TMT Thesis Notes

Biology

1. Show that cells can live on a chip
   • Stick yeast cells down in the channel, and flow media (at a slow rate) over them. Take a picture every 5 mintutes and compile into a movie of yeast cells growing (hopefully). Need to start with cells growing exponentially, and concentrate to OD 1.5-2. Try sonicating briefly to break up clumps (talk to Jeff).
   • I've proven to myself that cells can grow on a chip, though I still don't have a good movie demonstrating this. I will try to get this evidence so everyone will be a believer.
2. Show that you can control in ON/OFF fashion response of cells to alpha factor
   • Using strain with YFP driven by P_prm1 promoter. Show that cells won't react (ie fluoresce) when they are not in part of channel where alpha factor is flowing, and that they do react when they are exposed to alpha factor. I need to use casein in the media to block pheromone adsorption to the tubes and channel walls.
3. Find out if reset of receptor/G protein sub-system is limited by pheromone dissociation or Ste2 internalization.
   • Hit cells with a short dose of pheromone and see if reset is on the order of 4-5 mintutes (internalization) or 10 minutes (dissociation). See if Alejandro has already done this.
4. (Is yeast pheromone response the best model system for this project?)

Data Collection/Analysis

• Show that you can measure Ste5-YFP translocation to membrane
• This will involve either using or reimplementing the image analysis tools used by Alejandro and Andrew. Also, I might want to use/reimplement their autofocus routine. I should look into this soon.

Signal Design

Find out the extent of coupling and independence of parameters

• How can we use the model to get an idea of how well we're going to be able to estimate parameters? How many of the parameters are linked (eg, what if only the ratio of param1 to param2 matters)?
• SloppyCell appears to be useful for this purpose. I need to do some validation to prove to myself that SloppyCell is working as expected
  1. Compare species timecourses (vs Matlab, Jacobian & BNG2)to ensure input file and simulation engine are good.
     • DONE!
  2. Check sensitivities and normalized sensitivities to make sure they're as expected.
     • Can compare with Jacobian (generates automatically) and Matlab (numerical approximation of derivatives).
       • The sensitivitites in Jacobian and Matlab match.
       • So far, the sensitivities from SloppyCell don't match those from Jacobian and Matlab. I need to debug this. It looks like SloppyCell is having problems with sensitivitites. I've contacted Ryan and I'm waiting to hear what he thinks about this.
  3. Interpret parameter groupings.
     • Should be able to compare the stiffness of the parameter groupings (as given by the relative eigenvalues) with the sensitivities of the individual parameters in that grouping. Higher eigenvalue/stiffness should correlate with higher sensitivity?

Parameter Estimation

1. Get jacobian working
2. Would knowledge of parameter groupings affect parameter estimation? I want to think about this some more, maybe chat with some people in the lab, and try to talk to John Tolsma (@Jacobian) about this.

Meta/Administrative Issues

Thesis Committee
Q1. Do we need Thorner on the committee?

• I think so.

Q2. Do we need a yeast person on the committee?

• Thorner

Q3. Do we need a dynamic systems person on the committee?

• It's looking more and more like the answer is yes. I don't know enough to be efficient at guiding myself through the parameter estimation and dynamic system analysis. Figuring out who we can add should be a top priority.
• People to talk to:
  • Jacob White
  • Alan Oppenheim
    • He suggested that I talk to George Verghese. Looking at his research interests it looks like he could be the right guy to talk to.
  • Paul Barton

Should have a committee meeting ASAP to discuss current directions.
Should have another committee meeting mid/late Spring 2006 to update and plan.

Decision Points/Milestones
Much of my research depends on several programs working correctly (Jacobian, SloppyCell). Since neither of these programs are in their final releases yet (and may still contain bugs), I need to set decision points by which I need to have complete confidence in the program. If the program is not working by this decision point, then I will find a new program/method.