Cell Biology & Molecular Genetics Theses and Dissertations
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Item The Role of ErbB Receptors in Neisseria gonorrhoeae Invasion of Genital Epithelial Cells(2010) Swanson, Karen; Song, Wenxia; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Neisseria gonorrhoeae, the causative agent of the sexually transmitted infection gonorrhea, adheres to and invades genital epithelial cells. This study investigates host components that are used by the bacteria for their entry into epithelial cells. I found that the interaction of gonococci with the surface of HEC-1-B, a human endometrial carcinoma, and ME180, a human cervical epidermoid carcinoma, caused redistribution of both epidermal growth factor receptor (EGFR) and ErbB2, a related family member. Both EGFR and ErbB2 were translocated from the basolateral to the apical membrane in polarized HEC-1-B cells and concentrated under the microcolonies. Gonococcal infection increased EGFR and ErbB2 phosphorylation, indicating activation of the receptors. Kinase inhibitors of EGFR and ErbB2 inhibited and enhanced bacterial invasion, respectively, but had no effect on gonococcal adherence or the recruitment of EGFR and ErbB2 to the microcolonies. Gonococcal inoculation upregulated the transcription levels and matrix metalloproteinases (MMP)-mediated surface shedding of ligands of EGFR. Inhibition of the surface shedding of EGFR ligands by an MMP inhibitor and by heparin wash reduced gonococcal invasion without altering their adherence. N. gonorrhoeae induced the activation of the MAP Kinase ERK, PI3K/AKT and PLCγ signaling pathways in an EGFR tyrosine kinase-dependent manner. Blocking Ca2+ flux, the downstream pathway of PLCγ but not ERK and PI3K by inhibitors reduced gonococcal invasion. These data indicate that N. gonorrhoeae utilizes host signaling pathways to drive its invasion. The bacteria modulates host signaling by recruiting and activating EGFR and ErbB2. N. gonorrhoeae induces EGFR activation by increasing the expression and MMP-mediated shedding of EGFR ligands.Item The Role of Interleukin-19 in Interleukin-10 Production by Regulatory Macrophages(2010) Yahil, Ron Jonathan; Mosser, David M.; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Interleukin-19 (IL-19) is a recently discovered member of the IL-10 family of Class II cytokines. Although it is known to be secreted by monocytes and has been associated with various models of disease, the biological function of IL-19 remains largely unknown. IL-19 does share many important characteristics with IL-10. Because of this, we hypothesized that IL-19 may be regulated in a manner similar to IL-10, and may provide insight into the molecular mechanism of IL-10 regulation. In addition, IL-19 has been reported to increase IL-10 production in monocytes, and we theorized that it may be able to do the same in macrophages. Like IL-10, IL-19 is expressed in regulatory macrophages. Also, IL-19 is itself able to increase IL-10 production in regulatory macrophages, and the mechanism is independent of ERK phosphorylation. This work suggests that IL-19 can play a central role in the anti-inflammatory processes of IL-10.Item Role and Regulation of Autophagy During Developmental Cell Death in Drosophila Melanogaster(2010) McPhee, Christina Kary; Mount, Stephen M; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Two prominent morphological forms of programmed cell death occur during development, apoptosis and autophagic cell death. Improper regulation of cell death can lead to a variety of diseases, including cancer. Autophagy is required for survival in response to starvation, but has also been associated with cell death. It is unclear how autophagy is regulated under specific cell contexts in multi-cellular organisms, and what may distinguish autophagy function during cell survival versus cell death. Autophagic cell death is characterized by cells that die in synchrony, with autophagic vacuoles in the cytoplasm, and phagocytosis of the dying cells is not observed. However, little is known about this form of cell death. Autophagic cell death is observed during mammalian development, during regression of the corpus luteum and involution of the mammary and prostate glands. Autophagic cell death is also observed during development of the fruitfly Drosophila melanogaster, during larval salivary gland cell death. Drosophila is an excellent genetic model system to study developmental cell death in vivo. Cells use two main catabolic processes to degrade and recycle cellular contents, the ubiquitin/proteasome system (UPS) and autophagy. Here I investigate the role of the UPS and autophagy in developmental cell death using Drosophila larval salivary glands as an in vivo model. Proteasome inhibitors are being used in anti-cancer therapies; however the cellular effects of proteasome inhibition have not been studied in vivo. Here I demonstrate that the UPS is impaired during developmental cell death in vivo. Taking a proteomics approach