INBRE Faculty Associates in Cell Signaling


"The Role of TIMP-2 in Neural Crest Pathfinding"
Mark V. Reedy, PhD
Collaborator: Philip R. Brauer, PhD (CUMC)

Abstract: Neural crest cells detach from the embryonic neural tube and then migrate extensively throughout the embryo before differentiating into a number of derivatives. A subset of neural crest cells, called cardiac neural crest cells, contribute to several aspects of heart development, including septation, myocardial differentiation, and proper formation of the outflow tract. Many congenital heart defects are associated with abnormal cardiac neural crest cell development. However, very little is known about the factors that regulate cardiac neural crest migration in the embryo. We have shown the secreted protein TIMP-2 is expressed by chicken cardiac NC cells and required for their normal development. This project tests the hypothesis that TIMP-2 directs cardiac NC cells into the proper migratory pathway, and will elucidate the mechanism by which it does so.

"Intracellular and Epigenetic Mechanisms Underlying Neurotrophic Properties of Activated Microglia"
Annemarie Shibata, PhD
Collaborators: Howard E. Gendelman, MD (UNMC) and Scott D. Briggs, PhD (Purdue University)

Abstract: The nervous system was once considered to be "immune privileged" and isolated from immune system activity. However, a preponderance of evidence indicates that proper development and function of the central nervous system (CNS) relies on regulated interactions between nervous system and immune cells. Microglia are the resident immune cells of the CNS and respond rapidly to changes in the CNS environment. Microglia exhibit phagocytic activity following neuronal damage. Activated microglia produce neurotoxic molecules including inflammatory cytokines, chemokines, arachidonic acid, reactive oxygen and nitrogen species, and growth inhibiting proteins such as prostaglandins (Kim and Vellis, 2005; Lai and Todd, 2006). Conversely, emerging evidence suggests that, given specific activator(s), microglia may function to support neuronal survival, differentiation and potentially regeneration. Both in vitro and in vivo studies have shown that microglia produce neurotrophic factors such as nerve growth factor (NGF), neurotrophin 3 (NT3), and brain-derived neurotrophic factor (BDNF) (Kim and de Vellis, 2005; Morgan et al., 2004). Additional experiments have demonstrated that co-cultures of neurons and microglia increase neurogenesis in neural progenitor cells (Walton et al., 2006). Little is known about whether activated microglia are capable of producing neurotrophic effects in damaged neurons and which signaling and epigenetic mechanisms underlie these processes. Previous experiments have suggested that the PI3K/AKT and MAPK pathways could act as potential signaling mechanisms and it is likely that regulation of microglial signaling pathways determine their neurotrophic or neurotoxic phenotype. To investigate the signaling mechanisms and gene regulation involved in the immune response to neuronal damage, this proposal presents an in vitro model system employing state-of-the-art technology that is readily accessible to and utilized by undergraduate research students. Increasing our understanding of the mechanisms that drive neurotrophic verses neurotoxic phenotypes in microglia will provide insight into the intrinsic neuroprotective role of immune activity in the CNS and may aid in the development of methodologies to promote such activity during neurodegenerative disease or regeneration following injury.

"Zinc Finger-Inspired Fluorescent Chemosensors Operating via Confomational Restriction"
James T. Fletcher, PhD
Collaborator: D. David Smith, PhD (CUMC)

Abstract: Interactions between peptide residues and cations, such as that between Zn+2 and the zinc finger (ZnF) motifs, are vital constituents of cellular communication. The proposed investigation is relevant to the mission of Nebraska's INBRE program in that it will advance knowledge in the area of cell-signaling affected by interactions with zinc. Developing new sensor systems based on ZnF motifs will allow direct measurement of how zinc affinity varies with peptide identity and permit direct measurements to be made regarding the binding affinities of toxic metal ions relative to Zn+2 for a prescribed set of peptide chelating motifs. Such studies will lead to new insights as to how ZnF's exert selectivity for zinc and how toxic metals might lead to cellular damage. While using a synthetic chemical approach to better understand nature, this project will also exploit nature to develop new tools for ion and small molecule sensing, as the molecules developed for these studies will stand as new sensors for zinc detection. This study's first generation systems will be based on well-known ZnF chelator-analyte interactions (CCHH ZnF motif with Zn+2) in order to facilitate efficient structural optimization of the system's fluorescent output. Once optimized, point-mutation studies will be performed by modulating amino acid residue identities with the goal of discovering new derivatives selective for varying analytes. Uniquely, the proposed sensors have been designed to also allow the wavelength of arene fluorescence output to be modified independently of peptide variations, promising the ability to engineer multicolor readouts for varying analytes within a single chemosensor design. Because amino acid residues serve as the analyte recognition units in these systems, any analytes capable of noncovalently interacting with peptides could potentially be targeted using this general molecular approach (including cations, anions and small organic molecules). Hence, discoveries made in the proposed investigation will not only advance the understanding zinc's role in cellular communication but will also impact the field of fluorescence chemosensing.


