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Lintell, Nicholas Adrian, i n/a. "DNA Aberrations in Atypical Cancer Cohorts". Griffith University. School of Biomolecular and Biomedical Science, 2006. http://www4.gu.edu.au:8080/adt-root/public/adt-QGU20061009.164402.
Pełny tekst źródłaLintell, Nicholas Adrian. "DNA Aberrations in Atypical Cancer Cohorts". Thesis, Griffith University, 2006. http://hdl.handle.net/10072/365589.
Pełny tekst źródłaThesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Biomolecular and Biomedical Sciences
Full Text
Martorano, Laura. "The zebrafish orthologue of the human hepatocerebral disease gene MPV17 plays pleiotropic roles in mitochondria". Doctoral thesis, Università degli studi di Padova, 2018. http://hdl.handle.net/11577/3424883.
Pełny tekst źródłaLe sindromi da deplezione del DNA mitocondriale (MDS) sono un gruppo di malattie rare a carattere autosomico recessivo con esordio precoce e prognosi infausta. Le MDS sono causate da mutazioni a carico di diversi geni nucleari, coinvolti nel mantenimento del DNA mitocondriale (mtDNA), e caratterizzate da una forte riduzione del numero di copie del mtDNA nei tessuti interessati, nonchè da gravi difetti nella funzionalità mitocondriale. Le mutazioni a carico di MPV17, un gene nucleare che codifica per una proteina di membrana mitocondriale interna, sono state specificatamente associate a forme epatocerebrali di MDS. Tuttavia, la funzione della proteina MPV17 non è nota, sebbene sia stato ipotizzato un ruolo primario nel mantenimento del mtDNA. Zebrafish rappresenta un modello per rispondere a questa domanda biologica: un mutante nullo per il gene mpv17, detto roy orbison (roy), presenta una delezione di 19 bp che causa uno splicing aberrante tra gli esoni 2 e 3 del gene e, di conseguenza, l’assenza della proteina codificata. Dal punto di vista fenotipico, il mutante roy mostra un difetto di pigmentazione, caratterizzato dall’assenza di cellule, chiamate iridofori, le quali conferiscono alla pelle, grazie al loro contenuto di cristalli di guanina, la sua proprietà riflettente. Il presente lavoro di tesi ha avuto come obiettivo principale la caratterizzazione dettagliata del fenotipo mitocondriale delle larve mutanti per il gene mpv17, nelle quali abbiamo rilevato, già a stadi precoci, importanti alterazioni dell’ultrastruttura mitocondriale nel fegato e una significativa perdita della funzionalità dei complessi della catena respiratoria (OXPHOS). I nostri risultati suggeriscono, pertanto, una funzione essenziale del gene mpv17 nel mantenimento delle creste mitocondriali e della piena attività dell’OXPHOS, mentre il suo effetto sulla stabilità del mtDNA, ipotizzato in letteratura, sembra essere consequenziale, considerando che la deplezione del mtDNA è rilevabile solo in fasi tardive dello sviluppo. Inoltre, considerando che nei mutanti roy orbison era stata precedentemente ipotizzata una funzione di Mpv17 nel metabolismo delle purine e che, inoltre, l’inibizione della sintesi di pirimidine negli embrioni wild-type causa un fenotipo assimilabile a quello dei roy, sono stati somministrati precursori purinici e pirimidinici agli embrioni omozigoti mutanti, al fine di osservare o meno un miglioramento del fenotipo. È interessante notare che la somministrazione di acido orotico (OA), prodotto dell’enzima mitocondriale Diidroorotato deidrogenasi (DHODH) e precursore delle pirimidine, ha migliorato il fenotipo roy, collegando quindi la perdita di Mpv17 alla sintesi de novo dei nucleotidi. In particolare, il trattamento con OA, attualmente usato come integratore alimentare, ha aumentato significativamente non solo il numero di iridofori ma anche il contenuto di mtDNA nei mutanti nulli di mpv17, aprendo così nuove prospettive nel trattamento delle malattie da deplezione del DNA mitocondriale causate da mutazioni a carico di MPV17.
Komulainen, T. (Tuomas). "Disturbances in mitochondrial DNA maintenance in neuromuscular disorders and valproate-induced liver toxicity". Doctoral thesis, Oulun yliopisto, 2015. http://urn.fi/urn:isbn:9789526207230.
Pełny tekst źródłaTiivistelmä Mitokondrion DNA:n (mtDNA) kahdentumisesta ja ylläpidosta vastaavien tuman geenien mutaatiot voivat johtaa mtDNA:n määrän vähenemiseen (depleetioon) ja katkoksiin (deleetioihin). MtDNA:n kahdentumisesta vastaavaa entsyymiä koodaa tuman POLG1-geeni. POLG1-mutaatioihin liittyy kohonnut riski sairastua natriumvalproaatin (VPA) aiheuttamaan akuuttiin maksavaurioon. Tutkimuksen tavoitteena oli tutkia mtDNA:n depleetion ja deleetioiden molekyyligeneettistä etiologiaa ja kliinisiä taudinkuvia. Tutkimuksessa selvitettiin myös POLG1-mutaatioihin liittyviä taudinkuvia ja POLG1-mutaatioihin liittyvän akuutin maksavaurion patomekanismia. VPA:n vaikutusta mitokondrioiden toimintaan tutkittiin in vitro HepG2-solumallissa. Tutkimuksessa todettiin mtDNA:n depleetion liittyvän vaikeaan varhain alkavaan aivosairauteen. Depleetio todettiin myös sekundaarisena merosiini-negatiivisessa lihasdystrofiassa. Kahdella Kearns-Sayren syndroomaa sairastavalla potilaalla todettiin multippelit mtDNA:n deleetiot, mikä viittaa syndrooman geneettisen alkuperään. POLG1 p.R722H-mutaatiota on aiemmin pidetty neutraalina polymorfiana, mutta tutkimuksen tulokset viittasivat siihen, että homotsygoottisena tai yhdistelmäheterotsygoottisena mutaatio on tautia aiheuttava. Helsingin yliopistollisen sairaalan elinsiirtorekisteristä tunnistettiin retrospektiivisesti viisi potilasta, jotka olivat saaneet maksansiirteen VPA:n aiheuttaman maksavaurion vuoksi. Kaikilla viidellä potilaalla todettiin POLG1-geenin mutaatio, mikä vahvistaa käsitystä geenin yhteydestä VPA:n aiheuttamaan maksavaurioon. POLG1-mutaatioita on pidetty vasta-aiheena maksansiirrolle, mutta tutkimuksessa todettiin homotsygoottisena esiintyvän POLG1-mutaation ja nuoruusiällä tai varhaisella aikuisiällä alkaneen taudin liittyvän parempaan maksansiirron jälkeiseen ennusteeseen. HepG2-solumallilla tehdyt tutkimukset osoittivat VPA:n haittaavan mitokondrioiden solyhengitystä. Tutkimuksen tulokset tuovat lisätietoa mtDNA:n depleetioon ja deleetioihin liittyvistä taudinkuvista ja molekyyligeneettisestä taustasta. POLG1-mutaatiot eivät ole ehdoton vasta-aihe maksansiirrolle; potilaan geneettinen status ja ikä taudin alkamishetkellä vaikuttavat ennusteeseen, mikä tulisi huomioida potilaiden hoidossa. Tulokset myös osoittivat VPA:n olevan mitokondriotoksinen lääke, jonka käyttöä tulisi välttää mitokondriotautipotilaiden hoidossa
Hine, Donna Louise. "Mitochondrial DNA depletion and insulin secretion". Thesis, University of Newcastle upon Tyne, 2013. http://hdl.handle.net/10443/1906.
