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dc.contributor.advisorLahav, Galit
dc.contributor.authorGaglia, Giorgio
dc.date.accessioned2014-06-06T14:50:47Z
dc.date.issued2014-06-06
dc.date.submitted2014
dc.identifier.citationGaglia, Giorgio. 2014. Dynamics of p53 tetramers in live single cells. Doctoral dissertation, Harvard University.en_US
dc.identifier.otherhttp://dissertations.umi.com/gsas.harvard:11450en
dc.identifier.urihttp://nrs.harvard.edu/urn-3:HUL.InstRepos:12269874
dc.description.abstractProtein homo-oligomerization is the process through which identical peptides bind together to form higher order complexes. Self-interactions in many cases are constitutive and stable, used as building blocks for biological structures, such as rings, filaments and membranes. Further, homo-oligomerization can also be a regulatory process that influences the proteins' function such as change in transcriptional activities for transcription factors. Innovative methods to measure oligomerization in live cells are needed in order to understand regulation and function of homooligomerization in the native cellular context. This thesis examines the case of the tumor suppressor p53, whose homo-tetramerization greatly influences its activity as a transcription factor. We develop methods to quantify p53's self-interaction in individual living cells and follow it in time after DNA damage. The two methods we developed have complementary qualities and different applications. We first use fluorescent correlation spectroscopy to study the molecular events occurring in the first three hours of the p53 in response to double strand breaks. We find that in the absence of stress p53 is present in a mixture of, monomers, dimers and tetramers. When damage is sensed, oligomerization is rapidly induced and nearly all p53 is found bound in tetramers. We combine our data with a mathematical framework to propose the existence of a dedicated mechanism triggering p53 oligomerization independently of protein stabilization. Next, we use bimolecular fluorescent complementation to probe for tetramerization in the longer timescales of p53's response to ultraviolet radiation. In this context we find that even though the rate of p53 accumulation increases with the dose of radiation, p53 tetramers are formed at a steady rate. We hence propose the existence of an inhibitory mechanism that prevents the oligomerization reaction from following a linear input-output relation. We identify ARC, a known cofactor of p53, as part of this inhibitory mechanism. Downregulation of ARC restore the linear relation between to total and tetrameric p53. Finally, in both experimental setups higher oligomerization lead to an increase in p53 activity, underscoring the connection between regulation of oligomerization and the transcriptional activity of p53 in cancer cells. Collectively, this work emphasizes the importance of precise measurements to investigate the regulation and function of higher order complexes and provides generally applicable methods to quantify homo-oligomerization in live single cells.en_US
dc.language.isoen_USen_US
dash.licenseLAA
dc.subjectSystematic biologyen_US
dc.subjectDNA damageen_US
dc.subjectmathematical modelingen_US
dc.subjectp53en_US
dc.subjectprotein dynamicsen_US
dc.subjecttetramerizationen_US
dc.titleDynamics of p53 tetramers in live single cellsen_US
dc.typeThesis or Dissertationen_US
dash.depositing.authorGaglia, Giorgio
dc.date.available2014-06-06T14:50:47Z
thesis.degree.date2014en_US
thesis.degree.disciplineSystems Biologyen_US
thesis.degree.grantorHarvard Universityen_US
thesis.degree.leveldoctoralen_US
thesis.degree.namePh.D.en_US
dc.contributor.committeeMemberShah, Jageshen_US
dc.contributor.committeeMemberMitchison, Timothyen_US
dc.contributor.committeeMemberCluzel, Philippeen_US
dc.contributor.committeeMemberLee, Samen_US
dash.contributor.affiliatedGaglia, Giorgio


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