Robert C. Burghardt - US grants

The following proposal outlines our interest in the role of intercellular communication in the control of reproductive function in mammals. Evidence is provide for the regulation of ovarian and uterine cell surface structures termed gap junctions which are involved in the direct exchange of a variety of small regulatory and informational molecules between cells in contact. The anatomical basis for the establishment of coordinated uterine smooth muscle activity during labor is due to the appearance of myometrial cell gap junctions which facilitates electrical coupling and thereby a single electrical syncytium during parturition. The studies outlined in this proposal are designed to provide a unique experimental approach to the analysis of the mechanisms regulating intercellular communication in the mammalian uterus. The objective of the research are: (a) To study the action of estrogen and progesterone on the formation of myometrial gap junctions in vitro and compare the permeability properties of myometrial gap junctions with those of vascular smooth muscle cells: (b) to test the hypothesis that the permeability of gap junctions in myometrium can be modulated by cAMP and the inositol phospholipid signal transduction pathways; (c) to develop myometrial cell-granulosa co-culture systems in order to analyze differences in the regulatory mechanisms affecting gap junction channel permeability: (d) to test the hypothesis that electron dense deposits associated with gap junction membrane following receptor mediated activation off the cAMP pathway represents Ca++ trapped by adenylyl cyclase derived pyrophosphate anions. As prematurity is associated with over 60% of all infant deaths, reduction of this hazard requires research on the factors that maintain pregnancy and initiate labor. The results of these studies will lead to a better understanding of uterine myometrial contractility during parturition and hopefully, to the development of effective tocolytic agents.

The Superfund Basic Research Program (SBRP) at Texas A&M University has relied heavily on the facilities and staff support available in the Image Analysis Core. This Core provides a variety of support services to SBRP investigators with major emphasis placed on assessment of the effects of a variety of biological response modifiers (including toxicants) on cells and tissues using conventional optical and electron microscopy approaches. The Core is also working with SBRP investigators to integrate advances in vital analytical cellular imaging and fluorescence probe technologies to provide some of the most sensitive approaches for analysis of the molecular mechanisms of cellular toxicity. The Image Analysis Core fills a critical need for SBRP investigators by housing state-of-the-art technologies, by providing continued maintenance and upgrades to equipment, and by offering specialized instruction and/or technical advice to investigators and their trainees. This core provides a cost-efficient and highly effective way to provide advanced imaging technologies to six different projects. Because of its extensive utilization by the individual research projects and interactions with other facilities cores, the Image Analysis Core has a strong commitment to advancing SBRP goals and provides a forum for scientific interactions between SBRP investigators.

The Superfund Basic Research Program at Texas A&M University has relied heavily on the facilities and staff support available in the Image Analysis Laboratory due to advances in non-invasive imaging tools using biosensors and biomarkers for defining the function of living cells and tissues. Five different SBRP projects will exploit the analytical imaging technologies within this core to monitor numerous vital functional endpoints within cells exposed to a diverse array of relevant toxicants. Functional endpoints include assessment of intracellular glutathione, pH and Ca2+ homeostasis, intercellular communication, and GFP- tagged protein expression. Additional microscopy support with digital image acquisition and analysis for in situ hybridization and immunocytochemistry will be provided. This Core will provide the interface with the Field Services and Analytical Services Cores receive and/or distribute samples provided by the Field Services of Analytical Services Cores to investigators responsible for performing endocrine disruptor, genotoxicity, or pollutant-sensitive bioassays. Because of its extensive utilization by the different research projects and interactions with other facilities cores, the Image Analysis and Bioassays Core has a strong commitment to advancing the SBRP goals and serves as a forum for scientific interactions between SBRP investigators.

