Browsing by Subject "Genetic Phenomena"
Now showing 1 - 20 of 35
- Results Per Page
- Sort Options
Item A Systematic Screen of the Saccharomyces Cerevisiae Deletion Mutant Collection for Novel Genes Required for DNA Damage-Induced Mutagenesis(2008-07-01) Gong, Jinjun; Siede, Wolfram; Sheedlo, Harold; Reeves, RustinA Systematic Screen of the Saccharomyces Cerevisiae Deletion Mutant Collection for Novel Genes required for DNA Damage-Induced Mutagenesis. Jinjun Gong Department of Cell Biology and Genetics, University of North Texas Health Science Center, Fort Worth, TX 76107. Summary. Deoxyribonucleic acid (DNA) damage is common in a cell’s lifetime. DNA can be damaged by endogenous factors such as reactive oxygen species (ROS) or exogenous agents such as ultraviolet (UV) or industrial chemicals. DNA damage will trigger cell responses including cell cycle arrest, transcription activation, DNA repair or apoptosis. In addition to various DNA repair mechanisms including damage reversal, base excision repair, nucleotide excision repair, mismatch repair, homologous recombination and non-homologous end joining, translesion DNA synthesis is an important DNA damage tolerance pathway that can bypass the lesion on template DNA to finish the replication for cell survival but at the risk of potential mutation in the daughter cells. Accumulation of mutation may lead to cancer occurrence. Translesion DNA synthesis components are highly conserved from yeast to humans. Important players in trans-lesion synthesis pathway such as Rev1, Rev3 and Rev7 were first discovered in budding yeast. Saccharomyces cerevisiae. Homologues were found later in human cells. I used the Saccharomyces cerevisiae deletion mutant collection to do a systematic screen to search for novel genes required for DNA damage induced mutagenesis in yeast. After CAN1 forward mutation assay for the systematic screen and reverse mutation assay for further confirmation, two candidate genes SWI6 and DOA4 were detected. Deletion of SWI6 and DOA4 decreases mutagenesis of cells. At the molecular level, Swi6, a transcription cofactor, is involved in mutagenesis by regulating expression of REV7 at the mRNA and protein levels. Rev7 is a regulatory subunit of DNA polymerase zeta, which is essential for DNA damage induced mutagenesis as well as spontaneous mutagenesis. Rev7 is not UV inducible or cell cycle regulated. The regulation of Rev7 at the transcriptional level by Swi6 is essential. Future experimental approaches are planned to address the mechanism by which DOA4 is involved in mutagenesis.Item Approaches to mitochondrial genome sequencing using the oxford nanopore minion device(2017-05-01) Thorson, Kelcie C.; John V. Planz; Rance E. Berg; J. Thomas CunninghamCurrent DNA sequencing methods rely on polymerase chain reaction (PCR) to create sufficient copies of targeted DNA fragments to serve as a library. PCR and subsequent clean-up steps add considerable time and cost to the process and provide opportunity for introduction of amplification errors or contamination. The aim of this study was to develop a reliable method for mitochondrial genome sequencing sans PCR using the Oxford Nanopore MinION. Our hypothesis was that results generated from native DNA sequencing would be concordant to sequencing results generated from PCR-enriched libraries. Error-rates of 1.47% (enriched) and 1.26% (native) were observed when sequencing the control DNA. Consensus sequences generated from native and PCR-enriched libraries show an average of 98.7% identity between the two methods.Item Characterization of a Novel Extracellular Superoxide Dismutase Allele Discovered in Mouse Models of Atherosclerosis(2004-07-01) Pierce, Anson; Dory, Lad; Easom, Robert; Basu, AlakanandaAnson Pierce, Characterization of a Novel Extracellular Superoxide Dismutase Allele Discovered in Mouse Models of Atherosclerosis. Doctor of Philosophy (Biochemistry and Molecular Biology), July 2004, 128 pp., 3 tables, 22 illustrations, references, 230 titles. Many diseases display some involvement with oxidative mechanisms and could potentially benefit from antioxidant therapy designed to restore the balance between reductive and oxidative factors. Data presented in this dissertation explore and establish the protective effect hyperbaric oxygen (HBO) has on the development of atherosclerosis, an oxidation-driven inflammatory disease mediated through low-density lipoproteins in the vasculature. Atherosclerosis in the apolipoprotein