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University of Alberta
1.
Alford, Spencer Caleb.
Development of fluorogenic fluorescent protein
heterodimers.
Degree: PhD, Department of Chemistry, 2012, University of Alberta
URL: https://era.library.ualberta.ca/files/hh63sw19n
► Fluorescent proteins (FPs) are indispensible biochemical tools. The concerted efforts of protein engineers have produced FPs spanning the visible colour spectrum. This wide variety of…
(more)
▼ Fluorescent proteins (FPs) are indispensible
biochemical tools. The concerted efforts of protein engineers have
produced FPs spanning the visible colour spectrum. This wide
variety of FPs has greatly facilitated the development of FP-based
biosensors. However, researchers rely on relatively few fundamental
biosensor design templates. Förster resonance energy transfer and
bimolecular complementation are the principal FP-based technologies
for live cell imaging of physiological events, such as changes in
small molecule concentration, enzymatic activities, and
protein-protein interactions. Although widely used, these
techniques are often restrictive due to poor signal-to-noise ratios
and irreversible sensing, respectively. Furthermore, examples of
these biosensor strategies incorporating red FPs are limited. In
this thesis we describe our efforts to address this shortcoming in
the area of FP-based biosensors. We developed a
dimerization-dependent red FP (ddRFP) that serves as an alternative
template for biosensor construction. The prototype ddRFP was
engineered from a homodimeric variant of a Discosoma red FP.
Through extensive directed evolution the homodimer was converted
into a fluorogenic obligate heterodimer. The reversible changes in
fluorescence intensity that result from association of the ddRFP
monomeric constituents, or the irreversible decrease that
accompanies dissociation of covalently linked partners following
linker cleavage, provides a useful spectroscopic signal for
biosensing applications. Specifically, we demonstrated that ddRFP
is useful for detecting in vitro protein-protein interactions, as
well as imaging changes in calcium ion concentration and activation
of caspase-3 in live cells. We also report the expansion of the
ddFP colour palette through the development of green (ddGFP) and
yellow (ddYFP) ddFP variants. These variants have several
improvements relative to the ddRFP prototype including increased in
vitro contrast and brightness for ddGFP, and a reduced pKa for
ddYFP. While their utility for some live cell imaging applications
is restricted due to low dissociation constants, ddGFP proved to be
a useful fluorescent label of intermembrane contact sites between
the endoplasmic reticulum and the mitochondrial
network.
Subjects/Keywords: protein engineering; fluorescent protein; biosensor
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Chicago ·
MLA ·
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APA (6th Edition):
Alford, S. C. (2012). Development of fluorogenic fluorescent protein
heterodimers. (Doctoral Dissertation). University of Alberta. Retrieved from https://era.library.ualberta.ca/files/hh63sw19n
Chicago Manual of Style (16th Edition):
Alford, Spencer Caleb. “Development of fluorogenic fluorescent protein
heterodimers.” 2012. Doctoral Dissertation, University of Alberta. Accessed April 11, 2021.
https://era.library.ualberta.ca/files/hh63sw19n.
MLA Handbook (7th Edition):
Alford, Spencer Caleb. “Development of fluorogenic fluorescent protein
heterodimers.” 2012. Web. 11 Apr 2021.
Vancouver:
Alford SC. Development of fluorogenic fluorescent protein
heterodimers. [Internet] [Doctoral dissertation]. University of Alberta; 2012. [cited 2021 Apr 11].
Available from: https://era.library.ualberta.ca/files/hh63sw19n.
Council of Science Editors:
Alford SC. Development of fluorogenic fluorescent protein
heterodimers. [Doctoral Dissertation]. University of Alberta; 2012. Available from: https://era.library.ualberta.ca/files/hh63sw19n

University of Ottawa
2.
Eason, Matthew.
A GFP-Based Sensor to Detect Transiently Expressed Proteins
.
Degree: 2020, University of Ottawa
URL: http://hdl.handle.net/10393/40500
► Green fluorescent protein (GFP) fusion tags are commonly used to study protein expression and cellular localization in vivo. But, GFP must undergo an autogenic post-translational…
(more)
▼ Green fluorescent protein (GFP) fusion tags are commonly used to study protein expression and cellular localization in vivo. But, GFP must undergo an autogenic post-translational modification, known as chromophore maturation, to become fluorescent, a process that can have a half-time longer than 30 minutes inside research model organisms. The timescale of chromophore maturation in GFP is thus slower than many key biological processes, limiting its usefulness in measuring those processes. In this thesis, we discuss the creation and engineering of a sensor for transiently expressed proteins (STEP) based on a fully matured but dim GFP. Upon specific binding of STEPtag, a small (15.5 kDa) protein to the sensor, full fluorescence is restored. Thus, by genetically fusing STEPtag to a protein of interest, it can be detected as soon as folding is complete, without any maturation delay. Through a combination of rational design and targeted directed evolution, we describe the improvement of the original sensor, gSTEP0, into an optimized version, gSTEP1. The sensor has been validated in vitro and in E. coli cells, and we have found that for gSTEP1, the fluorescence signal increases more than three-fold upon binding, with a Kd of 120 ± 30 nM and a kon of 1.7 x 105 M-1s-1, allowing detection of the protein of interest on the second timescale. We have also created a yellow version of the biosensor, and provide preliminary attempts at developing orthogonal binding pairs, as well as red- and cyan-coloured STEPs, which could eventually be used in multiplex experiments. Our biosensor opens the door to the study of short-timescale processes in research model organisms, such as Drosophila and zebrafish embryogenesis, as well as in host-pathogen interactions, which we are currently investigating.
Subjects/Keywords: Biosensor;
Fluorescent Protein;
Protein Engineering
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APA (6th Edition):
Eason, M. (2020). A GFP-Based Sensor to Detect Transiently Expressed Proteins
. (Thesis). University of Ottawa. Retrieved from http://hdl.handle.net/10393/40500
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Chicago Manual of Style (16th Edition):
Eason, Matthew. “A GFP-Based Sensor to Detect Transiently Expressed Proteins
.” 2020. Thesis, University of Ottawa. Accessed April 11, 2021.
http://hdl.handle.net/10393/40500.
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
MLA Handbook (7th Edition):
Eason, Matthew. “A GFP-Based Sensor to Detect Transiently Expressed Proteins
.” 2020. Web. 11 Apr 2021.
Vancouver:
Eason M. A GFP-Based Sensor to Detect Transiently Expressed Proteins
. [Internet] [Thesis]. University of Ottawa; 2020. [cited 2021 Apr 11].
Available from: http://hdl.handle.net/10393/40500.
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Council of Science Editors:
Eason M. A GFP-Based Sensor to Detect Transiently Expressed Proteins
. [Thesis]. University of Ottawa; 2020. Available from: http://hdl.handle.net/10393/40500
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

University of Alberta
3.
Carlson, Haley J.
Development of cpRFP's for use as Ca2+ biosensors.
Degree: PhD, Department of Chemistry, 2013, University of Alberta
URL: https://era.library.ualberta.ca/files/5138jf10b
► The discovery of green fluorescent protein (GFP) from the Aequorea victoria jellyfish revolutionized many fields in the scientific community, including molecular biology, protein engineering, and…
(more)
▼ The discovery of green fluorescent protein (GFP) from
the Aequorea victoria jellyfish revolutionized many fields in the
scientific community, including molecular biology, protein
engineering, and neuroscience. The ability to genetically link a
fluorescent protein to a protein of interest has allowed scientists
to probe the exact structural localization of proteins. Another
important application of FPs is their design for use in biosensors,
whereby the fluorescence of the protein is intrinsically dependent
on a small molecule of interest, such as calcium ion (Ca2+) or a
physiological process such as phosphorylation or caspase activity.
In single FP-based biosensors of small molecules, the FP must be
circular permutated, whereby the original N- and C-termini are
linked together and new termini are introduced closer to the
chromophore. At the start of the work described in this thesis a
lot of work had gone into developing and improving GFP-based Ca2+
biosensors1,2, but there were no reports of a red FP-based
biosensor. The work in this thesis describes the engineering of an
RFP-based Ca2+ biosensor using a circular permutated RFP, mCherry.
The first step in this process was to engineer a cpmCherry variant
with termini near the chromophore3. mCherry required a lot of
engineering and optimization in order to identify a fluorescent
variant with termini near the chromophore. Ultimately, a cpmCherry
split at position 145 was found that, when fused to calmodulin
(CaM) and M13, showed a response to Ca2+. The initial construct had
limited response and was subjected to several rounds of mutagenesis
to improve both the brightness and fluorescence response. The final
variant CH-GECO3.1 shows a 250% signal increase with Ca2+ and could
be imaged successfully in mammalian cells to monitor Ca2+
fluctuations. To further our understanding of this biosensor,
site-directed mutagenesis was done to probe the structure-function
relationship. After mutagenesis a few residues stood out as key
residues that likely played a role in the mechanism of fluorescence
increase, such as Gln163 and Glu61 (linker). Other mutations were
introduced into the protein to determine whether the excitation and
emission wavelengths could be altered, while still retaining
function. The final section of this work describes the
reconstitution of split green and red Ca2+ biosensors using intein
technology. Inteins will spontaneously splice together protein
fragments that are genetically linked to them. To take advantage of
protein splicing several different Ca2+ biosensors were split into
and N-terminal and C-terminal fragments and attached to the
N-terminal or C-terminal intein, respectively. These fragments were
co-transfected into mammalian HeLa cells and imaged for
fluorescence signal and response to Ca2+
fluctuations.
Subjects/Keywords: biosensor; cpRFP; fluorescent protein
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❌
APA ·
Chicago ·
MLA ·
Vancouver ·
CSE |
Export
to Zotero / EndNote / Reference
Manager
APA (6th Edition):
Carlson, H. J. (2013). Development of cpRFP's for use as Ca2+ biosensors. (Doctoral Dissertation). University of Alberta. Retrieved from https://era.library.ualberta.ca/files/5138jf10b
Chicago Manual of Style (16th Edition):
Carlson, Haley J. “Development of cpRFP's for use as Ca2+ biosensors.” 2013. Doctoral Dissertation, University of Alberta. Accessed April 11, 2021.
https://era.library.ualberta.ca/files/5138jf10b.
MLA Handbook (7th Edition):
Carlson, Haley J. “Development of cpRFP's for use as Ca2+ biosensors.” 2013. Web. 11 Apr 2021.
Vancouver:
Carlson HJ. Development of cpRFP's for use as Ca2+ biosensors. [Internet] [Doctoral dissertation]. University of Alberta; 2013. [cited 2021 Apr 11].
Available from: https://era.library.ualberta.ca/files/5138jf10b.
Council of Science Editors:
Carlson HJ. Development of cpRFP's for use as Ca2+ biosensors. [Doctoral Dissertation]. University of Alberta; 2013. Available from: https://era.library.ualberta.ca/files/5138jf10b

