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Title: A novel assay to measure mitochondrial dysfunction in human skeletal muscle : implications for the diagnosis and treatment of mitochondrial diseases
Author: Rocha, Mariana Frota Cúcio De Moraes
ISNI:       0000 0004 5990 4506
Awarding Body: Newcastle University
Current Institution: University of Newcastle upon Tyne
Date of Award: 2016
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Mitochondrial dysfunction occurs in patients with mitochondrial disease, in neurodegenerative conditions and as part of the ageing process. It affects predominantly tissues with high metabolic requirements such as central nervous system and skeletal muscle. In patients with mitochondrial disease, both mitochondrial and nuclear genetic defects commonly cause a biochemical defect in muscle. However, due to the multi-copy nature of mitochondrial DNA, muscle displays a mosaic pattern of deficiency when the mitochondrial genome is affected. This particular pattern makes these defects challenging to quantify. Current standard methods to diagnose and investigate mitochondrial disease in affected tissues present several limitations. Biochemical studies are only suitable for cases with a high proportion of cells with mitochondrial respiratory chain deficiency. Moreover, histochemical assays only provide qualitative assessment of complex II and IV activities and are not capable of evaluating other complexes, such as complex I - the commonest affected respiratory complex in mitochondrial pathology. This project aimed therefore at developing a novel assay to accurately quantify mitochondrial dysfunction in human skeletal muscle. Once optimised, this assay was further used to explore: the mechanisms underlying mitochondrial pathology, its potential in helping the current diagnostic setting, as well as its potential to assess the effectiveness of therapeutic approaches aimed at treating mitochondrial dysfunction. This work described the development and validation of a novel quadruple immunofluorescent technique. This assay quantifies accurately key subunits of respiratory complexes I and IV together with mitochondrial mass, using a single 10μm section. The additional labelling of a cell membrane marker allows semi-automatic and computer-based sampling of large numbers of individual muscle fibres. Using this technique, this study characterised a variety of mitochondrial and nuclear genetic defects and demonstrated that specific genotypes exhibit distinct biochemical signatures in muscle. In patients with suspected mitochondrial disease, this assay provided clues on the possible genetic causes. Furthermore, this novel assay evaluated the effect of an endurance exercise program in patients with mitochondrial myopathy and was able to detect subtle changes in respiratory complexes levels.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available