The Effects of The Effects of PUGNAc on O-PUGNAc on O-

GlcNAC Levels and GlcNAC Levels and Dauer Formation in Dauer Formation in

C. elegansC. elegansBy Nicole KiessBy Nicole Kiess

NeedNeed

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

NeedNeed

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 BaseKnowledge 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 BaseKnowledge 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 BaseKnowledge Base

QuickTime™ and a decompressor

are needed to see this picture.

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 ReviewLiterature Review

Paradis, et al,(1998) showed that Paradis, et al,(1998) showed that mg144 does not activate AGE-1 mg144 does not activate AGE-1 P13K and that Akt/PBK is not P13K and that Akt/PBK is not dependent on AGE-1/P13Kdependent on AGE-1/P13K

QuickTime™ and a decompressor

are needed to see this picture.

Picture 6a: Shows normal growth conditions

Picture 6b: Shows dauer formation conditions

Literature ReviewLiterature Review

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

Literature ReviewLiterature Review Hanover, et al, 2005, findings show that OGT modulates Hanover, et al, 2005, findings show that OGT modulates

macronutrient storage and dauer formation in macronutrient storage and dauer formation in C. ElegansC. Elegans

Picture 7a: Insulin like signaling pathway

Picture 7b: Shows percentages of dauers in different strains

Literature ReviewLiterature Review Forsythe, et al, 2006 demonstrated that enzymes of O-Forsythe, et al, 2006 demonstrated that enzymes of O-

GlcNAc cycling perfect insulin-like signaling in response GlcNAc cycling perfect insulin-like signaling in response to nutrient fluxto nutrient flux

Picture 8a: Insulin like signaling pathway

Picture 8b: Dauer formation in different strains with temperature as a variable.

PurposePurpose

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

HypothesisHypothesis

NullNull- There will be no change in O-- There will be no change in O-GlcNAc levels and no change in GlcNAc levels and no change in dauer formationdauer formation

AlternativeAlternative- The levels of O-GlcNAc - The levels of O-GlcNAc will rise and dauer formation will will rise and dauer formation will rise rise

MethodMethodThe 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 100um 100um 100um50um

50um

50um

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

ProtocolProtocol

C. elegans C. elegans will be cultured in petri will be cultured in petri dishes with the Nematode Growth dishes with the Nematode Growth Medium (NGM) and will be fed Medium (NGM) and will be fed Escherichia coli Escherichia coli ((E. coliE. coli))

Picture 9: How the C. elegans will be cultured

Drawn by Victoria Wei

ProtocolProtocol

RL1 and RL2 dilated 1:400 and given RL1 and RL2 dilated 1:400 and given to to C. ElegansC. Elegans

Picture 10: Life cycle of C. eleganshttp://www.sfu.ca/biology/faculty/hutter/hutterlab/research/pictures/Ce_life_cycle.gif

ProtocolProtocol

Fluorescent microscope and camera Fluorescent microscope and camera used for pictureused for picture

Photoshop used for percentagesPhotoshop 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-AbilityDo-Ability Need to Purchase:Need to Purchase:

RL-1 RL-2RL-1 RL-2 C. elegans C. elegans strainsstrains E. coliE. coli PUGNAcPUGNAc

Already ObtainedAlready Obtained Fluorescent microscopeFluorescent microscope CameraCamera PhotoshopPhotoshop Petri DishesPetri Dishes

BudgetBudget

Vendor Cat. # Page Item Order # / Strain Quantitiy Unit Price Total PriceCaenorgabditis Genetics Center (CGC) - - RB653 ogt-1(ok430) 1 $7 $7Caenorgabditis Genetics Center (CGC) - - RB1169 oga-1(ok1207) 1 $7 $7

Caenorgabditis Genetics Center (CGC) - - CB1370 daf-2(e1370) 1 $7 $7

Caenorgabditis Genetics Center (CGC) - - VC2010 WT N2 1 $7 $7Caenorgabditis Genetics Center (CGC) - - E. coli OP50 1 $7 $7Sigma-Aldrich A7229 PUGNAc-10mg 1 $479 $479

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