Department: Molecular, Cellular, and Developmental Biology ![[clear]](close-x.png "Remove this limiter")
94 matches in the database.
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1.
Affara, Nesrine I.
The role of phosphoinositide 3-kinase/akt signaling pathway in tumor-associated angiogenesis, wound healing, and carcinogenesis.
Degree: PhD, Molecular, Cellular, and Developmental Biology, 2006, Ohio State University
► The goal of the present studies was to localize two proteins known…
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▼ The goal of the present studies was to localize two proteins known to be involved in regulation of cell proliferation and survival in specific cell populations in normal mouse skin, during multi-stage skin carcinogenesis, following skin injury, and during tumor angiogenesis. The proteins evaluated included activated Akt, as defined by phosphorylation of Akt at Serine-473 (pAkt) and mTOR, defined by phosphorylation of mTOR at Serine-2448 (pmTOR). Our laboratory previously identified a novel murine VEGF splice variant, VEGF205*, which was differentially expressed in mouse skin carcinomas, but not in normal skin. VEGF205* encodes for a truncated 145 amino acid polypeptide with a unique 7 amino acid carboxyl-terminal tail, YVGAAAV, that is significantly different from the carboxyl-terminal tail of mouse or human VEGF proteins previously identified. VEGF205* stimulated a PI3-K-dependent-increases in pAkt and pmTOR in human vascular endothelial cells, which were significantly higher than pAkt and pmTOR levels induced following incubation of endothelial cells with VEGF120. Immunochemical staining analysis as well as triple color immunofluorescence were used in combination with confocal microscopy to evaluate the presence of pAkt and pmTOR in keratinocyte stem cells (KSC) located within a specific niche in hair follicles defined as “the bulge”, as identified by expression of markers of mouse KSC, CD34 and K15. The location of CD34+/K15+ KSC remained restricted to the bulge niche within hair follicles during multi-stage skin carcinogenesis and cutaneous wound healing. Our results also provide the first evidence for the presence of pAkt and pmTOR in CD34+/K15+ KSC localized to the ORS niche of the bulge region in normal skin, during multi-stage skin carcinogenesis, as well as during wound healing. Using the highly invasive and metastatic human Hs578T breast tumor cell line and the slow growing and non-invasive human MCF-7 breast tumor cell line, the present studies also compared the effects of the selective PI3-K inhibitor, LY294002, alone and in combination with the selective mTOR inhibitor, rapamycin, on tumor cell proliferation and phosphorylation of downstream targets of PI3-K and mTOR, including, 4E-BP1 and cyclin D1. The combination of LY294002 and rapamycin inhibited hyperphosphorylation of the slower migrating (delta, gamma, beta)species of phospho-4E-BP1, and significantly decreased cyclin D1 protein levels.
Advisors/Committee Members: Robertson, Fredika M.
Keywords: angiogenesis; bulge region; breast cancer; CD34; cytokeratin 15 (K15); inflammation; skin; carcinogenesis; endothelial cells; stem cells; keratinocytes; Akt; protein kinase B; phosphoinositide 3-kinase (PI3-K); phosphorylation; proliferation; papilloma
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2.
Ai, Jing.
Molecular studies on G-CSF receptor signaling in granulocytes and regulation of FC gamma receptor function in macrophages (roles for a novel protein LRG and inositol phosphatase SHIP-2 respectively).
Degree: PhD, Molecular, Cellular, and Developmental Biology, 2006, Ohio State University
► Neutrophils and macrophages are two major cell types of innate immune system.…
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▼ Neutrophils and macrophages are two major cell types of innate immune system. They both have specific surface receptors, which transduce unique signals that are critical for their survival, differentiation, and functions. In Part I, we characterized the physiological roles of a novel protein, LRG, in G-CSFR signaling pathways in granulocytes. We showed that the transcription factors PU.1 and C/EBPepsilon that regulate the expression of multiple myeloid-specific genes also bind to the LRG promoter, and LRG localizes to the same cytoplasmic compartment as myeloperoxidase. Stable transfection of LRG into 32Dcl3 cells resulted in accelerated G-CSF-mediated neutrophil differentiation and induction of CD11b and CD13 expression, which was found to correlate with increased level of phospho-Stat3. In contrast, constitutive expression of LRG in 32Dwt18 cells expressing a chimeric Epo/G-CSF receptor consisting of the EpoR extracellular domain fused to the G-CSFR transmembrane and cytoplasmic domains failed to do so in response to Epo stimulation. Collectively, these findings suggest a role for LRG in modulating neutrophil differentiation and activation via non-redundant G-CSFR signals. In Part II, we studied the regulation of FcgammaR-mediated phagocytosis of IgG-coated particles in macrophages, which involves inositol phosphatase SHIP-1. We have analyzed the role of SHIP-2, with high level homology to SHIP-1, in phagocytosis using independent cell models that allow for manipulation of SHIP-2 function without influencing SHIP-1. We presented evidence that SHIP-2 translocates to the site of phagocytosis and downregulates FcgammaR-mediated phagocytosis. Our data indicated that SHIP-2 must contain both the N-terminal SH2 domain and the C-terminal proline-rich domain to mediate its inhibitory effect. The effect of SHIP-2 is independent of SHIP-1, as overexpression of dominant-negative SHIP-2 in SHIP-1 deficient primary macrophages resulted in enhanced phagocytic efficiency. Likewise, specific knockdown of SHIP-2 expression using siRNA resulted in enhanced phagocytosis. Finally, analysis of the molecular mechanism of SHIP-2 downregulation of phagocytosis revealed that SHIP-2 downregulates upstream activation of Rac. These data suggest that SHIP-2 is a novel negative regulator of FcgammaR-mediated phagocytosis independent of SHIP-1. Taken together, we concluded that specific receptors have unique downstream signaling events which are under tight control.
Advisors/Committee Members: Tridandapani, Susheela.
Keywords: SHIP-2; LRG; G-CSFR; SHIP-1; phagocytosis; cell; 32Dcl3
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3.
Alami, Nael H.
The Role of Myosin Va and the Dynein/Dynactin Complex in Neurofilament Axonal Transport.
Degree: PhD, Molecular, Cellular, and Developmental Biology, 2009, Ohio State University
► Neurofilaments are the major cytoskeletal elements in mature neuronal cells. They are…
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▼ Neurofilaments are the major cytoskeletal elements in mature neuronal cells. They are known for their space-filling properties and for forming an elastic network along the axons that is responsible for radial growth and maintaining proper caliber. Neurofilaments and other cytoskeletal polymers and macromolecular cargo are transported along the axon in one of two directions: in an anterograde fashion, or in a retrograde fashion. The regulation of this transport is vital for the functional and structural well-being of the neuron and is dependent on kinesins and dynein/dynactin.In 2002, a study by Rao et al. suggested a role for the actin-based motor protein myosin Va in neurofilament transport. Based on these observations, we hypothesize that myosin Va is involved in neurofilament transport and that in the absence of myosin Va, neurofilaments move less efficiently along the axons. Our results indicate that the absence of myosin Va from SCG neurons does not significantly alter neurofilament velocity or frequency of movement. We also used a fluorescence photoactivation pulse-escape technique to measure the rate of departure of neurofilaments from photoactivated axonal regions in cultured DRG neurons from two strains of dilute lethal mice. We observed a 48%-169% increase in the mean time for neurofilaments to depart the activated regions in neurons from dilute lethal as compared to wild type. We propose that myosin Va is a short-range motor for neurofilaments and that it can function to enhance the efficiency of neurofilament transport in axons by delivering neurofilaments to their microtubule tracks. We also studied the role of dynein/dynactin in neurofilament transport. Dynein/dynactin is a retrograde motor complex that was found to associate with neurofilaments in vivo and in vitro. We used SCG and cortical neuronal cultures to observe neurofilament transport in cells where dynein/dynactin activity has been disrupted using a number of different approaches that target different subunits of the complex. Using dynein heavy chain knock-down, dynein intermediate chain functional blocking antibody, dynamitin/p50 overexpression in SCG neurons and p150-coiled-coil1 overexpression in cortical neurons, we report an inhibition of retrograde transport. This clearly indicates that dynein/dynactin is indeed the retrograde neurofilament motor. We also observed a reciprocal inhibition of anterograde transport that mirrored the retrograde transport inhibition in every one of these manipulations. This suggests that a tight functional coupling exists between the retrograde and anterograde motors of neurofilaments, where the activity of one motor is needed for the activity of the other and vice versa. In conclusion, we propose that the transport and organization of neurofilaments may be orchestrated by the coordinated activity of at least three different motor proteins, kinesins, dynein/dynactin, and myosin-Va, which act together to convey and distribute these polymers along neuronal axons, and the disruption of any of these motors could lead to neurofilament transport defects and accumulations that could ultimately result in neuronal degeneration.
Advisors/Committee Members: Brown, Anthony.
Subjects: Biology; Neurology
Keywords: Neurofilaments; axonal transport; myosin; dynein; dynactin; kinesin
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4.
Anderson, Matthew David.
Studies with the human t-cell leukemia virus tax and rex positive trans-regulatory proteins.
Degree: PhD, Molecular, Cellular, and Developmental Biology, 2004, Ohio State University
► Retroviruses have highly organized genomes that encode a large number of proteins…
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▼ Retroviruses have highly organized genomes that encode a large number of proteins from a relatively short nucleic acid sequence. They utilize a variety of methods to achieve this, including the use of overlapping reading frames and the production of multiple RNAs through alternative splicing. In order to efficiently express RNAs encoding structural and enzymatic proteins, complex retroviruses like human T-cell leukemia virus (HTLV) must first express a doubly spliced RNA encoding both the Tax and Rex positive transregulatory proteins in separate but partially overlapping reading frames. As a result, functional studies of each of these proteins in the context of replicating virus, particularly mutational studies, present unique challenges. This dissertation both describes and utilizes experimental systems for the study of Tax and Rex that model in vivo in the context of replicating virus in human T cells, the natural target of HTLV infection. As a whole, the work described in this dissertation uses methods for the study of the HTLV regulatory proteins Tax-1 and Rex-2 that closely approximate conditions that occur in in vivo HTLV infections. Ideally this is an infection of primary human T-lymphocytes. This approach provides highly relevant insight into the molecular mechanisms utilized by these proteins, validates previous results obtained from in vitro studies as well as extends and refines our understanding of the way these proteins function in natural infection.
