2026-09-19T09:26:05Zhttps://dspace.mit.edu/server/oai/request

oai:dspace.mit.edu:1721.1/447462022-01-13T07:54:36Zcom_1721.1_7582com_1721.1_7581col_1721.1_131022

Roger D. Kamm. Abdul Rahim, Nur Aida Massachusetts Institute of Technology. Dept. of Mechanical Engineering. Massachusetts Institute of Technology. Department of Mechanical Engineering 2009-03-16T19:37:25Z 2009-03-16T19:37:25Z 2008 2008 http://hdl.handle.net/1721.1/44746 298561100 Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Mechanical Engineering, 2008. Includes bibliographical references (p. 99-108). Force, due either to haemodynamic shear stress or relayed directly to the cell through adhesion complexes, is transmitted and translated into biological signals. This process is known as mechanotransduction. Extensive studies have been carried out on the signaling pathways involved in mechanotransduction. However, the mechanism(s) of mechanotransduction has yet to be fully understood. This thesis focuses on the measurement of the intracellular binding constant between focal adhesion proteins of interest, GFP-Paxillin and FAT-mCherry, using two-photon excitation fluorescence microscopy and the utility of it as a measure of protein conformational change. The hypothesis tested is that force-induced changes in protein conformation alter inter-protein binding affinity. A comprehensive toolkit that utilizes fluorescence microscopy techniques, Forster Resonance Energy Transfer (FRET) and its corollary, Fluorescence Lifetime Imaging (FLIM), as well as Fluorescence Correlation Spectroscopy (FCS), was developed. A procedure by which low photon counts cell data from FLIM could be included in global analysis fits and be corrected for was developed. This results in the recovery of maximum information from cellular data. Successful intracellular FCS measurements were combined with FLIM global analysis data to calculate the free energy of binding between GFP-Paxillin and FAT-mCherry. Results demonstrate that inter-cell heterogeneity exists and likely gives rise to differences in measured AIG. The application of these measurement techniques to cells experiencing 10% step strain shows that inter-protein binding is tighter upon stretch application. The source of this change is not clear, though Tyr phosphorylation has been ruled out by biochemical disruption of kinase activity. by Nur Aida Abdul Rahim. Ph.D. 108 p. eng Massachusetts Institute of Technology M.I.T. theses are protected by copyright. They may be viewed from this source for any purpose, but reproduction or distribution in any format is prohibited without written permission. See provided URL for inquiries about permission. http://dspace.mit.edu/handle/1721.1/7582 Mechanical Engineering. Investigating the mechanotransduction by two-photon fluorescence microscopy measurement of intracellular free energy of binding Thesis application/pdf Publication unknown unknown <Publication xmlns="https://www.openaire.eu/cerif-profile/1.1/" id="e45539f7-e595-4e24-95fd-1b0b0315b9cf"> <Type xmlns="https://www.openaire.eu/cerif-profile/vocab/COAR_Publication_Types">http://purl.org/coar/resource_type/c_1843</Type> <Language>eng</Language> <Title>Investigating the mechanotransduction by two-photon fluorescence microscopy measurement of intracellular free energy of binding</Title> <PublishedIn> <Publication> </Publication> </PublishedIn> <PublicationDate>2008</PublicationDate> <Authors> <Author> <DisplayName>Abdul Rahim, Nur Aida</DisplayName> <Affiliation> <OrgUnit> </OrgUnit> </Affiliation> </Author> </Authors> <Editors> </Editors> <Publishers> <Publisher> <DisplayName>Massachusetts Institute of Technology</DisplayName> <OrgUnit /> </Publisher> </Publishers> <License>http://dspace.mit.edu/handle/1721.1/7582</License> <Keyword>Mechanical Engineering.</Keyword> <Abstract>Force, due either to haemodynamic shear stress or relayed directly to the cell through adhesion complexes, is transmitted and translated into biological signals. This process is known as mechanotransduction. Extensive studies have been carried out on the signaling pathways involved in mechanotransduction. However, the mechanism(s) of mechanotransduction has yet to be fully understood. This thesis focuses on the measurement of the intracellular binding constant between focal adhesion proteins of interest, GFP-Paxillin and FAT-mCherry, using two-photon excitation fluorescence microscopy and the utility of it as a measure of protein conformational change. The hypothesis tested is that force-induced changes in protein conformation alter inter-protein binding affinity. A comprehensive toolkit that utilizes fluorescence microscopy techniques, Forster Resonance Energy Transfer (FRET) and its corollary, Fluorescence Lifetime Imaging (FLIM), as well as Fluorescence Correlation Spectroscopy (FCS), was developed. A procedure by which low photon counts cell data from FLIM could be included in global analysis fits and be corrected for was developed. This results in the recovery of maximum information from cellular data. Successful intracellular FCS measurements were combined with FLIM global analysis data to calculate the free energy of binding between GFP-Paxillin and FAT-mCherry. Results demonstrate that inter-cell heterogeneity exists and likely gives rise to differences in measured AIG. The application of these measurement techniques to cells experiencing 10% step strain shows that inter-protein binding is tighter upon stretch application. The source of this change is not clear, though Tyr phosphorylation has been ruled out by biochemical disruption of kinase activity.</Abstract> <Access xmlns="http://purl.org/coar/access_right" > </Access> </Publication>