Pełny tekst źródłaLomartire, Laura <1982>. "Down Syndrome: Neuropsychological phenotype and mitochondrial DNA". Doctoral thesis, Alma Mater Studiorum - Università di Bologna, 2012. http://amsdottorato.unibo.it/4552/1/Lomartire_Laura_tesi.pdf.
Pełny tekst źródłaLomartire, Laura <1982>. "Down Syndrome: Neuropsychological phenotype and mitochondrial DNA". Doctoral thesis, Alma Mater Studiorum - Università di Bologna, 2012. http://amsdottorato.unibo.it/4552/.
Pełny tekst źródłavan, der Watt George Frederick. "Whole Blood Mitochondrial DNA Depletion in Human Immunodeficiency Virus-Infected Children". Master's thesis, University of Cape Town, 2010. http://hdl.handle.net/11427/2705.
Pełny tekst źródłaRusanen, H. (Harri). "Pathophysiological and clinical consequences of the mitochondrial DNA 3243A→G mutation". Doctoral thesis, University of Oulu, 2000. http://urn.fi/urn:isbn:9514255380.
Pełny tekst źródłaCupp, John D. "Characterization of the Cellular and Organellar Dynamics that Occur with a Partial Depletion of Mitochondrial DNA when Arabidopsis Organellar DNA Polymerase IB is Mutated". BYU ScholarsArchive, 2012. https://scholarsarchive.byu.edu/etd/3747.
Pełny tekst źródłaWheeler, Joel. "USING SOUTHERN BLOTTING AND NON-RADIOACTIVE PROBE HYBRIDIZATION AS A TOOL TO MEASURE 2’,3’-DIDEOXYCYTIDINE INDUCED MITOCHONDRIAL DNA DEPLETION IN HUMAN CELL LINES". OpenSIUC, 2019. https://opensiuc.lib.siu.edu/theses/2637.
Pełny tekst źródłaJärviaho, T. (Tekla). "Germline predisposition to childhood acute lymphoblastic leukemia and bone marrow failure, and mitochondrial DNA variants in leukemia". Doctoral thesis, Oulun yliopisto, 2018. http://urn.fi/urn:isbn:9789526220437.
Pełny tekst źródłaTiivistelmä Akuutti lymfoblastileukemia (ALL) on lasten yleisin syöpä. Vaikka nykyisin noin 90 prosenttia paranee, ALL aiheuttaa huomattavan paljon sairastavuutta ja on merkittävä lasten kuolinsyy. Vastikään on löydetty perinnöllisiä geneettisiä muutoksia, jotka altistavat lapsuusiän ALL:lle. Tutkimuksen kohteena oli kaksi perhettä, joissa vähintään kaksi lasta on sairastunut ALL:aan. Ensimmäisessä perheessä havaittiin lapsuusiän ALL:aan sairastuneilla kehityshäiriöisillä sisaruksilla äidiltä periytyvä heterotsygoottinen deleetio kromosomissa 7p12.1p13, jossa sijaitsee IKZF1-geeni. Toisessa perheessä perinnöllinen kahden kromosomin translokaatio todettiin kahdella lapsuusiän ALL:aan sairastuneella sekä seitsemällä perheenjäsenellä. Balansoitu translokaatio t(12;14)(p13.2;q23.1) aiheuttaa katkaisukohdan ETV6-geeniin kromosomissa 12 ja RTN1-geeniin kromosomissa 14. Tähän mennessä on julkaistu vain muutamia tutkimuksia potilaista, joilla on ollut perinnöllinen muutos joko IKZF1- tai ETV6-geenissä. Näillä geeneillä oletetaan olevan tärkeä merkitys perinnöllisessä alttiudessa sairastua lapsuusiän ALL:aan. Perinnölliset luuytimen toimintahäiriöt voivat altistaa leukemialle, kuten ALL:lle. Kahdella lapsella todettiin luuytimen toimintahäiriö, mutta ei muita oireita, jotka voisivat liittyä tyypillisiin perinnöllisiin luuytimen toimintahäiriöihin. Eksomisekvensoinnissa todettiin identtinen, homotsygoottinen mutaatio c.1457del(p.(Ile486fs)) ERCC6L2-geenissä. Kirjallisuuslähteiden mukaan vain muutamalla potilaalla on todettu ERCC6L2-geenin muutoksesta johtuva luuytimen toimintahäiriö. Osalla heistä on ollut synnynnäisiä kallon ja kasvojen anomalioita sekä kehityshäiriö, jollaisia tähän tutkimukseen osallistuneilla potilailla ei todettu. Potilaskohorttitutkimuksessa tutkittiin mitokondriaalisen DNA:n (mtDNA) muutoksia ALL:aan sairastuneilla lapsilla. Syöpäsolut eivät hyödynnä mitokondrion elektroninsiirtoketjua energian tuotantoon, ja tämä aineenvaihdunnan muutos on tunnustettu syövän ominaisuus. Tutkimuksessa havaittiin, että 22 prosentilla potilaista ilmeni diagnoosivaiheessa poikkeavia mtDNA:n muutoksia, jotka olivat elektroninsiirtoketjun entsyymien alayksiköitä koodaavissa geeneissä. Muutoksia todettiin useimmiten potilailla, joilla oli leukemiasoluissa huonon ennusteen geneettinen tekijä. Havaitut muutokset voivat mahdollisesti vaikuttaa leukemiasolun energia-aineenvaihduntaan
Schubert, Susanne, Sandra Heller, Birgit Löffler, Ingo Schäfer, Martina Seibel, Gaetano Villani i Peter Seibel. "Generation of rho zero cells". Universitätsbibliothek Leipzig, 2015. http://nbn-resolving.de/urn:nbn:de:bsz:15-qucosa-167888.
Pełny tekst źródłaPätsi, J. (Jukka). "Catalytic core of respiratory chain NADH-ubiquinone oxidoreductase:roles of the ND1, ND6 and ND4L subunits and mitochondrial disease modelling in Escherichia coli". Doctoral thesis, Oulun yliopisto, 2011. http://urn.fi/urn:isbn:9789514294723.
