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Title: Quantitative analysis of oxidative phosphorylation dysfunction in mitochondrial myopathy and ageing
Authors: Warren, Charlotte Frances
Issue Date: 2019
Publisher: Newcastle University
Abstract: Mitochondrial dysfunction not only occurs in patients with mitochondrial diseases, but in other neuromuscular conditions as well as the normal ageing process. In patients with mitochondrial disease, both nuclear and mitochondrial genetic defects cause a range of symptoms that are extremely heterogeneous. These symptoms predominantly affect tissues with a high metabolic demand such as the skeletal muscle, and often present as oxidative phosphorylation (OXPHOS) deficiency. This disease phenotype is further complicated by the multicopy nature of mitochondrial DNA (mtDNA), which means that mtDNA populations can be heteroplasmic, with variable mutation loads leading to a mosaic pattern of OXPHOS deficiency. This project aims to understand mitochondrial defects within skeletal muscle in both ageing and mitochondrial myopathy. An immunohistochemical investigation was undertaken to assess biochemical changes in skeletal muscle biopsies from participants aged 84-85. This study provided a more thorough understanding into what is happening to the muscle with age and revealed that active participants aged 84-85 have a preserved mitochondrial function comparable to young, healthy controls. However, although current methods such as immunohistochemistry and immunocytochemistry can reveal specific aspects of the mitochondrial OXPHOS defect, they fail to assess individual cell changes in all OXPHOS complexes within a single tissue cryosection. The advent of imaging mass cytometry (IMC) addresses this limitation and permits the investigation of more proteins at a single cell level than is possible with current techniques. Henceforth, this project was aimed at developing a novel workflow and bespoke analysis for applying IMC in skeletal muscle to investigate OXPHOS deficiency more thoroughly. Once optimised, this technique was used to investigate patients with different genetically characterised mitochondrial diseases, followed by a larger cohort of single, large-scale mtDNA deletion patients. There are several novel findings from this work: (i) IMC can be used to successfully quantify mitochondrial dysfunction in skeletal muscle; (ii) compared to controls and normal muscle fibres, patients with a deficiency of complex I present with an increase in unaffected respiratory chain complexes; (iii) complex III and V deficiency occur at low levels in muscle fibres from some patients with defects in mitochondrial translation; (iv) the successful optimisation and use of novel antibodies that target mtDNA-encoded subunits; (v) the amount of OXPHOS deficiency detected in single, large-scale mtDNA deletion patients correlates with the size and location of the deletions.
Description: PhD Thesis
Appears in Collections:Institute of Neuroscience

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