1. Genomics-based determination of nanoparticle toxicity: structure-function analysis Alex Hadduck – Biochemistry and Biophysics Dr. Alan Bakalinsky – Food Science and Technology

2. Fullerene  Buckyballs -1985 discovery by Kroto, Curl, and Smalley. 1996 Nobel

3. Oxygen Radical Scavenger  Alzheimer’s  Parkinson’s  Lou Gehrig’s

4. Drug Delivery  Osteoporosis  Tumor eradication

5. …And Much More  Gas absorption/storage/purification  Artificial muscles  Superconductors  Combat jackets  Air pollution filters  Bridge building

6. Toxicity of Buckyballs  Largemouth bass  DNA deformation  Eukaryotic cell growth inhibition  Antimicrobial activity  The how/why of fullerene toxicity is of great importance

7. Experimental Overview  S. cerevisiae submitted to many conditions in order to establish toxicity parameters, mimicking possible environmental exposure. Tests monitored either cell survival or growth rate.  Once parameters have been established, gene- deletion libraries used to screen for fullerene- protective genes – over 4800 non-essential genes.  Insight into toxicity mechanisms.  Expected human (and other) orthologs.

8. Toxicity Variables  Fullerene forms colloids – small uniform aggregates – in solution.  Aggregate size, and therefore function, very sensitive to solution chemistry.  pH, ionic strength (salts), preparation method, and organic matter (including cells) all play a role in how fullerene aggregates.

9. Deletion Library  Mutants of a single S. cerevisiae strain, each with a unique and non-vital gene missing.  Significant increase in sensitivity in a mutant signifies missing gene plays a role in fullerene protection.  Good way to quickly get to the mechanism of toxicity

10. 2007 – The Summer of Toxicity Parameters  Toxicity was not established early on  We struggled with finding assays that best met our needs.

11. No visible difference between test and control

12. If only…..

13. New Assays and Endpoints  Without being able to reliably recreate results (either positive or negative), our first goal was to re-think how we gather our data.  New assays had to be employed – we chose to use optical density and plating  We also added another possible route of toxicity – growth rate inhibition.

14. Plating

16. Plate Counts  Say we counted 100 cfu (colony forming units) in a plate after plating 100 microliters of a 10,000 fold dilution.  Formula: (cfu/mL plated) x dilution factor = cells/mL  So: (100/.1) x 10,000 = 1x10 7 cells/mL

17. Optical Density – Growth Rate  UV spectrophotometer used to measure the light scattering of each test – a direct correlation to cell count.

18. What We Have Learned So Far  Not toxic, but we aren’t finished  Colloidal stability of fullerene even more sensitive than we thought.  Crucial progress on proper procedures and assays for reproducible data

19. The Future  Continue to gather data either for or against fullerene toxicity in yeast.  The hardiness of yeast may be a blessing in disguise.

20. Thank You  Howard Hughes Medical Institute  College of Science Cripps Scholarship Fund  Dr. Alan Bakalinsky  Jeff Rowe  Vihangi Hindogalla  Dr. Qilin Li  Bin Xie