E-/- (apoE-/-) mouse is drastically reduced after 10 weeks of HBO treatment. Macrophages in HBO treated mice have an increased antioxidant capacity and reduced ability to generate oxidants. From this work, a new polymorphism of a key antioxidant enzyme, extracellular superoxide dismutase (ecSOD), is identified and characterized in mice. The new polymorphism is termed the “short” allele, and has the potential to alter the regulation of ecSOD mRNA and protein, as well as enzyme activity. Examination of its effect on the ecSOD phenotype in mice shows dramatic changes in enzyme levels and activity. In the plasma compartment ecSOD activity and mass are elevated, and indicate based on heparin injection studies that a change in ecSOD distribution results in tissues of mice expressing the short allele. Systematic examination of ecSOD in tissues of mice shows that its distribution is altered such that it is more accessible to heparin; this is most evident in the liver and kidney of mice expressing the short allele. The finding that HBO is protective against atherosclerosis highlights a potentially promising approach to treatment for this devastating disease, sheds light on the role oxidative processes play in atherosclerosis, and identifies potential targets for antioxidant therapy. This study also shows for the first time that two alleles for a major antioxidant enzyme exist in mice that display markedly different effects on the ecSOD phenotype, a finding that underlines the importance of genetic homogeneity in mouse models and adds to our knowledge concerning the role antioxidants play in human health and disease.Item Characterization of the Role of PKN in TGF-Beta 1-Mediated Differentiation of Vascular Smooth Muscle Cells(2004-05-01) Deaton, Rebecca Ann; Dillon, Glenn; Shepard, Allan; Mallet, Robert T.Rebecca Ann Deaton, Characterization of the role of PKN in TGF-beta 1-mediated differentiation of vascular smooth muscle cells. Doctor of Philosophy (Biomedical Sciences), May 2004, 178 pp, 5 tables, 34 illustrations, references, 197 titles. Differentiated vascular smooth cells (SMCs) exhibit a work phenotype characterized by expression of several well-documented contractile apparatus-associated proteins. However, when exposed to mitogens such as serum or growth factors. SMCs retain the ability to de-differentiate into an “immature” proliferative phenotype, in which they lack contractile myofilaments. Proliferation of SMCs is involved in the formation of atherosclerotic plaques as well as arterial restenosis following balloon angioplasty. Thus, understanding the mechanism involved in maintain SMC differentiation process is critical to the development of therapies and treatments for the abnormal growth seen in these disease states. In this study, the molecular mechanisms through which transforming growth factor-beta 1 (TGF-B1) induces differentiation of SMCs were examined. The data presented demonstrate that TGF-B1 stimulates actin re-organization, up-regulation of SM-specific marker gene expression and inhibition of cell proliferation of PAC-1 SMCs. These characteristics are indicative of the differentiated phenotype. The effects of TGF-B1 can be blocked by pretreatment of the cells with either HA1077 or Y-27632, which inhibit the functions of the kinases downstream of RhoA. Moreover, TGF-B1 induced differentiation is correlated with an increase in the activity of RhoA and its downstream target, PKN. Over-expression of active PKN alone is sufficient to increase the transcriptional activity of the SM a-actin, SM-MHC and SM22 promoters in PAC-1 cells. In addition, the activity of SRF-GATA and MEF2, three transcription factors that are known to regulate expression of SM-specific marker genes, are also increased by PKN. Finally, examination of MAPK signaling cascades demonstrates that TGF-B1 increases the activity of MKK3/6 and p38 MAPK and decreases the activity of ERK1/2 and JNK ½. Co-expression of dominant negative p38 MAPK is sufficient to abolish PNK-mediated activation of SRF, GATA and MEF2 as well as PKN-mediated activation of SMC marker gene promoters. Taken together, these results identify components of an important intracellular signaling pathway through which TGF-B1 activates RhoA and PKN to promote differentiation of SMCs.Item Comparison of Next Generation Sequencing Methodology on the Ion PGM™ System Performance versus that on the Sanger Sequencing Method for HV1 and HV2 Regions of mtDNA(2015-05-01) Argueta, Wendy C.; Arthur J. Eisenberg; Michael