Advisors/Committee Members: Green, Patrick L.
Keywords: HTLV; leukemia; Tax; Rex
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5.
Arnold, Joshua E.
THE ROLE OF HBZ IN HTLV-1 BIOLOGY.
Degree: PhD, Molecular, Cellular, and Developmental Biology, 2008, Ohio State University
► Human T-cell leukemia virus type 1 (HTLV-1) is a pathogenic retrovirus that…
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▼ Human T-cell leukemia virus type 1 (HTLV-1) is a pathogenic retrovirus that has the capacity to transform primary human T-lymphocytes in culture and infected individuals. After nearly 30 years of research, the exact mechanism by which HTLV-1 induces cellular transformation and ultimately disease still remains elusive. Tax has been identified as the major viral determinant and is essential to the HTLV-1-mediated T-cell transformation process. The HTLV-1 accessory proteins p12, p13, and p30 are dispensable in culture, but are required for the maintenance of viral loads in vivo. In this dissertation, we sought to broaden our knowledge of HTLV-1 using in vitro culture assays and two animal model approaches focusing our efforts on understanding the contribution of a novel antisense encoded gene, the HTLV-1 basic leucine zipper factor (Hbz), in virus biology. Chapter 1 reviewed important aspects of HTLV-1 pathobiology and highlighted insightful comparative studies between HTLV-1 and HTLV-2. Our work in Chapter 2 determined that the HBZ protein is dispensable for immortalization/transformation of T-lymphocytes in culture, but is required for efficient infectivity and persistence in inoculated rabbits. In Chapter 3, utilizing Hbz-specific short hairpin RNA lentiviral vectors, we showed that Hbz significantly contributed to tumor formation and neoplastic cell spread in the NOG mouse transplant model. Chapter 4 expanded on Chapter 3 to show that Hbz functions in two molecular forms, mRNA and protein, to synergistically increase cell proliferation in vitro. Collectively our results indicate that the Hbz gene negativly regulates Tax-mediated viral gene expression and dysrupts the cellular microenviroment to ultimately support virus survival. The data in this dissertation have allowed us to better understand the contribution of Hbz to HTLV-1 infection, and its involvement in the development of disease.
Advisors/Committee Members: Green, Patrick.
Subjects: Molecular biology
Keywords: HTLV-1, HBZ, antisense transcript, short hairpin RNA knockdown, ATLL, NOG mouse, rabbit
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6.
Baker, Patrick Ericson.
Genetic regulation of virulence factors contributing to colonization and pathogenesis of helicobacter pylori.
Degree: PhD, Molecular, Cellular, and Developmental Biology, 2003, Ohio State University
► The urease enzyme of the gastric pathogen Helicobacter pylori catalyzes the degradation…
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▼ The urease enzyme of the gastric pathogen Helicobacter pylori catalyzes the degradation of urea into ammonia and carbon dioxide. Urease is believed to protect the bacterium from gastric acid. Urease activity of H. pylori is essential for colonization of the stomach. An objective of this dissertation was to correlate the level of urease expression with loss of colonization of H. pylori isolated from infected piglets and subjected to rounds of in vitro passage. Using slot blot and reverse transcription-polymerase chain reaction (RT-PCR) in a thermocycler with real-time analysis, urease RNA was determined to be constitutively expressed regardless of the number of in vitro passages. Determination of urease activity of the isolates by the phenol-hypochlorite assay confirmed the RNA data. Lewis x (Lex) and Lewis y (Ley) antigens of Helicobacter LPS undergo phase variation during in vitro growth due to slip-strand mispairing of polycytidine (poly(C)) tracts in the alpha-3-fucosyltransferase genes, futA and futB, that specify the Lewis antigen phenotype. Hence, another objective of this dissertation is to evaluate whether the on/off status of futA and futB via slip-strand mispairing determines Lex and Ley phenotype. The lengths of the poly(C) regions were analyzed by PCR. The Lewis phenotypes of the samples were determined by immunoblots and enzyme-linked immunoabsorbent assay (ELISA) using anti-Lex and anti-Ley antibodies. This study failed to correlate futA/B expression with Lex expression. Because both futB and Ley were expressed in all strains, no relationship could be determined. However, futB is sufficient for Lewis x/y synthesis. The final objective of this dissertation was to evaluate the ability of H. pylori LPS and its O-antigen to modulate the CD4+-dependent host immune response to H. pylori. This was performed by culturing splenocytes from uninfected and SS1-infected C57Bl/6 mice and T-bet-deficient mice. The cultures were stimulated with sonicate preparations and LPS samples from the parental and O-antigen-deficient bacterial strains. The data from this study suggests that Lewis antigens cannot alone account for the strong stimulation of the immune system and may even down-regulate the inflammatory response to H. pylori based on the higher stimulation of interferon-ã of SS1:0826 LPS compared to SS1 LPS.
Advisors/Committee Members: Eaton, Kathryn A.
Subjects: Biology, Microbiology
Keywords: Helicobacter pylori; urease; alpha-3-fucosyltransferase; beta-1,4-galactosyltransferase; slip-strand mispairing; gene regulation; gram-negative bacteria; lipopolysaccharide; Lewis Histo-Blood group antigens; Lewis x; Lewis y; Immunology
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7.
Bakkar, Nadine.
Molecular Mechanisms of NF-kB Regulation of Skeletal Myogenesis.
Degree: PhD, Molecular, Cellular, and Developmental Biology, 2008, Ohio State University
► NF-kB is a ubiquitous transcription factor involved in the regulation of innate…
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▼ NF-kB is a ubiquitous transcription factor involved in the regulation of innate immunity, cellular survival, proliferation, as well as differentiation. Its deregulation is associated with various diseases, and have thus been the target of developing therapeutic strategies. Skeletal muscle diseases are one field where this transcription factor is receiving recent attention, owing to its implication in muscular dystrophy, wasting and regeneration. In this dissertation, we focused on NF-kB regulation of myogenic differentiation, in an attempt to further understand the complex ways this transcription factor follows to regulate muscle development and extrapolate it to disease.In chapter 2, we focused on Myostatin (Mstn) a potent negative regulator of myogenesis that can inhibit myoblast proliferation and suppress synthesis of MyoD. NF-kB is similarly able to promote myoblast growth and induce loss of MyoD message. Given the similarities of these phenotypes, we examined potential Mstn and NF-kB signaling crosstalks in myoblasts and differentiated myotubes. Results show that Mstn does not activate NF-kB, nor does activated NF-kB induce Mstn expression. Furthermore, Mstn inhibition of differentiation can still occur in cells devoid of NF-kB activity. Such findings were confirmed in proliferating muscle precursors as well as mature muscle fibers and thus highlight the intrinsic differences between those two signaling pathways in the regulation of skeletal myogenesis, In chapter 3, we examined NF-kB regulation of skeletal myogenesis using genetic knockout models. Previous studies had attempted to investigate such a regulation, yet results remained perplexing, with both pro- and anti-myogenic roles of NF-kB documented. Using primary myoblasts and muscles devoid of NF-kB classical pathway components p65 or IKKb, we show that this canonical signaling is a negative regulator of myogenesis and gets downregulated during differentiation. On the other hand, NF-kB alternative signaling, mediated by IKKa activating the RelB/p52 complex is turned on and regulates mitochondrial biogenesis. Such a pathway is hence involved in the energy production and subsequent maintenance of newly formed myotubes. Consequently, our findings help to resolve the conundrum of NF-kB signaling in myogenesis by showing the existence of two opposing NF-kB pathways that function at temporally distinct stages of differentiation: classical signals inhibit premature myogenesis while alternative pathway activation regulate energy production and maintenance of nascent myofibers. Collectively, results presented in this dissertation highlight the various branches through which NF-kB signals to regulate skeletal myogenesis, emphasizing the need to take this complex regulation into account in clinical strategies aimed to modulate its activity.
Advisors/Committee Members: Guttridge, Denis.
Subjects: Molecular biology
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8.
Bhupathy, Poornima.
Sarcolipin a novel regulator of the cardiac sarcoplasmic reticulum calcium ATPase.
Degree: PhD, Molecular, Cellular, and Developmental Biology, 2008, Ohio State University
► Sarcolipin (SLN) is a small 31- amino acid protein which co-localizes with…
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▼ Sarcolipin (SLN) is a small 31- amino acid protein which co-localizes with the calcium transport ATPase (SERCA2a) in the cardiac sarcoplasmic reticulum (SR) membrane. The main goal of this study was determine the functional relevance of SLN in cardiac physiology. Using a highly specific SLN antibody we showed that SLN levels are altered during muscle development and cardiac pathophysiology. Our protein data indicate that SLN is expressed in cardiac and skeletal muscle tissues. More importantly, in the heart SLN is predominantly expressed in the atria. To determine the role of sarcolipin in SR calcium handling and cardiac contractility, we have generated two mouse models 1) cardiac specific overexpression of SLN and 2) SLN knockout. Overexpression or complete loss of SLN did not affect the cardiac muscle development or induce any cardiac pathology. Overexpression of sarcolipin decreases calcium-dependent calcium uptake, whereas ablation of SLN increases the calcium uptake in both atria and ventricle. Our results showed that loss of SLN significantly increases the rates of contraction and relaxation, whereas an increase in SLN level decreases the contractility. SLN null hearts showed blunted response to isoproterenol treatment. On the other hand, the inhibitory effect of SLN was relieved upon high dose of isoproterenol in SLN overexpressing hearts, which may suggest that SLN also mediates beta-adrenergic response. However, the mechanism of SLN action on SERCA2a is not well understood. Invitro phosphorylation studies revealed that a highly conserved Threonine 5 (T5) residue in SLN can be phosphorylated by CaMKII. To determine the role of T5 phosphorylation in SLN function we mutated T5 to alanine (A) (a non-phosphorylatable mutant), or to glutamic acid (E) (which mimics the phosphorylated form of SLN) and studied their effects on myocyte contractility and calcium transients. Our data indicate that T5 plays a physiological role in affecting SLN function. In conclusion, our data suggest that SLN is an important regulator of SERCA2a activity in the heart and the inhibitory effect is regulated by SLN phosphorylation.