King Abdullah University of Science and Technology
4.
Fischer, Johannes.
A Pathway to Artificial Metalloenzymes.
Degree: Biological and Environmental Sciences and Engineering (BESE) Division, 2015, King Abdullah University of Science and Technology
URL: http://hdl.handle.net/10754/583809
► The advancement of catalytic systems and the application thereof has proven to be the key to overcome traditional limitations of industrial-scale synthetic processes. Converging organometallic…
(more)
▼ The advancement of catalytic systems and the application thereof has proven to be the key to
overcome traditional limitations of industrial-scale synthetic processes. Converging
organometallic and biocatalytic principles lead to the development of Artificial Metalloenzymes
(ArMs) that comprise a synthetic metal catalyst embedded in a
protein scaffold, thereby
combining the reactivity of the former with the versatility of the latter. This synergistic
approach introduces rationally designed building blocks for the catalytic site and the host
protein
to assemble enzyme-like structures that follow regio-, chemo-, enantio- and substrate-selective
principles. Yet, the identification of suitable
protein scaffolds has thus far been challenging.
Herein we report a rationally optimized
fluorescent protein host, mTFP*, that was engineered to
have no intrinsic metal binding capability and, owing to its robust nature, can act as scaffold for
the design of novel ArMs. We demonstrate the potential of site-specific modifications within the
protein host, use
protein X-Ray analysis to validate the respective scaffolds and show how
artificial mutant binding sites can be introduced. Transition metal Förster Resonance Energy
transfer (tmFRET) methodologies help to evaluate micromolar dissociation constants and reveal
structural rearrangements upon coordination of the metal centers. In conjunction with molecular
insights from X-Ray crystallographic structure determination, dynamics of the binding pocket can
be inferred. The versatile subset of different binding motifs paired with transition metal catalysts
create artificial metalloenzymes that provide reactivities which otherwise do not exist in nature.
As a proof of concept, Diels-Alder cycloadditions highlight the potential of the present mTFP*
based catalysts by stereoselectively converting azachalcone and cyclopentadiene substrates.
Screens indicate an enantiomeric excess of up to 60% and provide insights into the electronic and
geometric constitution of the first coordination spheres binding the catalysts.
We further apply two general principles to optimize selective conversions of the generated ArMs.
1) Utilizing site-specific mutagenesis, increased hydrophobicity is introduced to the second coordination sphere. 2) In-vitro post-expressional modification utilizing N-hydroxysuccinimide
esters is anticipated to introduce a sterically more demanding second coordination sphere that
influences substrate entry by favoring a particular stereoisomer. The latter approach however also
enhances the host proteins robustness under processing conditions.
The presented study investigates a novel approach to create artificial metalloenzymes based on
non-enzymatic precursor proteins. It illustrates means of modification and functionalization.
Further guidance to overcome the general problem of insufficient stereoselectivity and stability is
also presented. In view of the insights gained we see the importance of further mutagenic studies,
i.e. through means of guided…
Advisors/Committee Members: Eppinger, Jörg (advisor), Arold, Stefan T. (committee member), Groll, Michael (committee member), Hamdan, Samir (committee member), Uwe, Bunz (committee member).
Subjects/Keywords: Metalloenzymes; fluorescent protein; Diels-Alder
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❌
APA ·
Chicago ·
MLA ·
Vancouver ·
CSE |
Export
to Zotero / EndNote / Reference
Manager
APA (6th Edition):
Fischer, J. (2015). A Pathway to Artificial Metalloenzymes. (Thesis). King Abdullah University of Science and Technology. Retrieved from http://hdl.handle.net/10754/583809
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Chicago Manual of Style (16th Edition):
Fischer, Johannes. “A Pathway to Artificial Metalloenzymes.” 2015. Thesis, King Abdullah University of Science and Technology. Accessed April 11, 2021.
http://hdl.handle.net/10754/583809.
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
MLA Handbook (7th Edition):
Fischer, Johannes. “A Pathway to Artificial Metalloenzymes.” 2015. Web. 11 Apr 2021.
Vancouver:
Fischer J. A Pathway to Artificial Metalloenzymes. [Internet] [Thesis]. King Abdullah University of Science and Technology; 2015. [cited 2021 Apr 11].
Available from: http://hdl.handle.net/10754/583809.
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Council of Science Editors:
Fischer J. A Pathway to Artificial Metalloenzymes. [Thesis]. King Abdullah University of Science and Technology; 2015. Available from: http://hdl.handle.net/10754/583809
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

University of Rochester
5.
Dean, Kimberly Marie.
Selection, Identification, and Characterization of Codon
Pairs that Inhibit Translation and the Development of the RNA-ID
Method to Measure the Effects of Cis-Regulatory Elements.
Degree: PhD, 2013, University of Rochester
URL: http://hdl.handle.net/1802/27301
► It has been known for over 30 years that synonymous codon choice regulates translation, but the characteristics of codons that inhibit translation, and the factors…
(more)
▼ It has been known for over 30 years that synonymous
codon choice regulates translation, but the characteristics of
codons that inhibit translation, and the factors that restrict
their function, are unknown. In a systematic screen of 59 of 61
sense codons, our lab identified the arginine CGA codon as strongly
inhibitory, and then determined that CGA inhibition was primarily
due to A·I wobble decoding. Furthermore, I found that adjacent CGA
codons are synergistically inhibitory relative to separated CGA
codons, which presents the possibility that combinations of
non-identical codons may regulate translation in ways that are not
predictable from their individual codon behavior.
To search for
inhibitory codon combinations among the 3,721 codon pairs and
226,981 codon triplets, I developed the RNA-ID method to screen for
cis-regulatory elements. RNA-ID utilizes a fluorescence-based,
integrated reporter and coupled to flow cytometry to evaluate
effects of sequences on expression in Saccharomyces cerevisiae.
This method is useful because insertion of test sequences, using
ligation independent cloning, is simple, expression is detectable
over a 250-fold range, measurements are quantitative and
dose-dependent, separation of specific populations is nearly
complete, and results from a single sequence are reproducible. To
find inhibitory codon combinations, a library of sequences,
consisting of random 9 nucleotide inserts, was evaluated using
RNA-ID. Only a small fraction of yeast (<0.2%) exhibited GFP/RFP
at 6-10% the maximal level, indicating that very few sequences
inhibit expression to this degree. Ninety-two strains from this
group were studied.
Five novel sequences that inhibit translation
where identified based on suppression of the expression defect by
appropriate tRNAs. The inhibitory sequences all contain at least
one codon decoded by wobble, and in each case, the suppression of
the inhibitory effect requires expression of a mutated, exact
base-pairing tRNA. Thus, I conclude that wobble decoding is likely
to be a critical factor in their inhibition of translation. In two
cases, inhibition depends upon a pair of adjacent codons and upon
the order of these codons, thus indicating that the intact codon
pair is the inhibitory unit, consistent with the idea that they act
within a single round of translation.
Subjects/Keywords: Yeast; Genetic Code; Fluorescence, Green Fluorescent Protein; Red Fluorescent Protein
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❌
APA ·
Chicago ·
MLA ·
Vancouver ·
CSE |
Export
to Zotero / EndNote / Reference
Manager
APA (6th Edition):
Dean, K. M. (2013). Selection, Identification, and Characterization of Codon
Pairs that Inhibit Translation and the Development of the RNA-ID
Method to Measure the Effects of Cis-Regulatory Elements. (Doctoral Dissertation). University of Rochester. Retrieved from http://hdl.handle.net/1802/27301
Chicago Manual of Style (16th Edition):
Dean, Kimberly Marie. “Selection, Identification, and Characterization of Codon
Pairs that Inhibit Translation and the Development of the RNA-ID
Method to Measure the Effects of Cis-Regulatory Elements.” 2013. Doctoral Dissertation, University of Rochester. Accessed April 11, 2021.
http://hdl.handle.net/1802/27301.
MLA Handbook (7th Edition):
Dean, Kimberly Marie. “Selection, Identification, and Characterization of Codon
Pairs that Inhibit Translation and the Development of the RNA-ID
Method to Measure the Effects of Cis-Regulatory Elements.” 2013. Web. 11 Apr 2021.
Vancouver:
Dean KM. Selection, Identification, and Characterization of Codon
Pairs that Inhibit Translation and the Development of the RNA-ID
Method to Measure the Effects of Cis-Regulatory Elements. [Internet] [Doctoral dissertation]. University of Rochester; 2013. [cited 2021 Apr 11].
Available from: http://hdl.handle.net/1802/27301.
Council of Science Editors:
Dean KM. Selection, Identification, and Characterization of Codon
Pairs that Inhibit Translation and the Development of the RNA-ID
Method to Measure the Effects of Cis-Regulatory Elements. [Doctoral Dissertation]. University of Rochester; 2013. Available from: http://hdl.handle.net/1802/27301

