Research Grants

Molecular Genetics of Olfaction in Drosophila

Robert R. Anholt, Trudy F. MacKay, Taufika Williams

National Institutes of Health (NIH)

$4,622,227

04/01/99 - 05/31/17

Olfactory deficits in people can compromise quality of life and, consequently, adversely affect nutrition, especially in the elderly. In addition, olfactory decline can be an early indication of neurodegenerative disease. Furthermore, diseases transmitted by insects rely on olfactory cues for host finding. Studies on the genetic underpinnings of olfactory behavior require a model organism that has a well characterized olfactory system and is amenable to genetic manipulation. The fruit fly, Drosophila melanogaster, fulfills these criteria, since it has one of the best characterized olfactory systems and large numbers of genetically identical individuals can be grown under controlled environmental conditions rapidly, economically, and without regulatory restrictions, allowing exquisite control over both the genetic background and the environment. In this application we propose to take advantage of a recently developed genetic resource that enables genome-wide analysis in Drosophila to associate DNA sequence variants with natural variation in olfactory behavior. We propose to use innovative genetic strategies to identify causal variants and obtain mechanistic insights in the genetic basis of natural variation for olfactory behavior. Results from these experiments will not only set a new standard for systems genetics studies in Drosophila, but also advance our general understanding of the genetic basis of natural variation in complex traits, and uncover principles that will be broadly applicable to studies of complex traits, including studies on the genetics of human diseases.

Molecular Genetics of Olfaction in Drosophila

Robert R. Anholt, Trudy F. MacKay

National Institute of General Medical Sciences (NIGMS)

$1,779,351

08/01/13 - 05/31/17

Olfactory deficits can compromise quality of life and, consequently, adversely affect nutrition, especially in the elderly, and olfactory decline can be an early indication of neurodegenerative disease. Furthermore, diseases transmitted by insects rely on olfactory cues for host finding. We propose to use innovative genetic strategies using the powerful Drosophila genetic model to identify causal variants and obtain mechanistic insights in the genetic basis of natural variation for olfactory behavior. Results from these experiments will also advance our general understanding of the genetic basis of natural variation in complex traits, and uncover principles that will be broadly applicable to studies of complex traits, including studies on the genetics of human diseases.

Neurogenetics of Lead Toxicity in Drosophila

Robert R. Anholt, Trudy F. MacKay

National Institute of Environmental Health Sciences (NIEHS)

$414,352

08/29/13 - 07/31/16

Heavy metal toxicity is a world-wide health problem, which is of concern especially due to the adverse effects of low concentrations on cognitive development in children. Whereas neurotoxic mechanisms of lead have been well studied, information regarding genetic susceptibility to the effects of lead exposure in human populations and studies aimed at identifying genetic variants that may exacerbate or ameliorate exposure risk are lacking. This proposal will use a powerful genetic model to identify evolutionary conserved genes that harbor variants that influence susceptibility to lead exposure and provide insights in the genetic factors that may exacerbate or ameliorate exposure to lead neurotoxicity.

Epigenetics, Environmental Exposure, and Reproduction in the Collaborative Cross

David Aylor

National Institutes of Health (NIH)

$788,517

06/17/14 - 05/31/17

Diethylstilbestrol (DES) is a synthetic estrogen that causes cancer and reproductive defects in humans and mice. This research will identify genetic variants that control differential susceptibility to DES exposure. To achieve these goals we will bring to bear the power of the newest mouse resources. The Collaborative Cross (CC) is a panel of recombinant inbred mouse strains that was designed to have an extremely high level of genetic diversity and ideal properties for genetic quantitative trait locus (QTL) mapping. This enables us to recreate identical populations of DES-exposed and control mice, and identify gene-byenvironment (G×E) interactions. Infertility, developmental reproductive defects, and uterine cancer have all been linked to DES exposure in mice, and individual mouse strains vary in their susceptibility. This research will characterize 60 mouse strains and identify QTL linked to reproductive defects and cancer. The proposed systems genetics approach will not only link genetic variants to those reproductive traits, but also characterize genetic mechanisms at the molecular level. Estrogenic chemicals like DES cause epigenetic modifications that change gene expression patterns. Next-generation sequencing technology enables us to measure DNA methylation and mRNA expression in each individual. Differential gene expression or differentially methylated regions will be associated with genetic variants in the manner of a traditional trait (eQTL mapping). In addition to shedding light on molecular mechanisms, eQTL can help refine the location of disease QTL. These rich data will enable us to model genetic effects and G×E across three levels of genome function, and the resulting model will provide a foundation for future research relating genetic variation, epigenetic regulation, gene expression, reproduction, and environment exposure. This research will produce new biological knowledge of the genetic networks underlying the reproductive system, DES function at the molecular level, and genes underlying DES sensitivity.

New Metrics and Multivariate Processing Methods to Monitor System Suitability in LC MS/MS Proteomic Experiments

Michael Bereman

American Society for Mass Spectrometry

$35,000

06/01/15 - 12/31/16

With advances in LC MS/MS technologies combined with an ever expanding user-base and the continued goals of biomarker discovery, clinical applications of established biomarkers, and integrating large multi-omic datasets (i.e., “big data”), there remains an urgent need for robust tools to timely assess instrument performance (i.e., system suitability) in proteomic experiments. Recently our laboratory developed a tool termed Statistical Process Control in Proteomics that implemented two main charts used in statistical process control – namely the control chart and Pareto Analysis to monitor both the intra- and inter-experiment variability in a proteomics workflow. It has been implemented as an external tool in the freely available, vendor neutral, and widely popular Skyline proteomics software ) and has been downloaded 700 times as of October 2014. In this proposal, we will develop and implement new sensitive objective metrics combined with classical SPC techniques into the SProCoP algorithm to be used by the global proteomics community to aid in the identification of special cause variation in LC MS/MS experiments.

Towards an Unbiased Multiplex Exposure Assay – Coupling Ultra-High Pressure Liquid Chromatography and Data Independent Acquisition (DIA) Mass Spectrometry

Michael Bereman

NCSU Center for Human Health and the Environment

$25,000

01/13/14 - 12/31/14

To date, studies of peptide sequence site specific heterogeneity have been extremely limited by current analytical technologies. Studies often have focused on the presence, absence, or abundance of a single in-vivo enzymatic modification (e.g., phosphorylation) or chemical modification (e.g., glycation). However, as the field of proteomics continues to advance, it’s becoming critically important to develop methods to assess all modifications at a particular protein motif as these modifications can participate/alter protein signaling, function, structure, and stability. Previous methods have used a narrow list of potential modifications and searched for these modified peptides using a traditional database workflow. There are several fundamental limitations to this approach but most noteworthy is its targeted nature as all peptide modified identifications are limited to a small predetermined “list”. Herein, a method is proposed based on ultra-high pressure liquid chromatography coupled to data independent acquisition mass spectrometry to ask these questions in a different manner. First, the total number of modified peptide forms is determined by querying the data for characteristic fragment ions. Secondly, the mass of the modification is elucidated from the MS1 precursor information. This method provides an unbiased assessment of site specific heterogeneity and will fuel discovery of novel modifications. Although this method is directly applicable to various fields, it will be used to measure mixtures of protein adducts within the emerging field of exposomics.

The Selfish Genetic Element Medea as a Mechanism for Suppressing Arthropod-Vectored Agricultural Diseases

David A. Bidelspach, Sarah A Cash

US Dept. of Agriculture (USDA) - National Institute of Food and Agriculture

$38,030

09/01/13 - 08/31/14

Continued human population growth is putting further strain on the food supply, making food security a greater priority than ever before. By minimizing losses from arthropod-vectored agricultural diseases, we can increase the efficiency of food production without additional stress on natural resources. One exciting possible avenue for suppressing these diseases involves replacing pest populations with populations of individuals which carry a genetic construct that inhibits their ability to transmit disease. A unique genetic element (Medea) exists in the red flour beetle, Tribolium castaneum, which triggers lethality in offspring lacking a copy of the Medea allele. Through eliminating non-Medea offspring, the element is capable of driving its own spread at the expense of wild-type alleles. By coupling Medea with an anti-pathogen construct, the element could drive the desired anti-pathogen genes into populations, resulting in individuals incapable of transmitting diseases. In contrast to the rapid spread of Medea predicted by mathematical models, however, the natural distributions of Medea elements are puzzling - many populations have very low frequencies of Medea, while others lack the element altogether. Understanding how Medea spreads in natural populations will give us valuable insights for refining potential pest management strategies. The proposed research will focus on expanding the sampling and genotyping of Medea elements in the United States, and examining what factors are responsible for the current Medea distributions by testing the following hypotheses: (1) Medea distribution and Tribolium castaneum population structure are reflective of one another, and (2) the genetic backgrounds of wild Tribolium populations influence, and may even prevent, the spread of Medea elements.

Expanding Learner-Centered Approaches Using A Blended Learning Model: Proposal To Continue Redesign of Introductory Biology Courses

Betty L. Black, Miriam G Ferzli

UNC - General Administration

$48,586

10/30/14 - 10/29/15

We propose to continue our redesign of BIO 183 Introductory Biology: Cellular, Molecular and Developmental Biology and expand course redesign to BIO 181 Ecology, Evolution, and Biodiversity by employing a blended learning approach. Approximately 2,800 students per year are enrolled in these courses: 1,500 in BIO 181 and 1,300 in BIO 183. During the first year of this project, we collected potential active-learning exercises and focused on development of templates for the digital resources required to move toward a blended learning approach in Introductory Biology. These resources were tested on a small scale in two sections of BIO 183. For the second year, we propose to utilize our templates to create a large number of interactive learning objects (ILOs), expand use of these resources to most sections of BIO 181 and 183, and increase the amount of time devoted to active learning in the classroom of these sections. In the development of ILOs, we need to provide maximum flexibility so that instructors have a tool-box approach for maximum integration into already existing lessons and for embedding their own ideas of how to present the material. Providing faculty with the opportunity to develop these materials as a group will allow for everyone teaching these courses to feel ownership of the materials and process for implementation. Thus, our proposal provides technical support which participating faculty can leverage while designing ILOs and writing various types of embedded questions with feedback. We will use a comparison group design to measure the effectiveness student-centered blended learning approaches. This study will assess the impact of individual modules on student learning outcomes and overall conceptual understanding of the concepts being tested through active learning and supporting ILOs. Students using the learner-centered approaches with supporting ILOs will be compared to a section taught in a traditional format. Students in the study will also complete a pre- and post-test of conceptual knowledge. In addition, we will hold student focus groups at the end of the semester to determine affective variables associated with the usability and usefulness of this teaching strategy. To ensure summative evaluation of the project, we plan to enlist the help of University Planning data to track student achievement in subsequent courses that require competency of concepts learned in BIO 181 and 183. We will also track DFIW and course completion rates for BIO 181 and 183 each semester as new approaches are implemented for comparison to semesters prior to implementing course redesign. Successful implementation should decrease early withdrawal from these courses, increase course completion rates for both BIO 181 and 183, better prepare students for upper level courses in their major, and reduce time to degree.

Genetic Susceptibility to Nanoparticle-Induced Respiratory Disease

James C Bonner, Gregory N. Parsons

National Institutes of Health (NIH)

$1,684,077

09/01/12 - 05/31/17

This administrative supplement application to our existing NIEHS R01 grant entitled “Genetic susceptibility to nanoparticle-induced respiratory disease” (R01 ES020897) is requested in response to a unique opportunity to acquire mice from an ongoing National Toxicology Program inhalation study to address the adverse effects of multi-walled carbon nanotubes (MWCNTs). The proposed study described below is directly relevant to the aims of our current R01 grant and will also serve to complement the mission of NTP towards identifying health risks of inhaled MWCNTs. Specifically, our proposed work with eighty (80) mice from the NTP inhalation study will address susceptibility to allergen-induced lung disease and will thereby provide important information towards identifying individuals at risk for allergic lung disease who may have been pre-exposed to MWCNTs at occupationally relevant doses.

Improved Cost Effectiveness and Sustainability of Aquaculture in the Philippines and Indonesia

Russell J. Borski, Peter R. Ferket, Charles R. Stark, Harry V. Daniels, Luther Hatch

Oregon State University

$2,265,491

10/25/07 - 02/28/18

Bangladesh is the most densely populated country in the world, with 40% of the population living in poverty. Fifty percent of her women are undernourished and over 40% of children under 5 show moderate to severe stunting. Agriculture employs 48% of the Bangladesh population and due to its vast coastal and inland water resources, fisheries and aquaculture support a large majority of Bangladeshis in rural areas. Fish are also an important source of nutrition meeting 40-80% of total demand for protein. Accordingly, aquaculture in Bangladesh is a high food security priority needed to enhance nutrition for its people and to improve incomes of its poorest populations. This is particularly important in coastal plain areas of the Southwest where poverty is exceptionally high and frequent flooding, salt incursion, cyclones, seasonal droughts, and extreme temperature fluctuations linked, in part, to climatic change poses a threat to lives and livelihoods of fishers and farmers. Problems facing aquaculture households include limited production of nutrient rich aquafoods that could be used for farmer household consumption; excessive and costly feed inputs that reduce income potential; poor pond management, water quality, and underutilization of natural productivity that limit production potential, increase risks and enhance environmental degradation; and limited diversification of aquaculture products. We propose a series of studies to address some of these problems, including those centered in the high priority region of Southwest Bangladesh. We aim to develop technologies that enhance aquaculture production efficiency, intensification, sustainability and adaptive measures to climate change impacts in Bangladesh that are needed to improve household incomes and nutrition while reducing environmental impact of seafood farming.

Optimization of Ions For Low Salinity Culture of Southern Flounder and Black Sea Bass With Emphasis on Reduction of Sodium Chloride in Recirculating Aquaculture Systems.

Harry V. Daniels, Russell J. Borski

NCSU Sea Grant Program

$102,738

02/01/12 - 01/31/15

Five separate trials will be done over a two-year period on southern flounder (Paralichthys lethostigma) and black sea bass (Centropristis striata) to determine optimum ion ratios for survival and growth in low salinity waters. The objective of the first three trials is to establish the calcium and magnesium levels for southern flounder growout. The next two trials will be focused on the reformulation of the major ions in culture water after drastic reductions of sodium and chloride. The overall goal of the second series of trials is to reach Sodium Adsorption Ratio values (SAR) of 15 or lower to allow the effluents from recirculating systems to be applied to terrestrial crops. Chloride concentrations will need to be reduced to 165 mg/l or lower to avoid toxicity to plants when these effluents are land applied. This work will aid in establishing environmentally-sound production practices for marine fish or shrimp in low-sodium and low chloride recirculating systems. Furthermore, the information gained and the techniques developed from this work can be applied to other marine species suitable for culture in recirculating systems (Pacific white shrimp, summer flounder, baitfish, etc). Definitive insights on the mechanisms of osmoregulation at low sodium concentrations will be gained. We will have established the requirements of key ions essential to survival and growth of flounder and black sea bass in low saline waters along the inner coast. We anticipate that the information generated from the land application studies will lead to future selection of suitable terrestrial plants and break new ground on sustainable treatment methods of RAS effluents for aquaculture producers nationwide.

Optimizing All-Female Southern Flounder Culture in Low Saline Waters

Russell J. Borski, Harry V. Daniels

NCSU Sea Grant Program

$166,442

02/01/10 - 07/31/14

This proposal is to support a research stipend for a graduate student to perform research aimed to to optimize flounder culture through maximizing all female production and ability to grow fish in groundwater that lacks certain minerals. The southern flounder is a native fish in North Carolina's sounds and estuaries. The potential to culture these flounder in low salinity groundwater means that inland areas could be suitable for flounder farms. Our goal in this project is to leverage the considerable investment from Sea Grant and others into a project designed to jump start the first southern flounder farms in the U.S. and indeed the world. The flounder industry is at a critical point in its development. Significant advances in production have brought the promise of low salinity culture of flounder to fruition, but site-specific water quality issues and excess production of slower growing males are still constraining wider adoption of flounder culture in coastal areas. This proposal intends to optimize flounder culture through maximizing all female production and ability to grow fish in groundwater that lacks certain minerals.

Year-round Indoor Production of Hybrid Striped Bass Fingerlings

Harry V. Daniels, Russell J. Borski

NCSU Sea Grant Program

$93,608

05/01/11 - 12/31/14

We propose to investigate the use of an indoor tank system to extend the growing season for fingerling HSB as a means of gaining production time, eliminating the costly practice of annual draining to control yellow grubs, and reduce effluents by conserving culture water through water reuse technology. The HSB producers are at a critical point in their history. Current production practices are inefficient with respect to water use and are creating conflict with state regulatory agencies. However, new practices, such as RAS, have not yet been evaluated. New and innovative production practices need to be objectively evaluated before they can be responsibly recommended and adopted by the industry. This project will directly address those needs by providing real commercial-scale information and analysis to the producers, as well as new less-expensive feeds for better sustainability.

Coal Ash Constituents at the Base of Aquatic Food Webs: Processes Affecting Bioaccumulation and Trophic Transfer of Arsenic

David B. Buchwalter, Dean L. Hesterberg

NCSU Water Resources Research Institute

$60,000

03/01/15 - 02/28/16

The combustion of coal to meet demands for energy produces byproducts (“ash”) containing trace elements known to be toxic to fish, wildlife, and people. North Carolina ranks ninth among states in the annual generation of coal-derived ash. The recent spill of 39,000 tons of coal ash into the Dan River highlights the need to understand both the immediate and long-term impacts of trace element inputs at the base of aquatic food webs, which controls bioaccumulation in fish and higher organisms. The storage of another 102 million tons of ash in 33 basins throughout North Carolina is also of concern with regard to past and future releases of potentially toxic trace elements into ground and surface water resources. This project focuses on arsenic – one of the primary elements of concern related to coal ash. Arsenic is challenging because it exists in many forms that vary tremendously in their toxicity and bioavailability. As such, measurements of total arsenic in environmental media or biological tissues do not provide sufficient information to assess potential risk. Our research focuses on biological processes that play primary roles in determining the forms of arsenic present in the tissues of aquatic organisms. This project combines bioaccumulation and trophic transfer studies using radiotracers with detailed studies of arsenic speciation using X-ray Absorption Near Edge Structure (XANES) spectroscopy. This research spans different trophic levels (periphyton, invertebrates and fish) to explicitly assist in the interpretation of field monitoring efforts of the Dan River coal ash spill site.

Collaborative Research: Integration of Physiological, Life-history, and Macro-ecological Approaches for Understanding Thermal Limitation in Aquatic Insects: Implications for Freshwater

David B. Buchwalter

National Science Foundation (NSF)

$262,610

05/01/15 - 04/30/18

Freshwater ecosystems support a disproportionate percentage of earth’s biodiversity and are among the most threatened by human activities and global climate change in general and global warming in particular. Insects dominate fresh-water ecosystems in terms of biodiversity and ecological processes. Thus, many effects of global change and other anthropogenic activities on freshwater ecosystems will likely be manifested via thermal effects on aquatic insect performance (growth, survival, and reproduction). Temperature controls the developmental timing, species distributions, and community structure, but both the thermal limits of individual taxa and the underlying physiological mechanisms that determine those limits remain poorly understood. Thus we remain limited in our ability to predict how thermal variation shapes freshwater communities in space and time. The work proposed here takes major steps towards achieving our long-term goal of understanding how temperature drives the macro-ecology of aquatic insects by first linking rigorous, full life-cycle laboratory rearing experiments that quantify thermally-mediated life history outcomes/performance to physiological studies that define the mechanisms underlying those life history outcomes. We focus on thermal effects on energy allocation through integrated approaches (respirometry, calorimetry, molting frequency, gene expression) that ultimately tie physiological processes to fitness. Finally, we extend our physiological understanding to the macro level by determining if the physiological effects of temperature are consistent with the predictions of empirical species distribution models (ESDMs).

Experimental approaches to understanding temperature and flow responses of select North Carolina macroinvertebrates

David B. Buchwalter

NCSU Water Resources Research Institute

$60,000

06/01/13 - 12/31/14

The objectives of this proposed work are to understand the thermal and flow requirements of North Carolina aquatic macroinvertebrates. Macroinvertebrates are the primary faunal group used by the North Carolina Division of Water Quality in ecological monitoring programs because they predominate in aquatic ecosystems, and are responsive to environmental change. However, the scientific community lacks the ability to predict how communities will be altered due to changing thermal and flow regimes that result from human activities and predicted climate change. Both temperature and flow have the ability to directly affect respiration in water breathing species. This proposed research will use state-of-the-art respirometry techniques with carefully selected species to provide a major first step in giving resource managers the ability to predict which specific taxa are likely to be the most responsive to thermal and flow change.

SETAC/ICA Chris Lee Award for Metals Research: Monica Poteat

David B. Buchwalter

Society of Environmental Toxicology & Chemistry

$5,000

10/01/13 - 12/31/14

As a 3rd year doctoral student in Dr. David Buchwalter’s laboratory at North Carolina State University, Ms. Poteat has developed a dissertation project that encompasses mechanistic studies of metal bioaccumulation and interactions in aquatic insects as well as comparative/evolutionary aspects of metal bioaccumulation in aquatic organisms. Because aquatic insects form the backbone of biomonitoring programs in the United States and are the most commonly used bioindicators of environmental quality in freshwater ecosystems, it is important to understand their underlying physiologies. Ultimately, Ms. Poteat is interested in identifying how and why insect species are differentially responsive to metals. She is applying evolutionary physiology perspectives to better understand and predict how metal bioaccumulation parameters vary within and among different faunal groups. Her ultimate goal is to test whether phylogenetic methods can be used to enable prediction and extrapolation of metal bioaccumulation/toxicity in aquatic communities. Overall, her work has the potential to improve water quality criteria development, risk assessment and bioassessment by combining mechanistic and comparative methods to predict metal sensitivity parameters for data-poor species and decreasing the need for resource-intensive in vivo testing.

Water Quality Criteria Development for the Great Salt Lake **Prime Awarded as Project Title "Contract to Conduct Laboratory and in Situ on Brine Shrimp"**

David B. Buchwalter

University of Notre Dame

$208,151

10/01/14 - 06/30/16

Concerns about water quality in the Great Salt Lake (Utah) have prompted the need to evaluate the toxicity of priority pollutants to the dominant invertebrate fauna of the lake. In support of this goal, toxicity tests will be performed for methyl mercury, ammonia, copper, arsenic and lead in two species - brine shrimp and brine flies.

