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 Inactive and couch potato mutants of Drosophila : Locomotor activity vs metabolism

 Inactive and couch potato mutants of Drosophila : Locomotor activity vs metabolism. Sherry Lee Dr. Jadwiga Giebultowicz Dr. Natraj Krishnan HHMI Summer Program. Circadian Rhythm. Endogenous Period ( per ) and Timeless ( tim ) genes Entrained by exogenous cues – light

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 Inactive and couch potato mutants of Drosophila : Locomotor activity vs metabolism

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  1.  Inactive and couch potato mutants of Drosophila:Locomotor activity vs metabolism Sherry Lee Dr. Jadwiga Giebultowicz Dr. Natraj Krishnan HHMI Summer Program

  2. Circadian Rhythm • Endogenous • Period (per) and Timeless (tim) genes • Entrained by exogenous cues – light • Drosophila – crepuscular Dark Light Dark Days jneurosci.org/content/vol26/issue9/images/large/zns0080614060002.jpeg

  3. Feedback Loop From Dr. Jadwiga Giebultowicz

  4. Hypoactive Mutants • Inactive (iav): 70 and 78 • Couch potato (cpo): E023 flybase.bio.indiana.edu

  5. Metabolism • Intracellular – mitochondria • Cellular respiration converts glucose to ATP C6H12O6 (aq) + 6O2 (g) → 6CO2 (g) + 6H2O (l) • Waste products

  6. Activity Links to ROS Production ↑ activity = ↑respiration = ↑reactive oxygen species (ROS) www.sapphirebioscience.com

  7. Rhythmic ROS Production – wild-type END Krishnan N., et al. 2008. Biochemical and Biophysical Research Communications

  8. Objectives • Rhythmic activity compared to wild-type controls (CS) • Rhythmic ROS and ATP productions compared to wild-type controls (CS)

  9. Hypothesis Inactive and couch potato flies have less activity  less metabolism  less ROS and ATP production.

  10. Activity and Longevity • DAM activity monitor with computer data recording • Longevity cages Google images

  11. Mitochondrial H2O2 Production • Mitochondria isolated from whole bodies • Hydrogen peroxide microplate assay Google images

  12. Mitochondria isolated from whole bodies ATP bioluminescence ATP Production Google images

  13. Activity Results - LD • iav and cpo: not hypoactive Fig. 1 Activity graph of three genotypes, 78 iav, 70 iav, and E023 cpo. Horizontal activity vial

  14. Activity Results - LD Fig. 2 CS actogram Fig. 3 78 iav actogram Fig. 5 E023 cpo actogram Fig. 4 70 iav actogram

  15. Activity Results - DD Fig. 6 CS actogram Fig. 7 78 iav actogram Fig. 8 70 iav actogram Fig. 9 E023 cpo actogram

  16. Activity Results - DD % rhythmicity in constant darkness: • 78 iav: 50% (mean = 23.4) • 70 iav: 50% (mean = 23.6) • E023 cpo: 80% (mean = 27.6)

  17. Climbing Ability • iav and cpo: impaired negative geotropic movement iav CS

  18. H2O2 Production Results • iav: more H2O2 than CS • cpo: less H2O2 than iav 8 20 Fig. 10 Rate of H2O2 production for 78, 70, and E023 mutants and CS wild-type flies at ZT8 and ZT20.

  19. ATP Production Results • iav and cpo: more ATP than CS • cpo: more ATP than iav 8 20 Fig. 11 Rate of ATP production for 78, 70, and E023 mutants and CS wild-type flies at ZT8 and ZT20.

  20. Longevity Results • iav: shorter lifespan than CS • cpo: longer lifespan than iav TO BE CONTINUED Fig. 12 Kaplan-Meyer survival curves for 78, 70, and E023 mutants and CS wild-type flies.

  21. Discussion • iav compared with CS • cpo defects • Compare results with Hyperkinetic (Hk)

  22. Significance • Level of beneficial physical activity • ROS production affects longevity • Promotion of healthy aging

  23. Acknowledgements • Dr. Natraj Krishnan • Dr. Jadwiga Giebultowicz • Dr. Kevin Ahern • HHMI Summer Undergraduate Research Program

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