Michigan State University
6.
Assar, Zahra.
Elucidation of iLBP family folding pathway and study of reengineering them as fluorescent protein tags via structural analysis.
Degree: 2018, Michigan State University
URL: http://etd.lib.msu.edu/islandora/object/etd:6825
► "The intracellular lipid binding proteins (iLBP) family are found in the cells of mammals, birds, fish, amphibians and reptiles. They function to shuttle large insoluble…
(more)
▼ "The intracellular lipid binding proteins (iLBP) family are found in the cells of mammals, birds, fish, amphibians and reptiles. They function to shuttle large insoluble hydrophobic molecules, including retinal, various long chain fatty acids and etc. throughout the cell around the cytosol and nucleus. The combination of their small size and relatively large binding pocket make them suitable templates in a variety of protein design applications, including the study of an innovative class of fluorescent proteins. To pursue our goals, we used human Cellular Retinol Binding Protein II (hCRBPII). We were the first group to achieve the structure of an all-transretinal and the first bonafide structure of retinol-bound hCRBPII. In the course of these studies, we have discovered hCRBPII is surprisingly capable of folding as domain swapped dimer (DS), with single mutations able to shift the folding product from monomer to dimer. Structural analysis of both wild type and multiple mutant DS dimers led us to remarkable hypotheses regarding mechanism of this phenomenon, which is different from the previous studies on this family. We proposed that the N-terminal and C-terminal halves of hCRBPII are capable of at least partially folding independently, to form "open monomer." The dimer/monomer ratio depends on the relative rates of dimerization of the open monomers, versus closing of the two halves together to form the "closed monomer". In addition, by comparing structures of holo hCRBPII DS dimer variants, we identified an extremely large change in the relative orientation of the two domains upon ligand binding in dimers. This suggests the possibility that iLBP domain swapped dimers could be allosterically regulated forms of these proteins, at least in some cases. Fatty acid binding protein 5 (FABP5), another member of iLBPs, has also been reported to forms a very similar DS dimer, which makes it likely that other family members could also form DS dimers, and have physiological importance for some members of the family. As mentioned, a new class of fluorogenic proteins was created by binding fluorophore aldehydes in the binding pocket of hCRBPII via protonated Schiff base (PSB) formation. In this new system, emission of the designed solvatochromic fluorophore is flexible based on the polarity of the environment; therefore multicolor probes can be developed. More importantly, absorption/emission wavelengths can be tuned therefor; nonspecific labeling and background fluorescence can be reduced. By now, the absorption maxima are tuned from 501nm to 705nm and emission maxima from 613 nm to 744 nm. Covering both the red and far-red fluorescence wavelength regimes." – Pages ii-iii.
Online resource;
Advisors/Committee Members: Geiger, James H., Borhan, Babak, Blanchard, Gary, Henry, William.
Subjects/Keywords: Lipoproteins; Protein engineering; Protein folding; Fluorescent polymers; Fluorescent probes; Chemistry; Biochemistry
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❌
APA ·
Chicago ·
MLA ·
Vancouver ·
CSE |
Export
to Zotero / EndNote / Reference
Manager
APA (6th Edition):
Assar, Z. (2018). Elucidation of iLBP family folding pathway and study of reengineering them as fluorescent protein tags via structural analysis. (Thesis). Michigan State University. Retrieved from http://etd.lib.msu.edu/islandora/object/etd:6825
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Chicago Manual of Style (16th Edition):
Assar, Zahra. “Elucidation of iLBP family folding pathway and study of reengineering them as fluorescent protein tags via structural analysis.” 2018. Thesis, Michigan State University. Accessed April 11, 2021.
http://etd.lib.msu.edu/islandora/object/etd:6825.
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
MLA Handbook (7th Edition):
Assar, Zahra. “Elucidation of iLBP family folding pathway and study of reengineering them as fluorescent protein tags via structural analysis.” 2018. Web. 11 Apr 2021.
Vancouver:
Assar Z. Elucidation of iLBP family folding pathway and study of reengineering them as fluorescent protein tags via structural analysis. [Internet] [Thesis]. Michigan State University; 2018. [cited 2021 Apr 11].
Available from: http://etd.lib.msu.edu/islandora/object/etd:6825.
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Council of Science Editors:
Assar Z. Elucidation of iLBP family folding pathway and study of reengineering them as fluorescent protein tags via structural analysis. [Thesis]. Michigan State University; 2018. Available from: http://etd.lib.msu.edu/islandora/object/etd:6825
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

Hong Kong University of Science and Technology
7.
Jiang, Meijuan CHEM.
Development of novel AIEgens based on benzylidene-methyloxazolones and isoquinolinium salts and exploration of their biological applications.
Degree: 2017, Hong Kong University of Science and Technology
URL: http://repository.ust.hk/ir/Record/1783.1-95153
;
https://doi.org/10.14711/thesis-991012564469003412
;
http://repository.ust.hk/ir/bitstream/1783.1-95153/1/th_redirect.html
► To tackle world-challenging problems, luminescent materials have played a vital role in the promotion of scientific discoveries and technological innovations. Practically, traditional luminogens often face…
(more)
▼ To tackle world-challenging problems, luminescent materials have played a vital role in the promotion of scientific discoveries and technological innovations. Practically, traditional luminogens often face a problem of aggregation-caused quenching (ACQ), resulting a reduction or diminishment of luminescence and thus limiting their practical applications. Circumventing the notorious ACQ effect, luminogens with aggregation-induced emission characteristics (AIEgens) have become intriguing with a rapid expansion in applications ranging from optoelectronics, chemo/biosensing to bioimaging. However, to tailor for specific high-tech applications and deepen the mechanistic understanding of AIEgens, there is a high demand on the developments of novel AIEgens with easy preparation and functionalization. In this thesis, two series of AIEgens were developed. Their working mechanism and various applications, especially biological applications, were investigated. Based on green fluorescence protein chromophore analogues benzylidene-oxazolone, a series of AIEgens were synthesized with solid quantum yield of up to 50% and emission wavelength of up to 635 nm. Their working mechanism were carefully deciphered. Further, through minor modifications, a two-photon AIE bioprobe was developed for specific lipid droplet imaging in cells and tissues. Also, we prepared a series of isoquinolinium salts-based AIEgens with high structural stability via a simple one-pot reaction. With the merits of visible light excitation, large Stokes shift, high quantum yield, tunable color and sufficient two-photon absorption of near-infrared light, we found their various applications in mechanochromic rewritable paper, mitochondrial targeting cell imaging and bacterial imaging. Interestingly, these molecules exhibited high viscosity-sensitivity and were further utilized as sensors for extracellular and intracellular microviscosity sensing. Among them, a simple AIEgen was developed for image-guided two-photon excited photodymanic therapy for precise cancer treatment.
Subjects/Keywords: Electroluminescent devices
; Photoemission
; Aggregation (Chemistry)
; Green fluorescent protein
; Fluorescent polymers
; Isoquinoline
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❌
APA ·
Chicago ·
MLA ·
Vancouver ·
CSE |
Export
to Zotero / EndNote / Reference
Manager
APA (6th Edition):
Jiang, M. C. (2017). Development of novel AIEgens based on benzylidene-methyloxazolones and isoquinolinium salts and exploration of their biological applications. (Thesis). Hong Kong University of Science and Technology. Retrieved from http://repository.ust.hk/ir/Record/1783.1-95153 ; https://doi.org/10.14711/thesis-991012564469003412 ; http://repository.ust.hk/ir/bitstream/1783.1-95153/1/th_redirect.html
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Chicago Manual of Style (16th Edition):
Jiang, Meijuan CHEM. “Development of novel AIEgens based on benzylidene-methyloxazolones and isoquinolinium salts and exploration of their biological applications.” 2017. Thesis, Hong Kong University of Science and Technology. Accessed April 11, 2021.
http://repository.ust.hk/ir/Record/1783.1-95153 ; https://doi.org/10.14711/thesis-991012564469003412 ; http://repository.ust.hk/ir/bitstream/1783.1-95153/1/th_redirect.html.
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
MLA Handbook (7th Edition):
Jiang, Meijuan CHEM. “Development of novel AIEgens based on benzylidene-methyloxazolones and isoquinolinium salts and exploration of their biological applications.” 2017. Web. 11 Apr 2021.
Vancouver:
Jiang MC. Development of novel AIEgens based on benzylidene-methyloxazolones and isoquinolinium salts and exploration of their biological applications. [Internet] [Thesis]. Hong Kong University of Science and Technology; 2017. [cited 2021 Apr 11].
Available from: http://repository.ust.hk/ir/Record/1783.1-95153 ; https://doi.org/10.14711/thesis-991012564469003412 ; http://repository.ust.hk/ir/bitstream/1783.1-95153/1/th_redirect.html.
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Council of Science Editors:
Jiang MC. Development of novel AIEgens based on benzylidene-methyloxazolones and isoquinolinium salts and exploration of their biological applications. [Thesis]. Hong Kong University of Science and Technology; 2017. Available from: http://repository.ust.hk/ir/Record/1783.1-95153 ; https://doi.org/10.14711/thesis-991012564469003412 ; http://repository.ust.hk/ir/bitstream/1783.1-95153/1/th_redirect.html
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

