Gerald LeBlanc PhD

Structured population models have utility in estimating population-level responses to natural changes in the environment (e.g., global warming) as well as anthropomorphic influences on the environment (e.g., ecotoxicological risk assessments). We propose to develop a new mathematical framework that can be used to reduce parameter uncertainty for population models, and therefore enable more accurate predictions of population dynamics across many species. Our framework is based on mathematical tools developed by HT Banks and colleagues over the past two decades. The framework we propose to develop will be used to jointly estimate organismal- and population-level (density-dependent) parameter distributions from multi-scale data, i.e., aggregate population data and organismal-level data. Our mathematical modeling framework will provide a novel methodology that quantitatively connects and propagates the assessment of organismal responses, i.e., to environmental change, to the population level, thereby enabling the causal association of organismal responses to ecosystems adversity. To validate our methods, we will collect experimental data using a species of water flea, Daphnia magna. Our modeling results will provide a deeper understanding of fundamental processes underlying population response of Daphnia magna to changes in the environment. Our findings will have important implications for environmental sustainability, since D. magna is a toxicologically sensitive species that plays a vital role in freshwater ecosystems as feeders on phytoplankton and as a source of food for other invertebrates and fish.

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

The sponsor has awarded Maher Haeba a fellowship to undertake a 2015-2016 academic year placement at NCSU.

The high-throughput evaluation of toxicity pathways is an emerging paradigm for future toxicity characterization. In order to meet the goals of this paradigm, methods are needed to assess toxicity pathways using as few assays as possible. We propose the use of multi-sensor cell-based reporter assays to meet this need. The proposed assay will allow for the single-assay assessment of the impact of a chemical or chemical mixture on processes along a defined signaling pathway. This proposed developmental work will utilize the RXR:PPAR signaling pathway which contributes to the regulation of development, reproductive, and metabolism. The methods will have application in the characterization of innumerable pathways with simple modifications to the construction of the assay components. Results will define an experimental approach that can be used in a high-through format to evaluate the response of hormone signaling pathways and networks to individual chemicals or mixtures. The assay also will have application across species and would significantly reduce uncertainty related to species sensitivity differences.

The terpenoid hormone methyl farnesoate is emerging as a master morphogen in crustaceans. We propose to test the hypothesis that methyl farnesoate regulates gene expression through interaction with a nuclear receptor of the NR2B subfamily. The hypothesis will be tested by definitively establishing the identity of the methyl farnesoate receptor (MfR) and evaluating the complementary roles of methyl farnesoate and its putative receptor in regulating hemoglobin hb2 gene expression. We have revealed that the putative MfR binds to a nucleotide sequence in the promoter region of the hb2 gene that consists of a nuclear receptor half-site flanked on the 5? end by an array of adenines and thymines (designated the MfRE). We also have shown that the putative MfR is immunochemically related to the nuclear hormone receptor NR2B4. Jones et al. (2006, FEBSJ, 273:1) have recently shown that methyl farnesoate binds with high affinity to the NR2B4 ortholog in Drosophila known as ultraspiracle. Taken together, these results indicate that the MfR in crustaceans is a member of the NR2B nuclear receptor subfamily. Further, we have identified and cloned the NR2B4 receptor in Daphnia magna, the crustacean model used in this research. The first objective of this study will be to conclusively identify MfR by: a) sequencing the putative MfR that we have affinity precipitated with the MfRE, and b) determining whether protein from methyl farnesoate-treated daphnids that binds the MfRE during electrophoretic mobility shift assays is immunochemically related to NR2B4. Next, recombinant MfR protein will be expressed and used to characterize binding interactions between potential ligands (terpenoids and terpenoid mimics) and the receptor (objective 2), as well as, the ability of the ligand/receptor complex to bind to the MfRE (objective 3). Potential ligands will include methyl farnesoate and several compounds with varying abilities to mimic the action of methyl farnesoate in vivo. Finally, (objective 4) the ability of MfR-ligand complexes, as identified in objectives 2 and 3, to activate gene transcription will be evaluated. For these experiments, cells will be cotransfected with an expression plasmid containing the MfR and a plasmid containing an MfRE/luciferase reporter construct. Potency of the ligands to stimulate reporter gene activity will be compared to their potency to induce the hemoglobin hb2 gene in the intact organism. This research holds significant promise in: a) establishing new paradigms in arthropod endocrinology by identifying the receptor that mediates the action of one of the most influential hormones in crustacean endocrine signaling, and b) providing new insight into the evolution of sexual versus asexual reproduction since we have shown that methyl farnesoate contributes to the selection of reproductive strategy by branchiopod crustaceans. This program also will provide opportunities for training of students from communities that are underrepresented in graduate education and will provide a venue for high school students from minority communities to interact with professional scientists.

