Thesis (M.Sc.). / Written for the Dept. of Human Genetics. Title from title page of PDF (viewed 2008/07/30). Includes bibliographical references.

Dissertation (Ph.D.) --University of Texas Graduate School of Biomedical Sciences at San Antonio, 2008. / Vita. Includes bibliographical references.

Thesis (Ph. D.)--Case Western Reserve University, 2005. / [School of Medicine] Department of Genetics. Includes bibliographical references. Available online via OhioLINK's ETD Center.

BACKGROUND: Mitochondrial complex I deficiency often leads to a devastating neurodegenerative disorder of childhood. In most cases, the underlying genetic defect is unknown. Recessive nuclear gene mutations, rather than mitochondrial DNA mutations, account for the majority of cases. AIM: Our aim was to identify the genetic basis of complex I deficiency in 34 patients with isolated complex I deficiency, by studying six of the 39 nuclear encoded complex I subunit genes (NDUFV1, NDUFS1, NDUFS2, NDUFS4, NDUFS7 and NDUFS8). These genes have been conserved throughout evolution and carry out essential aspects of complex I function. METHODS: RNA was extracted from patient fibroblasts and cDNA made by reverse transcription. Overlapping amplicons that together spanned the entire coding area of each gene were amplified by PCR. The genes were screened for mutations using denaturing High Performance Liquid Chromatography (dHPLC). Patient samples with abnormal dHPLC profiles underwent direct DNA sequencing. RESULTS: Novel mutations were identified in six of 34 (18%) patients with isolated complex I deficiency. Five patients had two mutations identified and one patient had a single mutation in NDUFS4 identified. All patients with mutations had a progressive encephalopathy and five out of six had Leigh syndrome or Leigh like syndrome. Mutations were found in three nuclear encoded subunit genes, NDUFV1, NDUFS2 and NDUFS4. Three novel NDUFV1 mutations were identified (R386H, K111E and P252R). The R386H mutation was found in two apparently unrelated patients. Four novel NDUFS2 mutations were identified (R221X, M292T, R333Q and IVS9+4A&ltG). The novel NDUFS4 mutation c.221delC was found in two patients - one in homozygous form and the other heterozygous. Specific genotype and phenotype correlations were not identified. CONCLUSIONS: Nuclear encoded complex I subunit gene mutations are an important contributor to the aetiology of isolated complex I deficiency in childhood. Screening of these genes is an essential part of the investigation of complex I deficiency.

