1. Victoria Wei

2. Need Taken from Rajput AH, Offord KP, Beard CM, Kurland LT. Epidemiology of parkinsonism: incidence, classification, and mortality. Ann Neurol. 1984;16:278-282. Figure 1 The amount of Parkinson’s disease cases per 100,000 people in the United States as age increases

3. Knowledge Base Parkinson’s disease is a brain disorder involving the nerves. http://www.spinstudios.co.uk/sa/pa3.jpg Figure 2 The effects of Parkinson’s disease

4. Knowledge Base Figure 3 The life cycle of C. elegans http://www.wormatlas.org/handbook/fig.s/IntroFIG6.jpg

5. Knowledge Base http://www.vhl.org/gifs/hif-1.jpg Figure 4 The hypoxic response in normal and low oxygen environments

6. Knowledge Base Lipofuscin is an auto- fluorescent age pigment which is found in people with neurodegenerative diseases. (Gray, et. al., 2005) Figure 5 Lipofuscin in neurons of the human brain. http://www.innovitaresearch.org/news/res/06042501_01.jpg

7. Knowledge Base Exposure to heavy metals induced a higher chance of acquiring PD. (Bjorklund, 1995.) http://img.alibaba.com/photo/103547192/_Super_Deal_Spot_Electrolytic_Copper_Powder_Hig h_Purity_Copper_Powder.jpg Figure 6 Copper oxide

8. Literature Review Braungart, et. al. (2004) Figure 7 The C. elegans with and without MPP+ Taken from Braungart, Evelyn; Gerlach, Manfred; Riederer; Peter, Baumeister, Ralf; and Hoener, Marius C. “Caenorhabditis elegans MPP+ Model of Parkinson’s Disease for High- throughout Drug Screening.” Neurodegenerative Disease. 2004. Volume 1: pgs 175-183.

9. Literature Review Sutphin, et. al. (2009) Sutphin, George; M. Kaeberlein. “Measuring Caenorhabditis elegans Life Span on Solid Media” JOVE. 2009. Figure 8 Auto fluorescent pigments present in Day 4 and Day 8 C. elegans

10. Literature Review Gerstbrein, et. al. (2008) Figure 9 Fluorescence of the C. elegans using the lipofuscin as a biomarker for health span. Gerstbrein, Beate; G. Stamatas; N. Kollias; M. Driscoll. “In viv spectrofluorimetry reveals endogenous biomarkers that report healthspan and dietary restriction in Caenorhabditis elegans.

11. Literature Review Mehta, et. al., (2009). Mehta, Ranjama; K.A. Steinkraus; G. L. Sutphin, F. J. Ramos, L. S. Shamieh;A. Huh; C. Davis; D. Chandler-Brown; M. Kaeberlein. “Proteasomal Regulation of the Hypoxic Response Modulates Aging in C. elegans.” Science. 2009. Figure 10 Mutation of VHL-1 reduces accumulation of auto-fluorescent age pigments

12. Purpose The purpose of the experiment is to observe the effects of a hypoxic response in anoxia on the amount of lipofuscin present in C.elegans Hypothesis Null- the amount of lipofuscin present during the hypoxic response in anoxia will be the same as the amount present without the hypoxic response in anoxia. Alternate- the amount of lipofuscin present during the hypoxic response in anoxia will be less than the amount present without the hypoxic response in anoxia.

13. Methodology The Effects of the Hypoxic Response in Anoxia on the Amount of Lipofuscin Present in C. elegans C. elegans obtained from the Caenorhabditis Genetics Center- N=80 Wild type C. elegans: N=40 VHL-1(ok161) C. elegans strain: (vhl-1 deletion) Constitutive HIF-1 in normoxia; slow growth and reduced brood size: N=40 Copper oxide concentrations will be found through experimentation with lethal doses Use of 4',6-diamidino-2-phenylindole (DAPI) to observe the amount of auto fluorescent pigment- lipofuscin- in C. elegans Statistical analysis using SPSS and T-test Given copper oxide N=20 Control VHL-1 N=20 Given copper oxide N=20 Control Wild Type N=20

14. Protocol C. elegans are grown in petri dishes containing Nematode Growth Media (NGM) from Carolina Biological and fed U.V. killed Escherichia coli. Figure 11 Culturing the C. elegans in Petri dishes Picture by author