to identify proteins enriched in salivary glands during developmental cell death and in response to proteasome impairment, I identify several novel genes required for salivary gland cell death, including Cop9 signalsome subunit 6 and the engulfment receptor Draper. Here I show that the engulfment receptor Draper is required for salivary gland degradation. This is the first example of an engulfment factor that is autonomously required for self-clearance. Surprisingly, I find that Draper is cell-autonomously required for autophagy during cell death, but not for starvation-induced autophagy. Draper is the first factor to be identified that genetically distinguishes autophagy that is associated with cell death from cell survival.Item POLLEN TUBES FAIL TO TARGET OVULE IN THE ABSENCE OF TWO CATION/PROTON EXCHANGERS IN ARABIDOPSIS(2010) Lu, Yongxian; Sze, Heven; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Flowering plant reproduction requires precise delivery of the sperm cells to the ovule by a pollen tube. Guiding signals from female cells are being identified, though how pollen senses and responds to those cues are largely unknown. Here I provide genetic evidence that two predicted cation/proton exchangers expressed in Arabidopsis pollen play essential roles in pollen targeting of ovules. Male fertility was unchanged in single chx21 or chx23 mutant pollen; however, male-specific gene transmission was blocked in chx21chx23 double mutant. Wild-type pistil provided with a limited amount of pollen containing a mixture of single and double mutant produced ~60% less seeds compared to that produced with chx23 single mutant pollen, indicating that chx21chx23 pollen is infertile. The double mutant pollen, visualized by a pollen-specific promoter-driven GUS activity, germinated and extended a tube down the transmitting tract, but the tube failed to turn and target an ovule. Unlike wild-type pollen that targeted isolated ovules in a semi-in vivo assay, tube guidance in chx21chx23 pollen was compromised. As a first step to understand the cellular and molecular bases of tube guidance, membrane localization and activity of CHX23 was determined. GFP-tagged CHX23 was localized to endomembranes, predominantly endoplasmic reticulum (ER), in elongating pollen tubes. Furthermore, expression of CHX23 in E. coli resulted in enhanced K+ accumulation at alkaline pH, suggesting a role for CHX23 in K+ acquisition and pH homeostasis. Based on these studies and observations by others that ER oscillates and enters the apex, a simple model is proposed: Modification of localized pH by CHX21 or CHX23 enables pollen tube to sense female signals and respond by shifting directional growth at the funiculus and micropyle to target pollen tip growth towards the ovule.Item Molecular Mechanisms of the Inhibition of Apoptosis by Mycobacterium tuberculosis(2009) Miller, Jessica Lynn; Briken, Volker; Molecular and Cell Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The capacity of infected cells to undergo apoptosis upon insult with a pathogen is an ancient innate immune defense mechanism. Consequently, the ability of persistent intracellular pathogens, such as the human pathogen Mycobacterium tuberculosis (Mtb), to inhibit infection-induced apoptosis of macrophages is important for virulence and to achieve persistence in the host. The nuoG gene of Mtb, which encodes the NuoG subunit of the type I NADH dehydrogenase NDH-1, is important in Mtb-mediated inhibition of host macrophage apoptosis. Here I determine the molecular mechanisms of this host-pathogen interaction. Apoptosis induced by the nuoG deletion mutant (nuoG ) is caspase-8 and TNF-α dependent. This cell death was also reduced in the presence of neutralizers and inhibitors of reactive oxygen species (ROS) and in macrophages derived from NOX2 deficient mice, suggesting that DnuoG induced death is dependent upon NOX2 derived ROS. Correlatively, nuoG infected macrophages also produced more phagosomal ROS than those infected with Mtb, or cells derived from NOX2 deficient mice. NuoG also inhibited apoptosis in human alveolar macrophages in a NOX2 dependant manner. These data suggest that reduction of phagosomal ROS is important for inhibition of apoptosis. Consistent with this hypothesis, Mtb deficient in the ROS neutralizing catalase, KatG, also accumulated ROS in the phagosome and was pro-apoptotic in macrophages. The specific mechanism by which NuoG reduces phagosomal ROS is still unknown. We could not detect secretion of NuoG, so direct neutralization of ROS is unlikely. Interestingly, preliminary data suggests that nuoG may be defective in secretion of SodA and KatG, enzymes known to be important for neutralizing ROS. In conclusion, these studies revealed that Mtb inhibits macrophage apoptosis by neutralizing phagosomal ROS via the NuoG dependent secretion of SodA and KatG. Furthermore, this research suggests a novel function for NOX2 activity in innate immunity, which is the sensing of persisting intracellular pathogens and