Michigan State University
8.
Santos, Elizabeth Marie.
Development of fluorescent protein tags for live-cell imaging.
Degree: 2017, Michigan State University
URL: http://etd.lib.msu.edu/islandora/object/etd:6843
► "Our primary goal is to develop fluorescent proteins that span the entire visible spectra, to be used when conventional fluorescent proteins are inadequate. In our…
(more)
▼ "Our primary goal is to develop fluorescent proteins that span the entire visible spectra, to be used when conventional fluorescent proteins are inadequate. In our system, fluorescence is activated upon coupling of the protein and ligand, such that temporal control can be achieved, whereas intrinsically fluorescent proteins are constitutively on. Additionally, our system does not require oxygen and can therefore find potential uses in obligate anaerobes. Our lab has demonstrated the ability to effectively control the absorption profile of conjugated polyenes. The initial aim of this project was to regulate the emissive properties of bound fluorophores with the same degree of control. This was achieved by the coupling of the solvatochromic fluorophore ThioFluor to hCRBPII mutants. ThioFluor yielded mutants with absorption maxima varying from 501 nm to 705 nm and emission maxima from 613 nm to 744 nm. This is equivalent to regulation over 204 nm in absorption and 131 nm in emission, covering both the red and far-red fluorescence wavelength regimes. Furthermore, we have shown its utility in live-cell imaging in whole cells, and with targeting to the nucleus and extranuclear space; fortuitously, negligible background fluorescence is apparent. In the course of optimizing binding of ThioFluor to hCRBPII, we discovered that it was observed that not only is the protonated Schiff base (PSB) fluorescent, but the Schiff base (SB) is as well. However, while PSB emission wavelength could be altered over 204 nm, SB emission remains nearly constant at 500 nm. Interestingly, select mutants displayed a far-red emission upon SB irradiation, similar to that obtained upon irradiation of the PSB, presumably through protonation in the excited state. This serendipitous discovery leads to more than a 200 nm Stokes shift with high quantum yield (> 60%). One such hCRBPII/ThioFluor complex displays ideal spectroscopic properties including fast iminium formation with a half-life of 1.7 min, low pKa of 5.3 (rendering almost complete SB formation at physiological pH), high quantum yield (0.51) and large Stokes shift (208 nm). This fluorescent protein was successfully used to visualize whole cell fluorescence, as well as targeting to the nucleus. The last major endeavor was to develop a protein-based pH sensor, with the ability to report pH values with high accuracy. We have previously reported an absorptive system that was capable of ratiometric sensing of pH. However, we have now developed a single protein fluorescent ratiometric pH sensor, based on the titration of an acidic residue near the iminium. Standard curves were generated based on the ratio of emissions at two excitation wavelengths, allowing for concentration independent sensing of pH. We have been able to demonstrate its applicability as an in vivo fluorescent pH probe, obtaining a pH value of 6.7 when hCRBPII is targeted to the nucleus of HeLa cells." – Pages ii-iii.
Online resource;
Advisors/Committee Members: Borhan, Babak, Jackson, James, Geiger, James, Huang, Xuefei.
Subjects/Keywords: Fluorescent polymers – Research; Fluorescent probes; Protein engineering; Organic chemistry; Biochemistry
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APA ·
Chicago ·
MLA ·
Vancouver ·
CSE |
Export
to Zotero / EndNote / Reference
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APA (6th Edition):
Santos, E. M. (2017). Development of fluorescent protein tags for live-cell imaging. (Thesis). Michigan State University. Retrieved from http://etd.lib.msu.edu/islandora/object/etd:6843
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Chicago Manual of Style (16th Edition):
Santos, Elizabeth Marie. “Development of fluorescent protein tags for live-cell imaging.” 2017. Thesis, Michigan State University. Accessed April 11, 2021.
http://etd.lib.msu.edu/islandora/object/etd:6843.
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
MLA Handbook (7th Edition):
Santos, Elizabeth Marie. “Development of fluorescent protein tags for live-cell imaging.” 2017. Web. 11 Apr 2021.
Vancouver:
Santos EM. Development of fluorescent protein tags for live-cell imaging. [Internet] [Thesis]. Michigan State University; 2017. [cited 2021 Apr 11].
Available from: http://etd.lib.msu.edu/islandora/object/etd:6843.
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Council of Science Editors:
Santos EM. Development of fluorescent protein tags for live-cell imaging. [Thesis]. Michigan State University; 2017. Available from: http://etd.lib.msu.edu/islandora/object/etd:6843
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

University of Texas – Austin
9.
Hunt, Marguerite E.
Exploring the emerging properties of novel GFP-like fluorescent proteins.
Degree: MA, Cell and Molecular Biology, 2013, University of Texas – Austin
URL: http://hdl.handle.net/2152/22311
► In 2008 the Nobel Prize in Chemistry was awarded to the scientists who revolutionized biomedical technology by isolating, characterizing, and pioneering the use of a…
(more)
▼ In 2008 the Nobel Prize in Chemistry was awarded to the scientists who revolutionized biomedical technology by isolating, characterizing, and pioneering the use of a green
fluorescent protein (GFP) from a humble hydrozoan jellyfish. Now numbering in the hundreds of colors and applications,
fluorescent protein (FP) tools have facilitated the explosion of biological knowledge elucidated by a technology that can label DNA or RNA, track
protein expression, and identify
protein interactions. The development of the large variety of FP biotechnology available today has been due to the need for expanded color palettes and applications, and more efficient functionality. Yet, as our understanding of the biochemical and spectral characteristics of these genetically-encoded, self-assembling proteins has expanded, our comprehension of the biological function of FPs in the host organisms has remained inadequate. While the need for novel FP laboratory applications still continues, the new focus in the field of
fluorescent proteins is moving to also characterize their biological functions. In this research compilation, the identification of three groups of new
fluorescent proteins from marine copepods and hydrozoans has provided a collection of eleven FPs exhibiting previously uncharacterized colors, and biochemical and structural features. The green FPs from copepods are the brightest wild-type FPs identified and support the hypothesized biological function of fluorescence as counter-shading in the marine environment where these animals live. The FPs from the siphonophore and anthoathecate jelly, both hydrozoan animals, are comprised of tandemly expressed
fluorescent protein units, a solution to the oligomeric structure common to most FPs that suggests a novel structure-function relationship. The
fluorescent proteins from Obelia reveal a novel hydrozoan cyan FP, previously uncharacterized higher-order structural complexes, and have initiated the work to describe the biological function of these proteins as potential regenerators of their internal bioluminescent light sources. All eleven
fluorescent proteins may also be adapted for FP technology.
Advisors/Committee Members: Matz, Mikhail V. (advisor).
Subjects/Keywords: Fluorescent protein; Biotechnology; Fluorescent spectra; Genetically encoded proteins
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❌
APA ·
Chicago ·
MLA ·
Vancouver ·
CSE |
Export
to Zotero / EndNote / Reference
Manager
APA (6th Edition):
Hunt, M. E. (2013). Exploring the emerging properties of novel GFP-like fluorescent proteins. (Masters Thesis). University of Texas – Austin. Retrieved from http://hdl.handle.net/2152/22311
Chicago Manual of Style (16th Edition):
Hunt, Marguerite E. “Exploring the emerging properties of novel GFP-like fluorescent proteins.” 2013. Masters Thesis, University of Texas – Austin. Accessed April 11, 2021.
http://hdl.handle.net/2152/22311.
MLA Handbook (7th Edition):
Hunt, Marguerite E. “Exploring the emerging properties of novel GFP-like fluorescent proteins.” 2013. Web. 11 Apr 2021.
Vancouver:
Hunt ME. Exploring the emerging properties of novel GFP-like fluorescent proteins. [Internet] [Masters thesis]. University of Texas – Austin; 2013. [cited 2021 Apr 11].
Available from: http://hdl.handle.net/2152/22311.
Council of Science Editors:
Hunt ME. Exploring the emerging properties of novel GFP-like fluorescent proteins. [Masters Thesis]. University of Texas – Austin; 2013. Available from: http://hdl.handle.net/2152/22311

