This study investigated the effects of hydrogen peroxide (H2O2) on three species of protozoa: Volvox, Prorocentrum, and Porphyridium. The protozoa were exposed to varying concentrations of H2O2 for 24 hours. DNA was then isolated and analyzed via gel electrophoresis. Both Porphyridium and Volvox degraded their DNA at H2O2 concentrations below 4 mM, while Prorocentrum only degraded its DNA above 10 mM. This suggests the protozoa may undergo apoptosis when exposed to oxidative DNA damage, as their DNA is fragmented in a dose-dependent manner. Further experiments aim to test lower H2O2 concentrations and examine caspase

2. Volvoxwas grown in 50mL of soil water

3. Prorocentrumwas grown in 25mL of seawater and 25mL of soil water

6. The cells were centrifuged to form a pellet

7. They were then each resuspended in 1mL of their specific growth media

10. All three species degrade their DNA in the presence of H2O2

11. Porphyridium and Volvox degrade their DNA at H2O2 concentrations < 4 mM

12. Prorocentrum is tolerant of H2O2 at 4 mM but degrades its DNA at concentrations > 10 mM

13. Testing Volvox and Porphyridium at lower concentrations of H2O2 should deterimine the maximum tolerance of these species

15. Take samples from the exponential and stationary phase to determine if stage of lifecycle is important

16. Expose other species of protozoa to hydrogen peroxide to determine if they are also capable of degrading their DNA in response to oxidative stress

17. Develop a procedure for isolating protein from Prorocentrum and run a western blot to determine if they contain any caspases associated with the intrinsic pathway of apoptosis.10 mM H2O2 exposure for 24 hours Control 100x 100x Porphyridium Literature cited Volvox Prorocentrum 4 C 10 4 10 C 4 10 40 80 40 C 40 JékelyGáspár. The ICE family of cysteine proteases. April 2009. http://www.cryst.bbk.ac.uk/pps97/assignments/projects/jekely/caspase.htm LawenAlfons. Apoptosis - an introduction. BioEssays. 2003; 25:888-896. Research Apoptosis. Apoptosis regulation and execution signaling pathways. April 2009. http://www.researchapoptosis.com/apoptosis/pathways/index Results Previous research using western blotting to identify caspases indicates that Porphyridium and Volvox contain caspase 9 which is associated with DNA damage and the intrinsic pathway of apoptosis. Proteins from Prorocentrum were unable to be isolated so no predictions of its ability to undergo apoptosis could be made. If it can be shown that these protozoa contain caspases and undergo the process of apoptosis it would suggest that apoptosis is a highly conserved process which originated in unicellular organisms. C= no H2O2; numbers = concentration (mM) of H202 exposure for 24 hours. Gel Stained with SYBR Green Acknowledgments This independent project was executed for BIO 336 under the supervision of Dr. Michael Bumbulis. Experimental cells taken from day 14 and 21 of the 3/30 culture