"Transcriptional and Epigenetic Regulation of Human N-cadherin Gene Expression"
Katherine E. Marley, PhD
Collaborator: Keith R. Johnson, PhD (UNMC)

Abstract: Cadherins are a class of adhesion proteins required for tissue integrity. They are transmembrane proteins that form homodimers with cadherins on adjacent cells and attach either directly or indirectly to the cytoskeleton. E-cadherin is the most thoroughly studied cadherin due to its role in epithelial tissue maintenance. Most human cancers arise in epithelial tissues, and, during tumor progression, tumor cells may begin expressing N-cadherin inappropriately. N-cadherin is named for its role in neurite outgrowth, is found in more mesenchymal tissues, and tumor cells that begin expressing N-cadherin become more motile and invasive. The N-cadherin gene has only recently become the subject of study and promoter characterization in order to understand the regulation of transcription has been limited. The misexpression of N-cadherin in cancer cells appears to be very similar to the increase in N-cadherin expression that occurs during the normal developmental epithelial-to-mesenchymal transition (EMT), suggesting that insight into this metastatic transition can be gleaned from studies in EMT model cell culture systems. The overall goal of this research is to identify specific transcription factor binding sites, transcription factors, and epigenetic promoter modifications involved in regulating transcription of the human N-cadherin gene. Specific Aim 1 is to evaluate a putative repressor site between -462bp and -1896bp of the N-cadherin promoter in detail using luciferase reporters. Specific Aim 2 is to evaluate the proposed transcriptional role of a LEF-1 consensus binding site 60bp 3' of the first exon also using luciferase reporters. Specific Aim 3 involves using chromatin immuno-precipitation (ChIP) to detect in vivo binding of transcription factors including AP-1, LEF-1, any identified repressor, and potential epigenetic modifications including methylation. If ChIP assays indicate that AP-1, LEF-1 or other relevant transcription factors are binding to N-cadherin promoter fragments, Specific Aim 4 is to evaluate expression of N-cadherin promoter fragment-luciferase reporters in EMT model cell culture systems in which components of the EMT inducing signaling pathways can be interrupted.

"Genome Utilization and Plasticity during Root Gravitropism in Six Conditions"
Tessa L. Durham Brooks, PhD
Collaborator: Edgar P. Spalding (University of Wisconsin)

Abstract: The proposed research aims to develop tools and infrastructure to reliably describe gene function at the organismal level. While genome sequences of multicellular organisms are widely available, experimentally determined functions for the genes of any particular model are lacking. Understanding when, where and how genes are recruited in intact organisms goes hand-in-hand with the ability to define how gene networks are disrupted in a disease state. A first step in piecing together the gene networks involved in complex physiological processes is to develop a set of tools that can be used to detect the effects of single genes at the organismal level. This will be accomplished using a relatively simple model, the Arabidopsis root. This organ is simple in shape, which allows for more rapid development of image analysis platforms to characterize organ morphology, but also complex in its physiology; the root gravitropic response includes all of the classic components of a highly regulated stimulus-response system. Using high resolution imaging in tandem with computational approaches, subtle changes within the genome can be robustly detected. In order to scale these tools appropriately, the imaging platform will consist of high-resolution scanners capable of imaging many individuals continuously as they respond to gravity stimulus. The characterization of genome recruitment during this physiological response will use both course and fine association mapping techniques in addition to candidate gene studies. The proposed work also aims to begin mapping metabolomics data onto the genome data using a recently acquired 400 MHz NMR spectrometer. These efforts will be undertaken by undergraduates both in the lab and classroom and will require collaboration between a range of departments including biology, chemistry, physics and mathematics. The results of this study will allow for the construction of models that describe how the genome is recruited over the course of a given physiological response and how this recruitment changes in different environmental or developmental contexts. These data will be directly applied to the characterization of a newly discovered pathway within the gravitropic response that is dependent on Arabidopsis Glutamate Receptor-Like (AtGLR) genes.


