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Calibration and testing of FRET efficiency in a vinculin tension probe

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Title
Calibration and testing of FRET efficiency in a vinculin tension probe
Name (type = personal)
NamePart (type = family)
Stein
NamePart (type = given)
Jeremy L.
NamePart (type = date)
1996-
DisplayForm
Jeremy L. Stein
Role
RoleTerm (authority = RULIB)
author
Name (type = personal)
NamePart (type = family)
Boustany
NamePart (type = given)
Nada N
DisplayForm
Nada N Boustany
Affiliation
Advisory Committee
Role
RoleTerm (authority = RULIB)
chair
Name (type = personal)
NamePart (type = family)
Pierce
NamePart (type = given)
Mark C
DisplayForm
Mark C Pierce
Affiliation
Advisory Committee
Role
RoleTerm (authority = RULIB)
internal member
Name (type = personal)
NamePart (type = family)
Gormley
NamePart (type = given)
Adam J
DisplayForm
Adam J Gormley
Affiliation
Advisory Committee
Role
RoleTerm (authority = RULIB)
internal member
Name (type = corporate)
NamePart
Rutgers University
Role
RoleTerm (authority = RULIB)
degree grantor
Name (type = corporate)
NamePart
School of Graduate Studies
Role
RoleTerm (authority = RULIB)
school
TypeOfResource
Text
Genre (authority = marcgt)
theses
OriginInfo
DateCreated (encoding = w3cdtf); (qualifier = exact)
2019
DateOther (encoding = w3cdtf); (qualifier = exact); (type = degree)
2019-05
CopyrightDate (encoding = w3cdtf); (qualifier = exact)
2019
Language
LanguageTerm (authority = ISO 639-3:2007); (type = text)
English
Abstract (type = abstract)
As interest grows in the analysis of cellular level mechanical forces, including cell adhesion, cellular extension, and other microscopic-level physical phenomena, our goal is to use a previously developed Vinculin tension probe, VinTS, in conjunction with Fluorescence Resonance Energy Transfer (FRET) microscopy to quantify the mechanical forces that arise in growth cones during neuronal development. The VinTS tension probe consists of a donor and an acceptor fluorophore connected by an elastic linker inserted between the head and tail of vinculin. The work presented here aims to establish a protocol to properly correct, calibrate and convert the imaging data to FRET efficiency which is instrument-independent, and reports on nanometric changes in the distance between the donor and acceptor. For this calibration, we used control FRET constructs, with known FRET efficiency that we expressed in immortalized baby mouse kidney (iBMK) cells, bovine aortic endothelial cells (BAEC), and isolated cortical neurons. The calibration constructs consisted of either our donor, mTFP1, our acceptor, mVenus, or both connected by a long or short linker. The long linker protein was TRAF (TNF receptor associated factor) while the short linker was the amino acid sequence (GGSGGS)2. These constructs were transfected into the selected cell types using Lipofectamineâ LTX according to well-established protocols. All of the constructs were tested in the aforementioned cell lines. The methods involved imaging using a 3-channel “sensitized emission” FRET methodology, with the results analyzed in a lab-developed MATLAB Script. The collected images were also corrected for background. The calibrated system was then utilized to measure the FRET efficiency in the VinTS tension probe and comparing its efficiency to that of the unloaded tension module (TSMod). The efficiency of VinTS and TSMod was measured in iBMK cells and neurons. We investigated segmentation methods to isolate the focal adhesions so that the FRET efficiency could be measured in solely those areas. So far, the investigation has allowed for calibration within the BAEC and iBMK cells, with positive results matching published FRET efficiency values for the calibration constructs. The VinTS and TSMod constructs also appear to produce expected values within the target range in iBMK cells. Current data from neuronal cells appear to have significantly more variability than the other cells which have an epithelial cell morphology. Additional data acquisition and analysis in neuronal cells is currently underway and will pave the way for further study of neuronal growth cones on substrates with various surface properties.
Subject (authority = local)
Topic
FRET
Subject (authority = RUETD)
Topic
Biomedical Engineering
Subject (authority = LCSH)
Topic
Cells -- Mechanical properties -- Imaging
RelatedItem (type = host)
TitleInfo
Title
Rutgers University Electronic Theses and Dissertations
Identifier (type = RULIB)
ETD
Identifier
ETD_9810
PhysicalDescription
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InternetMediaType
application/pdf
InternetMediaType
text/xml
Extent
1 online resource (ix, 67 pages) : illustrations
Note (type = degree)
M.S.
Note (type = bibliography)
Includes bibliographical references
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TitleInfo
Title
School of Graduate Studies Electronic Theses and Dissertations
Identifier (type = local)
rucore10001600001
Location
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NjNbRU
Identifier (type = doi)
doi:10.7282/t3-hp4j-dp58
Genre (authority = ExL-Esploro)
ETD graduate
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Rights

RightsDeclaration (ID = rulibRdec0006)
The author owns the copyright to this work.
RightsHolder (type = personal)
Name
FamilyName
Stein
GivenName
Jeremy
MiddleName
L.
Role
Copyright Holder
RightsEvent
Type
Permission or license
DateTime (encoding = w3cdtf); (qualifier = exact); (point = start)
2019-04-11 18:50:32
AssociatedEntity
Name
Jeremy Stein
Role
Copyright holder
Affiliation
Rutgers University. School of Graduate Studies
AssociatedObject
Type
License
Name
Author Agreement License
Detail
I hereby grant to the Rutgers University Libraries and to my school the non-exclusive right to archive, reproduce and distribute my thesis or dissertation, in whole or in part, and/or my abstract, in whole or in part, in and from an electronic format, subject to the release date subsequently stipulated in this submittal form and approved by my school. I represent and stipulate that the thesis or dissertation and its abstract are my original work, that they do not infringe or violate any rights of others, and that I make these grants as the sole owner of the rights to my thesis or dissertation and its abstract. I represent that I have obtained written permissions, when necessary, from the owner(s) of each third party copyrighted matter to be included in my thesis or dissertation and will supply copies of such upon request by my school. I acknowledge that RU ETD and my school will not distribute my thesis or dissertation or its abstract if, in their reasonable judgment, they believe all such rights have not been secured. I acknowledge that I retain ownership rights to the copyright of my work. I also retain the right to use all or part of this thesis or dissertation in future works, such as articles or books.
Copyright
Status
Copyright protected
Availability
Status
Open
Reason
Permission or license
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Technical

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DateCreated (point = end); (encoding = w3cdtf); (qualifier = exact)
2019-04-12T14:36:27
DateCreated (point = end); (encoding = w3cdtf); (qualifier = exact)
2019-04-12T14:36:27
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