Epigenetic Mechanisms Linking the Environment to Metabolic Health

Michael Anthony Cowley

Oak Ridge Associated Universities

$5,000

06/01/15 - 05/31/16

The environment we are exposed to in early life influences our health as adults. Maternal diet, for example, can affect our risk of developing obesity or type 2 diabetes. Exposures in one generation can therefore affect the health of the next. Understanding the processes that underpin this relationship will be important for identifying therapeutic opportunities. Our work will help to unravel the biology behind these effects.

Mechanisms of Recurrent, Parallel Loss of X/Y Pairing and Recombination at Meiosis in Voles

Bethany Lynn Dumont

National Institutes of Health (NIH)

$164,395

08/15/14 - 07/31/16

I am proposing a training program that will prepare me for an independent research career in the field of evolutionary genomics. I received my PhD from the Laboratory of Genetics at the University of Wisconsin-Madison for my work on the genetic and evolutionary basis of mammalian species differences in global recombination rate. I then completed a 2-year postdoc in the Department of Genome Sciences at the University of Washington where I acquired considerable experience and proficiency in the computational analysis of large genomic datasets. Through the proposed training program, I will expand my experimental skill set to include genomic library construction, diverse cytogenetic methods, and functional analyses of candidate genes in mammalian model systems. I propose a 5-year research program composed of a 2-year phase of organized mentorship and training in the Initiative for Biological Complexity at North Carolina State University (NCSU), followed by a 3-year research phase as an independent investigator. The proposed training program will leverage cutting-edge sequencing technologies and innovative methods for chromosome visualization to elucidate the mechanisms of recurrent degeneration and loss of the pseudoautosomal region (PAR) in voles (genus Microtus). This research will draw on diverse research fields, including genomics, evolutionary biology, and cytogenetics. Dr. Trudy Mackay, William Neal Reynolds and Distinguished University Professor of Genetics, will act as the primary mentor for my scientific development. Dr. Mackay is a world-renowned for her work in evolutionary and quantitative genetics, and has an impressive record of successful students and postdocs as a testament to her commitment and enthusiasm for training young investigators. Dr. David Threadgill, Professor and head of the Department of Genetics at NCSU, will provide specific instruction in mouse functional genetics. Dr. Matthew Breen and Dr. Lisa McGraw, both at NCSU, will provide focused mentoring in cytogenetics and vole genetics, respectively. The remarkable breadth of faculty research interests and their overlap with the intellectual and experimental foci of the proposed training program make NCSU the optimal place for conducting the mentored phase of this research. The mammalian PAR promotes pairing, synapsis, and recombination between the heterogametic sex chromosomes at meiosis. These processes are integral to proper sex chromosome segregation. Failure to initiate pairing or recombination in the PAR can lead to spermatogenic failure, infertility, and sex chromosome aneuploidy, including Turner and Kleinfelter Syndromes in humans. Despite its important meiotic function, the PAR is structurally dynamic, rapidly evolving, and has even been completely lost in several exceptional mammalian species. Voles (genus Microtus) display particularly exciting trends in recent PAR evolution. Across the vole phylogeny, there is evidence for ≥3 parallel losses of X/Y chromosome pairing at meiosis, including multiple closely related species pairs with and without meiotic sex chromosome associations. I propose to study these recurrent evolutionary episodes to address 3 specific questions: 1. What structural and sequence-level changes precipitated the degeneration and loss of the PAR along these vole lineages? I propose cytogenetic assays and genome sequence comparisons between closely related vole species with and without meiotic X/Y pairing and recombination to answer this question. 2. How do unpaired, achiasmate sex chromosomes reliably segregate at meiosis? I propose cytological studies linking sex chromosome dynamics with the localization patterns of meiotic proteins to identify candidate achiasmate segregation genes. 3. Do candidate X/Y segregation genes identified in voles rescue sex chromosome segregation defects in organisms with obligate requirements for PAR pairing that harbor disruptive mutations across the region? I will carry out these functional tests using a mouse model with a high frequency of sex chromosome aneuploidy. The a

Career: Working with Student Scientists to Understand Global Patterns in the Present and Future Ecological Consequences of Ants

Robert R. Dunn

National Science Foundation (NSF)

$709,974

04/01/10 - 03/31/15

A team of graduate students, post docs and research associates affiliated with the Dunn Lab will engage attendees at the USA Science and Engineering Festival (including K-12 students, teachers, and families) in an interactive exhibit hosted by NSF. On April 24-26, 2014, the team will share information and opportunities to get involved in a number of citizen science projects related to the biodiversity in our daily lives, including School of Ants and the Camel Cricket Census.

Dimensions: Collaborative: The Climate Cascade: Functional and Evolutionary Consequences of Climatic Change on Species, Trait, and Genetic Diversity in a Temperate Ant Community

Robert R. Dunn, DeAnna Estella Beasley, Mary Jane Epps

National Science Foundation (NSF)

$563,916

01/01/12 - 12/31/15

Supplemental funds are requested to support the undergraduate research developed as part of Award # 1136717 (The climate cascade: functional and evolutionary consequences of climatic change on species, trait, and genetic diversity in a temperate ant community). Students will participate in field and laboratory studies to explore the effects of climate change on the prevalence of fungal pathogens in eastern forests and evaluate the extent to which social insects are susceptible to infection by these fungal pathogens. We will address the following questions: 1) How does species composition and the prevalence of entomopathogens vary with temperature, 2) How does the relative virulence of entomopathogens vary with temperature and 3) How does susceptibility to infection by entomopathogens in ants vary with temperature? Upon completion of the projects, students will present their findings at the 2016 Southeastern Branch Entomological Society of America meeting and the 2016 Shaw University undergraduate research symposium. Students will also publish their work in peer-reviewed academic journals.

Examining Links between Home Microbial Diversity and Human Health in Personal Genome Project Participants

Robert R. Dunn, Holly Lynn Menninger

PersonalGenomes.org

$28,200

08/06/12 - 11/01/14

We are conducting the first continental-scale study of house-associated microbial communities in homes throughout the United States (Wild Life of Our Homes). With samples collected by citizen scientists from four common surfaces in 1000 homes, we will test a suite of hypotheses about how regional factors (climate), local environmental factors (biodiversity outside of houses, pets, cleaning practices) and home design elements affect microbial community composition. Additionally, we aim to understand what, if any, consequences variation in home-associated microbial communities might have on human health and wellbeing (i.e., test the hygiene hypothesis). Here we propose augmenting our study population with 250-300 volunteers from the Personal Genome Project (PGP). In addition to expanding the sample size of our home microbiome study, PGP volunteers will provide unprecedented access to a rich data set that includes participants? genetic information, personal health records, and human body microbiome. This data will allow us to explicitly test the hygiene hypothesis and inform larger efforts to understand how genes and the environment interact to affect traits.

Integrated Assessment of Climate Change and Ecosystem Diversity

Ryan Boyles, Robert R. Dunn

US Geological Survey (USGS)

$85,000

01/15/15 - 09/30/17

Here we build on the world's largest warming experiment by integrating the research of scholars of many institutions into a single project. In short, as we prepare to shut-down these long-running chambers, we will coordinate the sampling of every kind of organism in order to understand the many different ways in which global warming will affect different groups of organisms. Importantly, because we are doing this work at the end of the project we can study, destructively, many things we wouldn't study during the project (in which each year's growth depended on the last). We will, for example, excavate plant roots, dig up ant colonies, consider the extensive networks of fungi and more. We will then produce a comprehensive analysis of the effects of warming on diverse taxa and whether these differential effects are predictable in ways that relate to the choice we make every day.

Integrating the Effects of Global and Local Climate Change on Wildlife in North America

Robert R. Dunn, Nadia D Singh, Nicholas M. Haddad, Steven D Frank

US Geological Survey (USGS)

$234,997

08/29/11 - 09/30/14

This project was originally planned as a one-year study to assess the effects of urban warming wildlife and integrate the effects of urbanization into models of climate change. This included two main goals: -Testing the effects of urban warming on tree pests and butterflies and biodiversity more generally -Integrating urbanization and global warming (both in the field and through models) The proposed expansion will evaluate the predictive capability of models developed during initial phase of research, and answer the following questions stemming from initial findings: -Does urban warming alter the life history and distribution of pests and native species? -How does urban warming alter the life history and distribution of pests and native species? -How do local patterns of urbanization scale up to affect North America wide patterns and how do they interact with global warming? -How will pests and pathogens and species of conservation concern move as a function of cold and hot corridors?

RAPID: Consequences of extreme weather events for urban arthropod communities: Effects of Hurricane Sandy on ecosystem processes and the spread of exotic species in New York City

Steven D Frank, Robert R. Dunn

National Science Foundation (NSF)

$157,768

02/15/13 - 01/31/15

Both chronic environmental stress and acute, high-intensity disturbances are known to alter the composition of animal communities and their susceptibility to invasion by exotic species (Sousa 1984, Diez et al. 2012, Schowalter 2012). However, these two dynamic processes almost certainly interact in ways that are impossible to predict based on studies of either one alone (Sousa 1984,Underwood 1989). Urban ecosystems may be particularly susceptible to such interactive effects because they are exposed to chronic, anthropogenic stresses (Pickett et al. 2001, McKinney 2002). Among animal groups, arthropods provide ideal systems to assess community and ecosystem responses to environmental change: They are diverse, they represent multiple trophic levels, and they shape ecosystem processes from the soil to the canopy.

Students Discover: Improving Middle School STEM Outcomes Through Scaling Citizen Science Projects.

Robert R. Dunn, Jason Painter, Jenifer O Corn

National Science Foundation (NSF)

$7,221,108

10/01/13 - 09/30/18

The major challenges of the coming decades are scientific. They require a citizenry educated in science and math, particularly as they relate to the real world. Unfortunately, by nearly any metric the average education of citizens in science and math skills is lagging, whether that be in terms of the knowledge of adults about scientific processes and issues or their precursors, the knowledge and standardized test scores for students in basic environmental science and related mathematical content. Fortunately, there is a strong desire among both teachers and, in many cases students, for more and better STEM education, particularly as it relates to science in practice. Our overall goal is to improve STEM education, in middle schools, by engaging students and teachers in course curricula built around citizen science projects. We will improve education and engagement by having students do real science.

Tree Eaters: Predicting the Response of Herbivores to the Integrated Effects of Urban and Global Change

Robert R. Dunn, Steven D Frank

US Geological Survey (USGS)

$33,074

09/19/13 - 09/18/14

The future of forests is threatened by herbivores and pathogens. The loss of trees due to these organisms has affected carbon emissions, fire risk, forest value and the conservation of rare species. By all estimates, the number of future pests and pathogens that pose risks to trees in North America is likely to increase due to global commerce, global warming and urban warming. Efforts to reduce the number of introduced pests and pathogens or to control them once they have arrived have been very modest in their success. An alternate approach (and ultimately complimentary) approach is to understand whether there are features of some forests that predispose them to more minor effects due to the invasion of pests and loss of individual tree species. Here we test the extent to which the composition and management of forests has historically affected the risk of those forests to tree pests. We focus on the species and phylogenetic diversity of forests, both of which can be actively managed (but are not typically a major focus).

Tree Eaters: Predicting the Response of Herbivores to the Integrated Effects of Urban and Global Change

Ryan Boyles, Robert R. Dunn, Steven D Frank

US Geological Survey (USGS)

$40,000

09/19/14 - 09/18/15

The future of forests is threatened by herbivores and pathogens. The loss of trees due to these organisms has affected carbon emissions, fire risk, forest value and the conservation of rare species. By all estimates, the number of future pests and pathogens that pose risks to trees in North America is likely to increase due to global commerce, global warming and urban warming. Efforts to reduce the number of introduced pests and pathogens or to control them once they have arrived have been very modest in their success. An alternate approach (and ultimately complimentary) approach is to understand whether there are features of some forests that predispose them to more minor effects due to the invasion of pests and loss of individual tree species. Here we test the extent to which the composition and management of forests has historically affected the risk of those forests to tree pests. We focus on the species and phylogenetic diversity of forests, both of which can be actively managed (but are not typically a major focus).

Understanding the Effects of Host Evolution and Skin Bacteria Composition on Disease Vector Choices

Robert R. Dunn

US Army - Army Research Office

$111,807

09/01/14 - 12/31/15

Many of the volatiles produced by the human body are actually produced by bacteria living on and in the body. These volatiles influence both human behavior and the response of other species, including disease vectors, to humans. Recently, several studies have found links between the composition of bacteria on human skin and the attractiveness of humans to mosquito vectors of disease. Key next steps are to understand how humans, or more generally, host genes, affect microbial composition and then, in greater detail, describe how microbial composition affects the choices of different species of mosquitoes. It is very likely that hosts vary in their bacteria in ways that relate to the historic influence of insect-borne pathogens and that mosquitoes differ from one species to the next in their preference for particular bacteria. Understanding these links could, in turn, inform the development of methods for camouflaging human odors in such a way as to reduce disease transmission. Here we build on our public science approach to link host genes, bacteria composition and mosquito choice in a comprehensive framework.

Control and Mitigation of Aquatic Invasive Species in Pacific Island Streams

James F. Gilliam

Tulane University

$75,134

06/02/15 - 01/01/18

Species invasions have resulted in the loss of native species and alteration of ecological processes on oceanic islands, including those with military missions and under Department of Defense stewardship. Cost-effective management solutions are needed by managers that can be implemented without comprising the military missions of such locations. This proposal will identify impacts of invasive fauna in insular, oceanic stream ecosystems, and compare efficacy of mitigation actions based on species suppression, species removal, or hydrological manipulation on military lands, including analyses of effects of alternative actions under alternative future climate scenarios.

UBM-Group: Integrated Undergraduate Training in Mathematics and Life Sciences at NCSU

Hien T. Tran, Alun L. Lloyd, James F. Gilliam, Aloysius G. Helminck

National Science Foundation (NSF)

$240,000

08/01/11 - 07/31/16

The overriding goal of the Integrated Undergraduate Training in Mathematics and Life Sciences at North Carolina State University (NCSU) is to attract and train undergraduates in mathematics and life sciences for academic and nonacademic careers at the interface between mathematics, computational science, and life sciences. Galileo was perhaps the first to clearly state that the laws of nature are mathematical. Indeed and nearly 40 decades later, cutting edge research at the forefront of life sciences has become more dependent on mathematical, computational, and statistical methodologies. The proposed UBM program uses a multi-faceted approach to prepare next generation of mathematicians and scientists that will meet the holistic, multi-disciplinary research problems of the 21st century. More specifically, our proposed UBM has four primary objectives. It (1) provides a focused environment to involve undergraduate students in mathematics and life sciences in cutting edge cross-disciplinary research involving a broad spectrum of applications in life sciences. For many undergraduates, this will be their first research experience and one that will encourage them to pursue interdisciplinary graduate studies in mathematics and life sciences. It (2) develops and integrates a number of research training and professional development to ensure successful training of a new generation of mathematicians and scientists. These include the development of two new courses: an applied differential equations course in which the theory and analysis of ordinary differential equations are introduced in the context of relevant biological applications and a novel course in model verification and validation. The context-rich material curriculum will be supplemented with UBM research seminars and Professional Development Modules (PDM) that will be held weekly. Students will also be encouraged and assisted by faculty mentors to make professional contacts, participate in tours of mentor's laboratories, attend conferences, and become members in student chapters of professional/academic societies. It (3) provides a team environment for interdisciplinary and collaborative research. The proposed UBM program will train a cohort of 8 undergraduates per year, divided into two groups, with joint mentoring of each 4-student group (two mathematics majors and two life sciences majors) by a pair of faculty from mathematics and life science disciplines at NCSU as well as with our collaborators who are external to NCSU . The long-term objective is to institutionalize a paradigm for training mathematics and life science students for academic and nonacademic careers that involve collaborative, interdisciplinary, team research. It (4) enhances cooperation among faculty in mathematics and life science disciplines. Intellectual Merit and Education. There has been an outburst in the last ten to twenty years in quantitative analysis of biological systems that requires new approaches at how we educate undergraduates. The NCSU UBM team is truly interdisciplinary, with members in mathematics, biomathematics, statistics, biology, chemistry, veterinary medicine and medicine. This powerful combination of areas of expertise offers a truly unique cross-disciplinary educational experience for undergraduates in life sciences and mathematics. Indeed, the project will produce future scientists with both biological skills and mathematical insight and facility. In addition, both mathematics and life science disciplines can expect to gain by this collaborative effort. To faculty in mathematics, the stimulation of biological applications will enrich the discipline of mathematics as it provokes refinements and further mathematical developments. Life science faculty will benefit from the power of mathematical tools as they provide insight available in no other way. Broader Impact. The results of the proposed UBM program will provide a vehicle for systemic institutional change in introductory mathematics and science education. Since project leadership includes key members of existing

A Genetic Engineering Approach to Rodent Pest Management

John R. Godwin, Lisa Anne McGraw, Kevin R. Gross, Fred L. Gould, Nicholas M. Haddad, Alun L. Lloyd, Martha Burford Reiskind

NCSU Research and Innovation Seed Funding Program

$14,929

01/01/15 - 12/31/15

Rodent pests cause major economic losses and threaten food security and biodiversity worldwide. The problem is particularly acute on islands where most vertebrate extinctions occur. We propose to test an innovative approach based on genetic engineering. This would also support graduate training in the NCSU Genetic Engineering and Society Center.

Collaborative Research: Kisspeptin Regulation of Reproductive Physiology of a Coral Reef Fish

John R. Godwin

National Science Foundation (NSF)

$260,000

09/01/13 - 08/31/17

Sexual differentiation of the brain and behavior typically results from an interplay of endogenous and enviromental cues influencing the timing, level and location of gene expression. The relative strength of endogenous and environmental influences on this process varies across species. It is also clear that multiple mechanisms leading to similar patterns of behavioral expression may exist within species. This is suggested by variation in neuroendocrine mechanisms underlying aggression across sexes and seasons and the results of gene knockout studies where the lack of a presumably critical gene product does not abolish or sometimes even affect behavioral expression. We are building a solid understanding of how gonadal steroids influence the sexual differentiation of brain and behavior. Much less well understood are the mechanisms by which environmental influences such as social interactions affect these processes and how these mechanisms overlap and act with or through steroid hormone mediated pathways. This project will explore the role of a recently discovered neuropeptide system, kisspeptin, in mediating socially-controlled sex change. We will use a model system, the bluehead wrasse, that exhibits two interesting and experimentally useful features of its reproductive biology and where the development of the male courtship behavior can be stimulated through both social interactions and steroid hormones. Bluehead wrasses exhibit discrete alternate male mating phenotypes and socially-controlled functional sex and role change. Sex changing females initiate behavioral change immediately and develop fully male behavioral phenotypes within a few days of becoming socially dominant. Importantly, we have shown that even gonadectomized females who cannot develop male morphological characteristics nevertheless develop fully male behavioral phenotypes on attaining dominant status. We have also induced female-to-male sex change and male courtship behavior through gonadal steroid hormone manipulations in nature. The aims of this project are to clone key genes in the kisspeptin signaling system in bluehead wrasses, compare expression of these genes across sexual phenotypes and experimentally induced sex change, and characterize the neuroanatomical locations of expression within the bluehead wrasse brain. Identifying mechanisms by which social interactions influence behavior-controlling mechanisms is of both basic and practical signficance. Aggressive behavior has enormous societal costs and a better understanding of social influences on the neural substrates of this behavior is needed. Also, the increasing importance of finfish aquaculture makes a better understanding of the control of sexual behavior in fishes useful for captive propagation. The bluehead wrasse system presents the opportunity to experimentally dissect gonadal and social influences on these mechanisms in the natural environment.

DISSERTATION RESEARCH: Androgen Regulation of Territorial Male-Typical Behavior and Neuropeptide Gene Expression During Socially-Induced Sex and Role Change in a Coral Reef Fish

John R. Godwin

National Science Foundation (NSF)

$18,772

07/01/14 - 06/30/16

This dissertation improvement project would test whether gonadal signals impact the speed and intensity (frequency and duration) of behavioral changes during female-to-male sex change and subordinate-to-dominant male role change. Furthermore, this project would test if gonadal presence affects gene expression of the systems hypothesized to control sex and role change: the vasotocin, kisspeptin, and gonadotropin-releasing hormone systems. The animal model that would be used in this project is the bluehead wrasse, Thalassoma bifasciatum, a diandric (two male phenotypes) protogynous (female-to-male) coral reef fish capable of changing male role and sex.

Improving Southern Flounder Management in the Southeastern United States through Characterization of Habitat Effects on Juvenile Sex Ratios

John R. Godwin, Russell J. Borski, Harry V. Daniels

National Oceanic & Atmospheric Administration (NOAA)

$226,576

07/01/14 - 06/30/17

Southern flounder support critically important recreational and commercial fisheries in the Southeast US, but these fisheries have also suffered substantial recent declines. Southern flounder also show strongly sexually dimorphic growth with females growing much larger than males. Due to this difference in growth and capture size limits, male southern flounder rarely grow large enough to enter the commercial or recreational fisheries and the catch is therefore heavily female-biased. Currently, managers must assume 50:50 sex ratios in Juvenile Abundance Indices (JAI). Departures from this assumed even sex ratio in juveniles could adversely influence estimates of Spawning Stock Biomass (SSB) and mortality (F), hindering efforts to manage and recover these stocks. Importantly, southern flounder also show strong environmental influences on sex determination, particularly with higher temperatures inducing a strong sex ratio skew towards males. Sampling in conjunction with the North Carolina Division of Marine Fisheries in the unusually warm spring/summer of 2012 strongly suggested elevated temperatures in flounder nursery habitats can also strongly skew sex ratios in wild populations and that this can occur without detectable differences in growth. The goal of the proposed studies is to better characterize sex ratios in juvenile flounder. This would benefit management of this economically and ecologically important fishery resource.