Advisors/Committee Members: Periasamy, Muthu.
Keywords: Sarcolipin,; SERCA pump,; Cardiac,; calcium homeostasis
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9.
Bolinger, Cheryl Giles.
Study of translation control by a RNA helicase A-responsive post-transcriptional control element in Retroviridae.
Degree: PhD, Molecular, Cellular, and Developmental Biology, 2008, Ohio State University
► Mechanisms governing retrovirus translation are a topic of great interest and controversy.…
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▼ Mechanisms governing retrovirus translation are a topic of great interest and controversy. Motifs located within the untranslated region (UTR) of retroviral mRNA have established roles in virus replication, and a growing volume of literature is revealing a necessary role of the UTR in control of viral protein synthesis. Two elements implicated in retrovirus translation control are a cap-dependent post-transcriptional control element (PCE) that is responsive to cellular protein RNA helicase A (RHA), or a cap-independent internal ribosome entry site (IRES). We have utilized stringent RNA and protein analyses to show that the 5’ UTR (specifically the RU5 region) of spleen necrosis virus (SNV), reticuloendotheliosis virus A (REV-A), and human T-cell leukemia virus type 1 (HTLV-1) exhibit PCE activity, but not IRES. PCE activity was also conferred by the 5’ UTR of HIV-1 and feline and bovine leukemia viruses, increasing the number of PCE-containing retroviruses to eight viruses spanning 6 genera. SNV, REV-A and HTLV-1 PCE require RHA for activity and it is expected that RHA regulates translation of all PCE-containing mRNA. Direct association of RHA with HTLV-1 and HIV-1 5’ RU5 has been demonstrated, and a combination of ribosomal profile analyses and metabolic labeling experiments determined that RHA is necessary for translation of HTLV-1 and HIV-1 gag from genomic mRNA. These results provide direct evidence that RHA is necessary for efficient HTLV-1 and HIV-1 replication. Targeted RHA mutational analysis identified specific amino acid residues that modulate HTLV-1 PCE activity. The conserved residues that define the redundant N-terminal double-stranded RNA binding domain (dsRBD), the C-terminal arginine-glycine-rich (RGG) bi-directional nuclear transport domain, and central ATPase domain are essential for HTLV-1 translation. Identified transdominant RHA mutants accumulate in cytoplasmic stress granules, presumably with stalled translation complexes. We propose that RHA operates a selective translation control switch of retroviral mRNAs; PCE interaction with RHA through the dsRBD and RGG motif in conjunction with RNP remodeling by the ATPase domain facilitates cap-dependent translation. In the realm of translational research, each new insight into retroviral protein synthesis offers a prospective target for antiviral therapy and strategic improvement of gene transfer vectors.
Advisors/Committee Members: Boris-Lawrie, Kathleen.
Subjects: Molecular biology
Keywords: Translation control; retrovirus; post-transcriptional control element; RNA helicase A; RHA; PCE; HIV-1; HTLV-1; IRES:stress granule
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10.
Buchmann, Cody.
Reversal of RNA-mediated gene silencing pathways by geminivirus AL2 and L2 proteins.
Degree: PhD, Molecular, Cellular, and Developmental Biology, 2008, Ohio State University
► RNA-directed gene silencing is an adaptive response by which plants defend against…
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▼ RNA-directed gene silencing is an adaptive response by which plants defend against virus invasion. RNA-silencing pathways can be divided into two basic categories: Post-Transcriptional Gene Silencing (PTGS) and Transcriptional Gene Silencing (TGS). PTGS occurs in the cytoplasm and results in mRNA degradation. In addition, a mobile signal circulates throughout the plant, is amplified, and triggers systemic silencing. The second pathway, TGS, takes place in the nucleus and results in methylation of cytosine in DNA, which encourages the formation of transcriptionally inactive heterochromatin by histone modification. TGS suppresses damaging sequences such as transposons, and also plays an important role in defense against DNA viruses.The AL2 and L2 proteins are related proteins encoded by geminiviruses of the Begomovirus and Curtovirus genera, respectively. AL2 is transcriptionally active, is required for late viral gene expression, and can also activate unknown host genes, whereas L2 is transcriptionally inactive. AL2 can suppress silencing by two mechanisms. The first requires host gene activation by AL2 (transcription-dependent mechanism). The second is shared by AL2 and L2 and involves inactivation of adenosine kinase (ADK), an enzyme required for efficient cellular transmethylation activity. Both AL2 and L2 can suppress local silencing via a transcription-independent mechanism. In this thesis AL2, but not L2, is also shown to block systemic spread of PTGS by a transcription-dependent mechanism. In addition, AL2 and L2 are shown to reverse established PTGS, which suggests interference with a maintenance step. However, while both proteins reversed silencing in immature plants, only AL2 did this in mature plants, suggesting the existence of multiple, developmentally regulated maintenance pathways. Finally, the role of methylation as a host defense was confirmed by studies which demonstrated that both AL2 and L2 can reverse TGS of a transgene and of native loci, and cause significant reversal of methylation throughout a plant genome. In this case, TGS reversal occurred by both transcription-independent and transcription-dependent mechanisms. These studies further our understanding of viral pathogenesis and the nature of innate host defenses by demonstrating that AL2 and L2 can suppress both TGS and PTGS by multiple mechanisms.
Advisors/Committee Members: Bisaro, David.
Subjects: Molecular biology; Plant pathology; Virology
Keywords: geminivirus; RNA silencing; virus counterdefense; TGS; PTGS; DNA methylation
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11.
Carrel, Tessa Lyn.
The use of the spontaneous Bn mouse mutant and targeted alleles of Smad2 and Tgif to understand axial specification and neural development.
Degree: PhD, Molecular, Cellular, and Developmental Biology, 2004, Ohio State University
► To understand the basis of axial specification and neural development, mouse models…
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▼ To understand the basis of axial specification and neural development, mouse models are commonly utilized. Bent tail (Bn) is a spontaneous mutation on the mouse X chromosome that produces tail deformities and open neural tube defects (NTDs). Analysis of progeny from an intraspecific backcross places the mutation between the microsatellites DXMit166 and DXMit140. Refined genetic and physical mapping of the Bn critical region demonstrate that the mutation is associated with a <170 kb submicroscopic deletion, including the entire Zic3 locus. Human mutations in ZIC3 are associated with left-right axis malformations. The presence of anal and spinal abnormalities in some patients and deletion of Zic3 in Bn mice support a key role for this gene in neural tube development and closure. However, mutations in the ZIC3 gene have yet to be identified in families with X-linked NTDs. Holoprosencephaly (HPE) results from abnormal development of the forebrain. One gene associated with HPE in humans, TG interacting factor (TGIF), was identified by its ability to bind to the retinoid X receptor response element, and has been shown to play a role in regulating TGF-β signaling. HPE is not evident in mice carrying the targeted null allele of Tgif. To elucidate whether Tgif in conjunction with reductions in TGF-β signaling can cause HPE, mice that have mutations in both Smad2 and Tgif were generated. Results show that of the Smad2+/-; Tgif+/- and Smad2+/-; Tgif-/- embryos, one third display HPE. Molecular characterization at E9.5 reveals that Shh, Fgf8, Six3 and Zic2 expression are not affected. The forebrain domain of Otx2 expression shows a modest to nearly complete reduction in affected embryos. Published data has shown that introduction of retinoic acid (RA) to cultured embryos or pregnant dams can induce HPE in embryos. Some of these studies have further shown a reduction in rostral Otx2 expression. The known interaction of Tgif and RA signaling led to the evaluation of the possibility that the HPE resulted from altering RA pathways. Analysis of developing embryos provided evidence that genetic deficiencies of Smad2 and Tgif allows for increased sensitivity to RA, suggesting a unique link between genetics and environmental teratogens.
Advisors/Committee Members: Weinstein, Michael.
Keywords: TGIF; SMAD2; embryos; HPE; Zic3; BN; gene
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12.
Chandra, Shweta.
Roles of immunoglobulin domain proteins echinoid and friend-of-echinoid in drosophila neurogenesis.
Degree: PhD, Molecular, Cellular, and Developmental Biology, 2004, Ohio State University
► During neurogenesis in Drosophila, groups of ectodermal cells are endowed with the…
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▼ During neurogenesis in Drosophila, groups of ectodermal cells are endowed with the capacity to develop into neuronal precursors. The Notch signaling pathway is required to limit the neuronal potential to one or two cells within each group. Loss of genes of the Notch signaling pathway results in a neurogenic phenotype: hyperplasia of the nervous system accompanied by a parallel loss of epidermis. Echinoid (Ed), a cell membrane associated Immunoglobulin C2-type protein has previously been shown to be a negative regulator of the EGFR pathway during eye development. Work from our lab has shown that Ed has a role in restricting neurogenic potential during embryonic neurogenesis. I have extended the functional and genetic analysis of Ed. Loss of ed suppresses the lack of neuronal elements caused by ectopic activation of the Notch signaling pathway. Using a temperature sensitive allele of ed, I show that Ed is required to suppress sensory bristles and for proper wing vein specification during adult development. In these processes also, ed acts in close concert with genes of the Notch signaling pathway. Overexpression of the membrane-tethered extracellular region of Ed results in a dominant-negative phenotype. This phenotype is suppressed by overexpression of Enhancer of split m7 {E(spl)}and enhanced by overexpression of Delta. Thus Ed interacts synergistically with the Notch signaling pathway. I have identified a paralog of Ed, Friend-of-echinoid (Fred). fred function was examined in transgenic flies using inducible RNAi. Suppression of fred in the developing wing disc results in specification of ectopic SOPs, additional microchaeta and cell death. These phenotypes can be suppressed by increasing the activity of the Notch signaling pathway. Dosage-sensitive genetic interaction suggests a close functional relationship between fred and ed. Microarray analysis of fred RNAi discs revealed a number of genes that are misregulated in the absence of Fred activity. Changes in the transcript levels predicted by the microarray data were validated by performing in situ hybridizations. One of the genes that is downregulated is pannier (pnr). Genetic interaction experiments suggest that Pnr cooperates with Fred to suppress neurogenesis in the apparently non-neurogenic regions of the wing disc.
