Danika Kusuma Dr. Dee Denver Summer of 2010 HHMI

1. The Effects of Vitamin E on the Lifespan of Caenorhabditiselegans Strains with Mitochondrial Mutations DanikaKusuma Dr. Dee Denver Summer of 2010 HHMI http://www.kiwicrossing.com/drrussell/images/worm.jpg

2. Background • Free-radical theory. • Organisms age because cells accumulate free radical damage over time. • Free radicals, reactive oxygen species (ROS) • Comes from food, mitochondrial dysfunctions, etc. • Antioxidants • Unpaired electron reacts with biological structures, causing damage; antioxidants prevent damage as reducing agents.

3. Background (con.) • Vitamin E • Fat-soluble, several different types. • Alpha tocopherol: often used as a supplement vitamin and main role is as an antioxidant although it may have cell signaling properties that are not well-known. • Used as a supplement vitamin and the main type of vitamin E that is held in the body. http://www.3dchem.com/imagesofmolecules/vitamine.jpg

4. http://www.cnsforum.com/content/pictures/imagebank/hirespng/MOA_VITE.pnghttp://www.cnsforum.com/content/pictures/imagebank/hirespng/MOA_VITE.png

5. Nematodes Model Organism Genome sequenced. Easily maintained. Short generation time Short lifespan Two Strains N2 wild type Caenorhabditiselegans mev-1 (genetic mutant strain of C. elegans) • mitochondrial electron transport chain mutant • higher ROS levels and shortened lifespan http://www.wormclassroom.org/ce/kahn/C.elegans_L4.jpg

6. Hypothesis • High levels of free radical damage result in a decrease in lifespan. • PREDICTION: • mev-1 strain will have a shorter lifespan than N2 wildtypeC. elegans. • Vitamin E reduces ROS damage and increases lifespan. • PREDICTION: • The introduction of antioxidant vitamin E to N2 wildtypeC. elegansand mev-1 strain will increase lifespan in both strains.

7. Methods • Assay lifespans of two genotypes of nematodes: N2 wildtype and mev-1 strain – after vitamin E treatments.

8. Approach Monitored 192 worms total. All vitamin E treatments in 100% ethanol. One treatment group exposed for 2 hours, another exposed for 24 hours.

9. Approach (con.) • Worms first developmentally synchronized. • Populations of worms suspended in 10 milliliters M9 buffer and then treated with 10 microliters of each treatment substance. • Put on a nutator for 2 hours or 24 hoursdepending on treatment group.

10. Treatments (con.) • Individual worms are monitored and lifespan is assayed. • Worms are determined alive by twitch response. • Lifespan assay recorded as number of days lived.

11. Results: 2 Hour Treatment

12. Results: 2 Hour Treatment (con.) • “mev-1 strain will have a shorter lifespan than N2 wildtypeC. elegans.” • Between N2-C and mev-1 –C: p-value= 0.00011 Between N2 treated and mev-1 treated: • p-value= 0.0034 • Between N2 –C and N2 treated: • p-value= 0.89 • Between mev-1 –C and mev-1 treated: • p-value= 0.023 • Trend shows an increase in lifespan.

13. Results: 24 Hour Treatment

14. Results: 24 Hour Treatment (con.) • “mev-1 strain will have a shorter lifespan than N2 wildtypeC. elegans.” • Between N2-C and mev-1 –C: p-value= 1.73 × 10-5 Between N2 treated and mev-1 treated: • p-value=4.19× 10-5 • Between N2 –C and N2 treated: • p-value= 0.42 • Between mev-1 –C and mev-1 treated: • p-value= 0.59 • Trend shows a decrease in lifespan.

15. Future Work • ANOVA • Further lifespan assays with increased replication. • Measuring mitochondrial ROS levels.

16. Acknowledgements • Dr. Dee Denver • Dana Howe • Kristin Gafner • Elizabeth Quimba • Sita Ping • Dr. Kevin Ahern • Howard Hughes Medical Institute • University Honors College • National Institutes of Health