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1
Errington, Fiona. „An investigation into the cytotoxic mechanisms of DNA topoisomerase II poisons and catalytic inhibitors : the role of DNA topoisomerase II alpha and beta“. Thesis, University of Newcastle Upon Tyne, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.340718.
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2
Padget, Kay. „Quantitative analysis and drug sensitivity of human DNA topoisomerase II alpha and beta“. Thesis, University of Newcastle Upon Tyne, 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.246093.
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3
McNamara, Suzan. „Topoisomerase II beta negatively modulates retinoic acid receptor alpha function : a novel mechanism of retinoic acid resistance in acute promyelocytic leukemia“. Thesis, McGill University, 2008. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=115693.
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Interactions between the retinoic acid receptor alpha (RARalpha) and coregulators play a key role in coordinating gene transcription and myeloid differentiation. In acute promyelocytic leukemia (APL), RARalpha is fused with the promyelocytic leukemia (PML) gene, resulting in the expression of the fusion protein PML/RARalpha. Here, I report that topoisomerase II beta (topoIIbeta) associates with and negatively modulates PML/RARalpha and RARalpha transcriptional activity, and increased levels and association of topoIIbeta cause resistance to retinoic acid (RA) in APL cell lines. Knock down of topoIIbeta was able to overcome resistance by permitting RA-induced differentiation and increased RA-gene expression. Overexpression of topoIIbeta, in clones from an RA-sensitive cell line, conferred resistance by a reduction in RA-induced expression of target genes and differentiation. Chromatin immunoprecipitation assays indicate that topoIIbeta is bound to an RA-response element, and inhibition of topoIIbeta causes hyper-acetylation of histone 3 at lysine 9 and activation of transcription. These results identify a novel mechanism of resistance in APL and provide further insights to the role of topoIIbeta in gene regulation and differentiation.Studies to determine the mechanism by which topoIIbeta protein is regulated found that levels of protein kinase C delta (PKCdelta) correlated with topoIIbeta protein expression. Moreover, activation of PKCdelta, by RA or PMA, led to an increase of topoIIbeta protein levels. Most notably, in NB4-MR2 cells, we observed increased phosphorylation levels of threonine 505 on PKCdelta, a marker of activation. Inhibition of PKCdelta was able to overcome the topoIIbeta repressive effects on RA-target genes. In addition, the combination of RA and PKCdelta inhibition led to increased expression of the granulocytic marker, CD11c, in NB4 and NB4-MR2 cells. These results suggest that PKCdelta regulates topoIIbeta expression, and a constitutively active PKCdelta in the NB4-MR2 cell line leads to overexpression of topoIIbeta.
In conclusion, these studies demonstrate that topoIIbeta associates with RARalpha, binds to RAREs and plays a critical role in RA dependent transcriptional regulation and granulocytic differentiation. In addition, I show that topoIIbeta overexpression leads to RA resistance and provide evidence that topoIIbeta protein levels are regulated via a mechanism involving the PKCdelta pathway. This work has contributed to an enhanced understanding of the role of topoIIbeta in gene regulation and brings novel perspectives in the treatment of RA-resistance in APL.
4
Wells, Nicholas James. „Phosphorylation of human topoisomerase II“. Thesis, University of Oxford, 1996. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.318833.
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5
Hochhauser, Daniel. „Transcriptional regulation of topoisomerase II“. Thesis, University of Oxford, 1993. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.333178.
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6
Lee, Ka Cheong. „Molecular pharmacology of DNA topoisomerase II drugs“. Thesis, University of Newcastle upon Tyne, 2016. http://hdl.handle.net/10443/3780.
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Topoisomerase II (TOP2) is an important anti-cancer drug target. This study demonstrates that proteasomal inhibition by MG132 or PS341 potentiates the effect of TOP2 poisons on cell growth inhibition. Mitoxantrone was potentiated the most. The presence of the proteasome inhibitor MG132 prolonged the half-life of drug-induced DNA-TOP2 complexes stabilised by mitoxantrone or etoposide. Genotoxicity was measured in K562 cells using in vitro micronucleus assays for combinations of a proteasome inhibitor (MG132 or PS341) and mitoxantrone and for each agent alone. Combinations that potentiated the cytotoxicity reduced the genotoxicity. This suggests that combining a proteasome inhibitor with a TOP2 drug has the potential to reduce late toxicities such as therapy related leukaemia. The genotoxicity of six TOP2 poisons was determined by high throughput in vitro micronucleus assays in three Nalm-6 cell lines with differing TOP2 levels. Lower genotoxicity was observed in TOP2B knock-out and TOP2A knock-down cells, suggesting both TOP2A and TOP2B have a role in genotoxicity triggered by TOP2 poisons.
7
Fry, Andrew Mark. „Phosphorylation of the human topoisomerase II protein“. Thesis, University of Oxford, 1992. http://ora.ox.ac.uk/objects/uuid:70d2dbb9-d3fe-43ed-8206-44a95202eeff.
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DNA topoisomerase II is an essential enzyme in eukaryotes and is required for many aspects of DNA metabolism including DNA replication, recombination, chromosome segregation and chromosome condensation. It is also a major component of the nuclear scaffold. Topoisomerase II from lower eukaryotes has been shown to be phosphorylated in vivo and this phosphorylation leads to a modulation of activity. However, unlike these lower eukaryotes, human topoisomerase II exists as two closely related, but genetically distinct, isozymes which have markedly different expression and localization patterns. Topoisomerase IIα is a 170kDa protein and topoisomerase IIβ is 180kDa. This study set out to analyse the phosphorylation of these specific isozymes and understand how this leads to the regulation of their distinct biological functions. In order to undertake this study, two polyclonal anti-topoisomerase II antibodies were generated and a series of other polyclonal and monoclonal antibodies characterized. Furthermore, the α isozyme of human topoisomerase II was purified to near homogeneity from cultured HeLa cells. A kinase activity with the biochemical characteristics of casein kinase II co-purified with and could phosphorylate the purified topoisomerase Hot protein. The α and β isozymes of human topoisomerase II were both shown to be phosphoproteins in vivo. The α isozyme is phosphorylated predominantly on serine residues but with a minor proportion of phosphothreonine. Both the α isozyme and a stable ISOkDa fragment of the β isozyme are phosphorylated in vitro by casein kinase II and the catalytic subunit of PKA (cAMP-dependent protein kinase). The α isozyme can also be phosphorylated in vitro by Ca2+-dependent and -independent isozymes of protein kinase C and the cell cycle-regulated p34cdc2 kinase. Two-dimensional tryptic phosphopeptide mapping suggested that the pattern of phosphorylation of human topoisomerase Ha protein in vivo is complex with phosphorylation occurring on multiple residues. Comparison with in vitro maps suggested that casein kinase II and PKA could account for most of the phosphorylation seen in vivo. Using a one- dimensional phosphopeptide mapping approach, a major site of phosphorylation in vivo appeared to be within the C-terminal 20kDa, and that casein kinase II, PKA and PKC may all phosphorylate this region. Phosphorylation of human topoisomerase Hoc protein by casein kinase II, PKA and PKC all led to a stimulation of activity as measured by plasmid relaxation and decatenation. In contrast, dephosphorylation led to a marked decrease in activity of the enzyme. The dephosphorylated enzyme could be reactivated by casein kinase II but not PKA phosphorylation. These data suggest that phosphorylation plays a crucial role in the control of DNA tertiary structure in human cells via regulation of the activity of topoisomerase II proteins.
8
Atwal, Mandeep. „Myeloperoxidase enhances DNA damage induced by drugs targeting DNA topoisomerase II“. Thesis, University of Newcastle upon Tyne, 2017. http://hdl.handle.net/10443/3956.
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Topoisomerase II (TOP2) poisons are effective anti-cancer agents used to treat a wide range of neoplasms. However, TOP2 poisons are subjected to enzymatic conversion which leads to metabolites with altered DNA damaging properties. Myeloperoxidase, present exclusively within developing myeloid progenitors and granulocytes, is capable of the biotransformation of TOP2 poisons to metabolites that are potentially more genotoxic in nature. Whilst TOP2 poisons are valuable chemotherapeutic agents, their use is associated with myelosuppression and with the risk of developing therapy related acute myeloid leukaemia. The reason that myeloid progenitor cells are particularly susceptible to TOP2 poison associated genotoxicity is unclear, however the presence of myeloperoxidase could potentially make myeloid progenitor cells more vulnerable to TOP2 poison mediated damage. This lead to the hypothesis that myeloperoxidase inhibition could protect developing hematopoietic cells from TOP2 poison associated genotoxic and/or cytotoxic damage. Data generated in this thesis supports the proposed hypothesis as expression of myeloperoxidase significantly enhanced the accumulation of TOP2 poison induced TOP2-DNA covalent complexes and the level of DNA breaks within myeloid cell lines. The use of two potential clinically relevant inhibitors of myeloperoxidase showed that reduction in myeloperoxidase activity reduced the abundance of TOP2 poison stabilised TOP2-DNA complexes and DNA breaks. Furthermore, depletion of glutathione to mimic conditions experienced during chemotherapy, resulted in a myeloperoxidase dependent increase in TOP2 poison mediated DNA damage. Taken together these results show inhibition of myeloperoxidase could protect developing hematopoietic cells from TOP2 poison mediated damage without compromising the effectiveness of these drugs as antineoplastic agents.
