1. Antioxidants & Obesity in C. elegans Manhasset Science Research Madelyn Sayed

2. Need: Obesity (American Heart Association, 2004) http://www.americanheart.org/downloadable/heart/1103 834132259FS16OVR5.pdf Figure 1: Prevalence of Obesity in adults ages 20-74 by sex. (1960-2004)

3. http://www.oxha.org/knowledge/backgrounders/overweight_and_obesity.gif/view Need: Obesity (2) Figure 2: Obese and overweight men and women in India, China, Mexico, & USA.

4. Need: Diabetes Statistics Figure 2.5: Diabetes in patients over 18 by race and sex. (Non- Hispanic Whites, Non-Hispanic Blacks, and Mexican Americans). http://www.oxha.org/knowledge/backgrounders/overweight_and_obesity.gif/view

5. Knowledge Base Hyperlipidemia Phytochemicals Antioxidant Free Radical Beta-carotene. Thiol Hypodermis Figure 3: Layers of the skin in the human body are shown. The hypodermis is the lowest layer in this diagram. http://www.daviddarling.info/images/hypodermis.jpg

6. Knowledge Base (2) Figure 4: The lifecycle of C. elegans in hours at a constant temperature of 25°C. http://thalamus.wustl.edu/nonetlab/ResearchF/elegans.html

7. Knowledge Base (3) Figure 5: C. elegans at various stages in its lifecycle in hours at a constant temperature of 25°C. http://thalamus.wustl.edu/nonetlab/ResearchF/elegans.html

8. Seymour et al., 1997 Rats fed low and high fat diets with and without blueberries Rats that received the blueberry-enriched powder ‘healthier’ Also true with grapes and cherries in other experiments

9. Jones et al., 2009 crucial regulators of lipid homeostasis in mammals serve similar functions in C. elegans. fat metabolism, & feeding/food-related behaviors intestinal and hypodermal cells in C. elegans (red) Systems transport fats between cells and tissues (green). Figure 6: C. elegans fat transport and storage areas.

10. Jones et al., 2009 (2) (A) Nile Red (B) Sudan Black B (C) LipidTOX neutral lipid staining (D) Nile Red and fatty acid lipid conjugated BODIPY staining (E) fatty acid lipid conjugated BODIPY staining (F) Both Nile red and BODIPY florescence Figure 7: C. elegans fat deposits shown-stained with Nile Red, Sudan Black B, and other stains.

11. Hildebrandt, 2004 Effect of thiol antioxidant on body fat and insulin reactivity 2 studies: obese & hyperlipidemic/normolipidemic treated with antioxidants such as x-lipoic acid and NAC Their studies showed that the effect of NAC was statistically significant, but the effect of creatine was not. Figure 8

12. Ashrafi et al., 2003 & 2007 (1) Figure 9: C. elegans stained with Nile Red in C, D, and E, with Sudan Black B in F and G. Fat appears fluorescent red (N.R.) and blue- black (S.B.)

13. Ashrafi et al., 2003 & 2007 (2) 10

14. Halliwell et al., 2009 Pine bark extract on metabolism and lifespan of C. elegans Figure 11: Antioxidant balance in body, affect on metabolism

15. Purpose / Hypothesis Purpose: to test the affect of antioxidants on fat deposition in C. elegans. Null Hypothesis: the blueberry polyphenol compounds will not reduce fat deposition in C. elegans. Alternate Hypothesis: the blueberry polyphenol compounds will reduce fat deposition.

16. Methodology Cultured axenically Grown under monoxenic conditions Pine Bark Extract E. coli strain OP50 Stain: Sudan Black B http://individual.utoronto.ca/proy/worms.htm

17. Flow Chart Caenorhabditis elegans Lifespan: (2-3 weeks) Rapid generation time Experimental flexibility Control Group Sample Size N = ? Not fed Pine Bark Extract Experimental Group Sample Size N = ? Fed Pine Bark Extract Measurement: Sudan Black B fat staining Motic Plus camera with Stereomicroscope Amount of fat (using Adobe Photoshop) deposited in the worm (cm 3 )

18. Do Ability Fluorescent Dissection microscope is available in the lab Pycnogenol® easily obtainable C. elegans easily cultured Sudan Black B reliable fat stain Motic plus camera with stereomicroscope for photography Adobe Photoshop available for fat measurement

19. Protocols for Measurement C. elegans will be fed E.coli OP50 Pycnogenol® 50 mg capsules Sudan Black: 0.7 gm Propylene Glycol: 100.0 ml Figure: E. Coli bacteria

20. Budget ItemVendorCatalogue #Pg.Unit PriceQtyTotal Price C. elegans N2 (wild type) Carolina Science 2009 NP-17-3500248$10.95 (per culture) 1 NGMCarolina Science 2009 NP-17-3520248$6.25 (per 135 mL bottle) 2 Sudan Black III (Fatstain Herxheimer Lab Grade) Carolina Science 2009 NP-89-2963821$6.65 (120 mL bottle) 2 Pycnogenol® (Pine Bark Extract) Vitacost http://www.vitacost.com/ Schiff- Pycnogenol?csrc=PPCA DWLT- pycnogenol&s_kwcid=T C|3001|pycnogenol||S|p| 2989626851 Item #: SCF 5127419 or 020525127419 $16.53 (30 capsules) (50 mg/ capsule) 1 $40.38

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