaCO3 and NaO2 nanoparticles suspended within soybean oil;
- create and optimize critically stabilized oil-in-water emulsions containing alkalinity release particles within a dispersed soybean oil phase;
- elucidate the influence of soybean oil composition on the rate of alkalinity release;
- analyze the ability of oil-in-water emulsions to deliver and release alkalinity for control of groundwater pH.
Emulsion based delivery and controlled amendment release are important concepts that are improving the science and engineering behind subsurface remediation. Many current approaches to managing groundwater contamination rely on further advances in amendment delivery to initiate and sustain contaminant degradation or immobilization. The research described in this proposal will integrate laboratory experiments across multiple scales to better understand how geoenvironmental engineers can control the placement and flux of remedial amendments within contaminated porous media. The specific ability to “tune” the delivery and release of alkalinity has significant potential to improve the performance of chemical and biological remediation strategies. Anticipated deliverables include knowledge and constitutive relationships related to mass transfer kinetics, droplet coalescence, and control of droplet retention. Project results will also contribute to understanding the spatial and temporal requirements for pH control.
PI: Reijmers, Leon
Title: Molecular Analysis of Functional Neural Circuits
Abstract: A functional neural circuit consists of a group of connected neurons that collaborate to execute a specific function of the brain. The understanding of the molecular mechanisms that underlie the development, maintenance and experience-dependent modification of functional neural circuits is incomplete due to limitations of existing methods. I propose to generate a transgenic mouse that addresses these limitations by exploiting two recent methodological advances. The first advance is the TetTag mouse, which is a transgenic mouse that can be used to genetically tag a single functional neural circuit. This tag enables the selective molecular analysis of neurons that have a shared function. This method is more sensitive to changes within a single functional neural circuit than other available methods, which have to rely on spatial criteria and thereby include neurons that do not participate in the circuit of interest.
The second advance is the Translating Ribosome Affinity Purification (TRAP) method, which enables purification of actively translated messenger RNA from a genetically defined group of neurons. TRAP analysis reflects changes at both the transcriptional and translational level, while other available methods only detect transcriptional changes. I will combine TetTag and TRAP within a single transgenic mouse to generate the first tool that enables the comprehensive analysis of all translational events within a single functional neural circuit. Neurons tagged with the TetTag mouse during fear conditioning provide a stable neural correlate of the fear memory. I will use the TetTag/TRAP mouse to purify actively translated messenger RNA from these tagged neurons in order to detect the protein synthesis events that underlie the storage of a memory. The TetTag/TRAP mouse can be used for the molecular analysis of various functional neural circuits, including those involved in memory, addiction, epilepsy, circadian rhythms, spinal cord regeneration, pain, brain development, and neuronal cell death.
PI: Ridge, Jack
Title: Collaborative Research: Synchronizing the North American Varve Chronology and the Greenland Ice Core Record Using Meteoric 10Be Flux
Abstract: The North American Varve Chronology (NAVC) is a nearly 6000-year-long sequence of annual sediment layers that was deposited in glacial lakes adjacent to the margin of the Laurentide Ice Sheet, which covered much of North America during the last ice age. At the time these sediments were deposited, between approximately 12,500 and 18,000 years ago, climate was warming and the Laurentide Ice Sheet was shrinking rapidly. The sediments that make up the NAVC are an annually resolved record of events that took place at the retreating ice margin, including the position and retreat rate of the ice margin, the flux of water derived from summer melting of the ice sheet, and large glacial-lake outburst floods that occurred periodically as ice-marginal lakes drained.
The goal of this proposal is to understand the relationship of these events to climate changes that were occurring at the same time and are recorded in ice core records from Greenland, which is important because i) it will help to understand how large ice sheets responded to rapid climate warming, and ii) because this time period is a potential analog for the current period of rapid warming and likely shrinkage of glaciers and ice sheets. The obstacle to comparing the NAVC with Greenland climate records is that, although both records have been independently dated, the precision of these dates is a few hundred years, which is not precise enough to accurately determine which climate changes are related to which glacial events.
This project will try to resolve this issue by comparing records of atmospherically-produced Beryllium-10 in both NAVC sediments and Greenland ice cores. Be-10 is a naturally occurring rare isotope of Beryllium that is created by cosmic-ray bombardment of the upper atmosphere and falls to earth in precipitation. Be-10 production changes through time due to changes in the Earth's magnetic field, and these changes occur everywhere on Earth at the same time. Be-10 is captured and preserved in sedimentary archives such as ice cores and lake sediments. Thus, Be-10 concentrations in the Greenland ice cores and in the NAVC lake sediments should show the same pattern of changes. By aligning these changes with each other, we can place the two records on a common timescale. Thus, even if we do not know the exact time that a particular climate change recorded in the Greenland ice cores took place, we will know exactly what ice-marginal events recorded in NAVC sediments were occurring at that time. We anticipate that we will be able to match these two records with a precision of several decades, which should be precise enough to determine the relationship between important climate changes and ice sheet change.
PI: Rife, Jason
Title: Risk Analysis to Enhance GBAS Performance for NextGen Applications
Abstract: Plans for the Next Generation Air Transportation System (NextGen) call for the certification of an enhanced Ground Based Augmentation System (GBAS) which will enable zero visibility (Category III) precision landing. Such a high-precision, high-integrity GBAS would also enable safe and efficient surface operations in low visibility conditions, including taxiing and take-off. It is expected that the Category III (Cat III) GBAS architecture will be based, in large part, on the Category I (Cat I) GBAS architecture, which was certified in late 2009. Although prior experience with Cat I will accelerate the certification of Cat III, research will be required to resolve remaining open questions regarding the practical implementation of a Cat III system.
Our proposed research will address two open questions which are central to the development of Cat III GBAS. The first of these questions is whether or not existing Cat I integrity monitors, such as the Excessive Acceleration and Signal Deformation Monitors, can be adapted for use with a Cat III system, given that the Cat III integrity risk requirements are stricter by two orders of magnitude. To make such an adaptation possible, our research will mitigate excess conservatism that exists in prior integrity analyses, specifically, in assumptions made regarding time-correlated random noise and its impact on monitor missed-detection probabilities.
Our research will also address a second key open question for Cat III GBAS implementation: how to make effective use of modernized, multi-constellation GNSS signals in GBAS. In the long term, it is desirable that Cat III GBAS be extensible for use with these emerging signals, in order to further tighten integrity bounds and increase system availability. We believe that a major obstacle to incorporating new signals into GBAS will be the labor-intensive process of certifying the error models associated with each new signal. Accordingly, we will pursue a novel integrity algorithm that leverages super-redundant satellite constellations (constellations with 25 or more satellites visible at any one time) to reduce the need for detailed characterization of error distribution models.
PI: Rios, Maribel
Title: BDNF and TrkB-Containing Neuronal Circuits Mediating Energy Balance
Abstract: Close to a third of the population in the United States is obese and over 60% is overweight. This is an alarming trend as obesity significantly increases susceptibility to type 2 diabetes, cardiovascular disease and other medical disorders. Mounting evidence indicates that the brain-derived neurotrophic factor (BDNF)/TrkB pathway plays a critical part in energy balance regulation and is a promising target for novel therapies. Accordingly, reduced BDNF signaling in mice and humans results in hyperphagic behavior and dramatic obesity. It remained unclear whether BDNF, which supports neuronal survival, differentiation and synaptic plasticity, acted as a required satiety factor in the adult brain or as a developmental facilitator of feeding neural circuits. As part of the previously funded project, we showed that BDNF acts in the adult animal to promote satiety and that the ventromedial hypothalamus (VMH) is a critical source of this neurotrophin. Supportive evidence includes the robust effects of energy status on expression of BDNF and TrkB in the VMH and the hyperphagia and obesity elicited by selectively deleting BDNF in the VMH of adult mice. The cellular and molecular mechanisms underlying the anorexigenic effects of BDNF in the VMH remain to be elucidated.
Our recent analysis of the transcriptome of cells laser-captured from the VMH of mice with central (BDNF2L/2LCk-cre) or VMH-specific depletion of BDNF revealed decreased expression of α2δ-1 and of two other genes associated with obesity susceptibility in a recent large-scale human study. Our preliminary studies show that BDNF's anorexigenic effects are mediated by α2δ-1, a high voltage-gated calcium channel subunit that enhances calcium currents and mediates excitatory synaptogenesis. We found that: i) selective α2δ-1 inhibition by chronic gabapentin infusion into wild type VMH increased food intake and body weight and ii) viral-mediated α2δ-1 delivery to the VMH of BDNF2L/2LCk-cre mutants ameliorated their hyperphagia and body weight gain.
This competitive renewal proposal seeks to build on these findings by ascertaining cellular mechanisms underlying the effects of BDNF and α2δ-1. Because calcium currents in VMH cells of BDNF mutants are normal, the satiety effects of α2δ-1 might be related to its ability to induce excitatory synaptogenesis in a calcium-independent manner. Thus, Aim 1 will investigate the role of BDNF and α2δ-1 in dynamic changes in VMH excitatory drive onto anorexigenic POMC neurons induced by nutritional cues using anatomical and electrophysiological approaches. In Aim 2, we will investigate whether BDNF and α2δ-1 also regulate excitatory drive onto anorexigenic VMH neurons. In Aim 3, we will test whether the reduced expression of two other gene candidates in the BDNF mutant VMH contributes to the emergence of hyperphagic behavior and obesity and whether they act in common pathways with α2δ-1 in the VMH. These studies will provide a mechanistic understanding of anorexigenic actions of BDNF and reveal novel avenues for the treatment of obesity.
PI: Rios, Maribel
Title: Investigations of Cellular and Molecular Mechanisms Underlying the Effects of BDNF and Restricted Food Intake on Binge Eating
Abstract: Binge eating is a cardinal symptom of binge eating disorder (BED) and bulimia nervosa (BN), which afflict a significant number of individuals with considerable medical consequences. Dieting or restricted intake of palatable, energy-rich foods is considered a high risk factor but the underlying pathological mechanisms are poorly understood. Involvement of the mesolimbic dopamine pathway was suggested because intermittent access to palatable food elicits both bingeing and alterations in this reward circuit in rodents. Evidence suggests that diminished mesolimbic activity produces reward deficiency syndrome and compensatory overeating. Similar to drugs of abuse, palatable food withdrawal might induce synaptic modifications in the mesolimbic reward circuitry that impair dopamine secretion and elicit binge eating as a maladaptive behavior. As not all individuals that diet binge eat, genetic factors might also contribute. Indeed, humans afflicted with BED and BN that also carried the Val66Met polymorphism in the brain-derived neurotrophic factor (Bdnf) gene exhibited more severe binge eating compared to wild type carriers. This mutation impedes regulated secretion and signaling of BDNF, a prominent regulator of neuronal synaptic plasticity. We found that central BDNF depletion impairs mesolimbic dopamine secretion and interacts with limited palatable food access to induce severe binge-like behavior in mice. We seek to test the hypothesis that deficient BDNF cooperates with intermittent palatable food access to induce synaptic modifications that decrease mesolimbic dopamine tone and drive binge eating.
In Aim 1, we combine genetic, electrophysiological and biochemical approaches to quantify changes in excitatory and inhibitory transmission in VTA dopamine neurons induced by palatable food restriction and how loss or gain of BDNF function influences these responses.
Aim 2 will identify molecular mechanisms activated by deficient BDNF signal and restricted palatable food access, leading to reduced mesolimbic activity. These investigations will inform cellular and molecular mechanisms underlying binge eating and novel therapeutic avenues.
PI: Rogers, Chris
Title: DRK12 – Integrating Engineering and Literacy
Abstract: The Integrating Engineering and Literacy (IEL) project is developing and testing curriculum materials and a professional development model designed to explore the potential for introducing engineering concepts in grades 3-5 through design challenges based on stories in popular children's literature. The project research and development team at Tufts University is working with pre-service teachers to design and test the curriculum modules for students and the teacher professional development model. Then the program is tested and refined in work with 100 in-service teachers and their students in a diverse set of Massachusetts schools. The research team hypothesizes that professional development for elementary teachers using an interdisciplinary method for combining literature with engineering design challenges will increase the implementation of engineering in 3-5 classrooms and have positive impacts on students.
The driving questions behind this proposed research are: (1) How do teachers' engineering (and STEM) content knowledge, pedagogical content knowledge, and perceptions or attitudes toward engineering influence their classroom teaching of engineering through literacy? (2) Do teachers create their own personal conceptions of the engineering design process, and what do these conceptions look like? (3) What engineering/reading thinking skills are students developing by participating in engineering activities integrated into their reading and writing work? The curriculum materials and teacher professional development model are being produced by a design research strategy that uses cycles of develop/test/refine work. The effects of the program are being evaluated by a variety of measures of student and teacher learning and practice.
The project will contribute materials and research findings to the ultimate goal of understanding how to provide elementary school students with meaningful opportunities to learn engineering and develop valuable problem solving and thinking skills.
Mentor: Romero, Michael
Fellow: Lattin, Christine
Title: Evaluating the Stress Response of Wild Birds as a Bioindicator of Sub-Lethal Acute and Chronic Effects of Crude Oil Exposure
Abstract: One of the most important yet least understood of oil's biological impacts is on the stress response. Studies show petroleum can interact in an additive fashion with other stressors to cause increased mortality, but it is not clear exactly why — does petroleum change stress hormone receptor concentrations in the brain or peripheral tissues, does it disrupt feedback mechanisms or stress hormone production, or both? This study uses both lab and field components to systematically quantify the effects of Gulf of Mexico crude oil on the stress response and stress hormone receptors of wild birds. This will allow an evaluation of whether these physiological measures can be used as bioindicators of sub-lethal acute and chronic effects of crude oil exposure. Potentially this could allow us not only to identify exposure in wild shorebirds from contaminated areas, but also in humans.
PI: Romero, Michael
Title: Physiology and Behavior of Stress in Wild Animals
Abstract: All organisms are faced with challenges (such as predators or storms) from the environment. Animals mount a stress response in order to survive these challenges, but even after nearly 100 years of studying stress, there is only a basic understanding of how the stress response helps wild animals to survive. This lack of knowledge is of growing significance because man-made noxious events (such as habitat degradation or human disturbance) could cause chronic stress. Although a short-term stress response to a predator or storm is necessary for survival, chronic stress can be very harmful, and if it affects enough individuals, can cause population declines.
This project will use wild European starlings as a model to test two hypotheses: (1) That increased frequency of normal environmental events can result in chronic stress in wild animals; and (2) There will be long-term impacts on offspring whose mothers were exposed to chronic stress. Testing these hypotheses will require an integrated approach of laboratory and field studies on the physiology and behavior of both captive and free-living starlings. The ultimate goal is to create a physiological and endocrinological profile of chronically stressed wild animals. This will provide criteria for identifying chronically stressed individuals in the wild and determining the long-term impact on those individuals and their offspring.
PI: Rothbaum, Fred
Title: Child and Family WebGuide (Minnesota)
Abstract: The Child and Family WebGuide is a non-profit project coordinated by Fred Rothbaum, a Professor in the Eliot Pearson Department of Child Development at Tufts University. It is supported by the Child Development Department and the University. The goal of the WebGuide is to help parents, professionals, students and others interested in child development find trustworthy, research-based information about infants, children adolescents, and their families and communities. The WebGuide addresses one of the major concerns of the internet: how to obtain credible, research-based information. For example, when searching on the internet for the topic of spanking children, a user would instead find sites relating to personal opinion, pornography, and religious organizations. The WebGuide selects only those sites and videos that provide non-technical, user-friendly, research-based information. The information addresses specific medical conditions and treatments as well as a broad range of child development information such as obesity, self esteem, discipline, anxiety and sibling relationships. All sites and videos have undergone a rigorous evaluation process by graduate students who are supervised by faculty in Child Development. The evaluations are based on criteria that have been recommended by a panel of scholars and research practitioners as well as findings from focus groups and interviews with parents. Both modes of research indicated the need for credible and non-technical information, criteria that the WebGuide relies upon in selecting sites and videos. As a result of its Google powered search tool, the WebGuide can help visitors instantly find the specific information about children that they need.
PI: Rozanski, Elizabeth
Title: Multiple Organ Dysfunction in Gastric Dilatation Volvulus (GDV); The Role of Cardiac Dysfunction and Coagulopathy
- Multiple organ dysfunction syndrome (MODS), including cardiac dysfunction and coagulopathy is responsible for the death of dogs which do not survive an episode of GDV.
- Myocardial dysfunction as assessed by echocardiography, elevated tropinin I, and increased pro-BNP will be more common in non-survivors.
- Coagulopathy, as assessed by thromboelastography (TEG), will be more common in non-survivors.
- Treatment of dogs with documented hypercoaguablity with a continuous infusion of heparin will result in normalization of thromboelastrography.
- A scoring strategy will be useful for stratifying affected dogs, and will provide a springboard for future studies.
- Retrospective medical record review of 200 cases of canine CDV for assessment of MODS and for initial creation and validation of a proposed scoring strategy (Functional Assessment of Bloat; FAB)
- Validation of the role of cardiac dysfunction by a combination of echocardiography, and measurement of Pro-BNP and troponin
- Determination of the incidence of coagulopathy, including hypercoagulability, by thromboelastography (TEG)
- Determination dose requirements for a continuous rate infusion (CR1) of heparin to result in a therapeutic heparin level in clinically normal dogs.
PI: Ruskai, Mary Beth
Title: Quantum Information Theory
Abstract: It was recently proved that there are situations in which the quantum phenomenon called entanglement can increase the capacity of a quantum channel used to transmit classical information. Such channels are called non-additive; however, no explicit examples are known. Even more recently, it was observed that the anti-symmetry associated with the Pauli exclusion principle can be used to give an example of a channel which is non-additive for a related quantity, called the minimal output Renyi entropy, when p > 2. This grant is intended to construct more examples of non-additive channels and clarify our understanding of this phenomenon. In connection with this, the PI will closely examine the role of permutational symmetry in quantum information theory. The PI will also study entropy inequalities and cones of entropy vectors for composite systems.
This highly interdisciplinary work will clarify the role of the Pauli exclusion principle, which is often suppressed in quantum information theory. It will also be of interest to quantum chemists and condensed matter physicists, particularly those who used reduced density matrices. The work should help identify situations in which quantum systems have advantages over classical ones for communication, as well as computation.
PI: Rybak-Akimova, Elena
Title: Mechanism-Based Design of Biomimetic Green Oxidation Catalysts
Abstract: The long-term goal of the proposed project is to develop efficient and selective catalytic systems for green oxidations. Dioxygen and hydrogen peroxide are ideal oxidants because they are readily available and environmentally clean, producing water as the only byproduct. These reagents allow for the most efficient, atom-economy approach to oxidative functionalization of organic molecules, thus saving energy in chemical synthesis. Non-toxic, biocompatible iron complexes will be used as catalysts acting similarly to natural iron-containing oxidative enzymes. Identification of kinetically competent intermediates (transient, very reactive species) is a critical component of a rational design of useful, selective and efficient oxidation catalysts utilizing dioxygen and hydrogen peroxide as terminal oxidants. Proposed detailed kinetic and mechanistic studies will take advantage of multi-mixing stopped-flow techniques that allow us to watch the intermediate formation, and their subsequent reactions with substrates by time-resolved spectrophotometry on a millisecond time scale. Short-lived, unstable intermediates will be observed at low temperatures. This program will provide much needed information about the details of individual reaction steps in oxygen activation.
The project will focus on non-heme iron(IV)-oxo intermediates supported by amino- or amidopyridine ligands. Oxygen atom transfer reactions to olefins and aromatic substrates will be developed and kinetically characterized. The role of proximity effects and intramolecular proton delivery pathways in generating high-valent iron intermediates and their reactivity in subsequent two-electron oxidations will be determined. The reactivity of diiron(IV) systems will be compared to their mononuclear counterparts. Complete kinetic description of catalytic cycles will become possible for the reactions with kinetically competent iron(IV) intermediates. Even more interestingly, the lack of agreement between kinetic models and experimental catalytic behavior will indicate the presence of new, more active intermediates, such as elusive Fe(V). Conditions for trapping these new species for spectroscopic studies will be identified on the basis of kinetic and mechanistic information. Strongly electron-donating deprotonated amide groups will stabilize high-valent iron species, and facilitate activation of molecular oxygen.
As an outcome of the proposed studies, detailed picture of the preferential reactivity patterns of various iron-peroxo and high-valent iron-oxo intermediates will emerge. This knowledge will allow researchers to rationally select a suitable intermediate for specific organic transformations (as exemplified by epoxidations and aromatic hydroxylations in the proposal). The ligand structural features that stabilize particular intermediates will be determined, along with the chemical ways of harnessing the oxidizing potential of less reactive, yet possibly more selective intermediates. The understanding of reactivity profiles of various iron-based intermediates will help to interpret the mechanisms of known catalytic oxidations, and provide guidance in future catalyst design.
Mentor: Sacheck, Jennifer
Fellow: Au, Lauren
Title: Dietary Vitamin D and Response to Vitamin D Supplementation on Serum Vitamin D Status in Overweight and Obese Urban Schoolchildren
Abstract: During childhood and adolescence, vitamin D is important for calcium absorption and bone growth. In addition to skeletal effects, vitamin D may also be associated with a variety of diseases, including diabetes, hypertension, common cancers, and cardiovascular disease. The growing number of studies on vitamin D has intensified interest in improving vitamin D status in populations at risk for deficiency. These populations include children who do not consume vitamin D-rich foods or supplements, live in northern latitudes, and have darker skin pigmentation. More recently, it has been noted that overweight and obese children may also be at heightened risk for deficiency, although the causes of this are still not entirely clear. Excess adipose tissue may sequester vitamin D, but research also suggests that these children consume less dietary vitamin D than normal-weight children potentially contributing to lower serum 25-hydroxyvitamin D (25OHD).
The overall objective of the proposed work is to determine the necessary vitamin D intake to meet recommended levels of serum 25OHD (>20 ng/mL) for overweight and obese schoolchildren. Our overarching hypothesis is that overweight/obese schoolchildren (BMI >85th percentile for age) will have lower baseline serum 25OHD levels than normal-weight children (BMI <85th percentile for age); and that this could be attributed to consuming less dietary vitamin D and excess adiposity. Consequently, overweight/obese children will need higher doses of vitamin D to achieve recommended 25OHD levels compared to normal-weight children. We propose to examine this concept in 4th – 8th grade children from socioeconomically disadvantaged and racially diverse communities with high obesity rates in the northeastern U.S. Our pilot data suggest this population has very high rates of vitamin D deficiency. Our specific aims and hypotheses are:
Aim 1: To examine dietary and serum 25OHD differences between overweight/obese and normal weight schoolchildren.
Hypothesis 1: Overweight/obese schoolchildren consume less vitamin D-rich foods (milk, milk products, fortified cereals, oily fish, etc.) compared to normal-weight children. Overweight/obese schoolchildren will also have lower baseline 250HD levels compared to normal-weight children, while controlling for covariates.
Aim 2: To examine the impact of three different doses of vitamin D (400 IU, 1000 IU, or 2000 IU/d) for six months on serum 25OHD in overweight/obese versus normal-weight schoolchildren.
Hypothesis 2: Due to dietary habits and adiposity, overweight/obese schoolchildren will need higher doses of vitamin D to effectively achieve recommended 25OHD levels compared to normal-weight children who will need lower doses, while controlling for covariates.
PI: Sacheck, Jennifer
Title: Impact of Vitamin D Supplementation on Cardiometabolic Risk in School Children
Abstract: Vitamin D deficiency has been linked to the development of cardiovascular disease and it is estimated that six million children are currently vitamin D deficient. As it is difficult for many children to consume the new recommended intake of 400 IU/d set forth by the American Academy of Pediatrics, many have suggested that vitamin D supplementation is necessary. Vitamin D deficiency is even more common in northern latitudes, amongst some minority groups with darker skin pigmentation, and in those who are overweight or obese. In these groups, higher supplemental doses may be needed to maintain optimal serum levels and to prevent cardiovascular risk.
The overall objective of the proposed work is to determine the appropriate vitamin D supplementation requirements for children living at northern latitudes (above 42°) to maintain optimal serum levels of 25-hydroxyvitamin D [25(OH)D] and minimize cardiometabolic risk. We will also investigate for the first time in a randomized controlled trial what happens to serum 25(OH)D and cardiometabolic risk factors after supplementation is discontinued. Fourth-eight grade children from socioeconomically disadvantaged and racially diverse communities with high obesity rates in the northeastern U.S. will be randomized to 400 IU, 1000 IU, or 2000 IU/d for six months starting in the early winter. Serum 25(OH)D and cardiometabolic risk factors (blood lipids and glucose) will be measured at baseline prior to supplementation, at 3- and 6-months, and then again 6 months following the discontinuation of supplementation. Adiposity, physical activity, dietary intake of vitamin D, and sun exposure will also be assessed at these times.
