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Showing 1 to 8 of 8 (0.041 seconds)Spelling suggestions: "subject:"circular DNA."" "subject:"dircular DNA.""
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Quantitation of hepatitis B virus covalently closed circular DNA in chronic hepatitis B patientsWong, Ka-ho, Danny., 王嘉豪. January 2004 (has links)
published_or_final_version / abstract / toc / Medicine / Doctoral / Doctor of Philosophy
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The design and application of a real-time PCR assay to assess rcDNA and cccDNA produced by HBV during infectionBloom, Kristie Michelle 30 August 2010 (has links)
Chronic hepatitis B virus (HBV) infection is endemic to sub-Saharan Africa, and despite the
availability of anti-viral agents, there is currently no cure. This double stranded DNA virus is
hepatotropic, and active viral replication results in two genomic equivalents, the relaxed circular
DNA (rcDNA) and covalently closed circular DNA (cccDNA). The virion encapsulated rcDNA
contains a partially synthesised positive DNA stand and a gap region within the negative strand.
After infection of hepatocytes, the rcDNA is repaired in the nucleus to form cccDNA. An
important objective of HBV therapy is the elimination of cccDNA, as its persistence within
hepatocytes has been attributed to chronic HBV infection. Therefore a reliable assay for this
replication intermediate is crucial. The objective of this study was to develop a method based
on real-time PCR to detect and quantify HBV cccDNA. PCR primers which flank the rcDNA gap
were designed to amplify cccDNA whilst primers flanking the pre-S1 region quantify total HBV
DNA. Viral DNA was extracted from HepG2.2.15 cells, along with serum and livers from HBV
transgenic mice. According to this assay, cccDNA was readily detectable in transgenic mouse
livers, but was present at low concentrations in serum samples. The intrahepatic HBV DNA
profile of transgenic mice was found to be 40% cccDNA to 60% rcDNA. In HepG2.2.15 cells,
only 2% of HBV DNA was cccDNA whilst the majority was in the form of rcDNA. These results
were validated using non-radioactive Southern blothybridisation. Additionally, it was established that although RNAi-based effecters inhibit HBV replication, established cccDNA
pools were not eliminated. Real-time PCR provides a convenient platform for HBV cccDNA
detection as it allows for the rapid simultaneous amplification and quantification of a specific
DNA target through either non-specific or specific DNA detection chemistries. In conclusion, this
HBV qPCR assay should enable improved monitoring of patients’ responses to antiviral therapy
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Novel methods for specific detection and quantification of covalently closed circular DNA in sera and biopsies of hepatitis B patients. / CUHK electronic theses & dissertations collectionJanuary 2011 (has links)
In conclusion, two new methods of cccDNA quantitation were developed and validated. The two assays are complementary to each other and may be used in patients with extreme HBV DNA levels. These cccDNA assays should be further validated in larger studies and may become important tests for diagnostic, prognostic and treatment monitoring purposes. / Over 350 million people worldwide suffer from chronic hepatitis B virus (HBV) infection, which leads to many cases of cirrhosis and hepatocellular carcinoma. HBV covalently closed circular DNA (cccDNA) is a critical intracellular replicative intermediate and cannot be eliminated during antiviral therapy. Current methods for cccDNA detection are limited by false positive detection due to the interference by HBV relaxed circular DNA (rcDNA). The tests also have limited sensitivity to detect cccDNA at low concentrations. Hence, we aimed to develop a highly sensitive and highly specific assay for cccDNA detection with wide linear range. / The modified Bowden's assay had the highest intrahepatic cccDNA detection rate (60 positive results out of 61 cases). The detection rate of the modified Bowden's assay is significantly higher than that of the Bowden's assay. On the other hand, the cccDNA detection rate in serum samples was low at 20--27% by all 3 assays. In 5 samples in which cccDNA was undetectable by the Bowden's assay but detectable by the other two assays, a point mutation in the HBV genome was found in the forward primer binding site of the Bowden's assay. This partly explained the false negative results. / The quantification result of cccDNA by the bisulfite conversion assay was significantly lower than that by the Bowden's assay assay (P=0.001) and the modified Bowden's assay (P=0.003). When the total HBV DNA was higher than 107 copies/ml, the serum cccDNA level detected by the bisulfite conversion assay was significantly lower than that detected by the Bowden's assay (P=0.008) and the modified Bowden's assay (P=0.046). When the total HBV DNA is less than 107 copies/ml, there were no significant differences. This suggests that the bisulfite conversion assay was less affected by rcDNA even in samples containing a high viral load. / With this background, two new cccDNA assays were developed and optimized. Bowden's assay was used as a standard to evaluate the performance of new assays. The first new assay (modified Bowden's assay) involved the use of new primers and probes that targeted more conserved regions in the HBV genome. The second assay adopted the bisulfite conversion method, which introduced gene sequence changes into the HBV genome and thereby enhance the specificity of the assay. Capillary sequencing was performed to find mutations in primers and probe range of different assays. / Yu, Ling. / Advisers: Vincent Wai-Sun Wang; Joseph Jao-Yiu Sung. / Source: Dissertation Abstracts International, Volume: 73-06, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 105-111). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.