9
Goldstein, Eric D. „Analysis of the repair of topoisomerase II DNA damage“. Honors in the Major Thesis, University of Central Florida, 2011. http://digital.library.ucf.edu/cdm/ref/collection/ETH/id/385.
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A large number of anti-cancer chemotherapeutics target DNA topoisomerases. Etoposide is a specific topoisomerase II poison which causes reversible double strand DNA breaks. The focus of this project is to analyze the repair of DNA damage induced by etoposide.. Double strand DNA break repair is mediated by through either non-homologous end joining (NHEJ) or homologous recombination. NHEJ repairs through direct ligation of a double stranded break while homologous recombination utilizes a homologous template to recover the wild type sequence. A reporter cassette, RYDR-GFP, has been stably integrated into HeLa cells. This reporter contains an ultra-high affinity topoisomerase II cleavage site (RY) placed in the middle of a mutant GFP sequence. Flanking this sequence is a corresponding stretch of wild type GFP that is used as template to repair the break and restore gene function yielding GFP positive cells. Titrations with etoposide have shown that a logarithmic increase in drug concentration yields a corresponding increase in repair through homologous recombination (HR). This result demonstrates that topoisomerase II mediated damage is efficiently repaired by the process of HR. To examine NHEJ repair, a doxycycline inducible, stably integrated NHEJ HeLa cell reporter cassette was also evaluated. The data indicates that repair of topoisomerase II mediated DNA damage occurs more efficiently through the HR pathway. Collectively, the data suggests that tumor cells proficient in HR repair may effectively elude treatment by topoisomerase II targeting drugs.B.S.
Bachelors
Medicine
Molecular Biology and Microbiology
10
Fox, Mary Elizabeth. „Mechanisms of action of anticancer DNA topoisomerase II poisons“. Thesis, University of Cambridge, 1991. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.239716.
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11
Marsh, Katherine Laura. „Structural and functional analysis of recombinant human topoisomerase II#beta#“. Thesis, University of Newcastle Upon Tyne, 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.336758.
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12
Zhou, Rong. „Topoisomerase II and drug resistance in leukemic cells /“. Stockholm, 2001. http://diss.kib.ki.se/2001/91-628-4738-4/.
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13
Kwok, Yan. „Proposed models for quinobenzoxazine and psorospermin-topoisomerase II-DNA complexes /“. Digital version accessible at:, 1998. http://wwwlib.umi.com/cr/utexas/main.
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14
DEREUDDRE, SOPHIE. „Implication de l'adn topoisomerase ii beta dans la cytotoxicite et la resistance cellulaire aux agents anticancereux inhibiteurs d'adn topoisomerase ii“. Paris 6, 1997. http://www.theses.fr/1997PA066302.
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La lignee cellulaire dc-3f/9-oh-e, selectionnee pour sa resistance a la 9-oh-ellipticine a partir d'une lignee de cellules de poumon de hamster chinois (dc-3f), presente une resistance croisee (superieure a 100 fois) aux inhibiteurs de l'adn topoisomerase ii. Grace au clonage et au sequencage des cdnas codant pour l'adn topoisomerase ii beta des lignees dc-3f et dc-3f/9-oh-e, nous avons pu mettre en evidence une mutation non sens en position 1710 du cdna codant pour l'adn topoisomerase ii beta de la lignee dc-3f/9-oh-e qui convertit un codon trp (tgg) en un codon stop (tga). Cette mutation conduit a la disparition de l'adn topoisomerase ii beta dans les cellules resistantes. Afin d'analyser le role de la disparition de cet enzyme dans le phenotype de resistance des cellules dc-3f/9-oh-e, nous avons restaure dans ces cellules une quantite normale et une activite catalytique normale adn topoisomerase ii beta. Cette restauration conduit a une restauration de l'induction des complexes de clivage et a une reversion du phenotype de resistance tres variable selon le type d'agent etudie. Ces resultats demontrent que l'adn topoisomerase ii beta est une cible pharmacologique in vivo de ces agents et qu'elle est impliquee dans leur cytotoxicite. Par ailleurs, le m-amsa apparait comme la cible preferentielle de l'adn topoisomerase ii beta dans le systeme cellulaire etudie. Enfin, la disparition de l'adn topoisomerase ii beta contribue au phenotype de resistance de la lignee dc-3f/9-oh-e aux inhibiteurs d'adn topoisomerase ii etudies. En accord avec des experiences similaires conduisant a la transfection des cellules dc-3f/9-oh-e avec l'adn topoisomerase ii alpha, d'autres alterations genetiques sont impliquees dans le phenotype de resistance.
15
Woo, Sung Ho. „Mechanistic studies of topoisomerase II and DNA polymerase inhibitors using SV40 DNA replication /“. The Ohio State University, 1998. http://rave.ohiolink.edu/etdc/view?acc_num=osu1487949508372332.
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16
Spitzner, Jeffrey Richard. „Characterization of recognition elements for DNA cleavage by topoisomerase II /“. The Ohio State University, 1990. http://rave.ohiolink.edu/etdc/view?acc_num=osu1487681788252562.
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17
Chung, In Kwon. „Reactivity of eukaryotic type II topoisomerase with unusual DNA structures /“. The Ohio State University, 1991. http://rave.ohiolink.edu/etdc/view?acc_num=osu1487758178238665.
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18
Gupta, Ranjan Brockman Herman E. „Effect of DNA topoisomerase II-targeting antitumor drugs in Neurospora crassa similarities to prokaryotic type II DNA topoisomerases /“. Normal, Ill. Illinois State University, 1990. http://wwwlib.umi.com/cr/ilstu/fullcit?p9115225.
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Thesis (Ed. D.)--Illinois State University, 1990.Title from title page screen, viewed November 28, 2005. Dissertation Committee: Herman E. Brockman (chair), Alan J. Katz, Lynne A. Lucher, Radheshyam K. Jayaswal, David F. Weber, Anthony E. Liberta. Includes bibliographical references (leaves 114-131) and abstract. Also available in print.
19
Bakshi, Rahul P. „Developmental And Functional Regulation Of DNA Topoisomerase II in Postnatal Rat Testis“. Thesis, Indian Institute of Science, 2000. http://hdl.handle.net/2005/166.
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Characterization of the polyoma virus chromosome as a circular, double-stranded, supercoiled DNA (Weil and Vinograd, 1963; Vinograd et al.,-1965) made it apparent that the DNA enzymatic machinery faces a formidable problem in ensuring faithful replication of genetic material. These studies initiated a search for enzymatic activities that are capable of overcoming this topological barrier and led to the discovery of DNA topoisomerase I, originally denoted as omega protein (Wang, 1971), followed by DNA gyrase (Gellert et al., 1976) from Escherichia coli. It is now established that Escherichia coli encodes four distinct topoisornerases. These include topoisomerase I (topA) (Wang, 1971), topoisomerase II (gyrA and gyrB) (Gellert et al., 1976), topoisomerase III (topB) (DiGate and Marians, 1988) and topoisomerase IV (parC and parE) (Kato et al, 1990; 1992). These enzymes perform various non-overlapping functions in vivo (reviewed in Roca, 1995). Additionally, a 'reverse gyrase", capable of introducing positive supercoils into DNA, in an ATP dependent manner, has been characterized from hyperthermophiles (Kikuchi and Asai, 1984; Kozyavkin et al., 1994).
20
Warsi, Tariq Hussain. „Centromeric functions and dynamics of DNA topoisomerase II in S. cerevisiae“. Diss., UC access only, 2009. http://proquest.umi.com/pqdweb?index=27&did=1907174001&SrchMode=1&sid=3&Fmt=2&VInst=PROD&VType=PQD&RQT=309&VName=PQD&TS=1270137471&clientId=48051.
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Thesis (Ph. D.)--University of California, Riverside, 2009.Includes abstract. Includes bibliographical references (leaves 227-256). Issued in print and online. Available via ProQuest Digital Dissertations.
21
Curtis, Hannah. „An investigation into the removal of human topoisomerase II-DNA adducts“. Thesis, University of Newcastle Upon Tyne, 2008. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.556060.
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Human topoisomerase 11 (topo 11) is an essential enzyme that controls DNA topology by relieving positive and negative supercoiling. It does this by creating a transient double-stranded break in one DNA duplex to allow a second duplex to pass through. The DNA double-stranded break is bridged by the topo 11 enzyme which is attached to each DNA end via a 5' covalent phosphotyrosyl bond. Human cells express two distinct topo JI isoforms, topo JI a and topo JI ~. The anti-cancer drug etoposide is extensively used in the treatment of malignancies. Etoposide traps the topo II homodimer in its normally transient cleavage state, a permanent DNA double stranded break can be produce through cellular processing. Non-homologous end joining (NHEJ) is a major pathway for the repair of to po II- mediated DNA damage, and inhibition of DNA-PK potentiates the cytotoxicity of topo JI poisons. The topo II enzyme remains covalently attached to the broken DNA and must be removed for NHEJ to take place. No mechanism to date has been elucidated for the removal of covalently attached human topo II-l)NA adducts. Inhibition of this process would slow the repair of to po II damage, thus potentiate the effects of to po IJ-targeting drugs. The Irapped in AgaRose DNA [mmunojitaining (TARDIS) assay was successfully adapted to incorporate a protein incubation phase to test candidate proteins for their ability to remove etoposide-stabilised human topo U-DNA adducts. Mrell and Tdp 1 immunoprecipitates from human chronic myelogenous leukaemia cells were identified that were capable of removing topo 11 complexes. Proteomic analysis identified nucleases and DNA repair proteins that were present in the two immunoprecipitates. Results provide evidence for the first time that Mre I I can facilitate the removal of human topo IJ a DNA adducts. In addition, results suggest that human topo II-DNA adducts are also removed by a 5' phosphodiesterase. These results may provide exciting new targets for future leukaemia drugs.
