TMT Thesis Project

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Thesis Topic

The main objectives of my work is to develop the tools to perform time-dependent stimulation and analysis of signaling pathways, and show that this is more powerful than traditional time-independent or step response analysis. I am using a computational model of the prototype system, the yeast pheromone response pathway, to generate hypotheses about the pathway. In order to test these hypotheses, time-dependent stimuli will be delivered to cells via a microfluidic device, and in vivo fluorescent reporters will be used to observe the system state. In addition to showing the strengths of this new approach to studying biological systems, I would like to use it to further our understanding of the pheromone response pathway.

Research Goals

My research can be broken down into 4 main goals that follow (for the most part) chronologically.

  1. Build a model of the pheromone response pathway
    • Develop a model of the pheromone response pathway that can be used in conjunction with time-dependent stimulation and analysis of the pathway to propose and test hypotheses. Once completed, this model can be used as a predictive tool for pathway response.
      • This model is largely already built (with instances in Matlab and Moleculizer). It needs to be further refined using data from the literature, and data that I will generate myself.
  2. Build a microfluidic device for time-dependent stimulation of cells
    • Design, build and characterize a device to allow for rapid variation of extracellular conditions for cells fixed in a microfluidic channel.
      • This chip has been designed using the technology out of the Quake Lab at Stanford (formerly Caltech). See protocols for more info on chip design. The most recent design of the Stimulator is currently being made by the Microfluidic Foundry (Caltech). Previous instances have shown great promise for my purposes. Preliminary tests have shown that I can vary the extracellular environment (with NO cells in the channel) on a sub 100ms timescale. I've also successfully adhered cells to the bottom of the channel, and had them resist detachment under fluid flow, though this needs further characterization.
  3. Investigate the pathway with time-dependent stimulation
    • Examine the frequency filtering characteristics of the pheromone response pathway in order to study the limits of propagation of time-varying signals through the pathway. Use the model to form and test hypotheses generated by studying the response of the pathway to time-dependent stimulation.
  4. Identify and apply techniques for non-linear system identification
    • Identify and apply tools developed for other fields to the analysis of signaling pathways, particularly with respect to time-dependent stimulation.
    • Notes on Parameter Estimation in Matlab

Q. What will determine if using time-varying stimuli is a success?
A. I think that showing it would be sufficient for me to show that you can better parameter estimates using time varying stimuli than with step increase. When I say better estimate, I mean that we can decrease the error bounds on parameters. This hinges on some intelligent way to put bounds or confidence limits on parameters. This is probably linked to the independence/coupling of paramters topic listed below under Signal Design.

Q. I say that a time varying stimulus can drive a system to a state that it won't normally attain in response to a step increase stimulus. For what types of systems is this true? A. I think that I can concoct systems that this is true for, but I should try to show that this is indeed true for the pheromone response pathway.

Near Future Plan


  1. Show that cells can live on 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).
  2. Show that you can control in ON/OFF fashion response of cells to alpha factor
    • Using strain with YFP driven by Pprm1 promoter. Show that cells won't react (ie fluoresce) when they arenot in part of channel where alpha factor is flowing, and that they do react when they are exposed to alpha factor.
  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

  1. 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

  1. coupling / 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)? I need to think about how to do this.

Parameter Estimation

  1. Get jacobian working
  2. Look into 'modulation spectroscopy'
    • Suggested by Dan Ehrlich(?)
    • Try introductory physical chemistry text book.

Thesis Committee
Q1. Do we need Thorner on the committee?
Q2. Do we need a yeast person on the committee?
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.

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