1. Investigating Intestinal Alkaline Phosphatase as a Risk Factor for Inflammatory Bowel Disease Vanessa Danquah Guillemin Lab Guillemin Lab

2. What is Inflammatory Bowel Disease ?

3. Intestinal Alkaline Phosphatase Detoxifies Lipopolysaccharide in Zebrafish IAP is an enzyme that interacts with the microbiota in the gut. IAP is an enzyme that interacts with the microbiota in the gut. It was proven that decreased IAP activity caused inflammation in zebrafish intestine. It was proven that decreased IAP activity caused inflammation in zebrafish intestine.

4. Is there a correlation between IAP and Inflammatory Bowel Disease ? Is the function of intestinal alkaline phosphatase in mediating bacterial-host interactions evolutionarily conserved? Is the function of intestinal alkaline phosphatase in mediating bacterial-host interactions evolutionarily conserved? Do polymorphisms in human IAP alter enzymatic activity and contribute to IBD? Do polymorphisms in human IAP alter enzymatic activity and contribute to IBD?

5. Measuring activity of IAP in Drosophila gut

6. Gender and Age Experiments Gender Gender Age Age

7. L-Phenylalanine-IAP Inhibitor

8. Searching for the gene or genes that encode IAP in Drosophila Each deficiency stock deletes approximately 50 genes, one of which is the candidate for encoding IAP. Each deficiency stock deletes approximately 50 genes, one of which is the candidate for encoding IAP. Female X 2L 2R 3L 3R 4 X

9. Two chromosomal regions encode IAP genes

10. Human IAP Polymorphisms Different people have different amino acids within their IAP proteins at certain locations Different people have different amino acids within their IAP proteins at certain locations We will investigate whether these amino acid changes alter the enzyme’s activity We will investigate whether these amino acid changes alter the enzyme’s activity P65L R125H H279L

11. How did we create known IAP variants? Make IAP variants via site directed mutagenesis Transfect and express them in Chinese Hamster Ovary Cells (CHO ) Assay for activity with pNPP substrate Clone IAP variants and wild type construct into an expression vector

12. Initial results of human IAP enzyme activity in vitro 3:2 4:2 5:2 6:2 7:2 8:2 UT 24 48

13. Conclusion and Future Directions Further characterize the genes within chromosomal deletions that reduce IAP activity Further characterize the genes within chromosomal deletions that reduce IAP activity Continue to test IAP variants and whether they alter activity in vitro Continue to test IAP variants and whether they alter activity in vitro Test whether expression of human IAP variants in Drosophila alter bacterial- host interaction in flies Test whether expression of human IAP variants in Drosophila alter bacterial- host interaction in flies Drosophila intestines have high alkaline phosphatase activity that is inhibited by the mammalian specific IAP inhibitor L-Phenylalanine We have identified two chromosomal regions in the Drosophila genome that are likely to encode Drosophila IAP genes We have established a cell culture system to study human IAP polymorphisms

14. Acknowledgements Guillemin Lab Dr. Karen Guillemin Ms. Anica Wandler Dr. Peter O’Day SPUR 2009