22
Petruti-Mot, Anca. „Genetic and functional analysis of topoisomerase II in vertebrates“. Thesis, University of Edinburgh, 2000. http://hdl.handle.net/1842/8985.
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The degree of DNA supercoiling in the cell is carefully controlled by DNA topoisomerases. These enzymes catalyze the passage of individual DNA strands (Type I DNA topoisomerases), or double helices (Type II DNA topoisomerases) through one another. The purpose of the present study is to conduct a detailed analysis of the topo llα and β mRNAs expressed in several vertebrate cell lines. The final aim of this project is to analyze the relative roles of topo llα in chromatin condensation and chromosome segregation during mitosis, by performing topo llα gene targeting experiments in the DT 40 chicken lymphoblastoid cell line. The knock-out strategy was based on the observation of a high rate of homologous recombination versus random integration in the DT40 cell line. The topo llα gene was shown to be located on the chicken chromosome 2 (APM unpublished), for which the DT40 cell line is trisomic. The targeting vector completely replaced the 32 kb topo IIα genomic locus, generating a topo llα (-/+/+)cell line, which showed an increased resistance to topo II inhibitors. Paradoxically, 150 uM etoposide or 100 uM mitoxanthrone induced apoptosis within 5 hours in the topo llα (-1+1+) cell line, more rapidly as compared to the normal DT 40 cells. A topo IIα (-I-I+) cell line has also been generated. This study revealed the presence of evolutionarily conserved alternatively spliced forms of both topo llα and β isoforms between birds and humans. Hybridization screening of two chicken cDNA libraries, MSB-1 and DU249, revealed the presence of two distinct forms of both topo llα and β cDNAs. One form of topo llα, designated topo llα-1, encodes the chicken topo llα amino acid sequence previously reported (dbjiAB007445) in the database (unpublished). The second form, designated topo llα-2, encodes a protein containing an additional 35 amino acids inserted after Lysine-322 of chicken topo IIα-1 protein sequence. In the case of topo 11(3 mANA, one form, designated topo IIβ-1, encodes the protein already described (dbjiAB007446). The second form, tapa IIβ-2, would encode a protein missing the next 86 amino acids after Valine-25 in tapa II β-1 protein sequence. The tapa 11β variant is positioned similarly to one previously described in human (Hela) cells. The 5 amino acid insertion in the human tapa 11β-2 variant follows v23. In chicken cells, a longer insertion of 86 amino acids sequence follows v25, the homologous position in the tapa 11β protein. In human cells, the situation with tapa llα is more complex, as revealed by RT-PCR experiments (APM, unpublished) which generated several bands. One of these amplified species was found to contain a 36 amino acids insertion, positioned after residue K321 in the human tapa IIα cDNA, similarly to chicken tapa IIα-2 variant. The second human tapa llα spliced form cDNA was shown to contain a 26 amino acids insertion after residue A401 in the canonical human tapa llα protein sequence. The third cDNA variant isolated from human cells was described to encode a 81 amino acids insertion after residue Q355 positioned within the known human tapa IIα protein. It appears possible that the posttranslational modifications of the a-2 and β-2 isoforms may differ substantially from those of the canonical a-1 and β-1 isoforms. Such variant proteins could fulfil specialized functions, which might be tissue or cell-type specific. In summary, two novel forms of tapa llα and β cDNAs have been identified in three chicken cell lines. These spliced versions of both tapa llα and 13 isoforms seem to be evolutionary conserved, with similar forms occurring in their human counterparts. Future functional analysis of vertebrate tapa IIα and β will have to account for the existence of these novel isoforms, which might encode proteins that may exhibit different regulation of their subcellular localization, interaction with other proteins, or catalytic activity.
23
Tsai, Francis T. F. „Crystallographic studies of DNA gyrase B protein“. Thesis, University of Oxford, 1996. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.390473.
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24
Corless, Samuel. „Role of DNA supercoiling in genome structure and regulation“. Thesis, University of Edinburgh, 2014. http://hdl.handle.net/1842/9623.
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A principle challenge of modern biology is to understand how the human genome is organised and regulated within a nucleus. The field of chromatin biology has made significant progress in characterising how protein and DNA modifications reflect transcription and replication state. Recently our lab has shown that the human genome is organised into large domains of altered DNA helical twist, called DNA supercoiling domains, similar to the regulatory domains observed in prokaryotes. In my PhD I have analysed how the maintenance and distribution of DNA supercoiling relates to biological function in human cells. DNA supercoiling domains are set up and maintained by the balanced activity of RNA transcription and topoisomerase enzymes. RNA polymerase twists the DNA, over-winding in front of the polymerase and under-winding behind. In contrast topoisomerases relieve supercoiling from the genome by introducing transient nicks (topoisomerase I) or double strand breaks (topoisomerase II) into the double helix. Topoisomerase activity is critical for cell viability, but the distribution of topoisomerase I, IIα and IIβ in the human genome is not known. Using a chromatin immunoprecipitation (ChIP) approach I have shown that topoisomerases are enriched in large chromosomal domains, with distinct topoisomerase I and topoisomerase II domains. Topoisomerase I is correlated with RNA polymerase II, genes and underwound DNA, whereas topoisomerase IIα and IIβ are associated with each other and over-wound DNA. This indicates that different topoisomerase proteins operate in distinct regions of the genome and can be independently regulated depending on the genomic environment. Transcriptional regulation by DNA supercoiling is believed to occur through changes in gene promoter structure. To investigate DNA supercoiling my lab has developed biotinylated trimethylpsoralen (bTMP) as a DNA structure probe, which preferentially intercalates into under-wound DNA. Using bTMP in conjunction with microarrays my lab identified a transcription and topoisomerase dependent peak of under-wound DNA in a meta-analysis of several hundred genes (Naughton et al. (2013)). In a similar analysis, Kouzine et al. (2013) identified an under-wound promoter structure and proposed a model of topoisomerase distribution for the regulation of promoter DNA supercoiling. To better understand the role of supercoiling and topoisomerases at gene promoters, a much larger-scale analysis of these factors was required. I have analysed the distribution of bTMP at promoters genome wide, confirming a transcription and expression dependent distribution of DNA supercoils. DNA supercoiling is distinct at CpG island and non-CpG island promoters, and I present a model in which over-wound DNA limits transcription from both CpG island promoters and repressed genes. In addition, I have mapped by ChIP topoisomerase I and IIβ at gene promoters on chromosome 11 and identified a different distribution to that proposed by Kouzine et al. (2013), with topoisomerase I maintaining DNA supercoiling at highly expressed genes. This study provides the first comprehensive analysis of DNA supercoiling at promoters and identifies the relationship between supercoiling, topoisomerase distribution and gene expression. In addition to regulating transcription, DNA supercoiling and topoisomerases are important for genome stability. Several studies have suggested a link between DNA supercoiling and instability at common fragile sites (CFSs), which are normal structures in the genome that frequently break under replication stress and cancer. bTMP was used to measure DNA supercoiling across FRA3B and FRA16D CFSs, identifying a transition to a more over-wound DNA structure under conditions that induce chromosome fragility at these regions. Furthermore, topoisomerase I, IIα and IIβ showed a pronounced depletion in the vicinity of the FRA3B and FRA16D CFSs. This provides the first experimental evidence of a role for DNA supercoiling in fragile site formation.
25
Muftic, Diana. „The role of topoisomerase II in replication in mammalian cells“. Thesis, University of Oxford, 2011. http://ora.ox.ac.uk/objects/uuid:8c2399d3-5cca-4c50-b1ca-8a5b96890f83.
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Topoisomerase 2α (Topo2α) is an essential protein with DNA decatenating enzymatic properties, indispensable for chromosome decatenation and segregation. It is a target for a plethora of antitumour drugs and Topo2α protein levels have been associated with the success of treatment, but also drug resistance and secondary malignancies. Although unique in its ability to resolve catenated chromosomes, the role of Topo2α in other steps of DNA metabolism, such as DNA replication elongation and termination have been elusive. A thorough understanding of the role of Topo2α in the cell will not only allow for increased insight into the mechanisms it is involved in, but it will also shed light on proteins and pathways that can act as back-up in its absence, and therefore hopefully expand the basis on which to improve treatment options. Through a synthetic lethal interaction (SLI) screen with an siRNA library targeting 200 DNA repair and signalling genes, Topo2α emerged as being synthetic lethal to Werner protein (WRN), a RecQ helicase involved in maintaining genome integrity mainly in S phase, and the loss of which leads to Werner Syndrome (WS), a segmental progeroid syndrome. The screen was performed in WRN deficient cells, with the initial aim to find proteins that act to buffer against loss of viability, which is the central idea in the concept of synthetic lethality in the absence of WRN. The screen revealed an SLI between WRN and Topo2α and although we were unable to fully validate this, it spurred the question of Topo2α’s role in DNA replication. The findings in this thesis suggest that Topo2α is not required for DNA elongation and timely completion of S phase, and that simultaneous loss of the closely related isoform Topo2β does not affect replication, suggesting that these proteins do not act in parallel back-up pathways during replication. Interestingly, cells accumulate in the polyploid fraction after both depletion and inhibition of Topo2α, albeit with different kinetics. The mechanistic basis of this phenotype remains to be understood through further research, but it is highly interesting as aneuplidity and polyploidy are implicated in the initial stages of tumour development.
