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

Need Picture 1: Shows the rate of Type 2 Diabetes in relationship to obesity over 10 years. http://www.ndep.nih.gov/diabetes/WTMD/images/figure2.gif

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. http://www.cdc.gov/diabetes/pubs/estimates07.htm

Knowledge Base Picture 3: Is a model of what happends in the body of a Type 2 Diabetic http://savvyhealthfitness.com/get-healthy/diabetes/type-2-diabetes-lifestyle-diabetes

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. http://www.nature.com/nature/journal/v408/n6809/fig_tab/408255a0_F3.html

Knowledge Base Picture 5: (Top) Insulin-like signaling pathway. (Bottom) C. Elegnas life cycle. http://www2.niddk.nih.gov/NR/rdonlyres/6B1E037B-3958-485C-8CFE-A448E43814DD/0/dauer2.jpg

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

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

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

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.

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

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

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

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

Protocol RL1 and RL2 dilated 1:400 and given to C. Elegans Picture 10: Life cycle of C. elegans http://www.sfu.ca/biology/faculty/hutter/hutterlab/research/pictures/Ce_life_cycle.gif

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

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

Budget

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