Pełny tekst źródłaTiivistelmä 45 alayksiköstä muodostuva NADH-ubikinoni oksidoreduktaasi (kompleksi I) on nisäkkäiden suurimpia entsyymejä. Sen mitokondriaalisessa DNA:ssa koodattujen alayksiköiden ND1-ND6 ja ND4L geeneihin liittyvät mutaatiot ovat yleisiä mitokondriosairauksien, kuten Leberin perinnöllisen näköhermoatrofian (LHON) ja MELAS-oireyhtymän, syitä. Bakteerien vastaava entsyymi (NDH-1) koostuu vain 13–14 alayksiköstä. Tästä huolimatta sen katalysoima reaktio on samankaltainen kuin kompleksi I:n. NDH-1:n katsotaankin edustavan entsyymin katalyyttistä ydintä. Tässä työssä tutkittiin ND1, ND6 ja ND4L alayksiköiden tehtävää kompleksi I:ssä niiden Escherichia coli bakteerissa olevien vastineiden (NuoH, NuoJ ja NuoK) kohdennetun mutageneesin avulla. Samaa lähestymistapaa käytettiin LHON/MELAS-oireyhtymässä todettujen ND1 alayksikön mutaatioiden, m.3376G>A ja m.3865A>G, ja LHON:ssa havaitun ND6:n m.14498T>C mutaation aiheuttamien aminohappomuutosten seurauksien selvittämiseen. Tehtyjen mutaatioiden vaikutuksia arvioitiin entsyymiaktiivisuus-mittauksin ja kasvukokein. NuoK:n solukalvon läpäisevissä rakenteissa olevien kahden glutamaatti-aminohappotähteen sijainti muistuttaa protoneita kalvon läpi kuljettavissa proteiineissa todettua. NuoK:n glutamaattien havaittiinkin olevan tärkeitä elektronien ja protonien kuljetukselle kompleksi I:ssä. m.3376G>A ja m.14498T>C mutaatioiden aiheuttamien aminohappomuutosten vastineet NDH-1:ssä alensivat NDH-1:n elektroninsiirtoaktiivisuutta ja heikensivät ubikinonin sitoutumista, kun taas m.3865A>G mutaatiolla ei ollut vaikutusta. Muut NuoH ja NuoJ alayksiköihin tehdyt aminohappovaihdokset johtivat huonontuneeseen ubikinonin ja kompleksi I:n inhibiittoreiden sitoutumiseen. Saadut tulokset vahvistavat m.3376G>A ja m.14498T>C mutaatioiden patogeenisyyden. Ne myös osoittavat, että LHON/MELAS-oireyhtymään liitetyn mutaation biokemiallisissa vaikutuksissa yhdistyvät sekä LHON:ssa että MELAS-oireyhtymässä todettujen mutaatioiden seuraukset. Esitetyt tulokset tukevat näkemystä siitä, että ubikinonin ja kompleksi I:n välisessä vuorovaikutuksessa tapahtuva muutos on kaikille LHON:aan liitetyille mutaatioille yhteinen vaikutusmekanismi. NuoH:n, NuoJ:n ja NuoK:n kohdennetusta mutageneesista saatujen tulosten perusteella ND1 ja ND6 alayksiköt ovat osa ubikinonin sitoutumispaikkaa entsyymikompleksissa, kun taas ND4L osallistuu protoninkuljetukseen
Duarte, Sara Filipa Rosa. "Genotype-phenotype correlation in mitochondrial depletion syndrome due to DGUOK deficiency". Master's thesis, 2014. http://hdl.handle.net/10316/30981.
Pełny tekst źródłaO défice da desoxiguanosina cinase (dGUOK), causado por mutações no gene DGUOK, leva ao desenvolvimento da forma hepatocerebral da Síndrome de depleção de DNA mitocondrial (MDS), ligadas a uma redução drástica no número de cópias do genoma mitocondrial (mtDNA), associada a elevada mortalidade e sem estratégias terapêuticas disponíveis, na criança. Apesar de se terem vindo a identificar novas mutações no gene DGUOK ao longo dos últimos anos, uma correlação genótipo-fenótipo precisa e exequível, nunca foi estabelecida. Um padrão reprodutível de associação entre o tipo e localização das mutações genéticas no gene DGUOK e a clínica apresentada pelo doente facultaria aos clínicos a capacidade de estimar, num estádio neonatal, ou mesmo pré-natal, o diagnóstico e a gravidade da doença, provendo os clínicos com ferramentas mais racionais para aconselhamento genético adequado às famílias e aos futuros pais. Procedeu-se a uma busca extensiva na base de dados de referências MEDLINE para procurar todas as mutações no gene DGUOK, publicadas até à data. Estas alterações foram analisadas quanto à sua localização no gene e o seu impacto na função e estrutura da proteína, assim como nas manifestações clínicas apresentadas pelos doentes e foi efetuado um estudo da correlação genótipo-fenótipo. Verificou-se a inexistência de uma correlação genótipo-fenótipo precisa e clara em doentes com MDS causado por mutações no gene DGUOK, revelando a existência de mecanismos mais complexos e ainda desconhecidos, que estarão subjacentes ao início e à progressão da doença
Deoxyguanosine kinase (coded by DGUOK) deficiency, caused by DGUOK gene mutations, leads to development of the hepatocerebral form of mitochondrial DNA depletion syndrome (MDS), linked to mitochondrial genome (mtDNA) copy number severe decline, associated to high mortality and absence of available therapeutic strategies in infants. In spite of the continuous identification of new DGUOK mutations over the years, an accurate and feasible genotype-phenotype correlation in DGUOK patients is lacking. A reproducible pattern of association between the type and localization of DGUOK gene mutation and its effect on patients´ clinic would ground physicians with the capacity to foresee, at a neonatal or even pre-natal stage, the disease diagnostics and severity, providing the clinicians, therefore, with more rationale tools for adequate genetic counselling in families and future parents. An extensive search on MEDLINE database was performed in order to search for all published DGUOK gene mutations to date. These alterations were analysed for their localization in the gene and their impact on protein structure and function and the clinical manifestations and a genotype-phenotype correlation study has been carried out. It was evident the absence of a clear and accurate genotype-phenotype correlation in patients with MDS caused by DGUOK mutations, unmasking, likely, more complex and unknown mechanisms underlying disease onset and progression
TSUN-YING, HUANG, i 黃純英. "Mitochondrial DNA Variations in Sudden Infant Death Syndrome". Thesis, 2005. http://ndltd.ncl.edu.tw/handle/84094129636137566490.
