Randall S. Hewes, Ph.D. - US grants

This award provides partial support for the purchase of three major equipment items: a 16-capillary DNA sequencer, a real-time quantitative PCR machine, and a digital phosphor/fluorescent imager. All three of these items will enhance an existing multi-user core facility for molecular biology research in the Department of Zoology at the University of Oklahoma-Norman Campus. The use of molecular methods has become essential in nearly all areas of biology. Beyond being central to molecular genetics, cell, and developmental biology, there has been rapid growth in the use of molecular techniques in areas such as ecology, systematics, evolution, and behavior. Molecular biology itself has recently been driven by large-scale genome sequencing and proteomics projects, with emergent new technologies and associated instruments. Studies in nearly all areas of biology increasingly focus on analysis of DNA sequence, DNA fragment (e.g., single nucleotide polymorphisms [SNPs] and microsatellites), and gene expression data, requiring access to sophisticated equipment frequently beyond the resources of individual investigators or labs. The instrumentation will also enhance undergraduate, graduate, and post-graduate research training opportunities at the University of Oklahoma.

Neuropeptides are chemical signals that are secreted by nerve cells to control the functioning of the brain and many other tissues. They are key regulators of diverse processes, including growth, reproduction, stress, sleep, body weight, pain, and circadian (daily) rhythms. Disruptions in neuropeptide signaling are associated with human diseases, such as obesity, diabetes and cancer. Thus, there is a critical need for basic research on neuropeptide systems.

The major goal of this project is to understand complex molecular mechanisms underlying long-term regulation of neuropeptide secretion. To support normal signaling, peptidergic (neuropeptide-secreting) cells must develop the capacity to produce and store large quantities of neuropeptides. In mature cells, neuropeptide synthesis is regulated in response to diverse internal and external cues in order to adjust the strength of neuropeptide signaling. For many cell types, the genes and cell signaling pathways underlying these processes, and their physiological and developmental regulation, are almost completely unknown. With the powerful tools recently developed for the genetic manipulation of peptidergic cells in the fruit fly, Drosophila melanogaster, these mechanisms are now experimentally accessible. In previous work, the Drosophila gene dimmed (dimm) was shown to control accumulation and/or storage of secretory proteins in diverse peptidergic cells. This result suggests that dimm is an essential component of a cell signaling pathway controlling the robustness and timing of neuropeptide synthesis. To test this hypothesis, this project will address two key questions. First, what are the roles of dimm in developing versus mature cells? Second, what other proteins interact with Dimm, the protein product of the dimm gene, to control neuropeptide levels?

To address the first question-what are the roles of dimm in developing versus mature cells-the first aim of this project is to define the contributions of the dimm gene in the regulation or maintenance of neuropeptide synthesis throughout the life cycle of the fly. Using transgenic animals, and a new temperature-dependent gene expression system, a re-introduced copy of the dimm gene will be turned on or off during specific stages of development. This system also allows peptidergic cell-type specific control over the expression of the dimm gene. Thus, transient and spatially restricted expression of the dimm gene will be used to determine the effects of post-embryonic increases in dimm expression on neuropeptide levels in dimm mutant and in normal cells. To address the second question-what other proteins interact with Dimm to control neuropeptide levels-the second aim of this project is to perform genetic screens to identify mutations of genes encoding other elements in the Dimm molecular signaling pathway. Genes will be identified by virtue of their ability to modify the effects of dimm misexpression in transgenic animals. Genetic interactions will be detected using two dimm-sensitive processes: molting and neuropeptide synthesis. Selected mutants then will be tested for effects on neuropeptide levels in developing and mature cells, and the mutated genes will be identified using standard Drosophila molecular cloning methods. Together, the proposed experiments will lay a foundation for defining key molecular pathways controlling neuroendocrine signaling.

This project will continue to provide unique opportunities for several undergraduate and graduate students to participate in the research. Students in Oklahoma have few options for non-agricultural training in the sciences. This project provides hands-on experiences in molecular biology and genetics that are not otherwise readily available and that complement traditional lecture-format courses. These research experiences will continue to enable and inspire talented students from the State of Oklahoma to pursue professional careers in industry, medicine, and scientific research.