Advisors/Committee Members: Vaessin, Harald.
Keywords: Drosophila neurogenesis, SOP; Echinoid and FRiend-of-echinoid; Notch signaling pathway; Immunoglobulin domain
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13.
Chang, Xing.
X-Linked FOXP3 and OTC in immune tolerance and autoimmunity.
Degree: PhD, Molecular, Cellular, and Developmental Biology, 2006, Ohio State University
► Self and non-self discrimination is one of the fundamental tenets of immunology.…
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▼ Self and non-self discrimination is one of the fundamental tenets of immunology. For the immune system, to avoid devastating self constituents is as important as to respond effectively to evading pathogens. Failure of self tolerance often leads to autoimmune disease, in which the immune system is attacking and disrupting self tissues. Self tolerance is maintained by the discrimination of self antigens against non-self antigens. The main focus of this thesis is to study molecular and cellular mechanisms of self and non-self discrimination and how its failure leads to autoimmune disease. The thymus anatomically separates self antigens from non self antigens. Antigen expression in the thymus leads to the deletion of self-reactive T cells and generation of regulatory lymphocytes, including regulatory T cells(Treg) and NKT cells. Our data presented in Chapter 5 show an Ornithine transcarbamylase(OTC) mutation causes ineffective presentation of self antigens in the thymus. As such, deletion of self reactive T cells is compromised and introthymic production of Treg and NKT cells is reduced in the OTC mutant mice. More importantly the heterozygous mice have increased susceptibility to autoimmune diseases, including the generation of autoantibodies and more severe EAE induced by MOG immunization. Even with efficient introthymic deletion, some autoreactive T cells are still exported to the periphery. Another difference between self antigens and non self antigens is that the former are constantly expressed in the secondary lymphoid organs. High affinity interaction with antigens usually induces activation induced-cell death of reactive T cells. In Chaper 4, we present evidence that the activation induced cell death by high affinity self antigens is enhanced by regulatory T cells. By tracking endogenous superantigen reactive T cells in Scurfy mutant mice, which lack regulatory T cells altogether, we demonstrate that the absence of Treg causes the accumulation of autoreactive T cells in the periphery that evade normal clonal deletion in the thymus. Adoptive transfer experiments further reveal that Treg does not directly inhibit the proliferation of autoreactive T cells, but instead induce their programmed cell death. We also demonstrate that Treg mediated cytotoxity is specific to the autoreactive T cells, and such specificity is due to the higher sensitivity of autoreactive T cells to apoptosis. Although T cells with high affinity against self antigens can be eliminated from the repertoire, T cells with low affinities against self antigens are still unavoidable. Under physiological conditions, due to their low affinities those T cells are not able to be activated. However, in the lymphopenic environment, such low affinity interaction can drive T cell activation and expansion through homeostatic proliferation. In Chapter 2 and 3, we provide evidence that the FoxP3 gene mutation in Scurfy mice leads to reduced thymopoeisis and subsequently less T cell output from the thymus. The FoxP3 mutation in the thymic stroma causes defective T cell output from the thymus and lymphopenia in neonatal mice, which leads to extensive homeostatic proliferation that precedes antigen driven proliferation. The homeostatic proliferation is independent of the loss of Treg and FoxP3 mutation in the T cell lineage. We propose that the combined deficiency in T cell production and development of functional Treg may explain the severe autoimmunity associated with the FoxP3 mutation.
Advisors/Committee Members: Liu, Yang.
Subjects: Health Sciences, Immunology
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14.
Chang, Ya-Wen.
The Ras/PKA pathway controls transcription of genes involved in stationary phase entry in Saccharomyces cerevisiae.
Degree: PhD, Molecular, Cellular, and Developmental Biology, 2003, Ohio State University
► Upon nutrient deprivation, Saccharomyces cerevisiae cells arrest division and enter into a…
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▼ Upon nutrient deprivation, Saccharomyces cerevisiae cells arrest division and enter into a specialized resting state, known as stationary phase. The entry into this resting state is regulated, in part, by the Ras/PKA (cAMP-dependent protein kinase) signaling pathway. We are interested in understanding the mechanisms regulating stationary phase biology in S. cerevisiae, with an ultimate goal of defining the targets of PKA that are responsible for this growth control. To this end, we have identified a collection of mutants that exhibited a defective transcriptional response to nutrient limitation and failed to enter into a normal stationary phase. These rye mutants were isolated on the basis of defects in the regulation of YGP1 expression. We found that the levels of YGP1 were significantly elevated in the rye mutants during the log phase of growth. The rye defects were not specific to this YGP1 expression defect because these mutants also exhibited multiple defects in stationary phase properties, including an inability to survive periods of prolonged starvation and a failure to tolerate a mild heat shock. These data indicated that the RYE genes may encode important regulators of yeast cell growth. Interestingly, three of these RYE genes encoded the Srb proteins, Srb9p, Srb10p, and Srb11p. These Srb proteins are components of the Srb complex associated with the RNA polymerase II holoenzyme. We found that specific transcription defects associated with these srb mutations were suppressed by RAS2val19, a hyperactive allele of RAS2. However, increased Ras signaling was not able to correct the expression defects associated with an srb9 null mutant, suggesting that the Srb9 protein is essential for the Ras suppression. Moreover, there are two potential PKA consensus sites in Srb9p. Our results showed that the suppression of the srb9 defects required the presence of these two PKA sites. In addition, we have found that Srb9p was phosphorylated by PKA in vitro and in vivo. In all, our results suggest that Srb9p is a substrate for PKA, and that this phosphorylation of Srb9p modulates the in vivo activity of the Srb complex to regulate transcription of a subset of genes involved in stationary phase entry.
Advisors/Committee Members: Herman, Paul K.
Keywords: Ras; Srb; RNA polymerase II; Transcription; Stationary phase
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15.
Chen, Wei-Kang.
Analysis of neural gene expression: glutamine synthetase and nitric oxide synthas 1.
Degree: PhD, Molecular, Cellular, and Developmental Biology, 2003, Ohio State University
► Expression of chicken glutamine synthetase (GS) is restricted to the retina Müller…
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▼ Expression of chicken glutamine synthetase (GS) is restricted to the retina Müller glial cells. There are two consensus elements, a noncanonical TATA box and a CCAAT box, essential for the 95 bp chicken GS core promoter. The ubiquitous transcription factor NF-Y activates GS relying on its interaction with a single CCAAT box element located at position -62 to -58 relative to the transcription start site. In addition, a transcription factor AP-2 alpha activates expression of a GS-luciferase fusion gene in transfected HepG2 cells but inhibits fusion gene expression in retinal primary cultures. Inhibition by AP-2 alpha requires a functional NF-Y binding site but does not involve direct binding of NF-Y to the CCAAT in the GS promoter. CCAAT displacement protein (CDP/cut), a factor known to diminish gene expression upon binding to CCAAT elements of target genes, has recently been shown to interact with AP-2 alpha, an interaction that diminishes the functioning of the histone H3.2 promoter. Thus, cell specific expression of GS might be affected by the availability of both CDP/cut and AP-2 alpha via their concerted inhibition of NF-Y mediated activation of the core promoter in neuronal cells of the neural retina. The human nitric oxide synthase 1 (hNOS1) gene is transcribed by at least 10 distinct promoters and is expressed in neuronal cells of the central nervous system. Expression of NOS1 is enhanced in response to neuronal injury in several models. Because cAMP mediated signaling may play a role in activation of hNOS1 transcription, we examined cAMP inducibility of hNOS1 promoter-reporter fusion genes in PC12 cells. A novel promoter, designated as hNOS1/E2, was found within exon 2 of the hNOS1 gene and shown to be cAMP inducible with two functional cyclic AMP response elements (CREs). Mutation of either CRE abrogated cAMP inducibility. Interestingly, although PKA-mediated activation of CREB is clearly important, activation of MEK/ERK signaling was also found to play a role in cAMP mediated activation of the hNOS1 E2 promoter. Finally, although inducible expression of NOS1promoter-reporter fusion genes encoded within plasmids could be observed in transient assays, cells stably transformed with the same plasmids proved refractory to this type of analysis. In response to this problem, we transduced PC12 cells using retroviruses containing the green fluorescent protein (GFP) reporter under transcriptional control by different hNOS1 promoters. In sharp contrast to results obtained using plasmids, inducible expression of GFP in response to treatment with forskolin (a cAMP inducer) and nerve growth factor (NGF) could be observed. An NGF responsive element was localized to a 565 bp region within the 5'2 promoter through this analysis. In general, these results demonstrate that retrovirus vectors provide a useful tool in characterization of inducible expression of weak promoters such as those of hNOS1.
Advisors/Committee Members: Young, Anthony Peter.
Keywords: neural gene expression; glutamine synthetase; nitric oxide synthase 1; transcriptional regulation; signal transduction; retroviral vectors
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16.
Colakoglu, Gulsen.
Assembly Dynamics of Intermediate Filaments.
Degree: PhD, Molecular, Cellular, and Developmental Biology, 2009, Ohio State University
► The cytoskeleton of eukaryotic cells comprises three classes of protein polymers: actin…
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▼ The cytoskeleton of eukaryotic cells comprises three classes of protein polymers: actin filaments, microtubules and intermediate filaments. Actin filaments and microtubules are known to lengthen and shorten by addition and loss of subunits at their ends. In contrast, several studies suggest that intermediate filaments may lengthen by end-to-end annealing of pre-existing filaments and that addition and loss of subunits is not confined to the filament ends. To address these hypotheses, we expressed mCherry-tagged, GFP-tagged or photoactivatable GFP-tagged neurofilament or vimentin proteins in cultured SW13 vim- cells, which lack endogenous cytoplasmic intermediate filaments. To test for annealing, we used polyethylene glycol-induced cell fusion to create cells containing a mixture of red and green filaments. Within five hours, we observed many examples of single intermediate filaments comprised of alternating red and green segments, indicating that end-to-end annealing is a common event. To determine the site of subunit incorporation, we used cell fusion to create cells containing a mixture of red and photoactivatable green fluorescent filaments and then used whole-cell photoactivation to mark those filaments that lacked red fluorescence. Alternatively, we co-expressed red and photoactivatable green fluorescent neurofilament proteins in the same cells to make filaments containing both red and green fluorescence, then photobleached the red fluorescence and used partial-cell photoactivation to mark a sub-population of the bleached filaments. In both cases, we observed the incorporation of newly synthesized or pre-existing red fluorescent intermediate filament subunits along the length of the pre-existing photoactivated green fluorescent filaments over a period of eight to twenty hours, and no preferential addition of subunits to the filament ends. We confirmed our results in SW13 vim+ cells with endogenous vimentin networks and in MFT-16 fibroblasts derived from vimentin knockout mice. These data provide the first direct evidence that intermediate filaments in cells can elongate by end-to-end annealing and that filaments can incorporate subunits along their length, a process that we term intercalary subunit exchange.
