Cell Biology & Molecular Genetics Theses and Dissertations
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Item Different Mechanisms of ras Proto-Oncogene Overexpression Detected by a Sib-Selection Tumorigenesis Assay(1990) Bachurski, Cindy J.; Hetrick, Frank M.; Cell Biology and Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md)Human insulinoma and renal cell carcinoma DNAs were analysed by a sib-selection tumorigenesis assay to detect the presence of dominant acting oncogenes. Although no novel oncogenes were detected in the five tumor DNAs tested, the increased sensitivity of the tumorigenesis assay allowed detection of overexpressed ras proto-oncogenes activated during transfection. Tumorigenic overexpression of ras proteins occured by two different mechanisms: gene amplification, and increased translation efficiency of a fusion protein. Thirty to fifty fold amplification of a human N-ras gene was detected in primary and secondary mouse tumors. All tumors containing amplified copies of N-ras overexpressed p21 and the cloned gene had no coding sequence mutations. Since DNA from the original human tumor and several metastases did not show N-ras gene amplification, it was concluded that amplification had occured during the primary tumorigenesis assay. In another series of tumors co-integration of the mouse c-H -ras gene during the secondary transfection resulted in tumorigenic overexpression of ras protein without elevation of mRNA levels. Three c-H-ras cDNA clones from secondary tumor RNA contained no coding sequence mutations, but were divergent at the 3'end. Polymerase chain reaction amplification of RNA showed novel alternative splicing at intron E of mouse c-H-ras mRNA in both tumors and untransfected cells. An upstream ATG was identified that potentially initiates translation of an open reading frame (ORF) overlapping the H-ras p21 translation initiation site. A single base deletion within exon -1 of the cDNA clones placed this upstream ATG in frame with the ras coding sequence, creating a potential fusion protein. Translation of this mRNA in rabbit reticulocyte extracts and Xenopus oocytes showed exclusive production of a p23 ras fusion protein. When the upstream ATG was deleted, only p21 ras was translated in both systems. Based on these results it is proposed that in vivo recognition of the upstream ATG and translation of the ORF overlapping the p21 start site might serve to modulate the translation of p21. The single base deletion and resultant ras fusion protein may constitute a novel mechanism of ras overexpression by circumventing this translational regulation .Item Studies on the Root Growth of Willow Cuttings at Controlled Temperatures(1931) Baker, Henry H.; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md)Item CHARACTERIZATION OF THE ROLE OF MAPKS IN LEISHMANIA INFECTED MACROPHAGES.(2009) Yang, Ziyan; Mosser, David M.; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)In the current study, we examined the role of the Mitogen Activated Protein Kinases (MAPKs) on the biological responses of macrophages infected with Leishmania. The first section examined the role of MAPK/ERK in IL-10 production by Leishmania-infected macrophages. The macrophage-derived IL-10 has been shown to exacerbate Leishmaniasis. However, the molecular mechanisms whereby Leishmaniasis prompts IL-10 induction are poorly understood. A combination of two signals was necessary for IL-10 induction by the Leishmania amastigotes-infected macrophages. The first signal is mediated by TLR ligation whereas the second signal is mediated by FcgammaR ligation, which yields a population of regulatory macrophages that produce high levels of IL-10. Infection of macrophages with L. amazonensis amastigotes from the lesion sites sparked MAPK/ERK activation, which was required, but not sufficient for IL-10 induction. In combination with an inflammatory stimulus, LMW-HA from the extracellular matrix, these parasites triggered the macrophages to highly produce IL-10. MAPK/ERK activation initiated an epigenetic modification of chromatin at the IL-10 locus, which allowed for transcription factor Sp1 binding to drive IL-10 transcription and subsequent production. U0126, an inhibitor of MAPK/ERK activation, decreased lesion progression in Leishmania infected mice. The second section examined the role of MAPK/p38 in cytokine production and vaccination against Leishmaniasis. TLR agonists activate macrophages to produce pro-inflammatory cytokines and reactive oxygen intermediates. Inhibition of MAPK/p38 reciprocally increased IL-12 but decreased TNFa production from LPS-stimulated macrophages, which also occurred following stimulation by a variety of other TLR agonists, and using different APCs. MAPK/p38 inhibition induced IL-12p40 mRNA accumulation mainly due to enhanced mRNA stability, which was independent of IL-10. Similar results were observed by knocking down MAPK/p38 using specific siRNAs or by targeted deletion of MKK3. IL-12 production following the inhibition of MAPK/p38 skewed antigen-specific T cells to produce more IFN-gamma and less IL-4 in vitro. A MAPK/p38 inhibitor was applied as an adjuvant to vaccine mice against