Pełny tekst źródła臺北醫學大學
醫學技術學系
93
Abstract The Sudden Infant Death Syndrome (SIDS) is one kind of leading cause of postneonatal infant death which also rises a difficult problem between the legal and medical systems. SIDS is defined as the sudden death of an infant less than 12 month old that remains unexplained after a complete clinical review, autopsy and death scene investigation. Despite the fact that many hypotheses have been proposed, the causes of SIDS are still uncertain. Although a number of coding region mtDNA mutations involving SIDS have been reported, the role of mtDNA variations in SIDS victims is still not known. The purpose of this study was to investigate whether the mtDNA variations or large-scale deletion exert any effect on the etiology of SIDS. Moreover, the biologic significance of the hOGG1 1245C"G polymorphism for SIDS has not yet been elucidated. The polymorphism of the hOGG1 gene was assessed by using a PCR-restriction fragement length polymorphism (RFLP) method. Seven SIDS and nineteen non-SIDS victims were enrolled. We determined the relative amount of mtDNA copy number and the occurrence of mtDNA deletion in blood, skeletal muscle and cardiac muscle from both SIDS and non-SIDS group using real-time quantitative PCR, primer-shift PCR analysis and DNA sequencing. Buccal epithelial samples from twenty age-matched live health control subjects were also examined. In this study we also explore the DNA sequence variation of D-loop hypervariable region I(HVR I) and HVR II. D-loop sequence of all subjects were sequenced and compared with the Cambridage sequence. The mean number of substitutions in HVR I and HVR II between SIDS, non-SIDS and live health control were no significant difference. Three types of mtDNA deletions were found in this study such as 4977, 5335 and 7599 bp deletion. The breakpoints of deletion were identified by sequencing method. In live health control group only one subject was found with 7599 bp deletion from buccal epithelial sample. Only one specimen was found with 4977 bp mtDNA deletion from cardiac muscle in congenital heart malformation subject. The frequencies of occurrence of 5335 and 7599 bp deletions in SIDS and non-SIDS were much higher than live health control group. There were no statistically significant difference associated with the frequencies of occurrence of 5335 bp and 7599 bp mtDNA deletions between SIDS and non-SIDS victims was found. The frequency of occurrence of 5335 and 7599 bp mtDNA deletion in blood from SIDS were four and two fold to non-SIDS, respectively. The frequency of occurrence of 5335 bp mtDNA deletion in skeletal muscle was 1.8 fold to non-SIDS. No significant correlation between the relative amount of mtDNA copy number and the frequencies of occurrence of mtDNA deletions in SIDS and non-SIDS. The allelic frequency of hOGG1 1245G was 78.6 % in SIDS, 61.1 % in non-SIDS and 45.0 % in live health control. The genotypic frequencies were no statistically significance between SIDS and live health control(p=0.062) and between non-SIDS and live health control(p=0.246). These defects in mtDNA may result in impaired production of ATP or sensitized cells to apoptosis. We proposed that mtDNA deletions in themselves do not cause SIDS but may cause energy deficiency or hypoxia in stressful situation during a vulnerable developmental stage. So the primitive results of mitochondrial DNA deletion might predispose infant to death in critical situations.
高淑慧. "= Mitochondrial DNA mutation and depletion in human sperm with declined motility and fertility". Thesis, 1998. http://ndltd.ncl.edu.tw/handle/21522626606238103090.
Pełny tekst źródłaChing-Hui, Huang, i 黃靜惠. "Low Copy Number of Mitochondrial DNA Is Associated with Metabolic Syndrome". Thesis, 2009. http://ndltd.ncl.edu.tw/handle/83313874695102835183.
Pełny tekst źródła長榮大學
醫學研究所
97
Objective: The metabolic syndrome (MetS) is a group of risk factors of metabolic origin that are accompanied by increased risk for type 2 diabetes mellitus and cardiovascular disease. One of the defects in MetS and its associated diseases is excess cellular oxidative stress and mitochondrial alternation. A low copy number of mitochondrial DNA (mtDNA) has been found to be associated with enhanced risk for the development of diabetes and cardiovascular disease. Therefore, the objective of this study was to clarify the characteristics of mtDNA copy number of leukocytes in MetS. Methods: Seventy nine control subjects and 42 metabolic syndrome subjects of Taiwanese were recruited and Quantitative polymerase chain reaction (QPCR) and real time PCR were applied in present study. A student t test, multi-variate ANOVA, and Spearman''s rho correlation analysis were used for statistic analysis. Results: The results revealed that compared to control subjects, a significant lower copy number of mtDNA in the leukocytes of MetS subjects and mtDNA copy number was associated with a significant increase in HDL and inversely correlated with weight, SBP, DBP, Urate and RIFACTOR (cholesterol/HDL). MetS group had higher insulin, creatinine, urate, oxLDL, ApoB, RIFACTOR, and homocystein and lower HDL, ApoA and adiponectin in the plasma. Conclusion: The present study shows that a low mtDNA copy number of leukocytes play an important role in the progression of MetS. The results may provide new diagnostic and therapeutic strategies to MetS and other associated disorder.
CHIN, WANG LI, i 王儷瑾. "ALTERATIONS OF BIOGENESIS GENES IN RESPONSE TO DEPLETION OF MITOCHONDRIAL DNA IN HUMAN 143B CELLS". Thesis, 2007. http://ndltd.ncl.edu.tw/handle/56607578934734061516.
Pełny tekst źródła長庚大學
醫學生物技術研究所
96
Mitochondrial biogenesis is regulated by the coordinated expression of nuclear genome and mitochondrial genome. Among them, three important transcriptional factors are mitochondrial transcription factor A (TFAM), nuclear respiratory factors (NRFs) and PPARγ coactivator-1α (PGC-1α). TFAM, which is localized in the mitochondria, regulates factors involving the replication and transcription of mtDNA. NRFs and PGC-1α, which are transcriptional factors in the nucleus, can interact and activate TFAM, and subsequently regulate the mitochondrial biogenesis genes. Previous studies shows that mitochondrial dysfunction could lead to cross-talk between nuclear genome and mitochondrial genome and increase the expression of mitochondrial biogenesis, but the exact mechanism of cross-talk has not been studied clearly yet. Based on this, we have used the mtDNA-depleted 143B (143B-ρ0) cell to investigate whether the defect of oxidative phosphorylation (OXPHOS) may result in alterations of expression of mitochondrial biogenesis genes, the activity of citrate synthase in tricarboxylic acid cycle (TCA cycle), and the activity of respiratory complex II. In addition, in order to exclude the possibility that the mtDNA-depletion process which might cause unknown nuclear DNA alterations could result in the changes of expression of mitochondrial biogenesis genes, we utilized the 143B-cybrid (cybrid) which was generated from 143B-ρ0 cell replenished with wild-type mtDNA for comparison to compare the alterations of mitochondrial biogenesis genes in 143B, 143B-ρ0, and cybrid cells. We used Western blot to analyze the protein levels of the genes related to mitochondrial biogenesis, employed real time RT-PCR to analyze mRNA levels of PGC-1α, and finally determined the activities of citrate synthase and complex II. The result showed that the expression of cytochrome c and ATP synthase β subunit of complex V in 143B-ρ0 cell were higher than that in 143B and cybrid cells, while levels of succinate dehydrogenase subunit B of complex II and TFAM were decreased in 143B-ρ0 cells. However, there was no difference in the level of core 1 protein of complex III among three cell lines. Taken together, we speculate that NRFs may not be the major or only factor that control mitochondrial biogenesis genes from results of Western blot analysis. Moreover, the mRNA level of PGC-1α was higher, the activity of complex II was lower, and the activity of citrate synthase was higher in 143B-ρ0 cells compared to 143B or cybrid cells. Therefore, mitochondrial biogenesis-related genes, proteins, or enzyme activities were indeed altered in mtDNA-depleted cells.