"TLR3 Signaling in Pulmonary Mucosal Epithelial Cells"
Therese M. McGinn, PhD
Collaborator: Debra J. Romberger, MD (UNMC)

Abstract: Pulmonary mucosal epithelia serve as barriers from the external environment and as targets for infection with RNA viruses such as respiratory syncytial virus (RSV) and influenza. Virus infection of pulmonary epithelial cells triggers inflammatory responses that culminate in production of antiviral cytokines and chemokines. While often a crucial component of a successful immune response to airway pathogens, these responses, if not appropriately controlled, can lead to pathological complications. Production of inflammatory cytokines by airway epithelial cells is initiated upon engagement of Toll-like receptor 3 (TLR3) by double stranded RNA (dsRNA) produced during RNA virus replication. Numerous published reports have indicated that TLR3 ligation in dendritic cells (DCs) promotes DC maturation and may contribute to development of virus-specific acquired immunity. However, the role of TLR3 signaling in the interaction between RNA virus-infected airway epithelial cells and underlying DCs has not been defined. Current models for investigating DC maturation in the context of mucosal tissues rely on the use of animals. An in vitro system that facilitates discrete analysis of epithelial cells or DCs would promote efforts to elucidate mechanisms of communication between these cells. The long-term goal of the proposed work is to define the paracrine signals between airway epithelial cells and submucosal DCs that occur in response to external stimuli, including TLR agonists, and to understand how such signaling pathways participate in viral pathogenesis. We anticipate that such expanded understanding will potentiate identification of treatment strategies for viral and other pulmonary conditions. We hypothesize that: a) TLR3 signaling in pulmonary epithelial cells promotes maturation of DC and provides a link between innate and adaptive immunity in the respiratory tract; b) interaction between TLR3-activated respiratory epithelial cells and submucosal DCs is mediated through soluble factors that promote promote DC maturation.

"Performance and Energetics of Multiple Modes of Limbless Locomotion in Lizards and Snakes"
Gary Gerald, PhD
Collaborator:  Paul Schaeffer, PhD (Miami University)

Abstract: Limb reduction and limblessness among vertebrates is a fascinating evolutionary paradox and has been shown to occur in snakes and multiple lineages of lizards and amphibians.  However, investigations into the biomechanical consequences of possessing a limbless, tubular body in the scope of evolutionary biology are somewhat limited.  The primary goal of this research is to investigate variation in locomotor performance and energetics of multiple ways of moving with an elongated, limbless body.  The data collected will provide information on the major factors (e.g. morphological, behavioral, and ecological) that have favored the reduction and eventual loss of lizards and early snakes.


"Candidate Aging Gene Analyses of Large Drosophila melanogaster Populations"
Kimberly A. Carlson, PhD
Collaborator: Lawrence G. Harshman, PhD (UNL)

Abstract: Aging is characterized by a steady decline in an organism's ability to perform life-sustaining tasks. All organisms age, and this process is partially controlled by the regulation of gene expression. The lifespan of an organism is based on extrinsic factors, genes, and gene-environment interactions. Gene–environment interactions are of considerable interest because they are especially relevant to aging in human populations that are not environmentally controlled. Little is known about the genetics of aging in most animals and even less is known in natural populations of species used as models for genetic research. For genetic analyses of aging, Drosophila melanogaster (fruit fly) is a useful model organism due to the genetic and genomic tools available, and ability to compare to humans because of the similarity in genes. Transcriptome studies have been conducted on D. melanogaster, but not in large populations that allow for many samples to be taken and for flies to be sampled at very old ages when all but a very small proportion of a cohort has died. The value of taking many samples is that temporal trends in gene expression become much clearer allowing for the identification of candidate genes that may be acting in a similar manner and, in fact, might allow for the identification of networks of gene expression. Replicate populations are valuable because they allow for identification of candidate genes whose pattern of expression is robust across populations. The main goal of the proposed research is to utilize use large laboratory populations of D. melanogaster recently derived from a natural population and thereby representing natural genetic variation to obtain comprehensive transcriptome profiles throughout the adult life span. The goals of this project are: 1) conduct a transcriptome analysis of large replicate populations recently derived from the field 2) validate the pattern of expression of candidate genes, 3) use mutations and P-element lines to test the effect of candidate genes. The overall goal of this research is to better understand the role of genes representing natural genetic variation throughout adult life including the very oldest ages, which is expected to provide unique insight into aging and longevity.