subsequent induction of host cell apoptosis as a second line of defense for pathogens resistant to the respiratory burst.Item EXPLORING THE ROLE OF NFκB HOMOLOGS IN AUTOPHAGIC CELL DEATH IN THE DROSOPHILA SALIVARY GLAND(2009) Ivory, Adrienne; Wu, Louisa P; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The innate immune response is an ancient, highly conserved means of defense against pathogens. An important mediator of innate immunity is the NF-κB (Nuclear Factor-Kappa B) family of transcription factors. Activation of immune-signaling pathways leads to the nuclear translocation of NFκB proteins which initiate the transcription of antimicrobial peptides (AMPs) that circulate and destroy microbes. In Drosophila, these AMPs are up-regulated during the destruction of larval salivary glands. Salivary gland cells are destroyed via autophagy during metamorphosis. This project sought to determine what, if any, role the NFκB transcription factors have in autophagic cell death. Using the Drosophila model, it was determined that a loss of AMP activity during metamorphosis results in a failure to completely degrade larval salivary glands, and this defect appears to be due to an inability to remove autophagic vacuoles. It is suggested that AMPs may serve to degrade the membranes of autophagic vacuoles.Item THE ROLE OF SPERMIDINE IN THE REGULATION OF DEVELOPMENT AND DIFFERENTIATION IN SPERMATIDS OF MARSILEA VESTITA(2009) Deeb, Faten; Wolniak, Stephen M; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Spermiogenesis in the microspore of the water fern, Marsilea vestita, is a rapid process where a dry microspore containing a single cell undergoes nine successive mitotic divisions to produce 32 spermatids and seven sterile cells. Immediately after the dry microspore is placed into water, cytoplasmic movements precede the first mitotic division; a number of proteins and mRNAs aggregate into zones that later become the spermatogenous initials of the gametophyte. Development is driven by the regulated translation of stored mRNA with little or no new transcription (Hart and Wolniak, 1999). The pattern of translation in the gametophyte is ordered precisely spatially and temporally, which indicates that certain proteins are required in specific locations at specific stages of development. Spermatid differentiation involves the de novo synthesis of 140 basal bodies, the remodeling and condensation of nuclear chromatin and then, nuclear elongation. A complex cytoskeletal structure, the multilayered structure (MLS), is formed at the anterior end of the cell and extends the length of the elongated gamete, and apparently functions in cell and nuclear elongation. This document focuses on the role of kinesin motor proteins and spermidine in the regulation of gametophyte development and spermatid differentiation. Blocking the translation of various kinesin isoforms with RNA interference (RNAi) resulted in arrested development at distinct time points. Centrin and tubulin immunolabeling showed different defects in basal body and microtubule ribbon formation, respectively, with the silencing of specific kinesins. The reduction of spermidine levels in the gametophytes by silencing proteins responsible for its synthesis and transport reveals the involvement of the polyamine in gametophyte cell cycle regulation and in spermatid maturation. In addition, drug inhibition of spermidine synthesis later in development highlighted the importance of its involvement in chromatin remodeling and nuclear elongation. Immunolabeling of spermidine and in situ hybridization assays for its synthesizing enzyme, spermidine synthase, indicated that spermidine levels are controlled in gametophytes by the regulated translation of spermidine synthase. The regulated levels of spermidine in the gametophyte is key to its function as a developmental regulator; spermidine appears to participate in multiple cellular processes in a concentration dependent manner.Item THE RELATIONSHIP BETWEEN AUTOPHAGY, CELL SURVIVAL AND CELL DEATH IN A MODEL OF NEURODEGENERATION AND DEVELOPMENT.(2009) Batlevi, Yakup; Pick, Leslie; Baehrecke, Eric H; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The catabolic degradation of proteins is vital for the proper function and homeostasis of all cells. Autophagy is one of the major catabolic systems, and it is involved in processes that are as diverse as cell survival, cell death, immune reaction, cancer and neurodegeneration. Neurodegenerative diseases often have the pathology of protein accumulation in inclusions, but it is unclear whether these inclusions cause cell toxicity. Here I show that autophagy has protective functions in a model of a polyglutamine neurodegenerative disease in Drosophila. Inhibition of autophagy in this model enhances polyglutmine-induced degeneration, while activation of autophagy suppresses degeneration. Moreover, I observed similar protein aggregates in the larval salivary glands of a Drosophila dynein light chain mutant. This dynein light chain mutant