Wild-derived Zebrafish as Models for Anxiety and Stress Responsiveness

John R. Godwin

National Institutes of Health (NIH)

$403,380

05/20/11 - 04/30/15

These studies are aimed at comparing gene expression in the brain of males and females across lines of zebrafish lines derived from wild populations that exhibit substantial variation in anxiety-related behavior. We have gathered samples for these neural gene expression comparisons at two ages including brain tissues for RNAseq-based gene expression comparisons and bodies to allow measurements of the key teleost stress hormone, cortisol. We also had the opportunity to compare neural gene expression profiles between male reactive line fish treated for two weeks with fluoxetine (trade name Prozac) and saline-treated controls in a small experiment. We found that sequencing to a depth of 20 million reads per library was optimal in terms of balancing statistical power and number of biological replicates possible given our budget. This dataset will also be useful for comparing to gene expression differences between proactive and reactive line fish. The second aim of this project is to profile the display of anxiety-related behaviors and neural gene expression across developmental age with contrasts between the proactive and reactive lines as well as between males and females. This will allow profiling of potentially important events during neural development that play a role in later development of proactive and reactive behavioral phenotypes.

ARI-R2: Renovation of the North Carolina State University Phytotron For Improved Environmental Control and BSL-3 Containment

Steven A. Lommel, Jose M. Alonso, Steven D. Clouse, Nicholas M. Haddad, Linda K. Hanley-Bowdoin

National Science Foundation (NSF)

$1,793,916

10/01/10 - 09/30/14

ARI-R2: Renovation of the North Carolina State University Phytotron for Improved Environmental Control and BSL-3 Containment. North Carolina State University (NCSU) faculty and students have a compelling need for a state-of-the art controlled, environmental facility (Phytotron) with containment for investigation of high-risk plant pathogens and other microbes. The four-story Phytotron was built in 1968 with most of the construction (~$1.5M) and first eight years of operating funds provided by the NSF, and 33 years of continued maintenance and staffing provided by NCSU. After 41 years however, the NCSU Phytotron is in dire need of upgrades to meet present and growing demands for controlled environmental research facilities for plants, insects and small mammals. This application proposes critical infrastructural upgrades to (1) renovate the core environmental system by replacing chillers and pumps, connecting to university chilled water lines, re-insulating chilled water and glycol lines, and applying epoxy coating to chamber and greenhouse floors; (2) replace controllers that modulate the environmental conditions for individual environmental chambers, and modernize and increase the precision of CO2 controls; and (3) convert a plant dark room and greenhouse into a BLS-3 facility for investigations with highest risk viral, bacterial, fungal and nematode plant pathogens and select agents. Intellectual Merit. Biologists in the post genome era face a major challenge in determining the function of the thousands of identified genes, many of which are under environmental control. Often, the phenotype of a mutant and the expression pattern of responsible gene(s) can be properly defined only in precise environmental conditions. Renovation of the Phytotron will provide researchers with the large-scale controlled environment capacity to take full advantage of publically accessible genetic tools not only to accelerate information-rich phenotyping in model plants but to translate the same high-throughput screens to crops, wild relatives and non-native species. Control over CO2 concentration will open new opportunities for NCSU researchers involved in global change research and predicting the responses of individual organisms and ecosystems to environmental change. These demands, as well as the growing need for studies of phenotypic plasticity needing reproducible combinations of climatic factors and ecological studies that require the capacity to simulate a variety of environments, make this facility an essential resource for future, competitively funded investigations. Similarly, upgrading a designated area of the facility for BSL-3 containment will provide a research environment and foster international collaborations to better understand the fundamental biology of plant pathogens that already cause severe disease problems in subtropical/tropical regions and pose an increasing threat to the U. S. and global food supply chain. Broader Impacts. The NCSU Phytotron is an outstanding testament to the value of investing in research infrastructure. This NSF-funded legacy is a valuable University-wide facility and focal point for training and research at all levels. The infrastructure renovations proposed here will allow this unprecedented resource to continue to fulfill this mission by becoming a high-throughput public facility for phenotypic analyses of many different plants. Graduate and undergraduate students from NCSU, summer REU programs involving students from non-Research I universities and other outreach programs for underrepresented groups use the Phytotron for independent research projects under the direction of a diverse representation of faculty who serve as role models and mentors. The renovations will have immediate scientific impacts as NCSU researchers can acquire pathogens and microbes from collaborators around the world by having a centralized facility inspected and known to meet or exceed containment guidelines. Unemployment in the State of North Carolina is over 11% and above the national average.

Connectivity for Climate Change in the Southeastern US

Nicholas M. Haddad, Jennifer K Costanza

US Geological Survey (USGS)

$299,897

09/17/12 - 03/31/15

Our objective is to create a map of landscape connectivity for the southeastern United States that will identify key linkages for wildlife and key targets for conservation to facilitate range shifts as climate changes. Connectivity has been identified as a focal element of conservation as climate changes by most state and federal agencies, conservation NGOs, and scientists. In identifying high-priority connections, we will advance Theme 2 of the SECSC Science Plan, specifically Task 3: Biological responses to changing land use and climate and Task 4: Develop land cover and habitat projections for the southeastern US. Our research will address the following questions: 1. When connecting landscapes, can we do better at conservation when we consider the potential effects of climate change? 2. How will connectivity after climate change differ for species that vary in their dispersal ability, habitat affinity, and home range sizes? 3. How can we connect landscapes in the face of rapid urbanization and climate change? 4. How will sea level rise affect the location of key connections?

Endangered Butterflies As A Model System For Managing Source-Sink Dynamics On Department Of Defense Lands(previous title: Endangered Butterflies As A Model System For Detecting And Managing Source-Sink Dynamics In Remnant And Restored Habitat On Depa

Nicholas M. Haddad

Harvard University - Harvard College

$469,542

05/03/11 - 04/30/16

We propose to use habitat management and restoration for at-risk butterflies as a model system for understanding the role of source-sink dynamics in metapopulation viability, and to evaluate what kinds of information resource managers need to assess the source-sink status of local populations. In the past, butterflies have proven to be key model species for understanding spatial ecology, including metapopulations, source-sink dynamics, and climate-induced range shifts. Butterflies are an ideal model system for studying source-sink dynamics because their short life-spans and relatively limited dispersal make it feasible to monitor their population dynamics and movement over multiple generations within the timeframe of a single study. In addition to their general suitability as model systems, understanding effects of habitat restoration on source-sink dynamics of butterflies is directly relevant to DoD concerns for 4 reasons. First, DoD actively manages habitat for numerous butterfly species (Table 1), both by controlled disturbance and by de novo habitat creation. Second, although the basic processes of local population growth and dispersal behavior that underlie source-sink dynamics of butterflies are the same ones that govern the dynamics of other species for which DoD has management responsibility (including amphibians, birds, and large carnivores), they are far more amenable to study in butterflies. Third, because managers deliberately manipulate habitat quality, and may at times push it outside the range of natural variation, source-sink dynamics may be even more relevant in a restoration context than it is in unmanaged settings. Finally, although DoD invests large amounts of money and labor annually in restoration and management actions, the consequences of these actions for species of conservation concern are often poorly monitored or difficult to assess. For cost-effective land management, it is vital to understand and monitor the population- and metapopulation-level consequences of restoration and management activities. Our proposed work will do this by providing decision support tools that can be applied to a wide range of TERS species that DoD manages.

IGERT: Genetic Engineering and Society: The Case of Transgenic Pests

Fred L. Gould, Alun L. Lloyd, Nora Haenn, Nicholas M. Haddad, William J Kinsella

National Science Foundation (NSF)

$2,998,062

08/15/11 - 07/31/17

This IGERT project will create a transformative graduate education program that trains students in technologies needed for manipulating pest genomes as well as methods needed to assess the environmental and social appropriateness of specific products of these manipulations. The concept of genetically manipulating a pest species to destroy or render it benign dates back to the 1940's, and there have been several major successes in using this approach. However, restricted tools of classical genetics limited the broader application of Genetic Pest Management. Recent advances in molecular genetics have provided much more precise techniques for manipulating the genomes of pests, and efforts are now underway for development and potential release of transgenic mosquitoes and transgenic agricultural pest species aimed at achieving Genetic Pest Management. The future of this pest management strategy will be determined by further technological advances, public attitudes to the novel technologies and products involved, and the creativity and wisdom of researchers and policy makers. Although esteemed scientific groups including the U.S. National Academy of Sciences have repeatedly emphasized that risk assessment for transgenic organisms should focus on the specific product and not the process, the legacy of genetically-engineered crop commercialization has made the logic behind this idea obscure to most people, including many scientists. For new applications of genetic engineering to be developed and judged appropriately, diverse social and cultural groups will need to more deeply examine the ramifications of each application. Broadly trained PhDs in biological and social sciences will facilitate this examination and help foster more sophisticated interactions among policy makers, academicians, and members of societies where Genetic Pest Management may be applied. Intellectual Merit of this IGERT derives from the fact that this could become the first graduate program in the world that is specifically training graduate students to understand, build, and assess impacts of transgenic organisms. All students will receive core transdisciplinary training that will encompass ethics, communication, economics, ecology, epidemiology, molecular biology, and population genetics. Each student will use expertise from at least two of these specialties in developing a dissertation. Our program is broad in integrating across diverse disciplines, but maintains the focus of students and faculty by specifically studying a small set of species that are targets for Genetic Pest Management. In each of the first years of the program, we will recruit graduate students in biological and social sciences. Each cohort of about six students, balanced across disciplines, will work together with faculty to choose a single target species as the focus of their dissertations. Focus on single species will challenge both student and faculty to work together, develop a common vocabulary, and understand how each other's disciplines operate. We are developing a set of core courses, which will provide all students with a basic toolkit for working in the field of Genetic Pest Management. Students specializing in the disciplines of a specific course will act as mentors to the other students taking the course. Broader Impacts of this IGERT fall into the following categories: 1) Improvement in the administration and extent of integrated graduate education at NCSU, 2) Impact on US integrated graduate education by evaluating a novel model of such integration, 3) Increased number of students from underrepresented groups that receive interdisciplinary education, 4) Improvement of methodologies for assessing and introducing new technologies, 5) Ph.D.s in biology and social sciences who have tools needed for future interdisciplinary, global work. 6) Education of local communities. Furthermore, most of the target pest species are of importance in poor nations, and we will use existing and newly developed partnerships to set up internships and dissertation projects in th

Support for ConservationCorridor.org (Vision for ConservationCorridor.org, 2014-5)

Nicholas M. Haddad

Wilburforce Foundation

$23,350

06/13/14 - 09/30/15

Since our launch just over a year ago, we have demonstrated that ConservationCorridor.org provides a useful platform for sharing information about connectivity and corridors – we now receive 1,400+ unique visitors/month and that rate is growing. As we have received feedback about our portal, two themes have emerged. First, our Toolbox has proven to be a popular entryway for users to other sites. This has caused us to recognize our potential role as a central clearinghouse that can make tools and information more accessible to managers. In this regard, changes to our site are needed to properly direct users to tools and information. This will come in the form of re-organizing our Toolbox, and in providing more direct and updated guidelines for management. This will help us to achieve our objective of expanding our content and audience to better capture conservation and challenges in working corridors. Second, we have until now focused on reporting corridor research and conservation, as we intend for the site generally to serve as an objective conveyor of information on corridor science and conservation. We have refrained from debates, discussions, and disagreements about corridor design, research, and implementation so far. However, we recognize that discussion is important for our topic, especially as the science and conservation are evolving. To these ends, we propose four new features for ConservationCorridor.org to facilitate engagement and interaction of site users, and to improve the science and practice of connectivity conservation.

Using Landsat Data to Quantify the Combined Effects of Habitat Loss and Climate Change on West African Wildlife

Nicholas M. Haddad, John William Wilson

National Aeronautics & Space Administration (NASA)

$90,000

09/01/11 - 08/31/14

The combined effects of habitat loss and climate change pose a great challenge to biodiversity conservation now and in the coming century. I propose to examine the combined and independent effects of habitat loss and climate change on the distributions of forest birds endemic to the Guinean Forests of West Africa. I hypothesize that the combined effects of habitat loss and climate change have a stronger influence on species distributions than the sum of their independent effects. I will address my hypothesis in four steps. First, using a time series of Landsat images, I generated land cover maps to quantify changes in the forest cover of a section of the Lower Guinean Forests from the pre-industrial era through 2010. Second, using an unprecedented historical dataset that enables me to quantify human land-use impacts, I predicted future forest loss as a function of the environment and human activity. Third, I will project changes in the area of occupancy of each study species over time by refining their distributions based on the historical, current, and future forest cover maps I generate. Last, I will use historic climate to model the climatic niches of species and use these niches and projections of climate change to model future species distributions that account for the effects of climate change both independent and combined with projected forest loss. The results I generate will allow me to estimate which endemic bird species will be most threatened by the combined effects of habitat loss and climate change, and which individual forest patches harbor more endemic bird species threatened by these combined effects at present and in future. These results will inform the conservation community of human impacts on the region?s unique biodiversity, and guide science-based policy and management by refining conservation priorities.

Center for Human Health and the Environment

Robert C. Smart, Jane A Hoppin, Carolyn Mattingly, Cathrine Hoyo, David Michael Reif, Philip L. Sannes, Heather B Patisaul, Michael Bereman, Fred Andrew Wright, Keith E. Linder, Heather R. Shive, Mary C. McGahan, James C Bonner, William G. Cope, David Baltzegar, Detlef R. Knappe, Katlyn May, Catherine Elizabeth LePrevost, Julia F. Storm, Jennifer Schaff

National Institutes of Health (NIH)

$2,238,387

04/20/15 - 03/31/20

This project brings together the unique expertise and resources of the NIEHS Environmental Health Sciences Core Centers (EHS CC) at the University of Michigan and North Carolina State University in a collaborative effort to address critical issues of human exposure, health threats, and community concerns from 1,4-dioxane (1,4-D). 1,4-D is water soluble solvent that is an emerging and potentially widespread threat to human health through contaminated drinking water. 1,4-D is a known liver and kidney toxicant and is classified as likely carcinogenic to human. 1,4-D is recognized by the US EPA as an emerging drinking water contaminant of concern. Communities in North Carolina and Michigan share significant concern over 1,4-D-contaminated drinking water. 1,4-D has been detected in municipal drinking water in Pittsboro, NC at some of the highest levels in the US due to contamination in the Cape Fear River basin. In Washtenaw County, MI, a migrating 1,4-D groundwater contamination plume at concentrations above benchmarks has contaminated numerous drinking water wells including a former municipal supply well. These contaminations threaten exposure to hundreds of thousands of residents, including communities with limited financial resources. Our long-term goal is to address critical gaps in our understanding of 1,4-D environmental contamination effects on human health. The central objective of this supplement is to assess exposure to 1,4-D via drinking water (Aim 1), biomonitoring of a urinary 1,4-D and it primary metabolite HEAA (Aim 2), and serum biomarkers of liver and kidney health (Aim 3) in a pilot study of residents potentially exposed to 1,4-D in Pittsboro, NC and Washtenaw County, MI,. Working with community groups, regulatory agencies, and publically available data, we will identify 20 adult participants from each of the potentially exposed areas and 20 participants from potentially unexposed areas in Michigan and North Carolina. Individual results will be reported back to the participating residents with the aid of each Center’s COEC. Through completion of our aims, this project will be the first assessment of associations of community exposure to 1,4-D via drinking water with urine levels of 1,4-D and HEAA and with health outcomes.

CEASE: Community Education About Smoke Exposure

Cathrine Hoyo

Duke University

$16,791

06/01/15 - 05/31/17

The Center for Study of Neurodevelopment and Improving Children's Health following EtS exposure (NICHES) is investigating the mechanistic relationships between environmental tobacco smoke (ETS) exposure and developmental neurocognitive impairments including Attention-Deficit/ Hyperactivity Disorder (ADHD). Dr. Hoyo co-founded the Newborn Epigenetics STudy (NEST) cohort, from which the sub-cohort members of NICHES are identified. Dr. Hoyo will assist with selecting and interpreting existing periconceptional, prenatal, peripartum and one-year risk factor data from NEST that we will merge with new data we are collecting as part of NICHES. She will participate in all meetings, assist with interpreting data and directing analyses, and will be a co-author on resulting scientific products and manuscripts.

Children's Exposure to SVOC Mixtures Indoors and Associations with Obesity

Cathrine Hoyo

Duke University

$47,727

07/01/14 - 06/30/19

Dr. Cathrine Hoyo will serve as the PI of the subcontract at North Carolina State University (NCSU). Dr. Hoyo will work with Dr. Stapleton on the children’s exposure project and will provide access to her birth cohort for the study. She will assist in generating the annual reports, publications and presentations related to this study.

Disparities in Cervical Cancer Percursors and Deregualtion of Imprinted Genes

Cathrine Hoyo

National Institutes of Health (NIH)

$512,912

05/01/14 - 04/30/17

Although screening for uterine cervical intraepithelial lesions (CIN) and its aggressive treatment has resulted in decreased cervical cancer incidence and mortality, an estimated 11,000 cases of invasive cervical cancer (ICC) and 40,000 cases of carcinoma in situ (CIS) continue to be diagnosed every year in the United States. ICC incidence is 60% higher and mortality over two times higher in African Americans compared to whites. Such disparate rates in incidence and mortality are despite comparable screening rates to detect precursor lesions, comparable prevalence of 'high risk' human papillomavirus (HPV) infection, the etiologic agent, and co-factors such as cigarette smoking. Reasons for such disparities are largely unknown. A more detailed understanding of the etiology of lesions that progress will improve risk stratification to distinguish women with a low risk of progression from those likely to rapidly progress to invasive cancer. We posit that deregulation of genomically imprinted genes, a group of approximately 50 known growth-regulatory genes where only one allele is normally active, may predict progression to cervical cancer. We found that a substantial group of imprinted genes exhibit altered expression in ICC versus normal cervical epithelium. In addition, methylation analysis of two regulatory regions controlling IGF2 expression shows deviation from normal in specimens with CIN2-CIN3 classification and more profound deviation in invasive cancers. Together, these findings suggest that imprinted genes are deregulated in ICC and these features may improve detection of CIN cases likely to progress. Our central hypothesis is that deregulation at imprint regulatory elements of imprinted genes, likely influenced by environmental exposures, increases risk of progression to cervical cancer in women infected with 'high risk' HPV. We have already shown racial variation on the effect of environmental exposures on imprint deregulation, and thus, further hypothesize that higher risk in African Americans and Hispanics is due to race/ethnic differences in deregulation of these genes. Our specific aims are to: (1) determine if deregulation of imprint regulatory elements of known imprinted genes is associated with increased risk of progression of CIN1 to CIN2+, and whether this association varies by race/ethnicity; (2) determine whether aberrant methylation of imprint regulatory elements in cervical cells is similar to that found in peripheral blood cells, suggesting an early event; and associated with (i) transcriptional expression, and (ii) loss of imprinting; (3) evaluate whether deregulation of known imprinted genes in cervical cells can be used to discriminate women with CIN2+ among 500 Atypical Squamous Cells of Uncertain Significance (ASCUS) cases. To address the Aims, we will recruit and follow 1,500 women with CIN1 and 500 with ASCUS, and examine the association between aberrant methylation markers at imprinted regulatory elements of 12 genes and progression from CIN1 to CIN2+. We will then test the performance of these markers to identify CIN2+ cases from among 500 ASCUS cases. Data from the proposed study will improve triage decisions and reduce disparities in ICC morbidity and mortality.

Early Child Care and Risk of Obesity

Cathrine Hoyo

Duke University

Unfunded

07/01/14 - 06/30/15

The need to prevent childhood obesity extends down to our youngest children. Obesity and excessive weight gain in the first year of life are independently associated with higher blood pressure, recurrent wheezing, higher hospital admission rates, and other adverse physical and psychosocial health conditions in childhood. Thus, effective approaches to obesity prevention in early childhood are needed. Because nearly two-thirds of U.S. children under 6 years of age are routinely cared for outside of the home, organized child care is an important setting for obesity prevention. Recent studies suggest that children who attend child care are more likely to be obese than children cared for at home by a parent or other caregiver, and younger children might be most at risk. Our own research found that infants who spent time in child care during the first 6 months of life were heavier at one yea and still heavier at 3 years of age than children cared for at home. These previous studies identified a relationship between child care and obesity, but failed to pinpoint specific causal pathways. The proposed longitudinal study will examine factors contributing to the development of obesity that may be influenced by the child care setting, including dietary behaviors, physical activity and inactivity, stress, and sleep duration and quality. To accomplish these aims, we propose a diverse southern cohort of 800 black, white, and Latino/a infants in various child care arrangements, followed regularly from birth to 12 months of age. Frequent in-home assessments throughout infancy will help pinpoint exactly when children begin to gain weight excessively and will identify risk factors related to energy intake, energy expenditure, stress, and sleep associated with obesity. To mitigate participant burden and enhance retention, we will use Interactive Voice Response (IVR) technology to supplement in-person visits through innovative telephone-administered assessments with parents and child care providers. Results of this study will provide new information on the relationship between child care attendance and obesity and may help determine causality in instances where the associations between these variables have been unclear. Findings will inform state and federal policy governing child care settings and will also guide intervention efforts to help prevent obesity in young children in child care.

