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Transcriptional repression by CTIP2, a C₂H₂ zinc finger protein /Topark-Ngarm, Acharawan Khamsiritrakul. January 1900 (has links)
Thesis (Ph. D.)--Oregon State University, 2007. / Printout. Includes bibliographical references. Also available on the World Wide Web.
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DNA binding and beyond : an investigation of proteins involved in PAH-induced carcinogesesisHooven, Louisa Ada 15 December 2003 (has links)
Exposure to polycyclic aromatic hydrocarbons such as benzo[a]pyrene (B[a]P) has
been determined to be a risk factor for various forms of human cancer. PAH DNA
adducts have been shown to cause mutations, but carcinogenesis is also
accompanied by alterations in gene expression. Inhibiting individual cytochrome
P450s could clarify the interaction of P450s and other enzymes in the activation of
polycyclic aromatic hydrocarbons to DNA binding intermediates.
Phosphorodiamidate morpholino oligomers (PMOs), a class of antisense agents
were targeted against cytochrome P450 1A1 (CYP1A1) and cytochrome P450 1B1
(CYP1BI). No significant inhibition of enzyme activity or expression was
observed with any PMO used as measured by ethoxyresorufin-O-deethylase
(EROD) activity and immunoblots. It was demonstrated that BPDE may react with
PMOs in vitro, and PMOs may be segregated in lysosomes, blocking their efficacy.
Nonspecific effects by the PMO on CYP1A1 activity were observed. These
observations indicate multiple confounding effects in the use of PMOs for this
purpose. Many of the genes regulated by histone deacetylases are involved in
proliferation, cell function, and differentiation, and HDAC inhibitors are of great
interest in cancer research. To probe epigenetic regulation of CYP1A1, MCF-7
cells were treated with two HDAC inhibitors, suberoylanilide hydroxamic acid
(SAHA) and trichostatin A (TSA). SARA and TSA increased EROD activity and
in RT-PCR. SARA and TSA reduced B[a]P induced CYP1A1 and CYP1B1
mRNA levels. B[a]P DNA binding was not significantly altered by SAHA or TSA
treatment. To assay global protein expression changes after treatment with PAR,
MCF-7 cells were treated with B[a]P, DB{a,1]P, coal tar extract (SRM 1597) and
diesel exhaust extract (SRM 1975), Proteins were separated by two-dimensional
electrophoresis, and analyzed using PDQuest. Spots of interest were excised and
identified by matrix assisted laser desorption/ionization time of flight time of flight
mass spectroscopy. Alterations in expression of heat shock proteins, cytoskeletal
proteins, DNA associated proteins, and glycolytic and mitochondrial proteins were
observed. Universally increased expression was observed for tubulin alpha and
myosin light chain alkali, cyclophilin B, heterogeneous nuclear riboprotein B1, and
alpha enolase. Additional proteins exhibiting change in expression included histone
H2A.1, heat shock protein 70-2, galectin-3, nucleoside diphosphate kinase, ATP
synthase, and electron transfer flavoprotein. / Graduation date: 2004
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Structural and biochemical characterizations of the CHD1 tandem chromodomains /Flanagan, John Francis. January 2006 (has links)
Thesis (Ph. D.)--University of Virginia, 2006. / Includes bibliographical references. Also available online through Digital Dissertations.
