1. Melody Clark Roger Worland Gordana Grubor-Lajsic Jelena Pilipovic Sleeping Beauty

2. cryoprotective dehydration anhydro / cryoprotectant (trehalose) Onychiurus arcticus – freeze avoiding

3. Fresh sample Sample cooled to –14°C

4. What has been done until now?

5. Determination of critical stages in the desiccation process samples cooled from +2 to ‑ 14°C (2°C per week) determined the freezing point and water content (DSC) polyol analysis (GC) the time taken to recover and return to normal water content was measured at each 2 degree interval we:

6. Going home

8. Onychiurus arcticus 1.Control live animals from +5°C 2.Desiccatingcooled to - 2°C (rate 2°C/week) 3.Fully desiccatedcooled to - 14°C (rate 2°C/week) 4.Recoveringfrom - 14°C, after 8 hours at RT with moisture 5.Fully recoveredfrom - 14°C, after 24 hours at RT with moisture

9. enzymes involved in synthesis of trehalose LEA proteins molecular analysis in Berlin (expecting 10 000 sequences) antioxidative enzymes

10. -desiccation is not characteristic -rapidly cold hardens, on a daily basis Cryptopygus antarcticus – freeze avoiding removing ice nucleators cryoprotectants (glycerol)

11. 1.Control live animals from +2°C 2.Cooled animals to -5°C (rate 0.5°C/min) 3.Heated animals to +15°C (rate of 1°C/min) 4.“High group” SCP when they were collected was above -15°C 5.“Low group” SCP when they were collected was below -15°C Cryptopygus antarcticus

12. microarray with cDNA of Cryptopygus antarcticus and Onychiurus arcticus together differential expression of genes in different treatment how closely related are the two species

13. Constructing 5 cDNA libraries (Stratagene pBluescript  II XR cDNA Library Construction Kit) Total RNA isolation with TRIZOL (300-400 animals, ~0.05mg) Purification of poly A+ mRNA (Qiagen Oligotex mRNA Kit- Midi) Transformation (Stratagene XL10-Gold® Ultracompetent Cells) Sequencing ~ 1000 clones (BIG Dye Terminator)

14. What are we planning to do…

15. continue work on final microarray submit these sequences to database microarray just with Cryptopygus antarcticus as a pilot experiment (about 800 clones have already been printed) general primers for HSP and AQP

16. Collecting samples in the Arctic (August 13 th to 24th)

22. Thank you!