Peptide hormones are chemical signals that are secreted by cells to control the functioning of the brain and many other tissues. They are key regulators of diverse processes, including growth, reproduction, stress, sleep, body weight, pain, and daily rhythms. The long-term goal of this proposal is to understand the mechanisms within cells that underlie the regulation of peptide hormone signaling by steroids. Specifically, this work will define mechanisms underlying the steroid regulation of the gene encoding the peptide hormone, ecdysis-triggering hormone (ETH). Toward this end, methods were established in the fruit fly, Drosophila melanogaster, for manipulation of steroid signaling in single cells. This is combined with tracking of ETH gene expression through genetic and molecular assays, and genetic manipulations to measure the contributions to this process from steroid receptors and other interacting proteins. The cellular sites of steroid receptor action underlying the regulation of ETH gene expression, and the roles of different DNA sequences within the ETH gene, will be determined genetically and biochemically. The regulation of gene expression by steroid receptors requires the participation of co-factors. These will be identified through genetic screens. Together, these experiments will examine how co-factors for a steroid receptor can mediate cell-type specific expression of steroid hormone-responsive genes. This basic research will contribute to our understanding of the disruptions in hormone signaling associated with human diseases, such as obesity, diabetes and cancer. This work will also be a unique opportunity for several undergraduate and graduate students to participate in research. Students in Oklahoma have few options for non-agricultural training in the sciences. The experiences provided by this project are not otherwise readily available, and they complement traditional lecture-format courses. These research experiences will continue to enable and inspire talented students from the State of Oklahoma to pursue professional careers in industry, medicine, and scientific research.

This Major Research Instrumentation award funds the acquisition of a multi-photon laser scanning confocal microscope for research and training in interdisciplinary fields on the University of Oklahoma (OU) Norman campus. The new instrument has higher sensitivity and faster speed and will significantly boost the capabilities for live and deep-tissue imaging by a diversity of life science and engineering researchers on the OU campus and at nearby research institutions. This new confocal microscope will be used by a number of NSF-sponsored laboratories that are already active in cell and molecular imaging as well as by new users who will incorporate confocal imaging in their research projects. The research topics include, but they are not limited to, neural crest migration in the primitive vertebrate lamprey, neurite outgrowth and synaptic vesicle trafficking in fruit flies, calcium imaging of deep nerve tissues, temporal and spatial patterns of protein complex formation in plants, and biofilm growth and morphology during corrosion of metallic surface. The new microscope will be an important part of education and research integration; students will be trained initially through a new upper undergraduate and graduate student course, Applied Confocal Imaging, taught collectively by the PIs. In addition, the PIs and their students will collaborate with rural and urban Oklahoma K-12 teachers in developing and implementing cutting-edge outreach and classroom projects. The results of these research, teaching, and outreach efforts will be broadly disseminated through participation of students and faculty at professional meetings and through peer-reviewed publications.

The National Science Foundation (NSF) Graduate Research Fellowship Program (GRFP) is a highly competitive, federal fellowship program. GRFP helps ensure the vitality and diversity of the scientific and engineering workforce of the United States. The program recognizes and supports outstanding graduate students who are pursuing research-based master's and doctoral degrees in science, technology, engineering, and mathematics (STEM) and in STEM education. The GRFP provides three years of financial support for the graduate education of individuals who have demonstrated their potential for significant research achievements in STEM and STEM education. This award supports the NSF Graduate Fellows pursuing graduate education at this GRFP institution.

The Louis Stokes Alliances for Minority Participation (LSAMP) program assists universities and colleges in diversifying the STEM workforce through the development of highly competitive students from groups historically underrepresented in STEM disciplines: African-Americans, Alaska Natives, American Indians, Hispanic Americans, Native Hawaiians, and Native Pacific Islanders. The goal of the LSAMP Bridge to the Doctorate (BD) Activity is to increase the quantity and quality of STEM graduate students from underrepresented populations, with emphasis on Ph.D. matriculation and completion. BD programs implemented in the nation's institutions of higher education contribute to addressing one of the objectives in NSF's 2014-2018 Strategic Plan, namely to "integrate education and research to support development of a diverse STEM workforce with cutting-edge capabilities." The University of Oklahoma, a member institution of the Oklahoma LSAMP alliance (OK-AMP), proposes an integrated model of social supports and community networks to support the graduate education for 12 graduate students from underrepresented groups as Bridge to the Doctorate (BD) Fellows for two years.

Bridge to the Doctorate: OK-LSAMP will integrate Social Cognitive Career Theory and Social Capital Theory into a research-based mentoring program for optimizing academic, personal, and professional development of 12 BD Fellows. The mentoring program focuses on helping Fellows develop multi-layered communities of support. The project seeks to institutionalize a culture that enhances the personal and professional development and improve retention to PhD for all STEM graduate students.