Allen; Raghu R. KrishnamoorthyAnalysis of mitochondrial DNA in forensic applications has enabled the identification of a missing person through comparison with additional maternal relatives. Most forensic applications are based on sequencing of both hypervariable regions of the mtDNA. Sequencing of these regions has been commonly done using Sanger-type sequencing (STS) methodology, which is expensive, time-consuming and laborious. Next Generation Sequencing (NGS) technology, such as the Ion Torrent PGM™ System platform, overcomes most of these issues. In this study, samples from the Guatemalan population (n=40) were sequenced with both Ion Torrent PGM™ technology and STS methods. A high level of consistency (98%) was observed among data derived from both methods. Most of the discrepancies were point heteroplasmy, which were more easily detected by PGM™ technology. In terms of performance, the NGS method was shown to be quick, with high-throughput and more efficient compared to the traditional STS method. More accurate and reliable sequencing data were obtained from the Ion Torrent PGM™ method due to its high level of coverage. Sequencing data for all individuals, representing 19 different family groups, were obtained using the NGS technology. Sequence polymorphisms were detected in 55 positions, from which 26 were observed only in relatives belonging to the same family and were not observed for any other family group. In a forensic context, haplotype specific polymorphisms are the basis for identification and comparison between evidence and reference samples purposes. Haplotypes between maternally related individuals were consistent in 18 family groups.Item Crime Scene Investigation: TV versus Reality(2013-08-01) ; Warren, Joseph; Budowle, Bruce; Eisenberg, Arthur J.; Pullin, Mike; Milligan, JessieJoseph Warren, Bruce Budowle and Arthur J. Eisenberg speak about crime scene investigation and forensic science as portrayed in popular television. They discuss how the shows distort and overstate the ways in which forensic scientists help solve crimes and identify victims, and they describe potential impacts on jurors' expectations. They also appreciate how these shows drive curiosity and bring better grant funding and more students to forensic science.Item Divergent Behavioral Phenotypes in Conditioned Place Preference(2017-12-01) Wagner, Alison N.; Forster, Michael J.; Gatch, Michael B.; Shetty, RituThe addictive properties of psychostimulants have been studied using a variety of animal models and behavioral paradigms. These studies consistently report individual variation in drug response that could reflect subgroups with different susceptibility to addiction. A place conditioning assay was used to assess the possibility that such divergent behavioral phenotypes explain variable outcomes in mice after conditioning with psychostimulants (cocaine, 3,4-methylenedioxypyrovalerone (MDPV), methamphetamine, and d-amphetamine). K-means clustering analysis partitioned individuals into groups (i.e. clusters) for analysis. The reliability of these phenotypes was supported with Pearson correlation analysis comparing adjacent time points, as well as Cronbach’s alpha and intraclass correlation coefficients for overall within-group relatedness at each time point. Furthermore, initial preference developing in a drug-naïve condition was reversed with drug conditioning, demonstrating that changes in salience were sufficient to reverse initial preference in some mice. By purposefully examining these behavioral phenotypes in place conditioning, we advance toward the development of pharmacological strategies for addiction and robust epigenetic outcomes.Item DNA-Prokids: Forensic Genetics and Human Trafficking(2013-08-01) Eisenberg, Arthur J.DNA - ProKids is a program jointly run by the Universidad de Granada (UGR) and the University of North Texas Health Science Center (UNTHSC). The Goal of DNA - Prokids is to return trafficked individuals, specifically young people and children to their families in their home countries. The FBI estimates that there are more than 100,000 children and teens in the United States that are being trafficked in the sex trade. Since Texas is a portal into the U.S. for Central and South America, the state government has been proactive in trying to address the problem. Part of the solution is the partnership between UNTHSC and UGR to work on DNA-Prokids. DNA-Prokids is an international humanitarian effort to help identify