Results of this work will inform the development of evidence-based recommendations and guidelines regarding vitamin D supplementation to maintain optimal 25(OH)D levels and to reduce cardiometabolic risk. If vitamin D repletion has the potential to improve the cardiometabolic risk profile during childhood, then we will ultimately be able to lower a child's risk of developing cardiovascular disease in adulthood.
PI: Saliba, Richard
Title: Mechanisms Underlying Activity-Dependent Modulation of GABAA Receptor Expression
Abstract: γ-aminobutyric acid (GABAA) receptors are the major sites of synaptic inhibition in the CNS and are drug targets for a variety of pharmacotherapeutic agents such as benzodiazepines, barbiturates, general anesthetics and neurosteroids. Compromised GABAergic transmission is one of the contributing factors in a number of neurological and psychiatric diseases such as epilepsy, anxiety, sleep disorders, addiction, autism, mental retardation, depression and schizophrenia. The number of post-synaptic GABAA receptors is highly regulated and is a major determinant in modulating the strength of synaptic inhibition. Chronic bi-directional changes in neuronal activity can lead to global, compensatory adjustments in excitatory and inhibitory synaptic strengths; a process known as homeostatic synaptic scaling. Modulating the number of GABAA receptors at post-synaptic sites is one of the mechanisms underlying activity dependent changes in inhibitory synaptic transmission. I have previously shown that chronic changes in neuronal activity regulate the endoplasmic reticulum (ER)-associated degradation of GABAA receptors, mediated by Ca2+ influx through L-type voltage gated Ca2+ channels, modulating receptor cell surface number and efficacy of synaptic inhibition. However, the signaling pathways linking changes in neuronal activity to the modulation of GABAA receptor turnover and abundance at inhibitory synapses is unknown. These phenomena together with my preliminary studies have allowed me to generate a central hypothesis driving the experiments in this proposal: Activity dependent phosphorylation of the GABAA receptor β3 subunit on serine 383 by CaMKII, modulates the insertion of these receptors and consequently their abundance at inhibitory synapses, modifying the efficacy of synaptic inhibition.
My work will focus on 3 distinguishable but complimentary experimental goals:
To test the hypothesis that the CaMKII phosphorylation site within the GABAA receptor β3 subunit (S383) modulates the synaptic expression levels of these receptors and synaptic inhibition.
To test the hypothesis that changes in [Ca2+]i regulate the CaMKII dependent phosphorylation of β3S383 and the numbers of GABAA receptors at the cell surface.
To test the hypothesis that changes in neuronal activity modulate the CaMKII phosphorylation of β3S383 and regulates the insertion and abundance of cell surface GABAA receptors.
PI: Sassaroli, Angelo
Title: Functional Near-Infrared Imaging Using the Phase of Hemodynamic Oscillations
Abstract: This project explores the potential of a novel approach to functional studies of the brain with near-infrared spectroscopy. We hypothesize that the phase relationship between oscillations of cerebral oxy-hemoglobin and deoxy-hemoglobin concentrations provides information about (1) the interplay of hemodynamic and oxygenation-related processes that are associated with brain activation, and (2) functional connectivity networks involving distinct cortical areas. Specifically, in this project we will perform a new analysis of data collected previously in a full-night sleep study using a novel phasor-based analysis and representation of hemodynamic oscillations. This new analysis will allow us to test the hypothesis that hemodynamic conditions associated with different sleep stages induce different phase relationship between spontaneous oscillations of oxy-hemoglobin and deoxy-hemoglobin concentrations at frequencies around 0.1 Hz (Mayer waves). We will also test the hypothesis that oxy-hemoglobin and deoxy-hemoglobin concentration changes have different relative phases depending on the cortical area activated and/or the stimulation protocol. Finally, we will perform pilot measurements to test the potential of this new phase-based method in studying functional connectivity networks in the brain. This project can lead to a novel approach to functional brain studies and explores signals that are not accessible with functional magnetic resonance imaging, involving both paramagnetic and diamagnetic hemoglobin species.
PI: Scheutz, Matthias
Title: Effective Human-Robot Interaction with USVs and UAVs through Robust Natural Language Dialogue and Dynamic Vehicle Autonomy
Abstract: Future urban or littoral missions by the US Navy will require an increasingly close and effective cooperation between humans and autonomous robotic systems. The particular roles of robots in these missions and their envisioned interactions with human team leaders, however, raise substantial challenges for the design of effective human-robot interaction (HRI). These challenges concern, among others, the robots' abilities to take directions in natural language about the tasks to be performed (taskability) and to use dynamic autonomy to fill in missing steps in the instructions which will make it more autonomous while reducing the cognitive load of an operator. Moreover, the goal for future operators of autonomous systems is for one operator to interact with multiple autonomous devices at the same time rather than requiring multiple operators to control one device, which presents a major research challenge.
The main goal of our research will thus be to design, implement, and evaluate computational mechanisms that improve human-robot interactions in natural language with multiple diverse autonomous vehicles, in particular, unmanned air vehicles (UAVs) and unmanned surface vehicles (USVs). These mechanisms will build on and complement the research already accomplished in our current ONR MURI project "Effective Human-Robot Interaction under Time Pressure through Robust Natural Language Dialogue and Dynamic Autonomy", extending it in two very substantial ways: (1) we will move from an indoor mobile robot to outdoor aircraft and boats, and (2) we will move from operator interactions with a single team member in natural language to interactions with two team members that can also interact among each other in natural language.
To enable interactions with UAVs and USVs, we will extend our DIARC architecture to integrate the necessary perceptual and motor primitives and add the corresponding natural language constructions (e.g., we will add a primitive for a UAV to take off together with the concept "taking-off" as well as the action verb 'to take off'). We will also add new control schemes as well as robot-robot natural language communication schemes. We will demonstrate the capability of the extended DIARC architecture to allow for the control of multiple outdoor robots through natural language interactions by one operator in three demonstration scenarios involving a single UAV, a single USV, and then both one UAV and one USV. The demonstrations will not only serve as a deliverable at the end of the two-year project, but will also prove that reliable natural language interactions with multiple diverse autonomous outdoor vehicles are feasible. A formal HRI evaluation will require subsequent funding, for which we intend to apply separately.
PI: Scheutz, Matthias
Title: HCC: Large: Collaborative Research: Human-Robot Dialog for Collaborative Navigation Tasks
Abstract: For humans and intelligent robots to collaborate on shared tasks, each agent must bring something useful to the partnership. In particular, the robot must have sufficiently rich knowledge of the domain to understand the requirements of the task, and that knowledge must be represented in a form that supports effective communication between the human and the robot.
We propose to address this problem, building on the Hybrid Spatial Semantic Hierarchy (HSSH), a human-inspired multi-ontology representation for knowledge of navigational space implemented on physical robots, and on our extensive experience in human-robot interaction (HRI). The domain of navigational space is distinctive because it is one of the few domains where the knowledge is sufficiently well understood for a physically embodied robot agent to be a useful collaborator, meeting genuine human needs.
The HSSH represents space within four different ontologies, representing different levels of abstraction of the spatial structure of the environment. The level represents local metrical knowledge of small-scale space (i.e., space within the sensory horizon of the agent), supporting efficient motion in the continuous environment, hazard detection and avoidance, and communication of local motion targets. The level abstracts continuous motion to discrete travel actions among decision points, the description of each decision point in terms of a small discrete set of choices for the next travel action, and supports communication like "Take the next left, and then the second right." The level represents environmental structure in terms of a graph of places, paths, and regions, and supports communication in terms of travel goals within the map. The level describes the environment in terms of locations within a single global frame of reference. This level of representation is useful to human navigators in the form of auxiliary cognitive aids, but it is neither necessary nor sufficient as part of the natural human cognitive map. These multiple levels make it possible to learn a sophisticated cognitive map by incremental assimilation of local travel experience. They also provide distinct representational targets for different types of communication acts.
Our project focuses on the basic issues of representation, inference, communication, and action in collaboration between a human and an intelligent robot, serving the needs of the human. To ensure generality, we will carry out this research with two different kinds of navigational robots. An intelligent robotic wheelchair carries the human driver to desired destinations. A telepresence robot transmits its perceptions to a remote human driver as it navigates within an environment, so the driver can achieve virtual presence and communicate with others within the environment. In both cases, the robot is an intelligent agent, learning from its travel experiences and building an increasingly sophisticated cognitive map. As the robot's cognitive map improves, the human can communicate with it at higher levels of abstraction.
PI: Schwob, James
Title: Anatomical Bases of Human Olfactory Dysfunction
Abstract: Improvements in diagnosis and treatment of olfactory disease will require first, a more comprehensive analysis of the anatomical status of the olfactory periphery (the 1cm2 areas on the nasal septum and the lateral nasal wall immediately inferior to the cribriform plate) with respect to the preservation of the olfactory epithelium (OE) and, second, a means of determining whether the OE's inherent capacity to reconstitute itself remains intact. We propose three specific aims designed to address our shortcomings with respect to the pathological bases of peripheral olfactory dysfunction.
Aim 1: We observe a better than expected preservation of OE in the olfactory area of autopsy specimens, but immature neurons are abnormally prominent. We will test the hypothesis that areas in which immature neurons predominate are disconnected from the OB by using immunohistochemical analysis and Dil/DiO tracing of axons.
Aim 2: The intermingling of OE and RE in the olfactory area and the occurrence of quiescent, aneuronal OE indicate that dysfunction of olfactory stem cells and progenitor cells contributes to peripheral olfactory pathology. Guided by our studies of the molecular profile of stem and progenitor cells in mouse and rat OE, we will identify markers for identifying neurocompetent stem and progenitor cells in human OE. We will screen for and analyze the expression of the human homologues of genes that are relevant in mouse using RT-PCR, IHC and in situ hybridization on human olfactory tissue collected by biopsy. We also plan open-ended gene profiling studies. The resulting panel can be used to assay for the stems and progenitors in human olfactory tissue.
Aim 3: To test the usefulness of the stem/progenitor marker panel and the analysis of axon trajectory for the diagnosis of dysosmic patients. Guided by our studies of the olfactory area in Aim 1 we will harvest biopsies of the olfactory area from patients rendered dysosmic as a consequence of aging, chronic rhinosinusitis, or prior viral URI, and use our panel to analyze the status of stem and progenitor cells as well as the pattern of axonal growth within the tissue.
Mentor: Schwob, James
Fellow: Krolewski, Richard
Title: Growth Factor Regulation of Olfactory Neurospheres
Abstract: The olfactory mucosa (OM) of both humans and rodents is an accessible tissue that exhibits ongoing neurogenesis and epithelial reconstitution following injury with demonstrated potential for cell-based therapies. To realize the OM's therapeutic potential requires a culture system that can be used to expand, understand the regulation of, and serve as a biomarker for engraftment potential of progenitor populations. The hypothesis will be tested that olfactory neurosphere formation serves as a biomarker for engraftment potential by varying the "state" of the stem and progenitor cells and also the growth factor influences that act on them. A subsidiary goal is to establish the use of neurospheres as a means of evaluating the network of interactions acting on stem and multipotent progenitor cells in vivo. OM-derived neurospheres will be tested as biomarkers for multipotency and stemness using the sphere-forming activity of cells isolated from in vivo conditions that involve different endogenous stem and progenitor cell activation: normal adult, selective neuronal regeneration, full reconstitution of the epithelium after methyl bromide (MeBr)-induced toxic injury, and early postnatal expansion. Resulting spheres will be quantified and their immunophenotype determined. Olfactory neurospheres will then cultured from constitutively expressing GFP mice and transplanted into MeBr lesioned host animals to determine whether the engrafted cells produce OM cell types. To test whether factors that act on globose basal cells (GBCs) or horizontal basal cells (HBCs) specifically enhance sphere formation and engraftment potential, olfactory neurospheres will be treated with a variety of growth factors with both known tropism as well as unknown function in the OM. Reporter mice that allow for live tracking of GBCs and their progeny will be used to obtain readouts of growth factor activity on olfactory neurospheres.
Growth factors that produce a significant change in the size distribution and fluorescence intensity of spheres will be tested via the aforementioned transplantation assay. The stem and progenitor cells of the OE are accessible in adult humans and undergo neurogenesis throughout adulthood. As in other systems that have translated stem cell biology to clinical practice, such as the hematopoietic system, it is apparent that the ability to expand the progenitor populations will be required for clinical applications. This work will expand on initial studies showing the clinical potential of olfactory mucosal-derived cells for treatment of spinal cord injury and neurodegenerative diseases by studying what factors can be used to enhance the proliferation and propagation of these stem cell populations.
PI: Schwob, James
Title: Medical Scientist Training Program at Tufts University
Abstract: The MD/PhD program at Tufts University School of Medicine and the Sackler School of Graduate Biomedical Sciences is designed to train the physician scientists of the future, and provide an outstanding cadre of investigators well-versed in both the practice of medicine and the conduct of basic biomedical research. Our goal is to bridge the communication and cultural divide that too often separates physicians and scientists and educate individuals who will be firmly rooted in both cultures and achieve highly productive careers that meld these two arenas. Medical training interfaces with eight basic science graduate programs in Biochemistry, Cellular & Molecular Physiology, Cell, Molecular & Developmental Biology, Genetics, Immunology, Molecular Microbiology, Neuroscience, and Pharmacology & Experimental Therapeutics Programs that have a combined faculty of 135.
The Tufts MSTP has been funded since 1991, but has undergone a major transformation in the past two years with the appointment of a new director and a new leadership team. A completely redesigned administrative structure, new admissions and advising processes, and major changes in the curriculum have been implemented. Our new curriculum effectively integrates research and clinical training. Research rotations prior to beginning medical school and research selective during the first two years of medical school allow students to make informed decisions about research mentors in a timely fashion. A new Clinical Connections program that provides an array of patient experiences during the years focused on research training has been put in place. A new course "Clinical Implications of Basic Research" that brings together MD/PhD trainees in all phases of their training to discuss the ways bench research is being translated in new clinical practices has also been added. A workshop that provides orientation and hands-on experiences for students transitioning from research to clinical education will be given for the first time in the spring of 2006. A retreat, seminars, and a career-oriented dinner program are also built into the training program. Our program has graduated 40 trainees since its inception and 32 are currently enrolled. Based on the changes that have occurred, we are requesting funding for 16 positions in this application.
Mentor: Schwob, James
Fellow: Schnittke, Nikolai
Title: Olfactory Epithelial Stem Cell Regulation by the Transcription Factor p63
Abstract: The mammalian olfactory epithelium (OE) is able to regenerate its cellular components (neurons, supporting cells, and duct/gland cells) throughout adult life under normal, maintenance conditions, as well as regenerative conditions after destruction of all differentiated cell types by exposure to the gas methyl bromide (MeBr). This ability together with its peripheral and therefore accessible location makes the CE an attractive system to utilize for purposes of regenerative medicine. Before such a potential can be realized one must first perform a complete structural, biochemical, and functional characterization of the cellular dynamics of the CE. At least two distinct populations of putative stem or progenitor cells have been identified among the basal cells of the epithelium that may be responsible for its regenerative capacity. Globose Basal Cells (GBCs) constitute a heterogeneous population of lineage committed and uncommitted progenitors situated below (basal to) the neuronal layer of the epithelium. Horizontal Basal Cells (HBCs) lie in the basalmost layer of the CE and correspond to an apparently homogeneous layer of cells in tight contact with the basement membrane of the CE. Both of these basal cell types have been shown by various methods to have the ability to generate all the differentiated cell types of the CE, however HBCs are not present until late in embryonic development whereas GBCs are the progenitor cells involved in the embryonic assembly of the tissue. The transcription factor p63, a member of the p53 family, is necessary for the embryonic appearance of HBCs and loss of p63 expression in the adult regenerative environment appears to be correlated with the ability of HBCs to give rise to differentiated CE cell types. The goal of the proposed research is to exhaustively characterize the functional role of p63 in HBC generation, quiescence, activation, and transdifferentiation.
First, nascent HBCs that express p63 but have not yet differentiated toward the HBC phenotype will be characterized based on expression of other markers previously identified in the lab as CE progenitor cell markers. Next, the sufficiency of p63 expression in HBC generation will be probed by forcing expression of p63 in the regenerative environment via retroviral infection. Finally the role of p63 loss will be assessed in the adult normal and post-lesion settings, via a conditional knock-out genetic approach. These studies will go a long way toward elucidating the nature of context dependent plasticity present in the CE specifically and adult stem cell systems in general, as well as provide a mechanistic framework in which to approach the therapeutic application of these cells.
PI: Schwob, James
Title: Regulation of Growth and Differentiation in 3-D Cultures of Olfactory Epithelium
Abstract: The capacity for regeneration of neuronal and non-neuronal populations in the olfactory epithelium (OE) is well-known and extends throughout life. By virtue of their accessibility, the neurocompetent stem and progenitor cells of the OE are attractive candidates for use in autologous cellular therapies. The regulatory signals and mechanisms that govern these processes are not well understood. An in-depth analysis of the regulation of olfactory epitheliopoeisis is needed in order to exploit the stem and progenitor cells fully, but progress has been slow. We have developed a tissue culture assay in which cells from the neonatal or adult lesioned OE grow within spheres that form at the air-media interface of culture well inserts in a condition-dependent manner. Remarkably, cells that are cultivated in 3-dimensions (3-D) retain the potency and plasticity to participate in the regeneration of the OE in the animal, which stands in sharp contrast to the failure of cells grown in 2-D to do so. Two Aims are proposed to explore the cues regulating cell generation and differentiation taking advantage of the in vivo-like properties of the sphere assay.
Aim 1 will test how growth factors, alone or in combination, will affect the growth of OE cells in the 3-D cultures. Some of the factors have a previously established role in the turnover and regeneration process in the OE; they will calibrate the assay, providing registration of the in vitro and in vivo results. For the majority, little is known of their role in the OE, although they function elsewhere in cell birth or differentiation.
In contrast, Aim 2 will determine the relevant cues within a complex molecular mix that is known to play an important role in regulating the OE. In this case, culture media conditioned by the growth of a lamina propria cell line (derived from the part of the mucosa that is deep to the OE) is required for the formation of spheres from adult lesioned OE on inserts. We will take both a candidate molecule and an open-ended approach to identifying the relevant factors.
PI: Selhub, Jacob
Title: Specific Fluorescent Labeling for Micronutrient Assessment
Abstract: The purpose of this project is to develop a proof of concept for a unique, new technology that allows, for the first time, measurement of multiple micronutrients simultaneously in biospecimens, rather than one at a time. Biofortification of staple foods, such as those studied by the Bill and Melinda Gates Foundation (BMGF)-funded HarvestPlus program, is one approach to address widespread micronutrient malnutrition. Some of the research emanating from HarvestPlus activity emphasizes the importance of seeking to address multiple nutrient deficiencies through biofortification. However to be cost-effective, it is critical to have analytical methods for nutrient assessment in biospecimens to identify those sub groups at risk for micronutrient deficiency. Similarly, laboratory assessment of absorption and bioavailability of micronutrients are essential to confirm efficacy of the delivery system.
We propose to establish a proof of concept for the use of fluorescent probes to simultaneously measure the following nutrients: vitamin A, folic acid, iron, iodine, and zinc. By taking advantage of the protein- binding capacity of each of these compounds, we propose to develop a single immunoassay that can be used to simultaneously determine each of these nutrients from a single blood sample. We propose to first develop fluorescent probes for vitamin A, folate, zinc, iodine and iron capacity and develop assays for individual nutrients (objective #1). Measures of success will be reproducible achievement of each sequential step of the process for each individual analyte, with validation of each method as compared to conventional methods. Once these individual assays are achieved, we will then combine use of individual fluorescent probes for simultaneous detection of vitamin A, folate, zinc, iodine and iron (objective #2). Measures of success will be the compatibility of each new analytes added to the combined assay already developed in the project. In spite of its innovative aspect of the proposed assay, the implementation of this concept will utilize traditional methods and equipment, thereby increasing likelihood of success. If successful, it will dramatically reduce the amount of biological sample required, increase throughput, and reduce costs.
PI: Selker, Harry
Title: Tufts Clinical and Translational Science Institute (CTSI - UL1)
Abstract: Leveraging Tufts University's excellent resources, traditions of multidisciplinary collaboration, generation of novel research methods, and academic innovation, including the first MS/PhD Clinical Research (CR) Program in a graduate school of biomedical sciences, we will establish the Tufts Clinical and Translational Science Institute (CTSI). This will unite eight Tufts Schools (Arts & Sciences, Citizenship and Public Service, Dental Medicine, Engineering, Graduate Biomedical Sciences, Medicine, Nutrition, and Veterinary Medicine), eight affiliated hospitals, the Tufts Center for the Study of Drug Development, the USDA Human Nutrition Research Center on Aging at Tufts, and the Tufts-New England Medical Center (Tufts-NEMC) Institute for Clinical Research and Health Policy Studies. This will:
Create a new home for clinical and translational clinical research (CTR), a cross-University, cross-affiliate Tufts CTSI, headed by an international leader in CTR who will report to the University Provost
Create eight research resource Components suggested by the CTSA RFA, plus four based on special Tufts strengths
Enhance and expand the "CR Portal" at Tufts-NEMC into a Tufts-wide "CTSI Portal" that provides coordinated and mentored University- and affiliate-wide access to CTSI Components and other resources
Create activities, processes, and new information systems that will support and promote collaborative cross-disciplinary full-spectrum translational research
Enhance our MS/PhD CR Program by:
Adding to the existing four Concentrations (Clinical Investigation, Epidemiology/Biostatistics, Health Services and Outcomes Research, and Medical Informatics), Concentrations in Bench-to-Bedside Translational Research and in Evidence-Based Medicine
Developing distance learning to extend the Program to fellows, K12 scholars, and other faculty at Tufts affiliates across Massachusetts
Creating the joint Pfizer Tufts Career Development Awards
Preparing predoctoral tracks for Tufts MD, DMD, and DVM students to also get a CR MS or PhD
Developing joint tracks for students to get an MS in Nutrition or in Biomedical Engineering with a PhD in CR
- Developing a program to generate interest in CTR among students from underrepresented minority group
In all aspects of training, research support, and organizational development, the Tufts CTSI will support and instill the culture of cooperative interdisciplinary research, a home without walls, for CTR Tufts-wide.
PI: Selker, Harry
Title: Tufts Clinical and Translational Science Institute (K12)
Abstract: The objectives are to:
Provide training based on didactic curricula that provide solid grounding in basic biomedical sciences, the essential concepts and methods of CR, a working knowledge of research subjects' protections and the regulatory structures that underlie these protections, and an appreciation for the ethical norms that frame the responsible conduct of research.
Present the trainees with a series of research experiences, both in the laboratory and in the clinic that will demonstrate the interdisciplinary character of clinical and translational research at CTSI institutions and that will form the basis for development of their dissertation projects.
Provide a robust system of mentoring that will guide trainees in all stages of graduate training from early interdisciplinary courses through decisions about the immediate postdoctoral experience.
Train a cadre of researchers who can lead the translation of basic research into practice at the community level.
- Strengthen CR collaborative ties between the Tufts CTSI and the affiliated hospitals.
PI: Selker, Harry
Title: Tufts CTSI Career Development Program in Comparative Effectiveness Research
Abstract: From its inception in 2008, a major goal of the Tufts Clinical and Translational Institute (CTSI) has been to develop a comprehensive infrastructure for the efficient conduct of comparative effectiveness research (CER), the development of innovative CER methods, and the training of fellows and scholars in CER. Based on a long tradition in this area, the Tufts CTSI has linked an extraordinary array of disciplines, novel methods and collaborative research opportunities focused on "bedside-to-practice" and "practice-to-policy" translational research and, based on these assets and this focus, has played a leadership role in coordinating the response of the National CTSA Consortium. It has been clear that the need for CER to provide readily available and actionable information on the relative benefits and costs of competing treatment options far outstrips the collective capacity of academic research centers like the Tufts CTSI, where the nation's expertise and capacity for CER largely resides. A large national investment to train the next generation of CER researchers, as well as a commitment on the part of academic centers to prioritize CER training, is urgently needed. The Tufts CTSI is exquisitely well prepared to expand our role in training leaders and innovators in CER, to grow our core CER capacity, to help meet these critical needs. With this application, we aim to establish a Mentored Career Development Program in CER that builds on the breadth and depth of our research expertise across the full CER spectrum, as well as on our long history of training CER scholars and on our robust links to community and industry stakeholders. The purpose of this training program is: 1) to produce the leaders and innovators to help meet the nation's enormous CER challenges and workforce needs of the coming decade, and 2) (since CER is performed collaborative in teams) to further strengthen the Tufts CTSI as a Center of Excellence in CER.