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Caractérisation du microDNome et sa modulation par le traitement anti-cancerMehanna, Pamela 11 1900 (has links)
Récemment, une nouvelle classe d'ADN circulaire extrachromosomique (eccDNA) appelée microADN a été identifiée dans des tissus humains et murins. Ces microADNs ont une longueur de 100 à 400 pb, sont dérivés de régions génomiques non répétitives uniques et présentent un enrichissement au niveau des régions géniques et riches en GC. Bien qu'il ait été proposé qu'ils puissent provenir du métabolisme de l'ARN ou des défauts de réplication, leurs mécanismes de production et leur éventuelle fonctionnalité restent à déterminer. Grâce à l'analyse des microADNs extraits d'une série de 10 lignées cellulaires lymphoblastoïdes humaines (LCL), nous avons confirmé la distribution nonaléatoire des microADNs vers les régions actives du génome. Les microADNs identifiés présentaient
des loci d'origine redondants et une périodicité de taille de 190 pb pouvant correspondre à la fragmentation de l'ADN lors de l'apoptose caspase-dépendante. L'apoptose induite de ces LCLs par des drogues chimiothérapeutiques (méthotrexate ou L-asparaginase) a entrainé la modulation de la diversité et de la taille des microADNs, suggérant qu'une partie de ces entités pourrait être des produits résiduels de la mort cellulaire apoptotique. Ainsi, bien que compatible avec l'observation initiale suggérant que les microADNs proviennent d'un processus physiologique normal, ces résultats impliquent une source de production alternative ou complémentaire. / Recently, a new class of extrachromosomal circular DNA (eccDNA) called microDNA was identified in mouse and human tissues. These microDNAs are 100 to 400 bp long, derive from unique nonrepetitive genomic regions and show an enrichment in GC rich and genic sequences. While it has been proposed that they could arise from RNA metabolism or replication defects, their production mechanisms and eventual functionality remain unclear. Through the analysis of microDNAs extracted from a series of 10 human lymphoblastoid cell lines (LCLs), we confirmed the non-random distribution of microDNA towards active regions of the genome. Identified microDNAs showed redundant loci of origin and a size periodicity of 190 bp that matched caspase-dependant DNA fragmentation of apoptotic cells. Strikingly, the chemotherapeutic drug-induced apoptosis (using methotrexate or Lasparaginase) of these LCLs modulated both diversity and size of microDNAs further suggesting that a part of microDNAs could represent circularized by-products of the programmed cell death. Thus, while compatible with the original observation that microDNAs originated from a normal physiological process, these results imply an alternative or complementary source of production.
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Instability and Extrachromosomal Circular DNA Formation at Microsatellites and Unstable DNA SequencesShanahan, Matilyn M. 02 September 2022 (has links)
No description available.
