TMT Thesis Project

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(Research Goals)
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(Identify and apply techniques for non-linear system identification)
 
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Back to [[Ty Thomson|Ty Thomson's user page]]
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== Thesis Topic ==
== Thesis Topic ==
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My research can be broken down into 4 main goals that follow (for the most part) chronologically.
My research can be broken down into 4 main goals that follow (for the most part) chronologically.
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#Build a model of the pheromone response pathway
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=== Build a model of the pheromone response pathway ===
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#*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.
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#**This model is largely already built (with instances in Matlab and [http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=15637632&query_hl=1 Moleculizer]).  It needs to be further refined using data from the literature, and data that I will generate myself. 
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#Build a microfluidic device for time-dependent stimulation of cells
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#*Design, build and characterize a device to allow for rapid variation of extracellular conditions for cells fixed in a microfluidic channel.
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#**This chip has been designed using the technology out of the Quake Lab at Stanford (formerly Caltech).  See [[Protocols#Microfluidics| protocols]] for more info on chip design.  Early versions of the chip (called the [[Stimulator]]) 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.  I made a [[Image:Cells_in_stimulator.avi|movie]] with the most recent version showing that I can change the fluid environment of cells in the channel (video in real time, with food dye used to color one of the fluids).
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#Investigate the pathway with time-dependent stimulation
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#*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.
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#Identify and apply techniques for non-linear system identification
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#*Identify and apply tools developed for other fields to the analysis of signaling pathways, particularly with respect to time-dependent stimulation.
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#*Notes on [[Parameter Estimation in Matlab]]
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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 [http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=15637632&query_hl=1 Moleculizer]).  Also, using [http://cellsignaling.lanl.gov/bionetgen/ BioNetGen2] I have generated an SBML version of my model, which can be read as input by [http://www.numericatech.com/jacobian.htm Jacobian], [http://sloppycell.sourceforge.net/ SloppyCell], and the [http://www.mathworks.com/products/simbiology/ SimBio] toolbox in Matlab.
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*The model needs to be further refined using data from the literature, and data that I will generate myself.
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Q. What will determine if using time-varying stimuli is a success?<br>
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=== Build a microfluidic device for time-dependent stimulation of cells ===
-
A. I think that it would be sufficient for me to show that you can get better parameter estimates using time varying stimuli than you can 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. 
+
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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 stimulusFor what types of systems is this true?<br>
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Design, build and characterize a device to allow for rapid variation of extracellular conditions for cells fixed in a microfluidic channel.
-
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.
+
*This chip has been designed using the technology out of the Quake Lab at Stanford (formerly Caltech).  See [[Protocols#Microfluidics| protocols]] for more info on chip design.  Early versions of the chip (called the [[Stimulator]]) have shown great promise.  Preliminary tests have shown that I can vary the extracellular environment (with NO cells in the channel) on a sub 100ms timescaleI've also successfully adhered cells to the bottom of the channel, and had them resist detachment under fluid flow, though this needs further characterization. I made a movie ([[Image:Cells_in_stimulator.avi]]) with the most recent version showing that I can change the fluid environment of cells in the channel (video in real time, with food dye used to color one of the fluids).  Please see the [[Stimulator]] page for the latest information.
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== Near Future Plan ==
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=== Investigate the pathway with time-dependent stimulation ===
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Biology
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Show that using time-varying stimuli increases parameter sensitivity, and that this leads to an improvement in parameter estimationThis is really just a specific instance of hypothesis testing (where the hypothesis is the particular parameter values).
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#Show that cells can live on chip
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#*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).
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#Show that you can control in ON/OFF fashion response of cells to alpha factor
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#*Using strain with YFP driven by P<sub>prm1</sub> promoterShow 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.
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#Find out if reset of receptor/G protein sub-system is limited by pheromone dissociation or Ste2 internalization.
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#*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.
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#(Is yeast pheromone response the best model system for this project?)
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=== Identify and apply techniques for non-linear system identification ===
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Data Collection/Analysis
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Identify and apply tools developed for other fields to the analysis of signaling pathways, particularly with respect to time-dependent stimulation.  This can be divided into into two thrusts, parameter estimation and other analysis tools.
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#Show that you can measure Ste5-YFP translocation to membrane
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#Parameter Estimation
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#*This will involve either using or reimplementing the image analysis tools used by Alejandro and AndrewAlso, I might want to use/reimplement their autofocus routine.  I should look into this soon.
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#*My first instinct was to try to do parameter estimation using Matlab.  This turned out to not be sufficient for my purposes.  See my notes on [[Parameter Estimation in Matlab]].
 +
#*I settled on using [http://www.numericatech.com/jacobian.htm Jacobian] for parameter estimation.  The newest version that I have is working well.
 +
#Model analysis tools
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#*One basic analysis of a model is parameter sensitivitySome people think that models should be robust to changes in parameters (reference to be filled in, since it's not cool to just state things and reference it blankly to 'some people').  I'm not so sure that is true, but either way the parameter sensitivity can in the very least tell you to what parameters your model's behavior is sensitive (critically depends on), and to what parameters it is insensitive (does not depend on).
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==Other==
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Signal Design
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''' Relevant questions '''
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#Find out the extent of coupling and independence of parameters
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#*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.
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 +
Q. What will determine if using time-varying stimuli is a success?<br>
 +
A. I think that it would be sufficient for me to show that you can get better parameter estimates using time varying stimuli than you can 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. 
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Parameter Estimation
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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?<br>
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#Get jacobian working
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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.
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#Look into 'modulation spectroscopy'
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#*Topic suggested by Dan Ehrlich (Director of BIOMEMS Laboratory in the Whitehead Institute)
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#*Try introductory physical chemistry text book.
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Thesis Committee<br>
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Q1. Do we need Thorner on the committee?<br>
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Q2. Do we need a yeast person on the committee?<br>
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Q3. Do we need a dynamic systems person on the committee?<br>
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*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.
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*I talked to Bree about this, and she had some advice (since she went through the ordeal of trying to find the best systems person for her committee).  One drawback to putting a systems person who doesn't know biology on your committee is that a lot of time is spent explaining why the system isn't Hamiltonian(?) and why you don't need to conserve engergy, and stuff like that.  So she is collaborating with math types, but she has van Oudenaarden on her committee as an in-between guy.  He understands the biology, and he can understand the math/analysis stuff, but he might not be the best guy to direct her in the math/analysis direction (hence collaborating with math-types).  Anyway, she suggested talking to Doug, who she called the great connecter.  She said that he would probably be very good at helping me find the appropriate person.  She also suggested talking to Jacob White, who is getting more and more interested in biological systems analysis, and is a pretty smart guy.  He doesn't know the bio really well, but she said that he is very good at giving you his full attention until he gets the biology and might be able to give some help.  So course of action would be to saet up meetings with Jacob and Doug.  Problem is that it might be ~2 weeks until Doug has time to meet. 
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*I can also try to meet with Gerry Sussman or Tom to see if they can point me towards anyone (suggested by Reshma).
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Should have a committee meeting ASAP to discuss current directions.<br>
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[[Parameter Sensitivity and Estimation]]
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Should have another committee meeting mid/late Spring 2006 to update and plan.
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Current revision