Advisors/Committee Members: Brown, Anthony.
Subjects: Cellular biology; Molecular biology
Keywords: intermediate filament; neurofilament; vimentin; assembly; dynamics
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17.
Copley, LaRae.
Investigation of the function of myotubularin through the examination of protein-protein interactions and exclusion of MTMR1 as a frequent cause of X-linked myotubular myopathy.
Degree: PhD, Molecular, Cellular, and Developmental Biology, 2004, Ohio State University
► X-linked myotubular myopathy (MTM1) is a rare neuromuscular disorder presenting at birth…
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▼ X-linked myotubular myopathy (MTM1) is a rare neuromuscular disorder presenting at birth with hypotonia, respiratory distress, and characteristic facies. In 1996, an MTM1 gene encoding a protein called myotubularin was isolated. Myotubularin acts as a lipid phosphatase by dephosphorylating phosphatidylinositol-3-phosphate, an intracellular membrane trafficking signal. To further elucidate the function of myotubularin, we employed a yeast two-hybrid system to screen for proteins with which it interacts. Our results yielded several clones, including the catalytic subunit of PP2A, and SCG10. Binding of the MTM1 protein with these candidates was determined to be specific in the yeast system, but confirmation of specific interactions using coimmunoprecipitation and GST pulldown strategies was unsuccessful. A second goal of this work was to develop antibodies to myotubularin. Regions of antigenicity were predicted in myotubularin and corresponding peptides were generated and injected into rabbits. An antibody that specifically detects human myotubularin but not mouse myotubularin was generated. Mutations in more than three hundred MTM1 families worldwide have been characterized. However, no mutation within this gene has been found in approximately 20% of “MTM1” patients. MTMR1, a gene highly homologous to MTM1, is located 50KB telomeric to MTM1 on the X chromosome. We have screened 16 exons of MTMR1 in genomic DNA from 14 males with biopsy proven myotubular myopathy in whom we found no mutation in MTM1. This includes two males with X-linked pedigrees and two with affected male siblings. No mutations were found in these patients. These results suggest that mutations in MTMR1 are not a frequent cause of X-linked myotubular myopathy in males for whom no mutation is found in MTM1. Finally, we evaluated changes in cell shape in the context of myotubularin overexpression. We developed cell lines that stably express Flag-tagged myotubularin in a doxycycline inducible fashion. We found a statistically significant decrease in spreading in cells with a high level of myotubularin overexpression compared to untreated cells. This system was also used to quantitate membrane ruffling. A decrease in membrane ruffling was observed in cells expressing high levels of the myotubularin, but statistical significance could not be established with two independent observers.
Advisors/Committee Members: Herman, Gail E.
Keywords: X-linked; myotubular myopathy; MTMR1; protein protein interactions; peptide antibodies; cell spreading; membrane ruffling
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18.
Davy, Brian Edwin.
Identification and characterization of hydin, a large novel gene disrupted in a murine model of congenital hydrocephalus.
Degree: PhD, Molecular, Cellular, and Developmental Biology, 2004, Ohio State University
► Congenital hydrocephalus is a frequent human birth defect, occurring with an estimated…
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▼ Congenital hydrocephalus is a frequent human birth defect, occurring with an estimated incidence of 1 in 1000 live births. While a significant portion of human hydrocephalus is genetic in origin, the molecular genetics of this disease remains poorly understood. The murine autosomal-recessive mutation hydrocephalus-3 (hy3) results in lethal communicating hydrocephalus with perinatal onset. A recently described transgenic insertional mutation, OVE459, represents a new allele of hy3. In previous work, the OVE459 transgene insertion site was cloned, and a wild-type BAC clone encompassing the transgene insertion site was subsequently identified. Here, we describe the isolation of expressed sequences on this BAC clone by direct cDNA selection. Selected cDNAs facilitated the identification of two novel candidate genes, Hydin and Vac14. Hydin consists of at least 86 exons spanning more than 340 kb on mouse chromosome 8. The full-length Hydin transcript is nearly 16 kb and encodes a putative 5099 amino acid protein. Hydin is expressed in compartments of the brain that are directly involved in CSF production and homeostasis. In situ hybridization revealed Hydin transcripts in the choroid plexus and ciliated ependymal cells lining the ventricles at various stages of development and in the adult animal. Outside the central nervous system, Hydin is specifically expressed in cell-types that possess 9+2 cilia or flagella. The OVE459 transgene insertion resulted in a rearrangement within Hydin, disrupting the order of Hydin exons in these mice. Northern analysis revealed a marked reduction of Hydin mRNA levels in OVE459 and hy3 homozygotes relative to wild-type littermates. Sequencing of all 87 Hydin exons from homozygous hy3 genomic DNA revealed a single CG base-pair deletion in exon 15, causing a premature termination signal two codons downstream of the deletion. From this evidence, we conclude that Hydin is the disrupted gene in the OVE459/hy3 mouse model of congenital hydrocephalus. The Hydin gene product does not resemble any previously identified protein with the exception of a 314 amino acid region with homology to caldesmon, an actin-binding protein. To facilitate future investigations concerning Hydin function, a conditional targeting construct generated via recombineering was used to target Hydin exon 15 in ES cells.
Advisors/Committee Members: Robinson, Michael Lee.
Subjects: Biology, Genetics
Keywords: hydrocephalus; hy3; Hydin; mouse models
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19.
Dearth, Lawrence.
Characterization of C/EBPs in Mammary Epithelial Cell Biology.
Degree: PhD, Molecular, Cellular, and Developmental Biology, 2002, Ohio State University
► Work from this dissertation has demonstrated that C/EBPbeta is the predominant C/EBPbeta…
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▼ Work from this dissertation has demonstrated that C/EBPbeta is the predominant C/EBPbeta isoform expressed in the normal mouse mammary gland and mouse mammary tumors in vivo. Specific analysis of the C/EBPbeta isoforms demonstrated that LIP protein levels are directly influenced by the protein isolation procedure, indicating that LIP levels are modulated by protein isolation induced proteolysis. C/EBPbeta LIP was not detected in the normal mammary gland, but was detected in mammary tumors. Analysis of the affect of C/EBPbeta LIP overexpression on mammary epithelial cell proliferation and differentiation demonstrated that LIP did not have a significant effect on proliferation, but abrogated differentiation by an extracellular matrix-independent mechanism. This work has lead to the model that disruption of the differentiation program by LIP allows mammary epithelial cells to undergo continued proliferation, which may result in breast cancer. In addition, we demonstrated for the first time that C/EBPdelta mRNA exhibits a relatively short half-life in G0 growth arrested mouse and human mammary epithelial cells. Results of oligo/RNase H cleavage analysis and RACE-PAT revealed a short C/EBPdelta mRNA half-life in addition to demonstrating that the C/EBPdelta mRNA poly(A) tail is relatively short. The poly(A) tail length is not modulated during C/EBPdelta mRNA degradation, which suggests a deadenylation-independent pathway. The C/EBPdelta protein also exhibited a short half-life in G0 growth arrested mouse and human mammary epithelial cells. Results of ubiquitination inhibitor studies demonstrated that C/EBPdelta protein is degraded in an ubiquitin-dependent manner, exclusively in the nucleus during G0 growth arrest. Finally, replacement of the C/EBPdelta 3’ untranslated region (UTR) with the bovine growth hormone 3’ UTR increased C/EBPdelta mRNA half-life, implying a role for the 3’ UTR in the regulation of C/EBPdelta mRNA stability. Analysis of the C/EBPdelta 3’ UTR identified two potential AREs. RNA electromobility shift assays (REMSAs) demonstrated that the C/EBPdelta mRNA 3’ UTR binds a trans-acting factor(s) present in G0 growth arrested, but not growing mammary epithelial cell lysates. Competition analysis revealed that the 5’ ARE is necessary for trans-acting factor binding. UV-binding analysis revealed that the specific RNA/protein complex observed upon REMSA analysis had a mass of approximately 35 kDa.
Advisors/Committee Members: DeWille, James.
Subjects: Biology, Molecular
Keywords: C/EBPs; mammary epithelial cells; posttranscriptional regulation; posttranslational regulation
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20.
Fisher, Ian Bradford.
Role of Ets-2 in lymphocyte development, function, and survival.
