Degree
Doctor of Philosophy (PhD)
Department
Biological Sciences
Document Type
Dissertation
Abstract
Embryos of Artemia franciscana survive harsh conditions in diapause and anoxia-induced quiescence for years by undergoing deep metabolic transitions. I investigated the relationship between phosphorylation of pyruvate dehydrogenase (PDH) and carbon flux to the tricarboxylic acid (TCA) cycle during metabolic depression. The difference in catalytic activity of the PDH complex (PDC) in maximally phosphorylated and dephosphorylated samples was 16.5-fold. Western blot analysis confirmed the changes in phosphorylation of PDH. Analysis of PDH expression in diapause and post-diapause embryos via phospho-PDH showed no significant differences. Investigation of post-diapause embryos under anoxia showed that phosphorylation of PDH decreased and activated the enzyme. These results show that PDH phosphorylation regulates PDC during diapause. However, inhibition sites in glycolysis are more important for regulating carbohydrate catabolism under anoxia.
Because brief metabolic disruptions in mammals result in bursts of reactive oxygen species (ROS) and oxidative damage during ischemia-reperfusion, I hypothesized mitochondria from Artemia embryos avoid similar oxidative distress during metabolic recovery. H2O2 efflux was identical between anoxia-reoxygenation (A/R) versus normoxia groups. Inhibition of peroxidase pathways increased H2O2 release by ~5-fold from normoxic and A/R-challenged mitochondria and confirmed that ROS-scavenging mechanisms substantially suppress routine ROS efflux. This maximal rate of H2O2 efflux was not significantly different between A/R-challenged and normoxic mitochondria. Treatment with rotenone, an inhibitor of Complex I and reverse electron transport (RET), produced a modest decrease in H2O2 efflux. This result indicates that RET, a major contributor to ROS bursts in mammalian mitochondria, is not stimulated by A/R in A. franciscana. Lack of oxidative damage markers demonstrated that A/R does not cause significant damage in Artemia mitochondria. The capacity to downregulate Complex I activity through active-deactive conformations was tested and is not operative. These data collectively suggest that Complex I from A. franciscana lacks the capacity for RET and prevents ROS surges.
Antioxidant pathways may also protect against oxidative stress upon metabolic reactivation. Analysis of antioxidant enzymes and small molecule antioxidants from diapause and post-diapause embryos showed alterations explicable by differences in metabolic rates and requisite ROS-scavenging capacities. A literature survey suggests A. franciscana embryos do not possess abnormally large antioxidant capacities; rather, avoiding ROS bursts during metabolic reactivation appears more important.
Date
4-2-2025
Recommended Citation
Arabie, Daniel Anthony, "Metabolic Transitions in Embryos of Artemia franciscana: Regulation and Consequences for Oxidative Stress" (2025). LSU Doctoral Dissertations. 6748.
https://repository.lsu.edu/gradschool_dissertations/6748
Committee Chair
Hand, Steven C.
Included in
Biochemistry Commons, Biodiversity Commons, Cell Biology Commons, Cellular and Molecular Physiology Commons, Comparative and Evolutionary Physiology Commons, Developmental Biology Commons, Integrative Biology Commons, Marine Biology Commons, Molecular Biology Commons, Other Biochemistry, Biophysics, and Structural Biology Commons, Other Cell and Developmental Biology Commons, Other Physiology Commons, Systems and Integrative Physiology Commons