University of Alberta
10.
Shen, Yi.
Engineering of red fluorescent proteins and red fluorescent
protein-based pH biosensors.
Degree: PhD, Department of Chemistry, 2014, University of Alberta
URL: https://era.library.ualberta.ca/files/c9z902z87p
► Fluorescent proteins (FP) and FP-based biosensors have become essential tools for cell biology and neuroscience research. This thesis describes efforts to engineer new FPs with…
(more)
▼ Fluorescent proteins (FP) and FP-based biosensors have
become essential tools for cell biology and neuroscience research.
This thesis describes efforts to engineer new FPs with red emission
and novel genetically encoded biosensors based on red FPs. Directed
evolution and semi-rational design are the main techniques used to
develop novel and improved red FP and red FP-based biosensors.
First, A new pH-sensitive red fluorescent protein based on mApple,
termed as “pHuji”, has been developed with high pH sensitivity,
exhibiting over 20-fold fluorescence intensity change between pH
5.5 and pH 7.5. In live cell imaging, cell surface-displayed pHuji
demonstrated high pH sensitivity when exposed to buffers with
defined pH values. Collaborators have used pHuji for successful
visualization of pH changes during exocytosis and endocytosis.
Following the engineering of intensiometric red pH sensors, we next
turned our attention to ratiometric red pH sensors. Through a
process of semi-rational design and directed evolution, the red FP
mApple was successfully engineered into a series of
dual-excitation, ratiometric pH sensors. These new red-shifted
ratiometric pH sensors, termed pHlorina, exhibit large ratio change
(over 70-fold) for pH changes from 5.0 to 7.5. A series of long
Stokes shift variants of mApple (λex = 450 nm and λem = 610 nm)
were also developed and characterized. A photochromic and
thermochromic red FP was serendipitously discovered during the
process of engineering the long Stokes shift red FP. This protein,
which we designated as switchable hypersensitive red FP (shyRFP),
was characterized in terms of light, temperature, and pH
dependence. A colour switching mechanism that involves protonation
coupled E-Z isomerization of the protein chromophore was proposed.
The monomeric RFP mCherry is widely used for live cell fluorescence
imaging experiments. Using semi-rational design and random
mutagenesis, two new mCherry variants were developed: Long Stokes
Shift mCherry (LSSmCherry; λex = 460 nm and λem = 610 nm) and
Red-Shifted mCherry (RDSmCherry; λex = 600 nm and λem = 630 nm).
These two proteins have distinctively different fluorescence
excitation and emission profiles from their predecessor mCherry2.
These new additions to the FP toolbox provide templates for the
engineering of new colour variants and fluorescent sensors with red
emission. Finally, a filter paper-based screening method has been
developed to screen for calcium ion (Ca2+) sensitive RFP variants
expressed in Escherichia coli colonies. These low-affinity Ca2+
sensors were semi-rationally designed by altering protein barrel
residues near the chromophore to form a Ca2+ binding pocket. By
combining high-throughput screening and rational design, a number
of Ca2+ sensitive variants were successfully
identified.
Subjects/Keywords: red fluorescent proteins; protein engineering; biosensors
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❌
APA ·
Chicago ·
MLA ·
Vancouver ·
CSE |
Export
to Zotero / EndNote / Reference
Manager
APA (6th Edition):
Shen, Y. (2014). Engineering of red fluorescent proteins and red fluorescent
protein-based pH biosensors. (Doctoral Dissertation). University of Alberta. Retrieved from https://era.library.ualberta.ca/files/c9z902z87p
Chicago Manual of Style (16th Edition):
Shen, Yi. “Engineering of red fluorescent proteins and red fluorescent
protein-based pH biosensors.” 2014. Doctoral Dissertation, University of Alberta. Accessed April 11, 2021.
https://era.library.ualberta.ca/files/c9z902z87p.
MLA Handbook (7th Edition):
Shen, Yi. “Engineering of red fluorescent proteins and red fluorescent
protein-based pH biosensors.” 2014. Web. 11 Apr 2021.
Vancouver:
Shen Y. Engineering of red fluorescent proteins and red fluorescent
protein-based pH biosensors. [Internet] [Doctoral dissertation]. University of Alberta; 2014. [cited 2021 Apr 11].
Available from: https://era.library.ualberta.ca/files/c9z902z87p.
Council of Science Editors:
Shen Y. Engineering of red fluorescent proteins and red fluorescent
protein-based pH biosensors. [Doctoral Dissertation]. University of Alberta; 2014. Available from: https://era.library.ualberta.ca/files/c9z902z87p

Northeastern University
11.
Salna, Bridget I.
Proton transport in proteins and the role of quantum tunneling.
Degree: PhD, Department of Physics, 2017, Northeastern University
URL: http://hdl.handle.net/2047/D20248978
► Proton transport is ubiquitous in biological systems and has been studied extensively, both with experimental and theoretical methods. A better understanding of this phenomenon is…
(more)
▼ Proton transport is ubiquitous in biological systems and has been studied extensively, both with experimental and theoretical methods. A better understanding of this phenomenon is sought because it underpins many fundamental mechanisms that sustain life, such as cellular respiration, photosynthesis, and drug metabolism. Many of these processes involve proton wires within proteins, in which protons are transported for use in biochemical reactions. This work examines the contribution of quantum tunneling to proton transport and proposes a primary role for tunneling in regulating this process.; This work discusses and improves upon the current theoretical models of proton tunneling in proteins and investigates the implications of considering tunneling to be the dominant transport channel. By including deep quantum tunneling under high potential barriers as a viable proton transport pathway, ionization-resistant residues such as serine and threonine can be considered as active constituents of proton wires. These residues have previously been found along many proton wires in proteins, but are seldom identified as transport elements. One notable exception is the green fluorescent protein (GFP), in which serine has been established as an active element of the well-characterized internal proton wire. This makes GFP an important model system to test the role of quantum tunneling in proton transport within proteins. This is one of the main projects discussed in this work, in which the biologically relevant ground state process is considered in depth. The rate-limiting step at room temperature is assigned to deep tunneling from the serine hydroxyl with a rate that is orders of magnitude faster than the classical pathway. This suggests how high pKa residues can act to stabilize and regulate proton flow along proton wires in proteins.; The role of quantum tunneling in enzymatic systems is also explored, in which proton coupled electron transfer (PCET) is often the critical process. The PCET in soybean lipoxygenase-1 (SLO), as well as its double mutant, is analyzed using a modified model of donor-acceptor atom interaction. This includes effects of electronic repulsion, external protein forces, and charge/bond polarization. Distinct mechanisms are proposed for the two species of SLO, in which the wild type forms an activated state with the substrate positioned close to the active site, while the double mutant is constrained to longer distances and transfer is only possible due to its increased flexibility.
Subjects/Keywords: green fluorescent protein; proton transport; tunneling
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❌
APA ·
Chicago ·
MLA ·
Vancouver ·
CSE |
Export
to Zotero / EndNote / Reference
Manager
APA (6th Edition):
Salna, B. I. (2017). Proton transport in proteins and the role of quantum tunneling. (Doctoral Dissertation). Northeastern University. Retrieved from http://hdl.handle.net/2047/D20248978
Chicago Manual of Style (16th Edition):
Salna, Bridget I. “Proton transport in proteins and the role of quantum tunneling.” 2017. Doctoral Dissertation, Northeastern University. Accessed April 11, 2021.
http://hdl.handle.net/2047/D20248978.
MLA Handbook (7th Edition):
Salna, Bridget I. “Proton transport in proteins and the role of quantum tunneling.” 2017. Web. 11 Apr 2021.
Vancouver:
Salna BI. Proton transport in proteins and the role of quantum tunneling. [Internet] [Doctoral dissertation]. Northeastern University; 2017. [cited 2021 Apr 11].
Available from: http://hdl.handle.net/2047/D20248978.
Council of Science Editors:
Salna BI. Proton transport in proteins and the role of quantum tunneling. [Doctoral Dissertation]. Northeastern University; 2017. Available from: http://hdl.handle.net/2047/D20248978

Brandeis University
12.
Yanding, Zhao.
Fluorescent labeled TAL protein as a tool to investigate single-molecule transcription.
Degree: 2015, Brandeis University
URL: http://hdl.handle.net/10192/31114
► TALEs are sequence specific transcription activator found in phytopathogenic bacteria. Interestingly, their specificity is defined by triplet amino acid repeat sequences, which can recognize individual…
(more)
▼ TALEs are sequence specific transcription activator found in phytopathogenic bacteria. Interestingly, their specificity is defined by triplet amino acid repeat sequences, which can recognize individual nucleotides in dsDNA. In crystal structures with mutants where the transcription effector domain has been deleted (TAL), we observe a fascinating structure where the TAL protein complete wraps around the dsDNA in a helical manner. There are a myriad of applications for an engineered site-specific DNA binding protein, with or without a transcription effector domain, such as genome editing and synthetic biology. Currently, however, there is no information regarding the kinetics or mechanism by which TALs bind to their respective sequences. Knowing the mechanism by which TALs find their targets and once there how long they remain bound will help in the design and application of TAL in monitoring transcription process. By engineering a SNAP-tagged TAL protein mutant, which can be fluorescently labeled with no transcriptional effector, used in co-localization of single molecules (COSMOS) experiment, we revealed that TAL, verified to be a highly specific target DNA binding protein, formed a very stable complex with target DNA. Moreover, We used this result as a springboard to incorporate fluorescent TAL with RNA polymerase (Holoenzyme) in single molecule experiments and found transcription process was blocked by TAL when TAL-DNA complex was formed. During the transcription, the RNA polymerase was stuck on the DNA with transcribed RNA fragments and cannot overpass the TAL protein.
Subjects/Keywords: Fluorescent; TAL protein; single-molecule transcription
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❌
APA ·
Chicago ·
MLA ·
Vancouver ·
CSE |
Export
to Zotero / EndNote / Reference
Manager
APA (6th Edition):
Yanding, Z. (2015). Fluorescent labeled TAL protein as a tool to investigate single-molecule transcription. (Thesis). Brandeis University. Retrieved from http://hdl.handle.net/10192/31114
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Chicago Manual of Style (16th Edition):
Yanding, Zhao. “Fluorescent labeled TAL protein as a tool to investigate single-molecule transcription.” 2015. Thesis, Brandeis University. Accessed April 11, 2021.
http://hdl.handle.net/10192/31114.
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
MLA Handbook (7th Edition):
Yanding, Zhao. “Fluorescent labeled TAL protein as a tool to investigate single-molecule transcription.” 2015. Web. 11 Apr 2021.
Vancouver:
Yanding Z. Fluorescent labeled TAL protein as a tool to investigate single-molecule transcription. [Internet] [Thesis]. Brandeis University; 2015. [cited 2021 Apr 11].
Available from: http://hdl.handle.net/10192/31114.
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Council of Science Editors:
Yanding Z. Fluorescent labeled TAL protein as a tool to investigate single-molecule transcription. [Thesis]. Brandeis University; 2015. Available from: http://hdl.handle.net/10192/31114
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