Degree: PhD, Molecular, Cellular, and Developmental Biology, 2004, Ohio State University
► Ets transcription factors have been implicated in the development, regulation, and survival…
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▼ Ets transcription factors have been implicated in the development, regulation, and survival of numerous hematopoietic derived cells, including B and T-lymphocytes. E26 avian leukemia oncogene-2 (Ets-2) is an Ets family member transcription factor. Strong expression of Ets-2 is observed in developing thymocytes from the DN1 stage and in B-cells from the pro-B stage. A conserved sequence within the Ets-2 pointed domain amino acids 69 to 73 PLLTP is able to interact with the Map kinase ERK. Threonine seventy-two is phosphorylated by ERK1, and permits transactivation by Ets-2. Mutation of this residue from a threonine to an alanine, Ets-2 T72A, abrogates Ras mediated transactivation of Ets-2. Based on this evidence, we hypothesized that Ets-2 was important in T- and B-cell development, activation, and function. To test this hypothesis we analyzed Ets-2 activation in T-cell lines, and created two transgenic mice lines that express Ets-2 T72A in either developing T-cells or B-cells. Transactivation of Ets-2 can be induced by both Ras activation and by phorbol 12-myristate 13-acetate and ionomycin stimulation in the Jurkat T-cell line. In-vitro analysis of Ets-2 activation by ionomycin results in a rapid, but transient Ets-2 band shift as evidenced by western blot analysis of protein extracts from thymocytes. Transgenic mice that over-express Ets-2 T72A in the thymus displayed a dramatic reduction in thymus size associated with hypocellularity. This reduction was associated with a partial developmental block at the double negative 2 and double negative 3 stage of development, a twenty-fold increase in c-Kit+ expression, and a five-fold increase the CD5low population. Further, thymocytes from the transgenic mice have increased apoptosis both in-vitro and in-vivo compared to non-transgenic littermates. Transgenic mice that over-express Ets-2 T72A in B-lymphocytes revealed a loss of the B220Hi population of B-cells in the bone marrow. This population consists of mature B-cells that are characterized to be IgM+, IgD+ and Cd49d+ mature lymphocytes. Analysis of transgenic Ets-2 T72A B-cells in other secondary lymph organs did not reveal any defects. This dissertation demonstrates that Ets-2 is an important target in lymphocyte development and function. It is involved in T-cell development and survival and in establishment of mature bone marrow B-cells.
Advisors/Committee Members: Muthusamy, Natarajan.
Subjects: Biology, Cell
Keywords: Ets transcription factors; Ets-2; Thymus; Thymocytes; T-lymphocytes; Bone marrow; Spleen; B-lymphocytes; Ras; MapK; Transgenic Mice
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21.
Fullenkamp, Amy N.
Functional Dissection of the Aristaless-related Homeobox Proteins, Arx and Rx.
Degree: PhD, Molecular, Cellular, and Developmental Biology, 2008, Ohio State University
► Transcriptional repression and activation are both necessary for the precise expression of…
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▼ Transcriptional repression and activation are both necessary for the precise expression of the genes involved in the proper development of an organism. Aberrations in transcription factors that lead to dysfunctional proteins result in disease. Understanding the mechanisms in which transcription is regulated is vital to the identification of the factors and genetic networks that contribute to normal development. This information also provides a better understanding of the role mutations play in causing disruption in transcription factor function that leads to the impairment of developmental processes. Homeobox genes are important for normal development. Arx is a member of the paired-type Aristaless-related family of homeobox proteins. Human mutations in Arx are associated with X-linked mental retardation and other neurological disorders. Findings from the studies of Arx deficient mice indicate the importance of Arx on brain development and neural cell migration. Therefore, Arx has emerged as a gene that is essential for normal brain development and is of major clinical significance. Despite what is known about Arx clinically, less is known about its molecular functions as a transcription factor and its downstream targets. It has been shown that Arx functions as a transcriptional repressor, but it is not clear how disease-associated mutations affect its function as a repressor. This dissertation describes the consequence of two disease-associated mutations on the function of Arx as a transcriptional repressor. Two independent repression domains: the octapeptide and a novel domain located in the C-terminus were identified in Arx as well as the co-factors that mediate the repression through these domains. This work also describes the DNA binding ability of Arx. Additionally, the transcriptional function of another Aristaless-related homeobox protein, Retinal Homeobox (Rx) is also reported. Rx has been shown to be an activator of transcription, however this work found that the Rx has the ability to mediate repression. Together, the goal of this dissertation is to provide insight into the transcriptional functions of two related but functionally different paired-type Aristaless-related homeobox proteins: Arx and Rx. Furthermore, this work provides evidence for the function of the octapeptide motif in the context of a transcriptional repressor and an activator.
Advisors/Committee Members: El-Hodiri, Heithem.
Subjects: Molecular biology
Keywords: Aristaless; homeobox; Arx; Rx; transcriptional repression
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22.
Gray, Michelle.
Zebrafish deadly seven: neurogenesis, somitogenesis, and neural circuit formation.
Degree: PhD, Molecular, Cellular, and Developmental Biology, 2004, Ohio State University
► Proper development of the vertebrate nervous system is essential for the overall…
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▼ Proper development of the vertebrate nervous system is essential for the overall function of the organism. The vertebrate nervous system is highly complex and contains an enormous number of distinct cell types. In order for the organism to carry out its normal behavior, it requires that all of the components of the nervous system be produced in appropriate numbers, at correct times, in the right locations and that they make the proper connections. The relative simplicity of the early zebrafish nervous system makes it an attractive model for studying vertebrate nervous system development. Mutagenesis screens in zebrafish have been undertaken to identify new genes involved in different aspects of nervous system development. We have characterized an ENU induced mutation in zebrafish deadly seven/notch 1a (des), which perturbs neurogenesis, somitogenesis, and motor axon outgrowth. The neurogenic defect is manifested as an increase in hindbrain interneurons and spinal motoneurons. In addition, we find a decrease in the number of spinal sensory neurons, and an increase in sensory neurons derived from neural crest cells. This data demonstrates that Notch signaling is important for determining the number of specific neuronal cell types during early nervous system development. The somite defect in des mutants is revealed by abnormalities in somite/myotome boundary formation and somite/myotome gene expression in the mid- and posterior trunk and tail. Each somite/myotome in wild-type embryos contains an anterior and posterior domain. This anterior-posterior somite patterning is disrupted in des mutant embryos. Our studies reveal that this patterning defect causes aberrant motor axon outgrowth. Motor axons in wild-types obey domain restrictions, never entering the posterior domain. However, in des mutant embryos, motor axons are seen in both domains. Thus, proper patterning of the somite is essential for stereotyped motor axon pathfinding. The des mutation results in a dramatic increase in the hindbrain interneuron, Mauthner (Mth). This neuron is an integral part of a relatively simple neural circuit driving the escape response in zebrafish and thus presents an excellent opportunity to study properties of neural circuit formation. Due to the presence of supernumerary Mth cells in des mutants; we analyzed the affect of having one cellular component of this circuit dramatically increased on circuit formation and behavior. Our results indicate that all of the supernumerary Mth cells are integrated into the circuit and the circuit is functional. The escape behavior of des mutants is very similar to wild-type embryos. We found, however, that individual Mth cells in des mutants contacted fewer target cells in the spinal cord than Mth cell in wild-type larvae. These data indicate that when there are more Mth cells present, they divide up the territory thus incorporating all cells into the circuit yet maintaining a normal escape response behavior. This study demonstrates that there is plasticity in the formation of the escape response circuit in zebrafish. Proper development of the vertebrate nervous system is essential for the overall function of the organism. The vertebrate nervous system is highly complex and contains an enormous number of distinct cell types. In order for the organism to carry out its normal behavior, it requires that all of the components of the nervous system be produced in appropriate numbers, at correct times, in the right locations and that they make the proper connections. The relative simplicity of the early zebrafish nervous system makes it an attractive model for studying vertebrate nervous system development. Mutagenesis screens in zebrafish have been undertaken to identify new genes involved in different aspects of nervous system development. We have characterized an ENU induced mutation in zebrafish deadly seven/notch 1a (des), which perturbs neurogenesis, somitogenesis, and motor axon outgrowth. The neurogenic defect is manifested as an increase in hindbrain interneurons and spinal motoneurons. In addition, we find a decrease in the number of spinal sensory neurons, and an increase in sensory neurons derived from neural crest cells. This data demonstrates that Notch signaling is important for determining the number of specific neuronal cell types during early nervous system development. The somite defect in des mutants is revealed by abnormalities in somite/myotome boundary formation and somite/myotome gene expression in the mid- and posterior trunk and tail. Each somite/myotome in wild-type embryos contains an anterior and posterior domain. This anterior-posterior somite patterning is disrupted in des mutant embryos. Our studies reveal that this patterning defect causes aberrant motor axon outgrowth. Motor axons in wild-types obey domain restrictions, never entering the posterior domain. However, in des mutant embryos, motor axons are seen in both domains. Thus, proper patterning of the somite is essential for stereotyped motor axon pathfinding. The des mutation results in a dramatic increase in the hindbrain interneuron, Mauthner (Mth). This neuron is an integral part of a relatively simple neural circuit driving the escape response in zebrafish and thus presents an excellent opportunity to study properties of neural circuit formation. Due to the presence of supernumerary Mth cells in des mutants; we analyzed the affect of having one cellular component of this circuit dramatically increased on circuit formation and behavior. Our results indicate that all of the supernumerary Mth cells are integrated into the circuit and the circuit is functional. The escape behavior of des mutants is very similar to wild-type embryos. We found, however, that individual Mth cells in des mutants contacted fewer target cells in the spinal cord than Mth cell in wild-type larvae. These data indicate that when there are more Mth cells present, they divide up the territory thus incorporating all cells into the circuit yet maintaining a normal escape response behavior. This study demonstrates that there is plasticity in the formation of the escape response circuit in zebrafish.
Advisors/Committee Members: Beattie, Christine E.
Keywords: Zebrafish; Neurogenesis; Somitogenesis; Neural Circuit Formation
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23.
Han, Linqu.
Molecular and genetic analysis of a novel f-box protein, seitlupe, in the arabidopsis circadian clock.
Degree: PhD, Molecular, Cellular, and Developmental Biology, 2006, Ohio State University
► The circadian oscillator of Arabidopsis thaliana builds upon interlocked transcriptional feedback loops…
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▼ The circadian oscillator of Arabidopsis thaliana builds upon interlocked transcriptional feedback loops onto which a diverse array of period-affecting factors are incorporated to culminate into a 24-hour periodicity. A subset of the period-affecting factors function in light input pathways which are specifically connected to the clock, thereby modulating circadian period in a light-dependent manner. A novel F-box protein, ZEITLUPE (ZTL) which controls circadian period in a fluence-rate dependent way, falls into this category. The primary focus of this dissertation is to characterize the light-regulated proteolytic pathway mediated by ZTL. At first we identified the SCFZTL complex in planta and established that the formation of SCFZTL complex is important for proper regulation of circadian clock. SCFZTL complex targets an essential oscillator component, TOC1 for its proteasome-dependent degradation. TOC1/PRR1 is the founding member of a small gene family of Pseudoresponse Regulators (PRRs) which consists of additional four genes (PRR3,PRR5,PRR7 and PRR9). The transcriptional regulations among PRR members partially define the interlocked transcriptional feedback loops which are at the core of central oscillator. In this study we characterized the expression pattern of all PRR members and identified two post-translational regulatory events which are shared by four PRR members (PRR1/3/5/7): phosphorylation and proteasome-dependent protein turn over. In addition, we identified PRR5 as a novel interactor of ZTL. In order to further characterize biochemical properties of ZTL and more broadly, to better understand the ZTL-mediated light signal transduction pathway which is specifically coupled to circadian clock, we took both forward and reverse genetic approaches. First, we dissected the roles of three distinctive domains of ZTL in the regulation of circadian period and photomorphogenesis. We found that the C-terminal F/KELCH domain could act together to alter circadian period, when overexpressed, similar to the effect of overexpression of full length ZTL protein. Second, we conducted a suppressor screen for intragenic and extragenic mutations which antagonize the gain-of-function effects mediated by high constitutive expression of ZTL. The suppressor screen identified key residues in ZTL function and suggested new loci in circadian clock function.