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Soares, Bruno Marques. „Hellebrigenina, um bufodienolídeo com potencial ação compatível de inibidor catalítico da topoisomerase II“. reponame:Repositório Institucional da UFC, 2013. http://www.repositorio.ufc.br/handle/riufc/5706.
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SOARES, Bruno Marques. Hellebrigenina, um bufodienolídeo com potencial ação compatível de inibidor catalítico da Topoisomerase II. 2013. 85 f. Dissertação (Mestrado em Farmacologia) - Universidade Federal do Ceará. Faculdade de Medicina, Fortaleza, 2013.Submitted by denise santos (denise.santos@ufc.br) on 2013-08-28T15:47:03Z No. of bitstreams: 1 2013_dis_bmsoares.pdf: 2334333 bytes, checksum: 332b2a9235ebad6d7da12ab6ac20ee16 (MD5)
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Bufodienolides are cardioactive steroids of 24 carbons, originally isolated from a frog’s skin extract of the family Bufonidae used in Chinese medicine. Bufodienolides shows many biological activities, including anticancer activities. Related to antitumor activity, the bufodienolídeos has been shown to inhibit the growth of several human cancer cell lines by inducing apoptosis and cell cycle arrest. This study evaluated the potential cytotoxicity and genotoxicity of six bufodienolides, in six human tumor cell lines, three normal murine lineages and PBMC (peripheral blood mononuclear cells). All six bufodienolides were cytotoxic to all cell lines and tumor PBMC with IC50 values ranging from 0.002 to 3.17 µM. Bufodienolides showed no cytotoxicity for normal murine strains. Thus, the compound hellebrigenin was chosen to determine the action mechanism involved, a sequence of in vitro experiments were performed using HL-60 leukemia cell line. Cells were treated at different concentrations of hellebrigenin (0.03, 0.06 and 0.12 µM) for 24 hours. Cell viability (viable cell number and membrane integrity) HL-60 assessed by flow cytometry showed that the number of cells decreased from the lower concentration (0.03 µM) tested and the percentage of cells with reduced membrane integrity from 0.06 µM concentration. Morphological analysis by flow cytometry revealed increased apoptotic cells starting at concentrations of 0.06 µM. The analysis of nuclear content, showed an increase in DNA fragmentation indicative of sub-G1 apoptosis and accumulation of cells in G2 / M phase from the concentrations of 0.03 and 0.06 µM, respectively. Other tests by flow cytometry revealed that there was an externalization of phosphatidylserine, mitochondrial depolarization, activation of caspase 8 and initiating subsequent activation of effector caspases 3 and 7. These data indicate a cytotoxic mechanism induced by over an apoptotic pathway. Hellebrigenin was not able to cause DNA damage in HL-60 and PBMC nor the emergence of chromosomal aberrations in PBMC. Through the studies of molecular docking was possible to predict the connection between hellebrigenina and human topoisomeraseIIα, showing a result that is compatible with a possible inhibition of this enzyme. Overall, the results indicate the potential cytotoxicity of hellebrigenin.
Os bufodienolídeos são esteróides cardioativos de 24 carbonos, isolados originalmente de um extrato de pele de sapos da família Bufonidae utilizado na medicina chinesa. Os bufodienolídeos possuem grande variedade de atividades biológicas, incluindo atividades antineoplásicas. Em relação à atividade antitumoral, os bufodienolídeos tem demonstrado inibir o crescimento de várias linhagens de células cancerígenas humanas por induzir apoptose e parada do ciclo celular. O presente estudo avaliou o potencial citotóxico e genetóxico de seis bufodienolídeos em seis linhagens tumorais humanos, três linhagens murinas normais e células mononucleadas do sangue periférico (CMSP) humano. Todos os seis bufodienolídeos foram citotóxicos para todas as linhagens tumorais e CMSP com valores de IC50 variando entre 0,002 e 3,17 µM. Os bufodienolídeos testados não apresentaram citotoxicidade para linhagens murinas normais. Desta forma, o composto hellebrigenina foi escolhido para se determinar o mecanismo de ação envolvido. Uma sequência de experimentos in vitro foram realizados utilizando-se a linhagem leucêmica HL-60. As células foram tratadas em diferentes concentrações da amostra hellebrigenina (0,03, 0,06 e 0,12 µM) por 24 horas. A viabilidade das células (número de células viáveis e integridade de membrana) HL-60 avaliada por citometria de fluxo, mostrou que o número de células reduziu a partir da menor concentração (0,03 µM) testada e a porcentagem de células com membrana integra reduziu a partir da concentração 0,06 µM. A análise morfológica por citometria de fluxo revelou aumento de células com padrão apoptótico a partir da concentração de 0,06 µM. Já a análise do conteúdo nuclear, nos mostrou aumento de fragmentação de DNA sub-G1 indicativo de apoptose e acúmulo de células na fase G2/M a partir das concentrações de 0,03 e 0,06 µM, respectivamente. Outros testes por citometria de fluxo revelaram que houve externalização da fosfatidilserina, despolarização mitocondrial, ativação da caspase iniciadora 8 e consequente ativação das caspases efetoras 3 e 7. Estes dados indicam um mecanismo citotóxico por indução de mais de uma via apoptótica. Hellebrigenina não foi capaz de causar danos ao DNA de HL-60 e de CMSP e nem o surgimento de aberrações cromossômicas em CMSP. Por meio dos estudos de docking molecular foi possível predizer a ligação entre hellebrigenina e topoisomeraseIIα humana, resultado compatível com a possível inibição dessa enzima. De forma geral, os resultados apontam o potencial citotóxico do bufodienolídeo hellebrigenina.
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Fagenson, Alexander. „Gold (iii) macrocycles are dna intercalators that inhibit topoisomerase i and ii“. Honors in the Major Thesis, University of Central Florida, 2012. http://digital.library.ucf.edu/cdm/ref/collection/ETH/id/1536.
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Human Topoisomerase IB (TOP1) and Topoisomerase II? (TOP2?) are essential nuclear enzymes that control DNA topology during DNA replication, gene transcription and cell division. These enzymes carry out their catalytic function by making transient single-strand (type I) or double-strand (type II) breaks in the DNA. In vivo, these complexes are short-lived but can be exploited by anti-cancer drugs to mechanistically kill cancer cells. Two general classes of compounds can kill cancer cells through a topo-targeted mechanism. Interfacial Poisons (IFPs) act at the enzyme-DNA interface to inhibit the religation reaction, resulting in the accumulation of DNA double-stand breaks (DSBs) in the genomic setting. Catalytic Inhibitor Compounds (CICs) act by interfering with other steps of the catalytic cycles such as DNA/protein binding or the cleavage reaction. In this work we identify new Au3+ macrocyclic gold complexes that act as CICs of both TOP1 and TOP2?. The complexes exhibit square planar geometry with an aromatic system that allows for DNA intercalation with binding affinities in the low micromolar range. A cytotoxicity screen across 60 human cancer cell lines performed by the National Cancer Institute (NCI, USA) reveals significant anti-tumor potential. Our lead compound (butyl gold(III) macrocycle, cmpd 3.) is currently undergoing further studies in animal models at the NCI. In vitro assays with purified DNA and enzyme reveal the Au3+ ion to be the quintessential switch that allows for DNA intercalation and subsequent inhibition of TOP1 and TOP2?.ID: 031908365; System requirements: World Wide Web browser and PDF reader.; Mode of access: World Wide Web.; Accepted in partial fulfillment of the requirements for honors in the major in DEPT HERE.; Thesis (B.A.)--University of Central Florida, 2012.; Includes bibliographical references.
B.S.
Bachelors
Burnett School of Biomedical Sciences
Molecular and Microbiology
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Gilroy, Kathryn Linda. „An investigation into the structure and mechanism of human DNA topoisomerase II“. Thesis, University of Newcastle Upon Tyne, 2007. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.443020.
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Cridland, Peter James. „An investigation of the protein-protein interactions of human DNA topoisomerase II“. Thesis, University of Newcastle Upon Tyne, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.364796.
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Soares, Bruno Marques. „Hellebrigenina, um BufodienolÃdeo com Potencial AÃÃo CompatÃvel de Inibidor CatalÃtico da Topoisomerase II“. Universidade Federal do CearÃ, 2013. http://www.teses.ufc.br/tde_busca/arquivo.php?codArquivo=10367.
