1. The Effects of PUGNAc on O-GlcNAC Levels and Dauer Formation in C
The Effects of PUGNAc on O-GlcNAC Levels and Dauer Formation in C. elegans
By Nicole Kiess

2. Need
Picture 1: Shows the rate of Type 2 Diabetes in relationship to obesity over 10 years.

3. Need
Estimated prevalence of diagnosed and undiagnosed diabetes in people aged 20 years or older, by age group, United States, 2007
                                                                                                                                                                                                                                                                                                       
Picture 2: Shows percent of people in different age groups with Type 2 Diabetes.

4. Knowledge Base
Picture 3: Is a model of what happends in the body of a Type 2 Diabetic

5. Knowledge Base
Picture 4a: Shows the pathway that regulate aging in C. Elegans.
Picture4 b: Shows the model for the regulation of life span and sensory perception.

6. Knowledge Base Picture 5: (Top) Insulin-like signaling pathway.
(Bottom) C. Elegnas life cycle.

7. Literature Review
Paradis, et al,(1998) showed that mg144 does not activate AGE-1 P13K and that Akt/PBK is not dependent on AGE-1/P13K
Picture 6a: Shows normal growth conditions
Picture 6b: Shows dauer formation conditions

8. Literature Review
Arias, et al, 2004 showed that PUGNAc increases O-GlcNAc levels in rat skeletal muscle

9. Literature Review
Hanover, et al, 2005, findings show that OGT modulates macronutrient storage and dauer formation in C. Elegans
Picture 7a: Insulin like signaling pathway
Picture 7b: Shows percentages of dauers in different strains

10. Literature Review
Forsythe, et al, 2006 demonstrated that enzymes of O-GlcNAc cycling perfect insulin-like signaling in response to nutrient flux
Picture 8a: Insulin like signaling pathway
Picture 8b: Dauer formation in different strains with temperature as a variable.

11. Purpose
Therefore the purpose of this experiment is to observe the O-GlcNAc levels, using densinometry, and dauer formations with PUGNAc as a variable.

12. Hypothesis
Null- There will be no change in O-GlcNAc levels and no change in dauer formation
Alternative- The levels of O-GlcNAc will rise and dauer formation will rise

13. Method
The effects of PUGNAc on O-GlcNAC levels and dauer fomation in the insulin-like signaling pathway of C. elegans
Control
Wild Type N2
Ogt-1 (ok430)
Oga-1 (ok1207)
Daf-2 (e1370)
50um
100um
50um
100um
50um
100um
50um
100um
C. Elegans will be cultured at 16 degrees C
O-GlcNAC levels will be observed by treating C. elegans with RL1 and RL2 antibodies
Pictures will be taken with camera under immunofluorescent microscope and photoshop will measure percentages
Adults of each strain lay embryos overnight at 15 degrees C. Cultures shifted to 22.5 degrees C and are scored 3 days later as either dauer or non dauer
Statistical Analysis will be run with SPSS and an ANOVA

14. Protocol
C. elegans will be cultured in petri dishes with the Nematode Growth Medium (NGM) and will be fed Escherichia coli (E. coli)
Picture 9: How the C. elegans will be cultured
Drawn by Victoria Wei

15. Protocol RL1 and RL2 dilated 1:400 and given to C. Elegans
Picture 10: Life cycle of C. elegans

16. Protocol Fluorescent microscope and camera used for picture
Photoshop used for percentages
Picture 11: A) Fluorescent picture to show O-GlcNAc levels with RL-2
B) Fluorescent picture to show O-GlcNAc levels with RL-2
Hanover, et al, 2005

17. Do-Ability Need to Purchase: Already Obtained RL-1 RL-2
C. elegans strains
E. coli
PUGNAc
Already Obtained
Fluorescent microscope
Camera
Photoshop
Petri Dishes

18. Budget

19. Bibliography
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"Introduction to C. Elegans." Genes, Genomes, and Human. 07 May 2009 <
Kao, G., C. Nordenson, M. Still, A. Ronnlund, S. Tuck, and P. Naredi. "ASNA-1 positively regulates insulin secretion in C. elegans and mammalian cells." Cell 128 (2007):
Kimura K.D., Tissenbaum H.A., Liu Y., Ruvkun G. (1997) daf-2, an insulin receptor-like gene that regulates longevity and diapause in Caenorhabditis elegans.Science 277:942–946.
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"Type 2 Diabetes: Conditions, Treatments, Resources - American Diabetes Association." American Diabetes Association Home Page. 04 May 2009 <
Twyman, Richard. "Model organisms: The nematode worm." The Human Genome. 07 May 2009 <
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