Sub-Project 3 Progress Report March 2009 Simon Moon, Anna Rose, Maggie Dallman and Jaroslav Stark

Recap TLR 4 Notch Interaction

Recap: Experimental Method + Jagged1 + control BMDC Macrophages +/- LPS RNA Real-time PCR Microarray Supernatant ELISA

Recap: Interaction

Modelling microarray data Rate of change of expression of a gene Basal rate Transcription factor activity Sensitivity Decay rate

Example Cluster: IL10 Jagged

Modelling IL-10 degradation Stimulate cells with our ligands Treat at 4 hours with Actinomycin D: an inhibitor of transcription. Observe decay of mRNA using RT-PCR Modelled using simple ODE models featuring mRNA stabilization and destabilization Unbound Protein Stable Protein mRNA Complex Unbound mRNA

Integration of the sub-projects Role of glycostructures of C. jejuni in the immune response DCs and macrophages are the one of the first cell types of the immune system to sense the presence of pathogenic bacteria They have a wide range of pattern recognition receptors, like the TLRs, that trigger expression of cytokines upon binding of a ligand. Investigation of the role of the glycostructures of C. jejuni in the immune response using C. jejuni mutants from sub- project 1.

Integration of the sub-projects Role of glycostructures of C. jejuni in the immune response Murine BMDCs were infected with various amounts of C. jejuni for three hours and changes in gene expression measured by real-time PCR. To date, WT, PglB (no N-linked glycosylation) and cj1439 (acapsular) were used. Cytokines like TNF , IL-6 and IL-10 were higher with the acapsular mutant than WT.

Prediction of Splice variants from Exon array data A collaboration with Sylvia Richardson Sylvia Richardson’s group developed a new algorithm to predict the presence of splice variants from Exon microarray data. Algorithm takes into account that some probes bind to more than one gene. Prediction should be more accurate than other methods. Used our microarray data (4hr time point) to predict splice variants. Predictions were verified with RT-PCR.

Prediction of Splice variants from Exon array data A collaboration with Sylvia Richardson Level of gene expression Probability

Prediction of Splice variants from Exon array data A collaboration with Sylvia Richardson Level of gene expression Probability Gel picture

Public Engagement in Science etc. Next Generation Project (NGP) Masterclass in Biomedical Sciences for A level students Sat 7th March: ERASysBio Workshop: Towards European Standards for PhD Training in Systems Biology

Future plans Continue Sub-project integration: Sub-project 1, 2 and 4 Continuation of work on IL10 degradation modelling Use Gaussian processes to obtain confidence intervals for parameter estimates. Investigation of phosphorylation states of proteins in signalling pathways

Recap: Notch Signalling S2 ADAM Metalloprotease S3  -secretase activity CoRs Fringe Numb MINT Nrarp Deltex ? RBP-J Target genes Maml CoA RBP-J p300