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Regulation of heat shock factor 1 (HSF1) DNA-binding and transcriptionMercier, Philippe Arthur 17 September 2003
Cellular stress invokes a protective response in which heat shock factor 1 (HSF1) is activated to increase heat shock protein (Hsp) expression. HSF1 exists as a latent monomer in unstressed cells. Upon stress HSF1 forms homotrimers, increasing its affinity for the heat shock DNA element upstream of all Hsp genes. A second conformational change is required for HSF1 to gain transcriptional competence. During prolonged heat shock or following the resumption of normal conditions HSF1 DNA-binding and transcriptional activities are reduced and HSF1 returns to the monomeric state in a process called attenuation. During the activation/deactivation cycle HSF1 is modified by small ubiquitin-related modifier (SUMO-1) conjugation and undergoes several phosphorylation and dephosphorylation events that modulate HSF1 activity. Hyperphosphorylation of HSF1 is hypothesized to trigger HSF1 transcriptional activity. HSF1 also interacts with a dynamic series of Hsp90/Hsp70-based chaperone heterocomplexes that negatively regulate DNA-binding, and transcriptional activity, and promote attenuation. This thesis was aimed at characterizing the mechanisms regulating HSF1 DNA-binding, and transcriptional activity. Expression of human HSF1 in Xenopus oocytes altered the set-point of DNA-binding in response to heat indicating that both the cellular environment and innate properties of the molecule allow HSF1 to set its activation/deactivation set-point in response to stress in vivo. HSF1 DNA-binding but not transcription was activated in oocytes treated with a high temperature heat shock. Further characterization of this observation determined that HSF1 activated by a brief high temperature heat shock inhibited transcriptionally competent HSF1 from activating transcription. It was hypothesized that this phenomenon exists to ensure the eventual death of the cell due to the accumulation of excessive damage and potential mutation caused by severe stress. The most significant observation made in this thesis is that Hsp expression was detected in oocytes injected with reporter plasmid only during recovery from a high temperature heat shock. These results led to the proposal of a model in which HSF1 trimers are either assembled in a transcriptionally incompetent form or one that has the potential to become transcriptionally competent during stress, prior to DNA-binding. The identity of HSF1-binding proteins that interact with HSF1 at different stages of activation/deactivation was characterized in an effort to assign regulatory roles to these proteins. HSF1 was detected in a high molecular weight complex (350-600 kDa) during all phases of the activation/deactivation cycle. HSF1 at different stages of activation was tested for interaction with specific molecular chaperones by electrophoretic mobility supershift analysis. Hsp90, p23, FKBP52, Hip and Hop are all associated with transcriptionally active and inactive HSF1 suggesting that interaction of HSF1 with any of these molecules does not activate HSF1 transcriptional activity. These results do not exclude the possibility that the function of these molecular chaperones may change during activation of HSF1 transcription or that post-translational modifications may be the primary mechanism that drives HSF1 from a transcriptionally inactive to active form.
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The role of protein phosphatase 5 (PP5) in the regulation of heat shock factor 1 (HSF1) in <i>xenopus laevis</i> oocytesMcLoughlin, Christine Louise 22 October 2003
Cells are continuously exposed to a variety of physiological and environmental stresses that can lead to protein aggregation and/or denaturation, and eventually cell death. In order to ensure survival, cells have evolved a stress response that monitors, detects, and responds to changes within the cellular environment. The stress response is characterized by the up-regulation of heat shock protein (hsp) genes whose products can mediate the assembly and/or degradation of misfolded or aggregated proteins within the cell. This stress-induced upregulation of heat shock protein encoding genes is under the regulation of heat shock transcription factor 1 (HSF1) and its associated proteins that together form what is known as the HSF1 heterocomplex. In eukaryotic cells, HSF1 exists as a non-DNA binding monomer in the absence of stress. Upon exposure to stress, HSF1 undergoes trimerization and acquires the ability to bind heat shock elements (HSEs) located upstream of all hsp genes and after further modification, can become converted into a transcriptionally active form. Following prolonged stress or after removal of stress, HSF1 loses its ability to bind DNA and transcription ceases in a process termed attenuation. <p>Several studies have suggested that the DNA-binding and transcriptional activities of HSF1 are regulated by phosphorylation and dephosphorylation events and by chaperone-based folding mechanisms similar to those involved in the regulation of glucocorticoid receptors. Protein phosphatase 5 (PP5) has been identified as a member of the glucocorticoid receptor chaperone complex and its phosphatase activity has been shown to regulate the maturation and activation of the receptor. It has been suggested that PP5 may regulate HSF1 in a manner similar to that of glucocorticoid receptors however it has not yet been determined how PP5 interacts with the HSF1 heterocomplex or if PP5 functions to regulate HSF1-DNA binding and/or HSF1 transactivation.<p>Utilizing the Xenopus model system, I tested the hypothesis that PP5 regulates the DNA binding and transcriptional activities of HSF1 through interactions with the HSF1 heterocomplex. Increasing the activity of PP5, either through the elevation of PP5 protein levels or by activating endogenous PP5, resulted in decreased HSF1-DNA binding as well as accelerated attenuation after the removal of stress. Conversely, inhibiting the phosphatase activity of PP5 using okadaic acid or by immunotargetting, where an antibody recognizing PP5 was microinjected into the nuclei of oocytes, resulted in delayed HSF1 attenuation. Transcription assays performed using activated PP5 also demonstrated that PP5 acts to decrease HSF1-mediated transcription. Immunoprecipitation and gel mobility supershift assays were also used to show that PP5 interacts with the HSF1 heterocomplex and PP5-HSP90 binding mutants illustrated that PP5 may exert its repressive effects independently of binding directly to HSP90.
