Spring+2012+Committee+Meeting+4-26-12

This presentation was a summary of the cDNA library construction and first round of screening, reminder about phage library progress, and summary of mining random space paper with the PQRE motif.

see F:\kurt\storage\CIM Research Folder\kwhittem\Presentations\2012\April 2012 events "F:\kurt\storage\CIM Research Folder\kwhittem\Presentations\2012\April 2012 events\presentation\Spring 2012 Committee Meeting 4-26-12.pptx" for most recent version of files

"F:\kurt\storage\CIM Research Folder\kwhittem\Presentations\2012\General Meeting Presentation 4-26-12\Spring 2012 Committee Meeting 4-26-12.pptx"

From the Cancer meeting in which I presented many of these things (Cancer Meeting Presentation 3-28-12), I had some suggestions. They felt I should know how many tumor cells I had, as well as what the titer of the SMC1fs mice was. They mentioned I could potentially use an antibody against actin as a positive control since many of the spots should have colonies which produce this protein. They also had some comments about the analysis. They felt that I could titrate the secondary, subtract the secondary signal, median normalize, and then do the t test. Note that I am titrating the secondary tomorrow. When I tried normalized and subtracted the secondary, the SMC1fs fell out of the top spot.

Quad Chart

"C:\kurt\storage\CIM Research Folder\kwhittem\Presentations\2012\General Meeting Presentation 4-26-12\Quad Chart Version 5 041612.zip"

Discovery of Tumor-specific Antigens Summary

"C:\kurt\storage\CIM Research Folder\kwhittem\Presentations\2012\General Meeting Presentation 4-26-12\Discovery of Tumor Specific Antigens Summary 4-17-12.docx"

Discussion with Kathy 1 day before presentation

She mentioned that I should highlight how my method is more distinct from other SEREX methods. One of the key things is that my method is more high-throughput than many other methods since spots are spotted onto a nitrocellulose array.

How do I justify how many clones are in my tumor cDNA library? Tumor cDNA Library Clone Number Justification How many antibodies are in the body?

Notes after committee meeting

4-26-12 Comments and Notes from Committee Meeting

In general it seems that I should know a lot of background details about my background slides.

I should make sure that I can draw the SMC1fs fusion information and what the protein as well as the peptide boost is. Kathy said the peptide was fused to KLH which surprised me! I think she just made a slight mistake here. I talked to Shen, and he said that it was just the peptide.

They thought I should have explained better why all 3 reading frames would not be expressed. The key reason that not all 3 reading frames are expressed is because the translation starts on the plasmid part not on the insert gene part.

There were a lot of comments on my "complexity of cDNA Library" slide. They felt that 53.3% was too low. I need to go back and check some of the details of my cDNA library.

Bert Jacobs was curious if there were any spots which increased from naive to SMC1fs in both the naive and tumor.

Make sure to change p values on heatmap slide!

There was a lot of discussion about why certain spots are decreasing. They wanted me to talk about how certain spots on our random peptide arrays decrease with the disease. Some peptides increase and some peptides decrease in the very same antigen.

There was a lot of discussion about why certain SMC1fs spots increase in intensity, especially if they don't have the tumor. Part of the reasons were that the peptide was fused to GST, so some of that reactivity could be against that. They were also curious about why I did not find SMC1fs reactivity in my library. Perhaps it was not in the library in the first place. They would like me to do several things to see whether it is in the library. They would like me to do a PCR of the whole library, to see if the same spots increase in the tumor and SMC1 from naive, and also do deep sequencing on some spots

With the phage library, Bertram Jacobs always has a problem with the random combination of heavy and light chains. Although this is a problem, it just gives me more to sort through, and phage libraries seem to have worked for many other people.

Stephen mentioned that I should check anti-IgA responses on the array as well.

They did not have any problem with my aims for graduation. They are skeptical that aim 1 is there. My committee seemed unified that I should focus on these two aims and not my "bonus work" right now. I might be able to look at the Gemini project after I have established that aim 1 is working.

To do list after Spring 2012 committee meeting

"C:\kurt\storage\CIM Research Folder\kwhittem\Presentations\2012\April 2012 events\Supervisory Committee Form Spring 2012.pdf"