Prenatal Tobacco Smoke, Genetic and Epigenetic Changes, and Respiratory Health

Cathrine Hoyo

University of Southern California

$21,790

07/01/14 - 06/30/17

Asthma is a leading cause of childhood morbidity and normal lung function development is essential for respiratory health during childhood and subsequent adult life. We have developed preliminary data that highlight the adverse effects of prenatal tobacco smoke (PTS) exposure. PTS exposure can permanently alter the developing lung and fetal immune function, increasing risk for respiratory disease, and understanding the mechanisms of these effects may shed light on the effects of other environmental exposures. Scientists have clearly demonstrated both early-life environmental and genetic factors contribute to the pathogenesis of asthma and lung function. Recent studies highlight the importance of genetic variants and epigenetic alterations underlying environmentally-related immune function and respiratory health. We present preliminary studies showing the potential importance of genetic and epigenetic variation in the TAM (TYRO3, AXL, and MER) family of Type I receptor tyrosine kinases in early life lung function and asthma pathogenesis in the context of PTS. To provide a conceptual framework useful for addressing this critical knowledge gap, we propose an integrated epigenetic-genetic analysis of the TAM (TYRO3, AXL, and MER) family of Type I receptor tyrosine kinases to better understand the biological mechanisms underlying the impaired lung function development and increased risk of asthma associated with PTS. Specifically, this application builds on the PI's K01 project to further evaluate 1500 subjects in the Children's Health Study (CHS), a longitudinal study of air pollutant and respiratory health in 16 Southern California communities. The study will leverage an existing comprehensive resource that includes genome-wide association data, linked birth records, and extensive respiratory assessments. The project focuses on the TAM gene family for the proposed integrated analysis because these genes are important in the development of chronic inflammatory and autoimmune diseases and our preliminary data suggest epigenetic mechanisms may play a role. The proposal will accomplish the following aims: (1) characterize the association between PTS exposure and TAM gene DNA methylation and validate observed associations; (2) explore whether haplotypes are associated with TAM gene DNA methylation or modify the PTS-associated DNA methylation; (3) characterize the association between PTS exposure and promoter DNA methylation of mi-R34a and miR-199a/b levels, three miRNAs known to regulate AXL expression, and further relate to their expression levels; and (4) build a comprehensive picture of the inter- relationships between PTS exposure, CpG methylation, gene or miRNA expression, and haplotypes in the TAM genes in association with asthma and lung function using methods based on canonical correlation. The proposed integrated epigenetic-genetic analysis coupled with the novel use of newborn bloodspots (NBS) to measure prenatal exposure and epigenetic alterations is expected to offer a powerful approach to studying the epigenetic-genetic mechanism for various complex human diseases with early-life environmental origins. Dr. Cathrine Hoyo will serve as the PI of the subcontract at North Carolina State University (NCSU). Dr. Hoyo will work with Dr. Breton providing her with data to accomplish the aims that will examine associations between prenatal tobacco smoke, child respiratory health, and genetic/epigenetic changes. Dr. Hoyo will supervise all work at NCSU including data processing and analysis. Dr. Hoyo will participate with Dr. Breton in the writing of manuscripts associated with this project, as well as in the public presentation of the work at research and community-based conferences.

A Comprehensive Examination of Endocrine Disrupting Compounds and Intersex Fish in North Carolina Water Bodies

David Derek Aday, Seth W Kullman, William G. Cope, Thomas J. Kwak, James A. Rice, Jerry M. Law

NC Wildlife Resources Commission

$489,788

07/01/11 - 12/31/16

The goal of this collaborative project is to better understand the causes and consequences of intersex in North Carolina water bodies and fish. This broad based project is truly multidisciplinary, with fishery biologists at NCSU and the NCWRC working with ecotoxicologists and veterinary pathologists to develop a mechanistic understanding of the biotic and abiotic factors that drive variation in the concentrations of Endocrine Disrupting Compounds (EDCs) in NC waterways as well as the extent and severity of intersex in fish. The project has six major components, all of which have links to the biology or management of sportfish. Objective 1: Develop a GIS-based map of potential Endocrine Disrupting Compounds (EDCs) in North Carolina water bodies . In this component of the project we will identify potential agricultural, municipal, and industrial sources of EDCs, ultimately producing a statewide reference map that would allow fishery biologists to identify areas of potential concern for game and nongame fishes. Objective 2: Conduct a statewide survey for the presence of EDCs in North Carolina water bodies and intersex in common fish. This aspect of the project develops a statewide assessment of intersex for key sportfish including black basses (Micropterus spp.), sunfish (Lepomis spp.), and catfish (Ictalurus or Ameiurus spp.). Specifically, we will measure the degree and severity of intersex in these fishes at 20 sites throughout North Carolina, at least ten of which are identified as ?hotspots? (agricultural, wastewater, or industrially-influenced sites that are expected to have significant concentrations of EDCs and, therefore, potentially high rates of fish intersex) from the GIS analyses in Objective 1. The remaining sites will be used as ?reference? sites (areas expected to have relatively low concentrations of EDCs and rates of intersex). We will also collect EDC data in water and sediment at each study site. This objective will directly assess the impact of EDCs in state waterways and how they impact important sportfishes. Objective 3: Quantify seasonal dynamics of EDCs and intersex in NC water bodies and fish. This objective builds upon the previous by specifically examining seasonal dynamics of EDCs in waterways and intersex in fish at four selected sites. One of those sites will be the Yadkin-Pee Dee River basin, where largemouth bass were found to have the highest incidence of intersex (64 ? 91%) of all species and systems studied in a recent national USGS study (Hinck et al. 2009). This component of the study asks if and how concentrations of EDCs, and rates and severity of intersex, change as a function of season in both impacted and reference systems. Once again, black basses, sunfish, and catfish will be the study organisms. Objective 4: Conduct intensive field research and experimental bioassays on intersex fish and EDC dynamics in the Pee Dee River system. This component of the investigation focuses on the highly-impacted Pee Dee River system and aims to collect longitudinal and caged-fish data to better understand EDC and intersex dynamics in fishes. Specifically, water, sediment, and fish will be collected from ten intensive study sites that span a broad range of longitudinal position, river and watershed size, land use patterns, and known point-source discharges. Water and sediment samples will be analyzed for estrogenic compounds and fish will be analyzed for intersex. Sportfish to be analyzed include black basses (Micropterus spp.) and sunfish (Lepomis spp). Objective 5: Laboratory assessment of endocrine disruption and intersex in Pee Dee River water mixtures. The purpose of this objective is to establish the direct relationship between exposure to endocrine disrupting compounds identified in Pee Dee river (as a representative ?hotspot?) to incidence of endocrine disruption and intersex in a model laboratory fish, the Medaka (Oryzias latipes). This analysis provides a controlled mechanism to establish a cause and effect relationship between EDC exposure and molecular an

Development/Use of Adverse Outcome Pathways-Establishing an AOP for the Role of the Vitamin D Receptor in Developmental Neurotoxicity

Seth W Kullman

US Environmental Protection Agency (EPA)

$799,496

08/01/13 - 01/31/18

Recent evidence points to important contributions of exposure to environmental neurotoxicant chemicals in the marked increase in neurodevelopmental disorders, including learning disabilities, attention deficit and hyperactivity disorder, autism spectrum disorders, and Parkinson?s disease. Despite the increasing recognition of the importance of evaluating developmental neurotoxicity in safety assessment, the fact remains that, of the tens of thousands of commercial chemicals in current use, neurotoxicity and or developmental neurotoxicity of any kind has been evaluated in very few compounds. A number of factors contribute to the dearth of information on developmental neurotoxicity relative to the demonstrated need for such knowledge. First, there is the essential dichotomy between the requirement to evaluate large numbers of compounds and the costly, cumbersome protocols prescribed for standard tests in animal. Second, testing compounds singly may produce results that are difficult to compare, in large measure because the presumed mechanisms and targets are based on systemic or central nervous system effects in adults that may be unrelated to developmental neurotoxicity. It is now evident that adverse effects on brain development actually involve multiple mechanisms, many of which are unrelated to cholinesterase inhibition. In this proposal we hypothesize that the vitamin D receptor (VDR), a neurosteroid with significant linkage to neuro-dopaminergic development and adult heath outcomes may serve as a putative target associated with developmental neurotoxicity. Here we propose four specific aims including Aim 1. Identify and validate VDR and Nurr1 as nuclear receptor targets for developmental neurotoxic receptor agonists and antagonists; Aim 2 Establish integrative neuronal cell response to select VDR/Nurr1 agonists/antagonists; Aim 3 Establish an in vivo zebrafish model to assess xenobiotic induced modulation of dopaminergic neurons though alterations in tyrosine hydroxylase expression and activity; and Aim 4 Establish a linkage between developmental exposures, modulation of VDR signaling and adult neural behavioral consequence. The overall goal of this proposal is to use an AOP approach to establish a VDR->Nurr1->tyrosine hydroxylase neuroendocrine axis as a mechanisms of developmental neurotoxicity and relate disruption of this pathway to the delineation of the relevant outcomes of toxicant exposure namely disruption of behavioral performance. Our strategy is to establish ?proof of principle? for VDR and Nurr as neurodevelopmental targets with the intent of defining a screening system that will enable testing large chemical libraries for receptor activities in relation to developmental neurotoxicity.

DISSERTATION RESEARCH: Does Phenotypic Integration Reflect Local Adaptation in Bahamas Mosquitofish?

R. Brian Langerhans

National Science Foundation (NSF)

$19,695

07/01/14 - 06/30/17

The proposed study will use a model system of adaptive diversification—Bahamas mosquitofish (Gambusia hubbsi) inhabiting blue holes—to (1) characterize phenotypic integration (phenotypic variance-covariance matrices) of diverse ecologically and sexually important traits (morphology, coloration, behavior, and physiology) in a livebearing fish, and test for divergence in trait correlations between populations that have evolved for several thousand years in the presence or absence of predatory fish, and (2) test for associations between fitness surrogates (ecological performance and mating success) and phenotypic trait correlations to assess the roles of natural and sexual selection in shaping phenotypic integration. This ambitious and integrative study will use a combination of field and lab studies to shed light on the stability of phenotypic integration and the importance of selection during evolution of whole-organism phenotypes.

IPA Position at USGS

Aranzazu Lascurain

US Geological Survey (USGS)

$18,415

12/01/14 - 03/08/15

Management and coordination of the NCCWSC federal advisory committee.

Design of Novel Therapeutics for Irritable Bowel Syndrome

Scott M. Laster, Joshua Glenn Pierce, Adam James Moeser

NCSU Research and Innovation Seed Funding Program

$20,000

01/01/14 - 12/31/14

Irritable bowel syndrome (IBS) is a chronic gastrointestinal (GI) disease that afflicts millions of humans worldwide. IBS arises from stress-induced intestinal barrier dysfunction that leads to inflammation and downstream clinical signs of diarrhea and abdominal pain. The goal of this proposal is to develop new compounds for the treatment of IBS based on the structure of a naturally occurring group of fatty acid-derived compounds known as alkamides. These molecules have been shown to suppress inflammation in a number of different cellular systems and their lipophilic nature would seem to make them well suited for the treatment of GI inflammation.

Unraveling Immunostimulatory and Immunosuppressive Effects of Echinacea purpurea

Scott M. Laster

UNC - UNC Greensboro

$163,155

05/01/12 - 04/30/16

Echinacea constitutes a greater percentage of the multibillion dollar US dietary supplements industry than any other herbal medicines, with annual sales of more than $100 million. Echinacea preparations are used primarily for the treatment of upper respiratory infections such as colds and flues, but there is considerable controversy over their efficacy for this purpose. The major goal of this project is to develop methods to produce Echinacea extracts with consistent, high levels of activity against respiratory viruses.

Environmental Nitrite and Endocrine Disruption

Gerald A. LeBlanc

National Science Foundation (NSF)

$603,847

05/15/14 - 04/30/17

Human activities have significantly modified the earth's natural nitrogen cycle. The amount of nitrogen that is biologically available in ecosystems has dramatically increased through commercial nitrogen fixation for the production of fertilizers. Graphic consequences of increased available nitrogen include the global occurrence severely compromised ecosystems known as dead zones. We propose that lower levels of nitrogen, as biologically available nitrite, target aspects of the endocrine system that regulate growth and development resulting in recruitment failure in a wide variety of species. Specifically, we hypothesize that: environmentally relevant levels of nitrite can alter endocrine circuitry of arthropods, and likely other species, resulting in the dysregulation of development. Nitrite acts through its intracellular conversion to the potent signaling molecule nitric oxide which binds to and alters the regulatory action of the nuclear receptor E75. The primary objective of this program is to evaluate the ability of environmentally-derived nitric oxide to alter the function of E75 with consequences on gene expression, physiology, and population dynamics. Aspects of this hypothesis will be rigorously tested in the proposed project using the model crustacean Daphnia pulex. The first aim of this program will be to characterize the regulatory activity of the nuclear receptor E75. E75 is a transcription factor along the ecdysteroid signaling pathway of arthropods that is ubiquitous within the animal kingdom. E75 is recognized as a negative regulator of another transcription factor called HR3 and the repressive activity of E75 is under the suppressive control of endogenous nitric oxide. Thus, E75 is a likely target of exogenous nitrite-derived nitric oxide. Regulatory interaction between E75 and HR3 will be definitively and mechanistically characterized. Next, the ability of nitric oxide, derived from nitrite, to modify this regulatory activity resulting in a shortening of the embryo-development cycle and consequential production of immature neonates will be mechanistically deciphered. Finally, population-level consequences of the action of exogenous nitrite on the endocrine physiology of the organism will be determined

Genetic Selection in Animals and Plants

Trudy F. MacKay

GenSAP

$278,979

01/01/13 - 12/31/18

The Danish Centre for Genomic Selection in Animals and Plants (GenSAP) will develop the next generation genomic breeding tools for genetic improvement schemes in agricultural plants and animals. Genomic selection (GS) is currently revolutionising cattle and pig breeding world-wide and has the potential to do so in all agricultural species. The method is recognised as the key genetic improvement technology of the future, and will be crucial in meeting the global challenge to efficiently increase production of high quality agricultural products while improving animal welfare, reducing environmental impact, and maintaining biodiversity. So far, research in GS has been driven by a need for fast industrial implementation resulting in a fragmented and species-specific research effort. GenSAP will bring together all relevant Danish breeding companies and research groups as well as leading international research groups to make a joint strategic research effort to build the foundation for next generation GS. GenSAP will develop GS methodologies across all agricultural species and integrate and extract relevant information from the massive amounts of data emerging from whole genome sequencing, functional genomics, epigenomics and complex phenotyping technologies. Improved GS models will be developed based on efficient algorithms accounting for e.g. gene-by-gene and gene-by-environment interactions. Advanced computational tools for optimising and evaluating selection decisions based on in silico data will be developed to ensure a sustainable long-term implementation of GS. As a result, next generation genomic breeding tools will be efficient across species and environments, and will be both economically and genetically sustainable. GenSAP will thus form the foundation of a globally competitive and sustainable food production

Genetics of Aggression in Drosophila

Trudy F. MacKay, Robert R. Anholt, Eric A. Stone

National Institutes of Health (NIH)

$1,252,734

08/01/12 - 03/31/17

Aggression is a near universal behavior. Among social animals, appropriately balanced aggressive behavior gives rise to a stable social organization by creating and maintaining dominance hierarchies. Inappropriate aggression has detrimental consequences for a society. Sociopathic and violent behaviors place a significant socioeconomic burden on human societies. Aggression can result from traumatic brain injury, neuro-degenerative diseases, and as a comorbid condition of drug or alcohol abuse. Aggressive behavior is a typical quantitative trait, with natural variation attributable to segregating variants at multiple interacting loci, the effects of which are sensitive to the environment. Despite substantial evidence for genetic predisposition to aggressive behavior in humans, only a handful of candidate genes associated with variation in aggression have been identified in human populations. Drosophila provides an excellent model for systems genetics analysis of naturally occurring variation in aggression. We generated the Drosophila Genetic Reference Population (DGRP), which consists of 192 fully sequenced inbred strains derived from the Raleigh, USA population as a public resource for genome-wide association (GWA) analysis of quantitative traits. This population harbors substantial genetic variation for aggressive behavior and provides an essential resource for this application. Our ultimate goal is to obtain a complete understanding of the genetic architecture of aggressive behavior and biological effects of natural variants on transcriptional genetic networks. The specific aims of this proposal are (1) to use the power of Drosophila genetics and genomics to map putative causal alleles associated with variation in aggression with high resolution and develop a statistical genetic model to predict individual aggressive behavior; (2) to derive causal transcriptional co-expression networks affecting aggressive behavior, placing novel loci identified by genetic mapping in appropriate biological context; and (3) to use mutations and RNAi to functionally test effects on aggressive behavior of genes implicated by the statistical analyses of natural variation and architecture of transcriptional networks, and to use the recently developed system for integrating transgenes in the same genomic location to perform tests for causal effects of natural alleles on aggressive behavior. Because aggression is a universal behavior and many genes in Drosophila have human orthologues, general insights derived from our proposed studies will have translational implications for human genetic studies on aggression; moreover, insights derived from systems genetic studies on aggression, will have a broad impact on our general understanding of quantitative traits, including the genetics of human behavioral disorders.

Genetics of Alcohol Sensitivity in Drosophila

Trudy F. MacKay, Robert R. Anholt

National Institutes of Health (NIH)

$3,982,370

01/01/07 - 06/30/17

Alcohol abuse and alcoholism are significant public health problems. In the United States, alcoholism affects approximately 14 million people at a health care cost of $184 billion per year. Human genetic studies on alcoholism have been challenging due to difficulties in quantifying alcohol-related phenotypes and obtaining large sample sizes together with co-morbidity of alcoholism with other behavioral and neuropsychiatric disorders, gender effects, population admixture, and the diversity of mechanisms of vulnerability and resilience to alcohol. The results of many studies indicate that, in addition to a few common alleles, many different rare alleles may contribute to vulnerability in different populations, suggesting a complex genetic architecture that underlies alcohol-related phenotypes. Understanding this genetic architecture requires an integrative systems genetics approach. Drosophila presents an ideal model system, because large numbers of genetically identical individuals can be reared at low cost and without regulatory restrictions, and a plethora of community resources is available for sophisticated genetic manipulations. Previously, we measured ethanol knockdown time in 40 wild-derived inbred Drosophila lines and constructed modules of correlated transcripts associated with alcohol sensitivity and induction of tolerance. In addition, we built computational networks of covariant transcripts around genes that affect sensitivity or resistance to alcohol exposure identified by P-element mutations. During the next phase of our research program we will utilize a novel resource, the Drosophila Genetic Reference Panel (DGRP), that will enable us to capitalize on naturally occurring variation by allowing genome-wide association (GWA) analyses to provide the missing link between DNA polymorphisms and transcriptional network structure, identify eQTL and designate directionality (i.e. cis and trans regulatory effects) to transcriptional networks associated with alcohol induced phenotypes. The DGRP is a community resource, which was generated in our laboratories and consists of 192 wild-derived inbred lines with fully sequenced genomes in which all segregating polymorphisms are known. The lines are genetically variable for all phenotypes measured to date. We will pursue the following Specific Aims: (1) Perform the first GWA study of alcohol sensitivity and tolerance in the 192 DGRP lines to identify polymorphisms associated with alcohol sensitivity and tolerance, use novel statistical models that combine GWA analyses and multiple regressions to generate genotype-phenotype predictions, and validate such predictions by genotyping individual flies from an advanced intercross population derived by crossing DGRP lines with extreme phenotypic values; (2) Combine transcript abundance data from extreme DGRP lines obtained by deep RNA sequencing with GWA information to derive causal genetic networks affecting alcohol sensitivity and tolerance and test network predictions using targeted disruption of hub genes through mutational analysis or RNAi knock-down; (3) Determine to what extent exposure to alcohol during development alters the genetic networks identified in Specific Aims 1 and 2, and identify causal variants associated with genotype by environment interaction for alcohol-related phenotypes. Aim 3 is motivated by our observation that previous chronic alcohol exposure as larvae modulates sensitivity to subsequent alcohol exposures as adults. The proposed studies will result in a comprehensive systems genetics blueprint of the genetic architecture of alcohol sensitivity in Drosophila on which human orthologues can be superimposed to generate novel hypotheses that designate candidate hub genes for future follow-up studies.

Initiative for Maximizing Student Diversity in Biomedical and Behavioral Sciences

Trudy F. MacKay, David M. Shafer, Erin Banks, Carrie Frederick McLean, Karrie Gibson Dixon

National Institutes of Health (NIH)

$5,197,369

04/01/08 - 03/31/18

Achieving a diverse biomedical research workforce is important to effectively advance knowledge and create innovative technologies and treatments. To reduce healthcare disparities and to bring critical social and cultural perspectives to bear on issues related to biomedical research among groups who are typically underrepresented in the research arena, diversity among research professionals is crucial. The Initiative for Maximizing Student Diversity (IMSD) uses a two-tier approach to increase the number of students from underrepresented groups who receive PhD degrees and engage in research in the biomedical and behavioral sciences. The first tier supports and trains 25 undergraduate students by: 1) providing courses and seminars to introduce students to explore potential careers in biomedical and behavioral sciences; 2) substantially enhancing our current educational and professional development activities; 3) expanding support for mentored research opportunities; and 4) training and preparing them to enroll in, and complete, a PhD program in biomedical or behavioral research. The second tier funds assistantships for 30 graduate students in biomedical and behavioral research. Graduate students receive two years of financial support from IMSD, with subsequent support coming from their respective departments, external funding, or individual research fellowships. IMSD fosters a positive exploratory environment with supportive programming, guidance and recognition for students from underrepresented groups to complete a PhD in biomedical and behavioral science research and ultimately become the faculty researchers and role models for the next generation.

Systems Genetics of Drosophila Life Span

Trudy F. MacKay, Robert R. Anholt

National Institutes of Health (NIH)

$1,503,637

09/01/13 - 05/31/18

The world population is rapidly growing older, and population aging will be one of the most important social and health problems in the coming half-century. Life span is a typical quantitative trait, with natural variation attributable to segregating variants at multiple interacting loci, the effects of which are sensitive to the environment to which the individuals are exposed. However, only a handful of candidate genes associated with variation in life span in human populations have been identified. In this application we propose to identify evolutionarily conserved genetic networks causally associated with life span by using the powerful Drosophila model system, which enables us to accurately measure life span while precisely controlling both genetic background and environmental conditions. We propose an integrated systems genetics analysis of life span using the Drosophila melanogaster Genetic Reference Population (DGRP), which consists of wild-derived inbred lines with sequenced genomes. The lines are genetically variable for all phenotypes measured to date, including life span. The Specific Aims of this application are: (1) To map causal alleles associated with variation in life span with high resolution using a combination of genome wide association and linkage mapping; (2) To derive causal transcriptional co-expression networks affecting life span, placing novel loci identified by genetic mapping in appropriate biological context, and to use allele specific expression to test the systems level predictions; and (3) To use mutations and RNAi to functionally test effects on life span of genes implicated by the statistical analyses of natural variation and architecture of transcriptional networks, and use the recently developed system for integrating transgenes in the same genomic location to perform tests for causal effects of natural alleles. Results from these studies are likely to uncover novel genes and evolutionarily conserved cellular pathways associated with variation in life span. Because many genes in Drosophila have human orthologues, general insights derived from our proposed studies will have translational implications for human genetic studies on life span; moreover, we argue that insights derived from systems genetic studies of life span will have a broad impact on our general understanding of quantitative traits.