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Évaluation d’une nouvelle approche vaccinale basée sur l’électroporation in vivo d’ADN pour le traitement des hépatites B chroniques / Evaluation of a new vaccinal approach based on DNA delivery by in vivo electroporation for chronic hepatitis B therapyKhawaja, Ghada 23 March 2012 (has links)
Malgré l’existence d’un vaccin préventif efficace, l’infection chronique par le virus de l’hépatite B (HBV) demeure un problème majeur de santé publique. La persistance de l’infection par HBV étant clairement associée à des réponses immunitaires insuffisantes, l’immunothérapie par le vaccin à base d’ADN nu, visant à stimuler les réponses humorales et cellulaires, apparaît comme particulièrement pertinente pour la thérapie des hépatites B chroniques. Toutefois, l’efficacité thérapeutique d’une telle stratégie reste limitée chez l’homme, d’où la nécessité d’optimiser cette approche vaccinale pour une utilisation ultérieure en clinique. Ainsi, l’objectif général de ce travail de thèse était d’explorer, avec le modèle du DHBV (« Duck Hepatitis B Virus »), étroitement apparenté au HBV humain, si l’administration du vaccin à ADN par électroporation (EP) pouvait davantage améliorer son efficacité prophylactique et thérapeutique. Nous avons montré, dans un 1er temps chez des canards naïfs, que l’administration du vaccin à ADN par EP permet de potentialiser le pouvoir neutralisant et d’élargir le répertoire épitopique de la réponse humorale dirigée contre la protéine d’enveloppe du DHBV, même avec des doses d’ADN relativement faibles. Dans un 2ème temps, nous avons montré chez des animaux chroniquement infectés par le DHBV, que l’administration par EP du vaccin à ADN ciblant les protéines structurales du DHBV et le DuIFN-γ améliore considérablement l’efficacité thérapeutique du vaccin, notamment au regard de la séroconversion et de la clairance virale. Les résultats ainsi obtenus confirment l’intérêt majeur de cette approche vaccinale pour la thérapie des hépatites B chroniques / Despite the existence of an effective prophylactic vaccine, chronic hepatitis B virus (HBV) infection remains a major public health problem. Since persistence of HBV infection is mostly associated with insufficient immune responses, therefore DNA vaccination capable of activating both humoral and cellular immune responses appears as a pertinent strategy for chronic hepatitis B therapy. However, the efficacy of such therapeutic approach remains limited in humans. Improvement of DNA vaccine efficacy is therefore needed for future therapeutic applications in clinic. The main objective of this thesis was to investigate in the duck hepatitis B virus (DHBV) model, whether the protective and therapeutic efficacy of DNA vaccine can be enhanced using EP-based delivery system. Firstly, we showed in naïve ducks that EP-based delivery was able to improve the dose efficiency of DNA vaccine and to maintain a highly neutralizing, multi-specific B-cell response even with relatively low DNA doses, suggesting that it may be an effective approach for chronic hepatitis B therapy at clinically feasible DNA dose. Secondly, we showed in chronic DHBV-carriers that in vivo EP is able to dramatically enhance the therapeutic potency of DNA vaccine targeting hepadnaviral proteins. Indeed, this approach was able to consistently restore humoral immune response and to sustainably decrease and even clear viral infection. Thus, these data strongly support the use of this approach for chronic hepatitis B therapy in humans
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ECCsplorer: a pipeline to detect extrachromosomal circular DNA (eccDNA) from next-generation sequencing dataMann, Ludwig, Seibt, Kathrin M., Weber, Beatrice, Heitkam, Tony 22 May 2024 (has links)
Backround:
Extrachromosomal circular DNAs (eccDNAs) are ring-like DNA structures physically separated from the chromosomes with 100 bp to several megabasepairs in size. Apart from carrying tandemly repeated DNA, eccDNAs may also harbor extra copies of genes or recently activated transposable elements. As eccDNAs occur in all eukaryotes investigated so far and likely play roles in stress, cancer, and aging, they have been prime targets in recent research—with their investigation limited by the scarcity of computational tools.
Results:
Here, we present the ECCsplorer, a bioinformatics pipeline to detect eccDNAs in any kind of organism or tissue using next-generation sequencing techniques. Following Illumina-sequencing of amplified circular DNA (circSeq), the ECCsplorer enables an easy and automated discovery of eccDNA candidates. The data analysis encompasses two major procedures: first, read mapping to the reference genome allows the detection of informative read distributions including high coverage, discordant mapping, and split reads. Second, reference-free comparison of read clusters from amplified eccDNA against control sample data reveals specifically enriched DNA circles. Both software parts can be run separately or jointly, depending on the individual aim or data availability. To illustrate the wide applicability of our approach, we analyzed semi-artificial and published circSeq data from the model organisms Homo sapiens and Arabidopsis thaliana, and generated circSeq reads from the non-model crop plant Beta vulgaris. We clearly identified eccDNA candidates from all datasets, with and without reference genomes. The ECCsplorer pipeline specifically detected mitochondrial mini-circles and retrotransposon activation, showcasing the ECCsplorer’s sensitivity and specificity.