missing children and if possible to reunite abducted and homeless children with their parents, and to provide law enforcement agencies a scientific methodology to help deter the human trafficking of children. The organization also works with law enforcement to ensure that the children are returned to their families or placed in safe environments. Since this video, 697 parent-child associations have been made, with the vast majority of those children being returned to their families. Out of those 697, approximately 12 children have not been able to return home due to crime, violence, and drugs in their biological families. DNA-Prokids has also detected and avoided 221 illegal adoptions, where the woman presenting the baby to be adopted was not the child's biological mother. The success of the program deters human trafficking and can prevent child abuse and slave labor by increasing coordination between efforts like DNA-Prokids and authorities.Item Dr. Arthur Eisenberg(2013-08-01) Eisenberg, Arthur J.; Milligan, JessieItem Dr. John Planz(2013-08-01)John V. Planz holds a B.S degree in Biology and Zoology (double major) from the State University of New York (Oswego, NY), a M.S. degree in behavioral ecology from Shippensburg University (Shippensburg, PA) and a Ph.D. in molecular evolutionary genetics and population genetics from the University of North Texas (Denton, TX). Dr. Planz studied as a postdoctoral fellow at the Carnegie Museum of Natural History, Section of Mammals (Pittsburgh, PA) in mammalian phylogenetic systematics. Dr. Planz entered the forensics field in 1993 at the Southwestern Institute of Forensic Sciences in Dallas, TX. He later served as the Director of Identity Testing at GeneScreen, Inc. in Dallas, TX and Biosynthesis, Inc. in Lewisville TX adding mitochondrial DNA typing and SNP development to the testing performed by those laboratories. Dr. Planz joined the faculty of the University of North Texas Health Science Center at Fort Worth in January 2000 were he serves as an Associate Professor in the Department of Forensic and Investigative Genetics and is the Associate Director of the UNT Center for Human Identification.Item Dr. Joseph Warren(2013-08-02)Item Effect of Phosphorylation on Muscle Physiology and Biophysical Characterization of Mutations Responsible for Familial Hypertrophic Cardiomyopathy(2016-08-01) Duggal, Divya; Borejdo, Julian; Gryczynski, Ignacy; Clark, Abbot F.Familial hypertrophic cardiomyopathy (FHC) is the most common cause of sudden cardiac death in young individuals. Molecular mechanisms underlying this disorder are largely unknown; this study aims at revealing how disruptions in actin-myosin interactions can play a role in the pathogenesis of this disorder. Cross-bridge (XB) kinetics and the degree of order were examined in contracting myofibrils from the ex vivo ventricles of transgenic (Tg) mice expressing FHC regulatory light chain (RLC) mutation K104E and Troponin I mutation, R21C. Because the degree of order and the kinetics are best studied when an individual XB makes a significant contribution to the overall signal, the number of observed XBs in an ex vivo ventricle was minimized to 20. Autofluorescence and photobleaching were minimized by using a relatively long-lived red-emitting dye. In case of K104E, mutated XBs were significantly better ordered during steady-state contraction and during rigor, but the mutation had no effect on the degree of order in relaxed myofibrils. The K104E mutation increased the rate of XB binding to thin filaments and the rate of execution of the power stroke. In case of R21C, differences were investigated in the left (LV) and right ventricle (RV) mutant where it was found that the mutation imposed significant difference in the distribution of angles that actin makes with thin filament axis: during contraction, actin angles from LV were more tightly distributed compared to actin angles from RV. Collectively, the data indicates that the mutation-induced changes in the interaction of myosin with actin during the contraction- relaxation cycle may contribute to altered contractility and the development of FHC. Phenotypic differences of the R21C mutation in the left versus right mouse ventricles, even though both ventricles express the same isoform of the cardiac highlights the importance of functional differences between the two ventricles of cardiac disease.Item Endothelin-1 Mediated Regulation of Extracellular Matrix Collagens- A Role in Pathology of