Orientador: Roger Frigério Castilho / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-24T17:18:02Z (GMT). No. of bitstreams: 1 Michelini_LuizGuilhermeBueno_M.pdf: 1602537 bytes, checksum: f58032fd3eaf359e8f094658a3612c02 (MD5) Previous issue date: 2014 / Resumo: A inibição parcial do Complexo I da cadeia respiratória mitocondrial em ratos tratados cronicamente com rotenona está associada com o desenvolvimento de características neuroquímicas, comportamentais e neuropatológicas da doença de Parkinson. Os objetivos deste trabalho foram (i) caracterizar os efeitos de uma inibição parcial do Complexo I por rotenona na produção de peróxido de hidrogênio (H2O2) por mitocôndrias de cérebro de ratos (MCR) em diferentes estados respiratórios e (ii) avaliar a suscetibilidade de MCR velhos (24 meses) à inibição do consumo de oxigênio (O2) e ao estímulo da produção de H2O2 por rotenona em comparação a MCR adultos (3-4 meses). A análise do potencial de membrana por citometria de fluxo em mitocôndrias isoladas indicou que a adição de rotenona promoveu uma inibição uniforme da respiração mitocondrial nestas organelas. Quando mitocôndrias foram incubadas na presença de uma baixa concentração de rotenona (10 nM) e de substratos geradores de NADH, o consumo de O2 foi reduzido de 45,9±1,0 para 26,4±2,6 nmol O2.mg-1.min-1 e de 7,8±0,3 para 6,3±0,3 nmol O2.mg-1.min-1 nos estados respiratórios 3 (respiração estimulada por ADP) e 4 (respiração de repouso), respectivamente. Nessas condições, a produção mitocondrial de H2O2 foi estimulada de 12,2±1,1 para 21,0±1,2 pmol H2O2.mg-1.min-1 e de 56,5±4,7 para 95,0±11,1 pmol H2O2.mg-1.min-1 nos estados respiratórios 3 e 4, respectivamente. Resultados similares foram observados ao comparar preparações mitocondriais enriquecidas com organelas sinápticas e não-sinápticas ou quando o íon 1-metil-4-fenilpiridina (MPP+) foi utilizado como inibidor de Complexo I mitocondrial. O estímulo da produção de H2O2 por rotenona nos estados respiratórios 3 e 4 foi associado a um aumento do estado reduzido de nucleotídeos de nicotinamida endógenos. Na respiração mitocondrial com succinato, onde a maior parte da produção de H2O2 se origina do fluxo reverso de elétrons do Complexo II para o I, baixas concentrações de rotenona inibiram a produção de H2O2. Rotenona não exerceu efeito sobre a eliminação mitocondrial de concentrações micromolares de H2O2. Em sinaptossomas intactos, observamos que rotenona 10 nM estimulou a liberação de H2O2 em 20,2±3,3% no estado respiratório basal. Ao compararmos MCR adultos e velhos, verificamos que o consumo de O2 no estado respiratório 3 e a atividade da citrato sintase foram 21,0±3,3% e 17,0±5,4% mais baixos em MCR velhos. Experimentos conduzidos na presença de diferentes concentrações de rotenona (5, 10 e 100 nM) demonstraram sensibilidade similar à inibição do consumo de O2 por rotenona no estado respiratório 3, com IC50 de 7,8±0,4 e 6,5±0,5 nM para MCR adultos e velhos, respectivamente. De acordo com esses resultados, o estímulo da produção de H2O2 observado foi similar em MCR adultos e velhos, tratadas com diferentes concentrações de rotenona. Concluímos que, uma inibição parcial do Complexo I pode resultar em uma crise energética e/ou estresse oxidativo mitocondrial, enquanto o primeiro evento predominaria numa situação de alta demanda de fosforilação oxidativa, o segundo ocorreria em condições de respiração de repouso. Em adição, os experimentos com ratos velhos indicaram que rotenona exerce efeitos similares no consumo de O2 e na produção de H2O2 em MCR adultos e velhos / Abstract: Partial inhibition of mitochondrial Complex I is associated with the development of neurochemical, behavioral, and neuropathological features of Parkinson's disease in rats chronically and systemically treated with rotenone. The aims of this work were (i) to characterize the effects of partial inhibition of respiratory Complex I by rotenone on H2O2 production by rat brain mitochondria in different respiratory states and (ii) to evaluate the susceptibility of brain mitochondria from old rats (24 month-old) to rotenone-induced inhibition of oxygen consumption and increased generation of H2O2 when compared with organelles from adult rats (3-4 month-old). Flow cytometric analysis of membrane potential in isolated mitochondria indicated that rotenone leads to uniform respiratory inhibition when added to a suspension of these organelles. When mitochondria were incubated in the presence of a low concentration of rotenone (10 nM) and NADH-linked substrates, oxygen consumption was reduced from 45.9±1.0 to 26.4±2.6 nmol O2.mg-1.min-1 and from 7.8±0.3 to 6.3±0.3 nmol O2.mg-1.min-1 in respiratory states 3 (ADP-stimulated respiration) and 4 (resting respiration), respectively. Under these conditions, mitochondrial H2O2 production was stimulated from 12.2±1.1 to 21.0±1.2 pmol H2O2.mg-1.min-1 and 56.5±4.7 to 95.0±11.1 pmol H2O2.mg-1.min-1 in respiratory states 3 and 4, respectively. Similar results were observed when comparing mitochondrial preparations enriched with synaptic or nonsynaptic organelles or when 1-methyl-4-phenylpyridinium (MPP+) ion was used as a respiratory Complex I inhibitor. Rotenone-stimulated H2O2 production in respiratory states 3 and 4 was associated with a high reduction state of endogenous nicotinamide nucleotides. In succinate-supported mitochondrial respiration, where most of the mitochondrial H2O2 production relies on electron backflow from Complex II to Complex I, low rotenone concentrations inhibited H2O2 production. Rotenone had no effect on mitochondrial elimination of micromolar concentrations of H2O2. In intact synaptosomes, we observed that 10 nM rotenone stimulated H2O2 release by 20.2 ± 3.3% under basal respiratory state. When comparing isolated brain mitochondria from adult and old rats we observed that oxygen consumption under respiratory state 3 and citrate synthase activity were 21.0±3.3% and 17.0±5.4% lower in mitochondria from old rats. Experiments conducted in the presence of different rotenone concentrations (5, 10 and 100 nM) showed that brain mitochondria from adult and old rats have similar sensitive to rotenone-induced inhibition of oxygen consumption in respiratory state 3, with IC50 of 7.8±0.4 and 6.5±0.5 nM for adult and old rats, respectively. In line with these results, similar stimulations in H2O2 production were observed in mitochondria from adult and old rats treated with different concentrations of rotenone. We conclude that partial Complex I inhibition may result in mitochondrial energy crisis and oxidative stress, the former being predominant under oxidative phosphorylation and the latter under resting respiration conditions. Rotenone exerts similar effects on oxygen consumption and H2O2 production by isolated brain mitochondria from adult and old rats / Mestrado / Biologia Estrutural, Celular, Molecular e do Desenvolvimento / Mestre em Ciências