15. Protocol Both Ampicillin and 5-Fluoro-2′-deoxyuridin will be used with NGM in the petri dishes with C. elegans http://upload.wikimedia.org/wikipedia/commons/b/b6/Ampicillin_structure.svg http://www.sigmaaldrich.com/structureimages/30/mfcd00006530.gif Figure 12 Ampicillin Figure 13 FUDR E.coli + NGM + Ampicillin + FUDR + copper oxide  symptoms of Parkinson’s disease starting with the L1 stage  using the DAPI filter to observe amount of lipofuscin present in both C. elegans groups

16. Protocol Sutphin, George; M. Kaeberlein. “Measuring Caenorhabditis elegans Life Span on Solid Media” JOVE. 2009. Figure 14 Age synchronization of C. elegans

17. Protocol Figure 15 Process of copper oxide application and observation amongst the four C. elegans groups Picture by author

18. Protocol Figure 16 4',6-diamidino-2- phenylindole (DAPI) http://upload.wikimedia.org/wikipedia/commons/7/7a/DAPI.png http://www.wormbook.org/chapters/www_intromethodscellbiology/cellfig3.jpg Figure 17 C. elegans as observed under DAPI filter

19. Budget VendorCat#ItemQty.Unit $Total $ Caenorhabditis Genetics Center CB5602 VHL-1(ok161) C. elegans 1$7 Caenorhabditis Genetics Center AB1 Wild type C. elegans1$7 SigmaD9542-5MGDAPI1$51.60 SigmaS2002Sodium azide1$21.20 SigmaF0503-100MGFUDR1$117 Sigma208825Copper Oxide1$28.30 Carolina Biological741270Petri dishes10$6.45$64.50 Carolina Biological216880Ampicillin dry powder1$43.25$43 Carolina Biological173520Nematode Growth Agar2$6.25$12.50 Carolina BiologicalOP50E. coli1$7 InvitrogenD21490DAPI1$116.00 Total Cost $359

20. Do-ability Available for Purchase: The VHL-1 and wild type C. elegans strains from CGC DAPI, Copper Oxide, and Sodium Azide from Sigma NGM and OP50 E.coli from Carolina Biological Equipment already Acquired: The DAPI filter (excitation filter centered at 365 nm and 445/50 nm emission band-pass filter), fluorescent microscope, UV lights

21. Bibliography "About Parkinson Disease." National Parkinson Foundation.. 1996-2007. Bjorklund, Geir. “Parkinson’s Disease and Mercury.” Journal of Orthomolecular Medicine. Volume 10. 1995. Braungart, Evelyn; Gerlach, Manfred; Riederer; Peter, Baumeister, Ralf; and Hoener, Marius C. “Caenorhabditis elegans MPP+ Model of Parkinson’s Disease for High-throughout Drug Screening.” Neurodegenerative Disease. 2004. Volume 1: pgs 175-183. Colleta, Susan. Introduction to C. elegans. Waksman Student Scholars.. 2009 Gerstbrein, Beate; G. Stamatas; N. Kollias; M. Driscoll. “In viv spectrofluorimetry reveals endogenous biomarkers that report healthspan and dietary restriction in Caenorhabditis elegans. Hall, D. H.; Z. F. Altun. “C. elegans Atlas.” Genetics Research, 90, pp 375-376. 2008. Hunt, Sara S. The Aging Process. Washington D.C. April 2004. Kenyon, Cynthia. “Environmental Factors and Gene Activities That Influence Life Span” C. elegans II. Cold Spring Harbor Press. 1997. Longo, V. “Oxygen? No thanks, I’m on a Diet” Science, Aging Knowledge Environment. Volume 2002. Pp. 10. 19 June 2002. Mc Naught, KS; P. Jenner. “Proteasomal function is impaired in substantia nigra in Parkinson's disease “ Neuroscience Letters. Volume 297. pp. 191-194. 2001. O'Riordan ; A.M. Burnell. Intermediary metabolism in the dauer larva. II. The glyoxylate cycle and fatty acid oxidation. Comp. Biochem. Physiol. Volume 95. pp. 125-130. 1990. Rajput AH, Offord KP, Beard CM, Kurland LT. Epidemiology of parkinsonism: incidence, classification, and mortality. Ann Neurol. 1984;16:278-282. Shen, Chuan; Daniel Nettleton; Min Jiang; Stuart K. Kim; Jo Anne Powell-Coffman. “Roles of the HIF-1 Hypoxia Inducible Factor during Hypoxia Response in Caenorhabditis elegans” The Journal of Biological Chemistry. Volume 280. pp.20580-20588. 2005. Sutphin, George; M. Kaeberlein. “Measuring Caenorhabditis elegans Life Span on Solid Media” JOVE. 2009. “What is Parkinson’s?” American Parkinson Disease Association West Coast Office.. 2009.