Dissertation support (Barrington and Kanavy)(Garbutt and Bryant)

James W. Mahaffey, Christopher R. Gould

Texas A&M University, Health Science Center

$140,334

01/01/14 - 08/31/17

David Threadgill recently moved his lab to Texas A&M University. Two students, whom had both completed their prelim exams in the genetics PhD program, moved with the lab to complete their research for their degree within the NCSU genetics program. Dona Kanavy and William Barrington will remain NCSU student but will be paid their stipend at Texas A&M as research assistants. Their tuition and fees will be paid through this subcontract. At Texas A&M, they will continue on their projects which are to develop a sterile mouse line (Dona Kanavy) and to investigate the role of ethnic diets on colon cancer incidence (William Barrington). Two other students, Shante Bryant and Tiffany Garbutt remained here at NCSU. They will continue their research into identifying genes governing naïve and primed stem cell states in two non-permissive mouse strains (Tiffany) and examining the effect of genetic background on a mouse model for inflammatory bowl disease. Their tuition, fees, benefits and stipends will be paid from this subcontract (Shante).

Integration of Appendage and Hox Networks During Drosophila Development

James W. Mahaffey

National Science Foundation (NSF)

$698,078

05/15/09 - 04/30/15

Understanding the genetic control of animal body patterning has been a major goal of developmental geneticists, and his is the long-term goal of the Mahaffey lab. Drosophila has proved to be an excellent model system to study development. Genes controlling many various aspects of animal development have been identified first from studies of the fly. Now, one major goal is to assemble these genes into pathways and networks to understand how they work together to bring into being a complete organism. Animal development is an interactive process. Recent results from the Mahaffey lab have led them to a new hypothesis integrating Hox- specification segment type and the proximal/distal (P/D) genetic network of segment development. All segments of an insect have a proximal/distal axis, most notable on segments that have appendages, though the centerline of the body outward must be included, yet each segment and appendage is different along the anterior to posterior axis Appendages can be tailored for feeding, walking, mating etc, a process controlled by the Hox genes. From a developmental standpoint, there must be an integration of the Hox and P/D developmental pathways, and this is true from a molecular standpoint as well. Many of the known and proposed ?Hox cofactors? are members of the P/D network. One hypothesis is that the Hox proteins are, in fact, the real cofactors. The P/D transcription network establishes the gene expression necessary to produce a (somewhat) generic segment and appendage, but the Hox proteins modify (tweak) this expression yielding the differences observed in each segment and appendage. Since many segments and their appendages are quite similar in overall design, similar genes would be activated, but the Hox genes would modify this to generate differences. The critical point is, that targets of the Hox genes (which have been elusive) are in fact genes regulated by the P/D transcription network. There are several predictions from this hypotheis that will be addressed. Ectopic co-expression will be used to test whether different combinations of P/D and Hox factors will have different but predictable consequences on development. Clonal analysis and ectopic expression will be used to examine where the Drosophila Disconnected and Disco-related proteins fit into the P/D network. Though functionally redundant, these proteins are quite different outside of the conserved zinc finger region. Since it is unlikely that these proteins function alone, we will take advantage of their differences to identify interacting factors. Finally, to identify genes that are responding to (targets of) the P/D network, a genome-wide population-based assay will be used to identify genes that cause variation in second leg morphology. These will then be assayed to determine if their expression is modified under different Hox conditions. Hox genes control body patterning in all metazoans, and the indicating conserved mechanisms of animal pattern formation. Many of the patterning genes are also implicated in cancers and developmental defects, so understanding these mechanisms will provide new clues to human health issues. This study provides an opportunity to learn about the interactive natures of transcription factors and the evolution of gene networks. The proposed experiments provide an opportunity for students at many levels, from high school to graduate students) to participate in an interactive research program. Results obtained from this study will be disseminated through publications and presentations at local and national meetings.

Advancing Mechanism-Based Studies with Cross-Species Chemical-Phenotype Data

Carolyn Mattingly

National Institutes of Health (NIH)

$1,005,881

01/01/15 - 12/31/19

Most human diseases involve interactions between genetic and environmental factors. While the environment is implicated in many common conditions and adverse toxicological outcomes such as asthma, cancer, diabetes, hypertension, and immune deficiency disorders, the molecular mechanisms underlying these environment-disease connections are not well understood. The Comparative Toxicogenomics Database (CTD; http://ctdbase.org) is the only freely available resource that provides high quality manually curated information key to informing hypotheses about chemical mechanisms and underlying etiologies of environmentally influenced diseases. It is a well-established resource with a strong and expanding user base. Because of its solid foundation, CTD has been able to evolve to meet the changing needs of the environmental health research community by expanding the scope of curation and ongoing development of analytical tools. With support form a previous Big Data to Knowledge (BD2K) supplement, we integrated CTD data with ToxCast/Tox21 high-throughput screening (HTS) data (from the Environmental Protection Agency and the National Toxicology Program) to expand the depth and biological context of each resource, respectively. This proposal will specifically build on that work. The aims of this project are to: 1) expand the depth and context of mechanism-based environmental health data by integrating CTD data, HTS data, and gene-phenotype information from select model organism databases (MODs); and 2) assess the added value of integrating gene-phenotype data from MODs with environmental health information through the adaptation of existing computational tools. We expect this project to provide environmental context to MOD data (something that is currently lacking), further expand the ability to conduct cross-species and mechanism-based toxicological comparisons, extend the value of the constituent data from these resources and existing analysis tools for understanding how environmental exposures affect human health, and facilitate broader use of MOD data and its integration and analysis within other Big Data initiatives.

Generation Of A Centralized And Integrated Resource For Exposure Data

Carolyn Mattingly

National Institutes of Health (NIH)

$1,169,136

01/16/12 - 12/31/16

Our objective is to provide a centralized, publicly available resource with comprehensive, well-annotated data and analysis tools that informs design and interpretation of environmental health studies and promotes novel insights into the etiologies of environmentally influenced diseases. Most human diseases involve interactions between genetic and environmental factors; however, the basis of these complex interactions are not well understood and limit improvements in toxicity prediction, risk assessment, research prioritization and therapeutic interventions. We developed the Comparative Toxicogenomics Database (CTD; http://ctdbase.org) to enhance understanding about environment-disease connections by providing manually curated data describing chemical-gene/protein interactions and chemical- and gene/protein-disease relationships from the peer-reviewed literature and integrating these data with select external data sets (e.g., pathways and biological process data) and novel data analysis tools. In this application, we propose to leverage our expertise and CTD infrastructure to: 1) enhance the capacity to identify environment-disease connections by curating and integrating exposure data into CTD; and 2) expand the capacity for prediction, analysis and interpretation of environment-disease networks by developing novel analysis and visualization tools that include exposure data. This proposal responds to the needs expressed by the NIEHS and partner agencies for inclusion of exposure data when prioritizing research and performing toxicity testing, it addresses the need for centralization of exposure data in a broader biological context and it will provide ?real-world? exposure context for existing data in CTD. The resulting resource will enable new opportunities for understanding and prioritizing human health effects from exposure and their underlying etiologies and coordinate data key to enhancing the capacity for toxicity prediction and risk assessment.

The Comparative Toxicogenomics Database (CTD)

Carolyn Mattingly

Mount Desert Island Biological Laboratory

$1,904,169

01/01/12 - 11/30/16

The long-range goal of the proposed project is to provide a centralized, freely available resource with comprehensive, well-annotated data and analysis tools that informs hypothesis development and interpretation of environmental health studies and promotes understanding about the etiologies of environmental diseases. Most human diseases involve interactions between genetic and environmental factors. The environment is implicated in many common conditions such as asthma, cancer, and diabetes; however, the etiology of these widespread diseases remains unclear. More than 85,000 chemicals are currently used in commerce, challenging elucidation about chemical mechanisms of action and prioritization of environmental research. Integration of critical data with novel analysis approaches is required to understand environment-disease associations and is essential for improving toxicity prediction, risk assessment, regulation and development of effective therapeutic interventions. We developed the freely available Comparative Toxicogenomics Database (CTD; http://ctd.mdibl.org) to address this need. CTD provides manually curated data describing cross-species chemical-gene interactions and chemical- and gene-disease relationships from the peer-reviewed literature and integrates this information with select external data sets (e.g., molecular pathways) and novel analysis tools. In this application we propose to: 1) comprehensively curate chemical-gene-disease interactions and expand the scope of phenotype curation to include cellular and diverse organism effects that will enable users to: a) identify biomarkers of environmentally influenced diseases and b) infer potential human health consequences from toxicological studies in model organisms and in vitro studies; and 2) design and implement new tools to facilitate development, analysis and interpretation of novel hypotheses focused on chemical-gene-disease interaction networks. This proposed project will leverage our cutting-edge software development, curation expertise and well-established, flexible infrastructure to facilitate increased understanding of critical environmental health issues in direct alignment with emerging research priorities.

Tox-Evaluator: An Integrated Computational Platform to Aid the Interpretation of Study-Related Findings

Carolyn Mattingly

Pfizer, Inc.

$146,050

12/15/12 - 06/30/15

Tox-Evaluator is envisioned as a web-based, hypothesis-generating resource to help pathologists and toxicologists investigate toxicity findings in exploratory and regulatory animal studies. Importantly, it will be designed to leverage existing computational tools (i.e., CTD, TargetScan, polypharmacology models, Tox Reporter, etc.) but will integrate them such that the underlying tools will be customized to communicate with each other to draw conclusions that otherwise cannot be generated by running the individual tools in isolation. The proposed solution will also make the tools readily accessible to users by providing a simple interface to enter information about the compound, the finding, and the intended target, with output that is collated in a simple, organized report format. Ultimately it could become a “smart” or Artificial Intelligence interface that generates plausible biological connections (i.e., hypotheses) among chemical structure, target modulation, and observed pathology. The activities outlined in the original research agreement have been completed. Additional functionality and modifications were identified by Pfizer users that have further improved the value of the tool.

The Role of Human DNA Polymerase Eta in the Mutagenic Response to Oxidative Stress

Scott McCulloch

National Institute of Environmental Health Sciences (NIEHS)

$1,620,162

08/01/09 - 07/31/15

Oxidative stress is caused by a number of environmental agents such as polycyclic aromatic hydrocarbons like dioxin, heavy metals, pesticides, and ultraviolet and ionizing radiation. Reactive oxygen species are created by normal cell processes and in elevated levels by oxidative stress. They cause multiple types of DNA lesions, the most common being 8-oxo-guanine (8-oxo-dG). This lesion is highly mutagenic due to its ability to base pair with the incorrect base adenine in a manner that escapes detection by the proofreading activity of the replicative polymerases. It is also blocks DNA synthesis by the replicative polymerases alpha, delta and epsilon, which have difficulty synthesizing past most DNA damage. Translesion synthesis (TLS) by specialized polymerases of the Y-family is therefore a necessary component of DNA replication. Direct bypass of damage allows completion of DNA replication in the face of lesions that would otherwise stall the replication fork, collapse of which can lead to strand breaks and other gross chromosomal changes. TLS past several DNA lesions by Y-family members occurs with higher efficiency compared to replicative polymerases, making them the preferred choice for ensuring replication is completed, even though the fidelity of many TLS polymerases is relatively low. Bypass of the UV-light induced thymine-thymine dimer is performed by the Y-family member polymerase eta and occurs with high efficiency but somewhat low fidelity, with errors generated during 3-5% of bypass events. These errors can be detected and removed by replicative polymerase proofreading activities, allowing pol eta multiple chances to insert the correct base, if needed. This allows the overall pol eta dependent bypass reaction to suppress mutations caused by UV light. A similar logic has been proposed for the suppression of mutagenesis during pol eta dependent bypass of 8-oxo-dG. Recently, we demonstrated that human pol eta does indeed perform highly efficient bypass of 8-oxo-dG, but also observed that the bypass occurred with remarkably low fidelity. Errors were generated during ~50% of all bypass events. The most frequent error was adenine misinsertion opposite 8-oxo-dG, which as noted above frequently goes undetected by the proofreading activities of replicative polymerases due to its structural similarity to a correct base pair. This implies that the mechanism by which pol eta suppresses mutagenesis caused during 8-oxo-dG bypass differs from that of UV-light induced damage. We hypothesize that the observed suppression of mutagenesis by pol eta during 8-oxo-dG bypass occurs by an increase in the fidelity of the polymerase by association with additional replication proteins. We aim to determine which factors may influence the fidelity of the bypass reaction and also to determine what role, if any, pol eta plays in the generation of mutations during times of oxidative stress when the number of 8-oxo-dG bypass events would be greatly elevated. The goals of this proposal are: 1) to determine the effects of replication proteins on the efficiency and fidelity of 8-oxo-dG bypass by human pol eta; 2) to reconstitute and characterize the ?complete? 8-oxo-dG lesion bypass reaction in vitro; 3) to identify and characterize human pol eta mutants, including known SNPs, that display altered fidelity for 8-oxo-dG bypass; and 4) to determine the mutation rate of wild type and pol eta deficient cells, and cells expressing mutant forms of pol eta, under conditions of oxidative stress. The long term objectives of this proposal are to determine the molecular mechanisms that modulate the efficiency and fidelity of 8-oxo-dG bypass in human cells, and ultimately the mutagenesis caused by oxidative DNA damage.

Collaborative MyD88 Pilot Study

Jennifer C Miller

University of New Haven

$6,012

07/01/14 - 08/31/14

: In the U.S, the spirochete bacteria Borrelia burgdorferi are transmitted to humans and other animals via the bites of Ixodes scapularis ticks. Infection with B. burgdorferi causes Lyme disease, clinical signs of which can include arthritis, carditis, and various neurological sequelae. If timely antibiotic therapy is not initiated, the bacteria cause a chronic infection that can persist for years. Unfortunately, in a subset of patients, clinical signs persist despite rigorous antibiotic therapy. The virulence mechanisms utilized by B. burgdorferi to evade the human immune response to establish a chronic, persistent infection are not well understood. One mechanism utilized by bacteria to shield themselves from both antibiotics and the host immune system is the formation of biofilms. A biofilm is an exopolysaccharide matrix secreted by an aggregated bacterial population that envelops them, thereby shielding the bacteria from the deleterious effects of antibiotics and the host immune system. B. burgdorferi was recently shown to form biofilms in vitro when grown to high densities. This interesting finding has generated much discussion and controversy, and it is currently unknown whether B. burgdorferi form biofilms when inside an animal host. Discovery of the mechanisms utilized by B. burgdorferi to cause persistent infection could lead to the development of novel targeted therapeutics for the treatment of patients suffering from the debilitating effects of “Chronic Lyme Disease,” as dubbed by the lay press.

Subcontract: A Proposed Pilot Study to Examine the in Vivo Effects of a Proprietary Agile Sciences, Inc Compound on Murine Lyme Disease

Jennifer C Miller

Bay Area Lyme Foundation

$87,351

10/16/13 - 07/15/14

This subcontract will utilize the mouse model of Lyme disease to evaluate the efficacy of a proprietary compound from Agile Sciences, Inc. to prevent/reduce bacterial multiplication/dissemination within host tissues or to mitigate the pathology/inflammatory response associated with Lyme disease.

Molecular Monitoring In Aiptasia, Rapids And Sds-page Exercises For Four Different Undergraduate Laboratories

Marianne Niedzlek-Feaver

Association for Biology Laboratory Education (ABLE)

$1,465

04/01/15 - 09/30/16

Purpose: I plan to use RAPDs (Random Amplification of Polymorphic DNA) and SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) electrophoresis to type Aiptasia populations. The DNA fragment or protein bands would initially be used as classification markers for accessing relatedness in Aiptasia and various Cnidarians kept in our salt water aquaria. The banding patterns would also be used in new laboratories being developed for three courses. In the laboratory being developed for our Parasitology course, “parasitized “ organisms (actually Aiptasia with mutualistic symbionts) would be compared to those “not parasitized” (Aiptasia without symbionts). In the Evolution (and Darwinian medicine) laboratory, clones of Aiptasia would be established, and the amount of genetic variability maintained over time monitored. All will serve to introduce molecular monitoring to students in traditional “oology” classes. In all of these classes, molecular tracking or identification is extensively discussed in lecture but no experience with such techniques is furnished in the laboratory portion of the course.

Determining the Cause and Control of Internal Necrosis in Sweet Potatoes

Jonathan R. Schultheis, Michael D. Boyette, Katherine Mary Jennings, George C. Yencho, Zvezdana Pesic-Van Esbro, Lina Maria Quesada , Anna Stepanova, Allan C. Thornton, Dahlia Nielsen

Golden Leaf Foundation

$127,931

07/01/14 - 03/31/16

Since 2006, a disorder has been found in sweetpotatoes that causes internal discoloring and necrosis. Outbreaks of this disorder has occurred in specific businesses in North Carolina where the incidence has exceeded 50% while other growers may have only 2% or less occurrence. Complicating this problem, is that with this disorder no visible symptoms occur on the outside of the roots, making it impossible to detect by evaluating the outside of sweetpotato roots. Thus, roots with the disorder can reach the customer without detection. This has occurred with customers of grocery stores and restaurants after sweetpotatoes are cooked. Already some sweetpotato buyers have refused to buy Covington sweetpotato variety from North Carolina in fear of this disorder. We believe that the approximately $200 million dollar North Carolina sweetpotato industry is threatened by this disorder and an emergency situation currently exits. Continued shipment and not knowing the manner to prevent and/or understand the cause of internal necrosis could very negatively impact the North Carolina sweetpotato industry if not solved. This disorder has been studied since its appearance to find the cause(s) and develop remedies. However, despite much work, the cause of internal necrosis (IN) has not yet been determined. Three hypotheses have been developed after preliminary research was completed in 2012: combination of pre‐harvest and post‐harvest factors, ethylene, and disease. The aim of this proposal is to investigate these hypothesis to help understand the cause and ways to remedy the situation.

Development and Application of Novel Glycan-Specific Reagents to Facilitate Early

David C. Muddiman, James N. Petitte, Dahlia Nielsen, Adam M. Hawkridge

National Institutes of Health (NIH)

$1,124,374

09/01/11 - 08/31/15

This proposal seeks support to develop and apply novel tags that will facilitate both global and targeted quantitative mass spectrometric analysis of N-linked glycans with significantly improved limits-of-detection. Once synthesized, the novel tags and ancillary methods will concurrently be applied to the experimental chicken model of spontaneous epithelial ovarian cancer for comparative analysis (i.e., biomarker discovery) and fully disseminated to the glycomics research community. The long-term application of these novel tags, as developed in our laboratory for this study, will be their use to elucidate glycan biomarkers for the early detection of epithelial ovarian cancer.

GTSPI: Genomic Tools for Sweetpotato Improvement

George C. Yencho, David Baltzegar, Lina Maria Quesada , Dahlia Nielsen, Zhao-Bang Zeng, Fred Andrew Wright, Jennifer Schaff

Bill and Melinda Gates Foundation

$12,409,389

08/29/14 - 08/31/18

This project will develop modern genomic, genetic, and bioinformatics tools to facilitate crop improvement and improve genetic gains in sweetpotato, an important food security and cash crop with highly recognized potential to alleviate hunger, vitamin A deficiency, and poverty in Sub-Saharan Africa (SSA), and predominantly grown in small plot holdings by poor women farmers.

The Identification of a Metastatic Signature in Canine Soft Tissue Sarcomas

Marlene L. Hauck, Dahlia Nielsen

Morris Animal Foundation

$141,751

10/01/09 - 05/31/16

Soft tissue sarcomas (STS) are a common solid tumor in dogs. Current treatment for the primary disease is surgical excision, with the addition of radiation in the case of incompletely excised tumors. The inclusion of chemotherapy is indicated when tumors are judged to be high grade. Several grading schemes have been developed for canine STS in an attempt to identify dogs at risk for developing metastatic disease. Reported rates of metastatic disease range from 15-41% for high grade tumors. Given the relatively high rate of metastasis in high grade STS, adjuvant chemotherapy is often recommended. Unfortunately, the application of histologic grading schemes can be highly variable between pathologists, lessening the usefulness of STS grading schemes in clinical recommendations. Difficulty in predicting which veterinary patients may benefit from adjuvant chemotherapy is thereby confounded by improper identification of patients at a risk. Currently, at North Carolina State University we offer adjuvant chemotherapy to all patients with high grade sarcomas as read by any pathologist. Our confidence in this recommendation is compounded by the lack of information as to the impact of adjuvant chemotherapy on the development of metastasis, as well as the knowledge that doxorubicin, the most commonly used agent, is not benign?with cardiotoxicity in addition to the routine GI toxicity and myelosuppression. In order to determine if adjuvant chemotherapy impacts disease free interval and survival, one must first be able to better identify the patients likely to develop metastasis. In human medicine, the use of gene expression signatures to better diagnose and prognose has been demonstrated in a number of tumors, including sarcomas. Importantly, gene expression signatures have been shown to independently predict the occurrence of metastasis. Recent studies have demonstrated that the metastatic signature is present in the ?bulk? of the tumor, and is not a characteristic of a minority of cells. Lately, a similar approach to identifying a prognostic signature has been successfully applied utilizing a proteomics approach. Currently, clinical trials lack the ability to precisely identify the dogs most likely to benefit from adjuvant chemotherapy if a trial to determine such a benefit were performed. Such a trial would require hundreds to thousands of patients in order to determine if any such benefit exists. Ultimately, identification of a gene and protein signatures predictive for the development of metastatic disease will not only improve the treatment of individual patients through the more appropriate administration of chemotherapy, but it would allow for the determination of the efficacy of adjuvant treatment and could also identify potential therapeutic targets for the prevention of metastasis.

