13. Testing Volvox and Porphyridium at lower concentrations of H2O2 should deterimine the maximum tolerance of these species
14.
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