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The Effect of the Hypoxic Response in Anoxia on the Amount of Lipofuscin Present in C. elegans

The Effect of the Hypoxic Response in Anoxia on the Amount of Lipofuscin Present in C. elegans. Victoria Wei. Need. Taken from Rajput AH, Offord KP, Beard CM, Kurland LT. Epidemiology of parkinsonism: incidence, classification, and mortality. Ann Neurol. 1984;16:278-282.

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The Effect of the Hypoxic Response in Anoxia on the Amount of Lipofuscin Present in C. elegans

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  1. The Effect of the Hypoxic Response in Anoxia on the Amount of Lipofuscin Present in C. elegans 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. Figure 2 The effects of Parkinson’s disease http://www.spinstudios.co.uk/sa/pa3.jpg

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

  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) http://www.innovitaresearch.org/news/res/06042501_01.jpg Figure 5 Lipofuscin in neurons of the human brain.

  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) Figure 7 Auto fluorescent pigments present in Day 4 and Day 8 C. elegans Sutphin, George; M. Kaeberlein. “Measuring Caenorhabditis elegans Life Span on Solid Media” JOVE. 2009.

  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. The Effects of the Hypoxic Response in Anoxia on the Amount of Lipofuscin Present in C. elegans Methodology 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 Given MPP+ N=20 Control Wild Type N=20 Given MPP+ N=20 Control VHL-1 N=20 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

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

  14. Protocol • Both Ampicillin and 5-Fluoro-2′-deoxyuridin will be used with NGM in the petri dishes with C. elegans http://www.sigmaaldrich.com/structureimages/30/mfcd00006530.gif http://upload.wikimedia.org/wikipedia/commons/b/b6/Ampicillin_structure.svg Figure 11 Ampicillin Figure 12 FUDR

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

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

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

  19. Budget

  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. <”http://www.parkinson.org/Page.aspx?pid=225”>. 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. <http://avery.rutgers.edu/WSSP/StudentScholars/project/introduction/worms.html>. 2009 • Gerstbrein, Beate; G. Stamatas; N. Kollias; M. Driscoll. “In vivspectrofluorimetry 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 substantianigra 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. <“http://www.apdawest.org/WhatIsParkinsons.html#2”>. 2009.

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