Conclusion:
The ECCsplorer (available online at https://github.com/crimBubble/ECCsplorer) is a bioinformatics pipeline to detect eccDNAs in any kind of organism or tissue using next-generation sequencing data. The derived eccDNA targets are valuable for a wide range of downstream investigations—from analysis of cancer-related eccDNAs over organelle genomics to identification of active transposable elements.
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Cis-regulation and genetic control of gene expression in neuroblastomaBurkert, Christian Martin 28 June 2021 (has links)
Genregulation beeinflusst Phänotypen im Kontext von Gesundheit und Krankheit. In Krebszellen regulieren genetische und epigenetische Faktoren die Genexpression in cis. Das Neuroblastom ist eine Krebserkrankung, die häufig im Kindesalter auftritt. Es ist gekennzeichnet durch eine geringe Anzahl exonischer Mutationen und durch häufige Veränderungen der somatischen Kopienzahl, einschließlich Genamplifikationen auf extrachromosomaler zirkulärer DNA. Bisher ist wenig darüber bekannt, wie lokale genetische und epigenetische Faktoren Gene im Neuroblastom regulieren. In dieser Arbeit kombiniere ich die allelspezifische Analyse ganzer Genome (WGS), Transkriptome und zirkulärer DNA von Neuroblastom-Patienten, um genetische und cis-regulatorische Effekte zu charakterisieren. Ich zeige, dass somatische Dosis-Effekte der Kopienzahl andere lokale genetische Effekte dominieren und wichtige Signalwege regulieren. Genamplifikationen zeigen starke Dosis-Effekte und befinden sich häufig auf großen extrachromosomalen zirkulären DNAs. Die vorgestellte Analyse zeigt, dass der Verlust von 11q zu einer Hochregulation von Histonvarianten H3.3 und H2A in Tumoren mit alternativer Verlängerung der Telomere (ALT) führt, und dass erhöhte somatische Kopienzahl die Expression der TERT Gens verstärken können. Weitere Erkenntnisse sind, dass 17p-Ungleichgewichte und die damit verbundene Herunterregulierung neuronaler Gene sowie die Hochregulierung des genomisch geprägten Gens RTL1 durch Kopienzahl-unabhängige allelische Dosis-Effekte mit einer ungünstigen Prognose verbunden sind. Die cis-QTL-Analyse bestätigt eine zuvor beschriebene Regulation des LMO1 Gens durch einen Enhancer-Polymorphismus und charakterisiert das regulatorische Potenzial weiterer GWAS-Risiko-Loci. Die Arbeit unterstreicht die Bedeutung von Dosis-Effekten im Neuroblastom und liefert eine detaillierte Übersicht regulatorischer Varianten, die in dieser Krankheit aktiv sind. / Gene regulation controls phenotypes in health and disease. In cancer, the interplay between germline variation, genetic aberrations and epigenetic factors modulate gene expression in cis. The childhood cancer neuroblastoma originates from progenitor cells of the sympathetic nervous system. It is characterized by a sparsity of recurrent exonic mutations but frequent somatic copy-number alterations, including gene amplifications on extrachromosomal circular DNA. So far, little is known on how local genetic and epigenetic factors regulate genes in neuroblastoma to establish disease phenotypes. I here combine allele-specific analysis of whole genomes, transcriptomes and circular DNA from neuroblastoma patients to characterize genetic and cis-regulatory effects, and prioritize germline regulatory variants by cis-QTLs mapping and chromatin profiles. The results show that somatic copy-number dosage dominates local genetic effects and regulates pathways involved in telomere maintenance, genomic stability and neuronal processes. Gene amplifications show strong dosage effects and are frequently located on large but not small extrachromosomal circular DNAs. My analysis implicates 11q loss in the upregulation of histone variants H3.3 and H2A in tumors with alternative lengthening of telomeres and cooperative effects of somatic rearrangements and somatic copy-number gains in the upregulation of TERT. Both 17p copy-number imbalances and associated downregulation of neuronal genes as well as upregulation of the imprinted gene RTL1 by copy-number-independent allelic dosage effects is associated with an unfavorable prognosis. cis-QTL analysis confirms the previously reported regulation of the LMO1 gene by a super-enhancer risk polymorphism and characterizes the regulatory potential of additional GWAS risk loci. My work highlights the importance of dosage effects in neuroblastoma and provides a detailed map of regulatory variation active in this disease.
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