L. major, which resulted in smaller lesions with fewer parasites. Our findings reveal an important role of MAPKs in the Leishmania pathogenesis, and suggest that the manipulation of these kinases may provide novel therapeutics for potential clinical applications.Item CHARACTERIZATION OF TWO HIGHLY CONSERVED POXVIRUS TRANSMEMBRANE PROTEINS OF UNKNOWN FUNCTION(2009) Sood, Cindy Leigh; Moss, Bernard; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The vaccinia virus I5L open reading frame encodes a 79-amino-acid protein, with two predicted transmembrane domains, conserved among all sequenced members of the chordopoxvirus subfamily. No nonpoxvirus homologs or functional motifs have been recognized, and the role of the I5 protein remains unknown. I5 synthesis was dependent on viral DNA replication and occurred exclusively at late times, consistent with a consensus late promoter motif adjacent to the start of the open reading frame. I5 was present in preparations of purified virions and could be extracted with nonionic detergent, suggesting membrane insertion. Transmission electron microscopy of immunogold-labeled thawed cryosections of infected cells revealed the association of an epitope-tagged I5 with the membranes of immature and mature virions. Viable I5L deletion and frameshift mutants were constructed and found to replicate like wild-type virus in a variety of cell lines, indicating that the protein was dispensable for in vitro cultivation. However, mouse intranasal challenge experiments indicated that a mutant virus with a frameshift resulting in a stop codon near the N terminus of I5 was attenuated compared to control virus. The attenuation correlated with clearance of mutant viruses from the respiratory tract and with less progression and earlier resolution of pathological changes. We suggest that I5 is involved in an aspect of host defense that is evolutionarily conserved although a role in cell tropism should also be considered. The vaccinia virus A43R open reading frame encodes a 168-amino acid protein with a predicted N-terminal signal sequence and a C-terminal transmembrane domain. Although A43R is conserved in all sequenced members of the orthopoxvirus genus, no non-orthopoxvirus homolog or functional motif was recognized. Biochemical and confocal microscopic studies indicated that A43 is expressed at late times following viral DNA synthesis and is a type-1 membrane protein with two N-linked oligosaccharide chains. Neither mature nor enveloped virions contained appreciable amounts of A43, which was detected in Golgi and plasma membranes. Loss of A43R expression had no discernible effect on plaque size or virus replication in cell culture and little effect on virulence in a mouse intranasal infection model. Although the A43 mutant produced significantly smaller lesions in the skin of mice than the control, the amounts of virus recovered from the lesions were similar.Item Resistance to Ionizing Radiation and Oxidative Stress in Halobacterium salinarum NRC-1(2009) Robinson, Courtney Kathryn; Dinman, Jonathan; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Oxidative stress results from environmental challenges that cause unchecked production of reactive oxygen species (ROS). We analyzed the cellular damage and stress response of the extremophile Halobacterium salinarum NRC-1 exposed to chemical oxidants and to ionizing radiation (IR). In contrast to IR, cellular damage from H2O2 and superoxide suggested that cell death resulted from interference with major metabolic pathways rather than generalized oxidative lesions. We found that essential ROS scavenging enzymes were not necessary for H. salinarum NRC-1 survival to IR. Protection assays using enzyme-free cellular extracts from H. salinarum NRC-1 demonstrated high level of protection for protein activity but not for DNA integrity against IR. Biochemical analysis of the extracts underlined an essential role in ROS scavenging for specific nucleosides and MnPO4 complexes. These studies contributed novel findings on the critical role played by non-enzymatic systems in IR resistance in H. salinarum NRC-1.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 Suppressors of etr1-2: I. etr1-11 is a loss-of-function mutation of the ETR1 ethylene receptor. II. REVERSION TO ETHYLENE-SENSITIVITY3 is a regulator of seedling growth.(2009) McClellan, Christopher Alan; Chang, Caren; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The plant hormone ethylene is an important regulator of plant growth and development, including senescence, abcission, fruit ripening, and responses to biotic and abiotic stresses. To find new members of the ethylene signaling pathway, a genetic screen for suppressors of the ethylene-insensitive mutant etr1-2 was performed. One mutant identified in this screen, etr1-11, is an intragenic mutation within ETR1. etr1-11 is a unique missense mutation that appears to eliminate ETR1-2 signaling. Mutant analysis further revealed that etr1-11 is a partial loss-of-function allele. The rte3 (reversion to ethylene sensitivity3) mutant was another mutant isolated in a genetic screen for suppressors of etr1-2. After testing other ethylene