PI: Selsing, Erik
Title: Testing a Role for Activation-Induced Deaminase in Omenns Syndrome B-Cell Autoimmunity
Abstract: Omenn syndrome is a human disease that exhibits an unusual combination of immunodeficiency coupled with autoimmunity. Patients have few mature T cells or B cells, and have poor immune responses. On the other hand, patients show substantial serum antibodies with elevated levels of self-reactive antibodies. Hypomorphic mutations in recombination-activating genes (RAG) are some of the best-characterized genetic defects associated with Omenn syndrome. RAG proteins are critical for recombination of receptor genes during development of B and T lymphocytes and hypomorphic RAG mutations lead to limited generation of mature lymphocytes. However, the causes of autoimmunity in the disease are not clear.
During the past several years, our research groups have collaborated on studies of B cell differentiation and tolerance during early B cell development. We have recently shown that isotype switching occurs even in immature B cells in the bone marrow, counter to the prevailing paradigm that switching was limited to activated mature B cells. We have also found that immature B cell switching is greatly elevated in a mouse strain (564Igi strain) that models human System Lupus Erythematosus (SLE), and develops autoimmune disease. Because isotype switched B cells respond more strongly to stimulation, we have hypothesized that isotype switching in immature B cells can lead to a breakdown of central tolerance checkpoints and can result in autoimmune disease. We have also developed mutant 564Igi variants that either can or cannot undergo isotype switching in immature B cells. Preliminary studies indicate that autoimmunity is greatly reduced in 564Igi mice that lack early B-cell isotype switching, supporting our hypothesis that early B-cell switching can circumvent tolerance and can lead to autoimmune disease.
The SLE-like 564Igi mouse model effectively reproduces many features of human SLE, but the gene- targeted anti-RNA antibody genes in the 564Igi strain are a genetic construct that is not found in human SLE patients. We want to test the role of early B-cell switching in the development of autoimmunity arising from a genetic mutation known to be involved in human autoimmunity.
Recent reports have described mouse models of the human Omenn syndrome disease. These have a gene-targeted hypomorphic RAG gene and, thereby, are analogous to patients with hypomorphic RAG. The mouse models replicate the lymphopenia, the serum antibody levels, and the autoimmunity of the human disease. Furthermore, some features of Omenn syndrome, such as anti-RNA, anti-chromatin and anti-DNA antibodies, as well as elevated serum cytokines that regulate isotype switching, are shared with SLE. We will use Omenn syndrome mouse model variants that cannot undergo isotype switching in immature B cells to test whether these exhibits reduced autoimmunity. Our tests will indicate whether or not tolerance breakdown through isotype switching might be a common feature of various B cell autoimmune diseases.
PI: Shang, Fu
Title: Impairment of the UPP and Choroidal Neovascularization
Abstract: Age-related macular degeneration (AMD) is the leading cause of irreversible blindness among elderly in industrialized countries. There are ~13 million Americans suffering from AMD disease currently. With the imminent demographic right-shift the toll of this devastating disease will be even greater in near future. Although the etiology and mechanism of AMD remain elusive, a large volume of literature indicates that AMD is associated with malfunction of retinal pigment epithelial cells (RPE) and oxidative stress is on the top of list of risk factors for AMD. The ubiquitin-proteasome pathway plays critical roles in various cellular functions, including transcriptional control and removal of damaged proteins. Our previous studies demonstrated that the activity of the ubiquitin-proteasome pathway is modulated by cellular redox status. The preliminary data showed that inhibition of the ubiquitin-proteasome pathway in RPE cells resulted in molecular changes that are similar to those of AMD, such as death of RPE and increased expression and secretion of vascular endothelial growth factors. The long term goal of this project is to investigate the physiological function of the UPP in RPE and its relationship with AMD.
The specific aim of this pilot project is to test the hypothesis that impairment of the UPP in RPE is sufficient to trigger the development of AMD-like phenotypes. Specifically, we will examine the pathological changes in the retina and RPE of transgenic mice that express dominant negative ubiquitin (that impairs the functions of the UPP) by immunohistochemical molecular biology techniques. We will also test whether impairment of the ubiquitin proteasome pathway in RPE triggers neovascularization of choroidal endothelial cells in a co-culture model. Due to the limited scope of this pilot project and limited maximal fund allowance, this project will only analyze the eyes that have already been harvested in previous work. No additional animals will be utilized. All of the experiments will be performed in the Laboratory for Nutrition and Vision Research and no core facility will be used in this project.
Successful completion of this project will not only provide information regarding the pathogenesis of AMD, but also provide an animal model to testing the protective role of specific nutrients, such as lutein and zeaxanthin, against AMD.
The purpose of project is to address CRIS Objective 3 and Sub-Objective 3A of the Nutrition and Vision Laboratory:
- Objective 3: Identify mechanisms by which retina and lens functions are maintained throughout life.
- Sub objective 3A: Determine if a fully functional ubiquitin pathway (UPP) is required for proper lens and retina formation and maintenance.
PI: Shoemaker, Charles
Title: A Platform for Therapeutic Agents that Promote Rapid Recovery from Botulism
Abstract: Botulism is caused by exposure to toxins produced by Clostridium botulinum neurotoxin (BoNT), a CDC Category A biodefense threat agent for which no antidote currently exists. Seven different BoNT serotypes have been discovered to date (BoNT/A-G), many having numerous additional BoNT subtypes. To protect against all of these diverse BoNT bioterror threats, expensive conventional small molecule drug development would need to be separately performed for each of the seven different drug targets and perhaps others. This challenge, together with other extreme hurdles confronting BoNT small molecule drug development, particularly complicates efforts to develop small molecule drugs to treat botulism. New therapeutic paradigms are urgently needed to counter the enormous risks associated with these easy to obtain, easy to produce and extremely dangerous bioterror agents.
We have developed and then extensively optimized two distinct 'designer E3-ligase' agents that each accelerate the 'molecular cure' of neurons intoxicated by one of the two most dangerous Botulinum neurotoxins, serotypes A or B (BoNT/A, BoNT/B). These two lead agents consist of the F-box domain of TrCP fused to a camelid 'nanobody' domain with binding specificity for one of the BoNT proteases. These polypeptide agents, with a size less than 30 kDa, bind to the BoNT protease and cause its rapid, intraneuronal destruction leading to rapid recovery of the neuron. Because of the modular nature of this antidote, it will be simple and straightforward to develop similar agents to treat all other BoNT serotypes and subtypes simply by substituting the nanobody domains with another having the appropriate specificity.
In this proposal, we will develop a general delivery vehicle to deliver our two lead agents to the cytosol of intoxicated neurons within botulism patients and perform pre-clinical evaluation. As the vehicle, we propose to use an atoxic Clostridial toxin-based neuronal delivery vehicle (TNDV) due to its highly evolved capability to enter the body, survive in serum and deliver enzyme activities to the cytosol of targeted cells. We will develop three different and proven TNDV systems, each having unique and compelling features, and then select the best vehicle(s) for further development and animal testing. The three TNDV systems will be modified, atoxic forms of: 1) BoNT serotype C; 2) C. difficile toxin B and; 3) Clostridial C2 toxin. If successful, it is expected that similar agents could be developed to target the accelerated turnover of virtually any cytosolic neuronal protein for research or therapeutic applications.
PI: Shoemaker, Charles
Title: Reversing Botulism with Agents that Accelerate Intraneuronal Toxin Degradation
Abstract: Botulism is caused by Clostridium botulinum neurotoxin (BoNT), a CDC Category A biodefense threat agent for which no antidote exists. We have developed two distinct “designer E3-ligases” that target either the intracellular proteases of botulinum neurotoxin serotype A or B (BoNT/A, BoNT/B) for accelerated ubiquitin-mediated degradation. Our agents consist of the F-box domain of TrCP fused to a camelid VHH domain with binding specificity for the BoNT protease. These polypeptide agents, with a size less than 30 kD, lead to the rapid, intraneuronal destruction of the intoxicating BoNT proteases. Here we propose to develop a Clostridium difficile toxin B (TcdB) based vehicle for delivery of the BoNT designer E3-ligases to the cytosol of intoxicated neurons. We propose to test the engineered TcdB agents for reversal of botulism symptoms within cultured neurons and then in mice, likely with a single dose therapy.
TcdB has highly evolved features that make it ideal for delivery of BoNT directed designer E3-ligases to the cytosol of botulism intoxicated neurons within patients. TcdB naturally binds to a surface receptor that is broadly expressed in cells, then is internalized by endocytosis and delivers a glucosyltransferase (GT) that inactivates Rho GTPases leading to cell death. This toxin also contains a protease activity that cleaves its enzymatic “cargo” from the “delivery vehicle”, and releases it into the cytosol. TcdB has been shown to deliver significant quantities of functional glutathione-S-transferase (GST) to the cell cytosol when the GST is fused to the amino terminus of the toxin GT domain. We have successfully expressed large amounts of full-size, bioactive, recombinant TcdB, and an atoxic variant TcdB, in microbial host cells. In this proposed project, we will develop and test a neuron-specific TcdB by replacing the TcdB receptor binding domain (RBD) with the equivalent RBD domain from BoNT/A. Secondly; we will test whether BoNT/A protease turnover can be accelerated within intoxicated neurons by treatment with a fusion of BoNT/A designer E3-ligase to atoxic TcdB. Finally, an engineered TcdB targeting the BoNT designer ligase to neurons will be tested in the mouse hind limb paresis model for the ability to reverse the symptoms of botulinum toxin induced paralysis.
If successful, similar TcdB agents should be readily possible and rapidly available for all botulinum serotypes by simply switching the VHH domain to those having specificity for other BoNT proteases. Secondly, we will have demonstrated that TcdB is a general vector for the delivery of biomolecules to the cytosol of cells targeted by the specificity of the RBD. Finally, the biomolecular cargo we are delivering to these cells, designer E3-ligases, are simple fusions between a camelid VHH targeting domain and the TrCP F-box, and it is expected that similar agents could be developed to target accelerated turnover of virtually any cytosolic protein for research or therapeutic applications.
PI: Shultz, Mary Jane
Title: Hydrogen-Bond Interactions and Imaging of Natural Ice
Abstract: Chemists have long used vibrational spectroscopy to “see” inside molecules to deduce the spatial relationship among the atoms, as well as to determine interactions between molecules via changes in the infrared or vibrational spectrum when molecules collide or bind. One part of the infrared, the hydrogen-bonded region for aqueous solutions and ice, has thus far yielded few secrets due to the huge oscillator strength in this region and the enormous width, nearly 800 cm-1, a substantial fraction of the entire vibrational infrared. These two features, width and strength, undoubtedly imply that there are several overlapping and intermeshed resonances. Hence, despite over a half century of progress in vibrational spectroscopy, this blind spot remains.
Yet, water, aqueous solutions, and ice are enormously important to areas ranging from the atmosphere where they are involved in many climate determining processes, to biological systems where aqueous solutions constitute about 80% of most organisms. In geology, hydration determines phase transformations and water transports countless minerals and hydrocarbons. Cometary ice in outer space shuttles small molecules from region to region. In every sphere the tiny triatomic molecule, water, is a key player. Thus the inability to use the hydrogen-bonded region to unravel water’s role in these interactions represents an important gap.
The proposed work has three aims:
- To shed light on the resonances contained within the hydrogen-bonded region to associate features in this region with specific bonding motifs, so that changes in the shape of this region can be used to generate a molecular-level picture of water’s response to adsorbate molecules impinging on the surface from the gas phase.
- To probe for pairs of complementary dangling bonds, which promote donor-acceptor binding.
- To differentiate reactions at defects such as grain boundaries from those on the crystal faces.
PI: Shultz, Richard
Title: Sarah Scaife Foundation Grant 2011-2012
Abstract: With continued support from the Sarah Scaife Foundation during the 2011-2012 academic year, we propose to maintain and to build on more than forty years of experience and achievement in ISSP teaching and "outside-the-classroom" educational enrichment activities. In light of the formidable challenges facing the United States, including terrorism, ongoing conflict in Afghanistan, and escalating crises and conflicts elsewhere in the world, it is crucial to continue to educate young professionals who are equipped to deal with an increasingly complex and uncertain international security environment. During the 2011-2012 academic year, the ISSP faculty will continue to develop our curriculum of courses and seminars, together with our other programmatic activities.
Among these activities are high-level conferences. These conferences help to enrich the understanding of key national security issues on the part of our students, and also publicize The Fletcher School and the International Security Studies Program and Institute for Foreign Policy Analysis in the broader national security/foreign policy communities. We are beginning the planning process for the 40th Conference on National Security Strategy and Policy in this series to be held during the next academic year with appropriate official co-sponsorship and in association with the Institute for Foreign Policy Analysis.
The forthcoming 2011-2012 ISSP guest lecture series again will include speakers who will examine the spectrum of 21st century national security issues facing the United States. A series of timely topics will be addressed, such as national security strategy, regional security, issues of ethnic and religious conflicts, proliferation, low intensity conflict, missile defense, roles and missions of U.S. military forces, energy and strategic minerals, and homeland security.
On November 4-5, 2011, ISSP will hold our annual weekend simulation, Simulex 2011. As in previous years, we will bring together a group of about 200 participants from The Fletcher School and the official policy community for a crisis management simulation. The purpose of Simulex 2011 will be to sensitize students to the constraints and opportunities facing policymakers in crisis situations in which important interests of the United States and its allies and other friendly states are at stake. As in previous years Simulex 2011 will be conducted with an innovative scenario. We anticipate that this year’s game will again attract substantial interest and participation from the official policy community. The results will be disseminated within the U.S. government, including the Departments of State and Defense, as well as each of the military services. This exercise will be adapted for use in January 2012 in our internet-mediated GMAP Security Studies and Crisis Management course.
During the next academic year ISSP will continue its work with each of the military services. We expect to have at least nineteen U.S. military officers in residence at Fletcher either as degree students or as military fellows. Two Army National Guard Lieutenant Colonels, two Army Reserve Lieutenant Colonels and one Army Lieutenant Colonel will be attending The Fletcher School as senior research fellows in lieu of their studies at the Army War College as research fellows. The ISSP will benefit from the assistance of two senior officers as our U.S. Air Force National Defense Fellows. The Air Force National Defense Fellows are an invaluable resource for the Program. We are confident that next year’s Air Force National Defense Fellows, Lieutenant Colonel Charles Butler and Lieutenant Colonel Michael Rickard, will be no exception. Our Air Force National Defense Fellows together with other military fellows, will play an integral role in ISSP activities. Their responsibilities will include assistance within and outside the classroom. Our military fellows will take ISSP courses and will conduct research under our auspices. In addition, there will be at least thirteen degree candidates from the various military services attending The Fletcher School for the 2011-2012 academic year. Last but not least, there will also be military officers from abroad taking part in the activities of the ISSP.
PI: Sims Gallagher, Kelly
Title: Climate Change Policy, with a US and China Focus
Abstract: Kelly Sims Gallagher will have primary responsibility for U.S. and Chinese climate policy. This is a hugely dynamic and active area of policy in the USA and China currently and Gallagher has been and remains deeply involved in shaping the thinking and ideas around cooperation and collaboration between the USA and China on climate change. This work is directly related to Energy, Climate Change and National Security and is influenced by technology as well as impacting technology. Gallagher participates in a number of distinguished academic and government advisory panels, including PCAST and the American Academy for Arts and Science. Funds permitting, she will also collaborate on projects led by faculty at Harvard. In particular, she is committed to collaborate on the geopolitics research led by Meghan O'Sullivan. She will interact with Henry Lee on transportation and Wendy Jacobs on CCS, especially as their work pertains to U.S. or Chinese climate policy.
Gallagher served as the Director of ETIP from 2003-2009. Major accomplishments of the ETIP program between 2006-2009 included policy recommendations regarding tax incentives and loan guarantees for advanced energy technologies, annual analysis and policy recommendations regarding the U.S. Department of Energy's RDD&D budgets, analysis of the "real costs" of CCS in the United States, roadmaps for advanced coal and CCS in India and China, annual workshops convened with the Chinese government's Ministry of Science and Technology on clean and efficient transportation and advanced coal/CCS, conception of the idea of "Acting in Time on Energy Policy" (major conference, book published by Brookings) and, of course, including John Holdren's nomination to be President Barack Obama's Science Advisor at the end of 2008. ETIP's policy relevance, outstanding outreach, analytical rigor, and convening power were all demonstrated and proven during this initial period. Many of the ideas developed by ETIP have become policy during this time, in both the United States and China.
There are two primary objectives for the Climate Change Policy program. The first objective is to achieve substantial interaction between BP and the Climate Change Policy program so as to mutually inform each other's work. The substantive interactions will engage on climate change policy with a USA and China focus. The second objective is for the program to advance policy-relevant knowledge in these domains through excellent research, high-quality research products, outreach to policymakers, and the convening of meetings, conferences and events.
PI: Skelly, Patrick
Title: Functional Characterization of the Schistosome Tegument
Abstract: Schistosomes are parasitic flatworms that cause a chronic, debilitating disease afflicting over 200 million people in over 70 countries. The parasites live for years, sometimes decades, in what should be a very hostile environment – the blood of vertebrates – yet they appear to solicit little if any protective reaction from two of the host’s major defensive systems: the hemostatic system and the immune system. We hypothesize that proteins at the host-interactive surface are central to the parasites ability to dampen host immunity and hemostasis while, at the same time, permitting metabolite exchange. In this competing renewal, we propose to use new molecular methods such as RNA interference that were first developed for use with schistosomes under our previous grant to test several key hypotheses concerning:
- The role of tegumental ecto-enzymes in hemostasis and immunomodulation
- The ability of tegumental sphingomyelinase to alter permeability properties at the parasite surface
- The molecular mechanisms of transtegumental metabolite exchange
The functional genomics approach we adopt here coupled with independent and direct, follow-up experiments employing more traditional cell biology and biochemistry techniques are designed to provide significant new information concerning the schistosome host interactive surface. In addition the work is designed to identify tegumental proteins critical for parasite survival in the host and subsequent screens will be undertaken to discover drugs that inhibit these molecules. In this way, our planned experiments have the potential to reveal novel and valid targets, as well as new treatments, for intervention in a parasite that remains a widespread and major cause of human disease.
PI: Slonim, Donna
Title: Translational Bioinformatics for Human Developmental Genomics
Abstract: Fetal developmental disorders and pregnancy complications pose critical public health challenges. Yet our ability to monitor prenatal development and neonatal health has not fully entered the era of genomic medicine. Recent technological advances have allowed us to monitor global gene expression in the living human fetus or newborn, raising the possibility of creating revolutionary early diagnostics for a range of developmental disorders. Yet in order to realize this vision, we need to overcome two bioinformatics challenges. First, state-of-the-art interpretation of gene expression data requires information about how genes work together as systems. However, existing systems/pathway annotation is focused predominantly on adult disorders and is inappropriate for the analysis of fetal gene expression. Second, traditional methods for designing diagnostics from gene expression data require many examples of each sample type to be recognized. For rare diseases that cannot currently be detected in utero, however, the collection of a large number of fetal expression profiles representing each disorder is impractical.
Our proposal addresses these challenges via two specific aims. First, we will develop a method for discovering new, statistically-validated gene sets from expression data. We will use this method, along with literature mining, to develop new systems biology annotation specific to this developmental stage. Second, we will develop and validate a new computational paradigm for sample classification that can identify and determine the nature of rare abnormal developmental profiles. Our methods, though motivated by the study of human development, can be applied to any medical area where existing functional annotation is inadequate or the collection of typical classification data is infeasible.
PI: Sonenshein, Abraham
Title: Isolation of Early Sporulation Genes
Abstract: Despite many years of research and despite great advances in our understanding of operon-specific regulation, we still do not have a clear view of how bacteria sense general nutrient insufficiency and how they integrate the multiple nutritional signals that they are constantly exposed to. These mechanisms are responsible for the remarkable ability of bacteria to adapt to constantly changing environments and influence the interactions, both symbiotic and parasitic, of bacteria with host cells and host organisms. In gram-negative bacteria, the leucine-responsive protein (Lrp) plays an important role in governing the expression of a wide variety of cellular functions in response to deprivation of leucine or certain other amino acids. In gram-positive bacteria, such as Bacillus subtilis, CodY protein seems to play an analogous role, even though CodY and Lrp are unrelated proteins.
Prior work revealed that CodY has the unusual property of responding to two different, critical metabolites, GTP and isoleucine (or valine). CodY represses, directly or indirectly, the expression of hundreds of genes, most of which encode proteins that allow cells to adapt to limited nutritional resources. In collaboration with Dr. A. J. Wilkinson, the structure of CodY has been determined in its ligand-bound and ligand-free states, leading to a molecular model for the mechanism by which interaction with its ligands makes CodY an effective regulator. CodY has also been found to act as a positive regulator of genes of central metabolism. As a result, the CodY regulon overlaps in interesting and complex ways with other globally controlled regulons. Most CodY homologs are found in low G+C gram-positive bacteria, including major pathogens. Preliminary results indicate that CodY in such pathogens contributes in important ways to the control of virulence gene expression.
The goals of this proposal are to determine how B. subtilis CodY structure and function intersect, to reveal the overall physiological role of CodY, to assess the effects of constitutive CodY activity, and to extend our understanding of the function of CodY by determining its role in the growth, adaptation and virulence of Staphylococcus aureus.
PI: Sonenshein, Abraham
Title: Meeting on the Pathogenesis of the Clostridia
Abstract: Funds are requested to support the Seventh International Meeting on the Molecular Biology and Pathogenesis of the Clostridia. This meeting, which has been held every 2-3 years since 1995, has become the premier meeting in its field. The involvement of Clostridium spp. in human and animal disease has been increasing in incidence and severity. Luckily, the breadth and depth of research on these species has also been increasing. Thus, the need for the meeting is urgent and its timing is highly appropriate. The membership of the organizing committee reflects the stature of the meeting; the members are leaders in the field and cover specialties ranging from toxin biochemistry, cell biology and molecular epidemiology to gene regulation and genetic manipulation. The meeting will be held from October 25-29, 2011 on the campus of Iowa State University. Attendees are expected to number about 175 and to come from the US (about half the attendees) and from many countries in Europe, Asia, and Oceania. For each of the 11 scientific sessions, speakers working at the cutting edge of the subspecialty have agreed to present their latest work. In addition, 3-4 junior scientists chosen from among the abstract submitters will be invited to speak at each session. The meeting has been designed to maximize interactions among the participants, while providing family care and handicapped accessibility to those who need it. Participation by scientists from traditionally underrepresented groups will be actively solicited.
PI: Sonenshein, Abraham
Title: Molecular Genetics of Basic Cell Functions
Abstract: Continued support is requested for five predoctoral trainees per year for five years for an interdepartmental training program in genetics of basic cell functions. The emphasis is on rigorous training using the power of genetic analysis to study basic cell processes, such as chromosome replication and segregation, regulation of gene expression, cellular differentiation, and host-parasite interactions.
This training program has strongly benefited from continuous support from NIGMS since 1975. Supported students are almost exclusively in their first year, allowing them to have a broad exposure to the role of genetics as a science in itself and as a tool for solving important biological problems. The training faculty, from the Departments of Molecular Biology and Microbiology, Biochemistry, Pathology, and Medicine, is highly interactive and dedicated to close, joint supervision and mentoring of graduate students. Past trainees include leading researchers in academia and industry. The training program is administered by the Graduate Program in Molecular Microbiology.
Starting with a pool of 80-145 applicants, the graduate program annually admits 6-8 new students, most or all of whom are eligible for training grant support. The program has been successful in attracting a significant number of students from underrepresented minority groups (including 8 of the 41 current students), nearly all of whom have been supported by this training grant. Nearly all graduates of the program have obtained high-quality postdoctoral appointments and are still active as researchers, as teachers, or in allied fields. Entering students take required courses in Genetic Analysis and Biochemistry and pursue 9-week rotation projects in four different laboratories. At the end of the first academic year, they choose a thesis supervisor and begin thesis research. In the second and third years, the students complete their coursework and prepare a research proposal (unrelated to the thesis topic) as a qualifying examination. All students are required to complete a seminar course in scientific ethics.
Mentor: Sonenshein, Abraham
Fellow: McBride, Shonna
Title: Molecular Mechanisms of Clostridium difficile Resistance to Innate Host Defenses
Abstract: Clostridium difficile causes a potentially fatal intestinal disease that is increasing in incidence and severity. These infections are often chronic and incredibly difficult to eradicate. Though this organism presents an enormous public health burden, little is understood about how C. difficile evades host defenses to colonize the human intestine. In order to persist, the bacteria must cope with a continuous onslaught by host defenses. The long-term goal of this project is to reveal how C. difficile evades host innate immunity with the intent of opening new avenues for treatment. The specific objective of this application is to define the mechanisms C. difficile employs to resist cationic antimicrobial peptides (CAMPs) produced by the host. These peptides play a critical role in host innate defenses, preventing the growth and spread of bacteria. Preliminary data obtained thus far indicate that C. difficile can respond and adapt to the presence of CAMPs. I hypothesize that C. difficile has evolved multiple resistance mechanisms that allow the bacterium to bypass host immune responses, such as CAMPs, allowing it to colonize and replicate in the human intestine.