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CoordenaÃÃo de AperfeiÃoamento de NÃvel SuperiorOs bufodienolÃdeos sÃo esterÃides cardioativos de 24 carbonos, isolados originalmente de um extrato de pele de sapos da famÃlia Bufonidae utilizado na medicina chinesa. Os bufodienolÃdeos possuem grande variedade de atividades biolÃgicas, incluindo atividades antineoplÃsicas. Em relaÃÃo à atividade antitumoral, os bufodienolÃdeos tem demonstrado inibir o crescimento de vÃrias linhagens de cÃlulas cancerÃgenas humanas por induzir apoptose e parada do ciclo celular. O presente estudo avaliou o potencial citotÃxico e genetÃxico de seis bufodienolÃdeos em seis linhagens tumorais humanos, trÃs linhagens murinas normais e cÃlulas mononucleadas do sangue perifÃrico (CMSP) humano. Todos os seis bufodienolÃdeos foram citotÃxicos para todas as linhagens tumorais e CMSP com valores de IC50 variando entre 0,002 e 3,17 ÂM. Os bufodienolÃdeos testados nÃo apresentaram citotoxicidade para linhagens murinas normais. Desta forma, o composto hellebrigenina foi escolhido para se determinar o mecanismo de aÃÃo envolvido. Uma sequÃncia de experimentos in vitro foram realizados utilizando-se a linhagem leucÃmica HL-60. As cÃlulas foram tratadas em diferentes concentraÃÃes da amostra hellebrigenina (0,03, 0,06 e 0,12 ÂM) por 24 horas. A viabilidade das cÃlulas (nÃmero de cÃlulas viÃveis e integridade de membrana) HL-60 avaliada por citometria de fluxo, mostrou que o nÃmero de cÃlulas reduziu a partir da menor concentraÃÃo (0,03 ÂM) testada e a porcentagem de cÃlulas com membrana integra reduziu a partir da concentraÃÃo 0,06 ÂM. A anÃlise morfolÃgica por citometria de fluxo revelou aumento de cÃlulas com padrÃo apoptÃtico a partir da concentraÃÃo de 0,06 ÂM. Jà a anÃlise do conteÃdo nuclear, nos mostrou aumento de fragmentaÃÃo de DNA sub-G1 indicativo de apoptose e acÃmulo de cÃlulas na fase G2/M a partir das concentraÃÃes de 0,03 e 0,06 ÂM, respectivamente. Outros testes por citometria de fluxo revelaram que houve externalizaÃÃo da fosfatidilserina, despolarizaÃÃo mitocondrial, ativaÃÃo da caspase iniciadora 8 e consequente ativaÃÃo das caspases efetoras 3 e 7. Estes dados indicam um mecanismo citotÃxico por induÃÃo de mais de uma via apoptÃtica. Hellebrigenina nÃo foi capaz de causar danos ao DNA de HL-60 e de CMSP e nem o surgimento de aberraÃÃes cromossÃmicas em CMSP. Por meio dos estudos de docking molecular foi possÃvel predizer a ligaÃÃo entre hellebrigenina e topoisomeraseIIα humana, resultado compatÃvel com a possÃvel inibiÃÃo dessa enzima. De forma geral, os resultados apontam o potencial citotÃxico do bufodienolÃdeo hellebrigenina
Bufodienolides are cardioactive steroids of 24 carbons, originally isolated from a frogâs skin extract of the family Bufonidae used in Chinese medicine. Bufodienolides shows many biological activities, including anticancer activities. Related to antitumor activity, the bufodienolÃdeos has been shown to inhibit the growth of several human cancer cell lines by inducing apoptosis and cell cycle arrest. This study evaluated the potential cytotoxicity and genotoxicity of six bufodienolides, in six human tumor cell lines, three normal murine lineages and PBMC (peripheral blood mononuclear cells). All six bufodienolides were cytotoxic to all cell lines and tumor PBMC with IC50 values ranging from 0.002 to 3.17 ÂM. Bufodienolides showed no cytotoxicity for normal murine strains. Thus, the compound hellebrigenin was chosen to determine the action mechanism involved, a sequence of in vitro experiments were performed using HL-60 leukemia cell line. Cells were treated at different concentrations of hellebrigenin (0.03, 0.06 and 0.12 ÂM) for 24 hours. Cell viability (viable cell number and membrane integrity) HL-60 assessed by flow cytometry showed that the number of cells decreased from the lower concentration (0.03 ÂM) tested and the percentage of cells with reduced membrane integrity from 0.06 ÂM concentration. Morphological analysis by flow cytometry revealed increased apoptotic cells starting at concentrations of 0.06 ÂM. The analysis of nuclear content, showed an increase in DNA fragmentation indicative of sub-G1 apoptosis and accumulation of cells in G2 / M phase from the concentrations of 0.03 and 0.06 ÂM, respectively. Other tests by flow cytometry revealed that there was an externalization of phosphatidylserine, mitochondrial depolarization, activation of caspase 8 and initiating subsequent activation of effector caspases 3 and 7. These data indicate a cytotoxic mechanism induced by over an apoptotic pathway. Hellebrigenin was not able to cause DNA damage in HL-60 and PBMC nor the emergence of chromosomal aberrations in PBMC. Through the studies of molecular docking was possible to predict the connection between hellebrigenina and human topoisomeraseIIα, showing a result that is compatible with a possible inhibition of this enzyme. Overall, the results indicate the potential cytotoxicity of hellebrigenin
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Mak, Ka Man. „Topoisomerases II in the cell cycle of dinoflagellates /“. View abstract or full-text, 2005. http://library.ust.hk/cgi/db/thesis.pl?BIOL%202005%20MAK.
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Meczes, Emma Louise. „A study of human topoisomerase II#alpha# and #beta# as targets for anticancer agents“. Thesis, University of Newcastle Upon Tyne, 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.389599.
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Keller, Beatrice Astrid. „Candida albicans DNA topoisomerase II : cloning, expression and characterisation as a potential antifungal target“. Thesis, St George's, University of London, 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.286358.
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Iijima, Junko. „RAP80 Acts Independently of BRCA1 in Repair of Topoisomerase II Poison-Induced DNA Damage“. Kyoto University, 2011. http://hdl.handle.net/2433/142083.
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Roythorne, Ashleigh. „Exploring the role of topoisomerase II beta in macrophage maturation and pro-inflammatory cytokine production“. Thesis, Northumbria University, 2014. http://nrl.northumbria.ac.uk/21412/.
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Although it is known that DNA topo IIβ is required for the regulation of transcription during neural development and differentiation, it is not clear whether the enzyme is required during differentiation of human monocytes into macrophages and/or the subsequent transcription of cytokine genes. To test this, a robust model of differentiation of monocyte-like cells into macrophage-like cells using U937 and HL-60 cells treated with phorbol 12-myristate 13-acetate (PMA) and Lipopolysaccharide (LPS) was validated. Differentiation was determined by morphological and growth characteristics and CD11b surface antigen expression as determined by flow cytometry. qRT-PCR was also used to measure mRNA transcript levels of key genes known to be up-regulated during monocyte differentiation and the secretion of pro-inflammatory cytokines produced by differentiated cells were measured using ELISA. siRNA topo IIβknockdown did not hinder monocyte-like cells from undergoing differentiation, however experiments revealed a correlation between topo IIβknockdown and secreted TNFα, with the latter decreasing when topo IIβwas reduced. This pattern was also noted when measuring IL-1βsecretion. Similar results were seen using a Murine transgenic fibroblast cell line lacking topo IIβ, which when stimulated with LPS secreted significantly lower levels of IL-6 compared to the wild type cells. Thus topo IIβexpression is necessary for secretion of normal levels of the cytokines, TNFα, IL-1βand IL-6 in response to LPS at certain time points. In addition in the macrophage-like state of the two cell lines, the relative levels of the βisoform (mRNA and protein) were shown to be significantly increased compared to α, further outlining the importance of topo IIβin the differentiated state. Chromatin immuno-precipitation followed by qPCR showed however that topo IIβwas not associated at three defined proximal promoter regions of either the TNFαand IL-1βgenes, although further studies are required to rule out a direct association of topo IIβwith these and other regions of the genes. Down regulation of topo IIβprotein using the inhibitor ICRF-193 did not hinder monocyte-like cells from undergoing differentiation either. However, contrary to the knockdown results, a 6 h pre-treatment with 1 nM ICRF-193 increased TNFαlevels in these cells, both at the mRNA and the protein level, along with a slight increase in secreted TNFα. NF-κB, EGR2, TLR4 and TLR2 transcript levels were also increased under these conditions. Thus further studies are required to determine if these increases are due to additional cellular effects of the drug or whether topo IIβmay play an inhibitory effect on transcription. Thus it is clear that topo IIβplays an important role in expression of cytokines and understanding the exact nature of this requires further research that may yield potential new avenues for treatment of disease.
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Freewan, Mohammed Medical Sciences Faculty of Medicine UNSW. „Studies of the poisoning of DNA Topoisomerase II?? by Acridine-4-Carboxamides and related Cytotoxins“. Awarded by:University of New South Wales. Medical Sciences, 2008. http://handle.unsw.edu.au/1959.4/42615.