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Regulation of heat shock factor 1 (HSF1) DNA-binding and transcriptionMercier, Philippe Arthur 17 September 2003 (has links)
Cellular stress invokes a protective response in which heat shock factor 1 (HSF1) is activated to increase heat shock protein (Hsp) expression. HSF1 exists as a latent monomer in unstressed cells. Upon stress HSF1 forms homotrimers, increasing its affinity for the heat shock DNA element upstream of all Hsp genes. A second conformational change is required for HSF1 to gain transcriptional competence. During prolonged heat shock or following the resumption of normal conditions HSF1 DNA-binding and transcriptional activities are reduced and HSF1 returns to the monomeric state in a process called attenuation. During the activation/deactivation cycle HSF1 is modified by small ubiquitin-related modifier (SUMO-1) conjugation and undergoes several phosphorylation and dephosphorylation events that modulate HSF1 activity. Hyperphosphorylation of HSF1 is hypothesized to trigger HSF1 transcriptional activity. HSF1 also interacts with a dynamic series of Hsp90/Hsp70-based chaperone heterocomplexes that negatively regulate DNA-binding, and transcriptional activity, and promote attenuation. This thesis was aimed at characterizing the mechanisms regulating HSF1 DNA-binding, and transcriptional activity. Expression of human HSF1 in Xenopus oocytes altered the set-point of DNA-binding in response to heat indicating that both the cellular environment and innate properties of the molecule allow HSF1 to set its activation/deactivation set-point in response to stress in vivo. HSF1 DNA-binding but not transcription was activated in oocytes treated with a high temperature heat shock. Further characterization of this observation determined that HSF1 activated by a brief high temperature heat shock inhibited transcriptionally competent HSF1 from activating transcription. It was hypothesized that this phenomenon exists to ensure the eventual death of the cell due to the accumulation of excessive damage and potential mutation caused by severe stress. The most significant observation made in this thesis is that Hsp expression was detected in oocytes injected with reporter plasmid only during recovery from a high temperature heat shock. These results led to the proposal of a model in which HSF1 trimers are either assembled in a transcriptionally incompetent form or one that has the potential to become transcriptionally competent during stress, prior to DNA-binding. The identity of HSF1-binding proteins that interact with HSF1 at different stages of activation/deactivation was characterized in an effort to assign regulatory roles to these proteins. HSF1 was detected in a high molecular weight complex (350-600 kDa) during all phases of the activation/deactivation cycle. HSF1 at different stages of activation was tested for interaction with specific molecular chaperones by electrophoretic mobility supershift analysis. Hsp90, p23, FKBP52, Hip and Hop are all associated with transcriptionally active and inactive HSF1 suggesting that interaction of HSF1 with any of these molecules does not activate HSF1 transcriptional activity. These results do not exclude the possibility that the function of these molecular chaperones may change during activation of HSF1 transcription or that post-translational modifications may be the primary mechanism that drives HSF1 from a transcriptionally inactive to active form.