This proposal will identify the genetic mechanisms of CAMP resistance in C. difficile, identify and characterize the regulation of CAMP resistance mechanisms, and determine the role of these mechanisms in the virulence and colonization in vivo. CAMP resistance will be studied using traditional genetic and biochemical methods, along with new technologies, such as rapid, whole-genome sequencing. Successful completion of these aims will illuminate fundamental aspects of C. difficile infection and identify potential therapeutic targets for treatment of disease. Furthermore, this knowledge will better our understanding of how host-bacterial interactions in the intestine can lead to digestive diseases. The experiments outlined in this proposal will help me learn a wide range of molecular genetics, biochemistry, and animal research techniques I have not previously used, and also to master methods for use with this important hospital pathogen. Ultimately, this work will set the stage for my future research on C. difficile pathogenesis and provide the framework for my transition into an independent investigator position.
PI: Sonenshein, Abraham
Title: Regulation of Glutamate Synthesis in Bacillus subtilis
Abstract: Management of key metabolic intersections is a critical issue in bacterial economy. One such intersection, the interconversion of a-ketoglutarate and glutamate, is particularly important and interesting because it represents the point of coupling between central carbon metabolism and central nitrogen metabolism. Synthesis of the enzymes of the tricarboxylic acid branch of the Krebs cycle (citrate synthase, aconitase and isocitrate dehydrogenase) that produce a-ketoglutarate and the enzymes of nitrogen assimilation (glutamine synthetase, glutamate synthase and glutamate dehydrogenase) are encoded by genes that are subject to multiple forms of regulation. This project seeks to understand in detail the molecular mechanisms that regulate these genes in Bacillus subtilis, a well studied model Gram-positive bacterium, and Listeria monocytogenes, a model intracellular pathogen whose metabolic regulation is relatively unexplored.
The proposal focuses on three transcriptional regulators (CcpC, GltC and GlnR) and two enzymes (glutamate dehydrogenase and aconitase) that have post-transcriptional regulatory functions. The aims of this proposal are:
- To determine the molecular mechanisms that control the synthesis and activity of the tricarboxylic acid branch enzymes;
- To reveal the molecular mechanisms that control the synthesis of glutamate and glutamine; and,
- To determine the role of aconitase as a post-transcriptional regulator of sporulation in B. subtilis and of iron metabolism and virulence genes in L. monocytogenes.
These issues will be addressed using a combination of genetic and biochemical techniques. The phenotypes of mutants defective in each of the regulatory factors will be determined and the proteins will be purified and tested using in vitro transcription and DNA and RNA binding experiments. Since L. monocytogenes mutants that lack two repressors of the Krebs cycle genes have a significant defect in virulence in a mouse model, the molecular basis for this defect will be explored.
PI: Sonenshein, Gail
Title: Signaling Pathways in Stages of Mammary Tumorigenesis
Abstract: In previous studies conducted in Project 1, we demonstrated that: a) AhR expression is increased dramatically in rat and mouse mammary tumors following oral administration of a prototypic PAH and in “spontaneous” human mammary tumor lines, b) constitutive AhR activation is indicated by nuclear AhR localization in rat, mouse, and human mammary tumors and by AhR binding to gene promoters in the absence of environmental chemicals, and c) constitutively active AhR differentially regulates target gene transcription. From these findings we proposed a new central hypothesis: As mammary epithelial cells progress from normal to immortalized cells and then to invasive tumors, AhR activity is modified through interactions with environmental chemicals, other transcription factors, and cofactors to differentially regulate target gene transcription and to effect changes in cell growth and invasiveness.
Three aims were proposed to test this hypothesis:
- Assess AhR-mediated tumor invasion in vitro.
- Map differential cofactor recruitment by constitutively active AhR.
- Define the functional consequences of constitutively active AhR in vivo.
PI: Sonkusale, Sameer
Title: CAREER: Nanoelectrochemical Systems on Silicon
Abstract: We are ushering in the new era of nanotechnology with great promise for the future of electronics, photonics and sensing, enabled by exciting novel properties of a diverse palette of nanomaterials. However, this promise cannot be fully realized until functional devices and systems can be made from these nanomaterials in a reliable and low-cost manner. As a career goal, PI plans to propose a highly unconventional yet powerful candidate to fill this missing technology gap — that of top-down fabricated silicon CMOS die as an electroactive functional substrate for fabrication or bottom-up assembly of various nanomaterials. This new paradigm extends the role of CMOS technology beyond conventional areas of computation, signal processing and communication towards nanoassembly and nanofabrication. The underlying theme of the proposed career research is the ‘fusion of microelectronics and electrochemistry” to facilitate low-cost high-throughput benchtop nanofabrication. This novel “nanoelectrochemical systems” paradigm will spur growth of functional nanosystems for biomedical sensing, environmental sensing, medical diagnostics and the next generation of on-chip power sources and energy storage devices.
The proposed research will develop three key technologies:
- CMOS for nanoassembly (C4NA) based on controlled dielectrophoresis
- CMOS for nanofabrication (C4NF) based on controlled templated electrodeposition, and
- Solder reflow approach for contact formation.
PI: Sonkusale, Sameer
Title: Collaborative Research: Active Metamaterial/pHEMT Hybrid Devices for Terahertz Modulation and Detection
Abstract: The main objective of the proposed research is the demonstration of a suite of terahertz devices, specifically modulators and detectors based on the active control of metamaterial structures implemented in GaAs pHEMT processes. Metamaterials are engineered sub-wavelength metallic structures capable of achieving nearly any electromagnetic response at THz frequencies. Demonstrations of exotic electromagnetic response in metamaterials such as negative permittivity and permeability, and negative index of refraction, suggest that these new systems hold great promise for future technological applications. For active control of the metamaterial structures, we plan to use the resonant behavior of a gated two-dimensional electron gas (2DEG) in the channel of an HEMT transistor as a potentially selective and tunable terahertz solid-state detector. Compared with transit time limited (diffusive transport) of conventional field effect transistor devices, plasma wave devices (ballistic transport) can effectively detect and modulate terahertz frequency signals. However no practical THz monolithic integrated circuits have been realized yet because of the lack of efficient antennas and couplers of THz radiation into devices. The objectives of this proposal are to marry the outstanding propagation control and coupling of free-space THz waves using “metamaterial structures” and “plasma wave terahertz conductivity” of two dimensional electron gas in pHEMT devices. This will create a unique pHEMT-MM transistor array that collectively exhibits exceptionally attractive properties for the next generation of THz components.
PI: Sonkusale, Sameer
Title: Collaborative Research: IDBR: Metamaterial Enhanced Spectrometer for Terahertz Hyperspectral Imaging of Biological Specimens
Abstract: The objective of the proposed research is the development of novel instrumentation to facilitate highly sensitive, hyperspectral imaging of biological specimens, in the minimally explored yet information-rich electromagnetic spectrum of terahertz frequencies. This spectrum is broad in spectroscopic possibilities due to presence of the intermolecular vibrational, rotational and torsional modes, providing a distinct broadband molecular fingerprint and intrinsic contrast for biomolecular identification. However existing terahertz imaging approaches have limited sensitivity due to: (1) Lack of efficient coupling of terahertz radiation in and out of the biological sample, (2) Higher level of signal loss due to unwanted scattering at sample/spectrometer interface, and (3) Lack of efficient detectors in the terahertz frequency range, thus limiting their performance. Moreover the detectors are typically single pixel detectors that require scanning to obtain a 2D image and they fail to capture the spatiotemporal evolution of biomolecular dynamics.
To solve these issues, the proposed instrument brings two emergent technologies — (1) Metamaterials (MM) and (2) Plasma wave operation of GaAs pHEMT device. Metamaterials are artificial structures with subwavelength metallic inclusions that can be designed to exhibit exotic electromagnetic response such as negative permittivity and permeability, negative index, and ideal absorption or transmittance. Plasma wave operation of GaAs pHEMT device facilitates resonant detection of terahertz radiation with unparalleled sensitivity.
PI: Sonkusale, Sameer
Title: Metamaterial Embedded Terahertz Signal Processing: Novel Devices and Architectures
Abstract: Current electronics and photonics devices do not operate at the terahertz frequencies commonly referred to as the “terahertz gap” (0.1 - 10THz, 1 = 3mm - 30mm). This frequency range offers tremendous opportunities for sensing and communications. However, lack of efficient terahertz components for signal processing and computation has so far stunted any development in this field. There has been a renewed effort focused on the search for terahertz materials and novel processing techniques to enable the construction of device components at these frequencies, however no concrete solution has been found that will address the generation, processing, computation and detection of terahertz signals within a unified framework.
The vision of this research program is to perform fundamental research and to explore new ideas for terahertz signal processing and computation. This will be enabled using novel principles of precise dispersion engineering of surface electromagnetic waves (Goubau Lines) and free space electromagnetic waves, both made possible by metamaterials, and active electrical control of these waves using plasma wave properties of III-V transistor technology (e.g. GaAs pHEMT, InP and GaN HEMT technology). The synergistic combination of III-V technology and metamaterials facilitates control of terahertz propagation and processing and thereby provides a rich platform for fundamental theoretical and experimental investigations.
The specific objectives of the proposed three-year research effort are as follows:
Research Goal #1: Explore new fundamental modes of signal processing that are based on Maxwell’s equations for dispersion engineering on two platforms: surface (G-lines) and quasi-optical (free space). The goal is to perform computational and experimental investigation into phase-time encoding of terahertz pulses using these approaches.
Research Goal #2: Explore essentials of electromagnetic control of plasma wave response in III-V transistors. The research goal is to explore fundamental research in metamaterial enhanced Mach Zehnder Modulator for both surface waves (G-lines) and quasi-optical (free space) approach and realize a two-step delay line filter operating at terahertz frequencies.
Research Goal #3: Investigate the fundamentals for the realization of terahertz time encoded delta sigma time modulators based on the goals #1 and #2 above.
PI: Soto, Ana
Title: BPA as a Developmental Carcinogen
Abstract: Developmental exposure to bisphenol-A (BPA) at doses within the range of human exposure causes a complex array of adverse effects in animals. These outcomes are also known to be present in human populations and the rise in their occurrence coincides with the massive use of BPA and other endocrine disrupting chemicals (EDCs) in consumer goods. The main hormonal activity of BPA is as an estrogen mimic. Exposure to estrogens throughout a woman's life, including the period of fetal development, is considered a main risk factor for breast cancer. The investigators found that developmental exposure to environmentally relevant doses of BPA altered mammary gland morphogenesis in rodents during the period of exposure and led to the development of pre-neoplastic and neoplastic lesions appearing in adulthood. These results justify adding mammary gland related end points to the large and well-controlled GLP NTP-FDA study. The investigators propose to pursue the following Specific Aims:
To test the hypothesis that pre-pubertal mammary gland morphology assessed by morphometries at PND21 is an excellent predictor of pathological outcomes which manifest during adulthood. This aim is based on data obtained independently in the investigator's laboratory and that of Dr. S. Fenton (NIEHS). The investigators will compare subjective scoring methods with morphometric ones, and determine which are the features that best predict neoplastic outcomes. The investigators will assess whole mounts of animals exposed until PND21.
To test the hypothesis that DNA methylation profiles and concomitant alterations of gene expression in the mammary gland stroma and epithelium at PND21 are predictors of pathological outcomes that manifest during adulthood.
To test the hypothesis that perinatal exposure to BPA induces the development of pre-neoplastic and neoplastic lesions.
The investigators will assess the development of intraductal hyperplasias, carcinomas in situ and microscopic tumors and the appearance of palpable tumors in animals exposed from i) GD6 until PND 21 and ii) continuously from GD6 to sacrifice.
The realization of these aims will definitively test the hypothesis that perinatal BPA exposure predisposes individuals to mammary cancer and reveal the dose-response pattern. Additionally, it will identify candidate molecular markers and morphological signs as predictors of neoplastic outcomes. This knowledge is crucial for the toxicological evaluation of BPA. The success of this project would suggest the addition of some of these mammary gland end points to the toxicological assessment of chemicals.
Mentor: Soto, Ana
Fellow: Speroni, Lucia
Title: Development of an in vitro Bioassay to Screen Compounds for Breast Carcinogenicity
Abstract: In her book Silent Spring (1962), Rachel Carson asserted “For the first time in the history of the world, every human being is now subjected to contact with dangerous chemicals”. In 1991, experts at the Wingspread Conference concluded that the developmental alterations then observed in wildlife and in humans were due to exposure to environmental chemicals that disrupted the endocrine system; they called them endocrine disruptors (EDCs). Around 1970, defects in embryonic and fetal development as a result of exposure to diethylstilbestrol (DES), a synthetic estrogen that since 1940 and for over two decades was prescribed to pregnant women to prevent miscarriages became evident. DES was finally banned for clinical use because it induced clear cell carcinoma of the vagina in women exposed in utero prior to the 13th week of gestation. The women exposed prenatally to DES are now reaching the age in which breast cancer is manifested. An overall 40% increase in breast cancer risk for these women was recently reported. This confirms reports that prenatal exposure to estrogen mimics (xenoestrogens) plays an important role in the development of mammary cancer in animal models. Thus, prenatal exposure to xenoestrogens may be the underlying cause of the increased incidence of breast cancer observed in European and US populations over the last 50 years.
We are daily exposed to a number of environmental chemicals, a major percentage of which were never assayed for their role as breast cancer inductors. The task of assaying every new chemical in vivo is virtually impossible given the time it would take, its high cost and the number of experimental animals needed. Yet, their identification is an obligatory step in order to develop a policy to reduce exposure to women from developing breast cancer. This proposal aims at providing a tool to identify those chemicals.
The aim of this proposal is to develop an in vitro bioassay with which to screen large numbers of chemicals in a short period of time, while reducing experimental animals. We propose to establish an organ in culture method of the mammary gland (MG) in which suspected synthetic chemicals would be tested in their ability to alter MG development. Chemicals could then be sorted out in two categories: non-MG disruptor or MG disruptor. The list of MG disruptors would contain the potential MG carcinogens and therefore serve as a priority list of chemicals to be assayed in vivo. Once this first goal is realized, we will seek funds to undergo the validation of the bioassay testing the prediction of the assay using in vivo models.
PI: Soto, Ana
Title: Mechanism of Developmental Toxicity of Bisphenol-A
Abstract: Developmental exposure to bisphenol-A (BPA) at doses within the range of human exposure causes a complex array of adverse effects in animals. These outcomes are also known to be present in human populations and the rise in their occurrence coincides with the massive use of BPA and other endocrine disrupting chemicals in consumer goods. The main hormonal activity of BPA is as an estrogen mimic. Exposure to estrogens throughout a woman's life, including the period of fetal development, is considered a main risk factor for breast cancer. Developmental exposure to BPA altered mammary gland morphogenesis in rodents during the period of exposure and led to the development of pre-neoplastic and neoplastic lesions appearing in adulthood.
The goal of this proposal is to identify the molecular, cellular and morphogenetic mechanisms underlying BPA-driven altered mammogenesis that predisposes to neoplastic transformation. To achieve this goal, we will use innovative tools such as a fetal mammary gland explant culture model that allows testing for direct effects of hormones and real-time observation of organogenesis. The Specific Aims of this proposal are to test three hypotheses, namely:
- That the direct effect BPA on mammary gland development is mediated by ER1 and/or 2.
- That BPA causes altered ductal morphogenesis i) by altering the composition and physical properties of the ECM and ii) by inducing adipogenesis.
- That the different mammary gland phenotypes resulting from gestational and gestational plus lactational BPA exposure are due to alterations at the hypothalamic level.
PI: Sprague Martinez, Linda
Title: Nuestro Futuro: Applied Science Education to Engage Black and Latino Youth
Abstract: This project is a curriculum based educational opportunity implemented in an after-school enrichment setting in a Boston, MA public middle school. The project builds on the active NIMHD funded research intervention study, Nuestro Futuro Saludable. This academic enrichment program is focused on inquiry-based science education, public outreach and community-based information campaigns to educate students, families, and their communities on health research topics within a health equity framework. The program is designed to (1) cultivate a deeper, contextualized understanding of scientific concepts related to health disparities and the social determinants of health in their community; (2) teach students to foster their ability to "think" like scientists from various disciplines (e.g.: epidemiology, biomedicine) as they study diseases that present disparities in their community (e.g.: asthma, diabetes, HIV/AIDS); and (3) provide an opportunity for students to use what they have learned about health, science, and disparities to develop and carry-out local advocacy and outreach efforts. The project is designed to increase the scientific literacy of Latino and Black students, as well as student interest in the sciences and enthusiasm for studying the sciences by providing research career awareness opportunities, mentoring and information dissemination to increase the representation of underrepresented minority groups in the sciences.
PI: Stadecker, Miguel
Title: Immunoregulation in Schistosomiasis
Abstract: Schistosomiasis is a major neglected helminthic disease suffered by over 200 million people throughout the world. Critical to understanding schistosomiasis is the notion that it represents an immunologically mediated disease, that is, the damage to the affected tissues is inflicted by the host's own immune system, rather than by the parasite itself. Thus, morbidity and mortality are largely due to a pathogenic CD4 T lymphocyte-mediated immune response against parasite egg antigens. This results in granulomatous and fibrosing inflammation, which in the case of the species Schistosoma mansoni, takes place in the liver and intestines. The magnitude of disease varies greatly from individual to individual. In the majority of cases there is relatively limited immunopathology with good survival, however, in a minority of patients there is severe disease and death. S. mansoni infection in a mouse model similarly results in marked strain variation of immunopathology. In CBA mice, severe hepatic inflammation is associated a novel subset of pathogenic CD4 T cells producing IL-17 (Th17 cells), of which an expanded, clonally-restricted subpopulation is specific for the immunodominant epitope 234-246 of the major Sm-p40 schistosome egg antigen (Sm-p40234-246). In contrast, C57BL/6 mice develop milder lesions within the context of a largely anti-inflammatory immune response. The objective of the present application is to investigate the mechanisms that underlie the development of the severe form of immunopathology embracing the hypothesis that this is precipitated by a distinct, genetically determined innate antigen-presenting cell (APC) reaction to parasite products resulting in the development of a pathogenic adaptive Th17 cell response.
Aim 1 of the proposal is to ascertain by gene profiling, and functionally test by gene knock- down, the lectin receptors on APC that recognize specific parasite egg-derived glycoproteins leading to pathogenic Th17 cell differentiation in CBA mice.
Aim 2 is to understand the role of Sm-p40-reactive T cells in mediating severe pathology by making use of novel mice expressing a transgenic Sm-p40-specific T cell receptor, or rendered unresponsive to Sm-p40 by virtue of transgenic expression of this antigen in the thymus.
Aim 3 is a forward genetic analysis to identify relevant quantitative trait loci taking advantage of unique congenic strains between BL/10 and BL/6 mice, which differ in only 5% of their genome, and yet exhibit significantly dissimilar IL-17 production and hepatic egg-induced inflammation.
The proposed studies will provide new insights into the mechanisms leading to severe immunopathology in schistosomiasis. Their ultimate objective is the design of focused, realistic and practical strategies for amelioration of disease, which could be amenable for consideration and implementation in the human patient population.
PI: Staii, Cristian
Title: Combined Atomic Forces Microscopy/Fluorescence Spectroscopy Approach for Measuring Adhesion, Connectivity and Electrical Activity of Neurons Patterned on 2-Dimensional Protein Substrates
Abstract: The objective of this proposal is to gain a deeper understanding of the basic rules that neuronal cells use to form functional connections with one another. Understanding the brain is of tremendous fundamental importance, but it is immensely challenging because of the complexity of both its architecture and function. The central nervous system consists of many different spatially localized and yet highly interconnected regions. To date the processes involved in forming functional neuronal connections, the mechanisms of axonal navigation to their target region and their specific interactions with guidance factors such as chemical gradients and mechanical cues are still largely unknown. The scientific goal of the current project is to understand the fundamental processes governing the development of connections and communications between neurons in living systems by studying the growth and interconnectivity of small numbers of neurons patterned in simplified, well-controlled geometries. The central hypothesis is that simplifying the neuronal growth environment by creating highly controlled neuronal circuits in vitro will allow the basic rules that underlie neuronal development and the formation of neural connections to be elucidated.
Simple neuronal networks will be created on two dimensional substrates, guiding the formation of synapses and measuring their electrical activity using a) atomic force microscope nanolithography; b) atomic force imaging and atomic force based electrical force microscopy; c) fluorescence spectroscopy. Specifically, one aims to: 1) pattern different types of proteins/growth factors at precise locations on surfaces and use them as growth templates for fluorescently labeled neurons; 2) guide the formation of neuronal synapses by controlling the type and geometry of the underlying protein patterns; 3) systematically investigate the adhesion and growth of neuronal processes using both atomic force and fluorescence spectroscopy measurements; 4) map the electrical activity of the network by combined electrical force microscopy and fluorescence microscopy. The crucial aspect for this last step is the use of a voltage-biased atomic force tip as a movable electrode to both stimulate and record the electrical activity of patterned neurons, both at the synapse level and along the neuronal pathway. Simultaneous fluorescence monitoring will identify the specific signaling molecules released during synapse formation as well as during the propagation of the electrical signal. By performing these experiments one seeks to a) quantify the role that different types of biochemical and geometrical cues play in neuronal growth and development; b) measure under what conditions synaptic junctions are functional and c) learn to control the formation of functional synapses in neuronal circuits having well-defined geometries.
PI: Starks, Philip
Title: Tufts REU Site: Integrative Approaches to Studying Recognition Systems in Cells, Organisms, and Populations
Abstract: An award was made to the Department of Biology at Tufts University to provide research training for 10 weeks for 10 students per year, for the summers of 2010-2012. Our summer program is entitled, “Integrative approaches to studying recognition systems in cells, organisms, and populations” and will provide students the opportunity to work closely on collaborative, interdisciplinary projects combining faculty expertise in biochemistry, genetics, development, endocrinology, neurobiology, animal behavior, plant ecology, and conservation biology. Students will work with two mentors, one serving as the primary mentor, to combine research techniques from different fields to design and conduct an independent project. In addition to research involvement, the program includes weekly discussions, seminars, field trips, and a student symposium.
The goals of this summer program are to increase students’ proficiency in biological research and collaborative skills, to educate and inspire students toward future research careers, and to develop students’ ability to effectively communicate scientific results. Accordingly, seminars are focused on proficiency in science/communication of scientific results, scientific ethics/responsible conduct of research, and career development. We select our participants from a broad undergraduate applicant pool, and we are committed to providing research opportunities for students traditionally underrepresented in science and those who might not have such opportunities at their home institutions (with an emphasis on community college students).
The Tufts University Department of Biology has a long-standing commitment to mentoring undergraduates in research, and a strong track record of highly productive interdisciplinary research collaborations. Student-mentor relationships typically extend long beyond the research experience and are facilitated via electronic networking tools. We solicit frequent feedback from participants, and use this to continuously assess and refine our program.
PI: Stites, Elizabeth
Title: Customary Law, Livelihoods Change and Conflict Mitigation in the Karamoja Cluster: The Case of Uganda
Abstract: The Karamoja Cluster is a cross-border region encompassing territory within northeastern Uganda, southeastern Sudan, northwestern Kenya and southwestern Ethiopia. It is an arid region inhabited by pastoral and agro-pastoral populations who rely on seasonal animal migration to varying degrees. These groups are minority populations within their respective countries and are often at odds with each other and the governments in the regions where they live and move with their herds. Human development index indicators rank these groups among the least developed populations within their countries, with the lowest rates of access to basic amenities such as health clinics, schools and clean water. Populations live in chronic poverty and many are dependent on food aid. The respective state governments largely marginalize the pastoralist groups politically, economically and socially, further limiting their resiliency to drought or external or internal shocks.
The livelihood systems of pastoral and agro-pastoral groups necessitate the seasonal movement of herdsmen and women with their livestock into areas where grazing land and water are present. This often requires migration into areas occupied or accessed for similar reasons by other groups. This sharing of resources can lead to increased tensions and the outbreak of violence between groups. Coupled with poor economic development and widespread poverty, many groups resort to cattle raiding to bolster herd populations or to benefit economically by quickly selling stolen animals. Additional factors that contribute to increased criminal activity include maladaptive coping systems in response to the erosion of traditional livelihoods, government policies limiting human and animal mobility, widespread insecurity and other "natural" phenomena such as climate change. In turn, these maladaptations towards criminal behavior lead to further insecurity, loss of livelihood assets and increased vulnerability. Combined with a general lack of law and order, criminal activity including cattle raiding, theft and murder often occurs with impunity. However, some pastoralist groups in the Karamoja Cluster seek to curb criminal behavior by their members and stem the spread of violence through the application of customary legal practices and use of traditional conflict prevention strategies.