Back to Ty Thomson's user page

Contents

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.

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). Also, using BioNetGen2 I have generated an SBML version of my model, which can be read as input by Jacobian, SloppyCell, and the SimBio toolbox in Matlab.
  • The model needs to be further refined using data from the literature, and data that I will generate myself.

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. Early versions of the chip (called the Stimulator) have shown great promise. 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. I made a movie (Image:Cells in stimulator.avi) with the most recent version showing that I can change the fluid environment of cells in the channel (video in real time, with food dye used to color one of the fluids). Please see the Stimulator page for the latest information.

Investigate the pathway with time-dependent stimulation

Show that using time-varying stimuli increases parameter sensitivity, and that this leads to an improvement in parameter estimation. This is really just a specific instance of hypothesis testing (where the hypothesis is the particular parameter values).

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. This can be divided into into two thrusts, parameter estimation and other analysis tools.

  1. Parameter Estimation
    • My first instinct was to try to do parameter estimation using Matlab. This turned out to not be sufficient for my purposes. See my notes on Parameter Estimation in Matlab.
    • I settled on using Jacobian for parameter estimation. The newest version that I have is working well.
  2. Model analysis tools
    • One basic analysis of a model is parameter sensitivity. Some people think that models should be robust to changes in parameters (reference to be filled in, since it's not cool to just state things and reference it blankly to 'some people'). I'm not so sure that is true, but either way the parameter sensitivity can in the very least tell you to what parameters your model's behavior is sensitive (critically depends on), and to what parameters it is insensitive (does not depend on).

Other

Relevant questions

Q. What will determine if using time-varying stimuli is a success?
A. I think that it would be sufficient for me to show that you can get better parameter estimates using time varying stimuli than you can 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.


Parameter Sensitivity and Estimation

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