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The central role of topoisomerase II in DNA transactions such as replication, transcription, and chromosome segregation, has led to the development of antimicrobial and anticancer agents that inhibit its DNA cleavage and religation functions by a mechanism known as topoisomerase poisoning. Poisoning involves the formation of a ternary complex between drug, DNA, and enzyme that stabilizes the cleavage complex, thereby inhibiting religation. In this work, we have prepared purified recombinant human topo II??, and used enzymatic and sequencing methods to probe the molecular interactions that stabilize the ternary complexes of several classical and acridine-4carboxamide topo II poisons. These studies used agarose gel techniques to assay inhibition of the relaxation of supercoiling, and the production of single and double strand DNA breaks, in pBR322 DNA. The findings are supported by acrylamide gel measurements to determine the nucleotide sequences at which the compounds trap topo II??. The results confirm the biologically active acridine-4-carboxamides DACA, ASDACA, 9-amino-DACA, and the threading 9-anilino-acridine-4-carboxamide SN 16713, as topo IIa poisons. By contrast, inactive analogues bearing morpholino and piperidine side chains fail to trap the cleavable complex, despite the fact that their side chains form hydrogen bonding interactions with guanine in DNA in the same manner as 9-amino-DACA. These findings emphasize the specific nature of the molecular interactions between drug, DNA, and protein in the ternary complex, and suggest that the N,N-dimethylamino groups of the active compounds occupy a cavity that is too small to accommodate the larger cyclic side chains. We have made a detailed comparison of the consensus sequences for enzyme trapping by DACA, AS-DACA, 9amino-DACA, SN 16713, amsacrine, etoposide, and mitoxantrone. The data have been interpreted in terms of a model for the trapped cleavable complex based on the crystal structure of the topo II-DNA complex of Dong and Berger (Nature, 2007, 450:12011205). The model proposes that amsacrine and etoposide trap the enzyme by binding in the DNA minor groove, with their side chains interacting with amino acid residues of the toprim domain that contribute to the active site. By contrast, it is proposed that mitoxantrone and the acridine-4-carboxamides bind to DNA in the complex with their side chains in the major groove, making bonding interactions with guanine bases and the a4 helix of the topo IIa CAP domain. SN 16713 poisons topo II more in the manner of amsacrine than DACA, implying that its anilino ring dominates over its carboxamide side chain in stabilising the cleavable complex.
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Feeney, Katherine M. „Investigations of G2/M decatenation checkpoint control, using the DNA topoisomerase II inhibitor ICRF-193“. Thesis, University of Ulster, 2007. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.529562.
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Zheng, Hui. „Stereoselective pharmacokinetics and metabolism of XK469, a new quinoxaline topoisomerase II beta poison, in the rat“. Columbus, Ohio : Ohio State University, 2004. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=osu1080257372.
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Thesis (Ph. D.)--Ohio State University, 2004.Title from first page of PDF file. Document formatted into pages; contains xxi, 190 p.; also includes graphics. Includes abstract and vita. Advisor: Kenneth K. Chan, Dept. of Pharmacy. Includes bibliographical references (p. 182-190).
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Anderson, Robert James. „Development of an affinity partitioning method for DNA/protein complexes and its application to interactions of topoisomerase II with DNA“. Thesis, Open University, 1992. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.386626.
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Ogbu, Stella Chinyere. „Role of Topoisomerase II alpha in DNA Topology and T cell responses during Chronic Viral Infections“. Digital Commons @ East Tennessee State University, 2019. https://dc.etsu.edu/etd/3661.
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The clearance of viruses is largely dependent upon the activation of T cells to generate a robust immune response. However, host responses are suppressed during chronic viral infections. In this thesis, we explored the role of Top2α in DNA topology in individuals with chronic HBV, HCV, and HIV infections. We found that Top2α protein expression and activity were low in T cells derived from chronically virus-infected individuals compared to healthy subjects. Using CD4+ T cells treated with Top2α inhibitor or poisoner as a model, we demonstrated that Top2α inhibition disrupts the DNA topology, suppresses DNA repair kinase (ATM), and telomere protein (TRF2) expression, and induces T cell dysfunction. These findings reveal that Top2α inhibition is a mechanism by which viruses evade the host responses and establish persistent infection, and thus, restoring Top2α levels could be a way of boosting immune responses during chronic viral infections.
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Chang, Chih-Jui. „Genetic and RNAi analysis of INCENP (inner centromere protein) and DNA Topoisomerase II in Drosophila melanogaster“. Thesis, University of Edinburgh, 2003. http://hdl.handle.net/1842/13356.
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This thesis is divided into two parts. In part I, I carried out a genetic screen to obtain new INCENP alleles and interacts. After screening 5000 chromosomes, I isolated 18 mutants, one of them was a new allele of INCENP that we called incenp3747. Two mutants (3322, 4330) were dominant female sterile. All of the mutants were homozygous lethal. Some of the mutants showed defects in eyes, wings, bristle when combined with incenpP(EP)2340. The sequence of the coding region of INCENP in incenp3747 homozygous mutants showed a point mutation. A single base change in exon 4 resulted in a stop codon. The predicted molecular weight of the putative truncated gene product would be 61kD. The truncated protein was not detected by Western blot, which could suggest that it is unstable although I cannot rule out the unlikely possibility that our antibody failed to detect the truncated product. The phenotypic analysis of incenp3747 showed that the homozygous embryos exhibit chromosome segregation defects. In mixed populations of early embryos (incenp3747/CyO-Ftz-LacZ) I found chromosome segregation defects as early as cycle 3. Some of the nuclei also failed to follow the globally synchronous mitotic oscillator in the early divisions. My analysis revealed that these early defects resulted from a maternal contribution to the phenotype, due to genetic interaction with the balancer chromosome. Later in development, homozygous mutant embryos showed aberrant neuronal morphology and the homozygous mutant first instar larvae escapers exhibit abnormal behaviour. In part II, I used dsRNAi in Drosophila S2 cells to carry out a detailed functional analysis of the role of Topoisomerase II (Topo II) during mitosis. I found that Topo II was not required for the assembly of a functional kinetochore or the targeting of chromosomal passenger proteins, nonetheless, it was essential for anaphase sister chromatid separation.
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Amir, Aslani Arsia. „Étude des propriétés structurales d'un site de coupure préférentiel de la topoisomerase II“. Paris 6, 1996. http://www.theses.fr/1996PA066454.
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La grande variete des structures d'adn repertoriees, nous permet de penser que ce sont les proprietes conformationnelles de l'adn qui se trouvent a la base des evenements gouvernant la regulation genetique. A l'origine de celle-ci se trouvent les phenomenes de reconnaissance moleculaires : liaison d'un ligand, que ce soit une proteine ou un agent antitumoral, a un domaine particulier de l'adn. L'analyse des sequences au voisinage des principaux sites de clivage de l'adn de pbr322 par la topoisomerase ii de thymus de veau couplee a l'ellipticine a permis de mettre en evidence une sequence consensus dont le prototype est le site (fort) 22 (fosse et al. , 1991). Cette coupure est egalement stimulee par l'etoposide vp-16 qui est sense agir sur la topoisomerase ii par un mecanisme different de celui de l'ellipticine. La question est de savoir si le site de coupure 22 de la topoisomerase ii peut adopter une conformation differente de celle de la double helice lineaire ? au cas ou une telle conformation existe, est-elle un site de reconnaissance pour la topoisomerase ii couplee aux principaux agents antitumoraux ? la sequence comprenant le site 22, 5'-d(agcttatc-atc-gtattgct)-3' (-atc-) et 5'-d(agcttatc-gat-gtattgct)-3' (-gat-) de par sa symetrie, est susceptible d'adopter une structure repliee cruciforme probablement en equilibre avec la structure en duplex lineaire. Pour analyser un tel systeme, nous avons utilise les techniques de resonance magnetique multinucleaire (#1h et #31p) et multidimensionnelle (1d, 2d) combinees a celles d'absorption-uv et de dichroisme circulaire (dc). Chaque brin a ete etudie independamment. Nous montrons que le brin -atc- adopte une structure exclusivement en epingle a cheveux, tandis que le brin -gat- est present sous forme d'un equilibre monomere-dimere, ou le monomere (forme repliee en epingle a cheveux) est preponderant. La mesure de la thermostabilite des formes en epingle a cheveux -gat- et -atc- montre que -atc- est plus stable que -gat- de 4c. Ceci est en accord avec les spectres de dc qui indiquent que les interactions d'empilement sont plus importantes dans -atc- que dans -gat-. Les spectres de rmn noesy montrent que le reseau des interactions noes est identique dans la tige des deux oligonucleotides mais les connectivites interresiduelles sont bien plus nombreuses dans la boucle -atc- que dans -gat-. Des experiences #1h-#31 cosy avec detection proton ou #31p-#1h (doc) ont ete realisees dans le but d'etudier la conformation du squelette phosphodiester des brins -atc- et -gat-. Pour -atc- des distorsions du squelette phosphodiester se produisent, sur l'ensemble de la boucle, alors que dans le cas de -gat- elles apparaissent beaucoup plus faibles en conclusion, il est necessaire de souligner la convergence des resultats obtenus avec des approches tres diverses : rmn, dichroisme circulaire mecanique moleculaire. La capacite de chaque brin -atc- et -gat- d'adopter une structure repliee est en faveur d'un equilibre conformationnel dans le site 22, in vivo. Ce dernier est certainement necessaire a la reconnaissance de l'adn par les enzymes telles que la topoisomerase ii.
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Lehmann, Maurício. „Toxicidade genética das antraciclinas : associação entre estrutura química e ação inibitória sobre a topoisomerase II“. reponame:Biblioteca Digital de Teses e Dissertações da UFRGS, 2003. http://hdl.handle.net/10183/2367.