Our proposed research seeks to understand the ways in which customary mechanisms are addressing the rapidly changing social, political and economic order in the Karamoja Cluster. We will focus on the Karamoja region of Uganda and use the Toposa in South Sudan as a comparative case. We will focus on Karamoja due to the profound and widespread changes that are occurring on multiple levels, including shifts away from traditional pastoral livelihoods and a radically changed security environment due to the on-going disarmament campaign. We will seek to understand the ways in which customary mechanisms respond (or do not respond) to these changes and how the activities of different groups compare.
PI: Swan, Chris
Title: Collaborative Research: Engineering Faculty Engagement in Learning Through Service (EFELTS)
Abstract: This project focuses on Learning Through Service (LTS), a pedagogical method that combines academic learning with service. Engaging investigators from five diverse institutions, the project is invoking a 4D Process (Discover, Distill, Design, Disseminate) to evaluate the impacts on faculty currently engaged in LTS efforts and to empower additional faculty to implement LTS. Major activities that are being undertaken include surveying and interviewing engaged faculty; convening a meeting of experts in LTS program/course designs, implementations, and assessments; conducting intensive faculty training workshops on LTS that lead to new LTS efforts at course and program levels; and sustaining faculty engagement via a continued dissemination of efforts. Assessment research methodologies (development and use) are being integrated throughout these activities. The project engages faculty through systemic implementation and support for LTS in engineering education. The project expands the use of LTS in engineering education and highlights LTS as a viable research endeavor and scholarly activity. The project identifies challenges and facilitators to LTS for different faculty and institution types.
PI: Swan, Chris
Title: Collaborative Research: The Impacts of Service on Engineering Students (ISES) – A Longitudinal Study
Abstract: This engineering education research project will increase our understanding of learning through service (LTS) programs, specifically aiming to determine whether international service learning is a viable curricular approach with respect to developing desired engineering attributes. The research questions addressed in the proposal are timely since an increasing number of engineering students participate in international service projects at some personal and institutional expense. The project hypothesizes that LTS improves holistic thinking without detracting from technical ability, and this hypothesis is tested through a well-defined set of research questions.
The broader significance and importance of this project is to validate service learning as an effective educational strategy for engineering students. Although such programs are popular, they can potentially add costs to college education and some questions about their effectiveness remain unanswered. This study may develop insights into LTS and a set of "best practices" that can inform programs that wish to adopt this pedagogy. There is the potential to demonstrate improved recruitment and retention of under-represented groups.
PI: Sykes, Charles
Title: A Single-Molecule Approach for Understanding and Utilizing Surface and Subsurface Absorption to Control Chemical Reactivity and Selectivity
Abstract: Heterogeneous hydrogenation reactions are among the most scientifically and technologically important chemistries and play a major role in the petrochemical, pharmaceutical, and food industries. While the reactions’ pathways themselves are often very complex, very basic steps often dictate important parameters like activity and selectivity. Recent research has hinted that the traditional picture of chemical reactions proceeding via only surface-bound species is totally inadequate in describing such systems. Evidence is gathering that subsurface species like hydrogen and carbon are sometimes more reactive and can even have a greater influence on the reaction outcome than surface-bound reactants. Therefore, elucidating the atomic-scale geometry, electronics and chemistry both on and under the surface of catalytically important metals and alloys will prove transformative in advancing current catalytic technology, cutting down on waste products, and helping facilitate more energy-efficient conversion to products.
This project will exploit and build on our recent findings that:
- Individual, isolated atoms like Pd in catalytically important alloys can be very active for key steps in hydrogenation chemistry
- Individual hydrogen atoms both on and under the surface of Pd metal can be imaged and manipulated at the atomic-scale and
- Single enantiomers of a chiral molecule can be distinguished based solely on scanning probe height measurements allowing enantiospecific reactions to be monitored in situ at the single-molecule level.
These unique capabilities will allow the roles of surface and subsurface hydrogen in both regular and enantioselective hydrogenation reactions to be interrogated at a new level of detail. The PI’s labs are well equipped with state of the art scanning probe and surface science instrumentation and the group has published twenty independent papers in the last four years.
The proposed work is aimed at:
- Quantifying hydrogen adsorption on and absorption in catalytically important Pd particles and alloys.
- Exploring the chemical reactivity and specificity of surface and subsurface hydrogen in a variety of industrially important hydrogenation reactions.
- Utilizing subsurface hydrogen to engineer a novel approach for asymmetric catalysis.
This work will be innovative in developing links between the atomic-scale composition, structure and electronic properties of catalytically important materials and their reactivity in industrially important reactions. The experiments will be the first to relate the atomic-scale details of subsurface reactants to the reactivity of the system as a whole. The work will also provide a framework for building theoretical models that predict reactivity trends. Knowledge garnered from these systems will address a critical need for structure-property-activity relationships that encompasses both the surface and near-surface regions of hydrogenation catalysts. As such, the results will have a significant impact on the chemical, clean energy and chiral catalysis fields.
PI: Sykes, Charles
Title: Collaborative Research: High Throughput Structure Sensitive Surface Chemistry
Abstract: In this project funded by the Chemical Structure, Dynamics and Mechanisms Program of the Division of Chemistry, Andrew J. Gellman (Carnegie Mellon University), E. Charles H. Sykes (Tufts University) and David S. Sholl (Georgia Institute of Technology) will collaborate to develop and apply methods for high throughput study of structure sensitive surface chemistry. The core of the experimental program is the preparation, characterization and study of curved single crystal metal surfaces that expose continuous distributions of surface orientations; i.e. regions of the surface that expose different step and kink densities. Spatially resolved experimental tools such as STM, XPS, and LEIS will be used to characterize the local structures of these surfaces and to measure surface reaction kinetics at each point. This effort will resolve the role of step and kink density in several surface reactions. Complementary computational modeling tools will be used to understand the role of surface orientation in surface reaction kinetics. These methods will greatly accelerate the study of structure sensitive surface chemistry. The impact of this work will be development of a fundamental understanding of several catalytically important surface reactions. The broader impact will include outreach to high school students, exposure of undergraduates to research and the development of short videos on surface science and nanoscience for public viewing.
PI: Sykes, Charles
Title: Turning Molecules into Motors and Mechanical Devices
Abstract: Molecular motors are ubiquitous in nature; they perform tasks as varied as organizing the cellular cytoplasm by vesicle transport (e.g. kinesin or dynein) to powering the motion of cells (e.g. the bacterial flagellar motor) and even driving whole body locomotion through muscle contraction. In stark contrast to nature current manmade devices, with the exception of liquid crystals, make no use of nanoscale molecular motion. This is due in part to a gap in the understanding of how individual molecular components behave in the face of opposing forces such as friction, thermal fluctuations, coupling to neighbors and lack of inertia. Understanding and actuating the rotation of individual molecules on surfaces is a crucial step towards the development of nanoscale devices such as fluid pumps, sensors, delay lines, and microwave signaling applications. Recently the Sykes group has pioneered the use of a new, stable and robust molecular rotor system based on surface-bound thioethers (RSR) to attack these issues. The research aims to achieve the directed electrically powered rotation of single molecules, organize functional molecular rotors in ordered 2D arrays, and develop methods for coupling mechanical motion between neighboring molecules.
PI: Tang, Guangwen
Title: The Study of Vitamin A Requirement in Chinese Children and the New Technology of Dietary Assessment
Abstract: This project relates to Objective 2 of the Carotenodis and Health Laboratory's approved CRIS: Determine the vitamin A requirement of health U.S. adults. However, to understand the vitamin A status in relation to the diets, it is necessary to know the vitamin A status with various dietary patterns and in various age and race groups including children in China. If funded, Dr. Tang will propose a revision to CRIS Objective 2: Evaluate vitamin A status in various populations in the world where vitamin A deficiency is still a health problem.
The CHL will help to train the scientist of Ph.D. graduate student from China to operate the HPLC and GC/MS for sample analysis and data analysis to determine the vitamin status in these Chinese adults.
This project is aimed to assess vitamin A status and needs of Chinese children consuming various levels of vitamin A capsules and the regular diets using stable isotope techniques. The study is conducted by the scientists and Ph.D. graduate students in China. The samples collected from the intervention trial will be sent to the CHL to be analyzed.
PI: Taylor, Allen
Title: Mechanistically Linking AMD, Glycemic Index and Protein Homeostasis
Abstract: Loss of sight is a major fear and significantly compromise to the quality of life among the elderly. Age-related macular degeneration (AMD) is the most common cause of irreversible blindness. There is no cure for this devastating disease. Costs associated with AMD are in the $billions per year in the US alone. It is imperative that means to delay the onset or progress of AMD be found soon because the number of people afflicted is growing so rapidly. New information from three large human cohorts indicates that consuming lower glycemic index diets (GI) is associated with a lower risk for all grades of AMD as well as for delayed onset or progress of early AMD. This information suggests that slightly limiting intake of readily digested carbohydrate, or simulating such dietary practice, may provide a means to delay the onset and progress of all stages of AMD or even prevent it. Slowing AMD progression, particularly at early stages, by as little as 10-20% can delay vision loss for 5-10 years. Prior to initiating costly intervention trials, it is essential to replicate these findings in controlled animal trials and learn about the mechanism of how consuming lower GI diets protects the retina.
Our pilot animal studies indicate that consuming lower GI diets results in delayed accumulation of early AMD-related retina lesions. This is also associated with less protein modification by sugars (glycation). Glycated proteins are toxic and related to AMD development. Furthermore, biochemical studies indicate that the cellular proteolytic capacities that normally eliminate cytotoxic proteins are compromised by glycation. In order to exploit these data, it is crucial to understand the patho-biochemical relationship between consuming higher GI diets, appearance of early AMD-related lesions, accumulation of cytotoxic glycated proteins, and the fidelity of the protein editing, proteolytic machinery.
In this work we will test the hypothesis that early AMD-like lesions will be delayed, glycative stress diminished, and proteolytic functions that remove glycated proteins retained in mice that consume lower GI diets or when activators of the ubiquitin or lysosomal proteolytic pathways are employed. Such etiologic and mechanistic information will substantiate the benefit of lower GI diets and pave the way for intervention trials.
The information is also essential for designing new interventions (dietary and pharmaceutical) that will diminish the AMD burden. The first Aim is to define the relationship between dietary GI, risk for early AMD lesions, and protein glycation. Because AMD is related to compromised protein quality in the RPE and its environs, the focus of Aim 2 will be novel experiments to define relationships between accumulation of glycated proteins and the fidelity of the protein quality control machinery, using RPE from the animal models and differentiated RPE. In Aim 3 we will try new drugs to diminish carbohydrate-induced stress and prolong retinal function. Due to the similarity of the response of many cells to glycative stress and the similar protein quality control in many cells it is anticipated that our observations and discoveries will impact many disciplines and have major health ramifications. This includes heart disease and type 2 diabetes, both of which have been related to dietary carbohydrate intake.
PI: Taylor, Allen
Title: Ubiquitin Function in Eye Lens
Abstract: Sight is our most valued sense. Cataract, or opacification of the lens afflicts virtually all the elderly and surgical extraction of the opacified lens is the most commonly performed surgery, accounting for among the largest line items in our Medicare budget. Fortunately, there is a successful procedure for removing cataracts. Unfortunately, the success is of limited duration because cells grow in the lens capsular bag that remains after cataract surgery causing re-opacification or "secondary cataract." It is essential to discover means to extend the duration of lens clarity after surgery and it would also be of invaluable benefit to delay cataract formation initially. To accomplish these objectives it is essential to understand how cell proliferation and differentiation are controlled in the lens. Cataract is due in part to production of abnormal genes and proteins. The ubiquitin proteolytic pathway (UPP) controls the expression of many genes and the levels of many proteins. We have shown that alteration of components of the UPP result in abnormal lens cell proliferation, differentiation and cataracts. Our findings clearly demonstrate a critical role for a fully functional UPP in regulation of lens formation, including cell proliferation and differentiation. There are many components to a ubiquitin pathway. Identifying essential components of UPPs and elucidating their function will identify specific molecules, the activity of which, if controlled, can be used to regulate proliferation and differentiation. The research proposed herein will identify functions of ubiquitin per se, and specific controllers (UbcH3, 7, 10) of lens cell proliferation and differentiation.
In accomplishing these objectives we will identify a myriad of new targets for pharmacologic intervention to delay formation of secondary cataract. Importantly, each of our hypotheses is testing a fundamental novel concept. Since much about the UPP is similar in many types of cells and tissues, the information we gather will provide understanding of how this pathway works in many other types of cells and tissue. Thus, our research will also inform about targets which should provide new therapeutics for many other tissues, in addition to lens, where controlled proliferation is desirable. This includes cornea, trabecular meshwork, retina and many cancers.
PI: Teixidor I Bigas, Monserrat
Title: The Poincaré Institute: A Partnership for Mathematics Education
Abstract: The Poincaré Institute for Mathematics Education, led by Tufts University, in collaboration with TERC, with Dover, NH, School District as core partner, and partner districts in Massachusetts (Fitchburg, Leominster, Medford, and Somerville), New Hampshire (Dover, Sanborn, and Timberlane), and Maine (Portland), seeks to transform and improve the teaching and learning of mathematics in middle school and the connections between the elementary, middle, and high school curricula. One hundred eighty in-service and 24 new teachers and, each year, more than 2200 students benefit from the project’s work.
Building on successful NSF-funded collaborations between Tufts and TERC — the Fulcrum Institute, the Early Algebra, Early Arithmetic Project, and the Inquiry Project — the Institute leverages expertise from mathematicians, physicists, educational researchers, and school districts to:
create graduate-level online courses on mathematical content, research in mathematics education, and mathematical knowledge for teaching, offered to three cohorts of in-service teachers (grades 5 to 9) from three states;
use algebra and the mathematics of functions and modeling for promoting coherence among topics in the mathematics curriculum;
implement permanent discussion forums where teachers plan, review (using video technology), and improve their lessons, drawing upon the critical mathematical topics and issues of learning and teaching;
strengthen and expand partnerships with schools by preparing teachers and researchers in Tufts’ graduate programs in Mathematics and Education, through internships with teacher leaders and participation in educational research;
conduct research on teacher development and student learning;
support school districts’ efforts to improve their mathematics curriculum; and
disseminate a teacher development model for adoption by other university-school partnerships.
Partners in this proposal recognize the importance of middle school, a period when many students lose interest in mathematics. The problem is acute not only in urban centers, where algebra has often served as an “engine of inequity” (Kaput, 1998) that widens the achievement gap between socio-economic, racial, and ethnic groups, but also a concern in rural areas and in areas of immigrant concentration. The project rests on the premise that to improve students’ learning one needs to broaden and deepen teachers’ understanding of mathematics, of how children think and learn, and of mathematics knowledge for teaching. The project’s research mathematicians and physicists identify and streamline a well-defined set of critical topics and physics phenomena for middle school mathematics, with input from mathematics education researchers and school districts. Mathematics education researchers identify issues of learning and teaching relevant to the chosen critical mathematics topics. Rather than re-teach the mathematics that the teachers already know or offer a set of pre-packaged lessons, the Institute offers them a broad, unified framework to re-envision the mathematics they already teach. Aided by online and face-to-face interactions with researchers, the schools create self-sustaining discussion groups focused on effective pedagogy for mathematics instruction. Experts in the use of software and classroom video in educational settings help bridge the Institute courses and teachers’ initiatives in the schools. Quantitative and qualitative measures are used to answer questions about impact and process.
PI: Terenzi, Gina
Title: Post-Doctoral Training in General, Pediatric and Public Health Dentistry
Abstract: The purpose of this project is to create an Advanced Education in General Dentistry (AEGD) program at Tufts University School of Dental Medicine (TUSDM) and to enhance the general practice training opportunities of the TUSDM’s General Practice Residency (GPR) in Dentistry training program, and will expand dental workforce training activities as a result of this new oral health workforce program. The development and implementation of the AEGD will be in accordance with the American Dental Association’s recommendation for the Commission of Dental Accreditation for new program development in post graduate dental education. The proposed AEGD and enhanced GPR, along with affiliated Primary Care Providers in medicine, will provide increased access to care for populations at risk for oral health disease, defined as patients with developmental disabilities, HIV/AID’s, elderly, victims of domestic violence and substance abuse, the homeless and immigrants. Emphasis will be made on the importance of oral health and its impact on general health and quality of life through the newly created and enhanced curriculum in conjunction with the services the funds requested will provide. The development and implementation of an AEGD and the enhancement of the GPR at TUSDM will use the distance learning principles of Internet based curriculum to foster curriculum enhancement for faculty and resident training. The grant funded project to create faculty development will provide the framework for comprehensive, quality patient care utilizing community and dental school-based facilities as clinical sites for pre-doctoral student, resident, and faculty education, development, and practice.
PI: Tesco, Giuseppina
Title: Role of BACE Stabilization in Alzheimer's Disease
Abstract: Alzheimer's Disease (AD) is the most common form of dementia that affects 5.3 million Americans. In a small percentage (>1%) of cases AD is inherited as an autosomal dominant trait (Familial AD), however the majority of cases are sporadic. A key neuropathological event in AD is the cerebral accumulation of Aβ, a ~4kDa peptide derived by serial proteolysis of the amyloid precursor protein (APP) by β- and γ-secretase. Beta-site APP-cleaving enzyme (BACE1) is a membrane-tethered member of the aspartyl proteases that has been identified as β-secretase. Several studies have shown that BACE1 protein levels and β-secretase activity are increased in AD brains. Thus, BACE1 elevation may be the first step in increasing Aβ and triggering AD pathology, at least in the sporadic cases.
Our studies have elucidated a novel post-translational mechanism of regulation of BACE1 mediated by the BACE1-interacting molecule, GGA3 (Golgi-localized γ-ear-containing ARF binding protein 3). We have determined that GGA3 depletion stabilizes BACE1 and increases β-secretase activity. We also found that levels of GGA3 are decreased in post-mortem AD brains and are inversely correlated with BACE1 levels. We have shown that BACE1 is degraded via the lysosomal pathway and demonstrated that GGA3 regulates the delivery of BACE1 to the lysosomes. The BACE1-C-terminal fragment (CTF) contains a specific di-leucine (DXXLL) sorting signal that has been shown to bind the VHS domain of the three members of the GGA family of proteins, GGA1, 2, and 3. We have found that, unexpectedly, direct binding of GGA3 VHS domain to the BACE1 di-leucine motif is not necessary for this regulation. Instead, GGA3 interaction with ubiquitin is essential for regulating BACE1 levels. Accordingly, we have found that BACE1 is mainly mono- and K63-linked polyubiquitinated at lysine 501.
The central hypothesis of this proposal is that the impairment of BACE1 degradation is the underlying mechanism of BACE1 elevation in the brains of subjects affected by AD. The overarching goal of this proposal is to determine the extent to which GGA- and ubiquitin-mediated regulation of BACE1 represent a potential target for the treatment of AD. Thus, we propose to specifically address the following aims: 1) To determine the extent to which BACE1 ubiquitination regulates BACE1 trafficking, activity and degradation via the proteasomal or lysosomal pathway; 2) To determine the extent to which over-expression of GGA3 reduces levels of BACE1 and A2 in a ubiquitin-dependent fashion in vivo; 3) To determine the extent to which GGA1, another member of the GGA family of proteins, regulates levels of BACE1 and Aβ independently and in association with GGA3 in vitro and in vivo.
PI: Tesco, Giuseppina
Title: The Role of BACE in the Pathogenesis of Alzheimer's Disease after Head Trauma
Abstract: Traumatic brain injury (TBI) is the strongest environmental risk factor for Alzheimer's disease (AD). Clinical and experimental TBI is associated with accelerated beta-amyloid (Abeta) deposition, a hallmark of AD pathology. The Abeta peptide is derived by serial proteolysis of amyloid precursor protein (APP) by beta-secretase at the N-terminus followed by gamma-secretase at the C-terminus. Beta-site APP-cleaving enzyme (BACE) has been identified as beta-secretase. BACE levels are elevated in AD brain, and BACE is induced as a stress-related protease following cerebral ischemia and TBI in rodents. We recently reported that BACE and beta-secretase activity increase following cerebral ischemia in vivo and caspase activation in vitro due to post-translational stabilization of BACE protein. We also found that the impaired degradation of BACE is due to caspase-mediated depletion of GGA3, an adaptor protein involved in BACE trafficking (Tesco et al. 2007). In the current proposal, we report that genetic ablation of GGA3 increases levels of BACE in the brain in vivo.
Furthermore, we have found that GGA3 is depleted following TBI while BACE protein levels increase with a pattern similar to the one observed following cerebral ischemia. These new findings indicate that GGA3 depletion, mediated by caspase cleavage, and consequent BACE upregulation may be the common underlying mechanism of increased Aβ production following cerebral ischemia and TBI. Since Abeta has been shown to impair synaptic transmission, increased Abeta levels may be responsible for impaired functional outcome after TBI. This mechanism may also explain how TBI leads to increased risk of developing AD over time.
In support of our hypothesis, we have found that GGA3 levels are decreased in both temporal cortex and cerebellum from AD subjects, suggesting that subjects with lower levels of GGA3 could be at greater risk of developing AD (Tesco et al. 2007). This may be particularly true for patients with stroke and TBI in which caspase activation occurs even in the chronic period after injury. The long-term goal of this proposal is to identify targets for novel treatments to prevent acute learning and memory deficits as well as development of AD following TBI.
We propose the following specific aims:
Determine the extent to which depletion of GGA3 regulates levels and activity of BACE and causes behavioral changes in mice.
Determine the extent to which decreased levels of GGA3 affect BACE levels and activity and functional outcome following TBI in mice.
Determine the extent to which lack or low levels of GGA3 exacerbate Aβ deposition in a mouse model of AD pathology (Tg2576 transgenic mice expressing human APP with the "Swedish" mutation (KM670/671NL)) in normal conditions and following TBI.
PI: Theoharides, Theoharis
Title: Brain Mast Cells and Chronic Fatigue Syndrome
Abstract: Chronic Fatigue Syndrome (CFS) is a complex disease with a prevalence as high as 1%. CFS involves the nervous, hormonal and immune systems with symptoms that include fatigue, sleep disturbances, malaise, muscle aches, migraines, gastrointestinal complaints and cognitive problems. There may be some mitochondrial “dysfunction” in CFS patients. Many CFS patients demonstrate abnormal hypothalamic-pituitary-adrenal (HPA) axis activity, while stress worsens symptoms. Central and peripheral cytokines produced in response to viral infections or other inflammatory stimuli may be implicated, but there is no distinct pattern. CFS is often comorbid with other disorders that include fibromyalgia, interstitial cystitis (1C), irritable bowel syndrome (IBS), migraines and post-traumatic stress disorder. Neuroimmune interactions in CFS are still unknown creating a vacuum in diagnosis and treatment. Mast cells and their mediators have been implicated in all diseases that are comorbid with CFS. Brain mast cells are abundant in the median eminence where they are juxtaposed to corticotropin-releasing hormone (CRH)-positive neurons and CRH is secreted under stress and we showed that CRH activates mast cells through CRHR-1 leading to release of vascular endothelial growth factor (VEGF), increased vascular permeability and blood-brain-barrier (BBB) disruption. We recently showed an inverse relationship between expression of the mitochondrial uncoupling protein 2 (UCP2), which also regulates production of reactive oxygen species (ROS) and cytosolic calcium, and mast cell activation. There are no effective therapies for CFS. Tricyclic antidepressants have been reported to be beneficial, and our preliminary results indicate that only the tricyclic amitriptyline, and certain natural flavonoids can inhibit mast cell secretion and reduce intracellular calcium ion levels.
Our hypothesis is that external triggers, along with CRH secreted by stress, activate diencephalic centers and mast cells, leading to release of proinflammatory and fatigue producing molecules, and these can be inhibited by select flavonoids. We will investigate:
The effect of CRH, viral poly(I:C), lipopolysaccharide (LPS), neurotensin (NT), substance P (SP) and thymus stromal lymphopoietin (TSMP) or restraint stress using female C57BL-6 mice on:
Fatigue using the forced water immersion test,
BBB disruption by measuring brain levels of the fluorescent marker AngioSense, as well as
Brain expression of histidine decarboxylase (HDC), CRN, beta-endorphin, IL-6, IL-8, IL-17, somatostatin, TNF, mouse mast cell protease (MMCP), urocortin 2, UCP2 and VEGF.
- The requirement for mast cells, for NT, SP, the CRHR involved, and the role of UCP2 in the endpoints studied in Aim 1 by using C57BL-derived WW mast cell deficient mice, NT -/- mice, SP -/- mice, CRHR-1; CRHR-2 -/- and UCP2 -/- mice.
Inhibitory effect of a flavonoid formulation containing luteolin/quercetin/olive kernel oil on endurance and brain biomarkers. The proposed research is hypothesis-driven, is based on strong preliminary evidence, and is innovative with high likelihood for novel findings with applicability to humans.