Objective: US EPA must be equipped with tools that allow for the comprehensive assessment of exposure to and effects of endocrine-disrupting chemicals (EDCs) in order to adequately evaluate risks associated with such chemicals. EDC exposure issues are particularly tenacious since EDC mixtures can elicit toxicity through a variety of mechanisms that cannot be discerned by analytical chemistry or reporter-gene approaches. The overall objective of this proposed research program is to develop a gene-expression based, whole-organism approach to evaluating cumulative exposure to EDCs. This will be accomplished using the water flea (Daphnia magna) as a sentinel of exposure and a systematic array of gene-expression based signals that will serve as a dosimeter of exposure to EDCs individually or in mixture. Approach: The first aim of the program will be to identify a suite of biological markers that specifically respond to chemical modulators of hormone-signaling pathways. Differential-display and targeted RT-PCR approaches will be used to identify changes in gene expression that are specific indicators of exposure to EDCs. Having identified a suite of biomarkers of exposure (the goal is to identify 12-50 functional signals), the sensitivity, dosimetry value, and strength in identifying EDC types of each signal will be established (Aim 2). These results will be used to refine the development of a sensor (sentinel/signal unit) that can be used to evaluate exposure to EDCs. The final aim (3) of the program will be to evaluate the ability of the sensor to detect and assess exposure associated with EDC mixtures. Predicted response of the sensor to at least 20 formulated mixtures in aqueous solution will be modeled using our Integrated Addition and Interactions model for chemical mixtures. The sensor then will be used to measure exposure to these mixtures and outcome will be compared to model results. Comparison results will be used to establish the functionality of the sensor as well as additional refinement needs. Expected Results Unlike other exposure assessment approaches that utilize single reporter gene assays, this sensor will exploit hormone signaling circuitry in an intact organism that encompasses processes of hormone synthesis, hormone-receptor interactions, hormone elimination, and hormone-receptor cross-communication. In addition, the sensor will provide for EDC-biotic interactions involving biotransformation processes and EDC-EDC interactions that may result in synergy or antagonism. Finally, results from this proposed research will provide the basic research necessary for validation of the sensor of EDC exposure under field conditions and in various sample matrices (i.e., using extracts from sediments, plasma from human blood samples, plasma from lab animals following EDC exposure experiments).