Hong, Chiung-Hui, i 洪瓊慧. "Effects of Mitochondrial DNA Depletion and 4977 bp Deletion on the Apoptotic Behaviors of Human Cells". Thesis, 2001. http://ndltd.ncl.edu.tw/handle/27311741329012720124.
Pełny tekst źródła國立陽明大學
生物化學研究所
89
Abstract The pathology of mitochondrial encephalomyopathies is associated with abnormal cell loss of neurons and muscle fibers. Is it possible that mitochondrial respiratory chain dysfunction results in cell loss, which is commonly found in patients with encephalomyopathies? The respiratory chain impairment may be caused by DNA mutations in mitochondrial or nuclear genome. Among the mitochondrial DNA (mtDNA) mutations associated with mitochondrial diseases, 4977-bp deletion is the most common one. In this study, the effects of mtDNA depletion on the apoptotic behaviors of human cells were first investigated. In order to exclude the possible contributions of nuclear background on the possible effect of mtDNA itself, r0 cells derived from 143B osteosarcoma cells were used. r0 cells that are devoid of mtDNA were obtained by long-term treatment of osteosarcoma cells with ethidium bromide (EtBr). Under the treatment of staurosporine (STS), the viability, DNA fragmentation, caspase 3 activities and ROS production of r0 and parental 143B cells were compared. The results showed that r0 cells exhibited lower viability, higher caspase 3 activity and increased cytochrome c release to the cytosol. These findings suggest that mtDNA depletion exerts damaging effects on an upstream effector of caspase activation. In addition, it was also found that r0 cells were not as sensitive to the inhibitors of scavenger enzymes as 143B parental cells. Moreover, r0 cells utilize pyruvate in culture media to provide energy needed and thus produce less ROS. Taken together, these findings suggest that it is not ROS increase per se that leads r0 cells to be more susceptible to apoptosis. By using cytoplasmic fusion techniques, 4977-bp mtDNA deletion from skin fibroblasts of a CPEO patient was introduced into r0 cells and created the so-called cybrids. The cybrids containing 0% and 85% of mtDNA with 4977-bp deletion as an experimental system were used as an experimental system to investigate the effects of high proportion of 4977-bp mtDNA deletion on apoptotic behaviors of human cells. Under the treatment of STS, both the wild-type cell line 1-3-16 and the high mutant cell line 51-10 exhibited high caspase 3 activities. However, they can be distinguished by cell viability. Moreover, by the treatment of antioxidant enzyme inhibitors, such as mercaptosuccinate and 3-amino-1, 2, 4-trizole, 51-10 cells were more susceptible to apoptosis than 1-3-16 cells. Taken together, these findings suggest that high proportion of 4977-bp mtDNA deletion may reduce the ability of cells to deal with ROS and result in apoptosis or necrosis. Based on the results of this study, it is proposed that 4977-bp mtDNA deletion renders human cells more vulnerable to oxidative stress and that antioxidant therapy can be useful for patients with CPEO syndrome. On the other hand, the apoptotic effects of mtDNA depletion in vivo are different from those observed in cultured cells because of the supplementation of pyruvate and uridine in the medium. It is warranted to better understand the pathological changes of muscles and neurons in relation to the role of mtDNA depletion in patients with MERRF syndrome.
Yen, Ju-Chen, i 顏汝珍. "Studies on the Effects of Mitochondrial DNA Depletion on the Sensitivity of Human Cells to Apoptosis". Thesis, 2007. http://ndltd.ncl.edu.tw/handle/31018427909023504623.
Pełny tekst źródła國立陽明大學
生化暨分子生物研究所
95
Mitochondria are the major energy suppliers in mammalian cells. They are also important initiators and regulators in apoptosis. Many studies have suggested mitochondrial DNA (mtDNA) alterations may affect susceptibility of human cells to apoptosis. However, whether mtDNA depletion affects the sensitivity of human cells to drug-induced apoptosis is still controversial. In this study, I used human osteosarcoma 143B cell line as the experimental model to investigate the effect of mtDNA depletion on the response of cells to doxorubicin or staurosporine-induced apoptosis and the underlying mechanisms. After treatment of cells with 0.4 mM doxorubicin for 24, 36 and 48 hours, the viability of 143B cells devoid of mtDNA (rho-zero cells) were lower than its parental cells (45.5 %, 17.9 %, 8.9 % vs. 63.5 %, 38.5 %, 28.0 %, respectively). Rho-zero cells were also more sensitive than 143B cells after treatment with 25 nM staurosporine for 8, 16 and 24 hours (78.3 %, 44.2 %, 17.9 % vs. 99.9 %, 81.6 %, 79.1 %, respectively). The proportions of rho-zero cells in sub-G1 phase (28.1 % and 45.5 %) were higher than those of 143B cells (2.8 % and 6.3 %) after treatment with doxorubicin or staurosporine. Apoptosis-related activation of caspases 8, -9, and -3 were more pronounced in rho-zero cells than that in 143B cells. As a result, rho-zero cells were more sensitive to drug-induced apoptosis as compared with 143B cells. Although the endogenous H2O2 level in rho-zero cells was lower than that in 143B cells, drug-induced H2O2 level was increased about 2 folds in rho-zero cells, which was higher than that in 143B cells. Besides, treatment of cells with exogenous H2O2 increased drug-induced apoptosis in both 143B and rho-zero cells. Therefore, reactive oxygen species (ROS) may be involved in the apoptosis of 143B cells. On the other hand, the phosphorylation level of Akt was found to be different between 143B and rho-zero cells. Upon treatment with doxorubicin or staurosporine, the phosphorylation level of Akt in 143B cells was upregulated at 4 hr and sustained to 24 hr, whereas it was remained unchanged in rho-zero cells. Inhibition of Akt phosphorylation by LY294002 could enhance doxorubicin and staurosporine-induced caspase 3 activation by 3-4 folds in 143B cells. By contrast, no such effect of LY294002 was observed in rho-zero cells. The activation of Akt may provide a protective mechanism in 143B cells, which rendered 143B cells more resistant to doxorubicin- or staurosporine-induced apoptosis. Furthermore, I reduced mtDNA content by RNA interference of the mitochondrial transcription factor A (mtTFA). The mtDNA content of 143B cells was reduced to 40 % after transfection with mtTFA siRNA for 4 days. After treatment of the cells with doxorubicin or staurosporine, no significant differences in the viability were observed between mtTFA knockdown cells and the controls. No marked differences in drug-induced H2O2 level and Akt phosphorylation were observed between mtTFA knockdown cells and the controls. This indicates that the degree of depletion of mtDNA may determine the sensitivity of 143B cells to doxorubicin and staurosporine. Taken these findings together, I suggest that ROS and Akt activation may play a role in the differential response to doxorubicin and staurosporine-induced apoptosis between 143B and rho-zero cells.