The Plant-Pathogen Interactome: Identifying Cross-Species Gene Networks

Dahlia Nielsen, Jennifer Schaff, David M. Bird

National Science Foundation (NSF)

$1,557,138

09/01/10 - 08/31/14

In this proposal, we will examine the relationship between a plant (Medicago truncatula) and a root knot nematode pathogen that infects that plant's roots. Our goal is to observe how the genetic code of the pathogen influences the biological responses of the plant, specifically at a gene transcription level. To address this question, we implement a technique known as expression quantitative trait locus (eQTL) mapping, which combines gene expression studies together with genetic linkage mapping. This technique is designed to investigate connections between DNA sequence changes and gene expression levels, in a way that compares each region of the genome to every expressed gene. We are transforming this approach to allow a cross-species investigation, in which each each region of the pathogen genome is compared with each expressed plant gene. We follow up on our results from this analysis with gene network identification, in order to characterize more complex cross-talk between species. The results of this study will be fundamental to furthering our understanding of not just plant defenses and susceptibility, but also the myriad of ways in which the pathogen influences the physiology and development of its plant host.

Evaluation of the Campylobacter Jejuni Multi-Copper Oxidase as a Drug Target

Jonathan W. Olson

NCSU Faculty Research & Professional Development Fund

$4,000

07/01/14 - 06/30/15

Campylobacter jejuni is responsible for the majority of foodborne gastroenteritis cases worldwide. Infection is typically attributed to consumption or handling of raw or undercooked poultry, in which C. jejuni is considered a commensal. Poultry have a high tolerance for copper, and C. jejuni has developed mechanisms to ensure cellular copper levels are kept low even in the face of high copper diets. We have found that disruption of genes responsible for copper homeostasis renders C. jejuni susceptible to copper and less able to deal with added reactive oxygen species. One gene in particular (cue:O) has shown a remarkable sensitivity to copper and an inability to successfully colonize chickens. The goal of the proposed research is to characterize the enzyme expressed via cueO (the multicopper oxidase MCO) and determine if inhibitors can successfully suppress host colonization of the wild-type organism. The knowledge gleaned from this proposal could be used for the development of novel antimicrobial alternatives to reduce or eliminate C. jejuni from poultry prior to slaughter

Interaction of BPA and Soy Isoflavones on Sociosexual Behavior

Heather B Patisaul

National Institutes of Health (NIH)

$435,357

05/08/12 - 12/31/15

The behavioral testing related to this project is ongoing.

Metabolic Disrupting Actions of the Flame Retardant Mixture Firemaster 550

Heather B Patisaul

NCSU Center for Human Health and the Environment

$21,320

01/01/14 - 12/31/14

Project Abstract: Although lifestyle factors are clearly primary contributors to rising obesity rates, fetal metabolic reprogramming by environmental chemicals called “obesogens” has been hypothesized to exacerbate risk. Data generated by the PI and her collaborators have identified the newly introduced fire retardant mixture Firemaster® 550 (FM 550) as an emerging contaminant in US homes, and shown that perinatal exposure to FM 550 (in rats) at environmentally relevant levels results in obesity, altered exploratory behavior, and hallmarks of metabolic syndrome including disrupted glucose sensitivity and cardiac hypertrophy. Building upon these findings, proposed experiments will test the hypothesis that these effects result from disregulation of metabolic set points. We will (1) characterize the activity of FM 550 and its four primary components at the thyroid and retinoic acid (RXR) receptors in vitro; and (2) establish how FM 550 impacts neuroendocrine pathways related to appetite, metabolic set points, and feeding behaviors in vivo by assessing exposure-related transcriptome changes (via RNAseq) in the arcuate nucleus; the brain’s “appetite control center.” Understanding the contributions of each FM 550 component is critical because some also have large volume applications as plasticizers in a wide variety of products (e.g. polyvinyl chloride (PVC), circuit boards, hydraulic fluids, adhesives). These projects are consistent with CHHE goals because they are interdisciplinary, novel, and could uncover new mechanisms of obesogenic activity. Importantly, the studies complement ongoing work by the PI and her collaborators seeking to understand FM 550 toxicokinetics, identify a biomarker of FM 550 exposure, and develop methods for modifying these compounds to preserve their fire retardant function but reduce their potential toxicity.

Neurobehavioral Effects of Bisphenol A Across Age and Sex

Heather B Patisaul

National Institute of Environmental Health Sciences (NIEHS)

$774,877

09/19/11 - 05/31/17

This is the required progress report for the conclusion of year 3 of a 5 year grant from the NIH exploring the neural and behavioral impacts of BPA exposure.

Toxicokinetics and Metabolic Disrupting Actions of the Flame Retardant Mixture Firemaster 550

Heather B Patisaul, Scott Belcher

National Institutes of Health (NIH)

$299,959

09/10/14 - 09/09/17

Obesity rates in the US are rapidly climbing and it has been hypothesized that early life exposure to chemicals which act as obesogens may be contributing. The goal of this research is to test the hypothesis that early life exposure to a newly discovered flame retardant mixture, Firemaster 550, induces sex-specific metabolic reprogramming which then results in a higher risk of obesity and cardiovascular disease later in life. We will also explore the mechanisms by which this might occur, and the individual contributions of each component of the mixture. This work will contribute new data regarding the potential health impacts of this fire retardant mixture and the mechanisms by which obesogenic chemicals induce metabolic disease.

Visiting Scientist Agreement for Scott Belcher

Heather B Patisaul

University of Cincinnati

Unfunded

01/01/15 - 06/01/15

Visiting Scientist Agreement for Scott Belcher

A Zebrafish Research Core at NCSU to Advance Basic, Environmental and Biomedical Research.

Antonio J Planchart, Carolyn Mattingly, John R. Godwin, Jeffrey A. Yoder, Seth W Kullman, Heather R. Shive

NC Biotechnology Center

$196,752

05/01/14 - 11/30/15

The goal of this proposed project is to establish a state-­‐of-­‐the-­‐art zebrafish research core facility at North Carolina State University (NCSU) to 1) advance basic, environmental and biomedical research; 2) increase the competitiveness of our faculty to secure extramural funding; 3) strengthen present and cultivate future collaborations; 4) provide opportunities to grow research programs and recruit new faculty; and 5) enable the potential for collaborative development of new technologies using the zebrafish model. The zebrafish (Danio rerio) has emerged as a critically important model for human biomedical research. Initially established as a model for genetics and developmental biology, its applications have extended to many cutting edge areas such as cancer biology, drug discovery, regeneration, epigenetics and understanding the underlying mechanisms of diverse human diseases from cardiovascular to neurodegenerative conditions. Zebrafish are highly fecund with short generation times of 3-­‐5 months. Eggs can be obtained in abundance (hundreds per female in a single day). Zebrafish eggs are fertilized externally, are relatively large (0.6 mm), transparent and readily manipulated by microinjection techniques. Rapid development from a zygote to the hatching period (~48 hours) provides many advantages over mammalian models for observation throughout organogenesis and for dissecting the molecular events that regulate diverse developmental processes. Zebrafish are significantly more economical to maintain than mammalian models (<1%), enabling many more experimental opportunities. Gene transfer procedures, in situ hybridization protocols, and morpholino-­‐based knockdown technology have been refined and are well-­‐accepted experimental approaches for zebrafish. Despite the evolutionary distance (~400 million years), its genetics and signaling pathways are highly conserved with humans. The combination of these features makes the zebrafish uniquely suited for high impact discovery-­‐based and technologically innovative biomedical research. NCSU recognizes the importance of this model for high impact research and technology development. In recent years, the University has been building a critical mass of faculty using zebrafish that currently focus on craniofacial development, neurodevelopment, immune system function and toxicology. Currently, these faculty and their resources are distributed across three colleges despite significant overlap in interests and resources. In order to coordinate research and training activities, this group self-­‐organized in 2012 and have been conducting cross-­‐ laboratory meetings on a monthly basis to develop plans to improve communication and resource sharing. This proposal stems from these activities and reflects the collaborative potential and commitment by each faculty member (including a recent recruit that will begin at NCSU in January 2014) to the importance of building a centralized resource at NCSU. Successful funding of this proposal and establishment of a centralized zebrafish core facility will have an immediate positive impact on the productivity and research potential of a critical mass of NCSU faculty. It will directly improve cost-­‐ and resource sharing and facilitate new collaborations that will better leverage the collective investments and expertise of our faculty.

Cardiotoxicity Adverse Outcome Pathway: Organotypic Culture Model and In Vitro/ In Vivo Extrapolation for High-Throughput Hazard, Dose-Response and Variability Assessments

Fred Andrew Wright, David Michael Reif, Yihui Zhou

Texas A&M AgriLife Research

$481,597

06/01/15 - 05/31/19

The central hypotheses of this proposal are that: (i) stem cell-derived cardiomyocyte cultures constitute an effective organotypic culture model for predictive toxicity screening of environmental chemicals; (ii) a population-based experimental design utilizing a panel of human iPSCs and mouse Collaborative Cross (CC) can assess variation in toxicity to better characterize uncertainties; and (iii) integration of dosimetry with screening provides an in vivo context to in vitro data and improves human health assessments. Project 1 will conduct population-based concentration-response high-content/-throughput in vitro screening of up to 200 ToxCast chemicals in iPSC-derived cardiomyocytes from 100 humans and collect pharmacokinetic data using hepatocytes. Project 2 will conduct mouse population-based in vitro screening of these chemicals in CC ESC-derived cardiomyocytes followed by in vivo validation in the CC strains. Project 3 will conduct dose-response modeling to establish appropriate point of departure, genome-wide association analyses and in vitro-to-in vivo extrapolation modeling.

Elucidating Risks: From Exposure and Mechanism to Outcome

Fred Andrew Wright, David Michael Reif

UNC - UNC Chapel Hill

$371,859

08/31/13 - 03/31/17

PROJECT SUMMARY The Biostatistics Core is designed to enhance the UNC Superfund Research Program's assessment and reduction of risks to human health associated with Superfund high priority chemicals. The five projects in the SRP present considerable biostatistical issues and bioinformatics challenges that are central to the success of the projects. Our overall aims are to (i) provide state-of-the-art biostatistics and bioinformatics expertise, end-user analytical support, and tool development; (ii) to develop new methods and tools as necessary to address project objectives; (iii) to foster a unique training environment for students, postdoctoral fellows, and faculty to prepare them for the new and complex challenges presented by modern datasets. The Core faculty and staff have a range of complementary skills, which will lead to a unified approaches to data interpretation, integration and cross-platform analysis. Collectively, the Core is highly relevant to the Superfund Program, as it will: • support interdisciplinary (toxicology, genetics, biostatistics, pharmacokinetic modeling) research to elucidate the genetic basis of dose-response and susceptibility; • develop and use state-of-the-art statistical techniques and analysis tools for systems biology approaches; • identify potential biomarkers linked to genetic differences in toxicant metabolism and/or response; and • generate knowledge that is directly applicable to quantitative elucidation of risk. The Core will be involved at the earliest stages of each project, assisting investigators in each step of planning and executing the projects. Each Core faculty member is assigned a role to two projects. A staff statistician with considerable bioinformatics experience will serve as a dedicated analyst, under the guidance of Core faculty members. The Core director will report to the SRP Director, and members, who already collaborate extensively, will meet regularly with the investigators and with each other to plan and exchange ideas.

System Toxicological Approaches to Define Flame Retardant Adverse Outcome Pathways

David Michael Reif

Oregon State University

$139,108

06/01/15 - 05/31/18

Objective: A team of researchers from Oregon State University and North Carolina State University proposes to conduct the first comprehensive in vivo toxicity studies of flame retardant chemicals (FRCs), including FRCs that EPA has banned, FRCs that companies manufacture now, and FRCs that companies have proposed as alternatives. We will test the hypothesis that the toxicity of FRCs will be highly dependent on their chemical structure. Experimental Approach: We will expose embryonic zebrafish to FRCs and observe their morphology and behavior for signs of toxicity. We will also grow exposed zebrafish to adulthood and observe their morphology and behavior for signs of toxicity. We plan to discover which FRCs have the lowest hazard potential and to rank the others according to their toxicity. With the identified phenotypic anchors, we will conduct whole-transcriptome analyses to define the early expression changes and pathways underlying the adverse outcomes to the toxic FRCs. in FRC outcomes across the levels of biological organization, i.e, chemical structure, similarity of gene expression profiles, early and adult life stage adverse outcomes, and thereby define adverse outcome pathways (AOP) for mechanistic FRC hazard prediction. We plan to bin FRCs according to their mode of action and to discover how an FRC’s mode of action depends on its structure, thereby gaining the ability to predict the toxicity of new compounds. We will develop new tools that enable manufacturers and risk assessors to determine the likelihood that a new compound is safe. Expected Outcome: The proposed toxicity screening results will help EPA prioritize chemicals for further testing and may also alert chemical manufacturers that some of their commercial products may be toxic. The identified AOPs will improve the research community’s ability to translate zebrafish results to other species. If high-throughput, low-cost zebrafish testing closely replicates the results of rodent testing, then zebrafish testing, in combination with other assays, may eliminate the need for rodent testing, at least for certain classes of chemicals. The proposed work will help to establish a base of knowledge that will lead to novel cell-based assays that can reliably predict chemical toxicity without in vivo testing.

Transgenerational Actions of the Endocrine Disrupting Compound Bisphenol A

Emilie Francesca Rissman

National Institutes of Health (NIH)

$737,028

03/01/15 - 02/28/19

Many endocrine disrupting compounds (EDCs) have immediate effects on exposed individuals, but more complex concerns surround their long term actions on subsequent generations. The limited data from humans concur with the animal work suggesting that exposure to EDCs, particularly exposure during development, produce subtle increases in disease states, along with evidence for transgenerational effects. The precise epigenetic and/or genetic actions of EDCs on specific target genes, which produce stable modifications in subsequent generations, are unknown. The first goal of this program is to understand the mechanisms underlying transgenerational inheritance produced by human-relevant levels of the ubiquitous EDC, Bisphenol A (BPA). The work proposed is the first to test transgenerational actions of human-relevant exposures of BPA on brain and behaviors. Pregnant inbred mice are exposed to EDCs via voluntary ingestion of maternal diet. At birth, control and BPA pups are fostered to dams on control diet to isolate the actions of BPA to gestation and control for any consequences of these compounds on maternal behavior. We have shown that several social behaviors in the first and fourth generation (F4) of mice the BPA lineage differ significantly from control mice. Moreover, mRNA for two genes that regulate social behavior, vasopressin (Avp) and oxytocin (Oxt), are decreased in brains of the F4 BPA-exposed mice. Experiment one will determine the parent of origin for transmission of BPA's actions. In addition we will assess a number of cognitive and emotional behaviors in these mice to improve our ability to predict which human diseases may be sensitive to BPA. In the next study brains regions are probed with next generation sequencing techniques to establish target genes. Brains from F3 mice will be dissected into the nuclei that compose the social behavior network. A combination of Chromatin Immunoprecipitation (ChiP-) and RNA-sequencing will be conducted. These data provide targets for the epigenetic modifications found in brain. The mechanisms revealed will be applicable to other target tissues and thus a variety of diseases. This research plan will provide the field with a model for transgenerational actions of BPA and an epigenomic map of the consequences of these exposures.

A Novel Genetic Model of Dietary Response and Host-Microbiota Interactions

Reade Bruce Roberts

Arnold & Mabel Beckman Foundation

$562,500

09/01/14 - 08/31/18

Maladaptive diets contribute to major, pervasive health issues worldwide. When diets are chronically insufficient or nutrient-poor, development and immunity is impaired, and ultimately mortality can result. In contrast, the Western pattern diet commonly eaten in the United States is maladaptive in its excess, and is associated with pressing national health concerns, including heart disease, diabetes, and obesity. Increasingly, obesity and other metabolic syndromes are found in children, and serve as a predictor of poor life-long health and early mortality. The digestive tract processes the diet and absorbs nutrients, and provides important immune functions. The digestive tract does not function in isolation, rather it works in concert with complex microbial communities in order to properly, or improperly, function. Links have been made between host genetic variation and both metabolic disease and gut microbial communities, demonstrating that response to a maladaptive diet is impacted by genetics, commensal organisms, and their interaction. While a number of these associations have been made, a substantial portion of the human genetic variation explaining diet-related disease syndromes remains unknown. New approaches and model systems are needed to study natural genetic variation and its impact on digestive development, gut microbiota colonization, and susceptibility to metabolic disease. The proposed experiments will develop multiple cichlid fish species as a comparative model for such studies. The chosen species are closely related and interfertile, but have divergent gut morphology and adaptations to a range of diets, including specialization for processing all-vegetable and all-animal diets. These species will allow us to examine a broad range of untapped natural genetic variation underlying the impact of maladaptive diets on development, homeostasis, and gut microbial communities. In the proposed work we will create baseline data for the model system, and link developmental and metabolic phenotypes with insufficient and excessive nutrient diets, as well as the make up of the gut microbiota. We will also produce a unique hybrid cross between herbivorous and carnivorous species to maximize phenotypic variance for future mapping studies. Mapping in these families will be combined with comparative genomics strategies in natural populations to identify novel genetic factors involved in response to maladaptive diets, and thus new avenues for therapeutic intervention for diet-related disease. The proposed experiments will serve as the foundation for an expansive future body of work exploring the genetic basis of gut development, dietary response, and host-microbiota interactions.

Complex Evolutionary Genetics of Gonadal Differentiation in Classic Model of Behavior Astatotilapia Burtoni

Reade Bruce Roberts

National Science Foundation (NSF)

$829,606

05/15/15 - 04/30/18

Abstract: For the greater part of the past century, genetic sex determination research has largely focused on a handful of ancient, chromosomal sex determination systems. More recently, genetic sex determination has been mapped in a broader set of species, revealing a diverse catalog of sex determination genes in a variety of chromosomal contexts. Polygenic sex determination (PSD) has also been confirmed in multiple taxa, where multiple genetic factors segregate and interact to direct sexual development. Since multiple genotypic types of each sex are produced in PSD systems, “sex” is no longer a binary trait, and different phenotypic classes can exist within a sex. While strides have been made in cataloging a range of genetic sex determination mechanisms, little is known about how new sex determination alleles evolve, and especially how they interact stably with existing sex determination mechanisms. Indeed, the integration of multiple sex determination alleles during development remains unstudied. We will use the cichlid fish behavioral model Astatotilapia burtoni as a functionally tractable model of polygenic sex determination that can be studied in both laboratory and natural population contexts. We have recently identified three novel sex determination alleles in this species, and a combination of genetic mapping and comparative genomics will be used to characterize these loci and identify the sex determination genes at each. Transgenic techniques have been optimized for A. burtoni, allowing direct testing of putative functional polymorphisms. Gene expression analyses through sexual development will supplement these mapping efforts, and provide understanding of how multiple genetic sex switches are integrated during development. Since A. burtoni is also a longstanding sexual behavior model, it provides excellent context for analysis of secondary sexual characteristics, including existing behavioral gene expression data, all in relation to the various modes of sex determination present in the species.

A Multi-Modal Method for Determining the Postmortem Interval in Juvenile Remains and Assessing Skeletal Health

Ann H. Ross

National Institute of Justice

$280,877

01/01/13 - 05/31/17

The Department of Health and Human Services reports that in 2005 there were 1460 cases of child maltreatment fatalities in the US. Child maltreatment crimes are difficult to investigate and resolve because little scientifically based research has focused on this specific issue. Because of the relatively small size of the victim, concealment of child maltreatment crimes are common and the victims may not be found for months allowing sufficient time for soft tissue decay and skeletonization. Using immature pigs as a proxy for human subadults, our intent is to identify specific changes in bone decomposition and weathering as key elements in estimating the post mortem interval (PMI). Seasonal data collection over two years will be conducted to document bone weathering and survivability of skeletal elements. A major contribution of this project is the assessment of bone mineral density loss during the decay process and the histological evaluation of diagenesis resulting from the postmortem environment. Although some normative data is available for infants and children, which have been developed by the instrumentation companies (e.g. Hologic, Lunar) and, to some degree, from population specific scientific research (.e.g. data for Turkish and Spanish children), data does not exist for the American juvenile population at large. Thus, in order to develop normative data for clinically healthy (e.g. well nourished) and ill or malnourished infants and children (0-4 years of age) that can be used to assess skeletal health in the medicolegal setting, bone mineral density (BMD) will be obtained via a Hologic DXA scanner from incoming cases at the North Carolina and New York City medical examiner offices. The proposed study will address many of the unknowns regarding juvenile decomposition (i.e., timing, patterns) and will develop baseline data on postmortem BMD loss, differential decomposition due to body disposition environment, and BMD data from human subadult forensic cases.

Photoactive Cellulose Materials for Anti-infective Applications: From Nanocrystals to Paper Sheets

Reza A Ghiladi, Dimitris S. Argyropoulos, Frank Scholle

NCSU Research and Innovation Seed Funding Program

$24,669

01/01/14 - 12/31/14

Adherence and survival of infective agents on surfaces leads to their transmission to new hosts and significantly contributes to pathogen proliferation, which in turn considerably increases the threat to human health, especially by drug-resistant strains. Consequently, more research into effective surface disinfection by alternative materials (fabrics, plastics or coatings) with anti-infective properties are needed. The research proposed herein describes the application of cellulose (nanocrystals and paper) that are modified with porphyrins, thereby leading to novel photoactive materials capable of rapid, efficient, and low-cost sterilization of a range of bacteria, fungi and viruses. The funding provided by this grant will enable our interdisciplinary research program between the Departments of Chemistry (Sciences), Forest Biomaterials (CNR), and Biological Sciences (Sciences) to develop anti-infective materials useful for the health care, textiles, and food preparation industries.