Primary Open Angle Glaucoma(2007-11-01) Rao, Vidhya Ramachandiran; Thomas Yoroi; Neeraj Agarwal; Raghu KrishnamoorthyEndothelin -1 Mediated Regulation of Extracellular Matrix Collagens –A role in Pathology of Primary Open Angle Glaucoma. Vidhya R. Rao, Doctor of Philosophy. (Pharmacology and Neuroscience), November, 2007, 157 pp., 3 tables, 18 figures. Summary. Primary Open Angle Glaucoma (POAG) is a progressive optic neuropathy characterized by loss of retinal ganglion cells, optic nerve degeneration and characteristic extracellular matrix (ECM) remodeling of the optic nerve head. An increase in collagen type I and VI is observed at the level of lamina cribosa (LC), a distinct connective tissue region of optic nerve in POAG subjects. Extensive ECM remodeling with enhanced collagen deposition observed in POAG is consistent with the pathology of fibrosis. Mechanisms contributing to ECM remodeling in POAG is not known. Endothelin-1(ET-1), a potent vaso-active peptide plays a key role in glaucoma pathology. Intra-vitreal administration of ET-1 in animal models results in optic neuropathy, RGC apoptosis, axonal transport block and ONA activation. An upregulation of ET-1 and ETB receptors is observed in glaucomatous LC and animal models of glaucoma and ET-1 mediated detrimental effects in POAG appears to be mediated by ETB receptors. ET-1 initiatives and maintains enhanced collagen synthesis and deposition in various tissues under pathological conditions and is recognized as a potent profibrotic factor. In the present study we hypothesized that ET-1 increases extracellular matrix collagen deposition in lamina cribrosa and this change in ECM contributes to optic nerve fibrosis. We have demonstrated that cells of lamina cribrose (LC) cells, express functional ETA and ETB receptors. ET-1 increases intracellular calcium mobilization via ETA receptors and increases NO release by mechanisms involving both ETA and ETB receptors. Consistent with POAG pathology we have observed an upregulation ETB receptors in LC cells in response to chronic treatment with ET-1. LC cells also express prepro-ET-1, the primary gene transcript of ET-1. We have demonstrated for the first time that ET-1 exerts its profibrotic effects by enhancing collagen type I and type VI mRNA, protein synthesis, deposition and secretion in LC cells. ET-1 enhanced collagen deposition in LC cells appears to involve both ETA and ETB receptors, as both of the receptor antagonist, individually inhibit ET-1 mediated collagen synthesis. We have demonstrated that ET-1 also exerts its profibrotic effects in vivo by enhancing collagen deposition in rat optic nerve head. We have also observed an apparent decrease in ET-1 mediated collagen VI deposition in optic nerve heads of ETB deficient transgenic rats suggesting that ET-1 mediated collagen VI synthesis involves ETB receptor activation. In conclusion, endothlein-1 stimulates collagen synthesis and deposition both in vitro in LC cells as well as in vivo at the level of rat optic nerve head. ET-1 mediated increase in collage synthesis at the level of optic nerve head could render a fibrotic mechanism that contributes to the progression of POAG.Item Genetic Diversity of Easter Island (Rapanui) Population from Identifiler® Plus autosomal, Y-filer®, and Y-Plex™ 6 Y-STR Loci(2015-05-01) Guadian, Laura; Chakraborty, Ranajit; Budowle, Bruce; Hodge, Lisa M.This study investigated the genetic diversity of the Easter Island (Rapanui) population using data on 15 autosomal Short Tandem Repeats (STRs) typed with the commercial STR kits Identifiler® Plus and 23 Y-chromosome STRs typed using Y-filer (17 loci) and Y- PLEX™ 6 (6 loci). The analysis was conducted using genotype and haplotype data of 122 presumably unrelated individuals that included 48 males and 74 females. This study: (i) examined if Easter Island population had reduced genetic diversity in comparison with cosmopolitan populations such as Mainland Chilean, Polynesian, European, and African; (ii) compared genetic affinity of the Easter Island population with historically related cosmopolitan populations; and (iii) investigated the forensic utility of autosomal STRs and Y-STRs in the Easter Island population.Item How do you get a job in Forensic Science?