TOWARDS MITOCHONDRIAL DNA MODIFICATION AND INVESTIGATION OF MOLECULAR ETIOLOGY OF THE INDUCED HETEROPLASMIC MITOCHONDRIAL DNA VARIANTS
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The growing interest in mitochondrial DNA (mtDNA) underscores its potential role in various degenerative diseases, including neurodegenerative disorders, diabetes, and cancer. However, the challenge of modifying the non-Mendelian mitochondrial genome has hindered definitive conclusions about causal rela- tionships and underlying mechanisms. This research aims to explore the onco- genic potential of mtDNA variants by introducing de novo mutations and ana- lyzing their cellular and mitochondrial impacts.Chapter One reviews the link between mitochondrial function and tumori- genesis, beginning with ’the Warburg effect’ and summarizing studies on tumor-specific mtDNA variants. The complexities of establishing causality for mtDNA variants in metastasis are discussed, highlighting the necessity for im- proved mtDNA editing tools. Chapter Two details our attempts to manipulate mtDNA, encompassing de novo mtDNA synthesis, circularization, and delivery of synthesized mtDNA. Specifically, we demonstrated our protocol in synthesizing the extinct Homo ne- anderthalensis mtDNA. We explored methods for mtDNA delivery, including TFAM-assisted direct mtDNA transfection, and electroporation with mitochon- drial redelivery, though these approaches did not yield successful outcomes. We suggest further directions for enhancing mtDNA delivery techniques. Chapter Three focuses on the effects of specific stop-gain mutations in MT- ND5, previously identified as positively selected in specific cancers. We intro- duced these mutations into HEK293 and MCF12A cell lines and observed cellu- lar and mitochondrial responses independent of the nuclear background. Our findings indicate consistent pro-metastatic phenotypes and provide insights into the mechanisms of increased tumorigenesis following MT-ND5 mutations. Chapter Four offers a summary and outlines future research directions.