Ni, Chyi-Yao, i 倪其堯. "Mitochondrial DNA mutation and oxidative damages in the pathogenesis of Down's syndrome". Thesis, 2004. http://ndltd.ncl.edu.tw/handle/83947040526306975910.
Pełny tekst źródła臺北醫學大學
生物醫學技術研究所
92
Down’s syndrome is the most common disease of chromosomal aberration on cytogenetics. The major form of chromosomal aberration is trisomy 21, an extra chromosome of chromosome 21. There are many studies in Down’s syndrome including clinical pathology, cytogenetics and molecular genetics. It is distinct why the germ cells are susceptible to trisomy formation at meiosis or mitosis stage. Recently, some researches reported that oxidative stress might play some roles in the formation of trisomy. Mitochondria are the major organelles that produce reactive oxygen species. In this study, we hypothesized the mitochondrial dysfunction might be contributed to the pathogenesis of Down’s syndrome. We investigated on mitochondria DNA (mtDNA) copy number, mtDNA deletion and oxidative damages in amniotic cells of Down’s syndrome patient. Decreased copy numbers of mtDNA were found in the patients of Down’s syndrome. Moreover, a novel 5335 bp mtDNA deletion was identified in amniotic cells from Down’s syndrome patient. Analysis of nucleotide sequences flanking the breakpoints of this deletion revealed a 10 nucleotides direct repeat “CCTATAGCAC” flanking the junction site of the 5335 bp deletion at nucleotide position 8263-8272 and 13598-13607. Increased ROS generation and oxidative damages were revealed in Down’s amniotic cells. Taken these data together, we suggested that the dysfunctional mitochondria might play a role in the pathogenesis of Down’s syndrome.
Hsieh, Rong-Hong, i 謝榮鴻. "Studies on multiple mutations of mitochondrial DNA associated with the MELAS syndrome and human aging". Thesis, 1997. http://ndltd.ncl.edu.tw/handle/66733499378791003172.
Pełny tekst źródłaProsser, Debra Olive. "Mitochondrial DNA (mtDNA) mutations in patients with suspected myoclonic epilepsy and ragged red muscle fibres (MERRF), Leigh syndrome (LS), and mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes (MELAS)". Diss., 2001. http://hdl.handle.net/2263/30471.
Pełny tekst źródłaDissertation (MSc (Human Genetics))--University of Pretoria, 2007.
Genetics
unrestricted
Lu, Ching-You, i 盧青佑. "Association of Imbalance of Free Radical Scavenging Enzymes with Mitochondrial DNA Mutations in Fibroblasts of Elderly Subjects and Patients with CPEO Syndrome". Thesis, 1999. http://ndltd.ncl.edu.tw/handle/28323173505249952059.
Pełny tekst źródła國立陽明大學
生物化學研究所
87
The cellular respiratory functions decline in aging human tissues and affected tissues of patients with mitochondrial diseases. This is accompanied by an increase of reactive oxygen species (ROS) production in mitochondria via the increased electron leak of the respiratory chain. As a result, an ever-increasing amount of ROS may escape from the defense systems and cause oxidative damage to biomolecules. To investigate the relationship between free radical scavenging enzymes and oxidative damage, a total of 76 normal subjects of different ages were recruited in this study. I found an age-dependent increase of 8-OHdG content and proportion of 4,977 bp mitochondrial DNA (mtDNA) deletion in skin tissues from subjects above the age of sixty years. Moreover, I also found an age-dependent increase of lipid peroxides in cultured fibroblasts of the subjects above the age of sixty years. To evaluate the antioxidant defense system from individuals of different ages, I measured the activities of Cu,Zn-superoxide dismutase (Cu,Zn-SOD), Mn-superoxide dismutase (Mn-SOD), catalase (CAT) and glutathione peroxidase (GPx). The activities of Cu,Zn-SOD, CAT and GPx were found to decrease with age, and the activity of Mn-SOD was increased with age before sixty years but was decreased thereafter. This indicated that elevated oxidative stress caused by an imbalance between the production and removal of ROS occurred in skin fibroblasts after sixty years of age. Taken together, I suggest that the functional decline of free radical scavenging enzymes and the elevation of oxidative stress may play an important role in eliciting oxidative damage and mutation of mtDNA during the human aging process. On the other hand, I investigated the alterations of age-dependent mitochondrial gene expression associated with mtDNA deletions. Total RNAs were isolated and analyzed by Northern hybridization. There were no significant differences of the mtRNAs among the skin fibroblasts from subjects of different ages. Moreover, I measured the activities of respiratory enzymes NCCR, SCCR and CCO in fibroblasts of subjects of different ages. There was an age-dependent decrease in the activities of the three enzyme complexes. More interestingly, I found a 75-year-old woman who had no mitochondrial disease but harbored 60% of the 4,977 bp deleted mtDNA in the fibroblasts established from her face skin. This deletion caused dramatic alteration in the mRNA levels and the activities of respiratory enzymes of mitochondria. In the second part of this study, skin and muscle fibroblasts were cultured from nine patients with chronic progressive external ophthalmoplegia (CPEO) syndrome for studying the relationship between mtDNA mutation and free radical scavenging enzymes. The skin and muscle fibroblasts from these CPEO patients carried different mtDNA mutations at different levels. The fibroblasts from patients had the common 4,977 bp mtDNA deletion, a 4,366 bp mtDNA, a 6,190 bp mtDNA deletion, a ~10 kb mtDNA deletion and the A3243G mutation in mitochondrial tRNALeu(UUR) gene. To examine whether these mtDNA mutations are associated with a defect or imbalance in the free radical scavenging enzymes. I assayed the enzyme activities of Mn-SOD, catalase and GPx of the fibroblasts. I found that the skin and muscle fibroblasts from the nine CPEO patients all had higher enzyme activity and mRNA level of Mn-SOD but those of catalase and GPx were not increased or even decreased. These results indicated an imbalance between the H2O2 generation and removal systems in the fibroblasts of the patients with CPEO syndrome. The ratios of activities of Mn-SOD/catalase in muscle fibroblasts (between 3.39 and 8.36) were about 1.4 to 3.5 fold higher than those of the control subjects. The ratios in skin fibroblasts of patients CPEO4, CPEO6, and CPEO8 were also significantly different from those of the control subjects. But the imbalance was much more pronounced in muscle fibroblasts. This tissue-specific imbalance of free radical scavengers was confirmed by analysis of the corresponding mRNA levels. The average mRNA ratio of Mn-SOD/catalase in fibroblasts of the nine CPEO patients was about 1.21 and that was 0.44 for control skin fibroblasts. By contrast, the average Mn-SOD/catalase mRNA ratio was 1.58-6.29 in muscle fibroblasts of these patients, which was much higher than those of the control fibroblasts (0.79). These results are consistent with the clinical observation that muscle is the main affected tissue of patients with CPEO syndrome. I suggest that the imbalance between free radical scavenging enzymes play an important role in the pathogenesis and age-dependent progression of the CPEO syndrome.