(2013-08-01)Item Identification and Characterization of Caveolins in Mouse Macrophages(2002-12-01) Gargalovic, Peter; Dory, Lad; Basu, Alakananda; McConathy, WalterPeter Gargalovic, Identification and Characterization of Caveolins in Mouse Macrophages. Doctor of Philosophy (Biochemistry and Molecular Biology), December 2002, 206 pp., 3 tables, 41 illustrations, references, 296 titles. The understanding of the mechanisms which control macrophage-lipid management, and their accumulation in atherosclerotic lesions, is of significant importance. Caveolins are proteins associated with cholesterol-rich membrane domains and are intimately linked to the regulation of lipid metabolism and transport. The expression and function of caveolin proteins in three macrophage cell types: thioglycollate-elicited mouse peritoneal macrophages, resident mouse peritoneal macrophages and the J774 macrophage cell line. Data in this work establish that the primary macrophages express caveolin-1 and -2, while J774 cells express only caveolin-2. Immunofluorescence microscopy studies indicate that caveolins in primary macrophages do not colocalize, with caveolin-1 being present on the cell surface and cavelon-2 in the Golgi compartment. Analysis of macrophages also showed that caveolin-1, but not caveolin-2, is present in detergent insoluble lipid raft membranes. While caveolin expression in macrophages is not regulated by sterols, both caveolin isoforms can be secreted from cholesterol-loaded macrophages in the presence of high-density lipoprotein (HDL). Secreted caveolins are part of the complex that has a density similar to HDL, which suggests their association with HDL and potentially a role in HDL-mediated reverse cholesterol transport. The examination of caveolin expression in macrophages shows that caveolin-1, but not caveolin-2 expression is highly upregulated by agents that induce apoptosis in these cells. Induction of caveolin-1 expression precedes DNA fragmentation, is independent of caspase activation, and correlates with the exposure of phosphatidylserine on the cell surface. Importantly, immunofluorescence analysis determined that caveolin-1 in lipid rafts colocalizes extensively with phosphatidylserine present on the surface of apoptotic cells. This study thus identifies caveolin-1 as a specific and early marker of the macrophage apoptotic phenotype. Findings here strongly implicate the involvement of caveolin-1 and lipid rafts in the changes of plasma membrane lipid composition as well as involvement in efficient clearance of apoptotic cells by a phosphatidylserine-mediated mechanism.Item Mechanistic Studies of the Sheep Liver 6-Phosphogluconate Dehydrogenase and cDNA Cloning(1996-07-01) Price, Nancy E.; Neeraj Agarwal; Robert Easom; Stephen R. GrantPrice, Nancy E., Mechanistic Studies of the Sheep Liver 6-Phosphogluconate Dehydrogenase and cDNA Cloning. Doctor of Philosophy (Biomedical Sciences), July, 1996, 124 pp., 5 tables, 28 Figures, 2 appendices, bibliography, 45 titles. A kinetic characterization of sheep liver 6-phosphogluconate dehydrogenase including product and dead-end inhibition patterns, primary deuterium isotope effects, and the pH dependence of kinetic parameters has been completed in order to determine the kinetic mechanism, and chemical mechanism of the enzyme. A rapid equilibrium random kinetic mechanism has been proposed, with product and dead-end inhibition patterns both being symmetric. Primary deuterium isotope effects were equal on V and V/K, confirming a rapid equilibrium mechanism, and indicate that hydride transfer is at least partially rate limiting in the overall reaction. The maximum velocity is pH dependent, decreasing at low and high pH with slopes of 1 and -1, respectively. The V/KNADP and V/K6PG also decrease at low and high pH with slopes of 1 and -1. The pH rate profiles are consistent with a general acid/general base mechanism where the catalytic residues are involved in binding. Reverse protonation states between the general acid and the general base is proposed where an unprotonated general base accepts a proton from the C-3 hydroxyl of 6PG concomitant with hydride transfer followed by decarboxylation of the resulting 3-keto intermediate to give an enediol which is protonated by the general acid to form ribulose-5-phosphate. The pH dependence of the pKi profile of the inhibitory analog 5-phosphoribonate decreases at low and high pH with slopes of 1, and -1 respectively, and suggests that intrinsic pKs are observed in the V/K profiles. The pKs of both the general base and general acid in the E:6PG complex appears to be perturbed such that the general base pK decreases slightly, and the pK of the general acid increases slightly, as a result of direct interaction with 6PG. Additionally, in preparation for site-directed mutagenesis, cDNA clones for sheep liver 6PHDH were obtained by RT-PCR.Item Molecular Regulation of Interferon Gamma in 2B4-Activated Natural Killer Cells: Functional Role in Tumor Rejection(2001-11-01) Johnson, Lori Ann; Mathew, Porunellor A.; Goldfarb, Ronald H.; Dimitrijevich, S. DanNatural killer cells are a third population of lymphocytes, distinct from T and B cells. NK cells are non-MHC-restricted cytotoxic effector cells which are effective against intracellular pathogens, virally-infected cells and tumor cells. 