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

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Presentation on theme: "The Effects of PUGNAc on O-GlcNAC Levels and Dauer Formation in C. elegans By Nicole Kiess."— Presentation transcript:

1 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 diabetes

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. R/rdonlyres/6B1E037B C-8CFE- A448E43814DD/0/dauer2.jp g

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 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 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 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 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. 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 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 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) 100um 50um 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) 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 RL1 and RL2 dilated 1:400 and given to C. Elegans Picture 10: Life cycle of C. elegans erlab/research/pictures/Ce_life_cycle.gif

16 Protocol Fluorescent microscope and camera used for picture Fluorescent microscope and camera used for picture Photoshop used for percentages 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: Need to Purchase: RL-1 RL-2 RL-1 RL-2 C. elegans strains C. elegans strains E. coli E. coli PUGNAc PUGNAc Already Obtained Already Obtained Fluorescent microscope Fluorescent microscope Camera Camera Photoshop Photoshop Petri Dishes Petri Dishes

18 Budget

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