PI: Theoharides, Theoharis
Title: Investigation of IL-9, IL-33 and TSLP in Serum of Autistic Children
Abstract: In spite of intense efforts, levels of pro-inflammatory cytokines (IL-6, TNF) in the serum of autistic children have been inconclusive. Yet, recent findings indicate that chemokines (eotaxin, MCP-1, RANTES) are elevated in autistic children. These chemokines are responsible for accumulation of mast cells and mast cell activation has been associated with autism.
Recent evidence indicates that three new cytokines, IL-9, IL-33 and thymus stromal lymphopoietin (TSLP) are secreted immediately after innate or external damage to various cell types. Interestingly, these cytokines appear to act as body "alarmins" through activation of mast cells (see references in chronological order under each category below). These cytokines have not been measured in autism, but they may be particularly relevant for a number of reasons. IL-9 has been associated with gut allergy, while IL-33 has been associated with glial activation and brain inflammation. These cytokines and TSLP appear to augment each other, as well as the action of neuropeptides present in the brain and gut such as neurotensin, in activating mast cells (References have been grouped under topic for ease of recognition).
We propose to measure levels of IL-9, IL-33 and TSLP in the serum of autistic children (n=25, 3-12 years old) and age/sex-matched normally developing controls. We believe that it not the absolute values, but interactions among these key molecules that may prove useful both as potential biomarkers, as well as novel targets for treatment. These molecules will be measured in serum being collected for measurement of neurotensin and extracellular mitochondrial DNA with support from Safe Minds.
PI: Theoharides, Theoharis
Title: Stress Induced Skin Mast Cell Activation and Vasodilation
Abstract: There is a fundamental gap in our understanding of why skin diseases, such as chronic urticaria and psoriasis, are aggravated by stress. The long-term goal of this research is to define the role of mast cells in inflammatory diseases. The objective of this application is to identify the contribution of the corticotropin-releasing hormone (CRH) receptors and neurotensin (NT) receptor-1 in stress-induced skin mast cell activation and increased vascular permeability, using both a mouse model and human skin biopsies. Restraint stress increases skin mast cell degranulation, CRH content and vascular permeability; these effects are unaffected in SP-/- and CRH-/- mice, but absent in W/WV mast cell deficient mice. CRH increases vascular permeability when injected intradermally in mice, but this effect is blocked by the NTR-1 antagonist SR48692 and is absent in NT-/- mice, demonstrating the critical role of NT. Stress may elicit release of CRH and NT from dorsal root ganglia (DRG) skin terminals and activate mast cells expressing functional CRHR and NTR-1. The clinical relevance of these findings is evidenced by increased expression of CRHR-1 and histidine decarboxylase (HOC) in affected skin from patients with chronic urticaria, indicating the involvement of CRH and mast cells. The central hypothesis is that CRH released in the skin by acute stress, alone or together with NT, activates mast cells leading to increased vascular permeability and neurogenic inflammation. Guided by strong preliminary data, this hypothesis will be tested by pursuing four specific aims:
Determine the importance of CRHR and NTR-1 on stress and intradermal CRH-induced skin mast cell activation and vascular permeability using CRHR-1-/-, CRHR-2-/-, double CRHR-/-, or NTR-1-/- mice.
Determine if CRHR or NTR-1 need to be expressed on skin mast cells by reconstituting W/WV mast cell deficient mice with bone marrow progenitors from the appropriate CRHR-/- or NTR-1-/- mice.
Investigate the expression of CRHR and NTR-1 in human skin biopsies from atopic dermatitis, chronic urticaria and psoriasis patients, and correlate findings with serum CRH levels and extent of stress by using the State-Trait Anxiety Inventory (STAI).
Investigate the effect of CRH and NT on release from human skin biopsy explants and human cultured mast cells of proinflammatory cytokines.
Our approach is innovative because it utilizes knockout mice and reconstitution techniques to understand how stress-derived neuropeptides contribute to skin inflammation, and employs novel methods of assaying mediator release. The proposed research is significant because it is expected to advance understanding of how acute stress increases skin vascular permeability and neurogenic inflammation. This is an important and under investigated area of skin pathophysiology that has potential applicability to understanding the pathogenesis of skin diseases and screening compounds that may develop into novel and effective treatments.
PI: Thomas, Samuel W.
Title: Enhanced Chromophore Stability Using Fluorescence Quenchers for Organic Photovoltaic Devices
Abstract: Organic photovoltaic (OPV) devices are a promising technology for the harvesting of solar energy for electricity, but show poor photolytic stability. The effect of molecular structure on this degradation remains speculative. Our long-term goal is to develop strategies for improving the useable lifetimes of these devices through an understanding of their decomposition. The overall objective of this application is to understand the effect of electron-accepting fluorescence quenchers, which are required for efficient OPV devices, on OPV photostability. Our central hypothesis is that electron-accepting quenchers used In OPVs protect conjugated organic molecules and polymers from photo-oxidation by increasing kinetic competition through non-radiative deactivation of excited states. The rationale that underlies this research is that once we firmly understand these effects on OPV degradation, rational strategies to minimize device degradation will follow. This fundamental research investigating the decomposition of conjugated organic molecules uses a physical organic approach for testing mechanistic hypotheses. We will test our hypothesis by following two aims:
- Aim 1: Determine the effect of electron-accepting fluorescence quenchers on the photostability of a conjugated oligomer.
- Aim 2: Extend our understanding of the effect of electron-accepting quenchers to conjugated polymer photostability.
The proposed research is significant, because it will provide the insight necessary for designing next-generation active layers of OPVs that will resist photodegradation and will result in devices with useable lifetimes longer than those currently available.
PI: Thompson, Eric
Title: Empirical Assessment of Site Amplification and Development of NEHRP Factors for CEUS: Collaborative Research with Pacific Engineering and Analysis, and Tufts University
Abstract: This proposal is for an analysis of Vs (30m) in the central and eastern United States, similar to and building on the NGA-west project. In the NGA-west project, Vs (30m) values were used by developers of GMPEs as one of the primary terms for site conditions. Following the NGA-west example, the PIs propose to expand the database of measured shear-wave velocity profiles, to compare measured velocity profiles with surficial geology, analyze residuals relative to GMPEs available in the CEUS and compare amplification factors in the western U.S. with those in the CEUS. This approach proved very valuable in developing new GMPEs in the western U.S. and should help in the CEUS.
Mentor: Tickle-Degnen, Linda
Fellow: Bogart, Kathleen
Title: Compensatory Expressive Behavior for Social Functioning with Facial Paralysis
Abstract: Although facial paralysis (FP) is a relatively common condition, affecting 127,000 Americans each year, there is a serious gap in understanding social functioning for people with this socially disabling disorder. My long-term goal is to develop interventions to improve social functioning for people with FP. The overall objective of this project, which is the next step toward attainment of my long-term goal, is to identify the compensatory expressive behaviors (i.e. gestures and prosody) that people with FP use to communicate and how other people interpret these behaviors to form impressions about their emotions and other attributes. My central hypothesis is that people with FP can compensate by using other expressive channels (body and voice) and that perceivers can improve the accuracy of their impressions of people with FP by focusing on their bodies and voices. The rationale that underlies the proposed research is that once compensatory expressive behaviors are identified and a perceiver training is tested, these can be used to develop interventions to improve social functioning.
The specific aims of the proposed research project are to: 1) Identify the expressive behaviors that people with FP use to communicate their emotions and attributes; 2) Determine the accuracy of perceivers' impressions of the emotions and attributes of people with FP and examine the relative contribution of different channels to accuracy; 3) Examine whether training perceivers to attend to channels other than the face improves accuracy of impressions.
The proposed research consists of three social perception studies. In Study 1, adults with various types of FP (targets), including both congenital and acquired conditions are videotaped while being interviewed about their experiences living with FP. Trained research assistants will rate the expressive behaviors and severity of paralysis of the targets. In Studies 2-3, participants without FP (perceivers) will view 1 minute clips of the Study 1 targets and rate their impressions of the targets' emotions and attributes. In Study 2, perceivers will be given one or more expressive channels to observe: voice only, body only, face only, body and voice, or all channels. In Study 3, which tests a training intervention to improve accuracy, some participants will be trained to attend to the body and voice when rating their impressions of targets with FP, and others will not receive training.
The proposed research is significant because it is expected to vertically advance understanding of how people with FP can use compensatory expressive behavior and how perceivers can form impressions based on this behavior. Ultimately, such knowledge will inform development of interventions that will improve social functioning for people with FP.
PI: Trimmer, Barry
Title: Neuromechanics of Soft-bodied Locomotion
Abstract: Animals can move through the world in remarkably complex and effective ways that are still difficult or impossible to replicate in machines. This is particularly evident in soft animals such as octopus, worms and caterpillars which can change shape as they move. A long-term goal of this research is to understand how boneless animals control their bodies and to apply this knowledge in the design and fabrication of entirely new types of robot. In effect, soft animals are working prototypes for all sorts of new devices. Using a well-established model animal (the caterpillar, Manduca sexta) these experiments will simultaneously collect data on the mechanical performance and neural signals driving behavior. New measuring devices (e.g., flexible electrode arrays, micro-force beams) and computational methods have been developed to carry out these studies. Working with engineers, these data will be used to build mathematical models that control and simulate soft animal movements. One outcome will be the first explanation of how a massively deformable structure can be controlled using a few simple commands.
In addition to developing a better understanding of animal movements, these studies will have an impact in the fields of engineering and robotics. One immediate effect will be new cross-disciplinary training opportunities for biology and engineering students. A broader impact will be through the public availability of these results for scientists and engineers to develop their own soft material research tools and applications for future technologies.
One potential outcome is the production of Soft Robots for diagnostic and therapeutic use inside the body or for other applications that need extreme mobility (e.g., search and rescue robots for disaster situations). These new machines could be cheap, safe, and biodegradable alternatives to current devices.
PI: Tzipori, Saul
Title: Evaluate the Impact of Tigecycline Treatment of Clostridium difficile Infection in the Newly Developed Animal Model with Normal Human Gut Microflora (NHGM)
Abstract: C. difficile infection (CDI), a serious nosocomial cause of diarrhea and intestinal inflammatory disease, has increased significantly in North America and Europe. The infection causes lengthy hospitalization and substantial morbidity and mortality (CDC, 2005). The severity of CDI is mainly mediated by exotoxins TcdA and TcdB. Standard therapy depends on antibiotic treatment, which in recent years has become less effective (Shah et al., 2010). Moreover, many patients who initially appear to have been cured suffer multiple relapses. While other interventions have been tried (probiotics, toxin-absorbing polymers, and toxoid vaccines), neither prevention nor treatment strategies have kept up with the increasing incidence of CDI, due in part to the epidemic strain, NAP-1/027.This strain produces larger quantities of toxins TcdA and TcdB, and also produces the binary toxin CDT, whose function is unclear. Vancomycin, the first drug to be used against CDI remains the most widely used and the only drug specifically approved by the FDA. Metronidazole is frequently prescribed off-label but has become increasingly ineffective leading to infection recurrence, attributed to several factors including older and sicker patients, an increasing tendency to prescribe multiple and/or broad-spectrum antibiotics, and the presence of the NAP-1/027 strain in hospitals. These limited options call for new therapeutic approaches. Paradoxically, the use of broad-spectrum antibiotic therapy, a precipitating factor for CDI, complicates the development of new therapies. Apart from being safe for the sick and elderly, ideal compounds for the treatment of CDI must be given orally and act locally with minimal or no systemic absorption.
Tigecycline is a broad-spectrum glycylcycline antibiotic with potent activity against C. difficile in vitro. When used clinically, this antibiotic has only infrequently been associated with CDI (Wilcox. 2007), and significantly, was successfully used intravenously as salvage therapy in 4 patients with refractory CDI (Herpers et al., 2009). These data suggest that tigecycline may achieve sufficient concentrations in the intestinal tract to inhibit the growth of C. difficile. Studies in mice showed that tigecycline did not promote the proliferation of C. difficile, yet it did not reduce concentrations of C. difficile in animals with established CDI. This suggests that tigecycline does not promote growth of C. difficile nor toxin production.
We propose to use gnotobiotic (germfree) piglets populated with normal human gut microflora (NHGM) to model human CDI and to investigate the impact of tigecycline, vancomycin and anti-toxin antibodies to study the relationship between drug treatment, NHGM, and CDI.
- Characterize the neonatal pig colonized with human gut microflora as a model for human Clostridium difficile infection.
- Evaluate in the pig model the impact on the human gut microflora oftigecycline, vancomycin or anti-toxin antibodies in the presence and absence of Clostridium difficile.
PI: Tzipori, Saul
Title: Evaluation of a New Class of Antimicrobial Agents against Clostridium difficile
Abstract: Clostridium difficile, a serious nosocomial cause of diarrhea and intestinal inflammatory disease, has increased significantly in both North America and Europe, causing lengthy hospitalization and substantial morbidity and mortality. Many patients who initially appear to have been cured suffer multiple relapses. While other interventions have been tried (probiotics, toxin-absorbing polymers, and toxoid vaccines), neither prevention nor treatment strategies have kept up with the increasing numbers of incidence of CDI, due in part to the epidemic strain, NAP-1/027, that produces larger quantities of toxins A and B and carries the binary toxin CDT, whose function is unclear. Vancomycin, the first drug to be used remains the most widely used and the only FDA approved drug, although metronidazole is frequently prescribed.
The goal is to develop a new class of antibacterials for treatment of CDI. MBX 259C, the prototype drug, is a dual acting DNA replication inhibitor which targets DNA polymerase through its anilino uracil (AU) component and gyrase/topoisomerase through its fluoroquinolone (FQ) component. In preliminary studies, this compound has both low oral bioavailability and a narrow spectrum of activity. It was found to specifically inhibit C. difficile, including the NAP-1/027 fluoroquinolone-resistant strains (MIC90 = 2 μg/mL), without affecting most anaerobic intestinal bacteria. MBX 259C was found to protect hamsters and mice from both acute CDI. The poor oral absorption of MBX 259C should maximize its gut concentration and minimize any systemic toxicity when dosed orally. For this work, we have developed the antibiotic-treated mouse model for screening analogs generated by chemistry, and effective inhibitors will then be tested in the chronic pseudomembranous colitis (PC) piglet model we have described recently (Steele et al. 2010) for preclinical evaluation. More specifically we will:
Specific Aim 1: Synthesize and optimize a library of MBX 259C hybrid analogs for in vitro potency testing against clinical isolates of C. difficile, with a view to: a) increase solubility and MIC in vitro potency (≤0.5μg/mL), b) increase bactericidal activity and inhibition of sporulation, and c) minimizing impact on other gut flora.
Specific Aims 2&3: Characterize MBX 259C and newly synthesized analogs in the infection mouse model to establishing the optimal effective dose in relation to frequency of drug administration and duration of treatment.
Specific Aim 4: Select two top ranking hybrid analogs, screened in mice, for preclinical evaluation in the diarrhea piglet model, including effective delivery (capsular formulation) of drugs to the large bowel.
Specific Aim 5: Conduct PK/Tox animal studies to establish a safety margin and determine systemic/oral doses.
In five years we will have one or more well-characterized compounds that have been preclinically evaluated in two animal models for safety and efficacy, with a well-established dose and duration of administration, packed into a suitable capsule, ready for IND submission and safety evaluation in phase I.
PI: Tzipori, Saul
Title: Evaluation of Shigella Vaccines in Mice and Pigs
Abstract: Shigella causes bacillary dysentery, an inflammatory gastrointestinal disease affecting the distal regions of the colon and the rectum. As few as 10-100 bacteria can cause disease in some volunteers that is characterized by acute abdominal pain, tenesmus, fever, nausea, vomiting, diarrhea and dysentery, which is small volume stools with blood and mucus. Human to human transmission of bacteria occurs via the fecal-oral route, but food-borne episodes have also been described. In healthy adults shigellosis is usually self-limiting and lasts for 7-10 days, but if left untreated, the disease can cause considerable morbidity and mortality in immunocompromised adults, in infants and children in developing countries where shigellosis is endemic. All four serogroups, S. flexneri, S. sonnei, S. boydii and S. dysenteriae can cause dysentery, provided they contain a large, non-conjugative ~215 kb virulence plasmid that encodes critical factors necessary for epithelial cell invasion and spread of the bacterium within the host tissue. S. sonnei, also known as Group D Shigella, is the serogroup most frequently responsible for sporadic and epidemic enteritis in developed countries. In the US, 70% of shigellosis cases are due to S. sonnei. In recent years, S. sonnei has also emerged as the most prevalent Shigella species in newly industrialized countries.
Owing to the wide range of Shigella serotypes and subtypes, there is a need for a multivalent vaccine representing prevalent species and serotypes. There are currently no licensed vaccines either in the US or elsewhere. Several different types of vaccines against Shigella have been experimentally tested in animal models and in volunteer trials. Each approach provides important information regarding the strategy used as well as safety, and immunogenicity data. Based on protection data, it is generally believed that repeated exposure to Shigella from the environment eventually produces serotype-specific immunity. The primary antigen is the bacterial lipopolysaccharide (LPS), which is an integral component of the gram negative bacterial cell wall. The variable O-antigen repeat region of the LPS provides serotypic specificity to the immune response.
Shigella traverses the gastrointestinal tract and colonizes and induces disease in the colon. It will be important to find immunization techniques for subunit and whole cell Shigella antigens that provide immunity to the mucosa of these surfaces. Whole cell vaccines are given orally, although the rectal route would also be possible. The ability of these 2 routes for antigen administered with or without a mucosal adjuvant would be important for guiding clinical development. The possible routes for administration of subunit antigens would not include the oral route unless the antigen were converted into a particulate delivery system, but could include sublingual or intradermal. These 2 routes for immunizing the intestinal mucosa need evaluation as they could simplify the vaccination process and make it possible to exploit new subunit vaccines.
Sublingual (SL) immunization is emerging as a novel alternative approach to mucosal vaccination against pathogens. Live vaccine vectors and adjuvants that cannot be used orally or intranasally might be safe and effective if administered by the sublingual route. The SL route has been used for many years as noninvasive and effective immunotherapy for the treatment of type I respiratory allergies to a variety of allergens. SL administration of ovalbumin as a model antigen, delivered with Cholera Toxin (CT), as an adjuvant, was shown to induce a broad range of immune responses in mucosal and in extra-mucosal tissues. Studies with papillomavirus administered SL induced protective specific antibody and T-cell responses in the genital tract, indicating an immune response at distant mucosal sites.
Studies with hepatitis B, rabies and influenza vaccines suggest that intradermal vaccination has potential for greater immunogenicity because the skin is populated with dendritic cells, which are efficient and potent antigen-presenting cells for induction of protective immunity. Therefore, ID vaccination can result in an efficient immune response with a lower dose as compared to IM application. Intradermal vaccination also has the advantages of being less painful than IM due to the much reduced volume which also results in reduced local lesions.
As an immunogen with strong adjuvant activity, dmLT is an appropriate antigen for evaluation of methods of vaccine delivery including mucosal and systemic. There is a great deal of information available on mLT in the literature, and in unpublished data, and is readily available through collaboration with Dr. John Clemens of Tulane University.
Little experience is available for immunizing the large intestinal mucosa. It is not clear how immunization at other mucosal surfaces might induce immunity in the colon. This becomes important because the major site for pathogenesis by Shigella is in the colon. Thus maximally effective vaccines against Shigella must be used in a way that induces strong immunity in this region of the intestine. The goal of the studies proposed below should provide immunologic data leading to a rational application of Shigella vaccines.
We will evaluate the ID in piglets comparing ID, given at high and low doses with IN. We will measure various immunological parameters at the systemic and mucosal levels to assess the immune response. We will likewise compare ID immunization with dmLT at 3 different doses with IN or SL immunization in mice.
PI: Utz, Arthur
Title: Energy Resolved Studies of Dynamics, Reactivity and Mechanisms at the Gas-Surface Interface
Abstract: In this project supported by the Chemical Structure Dynamics and Mechanisms Program of the Division of Chemistry, Prof. Arthur Utz of Tufts University will study how the motions (dynamics) of atoms and molecules can promote chemical reactivity at the gas-surface interface. Gas-surface reactions underlie industrial processes ranging from heterogeneous catalysis to materials deposition and semiconductor fabrication. The proposed studies will combine quantum state-resolved laser excitation of a gas phase reagent with ultra-high vacuum surface science techniques to show how specific molecular motions (bond stretches, bends, and surface vibrations) promote reactivity. Another line of inquiry will explore the rates and pathways for energy redistribution processes that dictate reactivity. The experiments will reveal how vibrational energy in the gas-phase reagent and in the surface impact reactivity. The study of surface vibrations will be a particularly significant focus as it has the potential to inform our understanding of the unique catalytic activity of nanoparticles. The ultimate goal of this work is to develop a better understanding of the important variables that influence gas-surface reactivity, with an aim towards designing better theories, identifying new strategies for chemical control, and revealing new practical applications. The results of this study will provide theorists with much useful data to test the latest theories.
The results of this research will also provide important guidance in designing practical catalytic reaction schemes, which are important to industry. Students and postdoctoral research associates who participate in this research acquire new knowledge and skills in preparation for advanced studies or entrance into the scientific/technological job market. The PI will also guide departmental efforts to incorporate active citizenship into the undergraduate and graduate curriculum at Tufts. These efforts will better prepare Tufts students to engage in public discourse, participate in outreach activities, and incorporate the principles of active citizenship in their professional careers.
PI: Utz, Arthur
Title: Training Tomorrow's Innovators: A GAANN Program for Interdisciplinary Doctoral Studies in Chemistry
Abstract: The steady decline in the numbers of graduating Ph.D. chemists must be reversed if the nation is to maintain its competitiveness in broad areas of industry, research, education, defense, and medicine, as recognized by the absolute priority for the FY 2009 GAANN competition. In fact, the need for chemists trained at the highest level is increasing as distinctions blur between chemistry and other fields such as biology, environmental science, or medicine. The Department of Chemistry at Tufts University is well positioned to help meet this national need. Our doctoral program has been in existence since 1904, and the faculty is well known for their cross-cutting research programs. Major facilities renovations and recent faculty hires support this emphasis on interdisciplinary research. Thus, with its excellent infrastructure and faculty, Tufts is well poised to train tomorrow’s innovators in chemistry and its related interdisciplinary fields.
This proposal requests funds for eight GAANN Fellows. All fellowships will be used to support students pursuing a doctoral degree. Training will include coursework in core areas of chemistry and will be complemented by interdisciplinary research in the laboratory, supervised teaching experience, and community outreach. The interdisciplinary training that the GAANN fellows will receive at Tufts will broaden their horizons by enabling them to enter other fields with fresh approaches to problem-solving that are rooted in chemistry.
Our department presents a special opportunity for a GAANN award to have significant long-term impact. GAANN support will attract an influx of highly qualified Ph.D. students at a crucial point in the department’s growth. GAANN fellows will explore new directions of inquiry that will attract external funding and lead to a permanent increase in the department’s capacity to train doctoral students. It will also increase the diversity of our graduate student body and enhance our ability to recruit students from traditionally underrepresented groups through partnerships with selected undergraduate institutions. In short, a GAANN program at Tufts will increase the number of Ph.D. chemists trained both in the short- and long-term.
PI: Vandervelde, Thomas
Title: CAREER: Metamaterial-Enhanced Thermal Energy Harvesters
Abstract: The objective of this research is to create a new class of thermophotovoltaic cells that convert thermal radiation (heat) into electricity that harnesses wide-ranging heat temperatures frequently occurring in home appliances, factories, and during electricity generation. The approach is to use recent advances in infrared photodetectors, developed in part by the PI, which maximize conversion of light into electrical current.
PI: Vilenkin, Alexander
Title: Fundamental Physics and Cosmology
Abstract: Inflationary cosmology, combined with recent developments in string theory, is pointing to a major paradigm shift: from a nearly homogeneous and isotropic universe to an extremely inhomogeneous "multiverse", where much of the volume is still in the state of explosive inflationary expansion. We live in a finite "bubble" where inflation has ended, and other bubbles with diverse properties are constantly being formed. A major goal of the proposed research is to learn how this multiverse scenario can be tested observationally. All possible events will happen an infinite number of times in an eternally inflating multiverse. Unless we learn how to compare these infinities, we will not be able to make any predictions at all. This is known as "the measure problem". To determine the probability measure of the multiverse, different measure prescriptions will be studied to check for internal inconsistencies and for conflict with the data. Some measure candidates have already been ruled out in this way, and the hope is that there aren't that many viable prescriptions satisfying some minimal set of requirements.
Another idea to be pursued has recently been suggested by Garriga and Vilenkin. They conjecture that the dynamics of the inflationary multiverse is holographically encoded at the future infinity, where it is described by a lower-dimensional Euclidean field theory. The measure is then defined by imposing an ultraviolet cutoff in that theory. This is an attractive approach, as it opens the possibility of deriving the measure from first principles. The most spectacular observational test of the multiverse scenario would be a direct observation of a collision of our bubble with another one. Observational effects of such a collision and the probability for us to be close enough to detect one will be investigated.