University of Kansas
13.
Lei, Ming.
Using Lysine-Reactive Fluorescent Dye for Surface Characterization of a Monoclonal Antibody.
Degree: MA, Pharmaceutical Chemistry, 2014, University of Kansas
URL: http://hdl.handle.net/1808/21645
► The last decade has witnessed a rapid growth in the development of protein pharmaceuticals for diagnostic and therapeutic purposes. The biopharmaceutical industry increasingly demands thorough…
(more)
▼ The last decade has witnessed a rapid growth in the development of
protein pharmaceuticals for diagnostic and therapeutic purposes. The biopharmaceutical industry increasingly demands thorough characterization of
protein conformation and conformational dynamics to ensure product quality and consistency. Here we present a chromatography-based method that is able to characterize
protein conformation and conformational dynamics at peptide level resolution in a high-throughput manner. The surface lysine residues of the
protein were labeled with a
fluorescent dye prior to trypsin and Glu-C digestion. The resulting peptide maps were monitored by fluorescence detection and the peak areas of the respective peptides were normalized to
protein concentration. The normalized fluorescence peak area for a specific peptide represents the individual lysine solvent accessibility. A higher normalized fluorescence peak area indicates higher solvent accessibility at a specific site. The identity of the peak of interested was determined by LC-MS/MS analysis. We first demonstrated this method is suitable for probing
protein surface/conformation by studying the effect of deglycosylation on a recombinant monoclonal antibody (mAb), IgG 1. The results from this method were consistent with previous results obtained by H/D-exchange. We then applied our method to study the interaction of the mAb with a common excipient, polysorbate-20 (PS-20). The interaction between PS-20 and the mAb was generally weak. The presence of PS-20 increased the
fluorescent labeling of several lysine residues on the mAb. These lysine residues localized near the
protein domains of relatively high hydrophobicity. This result provides a first insight into PS-20-mAb interaction at peptide level resolution.
Advisors/Committee Members: Schoneich, Christian (advisor), Stobaugh, John (cmtemember), Kao, Yung-Hsiang (cmtemember).
Subjects/Keywords: Chemistry; Fluorescent; labeling; mass spectrometry; protein structure
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❌
APA ·
Chicago ·
MLA ·
Vancouver ·
CSE |
Export
to Zotero / EndNote / Reference
Manager
APA (6th Edition):
Lei, M. (2014). Using Lysine-Reactive Fluorescent Dye for Surface Characterization of a Monoclonal Antibody. (Masters Thesis). University of Kansas. Retrieved from http://hdl.handle.net/1808/21645
Chicago Manual of Style (16th Edition):
Lei, Ming. “Using Lysine-Reactive Fluorescent Dye for Surface Characterization of a Monoclonal Antibody.” 2014. Masters Thesis, University of Kansas. Accessed April 11, 2021.
http://hdl.handle.net/1808/21645.
MLA Handbook (7th Edition):
Lei, Ming. “Using Lysine-Reactive Fluorescent Dye for Surface Characterization of a Monoclonal Antibody.” 2014. Web. 11 Apr 2021.
Vancouver:
Lei M. Using Lysine-Reactive Fluorescent Dye for Surface Characterization of a Monoclonal Antibody. [Internet] [Masters thesis]. University of Kansas; 2014. [cited 2021 Apr 11].
Available from: http://hdl.handle.net/1808/21645.
Council of Science Editors:
Lei M. Using Lysine-Reactive Fluorescent Dye for Surface Characterization of a Monoclonal Antibody. [Masters Thesis]. University of Kansas; 2014. Available from: http://hdl.handle.net/1808/21645

University of Kansas
14.
Egan, Chet.
Engineering GFP-Family Member Protiens to Achieve Novel Functions: A Class of Proteins Limited Only by the Imagination.
Degree: MA, Molecular Biosciences, 2015, University of Kansas
URL: http://hdl.handle.net/1808/19178
► The Green Fluorescent Protein (GFP) has single handedly revolutionized microscopy and molecular biology. A simple search for GFP in the NCBI Pubmed database yields over…
(more)
▼ The Green
Fluorescent Protein (GFP) has single handedly revolutionized microscopy and molecular biology. A simple search for GFP in the NCBI Pubmed database yields over 29,900 unique publications as of April 2015 demonstrating the importance and influence GFP has had on the scientific community. It is because of this impact that the 2008 Nobel Prize in Chemistry was awarded jointly to Osamu Shimomura, Martin Chalfie, and Roger Y. Tsien for their ground breaking work on the discovery and characterization of GFP family proteins. GFP has come a long way since it was first isolated from Aquorea Victoria, a bioluminescent jellyfish native to the Pacific Northwest. In the past 2 decades since it was first cloned countless variants have been engineered. There are now literally hundreds of variants of GFP family proteins with more continuously being engineered. The choice of emission colors spans the entire visible spectrum and even beyond with some reaching into the infrared wavelengths. It would be a daunting task to attempt to cover all the amazing achievements in engineering of GFP family proteins and their diverse applications. Researchers are continuously improving on the constructs available, increasing their efficiency, sensitivity, and stability. GFP family proteins have been adapted to be everything from reporters of gene expression to labels for entire organelles or even entire tissues. GFP can be used to visualize
protein binding and transient
protein-protein interactions. GFP can also have its fluorescence put under the direct control of a small molecule ligand paving the way for switchable and biosensor GFPs. The more one reads about GFP the more it becomes clear that the applications of this amazing
protein family are still expanding at a very rapid pace. Here, I will focus on a few of what I consider the most interesting, ambitious, and novel engineering applications of the GFP family of proteins.
Advisors/Committee Members: Karanicolas, John (advisor), Benedict, Steve (cmtemember), Richter, Mark (cmtemember).
Subjects/Keywords: Molecular biology; GFP; Green Fluorescent Protein
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❌
APA ·
Chicago ·
MLA ·
Vancouver ·
CSE |
Export
to Zotero / EndNote / Reference
Manager
APA (6th Edition):
Egan, C. (2015). Engineering GFP-Family Member Protiens to Achieve Novel Functions: A Class of Proteins Limited Only by the Imagination. (Masters Thesis). University of Kansas. Retrieved from http://hdl.handle.net/1808/19178
Chicago Manual of Style (16th Edition):
Egan, Chet. “Engineering GFP-Family Member Protiens to Achieve Novel Functions: A Class of Proteins Limited Only by the Imagination.” 2015. Masters Thesis, University of Kansas. Accessed April 11, 2021.
http://hdl.handle.net/1808/19178.
MLA Handbook (7th Edition):
Egan, Chet. “Engineering GFP-Family Member Protiens to Achieve Novel Functions: A Class of Proteins Limited Only by the Imagination.” 2015. Web. 11 Apr 2021.
Vancouver:
Egan C. Engineering GFP-Family Member Protiens to Achieve Novel Functions: A Class of Proteins Limited Only by the Imagination. [Internet] [Masters thesis]. University of Kansas; 2015. [cited 2021 Apr 11].
Available from: http://hdl.handle.net/1808/19178.
Council of Science Editors:
Egan C. Engineering GFP-Family Member Protiens to Achieve Novel Functions: A Class of Proteins Limited Only by the Imagination. [Masters Thesis]. University of Kansas; 2015. Available from: http://hdl.handle.net/1808/19178

University of Sydney
15.
Morin-Adeline, Victoria.
Molecular and Biological Investigations of Tritrichomonas foetus from Cattle, Domestic Cats and Pigs
.
Degree: 2016, University of Sydney
URL: http://hdl.handle.net/2123/15168
► Few organisms have the potential to shed light on aspects of parasitism as much as Tritrichomonas foetus. While it is a commensal organism in pigs,…
(more)
▼ Few organisms have the potential to shed light on aspects of parasitism as much as Tritrichomonas foetus. While it is a commensal organism in pigs, T. foetus is a urogenital parasite of cattle and an intestinal parasite of domestic cats. In this thesis, I show that a high prevalence of porcine T. foetus is maintained in domestic pigs on an Australian farm where pigs and T. foetus-free cattle are farmed in close proximity. A novel Australian reference isolate of porcine T. foetus with close association to bovine T. foetus is established. Baseline transcriptomes of T. foetus from cattle, cat and pig hosts are sequenced and compared to confirm that isolates from the three hosts represent the same T. foetus species. In addition, significant differences are identified in the transcription of virulence factors between the bovine and feline isolates. To understand how the environment of the organ niche in individual hosts influence host choice and epidemiology of the two parasitic T. foetus, tolerance to extracelluar pH was investigated in vitro. The feline T. foetus demonstrated an enhanced capacity to maintain viability when exposed to mild acidic pH in contrast to bovine T. foetus, implying that pH is a barrier to cross-infection between the two hosts. An in silico investigation into plausible drug targets for the parasitic bovine and feline T. foetus revealed that drug targets chosen for more focused investigation in T. foetus originating from cattle may not be ideal for T. foetus in domestic cats. Having made large sequences databases available, this thesis attempts to expand the molecular toolbox for parasitologists working on anaerobes by trialling miniSOG, a flavin-based oxygen-independent fluorescent protein (FbFP) tag in T. foetus. As a whole, this body of work has created a platform to advance future T. foetus research in the fields of epidemiology, drug target discovery and recombinant protein tagging.
Subjects/Keywords: Trichomonad;
Fluorescent protein;
parasite;
Transcriptome;
cattle;
cats
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APA ·
Chicago ·
MLA ·
Vancouver ·
CSE |
Export
to Zotero / EndNote / Reference
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APA (6th Edition):
Morin-Adeline, V. (2016). Molecular and Biological Investigations of Tritrichomonas foetus from Cattle, Domestic Cats and Pigs
. (Thesis). University of Sydney. Retrieved from http://hdl.handle.net/2123/15168
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Chicago Manual of Style (16th Edition):
Morin-Adeline, Victoria. “Molecular and Biological Investigations of Tritrichomonas foetus from Cattle, Domestic Cats and Pigs
.” 2016. Thesis, University of Sydney. Accessed April 11, 2021.
http://hdl.handle.net/2123/15168.
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
MLA Handbook (7th Edition):
Morin-Adeline, Victoria. “Molecular and Biological Investigations of Tritrichomonas foetus from Cattle, Domestic Cats and Pigs
.” 2016. Web. 11 Apr 2021.
Vancouver:
Morin-Adeline V. Molecular and Biological Investigations of Tritrichomonas foetus from Cattle, Domestic Cats and Pigs
. [Internet] [Thesis]. University of Sydney; 2016. [cited 2021 Apr 11].
Available from: http://hdl.handle.net/2123/15168.
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Council of Science Editors:
Morin-Adeline V. Molecular and Biological Investigations of Tritrichomonas foetus from Cattle, Domestic Cats and Pigs
. [Thesis]. University of Sydney; 2016. Available from: http://hdl.handle.net/2123/15168
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

