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. Literature Review “Caenorhabditis elegans MPP+ Model of Parkinson’s Disease for High-Throughput Drug Screenings” Braungart, et. al. (2004) Figure 6 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.

8. Literature Review “ Measuring Caenorhabditis elegans lifespan using solid media” Sutphin, et. al. (2009) Sutphin, George; M. Kaeberlein. “Measuring Caenorhabditis elegans Life Span on Solid Media” JOVE. 2009. Figure 7 Auto fluorescent pigments present in Day 4 and Day 8 C. elegans

9. Literature Review “ In vivo spectrofluorimetry reveals endogenous biomarkers that report healthspan and dietary restriction in Caenorhabditis elegans ” Gerstbrein, et. al. (2008) Figure 8 Fluorescence of the C. elegans using the lipofuscin as a biomarker for healthspan. Gerstbrein, Beate; G. Stamatas; N. Kollias; M. Driscoll. “In viv spectrofluorimetry reveals endogenous biomarkers that report healthspan and dietary restriction in Caenorhabditis elegans.

10. Literature Review “Proteasomal Regulation of the Hypoxic Response Modulates Aging in C. elegans” Mehta, Ranjama. 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 9 Mutation of VHL-1 reduces accumulation of auto-fluorescent age pigments

11. 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.

12. 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 2 hour timed egg laying for C. elegans. During the L4 stage, the C. elegans will be transferred to the Nematode Growth Media *(NGM) at 20°C. The C. elegans are fed U.V. killed E. coli stored at room temperature and grown overnight at 37°C. C. elegans are grown in liquid media at 16°C. 1.5 mM of 1-methyl-4-phenylpyridinium (MPP+) after the L1 stage is applied in the food. Use of 4',6-diamidino-2-phenylindole (DAPI) to observe the amount of auto fluorescent pigment- lipofuscin- in C. elegans- scale will be used to measure the amounts of lipofuscin. Statistical analysis using SPSS and T-test Given MPP+ N=20 Control VHL-1 N=20 Given MPP+ N=20 Control Wild Type N=20

13. Protocol C. elegans are grown in petri dishes containing Nematode Growth Media (NGM) from Carolina Biological and fed U.V. killed Escherichia coli. Figure 10 Culturing the C. elegans in Petri dishes Picture by author

14. 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 11 AmpicillinFigure 12 FUDR

15. Protocol http://upload.wikimedia.org/wikipedia/commons/a/a1/MPP%2B.svg Figure 13 1-methyl-4-phenlypyridinium (MPP+)

16. Protocol Figure 14 4',6-diamidino-2-phenylindole (DAPI) http://upload.wikimedia.org/wikipedia/commons/7/7a/DAPI.png

17. Protocol Sutphin, George; M. Kaeberlein. “Measuring Caenorhabditis elegans Life Span on Solid Media” JOVE. 2009. Figure 15 Age synchronization of C. elegans

18. Protocol Figure 16 Process of MPP+ application and observation amongst the four C. elegans groups Picture by author

19. Budget VendorCat#Pg.ItemQty.Unit $Total $ Caenorhabditis Genetics Center CB5602 VHL-1(ok161) C. elegans 1$7 Caenorhabditis Genetics Center AB1 Wild type C. elegans1$7 SigmaD048 MPP+1108 SigmaD9542-5MG DAPI151.6 SigmaS2002 Sodium azide121.2 SigmaF0503-100MG FUDR1117 Carolina Biological741270 Petri dishes106.4564.5 Carolina Biological216880 Ampicillin dry powder143.25$43 Carolina Biological173520 Nematode Growth Agar26.2512.5 Carolina BiologicalOP50 E. coli1$7 $439Total Cost

20. Do-ability Available for Purchase: The VHL-1 and wild type C. elegans strains from CGC DAPI, MPP+, 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, and petri dishes

21. Bibliography "About Parkinson Disease." National Parkinson Foundation.. 1996-2007. 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.