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The role of protein phosphatase 5 (PP5) in the regulation of heat shock factor 1 (HSF1) in <i>xenopus laevis</i> oocytesMcLoughlin, Christine Louise 22 October 2003 (has links)
Cells are continuously exposed to a variety of physiological and environmental stresses that can lead to protein aggregation and/or denaturation, and eventually cell death. In order to ensure survival, cells have evolved a stress response that monitors, detects, and responds to changes within the cellular environment. The stress response is characterized by the up-regulation of heat shock protein (hsp) genes whose products can mediate the assembly and/or degradation of misfolded or aggregated proteins within the cell. This stress-induced upregulation of heat shock protein encoding genes is under the regulation of heat shock transcription factor 1 (HSF1) and its associated proteins that together form what is known as the HSF1 heterocomplex. In eukaryotic cells, HSF1 exists as a non-DNA binding monomer in the absence of stress. Upon exposure to stress, HSF1 undergoes trimerization and acquires the ability to bind heat shock elements (HSEs) located upstream of all hsp genes and after further modification, can become converted into a transcriptionally active form. Following prolonged stress or after removal of stress, HSF1 loses its ability to bind DNA and transcription ceases in a process termed attenuation. <p>Several studies have suggested that the DNA-binding and transcriptional activities of HSF1 are regulated by phosphorylation and dephosphorylation events and by chaperone-based folding mechanisms similar to those involved in the regulation of glucocorticoid receptors. Protein phosphatase 5 (PP5) has been identified as a member of the glucocorticoid receptor chaperone complex and its phosphatase activity has been shown to regulate the maturation and activation of the receptor. It has been suggested that PP5 may regulate HSF1 in a manner similar to that of glucocorticoid receptors however it has not yet been determined how PP5 interacts with the HSF1 heterocomplex or if PP5 functions to regulate HSF1-DNA binding and/or HSF1 transactivation.<p>Utilizing the Xenopus model system, I tested the hypothesis that PP5 regulates the DNA binding and transcriptional activities of HSF1 through interactions with the HSF1 heterocomplex. Increasing the activity of PP5, either through the elevation of PP5 protein levels or by activating endogenous PP5, resulted in decreased HSF1-DNA binding as well as accelerated attenuation after the removal of stress. Conversely, inhibiting the phosphatase activity of PP5 using okadaic acid or by immunotargetting, where an antibody recognizing PP5 was microinjected into the nuclei of oocytes, resulted in delayed HSF1 attenuation. Transcription assays performed using activated PP5 also demonstrated that PP5 acts to decrease HSF1-mediated transcription. Immunoprecipitation and gel mobility supershift assays were also used to show that PP5 interacts with the HSF1 heterocomplex and PP5-HSP90 binding mutants illustrated that PP5 may exert its repressive effects independently of binding directly to HSP90.
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Molecular mechanisms in IL-10 production by macrophages during phagocytosis of apoptotic cells /Chung, Elaine Yee-Lin January 2007 (has links)
Thesis (Ph. D.)--Cornell University, January, 2007. / Vita. Includes bibliographical references (leaves 204-240).
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Analysis of the interactions between the 5' to 3' exonuclease and the single-stranded DNA-binding protein from bacteriophage T4 and related phages /Boutemy, Laurence S. January 2008 (has links)
Thesis (Ph. D.)--University of Toledo, 2008. / Typescript. "Submitted as partial fulfillment of the requirements for the Doctor of Philosophy in Chemistry." Includes bibliographical references (leaves 305-309).
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Mechanisms of Hairy-mediated transcriptional repression during Drosophila development /Phippen, Taryn Marie. January 2001 (has links)
Thesis (Ph. D.)--University of Washington, 2001. / Vita. Includes bibliographical references (leaves 91-109).
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