University of Texas – Austin
16.
Rodríguez Mendoz, Álvaro Eugenio.
Identifying mutations that enhance the evolutionary stability of fluorescent protein expression from a plasmid in Escherichia coli.
Degree: MA, Microbiology, 2014, University of Texas – Austin
URL: http://hdl.handle.net/2152/31999
► Synthetic biologists and metabolic engineers seek to design and create organisms with novel functions. A major difficulty with many designed genetic devices is that they…
(more)
▼ Synthetic biologists and metabolic engineers seek to design and create organisms with novel functions. A major difficulty with many designed genetic devices is that they lack evolutionary robustness. In this study, our aim was to identify mutations that could enhance the evolutionary stability of green
fluorescent protein (GFP) expression from a plasmid in Escherichia coli. To achieve this goal, we created a mutagenized strain library and performed an evolution experiment. To enrich potential mutants with improved GFP stability, we periodically sorted for cells that remained highly
fluorescent as this population was propagated for several hundred generations and less-robust strains accumulated inactivating mutations. Further testing of clones isolated from the final evolved population showed that GFP expression was more stable in these strains and suggested mutations in the chromosome were responsible. Re-sequencing the genomes of four of these strains found that, among other genetic differences from the ancestor, all had a mutation in either PolA or PolB. These two types of DNA polymerase mutations may enhance GFP stability by causing a lower point mutation rate in the E. coli host.
Advisors/Committee Members: Barrick, Jeffrey E. (advisor), Harshey, Rasika (committee member).
Subjects/Keywords: Evolutionary; Stability; Fluorescent; Protein; Mutations; Enhance
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APA ·
Chicago ·
MLA ·
Vancouver ·
CSE |
Export
to Zotero / EndNote / Reference
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APA (6th Edition):
Rodríguez Mendoz, . E. (2014). Identifying mutations that enhance the evolutionary stability of fluorescent protein expression from a plasmid in Escherichia coli. (Masters Thesis). University of Texas – Austin. Retrieved from http://hdl.handle.net/2152/31999
Chicago Manual of Style (16th Edition):
Rodríguez Mendoz, Álvaro Eugenio. “Identifying mutations that enhance the evolutionary stability of fluorescent protein expression from a plasmid in Escherichia coli.” 2014. Masters Thesis, University of Texas – Austin. Accessed April 11, 2021.
http://hdl.handle.net/2152/31999.
MLA Handbook (7th Edition):
Rodríguez Mendoz, Álvaro Eugenio. “Identifying mutations that enhance the evolutionary stability of fluorescent protein expression from a plasmid in Escherichia coli.” 2014. Web. 11 Apr 2021.
Vancouver:
Rodríguez Mendoz E. Identifying mutations that enhance the evolutionary stability of fluorescent protein expression from a plasmid in Escherichia coli. [Internet] [Masters thesis]. University of Texas – Austin; 2014. [cited 2021 Apr 11].
Available from: http://hdl.handle.net/2152/31999.
Council of Science Editors:
Rodríguez Mendoz E. Identifying mutations that enhance the evolutionary stability of fluorescent protein expression from a plasmid in Escherichia coli. [Masters Thesis]. University of Texas – Austin; 2014. Available from: http://hdl.handle.net/2152/31999

Rice University
17.
Pandey, Naresh.
Characterizing the tolerance of near infrared fluorescent bacterial phytochromes to random backbone fission and circular permutation.
Degree: PhD, Natural Sciences, 2016, Rice University
URL: http://hdl.handle.net/1911/96201
► Protein fission, fusion, and circular permutation have been used to convert green fluorescent protein (GFP) family members into biosensors that dynamically report on cellular processes,…
(more)
▼ Protein fission, fusion, and circular permutation have been used to convert green
fluorescent protein (GFP) family members into biosensors that dynamically report on cellular processes, ranging from
protein expression and metabolite concentrations to
protein solubility,
protein-protein interactions, and ligand-binding. Unfortunately, GFP are unsuitable for deep tissue reporting in animal models because the wavelengths of light used with these reporters is highly absorbed by tissues. In contrast, near infrared
fluorescent protein (IFP and iRFP) reporters derived from bacterial phytochrome proteins (BphP) are excited by light in the near-infrared spectrum (~700 nm, less absorptive) and are better suited for probing cellular processes within tissues. IFP and iRFP can report on biological processes under anaerobic conditions because it uses biliverdin (BV) as a chromophore and does not require oxygen for maturation, a requisite for GFP maturation. Unlike GFP, IFP and iRFP are not yet able to report on wide-range of biological processes beyond gene expression.
To report on gene expression, BphP must interlace its Per/ARNT/Sim (PAS) and cGMP phosphodiesterase/adenylcyclase/FhlA (GAF) domains into a topological knot. The extent to which this complex topology tolerates mutations (fission, fusion, and circular permutation) used to convert proteins into biosensors is not known. To better understand the tolerance of BphP to these types of mutational lesions, I have subjected IFP to random backbone fragmentation and iRFP to circular permutation using transposase mutagenesis. Screening a library of split IFP for
fluorescent variants yielded thirteen unique fragmented IFP and with parent like spectral properties. These two-fragment IFP all required assistance from associating proteins for maximal fluorescence. These split sites displayed AND gate logic behavior when the ORFs encoding the different fragment are placed under distinct transcriptional regulation. In addition, screening a library of circularly permuted iRFP led to the discovery of twenty seven permuted iRFP variants with near infrared fluorescence. These variants arose from backbone fission in both the PAS and GAF domains, although the brightest permuted iRFP initiated at residues near the domain linker and termini. Biochemical analysis revealed that permuted iRFP display similar oligomerizatoin, quantum yield, and stability as native iRFP. These proteins also retained sufficient BV affinity serve as reporters of gene expression in mammalian cells without the addition of exogenous BV.
The results described in this thesis represent the first study to map the tolerance of a BphP to random fragmentation and circular permutation. These results demonstrate that knotted BphP retain the ability to fold as their contact order changes, suggesting that these proteins can be further developed as reporters of biological processes like GFP. The split IFP represent a suite of assays that will be useful for monitoring the dynamics of a
protein-protein interactions under conditions…
Advisors/Committee Members: Silberg, Jonathan J (advisor).
Subjects/Keywords: near infrared fluorescent protein; circular permutation; knotted protein; protein engineering
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❌
APA ·
Chicago ·
MLA ·
Vancouver ·
CSE |
Export
to Zotero / EndNote / Reference
Manager
APA (6th Edition):
Pandey, N. (2016). Characterizing the tolerance of near infrared fluorescent bacterial phytochromes to random backbone fission and circular permutation. (Doctoral Dissertation). Rice University. Retrieved from http://hdl.handle.net/1911/96201
Chicago Manual of Style (16th Edition):
Pandey, Naresh. “Characterizing the tolerance of near infrared fluorescent bacterial phytochromes to random backbone fission and circular permutation.” 2016. Doctoral Dissertation, Rice University. Accessed April 11, 2021.
http://hdl.handle.net/1911/96201.
MLA Handbook (7th Edition):
Pandey, Naresh. “Characterizing the tolerance of near infrared fluorescent bacterial phytochromes to random backbone fission and circular permutation.” 2016. Web. 11 Apr 2021.
Vancouver:
Pandey N. Characterizing the tolerance of near infrared fluorescent bacterial phytochromes to random backbone fission and circular permutation. [Internet] [Doctoral dissertation]. Rice University; 2016. [cited 2021 Apr 11].
Available from: http://hdl.handle.net/1911/96201.
Council of Science Editors:
Pandey N. Characterizing the tolerance of near infrared fluorescent bacterial phytochromes to random backbone fission and circular permutation. [Doctoral Dissertation]. Rice University; 2016. Available from: http://hdl.handle.net/1911/96201