This project will develop and implement a novel integrative biology approach to investigate chemical-induced stress and injury mechanisms at the cell and tissue level caused by selected PAHs. Precision cut tissue slices of liver and kidney will be exploited to non-invasively detect and analyze well-chosen endpoints using a novel "cellulomics" approach in order to begin bridging the gap between in vitro and in vivo models for mechanistic analysis of cellular injury. Animal models of susceptible phenotypes including mice with polymorphic variations of the AhR that result in dioxin-sensitive (C57BL6J) and resistant (DBA2) phenotypes, a transgenic mouse possessing the human AhR (hAhR; the hAhR knock-in) and an AhR knock-out mouse will be utilized to identify PAH effects on altered cellular homeostasis and how this may contribute to disease risk. While the proposed investigations will initially focus on a few PAHs and two organ systems, the tools and approaches developed will have wide applicability to high throughput organ- and tissue-level assessment of cellular responses caused by model PAHs and complex mixtures as well as for new and/or untested chemicals. Model PAH compounds selected for this investigation include benzo[a]pyrene (BaP), benzo[e]pyrene (BeP), 5-methylchrysene (5MCr), and 3-methylcholanthrene (SMC). The inherent fluorescence of PAHs will be utilized to map the distribution and in situ metabolism of BaP, BeP, 5MCr and SMC in the precision-cut liver and kidney slices of the mouse models. Cellular homeostasis mechanisms will also be assessed and include analysis of intracellular Ca2+ homeostasis and signaling mechanisms as both a sentinel and an effector of cellular injury along with a combination of other functional homeostasis parameters including gap junction mediated intercellular communication, reactive oxygen species and nitric oxide production, glutathione and glutathione-S-transferase activity, mitochondrial function, lipid peroxidation, cytochrome P450 enzyme induction, P-glycoprotein induction and apoptosis/necrosis. Once optimized, these cellulomics investigations will be extended to binary and complex mixtures of PAHs.

The Superfund Basic Research Program (SBRP) at Texas A&M University has relied heavily on the facilities and staff support available in the Image Analysis Core. This Core provides a variety of support services to SBRP investigators with major emphasis placed on assessment of the effects of a variety of biological response modifiers (including toxicants) on cells and tissues using conventional optical and electron microscopy approaches. The Core is also working with SBRP investigators to integrate advances in vital analytical cellular imaging and fluorescence probe technologies to provide some of the most sensitive approaches for analysis of the molecular mechanisms of cellular toxicity. The Image Analysis Core fills a critical need for SBRP investigators by housing state-of-the-art technologies, by providing continued maintenance and upgrades to equipment, and by offering specialized instruction and/or technical advice to investigators and their trainees. This core provides a cost-efficient and highly effective way to provide advanced imaging technologies to six different projects. Because of its extensive utilization by the individual research projects and interactions with other facilities cores, the Image Analysis Core has a strong commitment to advancing SBRP goals and provides a forum for scientific interactions between SBRP investigators.

The Advanced Imaging Facility Core (AIFC) is part of an integrated Facility Core program consisting of hypothesis generating, testing, and translational resources within an Integrated Discovery Pipeline, designed to accelerate and advance innovative ideas from hypothesis to practice. The primary goal of the AIFC is to provide CTEHR investigators with state-of-the-art imaging technologies that can be used to visualize and quantify the effects of xenobiotics in live and fixed cells and tissues. By combining state-of-the-art technologies with a sophisticated program of informatics for data mining and sharing, this Core will also allow researchers to tackle some of the most challenging questions in EHS research. The AIFC will play an integral role in supporting the CTEHR mission by carrying out the following aims: Aim 1: Maintain a wide range of state-of-the-art microscopy technologies and expertise to address the diverse imaging needs of CTEHR research; Aim 2: Provide CTEHR investigators with cost effective, prioritized access to analytical microscopy tools, efficient and effective instrument training and consulting services for experimental design, applications development and data analysis; Aim 3: Educate CTEHR members about the capabilities of existing and emerging microscopy technologies and applications and support the career development and mentoring activities of Center investigators; Aim 4: Facilitate translational research activities and next generation optical microscopy initiatives through interactions with the other CTEHR Facility Cores and biomedical optics investigators in the Enabling Technologies Thematic Focus Area of the Center. The AIFC fills a critical need for CTEHR investigators by delivering advanced technologies, specialized training and extensive expertise in the analysis of the cytotoxic, genotoxic, mutagenic, and endocrine disrupting activities of a variety of environmental chemicals. A wide range of microscopy hardware/software for advanced light and electron microscopy and fully automated high throughput image-based screening facilities will make it possible for Center investigators to access expensive, rapidly evolving instrumentation and expertise in a cost effective fashion. Access to the powerful AIFC imaging infrastructure provides the opportunity to develop and test novel hypotheses about the critical interactions between cells and environmental factors that contribute to human health and disease.