responses, such as leaf senescence, and performing epistasis analysis with other ethylene signaling mutants, it was determined that RTE3 is unlikely to play a direct role in the ethylene signaling pathway. Instead, RTE3 appears to be responsible for promoting hypocotyl elongation in etiolated seedlings in the ethylene triple response assay. The RTE3 gene was identified by positional cloning, and is predicted to encode a protein with an annotated SAC3/GANP domain. SAC3/GANP domains are present in proteins that participate in large multi-peptide complexes, such as the 26S proteasome regulatory subunit and the eIF3 translation initiation complex. Similarities in protein composition between these two complexes and the COP9 signalosome (CSN) suggest that a SAC3/GANP domain-containing protein may interact with members of the CSN. Interestingly, yeast two-hybrid analysis reveals that RTE3 interacts with EER5 and EIN2, proteins that have been shown to interact with members of the CSN. In addition, rte3-1 ein2-1 seedlings show a synthetic phenotype of delayed growth. Protein localization using a GFP tag reveals that RTE3 and EER5 both localize to the nucleus. These interactions suggest that RTE3, EER5, EIN2, and the CSN form a protein complex that regulates seedling growth.Item Investigation of Ethylene Signal Transduction Mechanisms: Characterizing the Novel Gene AWE1 and Testing Hypothesis of Raf-like CTR1's Function In Vivo(2009) Kendrick, Mandy Danielle; Chang, Caren; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Ethylene is a gaseous plant hormone affecting multiple plant processes. Sixteen years ago the first components of the ethylene signaling pathway, the receptor ETR1 and Raf-like kinase CTR1, were identified. Since then many additional components of the pathway have been elucidated through genetic screens. Recent discoveries suggest ethylene signaling, once thought to be a linear pathway from ethylene perception at the endoplasmic reticulum to transcriptional activation at the nucleus, is more complex with multiple auto-feedback loops and potential parallel kinase cascades downstream of the receptors. Although the genetic backbone of the pathway is well established, the signaling mechanisms of the components remain unclear. ETR1 displays histidine kinase activity in vitro and physically interacts with the next-known downstream component of the pathway, CTR1. However the histidine kinase activity of ETR1 is mostly dispensable for signaling to CTR1. How then is CTR1 activated? I proposed that additional proteins, like AWE1, play a role in ETR1 to CTR1 signaling, and that the non-catalytic, amino-terminal region of CTR1 is required both for activation through direct interaction with the ETR1 receptor complex and for auto-inhibition of CTR1 kinase activity. ASSOCIATES-WITH-ETR1 (AWE1) was isolated in a yeast-two-hybrid screen for ETR1-interacting proteins and was of specific interest because the AWE1 clone also interacted with a portion of CTR1. Protein-protein interaction studies and genetic analysis of an awe1 mutant support a role of AWE1 in repressing ethylene responses. However double mutant analysis, over-expression analysis, and protein sub-cellular localization studies suggest that AWE1's function in hypocotyl elongation and cell expansion is more general. AWE1's function may require ETR1 for proper regulation but is likely to lie outside of the direct step from ETR1 to CTR1. To investigate a role of the CTR1 amino-terminal region in CTR1 regulation, I constructed transgenes consisting of truncated ETR1 receptors fused to truncated or full length CTR1 and examined how those transgenes carrying the truncated CTR1 (kinase domain only) affected Arabidopsis seedling growth compared to those transgenes expressing full length CTR1. I concluded that the CTR1 amino-terminal region may have a role in autoregulation, but additional components are required for regulation of CTR1 signaling.Item Phylogenetic analysis of swine influenza viruses isolated from humans in Alma-Ata, Kazakhstan(2009) Padmanabhan, Rangarajan; Perez, Daniel; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Continuous surveillance of influenza becomes important considering the economic, epidemic and pandemic implications of influenza infections. This study details phylogenetic & molecular analysis of the genes of four swine influenza viruses isolated from humans in Alma-Ata, Kazakhstan. Phylogenetic analysis placed the eight segments of the four viruses in the classical H1N1 swine clade, along with the isolate A/sw/Jamesburg/1942, except for the HA of A/Alma-Ata/32/98, which was placed in the human H1N1 lineage, along with the isolate A/WS/1933. On amino acid analysis, the viruses displayed mutations on HA and ribonucleoproteins which putatively disrupt antigenic recognition of the virus by the host immune system. The presence of these viruses relatively unchanged for 6 decades after their initial isolation could be speculated to be a combination of laboratory leaks in southern USSR in 1980s, low divergence of classical H1N1 viruses in pigs, and the low population density of Kazakhstan.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.