"Isolation and Characterization of Extremozymes from Alkaline Lakes in Nebraska"
Julie J. Shaffer, PhD
Collaborator: Vicki Schlegel, PhD (UNL)

Abstract: Extreme environments have been of considerable interest in recent years due to the discovery that these "uninhabitable" environments are colonized by a diverse community of microorganisms. These microorganisms have been shown to provide valuable extremozymes, enzymes adapted to catalyze reactions under extreme environmental conditions. One well-known extremozyme is Taq polymerase from the hyperthermophile Thermus aquaticus. This research focuses on the little studied, hypersaline, alkaline lakes in western Nebraska. We will isolate and identify potential microorganisms capable of producing extremozymes of interest for numerous biotechnology applications. We will look for esterases, xylanases, cellulases, proteases, and lipases that are active at high pH's. Microorganisms capable of producing these enzymes will be isolated, the enzyme activity characterized, the enzyme purified, and the enzyme gene sequences identified and cloned into Escherichia coli.

"Degeneration of Group II Introns"
Dawn M. Simon, PhD
Collaborator: Hesham Ali, PhD (UNO-IS&T)

Abstract: The origin of spliceosomal introns has been in contention ever since they were first discovered. The ubiquity of introns in eukaryotes, their importance for RNA processing, as well as their role in disease make their origin and evolution a fundamental issue in biology. While there are many unanswered questions, there is a general consensus that group II introns are the most likely precursors. However, it is not known whether degeneration of group II introns is an ongoing process that can lead to the formation of novel spliceosomal introns, or has only occurred a limited number of times in the distant past. Based on diversity of group II introns and the frequent observation of putatively non-functional introns, degeneration is thought to be frequent. However, this process has rarely been studied in detail. The proposed project will study group II intron degeneration using three complementary approaches. First, publically available sequence data will be utilized to develop a global model of the group II intron life cycle. This will include the use of several search strategies to gain a complete picture of group II intron composition in bacterial and organellar genomes. The introns will be characterized using phylogenetic and sequence analyses and then fit into a model describing intron origin, degeneration, and loss. Next, the model will be refined using phylogenetic methods to understand the evolutionary relationship of the group II-intron encoded ORF and RNA structures. This method will first be used to determine whether the ORF and RNA have strictly coevolved and then to examine common degeneration patterns in secondary structure. Finally, an exemplar intron in red algae will be studied to better understand the pattern and temporal components of degeneration. In summary, the proposed project ranges in scope from broad-scale genomic analyses to a narrow focus on a single exemplar intron. The combination of approaches is expected to provide a more complete understanding of group II intron degeneration.

"Hormonal Mechanisms of Alternative Reproductive Tactics"
Letitia M. Reichart, PhD
Collaborator: Jeff French, PhD (UNO-Biology)

Abstract: Variation in animal behavior is the product of gene by environment interactions. Specifically reproductive behaviors among vertebrates are often regulated by chemical signals (e.g., hormones) which are influenced by environmental cues. In many vertebrate species, variation in reproductive behavior exists as alternative reproductive tactics (e.g., male birds obtaining additional reproductive success through extra-pair mating, whereas other males assume a monogamous relationship). Individuals participating in alternative reproductive tactics likely exhibit physiological differences in hormone secretion and could potentially display unique variation in the genetic code. Indeed, unique genetic variation in hormone signaling pathways may provide valuable information for variation in reproductive physiology and development under certain environmental conditions. Currently, most research investigating alternative reproductive behaviors in animals have focused singly on either proximate mechanisms or ultimate explanations for behaviors. Few studies have combined approaches to address potential gene environment interactions influencing differences in reproductive tactics.

Thus, for this research we propose three specific aims to study variation in alternative reproductive behaviors in an avian model system. First, we will measure success for individuals pursuing alternative reproductive tactics using molecular genetic analyses of parentage. Second, we aim to identify proximate mechanisms associated with differences in reproductive behavior via measures of hormones in avian eggs and fecal samples. Third, we will use the information obtained from aims one and two to conduct an informed analysis (e.g., identify specific target tissues based on important hormone pathways identified in specific aim 2) of differences in gene expression. Specifically, we aim to identify the molecular basis of hormone action for observed phenotypic polymorphisms (e.g., reproductive tactics).


"Cell Signaling in Intestinal Epithelial Cells from Wound Healing"
Shawn D. Pearcy, PhD
Collaborator: Myron L. Toews (UNMC)

Abstract: Investigate the expression and role of some of the protein kinase C family of enzymes on wound healing in intestinal epithelial cells. Specific emphasis will be placed on the classical isoforms α and β and the novel isoform δ.

Examine the effect of corticosteroid and lysophosphatidic acid exposure on the expression of protein kinase C isozymes (α, β, δ) and their impact on rates of wound healing in intestinal epithelium.