Environmental cues dictate various events in the life history of organisms. Critical among these events is the timing of reproduction such that the likelihood of offspring survival is maximized. Human activities can disrupt environmental signaling with consequences to population sustenance. For example, the global contamination of the environment with tributyltin has resulted in disruptions of reproductive differentiation in Prosobranch neogastropods resulting in the local extinction of some populations. The overall goal of this research program is to understand the environmental-endocrine signaling processes that control reproduction in the Prosobranch mud snail Ilyanassa obsoleta. The specific hypothesis to be tested in this grant cycle is: Retinoids, androgens, and estrogens form a signaling web that regulates male reproductive differentiation (recrudescence) in the mud snail. Efforts during the previous grant cycle of this program provided evidence for the involvement of these three hormone classes in recrudescence. The first aim of this research will be to clone and sequence the receptors NR2B (retinoid receptor), NR3A (estrogen receptor), NR4A (retinoid-related receptor)), and the androgen receptor from the mud snail and to assess expression of these receptors through the reproductive cycle. Dr. LeBlanc?s team has partially cloned the first three receptors and will obtain full length cDNA sequences by 3?,5?-RACE technology. Their search for an androgen receptor in the mud snail using targeted RT-PCR techniques has thus-far been unsuccessful; however, these efforts will continue. The relative expression of the cloned receptors will be assessed in male and female snails through their reproductive cycle (recrudescence, egg-laying, senescence, reproductive dormancy). During the past grant cycle, androgen and estrogen levels were measured in snails through the reproductive cycle. In the next phase of this program, pertinent retinoids will be identified and changes in their levels of expression will be assessed through the reproductive cycle. The role of fatty acid esterification of retinoids in regulating free retinoid levels also will be determined. Results from these aims, in conjunction with results from the past grant cycle, will identify components of the endocrine signaling web that contribute to recrudescence. During aim 3, the precise role of the candidate signaling pathways in male recrudescence will be established. Environmental cues for initiation and promotion of male recrudescence, as established in the past grant cycle (photoperiod and temperature, respectively) will be varied and effects on endocrine signaling pathways (hormones, receptors, and transcription factors) will be determined. Finally, the mechanism by which tributyltin interferes environmental endocrine signaling of recrudescence will be determined. Specific emphasis will be placed on the hypothesis that tributyltin stimulates penis development in both sexes by inhibiting acyltransferase activities resulting in increased free retinoid and testosterone levels. This program will provide a detailed description of the endocrine-signaling web responsible for male recrudescence in a Prosobranch neogastropod. Results will identify the effects of tributyltin exposure on these regulatory processes that result in global ecological disturbances. This research also will reveal why molluscs - and perhaps other species - are so exquisitely susceptible to the toxicity of the ubiquitous environmental contaminant tributyltin. The infrastructure created by this program will provide for the hands-on and classroom training of post-doctorates, graduate students, and undergraduate students, in particular through the development and annual offering of a course in environmental endocrine signaling.

Pharmaceuticals, personal care products, and other chemicals have been shown to accumulate in surface waters of North Carolina and many of these contaminants have the potential to disrupt endocrine signaling processes. The hypothesis will be tested that mixtures of pharmaceuticals and other surface water contaminants, at levels found in the environment and individually considered to be safe, can adversely impact population biology of some crustaceans by modulating the endocrine signaling pathway responsible for sex determination. The hormone methyl farnesoate is responsible for sex determination, hemoglobin production, and other functions in many crustacean species. We have shown that some environmental contaminants can interfere with normal methyl farnesoate signaling by mimicking the action of the hormone or by potentiating the action of endogenous hormone. In the present study, several pharmaceuticals and other major surface water contaminants will be evaluated for endocrine disrupting activity (either as a methyl farnesoate mimic or potentiator). Chemicals will be evaluated for their ability to alter sex-determination in offspring and induce hemoglobin levels in the freshwater crustacean Daphnia magna. Combined effects of chemical mixtures of mimics and potentiators will be mathematically modeled in an effort to identify combinations and concentrations of chemicals that pose risk to crustaceans. Finally, the adverse effects of the identified mixtures on methyl farnesoate-regulated processes will be directly evaluated. Results will identify combinations of chemicals known to occur in the environment that may be threatening populations of crustaceans and other species.

The Department of Energy (DOE) is entrusted with managing hazard waste sites that contain mixtures of chemicals and radionucleotides. These wastes have the potential to elicit hazard to humans and to the environment. Understanding the hazards associated with these sites and effectively minimizing this hazard is termed Legacy Management. Under this subcontract, scientists from NCSU will participate in a DOE program funded to Tulane University that will provide tools for Legacy Management. Tasks at NSCU will involve assembling, collating, and formulating data on environmental signaling pathways. Environmental signaling pathways are means by which environmental signals (i.e. temperature, light, food, phermones, etc.) stimulate neuro-endocrine pathways in living things resulting in modification to metabolism, maturation, reproduction, etc. Potential adverse effects on hazardous waste site contaminants, individually or in combination, on environmental signaling pathways also will be explored and disseminated in formats that will service the funding agency, serve as teaching, learning, and research tools, and inform the general public. Internet publications utilizing neurolinguistic communication styles will be evaluated, and where appropriate, will be used as means of effectively communicating such information.