Advisors/Committee Members: Somers, David E.
Subjects: Biology, Plant Physiology
Keywords: Circadian clock; ZEITLUPE; F-box protein
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24.
Hao, Linhui.
Genimivirus AL2 And L2 Proteins Interact With And Inactivate SNF1 Kinase.
Degree: PhD, Molecular, Cellular, and Developmental Biology, 2003, Ohio State University
► The AL2 gene of begomoviruses such as Tomato golden mosaic virus (TGMV)…
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▼ The AL2 gene of begomoviruses such as Tomato golden mosaic virus (TGMV) encodes a transcriptional activator protein (TrAP) that is required for efficient expression from the viral coat protein (CP) and BR1 gene promoters. In contrast, L2, the positional homologue of AL2 in the related Beet curly top virus (BCTV, a curtovirus), is not required for CP expression, raising questions about the functional relationship between the AL2 and L2 gene products. AL21-83 and L2 were found to interact with an Arabidopsis SNF1-related kinase (hereafter SNF1) in a yeast two-hybrid screen. SNF1 is a serine/threonine kinase that plays a key role in the regulation of cellular stress responses and carbon metabolism in general. Using partially purified recombinant proteins expressed in E. coli or in insect (Sf9) cells, we have shown that AL2 and L2 are not phosphorylated by SNF1 in vitro. Instead, the consequence of AL2:SNF1 and L2:SNF1 interaction is that kinase activity, including autophosphorylation, is abolished. Since transgenic Nicotiana benthamiana and N. tabacum var. Samsun plants expressing a truncated AL2 gene (AL21-100, lacking the activation domain) or full-length L2 display a novel enhanced susceptibility (ES) phenotype following inoculation with RNA or DNA viruses, we hypothesize that ES conditioned by AL2 and L2 transgenes is due to inhibition of metabolic responses mediated by SNF1. This hypothesis is supported by the observations that transgenic N. benthamiana plants expressing an antisense SNF1 gene show ES, whereas plants that constitutively express a sense SNF1 transgene show enhanced resistance to virus infection. Arabidopsis SNF1 can complement yeast snf1 mutant allowing the cells to grow on media containing carbon sources other than glucose. However, co-expression of L2 with SNF1 in the snf1 mutant yeast blocks complementation, confirming functional L2:SNF1 interaction in vivo. To our knowledge, these studies provide the first evidence that SNF1 is targeted by viral pathogens, and implicate metabolic responses as a part of the plant’s defensive arsenal.
Advisors/Committee Members: Bisaro, David.
Keywords: SNF1; AL2; plants; PROTEINS; KINASE; transgenic plants; AMPK
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25.
Hartman, Matthew George.
The roles of ATF3 in stress-regulated signal transduction and cell death in pancreatic beta-cells.
Degree: PhD, Molecular, Cellular, and Developmental Biology, 2005, Ohio State University
► Activating Transcription Factor 3 (ATF3) is a member of the ATF/CREB family…
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▼ Activating Transcription Factor 3 (ATF3) is a member of the ATF/CREB family of bZip domain-containing transcription factors. Previous studies demonstrated that most cells contain minimal endogenous ATF3 levels; however, ATF3 upregulation was observed in most cell types following treatment with a variety of stress signals. Work presented in this thesis describes the induction, functional consequences of expression, and mechanism of action of ATF3 in pancreatic â-cells. Data presented in chapter 2 demonstrates ATF3 upregulation in â-cell lines by stress signals relevant to type 1 (pro-inflammatory cytokines) and type 2 diabetes (free fatty acids, hyperglycemia, oxidative stress). In addition, elevated ATF3 protein levels were detected in pancreatic islets from a type 1 diabetic mouse model (NOD) and both type 1 and 2 human patient tissues. Investigation of the signal transduction pathways involved in ATF3 induction revealed that both the JNK and NFêB pathways are required for optimal ATF3 expression following IL-1â treatment. Using gain-of-function and loss-of-function approaches, data presented in chapter 3 describes a pro-apoptotic role for ATF3 during the stress response. First, ectopic ATF3 expression targeted to pancreatic â-cells during development resulted in mice displaying decreased islet area, high blood glucose levels, decreased serum insulin levels, and markers indicative of â-cell failure (elevated ketone bodies). Second, primary islets purified from ATF3 knock out mice were partially protected from cytokine-induced apoptosis. Chapter 4 investigates the potential cross-talk and functional interaction between ATF3 and Akt in â-cells. Results reveal that inhibition of ATF3 expression by activated Akt represents a novel mechanism whereby Akt promotes â-cell survival. In support of this notion, expression of ATF3 almost completely reversed the anti-diabetic phenotype of the constitutively-active Akt transgenic mice. Insights into the mechanism of ATF3-induced cell death suggest that ATF3 may promote apoptosis by triggering activation of the intrinsic (mitochondrial) death pathway.
Advisors/Committee Members: Hai, Tsonwin.
Subjects: Biology, Molecular
Keywords: ATF3; diabetes; beta-cell death
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26.
Hartt, Gregory Thomas.
Regulation of the human neuronal nitric oxide synthase gene via alternate promoters.
Degree: PhD, Molecular, Cellular, and Developmental Biology, 2003, Ohio State University
► Nitric oxide is a gaseous, free radical molecule that functions in the…
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▼ Nitric oxide is a gaseous, free radical molecule that functions in the nervous system as an atypical neurotransmitter, second messenger, vasodilator, and potent neurotoxin, depending on cell type and level of expression. Physiologically, nitric oxide is thought to mediate long-term potentiation, the cellular correlate of learning and memory formation, as well as synaptic plasticity and remodeling. Under pathologic conditions such as cerebral ischemia, NOS1 overproduces nitric oxide, which is a key mediator of excitotoxic cell death and neurodegeneration. Our laboratory discovered that multiple, individually functioning promoters (5’1 and 5’2) regulate human NOS1 gene expression. The present work describes the discovery and cloning of a novel human NOS1 promoter, 5’3, and the demonstration of its unique, developmentally regulated expression pattern in the central nervous system. Promoter-specific alternative splicing contributes to NOS1 mRNA diversity in both the 5’ untranslated and coding regions of the gene. We generated several lines of transgenic mice that express reporter genes under the control of separate human NOS1 promoter complexes (PR(5'1+5'2) and PR(5'3+5'4)), and studied how these alternate promoters contribute to NOS1 expression in various physiologic and pathophysiologic states in vivo. To this end, transgene expression is documented throughout normal development, which demonstrates overlapping but distinct patterns of promoter use that contribute to the overall recapitulation of endogenous NOS1 gene expression. In a murine model of striatal neurotoxicity, systemic methamphetamine administration results in promoter-specific transcriptional activation inthe transgenic mice. NOS1 enzymatic activity is stimulated in motor neuron cell bodies after peripheral transection of the facial nerve; however, the transgenic NOS1 promoter complexes are insufficient to recapitulate NOS1 activation in this model. These findings help clarify how multiple promoters and mRN A diversity contribute to the complex regulation of the human NOS1 gene, and further our general understanding of transcriptional regulation of gene expression via alternate promoters. Nitric oxide is a gaseous, free radical molecule that functions in the nervous system as an atypical neurotransmitter, second messenger, vasodilator, and potent neurotoxin, depending on cell type and level of expression. Physiologically, nitric oxide is thought to mediate long-term potentiation, the cellular correlate of learning and memory formation, as well as synaptic plasticity and remodeling. Under pathologic conditions such as cerebral ischemia, NOS1 overproduces nitric oxide, which is a key mediator of excitotoxic cell death and neurodegeneration. Our laboratory discovered that multiple, individually functioning promoters (5’1 and 5’2) regulate human NOS1 gene expression. The present work describes the discovery and cloning of a novel human NOS1 promoter, 5’3, and the demonstration of its unique, developmentally regulated expression pattern in the central nervous system. Promoter-specific alternative splicing contributes to NOS1 mRNA diversity in both the 5’ untranslated and coding regions of the gene. We generated several lines of transgenic mice that express reporter genes under the control of separate human NOS1 promoter complexes (PR(5'1+5'2) and PR(5'3+5'4)), and studied how these alternate promoters contribute to NOS1 expression in various physiologic and pathophysiologic states in vivo. To this end, transgene expression is documented throughout normal development, which demonstrates overlapping but distinct patterns of promoter use that contribute to the overall recapitulation of endogenous NOS1 gene expression. In a murine model of striatal neurotoxicity, systemic methamphetamine administration results in promoter-specific transcriptional activation inthe transgenic mice. NOS1 enzymatic activity is stimulated in motor neuron cell bodies after peripheral transection of the facial nerve; however, the transgenic NOS1 promoter complexes are insufficient to recapitulate NOS1 activation in this model. These findings help clarify how multiple promoters and mRN A diversity contribute to the complex regulation of the human NOS1 gene, and further our general understanding of transcriptional regulation of gene expression via alternate promoters.
Advisors/Committee Members: Young, Anthony P.
Keywords: NOS1; human; neuronal nitric oxide synthase; transcriptional regulation; alternate promoters; alternate splicing; transgenic; methamphetamine; facial nerve axotomy
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27.
Hull, Stacey Lynn.