2B4 is a natural killer cell receptor originally identified in the mouse as a surface molecule involved in non-MHC-restricted killing and enhancement of IFN-γ secretion. The human and rat homologues of 2B4 have recently been cloned in our laboratory. Interferon gamma (IFN-γ) is a cytokine with potent anti-viral and anti-proliferative effects. In addition, this cytokine acts as a global immune regulator by regulating gene expression and serving to attract other immune cells. In this work, we establish the function of human 2B4 in a NK cell line, YT. We have shown that human 2B4 activation induces cytolytic function and enhances IFN-γ release in YT cells. Additionally we show that 2B4’s regulation of IFN-γ occurs at the transcriptional level, both through mRNA stability and increased promoter activity. We also demonstrate that several regions in the IFN-γ promoter respond to 2B4 activation and IFN-γ both separately and together in the rejection of metastatictumor cells in C57B7/6 mice. Our results confirm that both 2B4 and IFN-γ are critical in the rejection of metastatic tumor cells. Through the use of activating monoclonal antibodies, our studies indicate that 2B4’s anti-tumor activity is through IFN-γ as well as through cytolytic function of NK cells.Item Mutation in myocilin affect it's processing and secretion in the trabedular meshwork cell(2003-05-01) Jacobson, Nasreen; Robert Wordinger; Richard Easom; Neeraj AgarwalJacobson, Nasreen, Mutations in myocilin affect it secretion and processing in the cell. Doctor of Philosophy (Cell Biology and Ginetics), May 2003, 157 pp., 6 tables, 46 illustrations, 17 movies. Introduction. Myocilin is the protein product of the glaucoma gene MYOC whose function is unknown. Structural predictions of the protein indicate myocilin is secreted. This study uses several techniques to determine whether myocilin is synthesized and processed through the secretory pathway. Methods. Agents known to disrupt the secretory pathway at specific organelles will be used to examine the effect on myocilin secretion. Also, constructs for chimeric myocilin and fluorescent proteins (myoc.504DsRED and myoc.504EGFP) will be used in conjunction with EGFP directed to specific organelles to determine colocalization of myocilin in the cell. The disruption of wild type and disease-causing mutants (myocQ368X.DsRED, myocG364V.504DsRED and myocY437H.504DsRED) of myocilin will be compared. Then in vivo studies will be used to try to determine if myocilin is associated with increased intraocular pressure (IOP). Results. Myocilin appears as a doublet on SDS-PAGE western blots when probed with anti-myocilin antibody (AB129). Treatment of cells with tunicamycin prevents secretion of the upper band of the myocilin doublet, but not secretion of the lower band. Brefelden A prevents secretion of both bands of the myocilin doublet indicating that both bands are processed in the Golgi. Monensin treatment indicates there is no post-Golgi processing of myocilin prior to secretion. Colocalization of fluorescent myocilin with cellular organelles tagged with EGFP indicated that myocilin travels through the ER, Golgi and is secreted from the cell. Disease-causing mutations in myocilin are not secreted. The Q368X associates with wild type myocilin and appears to be degraded. The G364V and Y437H mutants can apparently be retained in the ER and also are closely associated with peroxisomes. Experiments designed to determine if myocilin can be correlated with increased IOP suggest an association of myocilin with increased IOP in an ex vivo human anterior segment perfusion system, but in vivo experiments gave inconclusive results. Conclusions. Myocilin is a secreted glycoprotein in the TM. Glaucomatous mutations in myocilin cause non-secretion. TM cells handle different myocilin mutations differently.Item Other Applications of Forensic Genetics(2013-08-01)