is defective in autophagy, and their salivary glands fail to execute developmentally regulated programmed cell death. Ectopic activation of autophagy is sufficient to suppress the protein accumulation in dynein light chain mutant salivary glands. Both neurons and salivary glands are long-lived post-mitotic cells, and these cells are likely to have unique catabolic needs. Our data indicate that defects in catabolism are responsible for the neurodegenerative and salivary gland cell death defects that I observed, and could explain the association of autophagy with neurodegenerative diseases.Item Identification and Characterization of HRG-1 heme transporters in eukaryotes(2008-11-21) Rajagopal, Abbhirami; Hamza, Iqbal; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Heme is a prosthetic group in proteins that perform diverse biological functions including respiration, gas sensing, xenobiotic detoxification, cell differentiation, circadian clock control and micro RNA processing. In most eukaryotes, heme is synthesized through a multi-step pathway with defined intermediates that are highly conserved through evolution. Despite our extensive knowledge about heme biosynthesis and degradation, the molecules and pathways involved in intracellular heme trafficking are unknown, primarily due to the inability to dissociate the tightly regulated processes of heme biosynthesis and degradation from intracellular trafficking events. Caenorhabditis elegans and related helminths are natural heme auxotrophs that rely solely on exogenous heme for normal development and reproduction. We performed a genome-wide microarray analysis and identified 288 genes that are regulated by heme at the transcriptional level in C. elegans. Here, we characterize two heme-responsive genes, hrg-1 and its paralog hrg-4, that are highly upregulated at low heme concentrations and demonstrate that HRG-1 and HRG-4 are heme transporters. Depletion of hrg-1 and hrg-4 in worms by RNAi results in the disruption of organismal heme homeostasis and abnormal response to heme analogs. HRG-4 traffics to the plasma membrane, and HRG-1 localizes to endo-lysosomal compartments. While hrg-4 appears to be specific to worms, hrg-1 has homologs in vertebrates. Knock-down of hrg-1 in zebrafish results in severe anemia and profound developmental defects, which are fully rescued by worm hrg-1. Human and worm HRG-1 proteins localize together. CeHRG-1, hHRG1 and CeHRG-4 all bind and transport heme. To further understand the in vivo functions of hrg-1 and hrg-4, we characterize the genetic deletions of these genes in C. elegans. Preliminary experiments suggest that the deletion mutants respond abnormally to heme analogs, although these results do not phenocopy the RNAi knock-down studies. We speculate that the deletion strains may have developed compensatory mechanisms in response to the genetic lesions in hrg-1 and hrg-4. Taken together, the studies described herein lay the foundation for identifying the molecular mechanisms for heme transport by the HRG-1 proteins in metazoans and delineating the heme trafficking pathways in C. elegans.Item Genetic regulation of autophagic cell death in Drosophila Melanogester(2008-11-20) Dutta, Sudeshna; Baehrecke, Eric H; Molecular and Cell Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Apoptosis and autophagic cell death are the two most prominent morphological forms of programmed cell death that occur during animal development. While much is known about the mechanisms that regulate apoptosis, relatively little is known about autophagic cell death. The steroid hormone ecdysone coordinates multiple cellular processes during metamorphosis in Drosophila, including cell differentiation, morphogenesis and death. E93 is necessary and sufficient for larval tissue cell death during metamorphosis, including autophagic cell death of salivary glands. Here we characterize new mutant alleles of a dominant wing vein mutation Vein-off (Vno), and provide evidence that E93 and Vno are related. Our data also indicate that E93 functions in steroid regulation of both cell development and death during metamorphosis. E93 encodes a helix-turn-helix DNA binding motif and binds to specific regions of salivary gland polytene chromosomes. We have used genetic and genomic approaches to identify downstream targets of E93. We have identified numerous candidate E93 target genes using DNA microarrays, and have generated transgenic animals to identify downstream target genes of E93 by chromatin immune precipitation. We show that one putative E93 target gene, hippo (hpo), is required for salivary gland cell death. The Wts/Hpo tumor-suppressor pathway is a critical regulator of tissue growth in animals, but it is not clear how this signaling pathway controls cell growth. Our data indicate that salivary gland degradation requires genes in the Wts/Hpo pathway. Wts is required for cell growth arrest and autophagy in dying salivary glands, and regulates the degradation of this tissue in a PI3K-dependent manner.