Characterizing Dinosaur Soft Tissues and Cells

Mary H. Schweitzer

David & Lucile Packard Foundation

$775,000

10/20/06 - 10/31/15

The morphological preservation of still-soft tissues and cells in limb bones of a 68 Ma Tyrannosaurus rex, was announced in 2005 (Science 307:1952). Because organic material is normally destroyed in a relatively short time period, these findings were at odds with current opinions regarding molecular and tissue degradation. However, while our preliminary studies documented morhological preservation; they did not identify chemical and molecular constituents of observed soft tissues, and did not address mechanisms resulting in this preservation. Packard Foundation funds will be applied to address these two issues. Investigation of informative biomolecules from dinosaur or other fossils older than a few million years is minimal, due in part to a general assumption that original molecular or chemical information is lost upon fossilization. This assumption is based largely upon both the altered condition of most fossils, and on theoretical kinetics or bench-top experiments designed to simulate molecular diagenesis using unrealistically harsh conditions. As a result, even though studies of molecular recovery in truly ancient fossils have the potential to elucidate evolutionary relationships and/or physiological strategies, or to provide direct evidence as to the rate and direction of evolution, both of organisms and of molecules they possess, molecular recovery in fossils tens of millions of years old are not routinely conducted. The dinosaur soft tissues and cellular remains we reported represent promising material for the investigation of preserved biomolecules. In addition to organismal characteristics, tissues and cells may shed light on molecular diagenetic pathways, and may elucidate chemical compositional changes that allowed these components to persist with original flexibility and transparency over millions of years. In order to fully address these issues, appropriate analytical methods need to be developed. Preliminary data shows that preservation varies greatly within a single specimen. These methodological challenges must be addressed, and consistency obtained across analytical techniques, before the extent of preservation can be determined and the potential of molecular analyses of well-preserved fossil remains can be fully realized. We propose to conduct in-depth examination and continued methodological development to determine how the various techniques and preparation methods affect the results obtained. Establishing this baseline and optimizing analytical techniques will be part of the research conducted with Packard Foundation funds.

INSPIRE Track 1: Interdisciplinary Approach to Discovery, Analyses and Applications of Informative Ancient Biomolecules

Mary H. Schweitzer, Michael B. Goshe

National Science Foundation (NSF)

$1,014,563

09/01/13 - 08/31/17

This project will demonstrate that biomolecules recovered from ancient fossils can be used to investigate evolutionary relationships, organism physiology, molecular modification, and environmental effects on molecular preservation. Integrating paleontology, evolutionary biology, bioinformatics, analytical chemistry, molecular biology, immunology and sedimentology will optimize methods to retrieve/characterize organic components preserved in progressively ancient fossils and develop state-of-the art proteomics methods to interpret molecular data. We will build on previously published protein sequences and evidence for DNA within isolated osteocytes from demineralized dinosaur bones. Going beyond exploratory stages, we will generate data needed for broad, higher-risk studies. Identifying/characterizing endogenous molecules from deep time through interdisciplinary research will transform molecular paleontology and evolutionary biology, generating: 1) a uniform approach to molecular analysis/interpretation of factors in preservation of organismal remains; and 2) predictive models for organismal responses to changing environments across time. We will elucidate factors contributing to biopolymer stability and molecular preservation and expand existing databases. We will also develop new extraction and purification methods, interpretive algorithms, and analytical tools and techniques. Our objectives are to: 1) Determine bone/tooth proteomes of extant non-mammalian vertebrates not found in existing proteome databases. Analyzing molecules requires searches against broad DNA/protein sequence databases; Crocodylia, Chelonia/Testudines, and Avialae are target taxa with long geological records. A baseline of extant sequence data will allow comparison to data from non-mammalian fossils found in progressively older sediments, attaining a goal with immediate benefits. 2) Exploit existing collaborations to recover Mesozoic/Cenozoic fossils and appropriate museum specimens; analyze associated sediments and depositional environments. 3) Test hypothetical temporal limits to molecular longevity by sampling progressively older fossils (~10,000, 100,000, 500,000, 1M and 10M yrs) of target taxa, providing the first direct test of longevity of original fossil molecules preserved under natural conditions. Broad sampling will identify environmental factors contributing to preservation. 4) Isolate soft tissue components of fossils; conduct chemical/molecular characterization using antibodies and MS to determine sequences and identify alterations. 5) Use increasingly specific antibody studies on isolated components for in situ, immunoblot, and ELISA analyses, and reactive antibodies to purify material for sequencing. 6) Collect MS profiles and search against existing and newly expanded protein sequence databases, conduct de novo sequencing on profiles that do not match databases, and develop new extraction/analytical methodologies to account for significantly modified, low abundance biomolecules. Identified peptides will: validate ancient DNA data (not as subject to contamination or artifacts); facilitate recovery of evolutionary sequence data if DNA is unrecoverable; and study protein durability and fossil preservation. 7) generate phylogenies from obtained sequences to determine originality and evolutionary significance. 8) Use actualistic experiments and chemical characterization to test organic preservation mechanisms, examine iron?s role, and test effects of iron removal on molecular recovery/analyses.

Advancing the Tools of Freshwater Mussel Conservation: Determining the Relative Sensitivity of in Vitro and in Vivo Propagated Juveniles

William G. Cope, Thomas J. Kwak, Damian Shea

US Geological Survey (USGS)

$122,348

07/10/14 - 06/30/17

Native freshwater mussels (family Unionidae) are one of the most rapidly declining faunal groups in the North America. About 70% of the nearly 300 freshwater mussel species found in North America are considered vulnerable to extinction or already extinct. These declines have been attributed to an array of factors associated with pollution and water quality degradation and habitat destruction and alteration. Over the past two decades, the U.S. Fish and Wildlife Service and other federal and state agencies have invested substantial funding nation-wide in the propagation and culture of native freshwater mussels for conservation purposes, but particularly in the Southeastern and Midwestern regions. These federal and state efforts have resulted in tremendous advances in culture and propagation techniques, aquaculture system design, nutritional needs, and long-term growth and maintenance. Most of this success has been attributed to improving standard host fish (in vivo) infection techniques for propagation, but recent advances have made it possible to produce thousands of juvenile mussels with in vitro propagation techniques that require less space and with less cost than with traditional host fish methods. However, no definitive side-by-side studies have been conducted comparing the chemical sensitivity of in vitro propagated juveniles to in vivo propagated juveniles. Therefore, the overall goal of this this study is to conduct a robust side-by-side assessment of the relative sensitivity of in vivo and in vitro produced juvenile mussels to selected chemical toxicants. This project will greatly expand the toxicity data base for native freshwater mussels and toxicants with different modes of action that have been produced with different propagation techniques. Successful completion of this project will provide federal and state natural resource managers and decision makers with the information needed to assess mussel sensitivity to contaminants, facilitate the refinement mussel testing guidelines, and improve the overall conservation and management of this valuable, but imperiled faunal group.

Assessment of Terrestrial and Aquatic Monitoring Programs in the Southeastern United States

Ryan Boyles, Damian Shea

US Geological Survey (USGS)

$174,251

09/20/12 - 06/19/15

A significant challenge faced by climate scientists in the public and private sector is the need for information about the historical status of ecological systems expected to be influenced by climate change. The need is especially acute for reliable and complete information about monitoring networks maintained by government and non-governmental organization and associated data. While many organizations monitor one or more aspects of aquatic and terrestrial ecosystems, these monitoring programs are seldom coordinated and information about both the networks and the associated data are not readily available. The DOI Southeast Climate Science Center is participating in an effort by multiple federal, state, and other organizations to develop a comprehensive and integrated assessment of monitoring networks associated with atmospheric, stream, and terrestrial ecosystems. The objective of this two year project is to support the development of this assessment. Tasks will include: Compile, inventory, and map geographically, sources (federal, state, local and non-governmental) of atmospheric, terrestrial, water quality and quantity information and analysis capacity in the region to address climate issues; Characterize the sources of information in terms of longevity and ?depth?, consistency over time, and types and quality of information; Assess other attributes of the information sources to be identified; Assess currently available information for use in tracking regional trends, or running scenarios of interest to federal, state and local resource managers; and, Identify key information gaps of concern to federal, state and local resource managers. The geographic scope of this effort will include the states of North Carolina, Tennessee, Mississippi, Alabama, Georgia, and Florida and the Commonwealth of Puerto Rico. Results from this proposed work will provide the region?s scientists and decision makers with accurate and comprehensive information about monitoring networks that can be used to assess the potential effects of climate change in the southeastern United States. NCSU and USGS scientists will cooperatively author reports and / or journal articles to communicate findings to the public.

Development of a SECAS Conservation Decision Guidance Library

Nils Peterson, Frederick W. Cubbage, Damian Shea

US Geological Survey (USGS)

$84,659

09/19/13 - 02/01/16

A priority of the Southeast Climate Science Center (SECSC) is to support the planning and implementation of a Southeast Conservation Adaptation Strategy (SECAS). The objectives of this project are to (1) develop a better understanding of the management decision context for important SECAS resource management themes using restoration of open pine ecosystems as a case study; (2) describe and synthesize management objectives related to this resource management theme; (3) improve understanding of how management decisions are being made and how this decision making process can be improved; and (4) design a questionnaire to evaluate socio-structural drivers of decision making associated with SECAS. Methods will include review and synthesis of material from existing plans of organizations and agencies in the SE and selected interviews with decision-makers and stakeholders. A final selection of resource management themes will be made in conjunction with LCC representatives. Expected products include: (1) white papers describing the decision making context, management objectives, and decision process, (2) database cross-tabulating decisions with contextual variables, objectives, and processes, (3) transcripts and qualitative analyses associated with decision maker interviews, and (4) a preliminary questionnaire and sampling protocol for evaluating socio-structural drivers of decision making associated with SECAS.

Increasing Engagement in Science through Authentic Practice K-20

Damian Shea, Miriam G Ferzli, Sharon Kay Schulze

Howard Hughes Medical Institute

$1,500,000

09/01/10 - 08/31/14

Our overarching goal is to increase student recruitment and retention in science, and student interest in pursuing careers in science research and education, by increasing the number of students engaged in scientific research and increasing the amount of time students spend in authentic science practice. Our rationale is that students will best develop as researchers and sustain an interest in science when provided enough time to practice their skills in a supportive and guided environment prior to conducting independent research. We plan to emphasize teaching through research at all levels of education, establish active- and collaborative-learning classrooms, and continue to develop teachers who teach using authentic science activities. In doing so, we will make significant strides in promoting learning for all learners by engaging them in authentic scientific practice through inquiry-based approaches.

Measuring Chronic Exposure to and Bioavailability of Organic Chemicals and their Metabolites in Water and Sediment with a Novel Universal Passive Sampling Device (uPSD)

Damian Shea

UNC - UNC Chapel Hill

$1,482,400

04/01/11 - 03/31/17

This project will develop a new and novel sampling technology to measure chemical exposure in water, sediment and soil by: 1) Examining whether technological improvements to our current non-selective passive sampling device (nsPSD) will solve critical exposure assessment problems; 2) Measure the external chemical exposome using PSDs; 3) Implement PSDs to bring real world mixtures of both organic and inorganic chemicals for the advanced assessment and mitigation of chemical risks.

Recent Precipitous Declines of Endangered Freshwater Mussels in the Clinch River: An in Situ Assessment of Water Quality Stressors Related to Energy Development and other Land-Use

William G. Cope, Thomas J. Kwak, Damian Shea

US Geological Survey (USGS)

$298,287

05/01/12 - 09/30/15

Native freshwater mussels (Order Unionida) are one of the most rapidly declining faunal groups in the North America. About 70% of the nearly 300 freshwater mussel species found in North America are considered vulnerable to extinction or already extinct. These declines have been attributed to an array of factors associated with pollution and water quality degradation and habitat destruction and alteration, including most recently, rapid expansion of energy development and other extractive land-uses. This research project will measure contaminant stressors in surface water, sediment, and sediment pore water and evaluate the relationship of the combined stressors to freshwater mussels, including federally listed endangered species, in the Clinch River in Virginia and Tennessee and Clinch River tributaries in Virginia. The successful completion of this project will provide the U.S. Fish and Wildlife Service and other federal and state natural resource management agencies and decision makers with the information needed to assess mussel sensitivity to contaminants in relation to these multi-faceted stressors, which will help to improve the conservation and management of this valuable, but imperiled faunal group.

Robust Redhorse Recovery and Habitat Restoration: Assessing Water Quality Stressors and Food Web Contaminant Dynamics

Thomas J. Kwak, William G. Cope, Damian Shea, Seth W Kullman, Jerry M. Law

NC Wildlife Resources Commission

$460,000

01/01/14 - 12/31/16

The robust redhorse (Moxostoma robustum) is a rare and imperiled, large catostomid fish found in only three regulated river drainages in the southeastern U.S. The species was originally described in 1870 from the Yadkin–Pee Dee drainage in North Carolina and is known to make seasonal migrations within freshwater systems. It has large pharyngeal teeth for crushing mollusks and other invertebrates known to sequester anthropogenic contaminants. The robust redhorse has been negatively affected by habitat modification and fragmentation from hydroelectric dams, introduced species, sedimentation, and water pollution and is protected by state endangered status in Georgia and North Carolina. Previous research by the Principal Investigators has shown that habitat suitability will be enhanced by prescribed flow augmentations from hydroelectric dam releases; however, the impacts of water quality and contaminant loads remain unknown. In addition to the unknown effects of traditional organic and inorganic contaminants, recent research suggests that the impact of emerging contaminants, such as endocrine disrupting compounds and pharmaceuticals, may be of significant detriment to fishes and other Wildlife Action Plan priority species in the Pee Dee River. These uncertainties must be resolved before additional and significant investments are made in population augmentation or other recovery efforts. To further elucidate the impact and potential threat of water quality and contaminant dynamics on the robust redhorse, the aquatic food web, and 53 priority aquatic species of the Pee Dee River, we propose six research objectives to pursue in the Pee Dee River of North Carolina and South Carolina. We will (1) conduct systematic field sampling of habitat and food web components, (2) conduct experimental field bioassay exposures with captively-propagated robust redhorse, (3) perform laboratory analyses of traditional and emerging contaminants on aquatic habitat and food web components and histopathology to identify intersex condition in fish (i.e., effects of endocrine disrupting compounds), (4) determine aquatic food web structure and pathways using stable isotope ratios, (5) develop population and food web models to describe and project effects of habitat and water quality modifications, and (6) synthesize results for robust redhorse recovery from population and ecosystem perspectives. This research is unique in that it will yield results and inference that are descriptive (systematic sampling), explanatory (experimental bioassays, food web analyses), and predictive (population and food web modeling) at multiple scales and across disciplines to inform decision making and management. The food web to be studied supports a total of 53 priority aquatic species (31 fishes, 21 mussels, 1 crayfish), 35 species in the NC Wildlife Action Plan and 26 species in the SC Comprehensive Wildlife Conservation Strategy that will benefit from this research. Our proposal addresses critical research needs identified by the Robust Redhorse Conservation Committee, South Atlantic Landscape Conservation Cooperative, and the State Wildlife Action Plans of North Carolina, South Carolina, and Georgia, and is supported by these consortia and state and federal natural resource agencies to guide management objectives and goals for species recovery and habitat restoration at the landscape level.

Southeast Climate Science Center

Nicholas M. Haddad, Damian Shea, Robert R. Dunn, David B. Eggleston, James F. Gilliam, Suzanne Kennedy-Stoskop, Christopher E. Moorman, Margery F. Overton, Fredrick H. Semazzi, Michael K. Stoskopf, Ryan Boyles, Scott M. Fitzpatrick, Daniel Robison

US Geological Survey (USGS)

$2,251,159

09/23/10 - 04/15/17

This proposal is a supplement for a base award to NCSU and its affiliation with the USGS Southeast Climate Science Center (SECSC). Per the USGS requirements, all money will come in through the University SECSC Director, Nick Haddad, and then be distributed to each specific project.

Species and Strain Differences in the Toxicity of Caribbean Gambierdiscus Species: Implications For Ciguatera Fish Poisoning in the Caribbean

Damian Shea

National Oceanic & Atmospheric Administration (NOAA)

$56,451

09/01/11 - 08/31/14

We will provide analytical support to NOAA ECOHAB program related to work on the Species and Strain Differences in the Toxicity of Caribbean Gambierdiscus Species: Implications for ciguatera fish poisoning in the Caribbean. We will oversee the extraction, fractionation/purification, and analysis of ciguatoxins including identification of any previously unreported toxins. Initial and subsequent routine LC/MS work will be conducted using the Thermo Discovery MAX LC/MS/MS that is jointly owned and operated by NOAA and NCSU and located in the Center for Marine Science and Technology in Morehead City, NC. If higher sensitivity and accurate mass analysis is required, we will perform that on either a Thermo LTQ linear ion trap LC/MS or Thermo LTQ Orbitrap XL LC/MS on the main NCSU campus in Raleigh, NC. All of our laboratory extraction, fractionation/purification, and analytical instrumentation will be available to support this project. Data will be provided in both hardcopy and electronic format and will include all quality control data.

Understanding Conservation Management Decisions In The Face Of Sea-level Rise Along The U.s. Atlantic Coast

Ryan Boyles, Damian Shea

US Geological Survey (USGS)

$289,532

09/23/13 - 09/22/15

This project addresses a complex local scale conservation problem: managing the impacts associated with sea level rise and coastal flooding on migratory waterbirds and their habitat. Decisions made by a conservation manager are complicated by three elements that can be expected to occur in almost any of these management situations. Interactions among dynamic physical and biological processes affect both waterbirds and their habitat and food resources; these processes operate at local to flyway scales and are challenging to represent and analyze. These natural physical and biological systems are coupled with human systems; decisions made by nearby landowners or jurisdictions can have an impact on conservation resources. Finally, decisionmakers are still developing the experience and expertise to perceive, understand, and deal with the implications of the first two elements in making timely and effective decisions. The objectives for year 2 modification are threefold. Project activities in year 1 have led to the insight that the problem faced by refuge managers is a resource allocation problem, with both short (e.g., how to keep the current refuge footprint productive in meeting refuge objectives) and long term (e.g., in the face of climate and land use change, where should the refuge be located in the future). The objective is to build on year 1 activities to characterize this more general refuge management problem. The second objective is to work with scientists and managers from at least two refuges located in the southeast to understand the short and long term refuge objectives. The third objective is to incorporate this information into one or more proposals to the Southeast Climate Science Center for projects in FY15 and beyond.

Regulation of Stem Cell Pluripotency by CTCF

Michael L. Sikes

NCSU Faculty Research & Professional Development Fund

$4,000

07/01/14 - 06/30/15

Stem cells may revolutionize medicine, but they are also providing critical insights on cancer development and progression. Recent studies have underscored the similarities between transcription networks in embryonic stem cells (ESCs) and cancer cells. ES pluripotency critically depends on a panel of factors including the core pluripotency factors Nanog, Oct4 and Sox2, and the early embryonic microRNA cluster (EEmiRC). Each of these genes is bound by CTCF, a multifunctional genome regulator that can block the spread of repressive DNA methylation. While knockdown studies have implicated CTCF in controlling some of these factors, we find that loss of CTCF alters both expression and DNA methylation. We hypothesize that CTCF coordinates pluripotency factor expression in ES cells by maintaining a permissive epigenetic environment at their promoters. Alternatively, given the strong degree of cross-regulation of these genes, maintenance of the pluripotency network may require methylation protection of one or two master genes. To fill this critical gap in our understanding of pluripotency control, we will dissect how CTCF regulates expression of the pluripotency factors and microRNAs. These studies will lay the foundation for more mechanistic investigations into the critical contributions of CTCF to stem cell and cancer development.

The Genetic Basis of Phenotypic Plasticity in Meiotic Recombination Rate

Nadia D Singh

National Science Foundation (NSF)

$620,000

08/01/14 - 07/31/17

The work proposed in this application will identify genetic and molecular mechanisms underlying phenotypic plasticity in meiotic recombination rate. Phenotypic plasticity, the capacity of a single genotype to produce different phenotypes in different environments, is pervasive in nature. In addition, phenotypic plasticity has been argued to play a major role in evolution, in the contexts of adaptation and speciation. In spite of its ubiquity and importance, the genetic and molecular underpinnings of phenotypic plasticity remain unknown. In particular, the extent to which genes underlying individual traits are the same genes underlying phenotypic plasticity in those traits remains controversial, in part because there are precious few examples for which the underlying genetic architecture of both the trait and any associated plasticity have been clearly worked out. Meiotic recombination rate is an important example of a trait exhibiting phenotypic plasticity. Rates of meiotic recombination vary widely among environments. Moreover, the magnitude of recombination rate plasticity (the difference in recombination rate observed between environments) depends on genotype. However, the genetic and molecular bases of this variation remain unknown. Using meiotic recombination rate as a prototypical phenotype capable of plasticity, the PI proposes to test the hypothesis that phenotypic plasticity for a given trait is mediated by the same genes underlying population-level variation in that trait. The specific questions that will be addressed are: 1) What genetic loci mediate recombination rate plasticity? The proposed work exploits a panel of artificial selection lines that show significant differences in recombination rate plasticity in response to temperature. Whole genome and whole transcriptome sequencing of these lines will be conducted using next-generation sequencing technologies. Candidate genes associated with recombination rate plasticity will be identified by determining loci showing significant differentiation in underlying sequence or in transcript abundance among lines. The proposed work will also query the extent to which recombination rate plasticity in response to one type of environmental condition (e.g. temperature) is correlated with recombination plasticity in response to other environmental stimuli. 2) What genes contribute to population-level variation in recombination rate? The extent to which genes underlying phenotypic plasticity in a trait also underlie population-level differences in a trait remains debated. To address this question, the PI proposes to determine the genetic basis of population-level variation in recombination rate. Recombination rates will be in a fully-sequenced panel of 192 inbred lines of D. melanogaster. Whole genome association mapping will then be used to identify candidate genes associating with variation in recombination rate among individuals. Comparison of the loci identified in the two aims will reveal the extent to which genes mediating recombination rate plasticity are the same as those mediating population-level variation in recombination rate. RNAi and mutant analysis will be used to genetically validate a subset of the identified candidate genes. This innovative combination of classical genetics and next-generation sequencing will transform our understanding of the genetic and molecular basis of recombination rate plasticity and population-level variation in recombination rate. This is significant because little is known regarding the genetic and molecular underpinnings of these phenomena. Also, though the results from each objective have strong and significant independent utility, together these results have greater impact still, as they will speak broadly to the genetic architecture of phenotypic plasticity. The proposed work will clearly illuminate whether the genes underlying population-level variation in a given phenotype are the same genes associated with plasticity in that phenotype.

