Doctor of Philosophy (Ph.D.)
Degree Granting Department
Biology (Cell Biology, Microbiology, Molecular Biology)
Patrick Bradshaw, Ph.D.
Meera Nanjundan, Ph.D.
Stanley Stevens, Ph.D.
Sandy Westerheide, Ph.D.
aging, ATP, genetics, respiration
This project was developed to identify novel methods for high-throughput culturing and screening of C. elegans to investigate age-related metabolic changes and to survey the proteomic and metabolomic factors associated with age-related changes. To accomplish these goals we developed a novel way to grow C. elegans in liquid culture in 96-well microplates for several weeks without suffering significant fluid loss due to evaporation and without needing to shake or unseal the plates for aeration. We also developed methods for assaying the total volume of live C. elegans in microplate cultures using a fluorescence microplate reader and for performing RNAi experiments with dead instead of live bacteria, which allows for the measurement of nematode metabolic parameters without bacterial interference. Using these methods, along with established methods for the global identification of metabolites and proteins by mass spectroscopy, we observed an integrated pattern of changes that occurred at the molecular level in aged C. elegans. Specifically, we found protein changes suggesting muscle dysfunction and sarcopenia, an increase in free fatty acids, a decrease in the S-adenosylmethionine cycle, altered or impaired protein synthesis, changes in free amino acid levels consistent with an increase in cell size, indications of epigenetic changes and alteration of DNA repair, and a shift toward a more oxidizing cellular environment, as well as a decrease in NAD+ relative to NADH. Through the use of an automated RNAi screen targeting potential EF-hand Ca2+ binding proteins, we identified genes that are associated with high culture medium Ca2+ toxicity. In addition, from a screen of X chromosome RNAi clones, we identified clones that partially prevented the age-dependent decline in nematode ATP levels, oxygen consumption, and reductive capacity. The set of genes targeted by these RNAi clones is enriched in both anti-longevity genes and negative regulators of cellular processes and are potential targets for anti-aging interventions.
Scholar Commons Citation
Copes, Neil, "High-throughput Screening of Age-related Changes in Caenorhabditis elegans" (2015). USF Tampa Graduate Theses and Dissertations.