Harr, Claudia Mareike. "Genetische Polymorphismen der mtDNA als Risikofaktoren für das SIDS (Sudden Infant Death Syndrome)". Doctoral thesis, 2013. http://hdl.handle.net/11858/00-1735-0000-0028-864E-0.
Pełny tekst źródłaGonçalves, Ana Cristina Pereira. "Oxidative stress versus epeginetics - Role in susceptibility, development, and progression of myeloid neoplasms". Doctoral thesis, 2016. http://hdl.handle.net/10316/29610.
Pełny tekst źródłaMyeloid neoplasms (MN) are a group of heterogeneous diseases that includes myelodysplastic syndromes (MDS), myeloproliferative neoplasms (MPN), and acute myeloid leukemia (AML). These diseases can simultaneously harbor changes in reactive oxygen species (ROS) levels and in DNA methylation pattern. The oxidative stress (OS) results from the imbalance between ROS production, mainly in mitochondria, and their elimination by antioxidants. Moreover, MN recurrently show abnormal hypermethylation of tumor suppressor genes (TSG), such as P15 and P16 genes, and in less extent global hypomethylation of repetitive sequences such as long interspersed nucleotide elements 1 (LINE-1). The present study aimed to analyze the involvement of OS and DNA methylation in the development and progression of MN, in order to evaluate their role as diagnostic and prognostic markers, as well as to identify susceptibility variants in genes involved in these mechanisms. Frist, we conducted a proof of concept study to investigated the involvement of OS and mitochondrial dysfunction in MDS pathogenesis, as well as to assessed their diagnostic and prognostic value, and the relation of OS parameters (intracellular levels of peroxides, superoxide, and reduced glutathione – GSH) with methylation status of P15 and P16 gene promoters. To that end we used two technique: flow cytometry and methylation specific-PCR (MSP). We observed that bone marrow cells from MDS patients had higher peroxide levels and lower GSH content than control cells. Moreover, OS levels were dependent of MDS subtype and risk group. GSH showed to be an accurate MDS diagnostic marker, while ROS, GSH, and superoxide/peroxides ratio were good survival markers. MDS patients had higher P15 and P16 methylation frequencies than controls. Moreover, patients with methylated P15, P16, and P15 or P16 had higher OS levels than patients without methylation. To confirm these results, we expanded the evaluation of OS (several enzymatic and non-enzymatic antioxidant defenses; oxidative damage) and DNA methylation parameters [localized DNA methylation – methylation status of P15, P16, TP53, MGMT, DAPK, and KEAP1 genes; global DNA methylation – levels of 5-methylcytosine (5-mC), 5-hydroxymetylcytosine (5-hmC), and LINE-1 methylation], as well as the studied pathologies (MDS and MPN). These biological parameters were evaluated in peripheral blood samples by colorimetry and fluorimetry assays, as well as by MSP and combined bisulfite restriction analysis (COBRA). MDS patients had lower levels of GSH and TAS (total antioxidant status), as well as higher levels of peroxide, peroxide/GSH, and peroxide/TAS than controls. Moreover, MDS and MPN patients had higher 5-mC levels and lower 5-hmC/5-mC ratio, as well as increased methylation of at least one methylated gene (P15, P16, DAPK, or KEAP1). TP53 and MGMT genes were unmethylated in these patients. Peroxide levels and peroxide/GSH ratio were higher in patients with methylated genes than in those without methylation. LINE-1 methylation and 5-mC levels were correlated with peroxide levels and peroxide/GSH ratio. Next, we assessed the association of variants of genes involved in OS, folate metabolism, DNA repair, and DNA methylation with the susceptibility and prognosis of MDS and AML. To that end, 16 SNPs (one per gene: CAT, CYBA, DNMT1, DNMT3A, DNMT3B, GPX1, KEAP1, MPO, MTRR, NEIL1, NFE2F2, OGG1, SLC19A1, SOD1, SOD2, and XRCC1) were genotyped by PCR techniques. We also analyzed OS (ROS/TAS), DNA damage (8-OHdG), and DNA methylation (5 mC) in a sub-cohort of MDS patients and controls. Results showed that five genes (GPX1, NEIL1, NFE2L2, OGG1, and SOD2) were associated with MDS, two (DNMT3B and SLC19A1) with AML, and two (CYBA and DNMT1) with both diseases. OS levels were correlated with CYBA, GPX1, and SOD2 genotypes, DNA damage with NEIL1 and OGG1, and 5-mC levels with DNMT1, DNMT3A, DNMT3B, and MTRR. Furthermore, DNMT3A, MTRR, NEIL1, and OGG1 variants modulated AML transformation in MDS patients. Additionally, DNMT3A, OGG1, GPX1, and KEAP1 variants influenced MDS and AML survival. Finally, we investigated if acute and chronic exposure to hydrogen peroxide (H2O2) affects the methylome of normal and malignant hematological cells. In this investigation, we used four acute myeloid leukemia cell lines and a normal B lymphocyte cell line, and analyzed the copy number and methylation status of several TSG, LINE 1 methylation, levels of 5-mC, 5-hmC, 8-hydroxy-2'-deoxyguanosine (8-OHdG), ROS, and GSH, as well as the gene expression of DNMT1, DNMT3A, DNMT3B, MECP2, MBD1, HDAC1, EZH2, EP300, and TET2. These analyses were performed by methylation-specific multiplex ligation dependent probe amplification (MS-MLPA), COBRA, colorimetry and fluorimetry assays, as well as real time PCR. Acute and chronic exposure to H2O2 increased TSG methylation (in cells with increased ROS/GSH ratio) and decreased LINE-1 methylation (in cells with increased GSH levels). TSG hypermethylation was cell line-dependent and accompanied by upregulation of DNMT1, DNMT3A, MECP2, HDAC1, and EZH2 genes. Moreover, the pre-treatment with N acetylcysteine, an antioxidant molecule, prevented these events. Overall, the present study points to a possible link between OS and DNA methylation, which besides the relevance in the development and progression of these MN, could also constitute new diagnostic and prognostic markers as well as new potential therapeutic targets.