Another major topic of the proposed research is the study of the evolution and observational signatures of topological defects, which can be copiously produced at phase transitions in the early universe. The main focus here will be on cosmic string networks and on "necklaces", consisting of monopoles connected by strings, which are the most promising defects from the observational point of view. Numerical simulations and analytic modeling will be used to study the evolution of string networks, and the results will be used to make accurate predictions for gravitational waves, high-energy particle fluxes, and other observable effects. Detection of topological defects would open a new window into the early history of the universe and into particle physics at extremely high energies. The broader impacts are that the proposed research will be done in collaboration with graduate students and postdocs. The PI will continue his efforts to communicate the results to the general public. This especially applies to the multiverse worldview, which has far-reaching implications beyond physics. The PI has already written a book for lay-persons called "Many Worlds in One" and his work has been featured in numerous newspaper and magazine articles in the US, Europe, Russia, and Japan, and in many popular books.
PI: Walker, Peter
Title: Livelihood Programming for Disaster Risk Reduction
Abstract: The program’s goal and anticipated impact are to improve livelihood outcomes in DRR programs by reducing the risk of negative outcomes from shocks, reducing the cost of humanitarian response, and enhancing community resilience in risk-prone areas. We will do this by increasing the aid community’s knowledge of the relationship between livelihoods and disaster risk reduction, and using this knowledge to enable more effective programming, and thereby enhance community and national DRR strategies.
Primary Beneficiaries are the agencies and donors who will be influenced by the proposed research and approaches developed from it. Secondary beneficiaries will be all those vulnerable individuals targeted by the agency’s future DRR programming.
Disaster Risk Reduction (DRR) programs seek to enhance the capacity of vulnerable communities to identify, reduce and manage risk, whether it be at the local, regional or national level. The recently published Global Assessment Report on Disaster Risk Reduction concludes, “The policy and strategy frameworks for disaster reduction... are not effectively integrated, are not focused on addressing the underlying risk drivers, and are insufficiently articulated to and supportive of effective local and sectoral actions. This is the missing link holding back progress.” The proposed research and development work seeks to address this gap through examining the linkages between livelihood strategies in risk prone environments and the ability of communities and intervening agencies to reduce those risks through DRR programming. By understanding better how disaster-affected communities themselves increase their resilience to disaster, we will identify a range of livelihood interventions, programs and practices that enhance community and national DRR. We will engage in strategic partnerships with key aid agencies with the goal of enhancing the effectiveness of their DRR programming. The work will be carried out through a literature review and a number of field based studies that explore particular risk communities and the specifics of their asset base. The knowledge we gather will generate publications, trainings and real time advice on DRR programming to operational agencies.
PI: Walker, Peter
Title: World Conference of Humanitarian Studies (IHSA Conference)
Abstract: Humanitarian crises and the responses they trigger are evolving rapidly. This conference looks at the opportunities and threats for addressing these crises, as well as at the strengths and weaknesses of the actual responses. The conference provides a unique forum for both scholars and practitioners to present research and debate these issues.
In particular, the conference focuses on four broad themes:
THEME 1: EMERGING FROM PROTRACTED CRISES
In many parts of the world, humanitarian crises have become protracted or even semi-permanent. Political and institutional fragility may have a long-term destabilizing effect and conflicts often turn into long periods of no-peace/no-war. Already marginal agricultural production, further weakened by demographic pressures, climate change, ecological deterioration and economic decline, is seen to tip over towards long term food insecurity. Crises appear to become self-sustaining. Panels under this theme will deal with three sets of questions: How can we deal analytically with and understand the causes of the semi-permanent character of such crises? How do long-term crises affect people’s lives and re-order their institutions and societies? How do different intervention mechanisms — including humanitarian action, institution building, peace building, reconstruction and development — work in these situations, and how do they (dis)engage with each other?
THEME 2: NEW DIRECTIONS IN POLICY
Humanitarian policy is developing quickly due to the changing nature of disasters, and new modalities for, and constraints upon, response. Climate change, volatile world food prices, and intractable conflicts in fragile states are among the challenges facing responders, as are water scarcity, extreme poverty and forced migration. The nascent participation of China, India and other non-traditional actors is reshaping the opportunities for policy alongside emergent international norms, including the Responsibility to Protect and criminal accountability for the most egregious crimes. New global humanitarian financing mechanisms and changing donor profiles also have implications for humanitarian action and recovery. The conference encourages panels and papers that deal with these and related issues, focusing on the challenges of the next decade.
THEME 3: INNOVATIONS IN HUMANITARIAN PRACTICE
Ensuring the efficient and effective delivery of humanitarian protection and assistance requires constant monitoring of developments in communications and information technology, nutritional science, water engineering, the social sciences and economics. At the same time, organizational systems and their collaborative approaches need to evolve and innovate if they are to cope with the future environment. Practitioners and academics alike are experimenting with new ways of organizing, sharing and adding value to knowledge, assessing needs, providing services, evaluating programs, stimulating social movements and enhancing accountability. Panels under this theme will be encouraged to accept papers which explore such innovations, going beyond description to include analysis. Papers should focus on innovations that are being field tested, including innovations in scaling up from pilot projects to regional and national programs.
THEME 4: ADVANCES IN PUBLIC HEALTH AND FOOD SECURITY IN CRISES
Ensuring survival through effective health services and achieving food security are dominant humanitarian concerns in disasters and war-related crises. The panels and papers within this theme will explore the latest clinical, institutional and operational research on humanitarian medicine, health and nutrition interventions, factors impacting on food security in crises and translational research which seeks to move from empirical research to changes in policy and practice. Panels may examine:
- the roles and use of medical and agricultural information and information technologies in humanitarian crises;
- adapting modern medical advances and current approaches to promoting food security to support more relevant response in humanitarian crises;
- integrating health and nutrition promotion with systems to support livelihoods, agricultural markets and income generation, and
- rebuilding and rethinking health and agricultural service systems in post-conflict settings.
Additional areas of interest include: community-based health promotion and nutrition interventions; famine/drought early warning systems; use of climate information and geo-location technologies for food distribution and targeted agricultural interventions; changing roles of public, private and civil society actors in health and food security; and conditional cash transfers and vouchers for health promotion and agricultural services. Papers documenting research in and on conflict-driven crises will be encouraged.
PI: Walt, David
Title: Development of Technologies for Early Detection and Stratification of Breast Cancer
Abstract: In the field of Chemistry, the fundamental unit is the molecule. In the field of Biology, the fundamental unit is the cell. Collections of molecules form pathways and systems; collections of cells form tissues and organisms. In order to understand Biology at its most fundamental level, it is essential to measure tissues with single cell resolution. Furthermore, if one could interrogate these tissues by literally counting the numbers of molecules of each protein, RNA transcript, and metabolite present in each cell, while also having a complete genetic profile of each and every individual cell in the tissue, one would likely have a much clearer picture than one does today. My vision is to develop the tools to enable the highest level understanding of biology in general and breast cancer in particular. If we can use our single cell and single molecule tools to detect molecules and measure cell populations with unprecedented resolution, we should uncover new biomarkers and indicators with diagnostic and predictive value. Breast cancer offers an important challenge because it is a serious medical problem that has the potential to be addressed by technologies that can get at the single cell level.
PI: Wanke, Christine
Title: Nutrition and HIV Progression
Abstract: There are more than 40 million individuals infected with HIV living throughout the world, the majority of these live within the resource-limited world. It has been clear throughout the HIV epidemic that the nutritional status of the host plays an important, independent role in HIV-associated outcomes particularly progression of HIV disease and mortality. Although it would appear to be intuitive that maintenance of or improvement in nutritional status would lead to improved outcomes in HIV infected individuals, few data are available to demonstrate the potential benefits of maintaining nutrition status at normal. There are data that suggest that the use of micronutrients could reduce CD4 count decline and delay death, however micronutrients alone will not support or maintain nutritional status. The overall hypothesis of this application is that the consumption of a nutrient dense protein supplement (NDPS) early in HIV infection will slow disease progression, and that the time from infection with HIV to the initiation of HAART will be prolonged.
If this hypothesis is proven to be correct, this type of intervention will result in benefit to the individual, as the need for the use of HAART would be delayed. It would also benefit the health systems, as cost savings would result from a delay in the initiation of HAART. Specifically we propose to enroll 740 HIV infected women in Kenya, with CD4 counts between 350 cells/µL and 500 cells/µL and no symptoms, opportunistic infections or AIDS defining illnesses or malnutrition (BMI<18.5 kg/m2) that would require the initiation of HAART. These individuals with early disease will be randomized to a group that will be provided with the nutrient dense protein supplement (NDPS) or standard of care (SOC) and followed until the initiation of HAART is necessary or a total of 2 years. Outcomes in this study will include the need for the initiation of HAART, the rate of decline of CD4 cell count, overall nutritional status as measured by BMI and lean body mass, and quality of life. We will determine the cost effectiveness of this intervention strategy.
In order determine if the dietary intake and the nutritional status (BMI) of the HIV-infected women with early disease is within the community norm, we need to evaluate the dietary intake and nutritional status of similar but non HIV-infected women in the local community. We propose to collect data on 200 women who are documented to be HIV-negative from Voi Division at a single visit, the Division that will also provide the HIV-infected women for the intervention study.
PI: Wanke, Christine
Title: Protease Inhibitor Related Dyslipidemia
Abstract: Our original project described predictors of abnormal surrogate markers of CVD (carotid intimal medial thickness and coronary calcium score) done at a 3 year interval. Antiretroviral regimens, markers of HIV disease activity, c-reactive protein and lipid profiles (including conventional lipid profiles, triglyceride-rich remnant lipoproteins, Lp(a), homocysteine, fasting blood glucose, free fatty acids and insulin levels), BMI, body composition by anthropometery, BIA and DXA as well as traditional risk factors (smoking, diet, blood pressure, age, gender, family history) were examined. Analyses from our original grant as well as others have suggested that traditional cardiovascular risk factors are the most common predictors for abnormal surrogate markers in this patient population; the impact of the dyslipidemia induced by PIs or other antiretroviral agents and the role of HIV and its associated inflammation on this increased risk remain unclear. To date no HIV surrogate marker studies have followed HIV-infected patients for the 6 year period that may be optimal detect the impact of the dyslipidemia or HIV on progression of disease in individuals. It remains clear that HIV infection itself induces low HDL-cholesterol levels and we have preliminary data that suggest that HIV infected individuals have an atherogenic HDL subpopulation profile. It appears that HDL-subpopulations may have a greater predictive value for risk of CVD than HDL-C alone. We now know that it is selected antiretroviral agents, not classes of agents, that are associated with more severe dyslipidemia in HIV-infected populations and propose to examine the impact of these agents (lopinavir/ritonavir, d4t, efavirenz) as well as traditional and emerging risk factors on HDL subprofiles and on surrogate markers of CVD. Markers of chronic inflammation are considered to be independent predictors of CVD; they also predict progression in HIV-infection and may reflect the cumulative burden of HIV disease in an infected individual.
We propose to expand our studies of chronic inflammation adding CRP isoforms and sPLA2 to the determination of CRP to examine the association of these with atherogenic lipid profiles and with abnormal surrogate makers in our cohort. We propose to extend the duration of our surrogate marker studies to study the progression of c-IMT and coronary calcium score in an HIV infected population over 6 years and to document the changes in HDL subpopulations and inflammatory markers over the new grant period in our HIV infected cohort. The longitudinal determination of HDL subpopulations, the simultaneous determination of both cIMT and coronary calcium scores at 6 years in an HIV infected cohort are novel to this project.
PI: Wanke, Christine
Title: The Impact of Omega-3 Fatty Acids on Vascular Function and cIMT in HIV Infected Individuals
Abstract: There is convincing evidence that there is an increased risk of cardiovascular disease in patients infected with HIV. There are multiple potential risks for CVD in HIV infected patients; atherogenic lipid profiles have long been associated with increased risk of cardiovascular disease; elevations in total and LDL cholesterol have been demonstrated to be associated with such increased risk in the general population. More recent data suggest that elevations in levels of triglycerides and decreased levels of HDL cholesterol may be equally important markers of CVD risk. There is also emerging data that CVD is an inflammatory disease, and that HDL-C levels and subprofiles are mediated by inflammation; in HIV-infected individuals these changes may be exacerbated by the HIV infection. While treatment of atherogenic lipid profiles is desirable, attempts to treat these abnormalities in HIV infection are complex in HIV infected individuals. In studies done in populations with and without HIV, intake of high doses of omega three fatty acids is demonstrated to decrease triglycerides and may have a beneficial effect on HDL-cholesterol levels. Intake of omega three fatty acids alters lipid metabolism and may decrease inflammation by decreasing production of arachidonic acid. At present, there are no data that extend these observations to determine whether intake of omega three fats over a more prolonged time period will also have a beneficial impact on vascular function and surrogate markers of CVD in HIV infected patients.
We propose a randomized, double blind trial of purified omega three fatty acids in HIV infected individuals with elevated levels of triglycerides. While the impact of omega three fatty acids on lipid profiles should be evident within 12 weeks, we propose to conduct this trial for a full 24 months to test our overall hypothesis that this intervention will not only improve triglyceride and HDL-C levels, improve HDL-subprofiles and membrane phospholipids and decrease inflammation, but will also improve brachial artery reactivity as a measure of vascular function at 24 weeks and lead to a reduced rate of progression of cIMT as a surrogate marker of CVD at 24 months when compared to controls. The specific aims of this proposal include:
To conduct a randomized, placebo controlled trial of omega three fatty acids over 24 months in HIV-infected individuals with elevated levels of triglycerides (>150mg/dl).
To demonstrate the impact of omega three fatty acid intake on TG levels and on HDL-C levels, HDL subprofiles, composition of membrane phospholipids, chronic inflammation as measured by CRP, sPLA2 and by levels of arachidonic acid.
To demonstrate the impact of omega three fatty acid intake on brachial artery reactivity at 24 weeks and on cIMT at 24 months.
PI: Wilson, Nancy
Title: Partnering for Economic Recovery Impact Through Service (PERIS)
Abstract: Building on 10 years of working relationships, Tufts University and our Somerville partners propose to provide community service for economic recovery in a new way. We will start with collaborative crisis response planning that engages students, faculty and community partners, and we will test and document that planning process to create a framework for responding to rapid change that is useful in other settings.
Until now, Tufts, like other universities and the service field in general, has supported a wide diversity of projects because we have emphasized student and adult voice and initiative. In the process, we have created powerful “social capital” in the form of effective working relationships and trust. But a time of crisis or rapid change is a time to seek new returns from that social capital. In Project PERIS we seek that new return with a new approach to service — starting with a foundation of shared planning in response to the economic crisis. Using academic and co-curricular strategies, we will start the collaborative planning process in Fall 2009 and deliver priority economic recovery services starting in Spring 2010. By marshalling key assets of the university and community — an annual research cycle, a proven model for service and reflection and innovative web tools — we will run a cumulative project that maximizes community impact on priority identified needs while still giving each cohort of students some degree of choice and voice.
We will conduct a rigorous mixed method evaluation to determine students’ development of civic competency and fulfillment of stakeholder’s objectives. Over the life of the project, we expect to have engaged between 350 - 525 Tufts students in 27,000 hours of crisis response planning and economic recovery services, 18 partners in planning and 70 partners as co-educators, 18 Tufts faculty and 85 Somerville youth. We expect to serve approximately 2,700 community members.
PI: Wilson, Nancy
Title: The Goldberg Initiative: Mitigating Obesity in Boston's Immigrant Communities
Abstract: The project will develop, pilot, evaluate and disseminate strategies that lead to more physical activity and improved nutrition across Boston's largest immigrant populations—Chinese, Haitian, Dominican and Vietnamese. This three-year initiative will include a strong research element, multiple intervention strategies, policy work and a communications plan, which will allow us to translate research into evidenced-based community programs and influence policy. All of this will be conducted using a trans-disciplinary, cross-sector team, with balanced participation from immigrant serving organizations alongside Tufts University faculty, staff and students. This process of co-creation between community partners and the university has been the cornerstone of the successful nutrition and obesity mitigation projects already undertaken by the principal investigators in this project, in other communities or on other health topics.
The Goldberg Initiative will produce results in four areas:
- Improved individual level health awareness and behavior for participants in direct intervention programs;
- Increased capacity to incorporate obesity mitigation strategies into the work of immigrant-serving organizations;
- Implemented and evaluated practices for obesity mitigation in immigrant communities that can be shared widely; and
- Influencing policymakers and the policy agenda.
PI: Wise, Timothy
Title: Global Economic Governance and Sustainable Development
Abstract: Over the next two years (July 2009-June 2011), GDAE will build on its impressive range of collaborations with researchers in the global South to ground the debate over policy space and alternative national development strategies. The goal is to contribute accessible, empirically-based research on the social, economic, and environmental impacts of globalization to shape a more sustainable economic future and promote a rights-based approach to international development in a climate-constrained world. The project has two principal areas of research. “New Models for Global Economic Governance” focuses on broad international trade, development and governance issues. “Sustainable Hemispheric Integration” deepens GDAE’s work on Mexico and Latin America.
PI: Wong, Peter
Title: Collaborative Research: Microscale Joining Using Nanoheater Structure
Abstract: The project combines the expertise of the collaborating PIs; metallurgical modeling and processing at Northeastern University (T. Ando), the systems and thermal processing expertise at Tufts University (P. Wong), and composite experimentation and modeling at UMass Lowell (Z. Gu and J. Chen). Built on a previous successful collaboration on NSF support “Industrial Safety of Nanoheaters” CMMI-0738253, the current collaborative project maintains regular research meetings and conference calls to share and discuss data/specimens, assess progress, and determine plans for future work. All graduate students present their work and get advice from the PIs at these meetings. The undergraduate student to be supported on the requested REU supplement will also attend and make presentations at the meetings.
The Tufts tasks for the project are to investigate the application of the composite nanoheaters in the context of microscale joining of components of dissimilar materials and complex geometries. The participating undergraduate student will be involved mainly in the work of application evaluation, modeling, and testing. The student will also assist in the ultrasonic consolidation of core-shell Al-Ni bimetallic nanopowders to be produced by galvanic replacement at UMass Lowell.
PI: Wortis, Henry
Title: Babesiosis as a Model of Age-Related Immunosenescence
Abstract: As people age, a greater number becomes susceptible to infections. Because the aged represent an ever-growing segment of the populations in the western world, morbidities due to infection are significant. We are interested in identifying alleles that underlie this age-acquired susceptibility. We developed a mouse model of human babesiosis due Babesia microti, an emerging infection particularly severe in the aged. We observed that young DBA/2 mice experience significant parasitemia but young C57BL/6, B10.D2 and BALB/c mice do not. In DBA/2 mice an early phase characterized by an intense and transient parasitemia is followed by a late phase of low grade, but persistent parasitemia. As age advances, both acute and chronic parasitemia become greater in DBA/2, but not in C57BL/6, B10.D2 or BALB/c mice. Using B10.D2 x DBA/2 (BXD) and BALB/c x DBA/2 (CXD) crosses as well as chromosome substitution mice, we have established that i) resistance is a complex trait in young and old mice, and ii) acute resistance in young mice maps to QTL that differ from those of old mice. We conclude that the greater susceptibility of old DBA/2 mice maps to age-restricted QTL.
We now propose to identify resistance and susceptibility alleles and to uncover how they modulate the response to B. microti infection. In Aim 1, we will identify the major genetic determinants of acute resistance in old mice of the BXD cross. In Aim 2, we will identify the major genetic determinants of acute resistance in old mice of the CXD cross. Both aims combine a positional cloning approach with bioinformatic analyses and gene expression studies. In Aim 3, we will identify the cells that contribute to resistance and are affected by the polymorphisms underlying the major QTL. Strain differences in cell function will be studied and used to guide the genetic analysis. We are hopeful that the identification of age-sensitive polymorphisms that modulate immune resistance of old mice to babesiosis may inform the search of alleles that predispose aged individuals to infection.
PI: Wortis, Henry
Title: Immunologic Aspects of Disease
Abstract: This is an application to support a well-established pre-doctoral training program in immunology at the Sackler School of Graduate Biomedical Sciences located on the Tufts University Health Sciences Campus in downtown Boston.
Currently, 21 faculty members are training 34 students of whom seven receive direct support from this grant. Highly qualified students are selected from a pool of over 100 applicants based on research experience, recommendations, coursework/grades, interviews, and GRE scores. Training is designed to provide an appreciation of clinical problems together with the technical expertise and scientific insight to successfully perform research on important immunological questions. It aims to stimulate originality, curiosity and the use of rational reasoning to understand biological mechanisms. The didactic component consists of required courses: Biochemistry, Introduction to Immunology, Immunogenetics, Immunochemistry, Immunological Mechanisms in Disease and an elective. These courses provide the foundation for required participatory courses: Journal Club, Advanced Journal Club, Seminars and Student Workshop. Students take four first year laboratory rotations and pass a qualifying examination before entering a thesis laboratory. Thesis research, considered the core of the Ph.D. training, is overseen by individual thesis advisory committees that meet with the student each semester. It is the thesis committee's role to critique the project proposal and to assess the student's progress towards producing an original and substantive contribution to scientific knowledge. While publication is not a formal requirement, graduates are expected to be first authors of peer-reviewed papers. Students are explicitly trained in oral and written presentation of scientific ideas and data and have opportunities to present their research findings at the weekly Student Workshop as well as the annual retreat and mini-symposium. Students also routinely attend and present at national and international meetings. A Program Student Adviser acts as student consultant and advocate. Requirements and expectations are established in writing and the Student Adviser keeps students well informed of their progress. The program successfully graduates a high proportion of all entering students (89%).
In the past 10 years there have been 65 graduates, three are in transition, twelve are in clinical training or service and of the remaining 53, a high proportion, 45 (85%), are actively engaged in research, including four in tenure track positions. The program has benefited from a recently increased University commitment of resources to graduate biomedical education, new research facilities and the hiring of new faculty members. Continued support for seven predoctoral positions is requested.
Mentor: Wortis, Henry
Fellow: Shapiro, Michael
Title: Light Weight Modeling of Epstein-Barr Virus
Abstract: Epstein-Barr virus (EBV) infects more than 90% of all humans, usually without symptoms. It can also be responsible for acute infectious mononucleosis (AIM) and is associated with fatal malignancies including immunoblastic lymphoma, Hodgkin's lymphoma, Burkitt's lymphoma, nasopharyngeal carcinoma and X-linked proliferative disorder (XLP). Our long-term goal is to understand these processes in sufficient detail to guide clinical intervention. Our overall model of normal and malignant EBV biology puts us in a good position to build computer models of EBV infection.
Our specific aims include the following:
- Evaluate the relative impacts of various factors known to play a role in EBV biology.
- Assess the probability of the varying fates of a cell once it has entered a particular infected state.
- Understand the overall dynamics of these models as dynamical systems. This includes distinguishing possible long-term behaviors and the transitory states that lead to them.
We will pursue these goals by building and analyzing multiple light weight computer models of EBV infection. By "light weight" we mean that these models are easy to write, modify and run. This will allow investigations not possible with larger agent-based computer models. Epstein-Barr Virus is widespread in the human population. While it is usually asymptomatic, it is also associated with fatal malignancies. Computer simulation is a way to study the normal asymptomatic course of this infection and the ways in which this turns malignant. We hope that a better understanding of these processes will show us how they can be controlled.
PI: Wortis, Henry
Title: Tufts Post-Baccalaureate Research Education Program
Abstract: This is a program designed to increase the number of post-baccalaureates from currently under-represented groups who become leading biomedical research scientists. It is based on the idea that an enriched hands-on experience in research is the best preparation for PhD training. The training typically consists of a one year academic research laboratory apprenticeship pursuing a hypothesis-driven research project. This core experience is complemented with interactive training to increase skills in scientific writing, oral presentation and the critical reading and interpretation of scientific literature. Trainees will be offered additional elective didactic training appropriate to their research project. Collegiality and cooperation will be fostered. The Sackler School of Graduate Biomedical Sciences and Tufts University School of Medicine propose a training plan that will be built on the foundation of well-funded and highly regarded research laboratories; highly successful experiences in PhD training in biomedical research and several successful training programs that target underrepresented groups. Assessment of the trainees' understanding of their own research, their bench skills, writing skills, presentation technique and leadership skills will be conducted regularly. Evaluation instruments are in place that will provide measurements for improving the training experience. The ultimate success of this PREP training program will be measured by outcomes: the aim is to have more than 80% of entering trainees go on to obtain an MD/PhD or PhD from a research-intense school.