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Considerando não apenas a importância das antraciclinas na terapêutica do câncer, mas também os efeitos colaterais associados ao uso destas drogas, o presente estudo procurou avaliar a atividade genotóxica de seis antraciclinas em uso clínico - doxorrubicina (DOX), daunorrubicina (DNR), epirrubicina (EPI), idarrubicina (IDA), além dos análogos de última geração, pirarrubicina (THP) e aclarrubicina (ACLA). Para tanto, foi empregado o Teste de Mutação e Recombinação Somática (SMART) em Drosophila melanogaster, que permite a detecção simultânea de mutação gênica e cromossômica, assim como de eventos relacionados com recombinação mitótica - possibilitando quantificar a contribuição deste último parâmetro genético para a genotoxicidade total induzida pelas drogas em estudo. Os dados obtidos a partir desta análise demonstraram que todas as antraciclinas estudadas induziram acréscimos significativos, relacionados tanto à mutação, quanto à recombinação nas células somáticas deste inseto. Além disso, a recombinação mitótica - entre cromossomos homólogos - foi o evento responsável por, aproximadamente, 62 a 100% da toxicidade genética observada. A comparação do potencial genotóxico dos diferentes análogos, através da padronização do número de danos genéticos por unidade de tratamento (mM), caracterizou a ACLA e o THP como as drogas mais potentes – sendo cerca de 20 vezes mais efetivas, como genotoxinas, do que a DOX, o análogo menos potente. Já que a principal ação genotóxica desta família de compostos está relacionada à inibição da topoisomerase II (topo II) – uma enzima que atua no relaxamento da supertorção da dupla hélice de DNA, através da quebra e posterior religação de suas fitas - as diferenças observadas podem ser atribuídas ao mecanismo envolvido neste bloqueio Enquanto os análogos DOX, DNR, EPI, IDA e THP atuam como venenos de topo II - tornando permanentes as quebras induzidas pela enzima - a ACLA inibe a função catalítica desta enzima, impedindo a sua ligação ao DNA. Cabe ainda ressaltar que a genotoxicidade da ACLA não está restrita à sua atividade catalítica sobre a topo II, mas também à sua ação como veneno de topo I e à sua habilidade de intercalar-se na molécula de DNA. Quando a potência genotóxica destas drogas foi associada a suas estruturas químicas, observou-se que substituições no grupamento amino-açúcar levaram a uma maior atividade tóxico-genética, quando comparadas a modificações no cromóforo. Cabe ainda ressaltar que as modificações estruturais, presentes nos análogos DOX, DNR, EPI, IDA e THP, não alteraram a sua ação recombinogênica. No entanto, no que se refere a ACLA, observaram-se decréscimos significativos na indução de recombinação mitótica - que podem ser atribuídas às múltiplas substituições presentes tanto no grupamento amino-açúcar quanto no cromóforo. O conjunto destas observações evidencia que a genotoxicidade total das drogas em estudo está centrada na indução de recombinação homóloga - um evento predominantemente envolvido tanto na iniciação, quanto na progressão do câncer. A alta incidência de tumores secundários, em pacientes submetidos ao tratamento com as antraciclinas, pode, pois, ser atribuída à ação preferencial destas drogas sobre a recombinação mitótica – embora a atividade mutagênica não possa ser desconsiderada.
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Lee, Ming-Ta. „Analysis of genome stability in mutants defective for the SUMO isopeptidase Smt4/Ulp2 /“. Diss., UC access only, 2009. http://proquest.umi.com/pqdweb?index=6&did=1907279801&SrchMode=2&sid=2&Fmt=2&VInst=PROD&VType=PQD&RQT=309&VName=PQD&TS=1270053784&clientId=48051.
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Thesis (Ph. D.)--University of California, Riverside, 2009.Includes abstract. Includes bibliographical references (leaves 213-243). Issued in print and online. Available via ProQuest Digital Dissertations.
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Lins, Antônio Claudio da Silva. „Estudo Químico e Atividade Antioxidante de Bauhinia Pentandra (Bong.) Vog. Ex Steud e Avaliação da Atividade Inibitória da Enzima Dna Topoisomerase II-a Humana de Substâncias Naturais e Semi-Sintéticas“. Universidade Federal da Paraíba, 2008. http://tede.biblioteca.ufpb.br:8080/handle/tede/6830.
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The species Bauhinia pentandra (Bong.) Vog. ex Steud (Leguminosae), known as mororó com espinho , is common in Northeast of Brazil. The enzyme DNA topoisomerase II-α (topo II) is responsible for the relaxation of the DNA during the processes of cellular division and is involved in diverse types of cancer, being considered a potential target in the treatment of the cancer. This work aimed to study the chemical composition and antioxidant activity of the bark roots of B. pentandra and the inhibition activity of topo II in natural and semi-synthetic compounds. In the chemical study of the vegetal species was isolated of the cyanogenic glycoside lithospermoside and a mixture of β-sitosterol and stigmasterol. The lithospermoside was acetylated and the structures had been identified through spectral data as IV and RMN1H and 13C including 2D. The Ethanolic extract and the fractions in hexane, AcOEt, MeOH and the substance lihtospermoside were submitted to antioxidant tests (DPPH, ABTS, quelatogenic activity of Fe2+ ion). The determination of total phenolic also was performed with the extract and fractions. All the samples showed antioxidant activity, being the AcOEt with highest activity and higher content of phenolic compounds, then this fraction was analyzed by HPLC-DAD having confirming the presence of phenolic. The inhibition of topoisomerase II-α was analyzed with natural compounds lithospermoside and γ-hydroxyferruginin A, isolated from Vismia guianensis and the EtOH extract of the latex of this species, and with semi-syntetic compounds: peracetyl lithospermoside and ten flavonoids derivated of retusin, isolated from Solanum paludosum. At 220 μM concentration all samples tested were actives, except lithospermoside and its acetylated derivative. In the concentration of 110 μM only two flavonoids did not inhibit the action of topo II. The γ-hydroxyferruginin inhibited the activity of topo II in a minimum concentration of 0,1 μM, being this anthranoid prenilated a promising candidate for posterior therapeutical studies for the treatment of the cancer.
A espécie Bauhinia pentandra (Bong.) Vog. ex Steud (Leguminosae), conhecida como mororó com espinho é comum do Nordeste brasileiro. A enzima DNA topoisomerase II-α (topo II) é responsável pelo relaxamento do DNA durante os processos de divisão celular e está envolvida em diversos tipos de câncer, sendo considerada um alvo potencial no tratamento desta doença. Este trabalho teve como objetivo estudar a composição química e atividade antioxidante das cascas das raízes de B. pentandra e a análise da atividade inibitória da topo II por compostos naturais e semi-sintéticos. O estudo químico da espécie vegetal resultou no isolamento do glicosídeo cianogênico litospermosida e de uma mistura dos esteróides β-sitosterol e estigmasterol. A litospermosida foi acetilada e as estruturas foram identificadas através de dados espectrométricos como IV e RMN1H e 13C, incluindo 2D. O extrato EtOH bruto e as frações hexânica, AcOEt, MeOH e a substância litospermosida foram submetidos aos testes antioxidantes (DPPH, ABTS, atividade quelatogênica do íon Fe2+). A determinação de fenólicos totais também foi realizada com o extrato e com estas frações. Todas as amostras mostraram atividade antioxidante, destacando-se a fração em AcOEt com melhor atividade e maior teor de fenólicos, então esta fração foi analisada por HPLC-DAD confirmando a presença de fenólicos. A atividade inibitória da topoisomerase II-α foi avaliada com as substâncias naturais litospermosida e γ- hidroxiferruginina A, isolado de Vismia guianensis e com o extrato EtOH bruto do látex desta espécie, e com as substâncias semi-sintéticas litospermosida peracetilada e dez flavonóides derivados de retusin, isolado de Solanum paludosum. Na concentração de 220 μM todas as amostras testadas apresentaram atividade, exceto a litospermosida e o seu derivado acetilado. Na concentração de 110 μM apenas dois flavonóides não inibiram a ação da topo II. A γ-hidroxiferruginina A inibiu a atividade da topo II em uma concentração mínima de 0,1 μM, sendo este antronóide prenilado um candidato promissor para estudos terapêuticos posteriores para o tratamento do câncer.
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Ding, Min. „Characterization of novel post-translational modifications of microtubule associated proteins and immunologically related modifications of dna topoisomerase II /“. The Ohio State University, 1996. http://rave.ohiolink.edu/etdc/view?acc_num=osu1487935958845204.
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Terry, Samantha Y. A. „A role for topoisomerase II alpha in chromosome damage in human cell lines“. Thesis, University of St Andrews, 2010. http://hdl.handle.net/10023/873.