Molecular Pathways to Pathogenesis in Toxicology

Robert C. Smart

National Institutes of Health (NIH)

$6,466,937

07/01/77 - 06/30/20

This NIEHS training grant has been active for 30 years and has supported 102 predoctorates and 15 postdoctorates. These trainees have gone on to successful careers in toxicology in academia, industry, government and the private sector. This training program has been extensively revised to be in accord with the NIEHS mission and the new NIEHS T32 guidelines. The objective of this training program entitled, Molecular Pathways to Pathogenesis in Toxicology, is to provide trainees with the skills and knowledge necessary to investigate and elucidate how environmental toxicants/cellular stressors contribute to toxicity and influence disease outcomes. The broad overarching research theme is to understand how toxicants/cellular stressors perturb cellular signaling pathways and deregulate gene expression and how this contributes to toxicity and human disease outcomes. Specific environmental-associated human diseases/conditions such as asthma, lung fibrosis, reproductive/endocrine abnormalities, developmental abnormalities and cancer as well as reactive oxygen species (ROS)-related toxicity will be the focal molecular/cellular research areas. Examples of types of environmental agents that will be investigated include environmental carcinogens, pesticides, particulates, metals, endocrine disrupters and nanoparticles. There are fifteen preceptors from 3 colleges and 5 departments that are participating in this interdisciplinary training program. Importantly, this training grant is administered through the Department of Environmental and Molecular Toxicology which provides a structured cohesive environment for trainees. Predoctoral trainees acquire a solid background by completing coursework in toxicology, molecular biology, pharmacology, statistics, and pathology. Pre/postdoctoral trainees receive training in ethics, grant writing and oral communication skills. Pre/postdoctoral trainees participate in a weekly seminar program both as members of the audience and as presenters. Each semester a former trainee is invited back to the department to present a seminar and to share their career experiences with current trainees. A plan is in place with the College of Veterinary Medicine that will enhance the recruitment of veterinarians (DVMs) interested in human health to postdoctoral positions on this training grant. We are requesting support for 6 predoctoral trainees and 3 postdoctoral trainee positions.

Role of C/EBPs in Cell Survival and Neoplasia

Robert C. Smart

National Institutes of Health (NIH)

$2,496,522

02/01/01 - 05/31/15

Recent studies have identified novel functional roles for the basic leucine zipper transcription factor CCAAT/enhancer binding protein-beta (C/EBPb) in cell survival and tumor development; however, the molecular mechanisms through which C/EBPb regulates these processes are poorly understood. C/EBP?Ò-/- mice are completely refractory to skin tumor development induced by carcinogens that produce tumors with oncogenic Ras mutations. In response to topical carcinogen treatment, C/EBPb-/- mice display a 17-fold increase in keratinocyte apoptosis compared to wild type mice. This abnormal increase in apoptosis in C/EBPb-/- mice requires p53 and is due to aberrant up-regulation of p53 protein. We hypothesize that C/EBPb mediates the survival of initiated tumor precursor cells through the repression of p53 and that the consequences of C/EBPb deficiency are de-repression of p53, p53-mediated tumor precursor cell apoptosis and ablation of tumorigenesis. Understanding the pro-apoptotic response in C/EBP?Ò-/- mice has important implications for tumor development. Exactly how C/EBPb represses p53 levels and whether this repression occurs in response to DNA damage and/or Ras-induced oncogenic stress is not known. If C/EBPb is critical for oncogenic Ras tumor cell survival then blocking C/EBPb function in a p53-proficient oncogenic Ras-containing tumor should result in tumor cell apoptosis and tumor regression, thus we propose that C/EBPb has potential as a molecular target for cancer therapy. The goals of this proposal are: 1) to determine how C/EBPb represses p53/apoptosis and detemine whether this repression occurs in response to DNA damage and/or oncogenic Ras; 2) to determine whether repression of p53 by C/EBPb is critical for tumorigenesis; 3) determine the importance of Ras and/or DNA damage-induced C/EBPb post-translation modifications in vivo; and 4) to determine whether C/EBPb is a potential molecular target for tumor regression. The long-term objectives of this proposal are to understand the molecular mechanisms through which C/EBPb influences the neoplastic process in epithelia and regulates tumor cell survival.

Core Facility Expansion at NCSU with the NextSeq 500

Eric A. Stone, Jennifer Schaff, Robert M. Kelly, Jeffrey A. Yoder, Heather B Patisaul, Tika B Adhikari, Deyu Xie, Chase Beisel, Michael R. Hyman, Heike W. Sederoff, George C. Yencho, Vincent Chiang, Benjamin Reading, Gina Brown-Guedira

NC Biotechnology Center

$400,000

04/01/15 - 03/31/16

The new equipment will serve a diverse user community. Because of its combined capability as a highthroughputb sequencer that generates reads ultra fast, it will be an essential tool in research requiring: 1) variant detection for genotyping breeding populations, 2) near real-time metagenomics remediation assays and other population surveys, 3) SNP detection, genotyping, and genetic mapping, 4) transcriptome sequencing and gene expression analysis, and 5) full genome and targeted resequencing (including ChIP seq and bisulfite-treated DNA applications)

Partially-supplied Graphs as a Unifying Concept in Biology

Eric A. Stone

National Science Foundation (NSF)

$200,000

10/01/11 - 09/30/15

Graphs are used throughout the biological sciences to represent how objects of interest are interrelated. A common problem, and the focus of this proposal, is how to elicit the structure of such a graph when only pairwise distances between objects have been supplied. A canonical example is phylogenetic reconstruction -- here the objects are species, distances may be measured in terms of pairwise genetic divergence, and the graph to be reconstructed is a tree of life. Other graphs of biological interest include, but are by no means limited to, family pedigrees, gene regulatory networks, and graphs of protein-protein interactions. A recurring feature of the graphs typical to biology is that often some of the objects of interest are missing; in phylogenetics, for example, the species tree includes extinct ancestral organisms for which no measurements can be obtained. Motivated by the ubiquity of graphs in biology and the frequency with which the biological objects of interest are missing, this proposal introduces and seeks to develop the concept of a partially-supplied graph.

Collaborative Research: A new Approach to Phylogenetic Analysis Using Structural Information

Jeffrey L. Thorne

University of California - Santa Cruz

$361,092

09/30/09 - 07/31/15

Our proposal focuses on the development of statistical models that use protein structural information to jointly construct the alignment and phylogenetic history of a collection of proteins. Similar approaches have been recently developed in the context of sequence-based phylogenetic analysis, with striking and extremely promising results. Because tertiary structure changes more slowly than does sequence, we aim to extend these sequence--based methods to include protein shape information by building on some of our recent work on Bayesian structural alignment.

Evolutionary Inferences From Protein-Coding Genes

Jeffrey L. Thorne, Eric A. Stone

National Institutes of Health (NIH)

$1,224,720

08/01/10 - 07/31/15

Because evolution occurs within populations, evolutionary inference is ideally framed with respect to population genetic parameters. Although elaborate statistical techniques exist for analyzing interspecific molecular sequence data, a population genetic basis for these techniques is often absent or unclear. This is unfortunate because interspecific comparisons are the only way to study most of evolutionary history. This project aims to make population genetic inferences from interspecific DNA sequence data. The focus is on the relative fitnesses conferred by protein-coding DNA sequences. A central quest of evolutionary study is to understand how phenotype affects survival of the genotype. Much biological research involves connecting genotype to phenotype, but evolutionary biology has an even more ambitious goal. It is the task of evolutionary biologists to connect genotype to fitness. The difficulty of this task is lessened when phenotype can be accurately predicted from genotype because the genotype-to-fitness mapping problem then becomes the slightly less intimidating phenotype-to-fitness mapping problem. We convert the former mapping problem to the latter by capitalizing on automated systems that make \textit{in silico} predictions about phenotype from gene sequence data. These systems for predicting phenotype solely from genotype can never be perfect because phenotype depends on both genotype and environment. However, systems for predicting some aspects of phenotype are reliable enough to be useful and they have not been sufficiently exploited by evolutionary biologists. The statistical techniques that we are developing can be employed to examine the evolutionary consequences of any phenotype for which an \textit{in silico} prediction system exists, but we are concentrating on the evolutionary role of protein tertiary structure. Our research stems from earlier work that we have done on assessing the evolutionary impact of protein tertiary structure from interspecific sequence data. We have now extended this work to a population genetic framework. We will continue our investigations via three interconnected lines of research: (1) More realistic descriptions of the evolutionary process: We will improve our treatment of protein tertiary structure and the natural selection that acts upon it. Sequences from the protein family of interest, empirically-derived information from a database of known tertiary structures, and covariates such as gene expression information will all influence our evolutionary models. We will also make our descriptions of the mutation process more realistic by having mutation rates depend on local sequence context and by having them vary among genomic regions. The more realistic models that result will yield improved estimates of relative fitness. These models will also provide the basis for more accurate inference of ancestral sequences and for characterization of the adaptive landscape. (2) Improved inference of population genetic parameters from interspecific data: Our current technique for making population genetic inferences from interspecific data requires a variety of restrictive assumptions, including the assumption that mutation rates are so low as to be able to neglect the possibility that multiple fitness-affecting polymorphisms are simultaneously segregating in a population. We intend to relax these assumptions as well as to explore their consequences via simulation. (3) Predicting health-related effects of nonsynonymous variation in humans: Because our interspecific models are framed in terms of population genetics, we can combine the interspecific and intraspecific data in a sensible way. PI Stone has developed a successful approach for predicting which nonsynonymous variation has health-related effects. Our carefully obtained estimates of the fitness consequences of genetic variation will allow further improvement of this already successful approach.

Transition Structures And The Evolution Of Protein Folds

Maria Celeste Sagui, Christopher M. Roland, Jeffrey L. Thorne

National Science Foundation (NSF)

$600,000

09/01/10 - 08/31/14

This proposal aims to investigate how protein native folds change over time, and how the existence of alternate folds affects the rates of protein evolution, and how new protein functionality can evolve from already existing proteins by means of bioinformatic and atomistic modeling techniques.

Effect of Cooperating Mutations and Genetic Background on Colon Cancer Metastasis

David Threadgill, Rachel Marie Lynch

National Institutes of Health (NIH)

$71,446

08/01/12 - 07/31/15

Colorectal cancer (CRC) is the second leading cause of cancer-related deaths in the United States, with mortality almost exclusively attributed to metastatic liver disease. Therefore, understanding the mechanisms governing how a primary tumor grows and disseminates to secondary sites is of great clinical importance. There is increasing evidence suggesting that genetic background is an important determinant of metastatic susceptibility, but little is known about the specific genetic factors that affect CRC. The proposed research will develop a mouse model of CRC that will allow the investigation of whether genetic background influences tumor development, progression, and metastases through interaction with specific transforming mutations. Tumor growth, histopathological analysis, and gene expression analysis will be used to characterize cancer development in the new model and compare it to an existing CRC mouse model that utilizes a different tumor initiating event. Similar analyses will be performed on mice with differing maternal genotypes and cooperating mutations to investigate how the mutations and genetic background interact to influence tumor progression. The long-term goal of this research is to be able to predict which patients have a higher risk of metastatic disease and develop more appropriate therapeutic strategies for these individuals.

Genetic Control of Colorectal Cancer Histopathology

David Threadgill

National Institutes of Health (NIH)

$1,384,280

09/28/09 - 07/31/14

Colorectal cancer (CRC) is the fourth most commonly diagnosed malignancy in adults. Although significant progress has been made in early detection, diagnosis and treatment, CRC remains the second leading cause of cancer-related deaths in the U.S. and first after smoking-related deaths are excluded. One factor contributing to successful outcome from CRC is early detection, largely performed by periodic colonoscopy. Recently, there is growing appreciation that cancer morphology at the time of presentation can greatly impact the frequencies of detection. In particular, classical polypoid lesions (polyps) are detected and removed during colonoscopy at a much higher frequency than flat or sessile lesions, although there is growing evidence that the latter group may have a higher propensity to become invasive at much smaller sizes. Little is known about how or why certain cancer pathologies form or even whether this is a stochastic process or if there are specific genetic or environmental factors contributing to distinct cancer morphologies. Fortuitously, we have discovered novel mouse models of human CRC that consistently and almost exclusively produce flat or sessile colorectal polyps compared to the much more frequent polypoid-type that occurs in the vast majority of mouse models. These results strongly suggest that there are specific genetic factors that determine CRC morphology. Using these new mouse models, we propose to identify the molecular characteristics that distinguish polypoid from non-polypoid CRCs and to identify the genetic factors responsible for determining cancer morphology. Although we will largely rely on a discovery-driven approach to achieve these goals, our underlying hypotheses are that a distinct set of cancer modifiers controls susceptibility to non-polypoid tumors and that there there exists specific host genetic factors that determine the type of CRC morphology an individual will develop. Investigation of this hypothesis will be achieved by 1) molecularly characterizing distinct histopathologies, 2) determining the number and genetic locations of histopathology modifiers, and 3) fine mapping and identifying candidate histopathology modifiers genes.

Modeling Heterogeneity for Safe Cancer Prevention and Detection

David Threadgill, Eric A. Stone, Deborah S Threadgill

National Cancer Institute

$3,117,501

09/01/09 - 07/31/14

Same as original grant.

Regulation and Role of CREB in Cellular Genotoxic Response to Xenobiotics

Yoshiaki Tsuji, Jun N. Tsuji

National Institutes of Health (NIH)

$1,191,272

09/01/11 - 08/31/17

The CREB (cAMP-response element binding) transcription factor is a stimulus-induced phospho-protein that is involved in numerous cell signaling pathways. Dysfunction and deregulation of CREB and CREB-interacting proteins cause human diseases such as cancer and neurodegeneration. CREB appears to play a key role in cell defense and survival in various tissues; however, the mechanisms through which CREB is involved in cell survival and the reason why deregulation of CREB function causes these human diseases remain incompletely understood. CREB phosphorylation at Ser-133 is the major posttranslational modification that enhances CREB activity in response to receptor-coupled stimuli. However, the status of CREB Ser-133 phosphorylation was not always correlated with CREB transcription function, suggesting that another event along with CREB Ser-133 phosphorylation seems to be involved in CREB regulation in a stimulus-specific manner. This research project may provide evidence and a critical answer to theses unsolved problems because we recently found that HIPK2 (homeodomain interacting protein kinase 2), a genotoxic stress responsive kinase, activates CREB via phosphorylation of a new serine site (Ser-271) but not Ser-133, resulting in activation of CREB transcription function. We will test our hypothesis that HIPK2 is a new regulator of the CREB transcription factor via phosphorylation of this new CREB site that induces a cell survival program in genotoxic and oxidative stress conditions. The proposed experiments will focus on characterization of molecular mechanism through which CREB phosphorylation by HIPK2 activates its transcription function as well as downstream events including expression of target genes and cellular susceptibility to genotoxic stress in in vitro and in vivo models. The scientific impact of this research will be broad and significant because CREB regulates essential cellular events such as cell growth, differentiation, metabolism, and immune response. Therefore the unveiled new CREB regulation from successful completion of this proposal will enhance our understanding in various physiological and disease conditions closely associated with the CREB activity.

Regulation of Antioxidant Genes and Oxidative Stress

Yoshiaki Tsuji, Jun N. Tsuji

National Institutes of Health (NIH)

$1,091,774

04/01/11 - 03/31/17

Iron is an essential element by serving as a constituent of vital cellular proteins involved in a variety of cellular functions; however, excess iron is detrimental because it catalyzes formation of reactive oxygen species (ROS). Disorder of iron homeostasis involving iron deficiency or overload is associated with various human health problems such as neurodegenerative disease, cancer and aging. Fine-tuning of intracellular iron levels is therefore essential for maintaining normal cellular function and physiological metabolic balance. Ferritin is the major iron-storage protein in eukaryotic cells and it plays a crucial role in regulation of iron metabolism by detoxifying and storing intracellular excess iron in a non-toxic but bioavailable form. Ferritin synthesis is regulated at both transcriptional and translational levels. Translational regulatory mechanism of ferritin by iron has been extensively studied and well characterized. In contrast, iron-independent transcriptional regulation of the ferritin gene under such conditions as cells need to limit iron availability remains incompletely understood. In particular, little is known about ferritin transcriptional regulation through chromatin remodeling mechanism under oxidative stress conditions. Transcription of ferritin and a battery of antioxidant genes are regulated by a conserved enhancer, termed the ARE (antioxidant responsive element). We hypothesize that chromatin remodeling and associated factors we have recently identified on the human ferritin ARE can serve as crucial proteins that regulate ferritin transcription and iron homeostasis. The proposed experiments will focus on characterization of these new ARE-interacting proteins and their roles in chromatin modifications adjacent to ARE-regulated ferritin and antioxidant genes. The scientific impact of this research will be broad and significant because it will not only provide new insight into the basic transcriptional mechanism of a group of antioxidant genes via coordinated regulation of transcription factors and chromatin-remodeling factors, but also define new regulatory proteins responsible for cellular antioxidant response and iron homeostasis under oxidative stress conditions that are associated with various iron- and ROS-involving human diseases.

Role of TAB1-TAK1 Signaling in Tumor-Associated Macrophage Survival

Jun N. Tsuji, September R Mihaly

National Institutes of Health (NIH)

$64,130

07/31/13 - 07/30/15

Cancer is characterized by the uncontrolled growth of tumor cells, and these tumor cells are known to affect neighboring cells in the stroma and specifically recruit macrophages to the primary tumor mass. As research in tumor-associated macrophages (TAMs) evolves, a picture of adaptive immune system “husbandry” is emerging, in which tumor cells recruit macrophages to the tumor microenvironment where they affect cell signaling pathways in macrophages to achieve growth and metastatic potential. TAMs are associated with poor prognosis and increasingly metastatic disease, and are coming to be recognized as important mediators of malignancy. The classically activated macrophage involves signaling through Toll-like receptors (TLRs), leading to downstream activation of NF-κB and the transcription of pro-inflammatory genes. Signals that promote TAM survival and persistence in tissues could contribute to tumor growth and metastasis. TGF-beta-activated kinase 1 (TAK1) is a kinase that is known in several cell types to be involved in pro-inflammatory and apoptotic cell signaling pathways, including the pro-inflammatory NF-κB and p38 pathways. TAK1 has a binding partner, TAK1-associated binding protein 1 (TAB1). Our recent results suggest that activity of TAK1 may be essential for macrophage survival, but TAB1-dependent TAK1 activity may be required only for activated macrophage survival. Because TAMs are known to be highly activated but with sustained survival, we hypothesize that TAK1 is activated through TAB1 in TAMs, resulting in escape from cell death. In the absence of TAB1, activated macrophages may undergo RIP1-dependent necrosis. This TAB1 dependency may represent a vulnerability in TAMs, and the inhibition of TAB1 may therefore be a potential target for anti-cancer therapy. Deleting TAB1 and TAK1 in tumor cell-activated macrophages and measuring and characterizing macrophage cell death will test this hypothesis. Transplanting wild type or Tab1-deleted bone marrow cells into tumor-bearing mice and measuring the effects on tumor size and multiplicity and on macrophage survival will help to determine the roles of TAB1 and TAK1 in tumorigenesis. TAMs have been implicated in metastasis, and may be essential for metastasis of some tumor types. To investigate the role of TAB1-TAK1 signaling in metastasis, cell lines established from highly metastatic human mammary will be injected in Rag1-deficient and Tab1- Rag1-double-deficient mice. This will allow for comparing mice having wild type or Tab1-deleted macrophages in the absence of mature T cells and B cells, thereby contributing specific knowledge of the pathways involved in TAM survival. Cell signaling and communication between macrophages and tumor cells promote macrophage survival and ultimately tumor growth and metastasis. This study will fill in gaps in our understanding of TAMs and how these processes contribute to malignancy. Disrupting macrophage-tumor cell interactions as well as signaling pathways within macrophages could lead to more effective and less toxic cancer therapies.

TAK1 Signaling Network in Tissue Homeostasis

Jun N. Tsuji, Robert C. Smart, Yoshiaki Tsuji

National Institutes of Health (NIH)

$2,440,806

04/01/04 - 03/31/16

TAK1 (MAP3K7) is an intermediate of cellular stress response signaling pathways and is activated by inflammatory cytokines, TNF and IL-1, and Toll like receptor (TLR) ligands through two adaptor/activator proteins, TAK1 binding protein 1 and 2 (TAB1 and TAB2). Ablation of TAK1 results in cell death in multiple tissues. However, TAK1 deficiency does not cause cell death in in vitro cultured cells. We have found that TAK1 deletion sensitizes cells to TNF- and TLR ligand-induced cell death in cultured cells, and that TNF-induced cell death is at least in part the cause of cell death in tissues, as evidenced by the fact that TNF receptor 1 deficiency rescues cell death in some tissues. Cells in the in vivo environment are constantly exposed to cytokines and commensal bacteria even without any exogenous stress. Our results indicate that TAK1 is activated by cytokines and bacteria (stressors) in vivo and plays an essential survival role in tissue homeostasis by protecting cells from cell death induced by these stressors. We have also demonstrated that TAK1 activators, TAB1 and/or TAB2, are required for the maintenance of TAK1 activity to prevent cell death in certain tissues. Thus, TAK1, TAB1 and TAB2 play a central role to prevent stressor-induced cell death in in vivo tissues. In this grant renewal, we will determine the mechanism by which the TAK1 complex blocks cell death in several tissues using genetically engineered mouse models. Specifically, we will determine: 1) how TAK1, TAB1 and TAB2 modulate the two major cell death pathways, i.e. apoptosis and necrosis; and 2) the specific roles of TAK1, TAB1 and TAB2 in cell survival in several different tissues. Our previous studies have revealed that, although deficiency of TAK1 complex in general increases cell death in tissues, impacts and consequences of the deletion of TAK1, TAB1, or TAB2 are different in different tissues, e.g. TAK1 but not TAB1 or TAB2 deficiency in epithelial tissues causes extensive cell death resulting in severe tissue damage that resembles chronic inflammatory disease conditions in humans. In contrast, TAB1 or TAB2 deficiency causes hypersensitivity and cell death in liver and macrophages when exposed to a TLR ligand. Thus, the objective of this proposal is to define the general mechanism of stressor-induced cell death in vivo, and to define the specific roles of TAK1, TAB1 and TAB2 in several tissues. Outcomes of this project could lead novel and potentially tissue-specific approaches to control tissue damage.