PI: Wu, Dayong
Title: Effect of the White Button Mushroom Supplementation on Resistance to Salmonella Infection and Immune Response in an Animal Model
Abstract: Food borne pathogen-caused infection is a global health concern. As immune function decreases with age, an efficient immune response to the pathogens, including both innate and adaptive immunity, as well as the cross-talk bridging both arms of immunity, is important in both preventing infection and reducing the severity and duration of infection. Components in commonly consumed mushrooms (including white button mushrooms), such as beta-glucans, and the extracts of certain mushrooms, have been shown to enhance innate immunity and the process bridging innate to adaptive immunity. Therefore, we hypothesize that consumption of white button mushrooms may positively impact the host’s resistance to local and systemic infection caused by food borne pathogens through enhancing the appropriate immune functions. We propose to test this hypothesis using an animal model of salmonella infection already developed in our laboratory. Mice will be fed two doses of mushrooms or control diet for 2-months and then subjected to use two infection models for localized and systemic infections, respectively, to determine the effect of mushroom consumption on the clearance of bacteria as well as selected markers for the host’s immune response. Two studies are designed to use localized and systemic infection model respectively as described below.
Study One: This study is designed to determine the impact of mushroom supplementation on acute enterocolitis caused by Salmonella (S. typhimurium) using streptomycin pretreated C57BL/6 mouse model 3. It will provide evidence for whether mushroom supplementation affects host resistance in early stages of S. typhimurium infection as indicated by bacterial loading in the gut, Peyer’ s patches (PP), and mesenteric lymph nodes (MLN) as well as the associated pathological changes. C57B/6L (3-mo) mice will be fed diets containing 0 (control), 2%, or 10% mushroom for 2 months. After the feeding period, mice will be pretreated with streptomycin 24 hours prior to inoculation with S. typhimurium and killed on days 0, 1, 4 post-infection (15 mice/time point). One group of mice fed the control diet and without streptomycin treatment will serve as control to determine any confounding effect that may be introduced by using the antibiotic. Fifteen mice will be needed for each diet group and time point based on previous studies. Thus, a total of 180 mice (15 mice/diet x 4 diet groups x 3 time points) will be needed for this study. The severity of infection will be assessed by bacterial colonization in the gut and other gut-associated lymphoid tissues, and pathological changes in cecum. Gene expression and production of the cytokines involved in the innate immune and inflammatory responses (IL-6, IL-I-beta, TNF-α, IL-12, IFN-g) and chemokines (IL-8, MCP-1) will be determined by RT-PCR and ELISA, immune cell phenotype in PP and MLN by FACS, and the neutrophil function by myeloperoxidase (MPO) assay.
Study Two: This study is designed to determine the effect of mushroom supplementation on systemic infection caused by S. typhimurium using a systemic infectious typhoid mouse model without pretreatment with streptomycin. Animal numbers in each group and feeding regime will be the same as those in Study One. Mice without streptomycin pretreatment will be infected with S. typhimurium and killed on day 0, 4, 7, 14 day post-infection. Selected indicators of the adaptive immune response will be determined, e.g., lymphocytes isolated from MLN and spleen will be re-challenged with the specific pathogen antigen (heat-killed Salmonella) in vitro and their proliferation and capacity to produce cytokines will be analyzed. The cytokines to be measured include inflammatory cytokines (TNF-α, IL-6, IL-I-beta, and T cell cytokines (IL-α, IFN-g, IL-l2). These cytokincs are involved in inducing adaptive immune response to eradicate Salmonella. A total of 180 mice (15 mice/diet x 3 diet groups x 4 time points) will be needed for this study. In addition to the mice needed for the two studies, 60 extra mice will be needed to conduct preliminary tests to confirm the optimal doses and time course established from the previous studies before the proposed study is conducted.
Successful completion of the proposed study will enable the identification of a new health benefit of consuming mushrooms as a functional food. Positive outcome would open a potential application of consumption of mushrooms as a nutritional approach to complement the preventive and therapeutic strategies in fighting food borne pathogen-induced infection.
PI: Wu, Dayong
Title: Green Tea EGCG and T-Cell Function in Autoimmune Inflammation
Abstract: This proposed study is directly related to the long term goals of the Nutritional Immunology CRIS “Nutrition, Aging, Immune and Inflammatory Responses in Health and Diseases”, and is in line with objective #4 of our previous CRIS, “to determine the effect and mechanisms of food components and their interaction with age on immune function and infectious diseases”. The studies proposed do not exactly match the objectives of our newly certified CRIS (April 2009-2014). However, if this proposed study is funded, Dr. Simin Meydani, will update the CRIS objectives by adding a new objective: “Determine the effect and mechanisms of food components and their interaction with age on immune function and infectious diseases”, to reflect the studies proposed in this LOI.
Autoimmune inflammatory diseases, such as arthritis, inflammatory bowel diseases, and multiple sclerosis, are common disabling diseases that affect millions of people. T cells, in particular CD4+ T (helper) cells, play a key role in mediating many aspects of autoimmune inflammation. Green tea and its active ingredient, epigallocatechin-3-gallate (EGCG) have been suggested to improve symptoms and reduce the pathological changes associated with autoimmune inflammatory diseases in animal models. However, the mechanisms for this effect of EGCG are not well understood.
New studies in the past two years have significantly reshaped our understanding of the roles of different lineages of T helper cells in development of autoimmune diseases. While, until recently, Th1 response was thought to be the main mechanism in development of autoimmune disease, recent evidence has pointed to a critical role for the newly discovered Th17 cells in these diseases. The relative contribution of each subset of Th cells is still a matter of debate, however, it is now widely accepted that Th1 and Th 17 cells promote while regulatory T cells (Treg) mitigate development of antoimmune diseases. Our preliminary results showed that EGCG supplementation at the physiologically relevant concentrations, 2.5 to 10 uM, dose-dependently inhibited CD4+ T cell proliferation and cell cycle progression. We also found that EGCG inhibited Th1 and Th17 response of CD4+ T cells and induced expansion of Treg cells. Moreover, EGCG treated antigen presenting cells (APC) had reduced ability to stimulate antigen specific T cell proliferation. Based on these results we hypothesize that EGCG ameliorates autoimmune inflammatory diseases by modulating expansion and function of different subsets of T helper cells. To test this hypothesis, we will utilize the experimental autoimmune encephalomyelitis (EAE), an animal model for human multiple sclerosis in the specific aims provided below. EAE is a well characterized and thus widely used autoimmune animal model.
Aim 1: To determine the effect of dietary EGCG supplementation on EAE symptoms, pathology, and T cell and APC functions. We will feed two sets of C57BL mice a diet containing 0, 1.5, 3, or 6 g/kg of EGCG for 1 mo and then induce EAE by immunizing mice with myelin antigen. The symptoms will be evulated in one set of mice daily from day 0 to 30 and the animals killed at day 30 to collect brain and spinal cord for pathology. Another set of mice will be killed at day 10 after immunization to determine T cell proliferation and cytokine production after re-stimulated with the specific myelin antigen. Dendritic cells will be isolated from the draining lymph nodes to measure the expression of APC maturation markers and the antigen presenting function.
Aim 2: To determine the cellular and molecular mechanisms for EGCG-induced suppression of autoreactive T cell function. We will determine effect of EGCG on CD4+ T cell differentiation into Th 1, Th17, and Treg cells under appropriate polarization conditions, using IFN-g, IL-l7, and Foxp3 as the marker of each cell type, respectively. We will further determine the signaling pathways involved in the EGCG-induced changes in Th subset polarization.
Successful completion of the proposed studies will help determine preventive potential of green tea/EGCG in improving autoimmune inflammatory disorders. It will also allow an insight into the mechanisms of EGCG’s action, which will help further define the role of EGCG in modulating immune cell function in general. Together, the information obtained can be incorporated into developing dietary recommendation for prevention of autoimmune and inflammatory disorders
PI: Xu, Jun
Title: X-linked Genes, Histone Modifications and Behavior
Abstract: Jarid1c is an X-linked gene expressed more highly in females than in males, suggesting that it may contribute to the effect of sex chromosome complement (XX vs. XY) on behaviors such as aggression. Jarid1c encodes a histone demethylase that represses expression of specific genes. The JARID1C enzyme plays an important role in brain development and function; patients with JARID1C mutations suffer from symptoms including mental retardation, elevated aggression, defective social interaction, language development, and seizure. We hypothesize that Jarid1c is implicated in the sex chromosome effect on aggression.
Three related aims are proposed to test this hypothesis. Aim 1 will identify genes that are differentially expressed between XX and XY mice, and genes that show different chromatin modifications in specific brain regions. These genes are candidates for causing the behavioral differences between XX and XY mice. Aim 2 will examine the effects of aggression on gene expression and chromatin modifications. Aim 3 will test the effects of knocking down Jarid1c expression in specific brain regions on behavior, gene expression, and chromatin modifications. The feasibility of siRNA treatment has been confirmed in our pilot study which detected changes in behaviors and gene expression. Hippocampal Jarid1c k/d mice were deficient in their performance in the object recognition test; furthermore, prefrontal cortical Jarid1c k/d mice were more anxious than controls in the open field test. Gene expression arrays indicated genes involved in GABAergic neurotransmission were up-regulated in both hippocampal and cortical Jarid1c k/d mice, suggesting GABA receptor signaling as a candidate mediating Jarid1c’s effects on brain and behavior.
A better understanding of Jarid1c’s involvement in brain function and behavior will provide insights in novel molecular mechanisms underlying brain sexual differentiation, aggression regulation, as well as behaviors and emotions (e.g. episodic memory and anxiety).
PI: Yang, Yongjie
Title: Role of Astroglial Exocytosis in the Pathogenesis of ALS
Specific Aim 1: Investigation of the exocytosis mediated astroglial gain-of-toxicity mechanisms in SODIG93A model in vitro.
Toxicity of exocytosis-inhibited SOD1G93A astrocytes to cultured motor neurons. Two approaches will be employed to inhibit the astrocyte exocytosis in SOD1G93A astrocytes: 1) Overexpression of dnSNARE in SOD1G93A astrocytes through transduction of lentivirus that overexpress dnSNARE; 2) Breeding of the GFAP-tTA, TetO-dnSNARE, and SOD1 (G93A/WT) mice. This breeding will generate a range of experimental and control mice. Primary spinal cord astrocytes will be prepared from A1, A5, B1, B5, and A8/B8 to form the astrocyte monolayer. Immunopanning procedures will be employed to remove potential microglia contamination. Embryonic stem (ES) cell-derived motor neurons will be purified and plated on the top of the astrocyte monolayer with different genotypes for 7d, 14d, and 21d. To facilitate identification of motor neurons in cultures, a HB9-eGFP mouse line (Jackson laboratory) that selectively expresses. eGFP reporter driven by the HB9 promoter in spinal cord motor neurons will be employed. ES cell-derived motor neurons will be purified by fluorescence activated cell sorting (FACS) of the embryold bodies (EB) that are formed from the spinal cord ES cells from HB9-eGFP mice. Following co-culturing with astrocyte monolayer, the number of motor neurons identified by the expression of eGFP reporter in co-cultures will be counted and compared.
Characterization and Identification of the secretory toxic factors from SOD1G93A astrocyte in vitro. Exosomes and exosome-free supernatant will be prepared through serial centrifugation steps from astrocyte conditioned medium (serum-free) collected from spinal cord astrocyte cultures. These spinal cord astrocyte cultures will be derived from mice with genotypes A1, A5, B1, B5, and A8/B8, respectively. Alternatively, astrocyte conditioned medium will be collected from dnSNARE expressing SOD1G93A astrocyte cultures. Prepared exosomes and exosome-free supernatant will be separately added into the motor neuron cultures derived from spinal cord ES cells from HB9-eGFP mice for 7d or 14d. Following treatment, the number of motor neurons will be counted by the expression of eGFP reporter and compared. These results will indicate which component of astrocyte conditioned medium is toxic to motor neurons and whether the selective inhibition of astrocyte exocytosis is sufficient to abolish the toxicity in SOD1G93A astrocytes. To identify the secretory toxic factors, exosomes/supernatant will be concentrated by using Amicon Ultra size filter (Millipore) and resolved on 2-D gels and silver-stained. Comparison of the 2-D gel images from different genotypic samples will identify particular candidate proteins and subsequent analysis by LCIMS/MS.
Because the sole expression of dnSNARE or tTA is not sufficient to induce the expression of dnSNARE in astrocytes, and no abnormality has been found in dnSNARE or tTA expressing mice (9-10), offspring with A3/A4, A6/A7 (from SOD1G93A breeding) or B3/B4, B6/B7 (from SOD1WT breeding) genotypes can be treated as control mice. Astrocytes from these genotypes will be used in some experiments when the offspring (genotypes AS, B5, and A8/B8) are not sufficiently produced. In addition, breeding of the GFAP-tTA, TetO-dnSNARE, and SOD1 (G93A/WT) mice will also generate offspring that allow investigation of the benefit of astrocyte exocytosis (A2, B2) to cultured motor neurons.
Specific Aim 2: Determination of the effect of astrocyte exocytosis inhibition on the disease phenotype in SODIG93A mice in vivo
- Analysis of disease phenotype in astrocyte exocytosis-inhibited SOD1G93AW/T mice in vivo. Specific binding of tTA to the TetO site is modulated by Dox, allowing temporal control of dnSNARE expression in astrocytes and investigation of the role of astrocyte exocytosis in pathogenesis of motor neuron degeneration at different disease stages. To suppress the expression of dnSNARE during prenatal and early postnatal development (until P30), Dox will be supplemented into food once the breeding is set up and will be removed at different stages: (1) Role of astrocyte exocytosis in modifying disease onset. Dox will be removed at P30 after early postnatal development. Both hindlimb and forelimb grip strength will be monitored weekly until disease onset, then daily till end-stage. Hindlimb and forelimb disease onsets will be determined individually for each mouse by a 20% loss in hindlimb or forelimb grip strength relative to each mouse’s own baseline grip strength level. All experimental mice with SDO1G93A genotype will be kept until mice reach end-stage (completely paralyzed); (2) Role of astrocyte exocytosis in modifying disease progression. Dox will be removed 2-3 weeks before the disease onset, which will be determined as described above. This will allow the induction of dnSNARE in astrocytes at the time of disease onset. Disease duration will be determined between hindlimb disease onset (20% loss of grip strength) and disease end-stage (completely paralyzed). Expression of dnSNARE in astrocytes after Dox removal will be confirmed by immunostaining of dnSNARE and GFAP, and the co-expressed eGFP reporter. End-stage mice with SOD1G93A genotype and control mice will be sacrificed and cryo-protected. Thin coronal sections (20μm) will be prepared and immunostaining of SMI-32 will be performed, and the number of motor neurons will be counted.
PI: Yee, Amy
Title: Epilepsy and the Wnt Signaling Pathways
Aim 1: Generate time course of Wnt activation following a prolonged seizure, status epilepticus (SE). (Months 1-6).
Task 1A: Develop time course for Wnt activation following kainate-induced SE (Months 1-3). Optimization of antibodies, Western blotting.
Task 1B: To confirm if Wnt activation is model dependent vs. status epilepticus dependent, develop time course for Wnt activation following pilocarpine-induced SE (Months 1-6).
Task 1C: Utilizing results from a, b, use real time PCR to determine Wnt target gene expression following SE. (Months 3-6).
Aim 2: To identify the role of Wnt signaling in two potential mechanisms of early epileptogenesis following SE. (Months 6-24).
Obtain in-vitro slices from control animals. Examine the role of bath-applied Wnt activators and inhibitors on CA3 burst frequency, a measure of seizure propensity. (Months 6-12).
b. Obtain in-vitro slices from SE animals. Examine the role of bath-applied Wnt activators and inhibitors on CA3 burst frequency, a measure of seizure. (Months 6-12).
Task 2B: To identify if changes in Wnt signaling alters synaptic plasticity in CA1 long-term potentiation (LTP), an in-vitro model of learning and memory in control vs. animals which have undergone SE.
Obtain in-vitro slices from control animals. Examine the role of bath-applied Wnt activators and inhibitors on CA1 long-term potentiation, a measure of change in synaptic plasticity (Months 12-18).
Obtain in-vitro slices from SE animals. Examine the role of bath-applied Wnt activators and inhibitors on CA1 long-term potentiation, a measure of change in synaptic plasticity (Months 12-18).
Aim 3: Animal Clinical Trail: Determine if Wnt inhibition modulates cell death and delays the onset to epilepsy in a whole animal model of SE (Months 24-36).
- Task 3A: Develop a delivery method for Wnt inhibitor, F8CDFr in CNS.
- Task 3B: Develop antibody assay for measurement of F8CDFr in serum.
- Task 3C: Develop time course for administration of F8CDFr into animals following SE.
- Task 3D: Optimize dosing regimen for F8CDFr into animals following SE.
- Task 3E: Examine if F8CDFr application alters cell death 7 days after SE
- Task 3F: Examine if F8CDFr application alters onset to epilepsy using Neurophysiology Core.
PI: Yelick, Pamela Crotty
Title: Dental Stem Cells and Tooth Tissue Engineering
Abstract: The ultimate goal of this research project is to develop novel biologically based, dental and craniofacial skeletal repair and regeneration therapies in humans. We hypothesize that improved knowledge of dental stem cell (DSC) properties, and characteristics, combined with improved knowledge of tissue engineering strategies to reliably generate mineralized dental and craniofacial tissues of predictable size and shape, will result in the development of novel and effective, clinically relevant therapies for humans. To address this hypothesis, four specific aims are proposed, all of which will exclusively use human DSCs harvested from extracted human teeth. First, we will characterize the properties of individually harvested human dental tissues, to establish the normal range of DSC properties, to correlate DSC properties with the age and overall health of the donor, and to correlate these properties with the ability to successfully use harvested dental stem cells for dental tissue regeneration therapies. Next, we will test four different scaffold materials, carefully chosen for their defined physical properties, for their ability to generate bioengineered human tooth crowns of predictable size and shape. Third, we will expand upon our results demonstrating that silk scaffolds can be used to generate osteodentin of predicted size and shape, to bioengineer full sized, functional human tooth root equivalents that can support a synthetic or bioengineered tooth crown. Silk scaffold fabrication methods, porosity, degradation rates, and added growth factor peptides, will be systematically evaluated. As recommended in prior review, Aims 2 and 3 will be performed in both subcutaneous and minipig mandibular implant models. Finally, in Aim 4, we will combine our tooth crown, root, and craniofacial skeletal regenerative strategies, again using the Yucatan mini pig mandibular implant model, to generate successive 1st, 2nd and 3rd generation replacement teeth. For each of the proposed aims, detailed developmental analyses of bioengineered dental implant tissues will be performed in order to better understand, and devise strategies to improve dental tissue regeneration.
Our novel approach to tooth repair and regeneration, using human DSCs, and state of the art scaffold fabrication methods, combined with our extensive expertise in Developmental Biology and Tissue Engineering, have the potential to provide new and improved, biologically based repair and regeneration strategies, using autologous tissues. The successful accomplishment of the proposed studies would dramatically alter the field of dentistry as it currently exists, extending clinically relevant dental repair therapies to include biologically based dental materials with properties closely matching those of naturally formed dental tissues.
PI: Yi, Hyunmin
Title: Collaborative Research: Hierarchically Assembled Viral-Synthetic Hybrid Microentities
Abstract: The ability to simultaneously assay for multiple target molecules in a complex mixture in a high-throughput capacity is an unmet challenge. Tobacco mosaic viruses (TMV) offer an attractive nanotemplate that can be readily functionalized with probes to detect targets in solution. To create a multiplexed sensing platform, viruses bearing different probes must be somehow assembled and encoded so that they can be mixed together and readily identified. Planar arrays can be created (spatial encoding), but in general these approaches are expensive, not easily customized and slow. Particles based arrays take advantage of better solution kinetics, are easily customized and have the potential to be rapidly scanned using flow-based systems. Viral “carpets” on particles would provide high probe densities and add the virus surface functionality. Thus, a critical challenge exists to develop methods for spatially patterning functionalized viruses on three-dimensional, encoded microparticle carriers.
The research goal of the proposed project is to develop and understand high throughput manufacturing of biomaterial-based microscale entities with their own biosensing capabilities assembled on discrete regions of each entity at high capacity. Our proposed approach is based on the hierarchical, high-throughput assembly of (bio)functionalized viral nanotemplates with biomaterial PEG-based microparticles via nucleic acid hybridization. This enables seamless integration of nanobio and synthetic elements with unprecedented density, selectivity and orientational control as well as unlimited programmability. We propose four aims to achieve this goal;
- Aim #1 Create encoded PEG hydrogel particles with TMV nanotemplates hierarchically assembled onto capture DNA region, and establish physical characteristics of the platform technology via fluorescence, confocal microscopy, AFM and FE-SEM.
- Aim #2 Investigate one-pot patterned assembly of multiple TMV species onto 5-plexed and complex geometry microparticles to create highly multiplexed, multifunctional hybrid materials.
- Aim #3 Study antibody-conjugated TMV assembly onto microparticles for ultrahigh capacity biosensing and optimize continuous microfluidic scanning.
- Aim #4 Develop benchmarking assays against commercially available technologies to establish the merits and sensing performance.
The outcome of the proposed research will be new multiplexed sensing platforms which integrate hydrogel barcoded particles with the biofunctionality of TMV viruses via highly programmable biochemical interactions to achieve features and performances beyond what is possible with individual technologies or mere addition of the two. In a broader context, the research will pave the way for moving beyond 2-D patterning of viruses on surfaces and move this into 3-D particle environments with complex functionalities capable of multiplexed sensing, catalytic activity and facile separation.
PI: Zeng, Li
Title: Muscle Cell-Enhanced Cartilage Tissue Engineering
Abstract: Arthritis is a leading cause of disability in the United States. Despite its prevalence in our ever-aging society, effective treatment options for arthritis are still limited. Arthritis is caused by the destruction of joint cartilage, which is accompanied by inflammation and pain. Cartilage tissue engineering offers a promising solution to regenerate cartilage and restore tissue function. However, the presence of pro-inflammatory cytokines at the host site inevitably leads to matrix degradation, causing the engineered cartilage to be unstable. Therefore, for this technology to be applied clinically, there is a critical need to engineer stable cartilage that is resistant to pro-inflammatory cytokine-induced degradation.
Our long-term goal is to gain critical knowledge of cartilage regulation and enhance the technology of cartilage tissue engineering for clinical applications. We are developing a novel strategy that integrates concepts and approaches from developmental biology with those of tissue engineering. During embryogenesis, muscle is one of the tissues that develop alongside the presumptive cartilage tissue.
Our extensive preliminary studies indicate a role of muscle cells in regulating cartilage homeostasis and inflammatory stimuli. Our central hypothesis is that muscle cells and optimal scaffold selection can be used to enhance the stability of engineered cartilage by enhancing cartilage matrix production and the resistance to pro-inflammatory cytokines. We plan to test this hypothesis by using primary human articular chondrocytes and mesenchymal stem cells seeded in 3D silk scaffolds.
We plan to:
- investigate the role of muscle cells in regulating cartilage matrix production,
- investigate the role of muscle cells in regulating the response to pro-inflammatory cytokines, and
- investigate the effect of scaffold material on muscle cell regulation of cartilage matrix production and the response to pro-inflammatory cytokines.
Our research team consists of experts in the fields of developmental biology, tissue engineering, immunology and orthopaedics. We believe that our synergistic efforts and interdisciplinary approach will result in deeper understanding of the regulation of cartilage homeostasis and the response to proinflammatory stimuli, providing the fundamental knowledge for modeling and treating arthritis. Thus, our study aspires to meet the critical need of improving tissue engineering technology, and may lead to the development of a novel strategy to engineer stable cartilage for clinical applications.
PI: Zeng, Li
Title: Novel Strategies in Cartilage Tissue Engineering: Enhancing Cartilage Stability Using Muscle-Derived Factors and Scaffold Section
Abstract: Cartilage is a tissue that has a poor capacity for self-repair. Current tissue engineering strategies aim to replace damaged tissue by seeding cartilage cells or chondrocytes into three-dimensional scaffolds. Within the scaffolds, these chondrocytes proliferate and secrete extracellular matrix, leading to the formation of regenerated cartilage. This regenerated cartilage will then be transplanted into the host, with the goal of assuming the function of native cartilage. The biomechanical strength of engineered cartilage is directly correlated to the amount of extracellular matrix produced by cartilage cells. Yet, even regenerated cartilage with good mechanical properties may be damaged by pro-inflammatory cytokines present in the host site, compromising the stability of the regenerated cartilage. Thus, there is a critical need for engineered cartilage to be resistant to pro-inflammatory cytokine-induced degradation.
PI: Zoukhri, Driss
Title: Effects of ENaC Blockers on Tear Production
Abstract: To determine whether ENaC blockers increase (preserve) the tear fluid on the ocular surface and whether ENaC blockers improve ocular hydration and decrease corneal staining in
an in IL-I induced dry eye model in BALB/c mice.