Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.647482
Title: Mitochondrial DNA replication and biogenesis during embryonic development and in disease
Author: Carling, Phillippa Julia
ISNI:       0000 0004 5367 1318
Awarding Body: University of Newcastle Upon Tyne
Current Institution: University of Newcastle upon Tyne
Date of Award: 2014
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Abstract:
Regulation of mtDNA content is critical to normal human health and is often abnormal in mitochondrial diseases. Only a proportion of a female’s mtDNA is used to populate the oocytes she forms during embryogenesis. This mtDNA bottleneck can cause rapid shifts in heteroplasmy levels between generations in families with mtDNA mutations. The heteroplasmy levels of several mtDNA mutations were analysed from mother-child pairs using previously validated pyrosequencing assays. Analysis of the shifts in heteroplasmy caused by the mtDNA bottleneck reveals that the inheritance of the pathogenic tRNA mutations m.3243A>G and m.8344A>G do not show selection bias. However, the distribution of m.8993T>G (in MT-ATP6) in offspring suggests this mutation is selected for during the mtDNA bottleneck. This finding is in agreement with meta-analysis performed on previously published data, which also reveals biased inheritance of the LHON mutations m.11778G>A and m.3460G>A, in Complex I genes. Selection for these mutations explains the higher prevalence of homoplasmy, as fixation can occur within fewer generations. Crucial to formation of the mtDNA bottleneck is the dramatic increase in mtDNA replication rate that allows primordial germ cells (PGCs) to repopulate their mitochondrial genomes. Gene expression analysis during pre-implantation embryonic development has not revealed any other biological processes associated with regulation of mtDNA copy number. Upregulation of Lrpprc correlates with the expression of transcription factors as pre-implantation development progresses at this stage. The limited number of PGCs in the early stages of post-implantation development prevented sufficient quantity of high-quality RNA to be isolated for use with gene expression analysis. The reduction of mtDNA copy number observed during the mtDNA bottleneck was modelled in myoblasts and fibroblasts, using ddC, a reverse transcriptase inhibitor. Although some gene expression changes were induced during repopulation of mtDNA, these were limited to below 3-fold change. Forcing reliance on oxidative phosphorylation through culture with galactose media could not increase the rate of mtDNA replication or induce greater gene expression responses. Ragged-red fibres (RRFs) are a common hallmark of many mitochondrial diseases, caused by proliferation of mitochondria in the subsarcolemmal region of muscle tissue. Multiplex immunohistochemistry allowed fibre typing and identification of RRFs in muscle tissue from a patient with the m.8344A>G mutation. A novel technique was iii established to ensure extraction of intact high-quality RNA from laser-microdissected muscle fibres. The RNA was used in a pilot microarray experiment to study the differences not only between control and patient tissue, but specifically within RRF, and has identified a multitude of potential signalling pathways involved in mitochondrial biogenesis and formation of RRFs. This thesis reveals the complexity of mtDNA copy number regulation and, the critical involvement it has to human health and the inheritance and pathogenicity of mitochondrial diseases.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.647482  DOI: Not available
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