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 Lipofuscin is an auto- fluorescent age pigment which is found in people with neurodegenerative diseases. (Gray, et. al., 2005) Figure 4 Lipofuscin in neurons of the human brain. http://www.innovitaresearch.org/news/res/06042501_01.jpg

6. Knowledge Base Centrophenoxine is an anti-aging medicine which slows the accumulation of lipofuscin. Schneider, et. al. (1977) http://commons.wikimedia.org/wiki/File:Centrophenoxine.svg Figure 5 Molecular structure of centrophenoxine

7. Literature Review Caldwin, et. al. (2008) Figure 7 Both images taken from Caldwin, Guy A.; K.A. Caldwell. “Traversing a wormhole to Combat Parkinson’s disease.” Disease Models and Mechanisms. Volume 1. pp.000- 000. 2008. Figure 6

8. 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

9. 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.

10. Literature Review Application of centrophenoxine to the C. elegans decreased the rate of lipofuscin accumulation by an average of 41.3%. (Shulkin, et. al., 1978) Figure 10 Fluorescence of the C. elegans using the lipofuscin as a biomarker for health span. Shulkin, D.J.; B.M. Zuckerman. “Spectrofluorometric analysis of the effect of centrophenoxine on lipofuscin accumulation in the nematode C. elegans.” Age. Volume 5. Pp. 50-53. 1982.

11. Purpose The purpose of the experiment is to observe the effects of centrophenoxine on the restriction of Parkinson’s disease symptoms in C.elegans Hypothesis Null- the symptoms of Parkinson’s disease will remain the same with or without the application of centrophenoxine. Alternate- the symptoms of Parkinson’s disease will lessen with the application of centrophenoxine.

12. Methodology The Effects of Centrophenoxine on the development of Parkinson’s disease in C. elegans C. elegans obtained from the Caenorhabditis Genetics Center- N=80 Wild type C. elegans: N=40 ham-1(ot339) C. elegans: N=40 6.8 mM centrophenoxine will be applied to the Nematode Growth Media for 21 days. Use of 4',6-diamidino-2-phenylindole (DAPI) to observe the amount of auto fluorescent pigment- lipofuscin- in C. elegans. GFP filter will also be used. Statistical analysis with SPSS Given centrophenoxine N=20 Control N=20 Given centrophenoxine N=20 Control 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 11 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 12 Ampicillin Figure 13 FUDR E.coli + NGM with centrophenoxine + Ampicillin + FUDR  using the DAPI and GFP filter to observe amount of lipofuscin and fluorescence present in both C. elegans groups

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

16. Protocol Figure 15 Process of centrophenoxine application and observation amongst the four C. elegans groups Picture by author

17. 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

18. Budget VendorCat#ItemQty.Unit $Total $ Caenorhabditis Genetics CenterGS1214ham-1(ot339) C. elegans1$7 Caenorhabditis Genetics CenterAB1Wild type C. elegans1$7 SigmaD9542-5MGDAPI1$51.60 SigmaS2002Sodium azide1$21.20 SigmaF0503-100MGFUDR1$117 SigmaSLC5377-25GCentrophenoxine Hydrochloride1$97.82 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$545

19. Do-ability Available for Purchase: The ham-1(ot339) and wild type C. elegans strains from CGC DAPI and Sodium Azide from Sigma NGM and OP50 E.coli from Carolina Biological Centrophenoxine purchaseable from Science Lab.com Equipment already Acquired: The DAPI filter (excitation filter centered at 365 nm and 445/50 nm emission band-pass filter), fluorescent microscope, UV lights

20. 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. Caldwin, Guy A.; K.A. Caldwell. “Traversing a wormhole to Combat Parkinson’s disease.” Disease Models and Mechanisms. Volume 1. pp.000- 000. 2008. 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. Kisiel, Marion J.; B. Zuckerman. “Effects of Centrophenoxine on the Nematode Caenorhabditis Briggsae” Age. Volume 1. Pp.17-20. January 1978. 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. Schneider, Howard F.; C. Nandy. “Effects of Centrophenoxine on Lipofuscin Formation in Neuroblastoma Cells in Culture” Journal of Gerontology. Volume 32. Pp. 132-139. 1997. Shulkin, D.J.; B.M. Zuckerman. “Spectrofluorometric analysis of the effect of centrophenoxine on lipofuscin accumulation in the nematode C. elegans.” Age. Volume 5. Pp. 50-53. 1982. 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.