The overall goal of the Career Development Program (CDP) of the CTEHR is to support training and professional development of new and transitioning environmental health investigators and physician scientists. Because the most important, scientifically interesting and challenging questions in EHS research are embedded at the interface of multiple disciplines, the CDP will play a key role in helping to facilitate the generation of new scientific knowledge, technical capabilities, and formation of inter- and trans-disciplinary groups to develop the future EHS research workforce. The approach will focus on the continuum of career development from post-doctoral fellow to junior faculty and clinician scientists to established scientists pursuing environmental health research questions. In order to accomplish this goal, we will carry out the following Specific Aims: Aim 1. Attract and engage a diverse group of junior investigators to enhance pursuit of EHS research through development of a formal mentoring program, targeted pilot project opportunities and access to advanced technology through CTEHR Facility Cores. Aim 2. Target and mentor selected post-doctoral trainees to provide focused mentoring and foster development of successful NIH K awards. Aim 3. Develop an Inter-Center Resource Alliance and Mentored Partnership Program to enhance inter- center collaboration and expand EHS research. Aim 4. Develop, in collaboration with the COEC and IHSFC, Inreach activities to provide information, strategies and expertise on engaging communities in community-based participatory and epidemiologic research. The CDP is in a unique position to accomplish these aims, due to connections with the CTEHR Thematic Focus Areas, and shared leadership with the Advanced Imaging, Quantitative Biology, Targeted Genomics and Integrated Health Sciences Facility Cores and the COEC.

Bio Science Facility Core (BSFC) ABSTRACT A major goal of the Texas A&M Center for Environmental Health Research (TiCER) is to evaluate the effects of toxicological agents present in the environment on target populations. The ability to quantify biological effects of different exposures is critical to understanding and interpreting the health impact of environmental exposures. The Bio Science Facility Core (BSFC) will provide Center members access to key enabling technologies and expertise to support the vision of the Center ?Enhancing Public Health by Identifying, Understanding and Reducing Adverse Environmental Health Risks.? This will be accomplished by providing access to state-of-the- art analytical technology, infrastructure, and expertise to perform mechanistic research on environmental factors that impact human health and disease. The BSFC will integrate imaging, molecular, and physiological phenotyping capabilities, which are currently distributed across the Texas A&M campus, to support multi-scale research into the underlying mechanisms of environmental health risks, from single cells and organoids to experimental animal populations. The BSFC will achieve this goal by: (1) Maintaining a wide range of state-of- the-art technologies and expertise to enhance environmental health research integration and translation; (2) Providing subsidized and prioritized access to expertise with analytical technologies through training, consulting services, applications development, and mentoring; and (3) Facilitating translational research activities to enhance the capacity, breadth, collaborative nature, and impact of environmental health research. The BSFC will provide expertise and analytical equipment to measure a continuum of phenotypic readouts ranging from the single-cell level to population-scale models. The leaders of the BSFC have an established track record of collaborative, interdisciplinary, and translational research, and are well positioned to facilitate the technology needs of Center members to increase research efficiency and facilitate sharing of experimental procedures.