As neoplasias mieloides (NM), de que são exemplo a síndrome mielodisplásica (SMD), as neoplasias mieloproliferativas (NMP) e a leucemia mieloide aguda, são um grupo heterogéneo de doenças clonais hematopoiéticas. A patogénese destas doenças pode envolver simultaneamente alterações nos níveis de stresse oxidativo (SO) e no padrão de metilação do ADN. O SO resulta do desequilíbrio entre a produção de espécies reativas de oxigénio (ROS), geradas principalmente na mitocôndria, e a sua eliminação pelas defesas antioxidantes. Além disso, estas doenças apresentam frequentemente hipermetilação nos genes supressores de tumor (GST), como os genes P15 e P16, e alguns casos podem também apresentar hipometilação nas sequências repetitivas, nomeadamente as long interspersed nucleotide elements 1 (LINE-1). O presente estudo teve como objetivo analisar o envolvimento do stress oxidativo e da metilação do DNA no desenvolvimento e na progressão das neoplasias mieloides, de modo a avaliar o seu papel como marcadores de diagnóstico e prognóstico, e identificar variantes genéticas de susceptibilidade em genes envolvidos nesses mecanismos. Inicialmente, efetuou-se um estudo de prova de conceito de modo a avaliar o envolvimento do SO e a disfunção mitocondrial na patogénese das SMD, o seu valor como marcadores de diagnóstico e de prognóstico, e a relação dos parâmetros OS (níveis intracelulares de peróxidos, anião superóxido e glutationa reduzida – GSH) com o perfil de metilação dos promotores dos genes P15 e P16. Para este efeito, recorreu-se a duas técnicas: citometria de fluxo e PCR específico de metilação (MSP). Os resultados demonstram que as células de medula óssea dos doentes SMD apresentam aumento dos níveis de peróxidos e diminuição de GSH, relativamente às células dos controlos. No entanto, estes resultados dependem do subtipo de SMD e do grupo de risco. Além disso, a GSH revelou-se um bom marcador de diagnóstico, enquanto os ROS, a GSH e a razão superóxido/peróxidos mostraram-se marcadores de sobrevivência. Estes doentes apresentam frequências de metilação dos genes P15 e P16 mais elevadas do que os controlos. Por outro lado, os doentes SMD com metilação nos gene P15, P16, e P15 ou P16 apresentam níveis de SO mais elevados do que os doentes sem metilação. De modo a confirmar estes resultados, realizou-se uma avaliação alargada dos parâmetros de SO (várias defesas antioxidantes enzimáticas e não enzimáticas; lesão oxidativa) de metilação do DNA [metilação localizada – perfil de metilação dos genes P15, P16, TP53, MGMT, DAPK e KEAP1; metilação global – níveis de metilação da 5-metilcitosina (5-mC), da 5-hidroximetilcitosina (5-hmC) e da LINE 1] não só em doentes SMD, mas também em doentes NMP. Estes parâmetros biológicos foram avaliados em amostras de sangue periférico por colorimetria, fluorimetria, MSP e COBRA (combined bisulfite restriction analysis). Os resultados demonstraram que os doentes SMD apresentam diminuição dos níveis de GSH e CAP (capacidade antioxidante total), assim como aumento dos níveis de peróxido e das razões peróxido/GSH e peróxido/CAP comparativamente aos controlos. Por outro lado, observou-se aumento dos níveis de 5-mC e diminuição da razão 5-mC/5-hmC nos doentes SMD e NMP. Além disso, este doentes apresentam aumento da frequência de metilação de pelo menos num gene (P15, P16, DAPK, ou KEAP1). No entanto, não se observou metilação dos genes TP53 e MGMT nestes doentes. Os doentes genes metilados apresentam aumento dos níveis de peróxido e da razão peróxido/GSH relativamente aos doentes sem metilação. Por fim, os níveis de metilação da LINE-1 e da 5-mC correlacionam-se com os níveis de peróxido e da razão peróxido/GSH. Seguidamente, avaliou-se os polimorfismos em genes envolvidos no SO, no metabolismo do folato, na reparação e na metilação do DNA e o seu papel na susceptibilidade e prognóstico dos doentes SMD e LMA. Com essa finalidade, genotiparam-se 16 SNPs (um por gene: CAT, CYBA, DNMT1, DNMT3A, DNMT3B, GPX1, KEAP1, MPO, MTRR, NEIL1, NFE2F2, OGG1, SLC19A1, SOD1, SOD2 e XRCC1) por técnicas de PCR. Analisaram-se, igualmente, os níveis de OS (ROS/CAP), da lesão (8-OHdG) e da metilação do DNA (5-mC) num subgrupo de doentes SMD e controlos. Os resultados sugerem que os genes GPX1, NEIL1, NFE2L2, OGG1 e SOD2 influenciam a susceptibilidade para SMD, os genes DNMT3B e SLC19A1 a susceptibilidade para LMA e os genes CYBA e DNMT1 o desenvolvimento das duas doenças. Além disso, os níveis de SO correlacionam-se com o genótipo dos genes CYBA, GPX1 e SOD2, a lesão do DNA com os genes NEIL1 e OGG1, e os níveis de 5-mC com os genes DNMT1, DNMT3A, DNMT3B e MTRR. Os polimorfismos nos genes DNMT3A, MTRR, NEIL1 e OGG1 influenciam o prognóstico (evolução para LMA) dos doentes SMD, enquanto os polimorfismos nos genes DNMT3A, OGG1, GPX1 e KEAP1 influenciam a sobrevivência dos doentes SMD e AML. Por fim, investigou-se a influência da exposição crónica e aguda ao peróxido de hidrogénio (H2O2) na metilação de células hematológicas normais e malignas. Para o efeito, utilizaram-se quatro linhas celulares de leucemia mieloide aguda e uma linha celular de linfócitos B normal. A variação do número de cópias e o perfil de metilação de vários GST, os níveis de metilação da LINE-1, da 5-mC, 5-hmC, da 8-hidroxi-2'-desoxiguanosina (8-OHdG), das ROS e da GSH, bem como a expressão dos genes DNMT1, DNMT3A, DNMT3B, MECP2, MBD1, HDAC1, EZH2, EP300 e TET2 foram avaliados por methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA), COBRA, colorimetria, fluorimetria e PCR em tempo real. A exposição aguda e crónica ao H2O2 induziu hipermetilação nos GST (nas células com aumento da razão ROS/GSH) e hipometilação nas sequências LINE-1 (nas células com aumento da GSH). A hipermetilação dos GST foi acompanhada da sobre-expressão dos genes DNMT1, DNMT3A, MECP2, HDAC1 e EZH2 de modo dependente da linha celular. Além disso, o pré-tratamento com N-acetilcisteína, uma molécula antioxidante, preveniu estas alterações moleculares. Em conclusão, o presente estudo sugere uma possível relação entre o stresse oxidativo e a metilação do DNA, os quais, além da relevância no desenvolvimento e na progressão das neoplasias mieloides, poderão igualmente constituir novos marcadores de diagnóstico e prognóstico, bem como potenciais alvos terapêuticos.
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