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Human response to ionising radiation (IR) shows a wide variation. This is most clearly seen in the radiation-response of cells as measured by frequencies of chromosomal aberrations. Different frequencies of IR-induced aberrations can be conveniently observed in phytohaemagglutin-stimulated peripheral blood T-lymphocytes from both normal individuals and sporadic cancer cases, in either metaphase chromosomes or as micronuclei in the following cell cycle. Metaphase cells show frequent chromatid breaks, defined as chromatid discontinuities or terminal deletions, if irradiated in the G 2 -phase of the cell cycle. It has been shown that the frequency of chromatid breaks in cells from approximately 40% of sporadic breast cancer patients, are significantly higher than in groups of normal individuals. This suggests that elevated radiation-induced chromatid break frequency may be linked with susceptibility to breast cancer. It is known that chromatid breaks are initiated by a double strand break (DSB), but it appears that the two are linked only indirectly as repair kinetics for DSBs and chromatid breaks do not match. Therefore, the underlying causes of the wide variation in frequencies of chromatid breaks in irradiated T-lymphocytes from different normal individuals and from sporadic breast cancer cases are still unclear but it is unlikely to be linked directly to DSB rejoining. My research has focused on the mechanism through which chromatid breaks are formed from initial DSBs. The lack of a direct association suggested that a signalling process might be involved, connecting the initial DSB and resulting chromatid break. The signal model, suggested that the initial DSB is located within a chromatin loop that leads to an intra- or interchromatid rearrangement resulting in incomplete mis-joining of chromatin ends during the decatenation of chromatids during G 2 . It was therefore proposed that topoisomerase II alpha (topo IIα) might be involved, mainly because of its ability to incise DNA and its role in sister chromatid decatenation. During my PhD research I have used a strategy of altering topo II activity or expression and studying whether this alters IR-induced chromatid break frequency. The first approach involved cell lines that varied in topo IIα expression. The frequency of IR-induced chromatid breaks was found to correlate positively with topo IIα expression level, as measured in three different cell lines by immunoblotting, i.e. two cell lines with lower topo IIα expression exhibited lower chromatid break frequency. Topo II activity in these three cell lines was also estimated indirectly by the ability of a topo IIα poison to activate the G 2 /M checkpoint, and this related well with topo IIα expression. A second approach involved ‘knocking down’ topo IIα protein expression by silencing RNA (siRNA). Lowered topo IIα expression was confirmed by immunoblotting and polymerase chain reaction. SiRNA-lowered topo IIα expression correlated with a decreased IR-induced chromatid break frequency. In a third series of experiments cells were treated with ICRF-193, a topo IIα catalytic inhibitor. It was shown that inhibition of topo IIα also significantly reduced IR-induced chromatid breaks. I also showed that lowered chromatid break frequency was not due to cells with high chromatid break frequencies being blocked in G 2 as the mitotic index was not altered significantly in cells with lowered topo IIα expression or activity. These experiments show that topo IIα is involved in IR-induced chromatid break formation. The final experiments reported here attempted to show how topo II might be recruited in the process of forming IR-induced chromatid breaks. Hydrogen peroxide was used as a source of reactive oxygen species (reported to poison topo IIα) and it was shown that topo IIα under these conditions is involved in the entanglement of metaphase chromosomes and formation of chromatin ‘dots’ as well as chromatid breaks. Experiments using atomic force microscopy attempted to confirm these dots as excised chromatin loops. The possible role of topo IIα in both radiation- and hydrogen peroxide-induced primary DNA damage was also tested. It was shown that topo IIα does not affect radiation-induced DSBs, even though it does affect chromatid break frequency. Also, topo IIα does not affect hydrogen peroxide-induced DNA damage at low doses. The results support the idea that topo IIα is involved in the conversion of DSBs to chromatid breaks after both irradiation and treatment with hydrogen peroxide at a low concentrations. I have demonstrated that topo IIα is involved in forming IR-induced chromatid breaks, most likely by converting the initial DSBs into chromosomal aberrations as suggested by the signal model.
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Liu, Jia, und 刘佳. „Luminescent cyclometalated platinum (II) complexes with isocyanide ligands as nucleic acid probes, topoisomerase poisons and anti-cancers agents“. Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2011. http://hdl.handle.net/10722/209595.
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Coelho, Raquel Autran [UNIFESP]. „Expressão de topoisomerase II alfa e de caspase-3 ativada em lesão intra-epitelial cervical escamosa de baixo grau“. Universidade Federal de São Paulo (UNIFESP), 2008. http://repositorio.unifesp.br/handle/11600/9620.
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Objetivos: Estudar a expressao imuno-histoquimica de topoisomerase IIƒ¿ e de caspase-3 ativada, marcadores de proliferacao e de apoptose, respectivamente, a deteccao de DNA HPV e a evolucao da lesao cervical em mulheres portadoras de lesao intra-epitelial escamosa de baixo grau (LBG). Metodos: Foram avaliadas 40 mulheres portadoras de LBG e 32 sem neoplasia cervical, diagnosticadas por exame cito-colpo-histopatologico, quanto a imunoexpressao de topoisomerase IIƒ¿ e de caspase-3 ativada e quanto a deteccao de DNA HPV por PCR consensual (GP5+/GP6+) em material de esfregaco cervico-vaginal. Os achados foram relacionados as variaveis clinicas das pacientes e a evolucao clinica das lesoes cervicais em 12 meses. As pacientes assinaram termo de consentimento livre e esclarecido. Resultados: A media percentual de celulas imunomarcadas por topoisomerase foi de 11,71% e 4,13%, no grupo com LBG e controle, respectivamente, com diferenca estatisticamente significante. Observou-se que houve expressao de caspase-3 em 17 (42,5%) e em 5 (15,63%) pacientes com e sem LBG, respectivamente, com diferenca estatisticamente significante. Foi detectado HPV DNA em 65% das pacientes com LBG e em 59,4% das pacientes sem lesao cervical, sem relacao com a expressao de topoisomerase IIƒ¿ ou caspase-3. Na presenca de DNA-HPV, a expressao de topoisomerase IIƒ¿ no grupo com LBG foi significativamente maior do que em fragmentos sem lesao. Nao foi observada diferenca quanto a evolucao da lesao cervical em 12 meses de acordo com a imunoexpressao de topoisomerase IIƒ¿. Com relacao a caspase-3 ativada, a maioria das pacientes com imuno-histoquimica negativa teve regressao da lesao cervical. Conclusoes: A imunoexpressao de topoisomerase IIƒ¿ e de caspase-3 ativada podem ser considerados marcadores de proliferacao e de apoptose em lesao cervical de baixo grau, sem relacao com a presenca de DNA-HPV.
Purpose: To evaluate the correlation between the expression of topoisomerase II alpha, active caspase-3 and infection with human papillomavirus in low-grade cervical intraepithelial lesion and in the normal cervix, and whether they might influence susceptibility to, or evolution of, cervical lesion. Patients and methods: Forty cervical biopsies patients with low-grade cervical intraepithelial lesion and thirty-two with normal cervix were stained by immunohistochemistry for topoisomerase IIá and active caspase-3 and were investigated for the presence of HPV on exfoliated cells by general primer GP5+/6+ PCR amplification of DNA. These findings were correlated with clinicopathological features of the patients including their clinical outcome after twelve months. Subjects provided written informed consent. Results: Low-grade CIN patients as a group had a significantly higher expression of topoisomerase II alpha compared to controls, without correlation to disease outcome at 12 months. Caspase-3 was expressed in 42.5% of CIN patients and in 15.63% without disease, and most of women without caspase-3 receded cervical lesion. HPV DNA testing was positive in 65% of the patients with cervical lesion, and in 59.4% of the control group and was not associated to the expression of topoisomerase IIá or active caspase-3. In the presence of a positive HPV DNA testing, women with cervical lesion had a significantly higher expression of topoisomerase II alpha compared to controls. Conclusion: Topoisomerase II alpha and active caspase-3 might be useful diagnostic and prognostic markers in low-grade cervical lesions, delaying a better follow-up.
CNPq: 134106/2005-9
TEDE
BV UNIFESP: Teses e dissertações
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Frota, Maria Zeli Moreira, und 92-98231-9393. „Avaliação de métodos de extração de DNA e de identificação de dermatófitos por análise de PCR-RFLP“. Universidade Federal do Amazonas, 2011. http://tede.ufam.edu.br/handle/tede/5883.
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Dermatophytes comprise a group of filamentous fungi of great interest on public health because of their ability to parasitize keratinized tissues, such as skin, hair and nails, and for their wide distribution in the world. As a consequence of this parasitism, an infectious process of dermatophytosis is established, from which a variety of clinical manifestations can occur, affecting people of both genders and all age groups. Laboratory methods for mycological diagnosis do not always allow a clear an especific definition of the agent. In this study, different strategies for extraction of DNA, and molecular typing by PCR-RFLP, of seven dermatophyte species were assessed. Two target regions: ITS/rDNA and the topoisomerase II gene were evaluated, by testing three PCR protocols and three restriction enzymes (DdeI, HinfI, HaeIII). For the DNA extraction, the glass bead shaking technique for cell lysis, followed by Gustincich (1991) based mehod for DNA separation, demonstrated more advantages. Our results has demonstrated that the topoisomerase II gene is a suitable target region for identification of the seven major pathogenic dermatophyte fungal species, reinforcing previous studies, and pointed to a new PCR-RFLP protocol, which is based on a PCR of this gene using dPsD2 primer, followed by digestion of PCR products with HaeIII restriction enzyme.
Os dermatófitos compreendem um grupo de fungos filamentosos de grande interesse na área da saúde, devido à sua capacidade de parasitar os tecidos queratinizados, como a pele, pêlos e unhas, e à sua ampla distribuição no mundo. Como conseqüência desse parasitismo instala-se um processo infeccioso de dermatofitose, a partir do qual pode ocorrer uma diversidade de manifestações clínicas, acometendo pessoas de ambos os gêneros e de todos os grupos etários. Os métodos laboratoriais para o diagnóstico micológico nem sempre permitem uma clara definição do agente em nível de espécie. No presente estudo foram analisadas diferentes estratégias para a extração de DNA e para a identificação molecular por PCR-RFLP das principais espécies de dermatófitos. Duas regiões alvo, a região ITS/DNAr e o gene da topoisomerase II foram analisadas, testando-se três protocolos de PCR e três enzimas de restrição (DdeI, HinfI, HaeIII). Na extração do DNA, o método de lise utilizando pérolas de vidro e a separação do DNA com base no método de Gustincich (1991) demonstrou importantes vantagens. Nossos resultados demonstraram que o gene da topoisomerase II é uma região alvo adequada para identificação das sete principais espécies de fungos dermatófitos patogênicos, reforçando estudos anteriores, e apontaram para um novo protocolo de RFLP-PCR, que se baseia em uma PCR desse gene utilizando o primer dPsD2, seguido da digestão dos produtos obtidos com a enzima de restrição HaeIII.