Identification and Characterization of New and Distinct Functional Roles of Posttranscriptional Control Elements in Cytoplasmic Expression of Retroviral RNA.
Degree: PhD, Molecular, Cellular, and Developmental Biology, 2002, Ohio State University
► The central focus of this dissertation is the identification and characterization of…
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▼ The central focus of this dissertation is the identification and characterization of retroviral posttranscriptional control elements that affect protein production from unspliced viral RNA. We identify and characterize a new posttranscriptional control element in the Mason-Pfizer monkey virus 5’ long terminal repeat (LTR) that modulates translational efficiency by augmentation of translational initiation. MPMV RU5 is necessary for cytoplasmic expression of HIV-1 gag-pol reporter RNA and also enhances cytoplasmic expression of intronless luc RNA by stimulation of ribosome loading. MPMV RU5 functions independently of any viral proteins and instead directs functional interaction with cellular posttranscriptional modulators to facilitate translational enhancement. This research has illuminated an essential step in viral gene expression and provides a new paradigm for understanding cellular control of the translation process. Secondly, we tested the hypothesis that combination of the MPMV constitutive transport element (CTE) and the MPMV or spleen necrosis virus (SNV) RU5 translational enhancer on a single RNA synergistically augments posttranscriptional gene expression. MPMV CTE functions compatibly with MPMV and SNV RU5 to increase cytoplasmic expression of HIV-1 gag-pol reporter RNA in monkey COS, but not 293 cells. The CTE-interactive cellular proteins, Tap and NXT1, are necessary and sufficient to rescue increased cytoplasmic expression of HIV-1 gag-pol reporter RNA in 293 cells. This work produced the realization that differences in cellular posttranscriptional modulators dramatically affect retroviral protein production. Thirdly, we evaluated the role of SNV RU5 on metabolism of homologous SNV RNA. SNV RU5 increases SNV Gag-GFP fusion protein production from unspliced genomic RNA. The increase in protein is attributable, at least in part, to increased cytoplasmic accumulation of the unspliced SNV transcript. RU5 exerts a distinct effect on the spliced env transcript. Deletion of RU5 has no effect on cytoplasmic accumulation of env RNA, but increases splicing efficiency. Therefore, SNV RU5 modulates metabolism of both unspliced and spliced SNV transcripts and is speculated to contain a RNA splicing suppressor. In summary, this dissertation has identified and characterized a new posttranscriptional control element in MPMV and synergistic interactions among functionally distinct retroviral posttranscriptional control elements and their cellular protein partners. This work also demonstrated an important role for SNV RU5 in SNV genomic RNA.
Advisors/Committee Members: Boris-Lawrie, Kathleen.
Subjects: Biology, Molecular
Keywords: SNV, MPMV, posttranscriptional control elements, cytoplasmic expression
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28.
Hu, Rong.
Regulation of osteoclast differentiation by transcription factors MITF, PU.1 and EOS.
Degree: PhD, Molecular, Cellular, and Developmental Biology, 2007, Ohio State University
► The microphthalmia-associated transcription factor (MITF), a basic helix-loop-helix leucine zipper (bHLH-Zip) transcription…
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▼ The microphthalmia-associated transcription factor (MITF), a basic helix-loop-helix leucine zipper (bHLH-Zip) transcription factor, regulates distinct target genes in several cell types including osteoclasts. Osteoclasts are terminally differentiated multinuclear cells responsible for bone resorption. CSF-1 and RANKL are two critical cytokines to induce osteoclast differentiation from bone morrow-derived precursors. In osteoclasts, MITF interacts with the Ets family transcription factor PU.1 to synergistically activate target genes like Cathepsin K (Ctsk) and Acid Phosphatase 5 (Acp5). The region of MITF required for the physical interaction with PU.1 is the bHLH-Zip domain. To finer map the interacting domain of MITF, we introduced point mutations into the loop region of MITF via in vitro site-directed mutagenesis. The properties of mutated MITF proteins were analyzed by transient transfection assays, EMSAs, and in vitro GST pulldown assays. We identified that specific amino acids including N235, D236, and W241 in the loop region of MITF are critical to mediate physical and functional interaction with PU.1. Both MITF and PU.1 are known transcriptional activators in osteoclasts. Results presented here demonstrate that they can also act as components of repressor complexes that suppress target gene expression in committed myeloid precursors. The direct interaction of MITF and PU.1 with the zinc-finger protein Eos, an Ikaros family member, appears to be necessary for repression of Ctsk and Acp5. In bone marrow-derived precursors treated with CSF-1 alone, Eos formed a complex with MITF and PU.1 at target gene promoters and suppressed transcription through recruitment of co-repressors CtBP and Sin3A. Eos expression was reduced during osteoclast differentiation initiated by combined CSF-1 and RANKL stimulation and Eos association with Ctsk and Acp5 promoters was significantly decreased. Subsequently, MITF and PU.1 recruited co-activators to these target promoters resulting in robust expression of target genes. Overexpression of Eos in bone marrow-derived precursors inhibited multinuclear osteoclast formation and repressed transcription of subset of osteoclast specific genes that are regulated by MITF and PU.1. This work provides a novel mechanism to account for the modulation of MITF and PU.1 activity in committed myeloid progenitors prior to the initiation of osteoclast differentiation in response to the appropriate extracellular signals.
Advisors/Committee Members: Ostrowski, Michael C.
Subjects: Biology, Molecular
Keywords: Cell differentiation, osteoclasts, transcriptional regulation
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29.
Ignatius, Myron Steve.
Zebrafish Hdac1 Is Reiteratively And Differentially Required During Neural Crest Cell Development And Hdac1 Is A Positive Regulator Of The Non Canonical Wnt Signaling Pathway.
Degree: PhD, Molecular, Cellular, and Developmental Biology, 2008, Ohio State University
► The neural crest is a transient embryonic cell population that contributes to…
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▼ The neural crest is a transient embryonic cell population that contributes to multiple cell types in the vertebrate embryo including chromatophores, craniofacial cartilages, neurons and glia of the peripheral nervous system. Neural crest cell (NCC) development has been used extensively to explore mechanisms of cell fate specification, differentiation and migration, which more broadly illuminate mechanisms in the development of multicellular organisms. Through a combination of forward and reverse genetic approaches using mutants, morpholinos and a chemical inhibitor, I identify in vivo requirements of hdac1 (histone deacetylase1) in NCC development. The zebrafish mutant colgate (col)/hdac1 exhibits defective NCC-derived melanophore, peripheral neuron and craniofacial development. Additionally, hdac1col mutants also display defects in the extension of the body axis and the migration of branchiomotor neurons. I demonstrate that hdac1 is specifically required for melanophore and branchial arch specification. In melanophores, I define a likely mechanism regulating specification. Characterization of the hdac1col mutant craniofacial defects, suggests that hdac1 is also required for proper terminal differentiation and migration of manidubular and hyoid arch cartilages. Similarly, hdac1 is required for the differentiation of PNS-derived dorsal root ganglion (DRG), enteric and sympathetic neurons. Specifically, in sympathetic neurons, hdac1 is required for acquisition of neurotransmitter characteristics. The HDAC inhibitor trichostatinA (TSA) inhibits multiple HDACs including Hdac1. Interestingly, TSA phenocopies multiple aspects of hdac1col development, suggesting that hdac1 is an important HDAC in embryogenesis. Using TSA, I define temporal requirements of HDAC/hdac1 function during craniofacial and sympathetic neuron differentiation. In hdac1col/tfap2a mutant/morphant embryos, there are severe reductions in melanophore and craniofacial derivatives at all stages analyzed, when compared to single mutants, suggesting a new additive or synergistic genetic interaction between hdac1 and tfap2a that is required for NCC-derived melanophore and craniofacial development. Finally, activation of the non-canonical Wnt/PCP pathway that is required for axis extension, in hdac1col mutant embryos, rescues axis extension defects. This suggests that hdac1 acts as a positive regulator of the non-canonical Wnt/PCP pathway. Additionally, hdac1 normally regulates the caudal migration of facial hindbrain branchiomotor neurons, independently of the non-canonical Wnt/PCP pathway.
Advisors/Committee Members: Henion, Paul D.
Subjects: Molecular biology
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30.
Iliopoulos, Dmitrio.
The role of the WWOX tumor suppressor in breast and lung cancer.
Degree: PhD, Molecular, Cellular, and Developmental Biology, 2006, Ohio State University
► The WWOX gene spans a genomic region of more than half million…
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▼ The WWOX gene spans a genomic region of more than half million nucleotide base pairs located at 16q23.3-24.1, a chromosome region commonly involved in LOH in many different types of cancer. Wwox is frequently down-regulated in breast and lung cancers due to DNA hypermethylation in its promoter region. We observed differential patterns of WWOX methylation in neoplastic vs. adjacent non neoplastic tissues, suggesting that targeted WWOX methylation specific amplification could be useful in following treatment or prevention protocols, and WWOX methylation analyses could enrich a panel of DNA methylation markers. Restoration of Wwox expression in Wwox-negative breast and lung cancer-derived cells suppressed tumor growth in vitro and in vivo and induced apoptosis, confirming that WWOX is a tumor suppressor gene that is highly effective in gene therapy of breast and lung cancer xenografts, whether transduced by adenovirus or re-expressed through epigenetic therapy. The preclinical studies that we have performed showed the therapeutic potential of restoration of tumor suppressor expression through epigenetic modulation and the promise of reexpressed tumor suppressors such as WWOX as markers and effectors of the responses. Although many patients benefit from tamoxifen treatment, half of breast tumors that recur after therapy are resistant to tamoxifen. Understanding mechanisms of tamoxifen resistance could lead to characterization of protein markers for identification of nonresponsive cancers, as well as tumors that are acquiring resistance, before emergence of more aggressive cancer cells. We have found that Wwox mediates the tamoxifen response through regulation of protein kinase A and ErbB2 signaling pathways and high Wwox expression level predicted tamoxifen sensitivity in a cohort of breast cancer cases. The results imply that epigenetic reactivation of Wwox could sensitize tamoxifen resistant cells, suggesting possible epigenetic and hormonal combination therapy in patients with acquired tamoxifen resistance.
Advisors/Committee Members: HUEBNER, KAY.
Subjects: Biology, Genetics
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