Investigation and validation of mammalian telomeres as biomarkers for health and disease
Telomeres play a crucial role in safeguarding chromosomal integrity and have emerged as promising biomarkers of aging, oxidative stress, and disease. However, accurately quantifying telomere length (TL) remains technically challenging, particularly in murine models whose telomeres are substantially longer and more heterogeneous than those of humans. This thesis investigates the biological and methodological determinants of murine TL, with a specific focus on the effects of DNA extraction methodology, tissue-specific telomere dynamics, and telomere-specific oxidative damage. A mouse adapted monochromatic multiplex qPCR (mmqPCR) assay was developed and validated to measure relative TL, complemented by a formamidopyrimidine DNA glycosylase (FPG) qPCR assay to quantify telomere-specific oxidative damage. Across multiple murine tissues, pronounced tissue-specific differences in TL and oxidative stress were observed, with kidneys exhibiting the longest telomeres and lowest oxidative burden, while testes and brown adipose tissue showed elevated telomeric damage. Contrary to expectations, Nnt genotype, despite its known role in mitochondrial redox regulation, did not significantly influence TL or oxidative stress across tissues. A major finding of this work is that DNA extraction method introduces substantial artefacts into TL measurement. Phenol/chloroform extraction preserved long telomeric fragments, whereas column based and magnetic bead based methods produced significantly shorter TL estimates due to DNA fragmentation. Finally, novel methodological advances were established for chromosome-specific telomere sequencing using Oxford anopore Technologies, including 3′ overhang targeting via probe ligation. Collectively, this work demonstrates that methodological variables can utweigh biological differences in murine telomere studies, underlining the need for rigorous tandardisation to advance telomere biomarkers in aging and disease research.
570 Life sciences; biology, 610 Medicine & health
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