University of Alberta
18.
Hoi, Hiofan.
Development of monomeric fluorescent proteins and
fluorescent protein-based biosensors.
Degree: PhD, Department of Chemistry, 2013, University of Alberta
URL: https://era.library.ualberta.ca/files/5712m723w
► Fluorescent protein (FP) technology is now an indispensible tool of biomedical research. Nevertheless, only a few members of the hundreds of existing FPs are generally…
(more)
▼ Fluorescent protein (FP) technology is now an
indispensible tool of biomedical research. Nevertheless, only a few
members of the hundreds of existing FPs are generally regarded as
the preferred options for most imaging applications. Accordingly,
new FPs with ever-improved properties are in demand and worth
pursuing, as these efforts may lead to variants that are closer to
the “ideal” FP. Also there remain a tremendous number of
opportunities for developing FP-based biosensors for probing
biological process in vivo. This thesis describes our effort on
engineering new FPs with improved properties, and further modifying
them to create novel biosensors. Directed evolution and
semi-rational protein engineering are the main techniques used to
develop these new FPs and FP-based biosensors. The first class of
FPs addressed in this thesis are the green-to-red photoconvertible
FPs (pcFPs). In an effort to overcome the limitations imposed by
the oligomeric structure of natural pcFPs, we created a new
monomeric pcFP based on consensus design. Subsequent optimization
yielded mClavGR2 and mMaple, two monomeric pcFPs displaying
superior performance in folding and maturation, brightness,
photoconversion efficiency and photostability. We demonstrate the
application of mClavGR2 for dynamic monitoring of protein
trafficking. Furthermore, in collaboration with researchers from
several other groups, mMaple was demonstrated to be a multi-model
probe that is suitable for use in several conventional and
super-resolution fluorescence imaging modalities. Using mMaple as a
template for single-FP biosensor design, we successfully combined
the two most important implementations of FPs, the “highlightable”
trait and the Ca2+ sensing capability, into one construct.
Optimization, characterization and live cell imaging of the
resulting green-to-red highlightable Ca2+ indicators are described.
Another class of FPs that are of interest in this thesis are the
true yellow emitting FPs that fill the spectral gap between
monomeric greenish-yellow FPs and monomeric orange FPs. By
disrupting the inter-subunit interfaces of zFP538, a FP with a
distinct three-ring chromophore and an emission maximum at 538 nm,
we successfully obtained its monomeric version and named it as
mPapaya1. Again, characterization and live cell imaging application
of mPapaya1 are described.
Subjects/Keywords: directed evolution; protein engineering; calcium indicator; genetically-encoded biosensor; fluorescent protein
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❌
APA ·
Chicago ·
MLA ·
Vancouver ·
CSE |
Export
to Zotero / EndNote / Reference
Manager
APA (6th Edition):
Hoi, H. (2013). Development of monomeric fluorescent proteins and
fluorescent protein-based biosensors. (Doctoral Dissertation). University of Alberta. Retrieved from https://era.library.ualberta.ca/files/5712m723w
Chicago Manual of Style (16th Edition):
Hoi, Hiofan. “Development of monomeric fluorescent proteins and
fluorescent protein-based biosensors.” 2013. Doctoral Dissertation, University of Alberta. Accessed April 11, 2021.
https://era.library.ualberta.ca/files/5712m723w.
MLA Handbook (7th Edition):
Hoi, Hiofan. “Development of monomeric fluorescent proteins and
fluorescent protein-based biosensors.” 2013. Web. 11 Apr 2021.
Vancouver:
Hoi H. Development of monomeric fluorescent proteins and
fluorescent protein-based biosensors. [Internet] [Doctoral dissertation]. University of Alberta; 2013. [cited 2021 Apr 11].
Available from: https://era.library.ualberta.ca/files/5712m723w.
Council of Science Editors:
Hoi H. Development of monomeric fluorescent proteins and
fluorescent protein-based biosensors. [Doctoral Dissertation]. University of Alberta; 2013. Available from: https://era.library.ualberta.ca/files/5712m723w
19.
Mythili, J Krishna.
Identification of a fluorescent protein from a marine
Zoanthid: Zoanthus Sansibaricus (Carlgren) from the intertidal
rocky shore of Anjuna (Goa); -.
Degree: Pharmacy, 2011, Jawaharlal Nehru Technological University
URL: http://shodhganga.inflibnet.ac.in/handle/10603/4505
► Zoanthids comprise an order of benthic, generally colonial Cnidarians, which can usually be distinguished from other hexacorallians by embedded sand and detritus in their mesoglea.…
(more)
▼ Zoanthids comprise an order of benthic, generally
colonial Cnidarians, which can usually be distinguished from other
hexacorallians by embedded sand and detritus in their mesoglea.
These animals are becoming increasingly important research subjects
in biochemistry and other research fields. Their inclusion of both
calcium and silica results in the need for both decalcification and
desilification for internal morphological examinations. With the
availability of the standardized techniques using HF treatment,
this is now possible. Recent investigations utilizing molecular
(16S mt rDNA) methods have brought a clearer understanding of
zoanthid diversity. Though collection and documentation of data has
started all over the world regarding clarifying zoanthid taxonomy,
zoanthids from Indain coast have not yet been explored in dual
prespective. This is the first report from India, based on
morphological, histological and molecular analysis, on identifying
a marine zoanthid from the intertidal, rocky shore of Anjuna (Goa)
as Zoanthus sansibaricus (Zoanthidae). A family of proteins named
Green Fluorescent Proteins (GFP)-like proteins (fluorescent
proteins) started a trend in biotechnological research, which is
ever expanding day by day. Not only is the occurrence of these
proteins so wide spread but also are their applications. Zoanthids
belonging to the phylum cnidarian are reported to have these
fluorescent proteins in them, though the zoanthids possessing them
were mentioned only to the genus level. This is first report from
India indicating the presence of a fluorescent protein in the far
red region of the visible spectrum and also mentioning the source
zoanthid to the species level.
References p.131-143, Appendices
p.144-149
Advisors/Committee Members: Ingole, Baban, Anjaneyulu, Y.
Subjects/Keywords: Green Fluorescent Protein; Chemistry; Protein; Zoanthid Taxonomy; Pharmacy
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❌
APA ·
Chicago ·
MLA ·
Vancouver ·
CSE |
Export
to Zotero / EndNote / Reference
Manager
APA (6th Edition):
Mythili, J. K. (2011). Identification of a fluorescent protein from a marine
Zoanthid: Zoanthus Sansibaricus (Carlgren) from the intertidal
rocky shore of Anjuna (Goa); -. (Thesis). Jawaharlal Nehru Technological University. Retrieved from http://shodhganga.inflibnet.ac.in/handle/10603/4505
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Chicago Manual of Style (16th Edition):
Mythili, J Krishna. “Identification of a fluorescent protein from a marine
Zoanthid: Zoanthus Sansibaricus (Carlgren) from the intertidal
rocky shore of Anjuna (Goa); -.” 2011. Thesis, Jawaharlal Nehru Technological University. Accessed April 11, 2021.
http://shodhganga.inflibnet.ac.in/handle/10603/4505.
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
MLA Handbook (7th Edition):
Mythili, J Krishna. “Identification of a fluorescent protein from a marine
Zoanthid: Zoanthus Sansibaricus (Carlgren) from the intertidal
rocky shore of Anjuna (Goa); -.” 2011. Web. 11 Apr 2021.
Vancouver:
Mythili JK. Identification of a fluorescent protein from a marine
Zoanthid: Zoanthus Sansibaricus (Carlgren) from the intertidal
rocky shore of Anjuna (Goa); -. [Internet] [Thesis]. Jawaharlal Nehru Technological University; 2011. [cited 2021 Apr 11].
Available from: http://shodhganga.inflibnet.ac.in/handle/10603/4505.
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Council of Science Editors:
Mythili JK. Identification of a fluorescent protein from a marine
Zoanthid: Zoanthus Sansibaricus (Carlgren) from the intertidal
rocky shore of Anjuna (Goa); -. [Thesis]. Jawaharlal Nehru Technological University; 2011. Available from: http://shodhganga.inflibnet.ac.in/handle/10603/4505
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

University of California – San Francisco
20.
Merksamer, Philip Ian.
The Development and Application of Fluorescent Protein Reporters to Measure Endoplasmic Reticulum Stress in Single Cells.
Degree: Cell Biology, 2010, University of California – San Francisco
URL: http://www.escholarship.org/uc/item/1dw4d3hd
► In eukaryotic cells, secreted and membrane proteins fold within the endoplasmic reticulum (ER). Various physiological or pathophysiological conditions can disrupt ER protein folding homeostasis and…
(more)
▼ In eukaryotic cells, secreted and membrane proteins fold within the endoplasmic reticulum (ER). Various physiological or pathophysiological conditions can disrupt ER protein folding homeostasis and cause unfolded proteins to accumulate within the ER. Unfolded proteins activate a conserved intracellular signaling pathway called the unfolded protein response (UPR) that increases the ER's protein-folding capacity in order to restore protein folding homeostasis. However, because it is infeasible to directly measure the concentration of unfolded proteins within the ER, any UPR-activated cell is generically described as experiencing "ER stress." To address this problem, I utilized an ER-targeted green fluorescent protein whose fluorescent output is responsive to its oxidation state (called eroGFP). I found that many stressors to ER protein folding homeostasis – both experimental and physiological – compromise oxidation of eroGFP in S. cerevisiae . By combining eroGFP with an additional fluorescent protein reporter to follow changes in UPR activity I was able to determine conditions in which the UPR is capable of promoting adaptation. Additionally, using high-throughput flow cytometry, I measured eroGFP oxidation in approximately 6000 yeast strains each with a deletion or hypomorphic allele of a single gene. Through this analysis, I was able to identify genes important for maintaining oxidative protein folding during normal growth conditions and during protein folding stress. The strategy of utilizing eroGFP as a proximal reporter for ER stress proved to be complimentary to UPR-based metrics to provide a more comprehensive understanding of ER protein folding. The tools and concepts developed here should be broadly applicable to other biological processes and should aid investigations of how ER stress affects human disease.
Subjects/Keywords: Cellular Biology; endoplasmic reticulum stress; fluorescent protein reporters; unfolded protein response
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❌
APA ·
Chicago ·
MLA ·
Vancouver ·
CSE |
Export
to Zotero / EndNote / Reference
Manager
APA (6th Edition):
Merksamer, P. I. (2010). The Development and Application of Fluorescent Protein Reporters to Measure Endoplasmic Reticulum Stress in Single Cells. (Thesis). University of California – San Francisco. Retrieved from http://www.escholarship.org/uc/item/1dw4d3hd
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Chicago Manual of Style (16th Edition):
Merksamer, Philip Ian. “The Development and Application of Fluorescent Protein Reporters to Measure Endoplasmic Reticulum Stress in Single Cells.” 2010. Thesis, University of California – San Francisco. Accessed April 11, 2021.
http://www.escholarship.org/uc/item/1dw4d3hd.
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
MLA Handbook (7th Edition):
Merksamer, Philip Ian. “The Development and Application of Fluorescent Protein Reporters to Measure Endoplasmic Reticulum Stress in Single Cells.” 2010. Web. 11 Apr 2021.
Vancouver:
Merksamer PI. The Development and Application of Fluorescent Protein Reporters to Measure Endoplasmic Reticulum Stress in Single Cells. [Internet] [Thesis]. University of California – San Francisco; 2010. [cited 2021 Apr 11].
Available from: http://www.escholarship.org/uc/item/1dw4d3hd.
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Council of Science Editors:
Merksamer PI. The Development and Application of Fluorescent Protein Reporters to Measure Endoplasmic Reticulum Stress in Single Cells. [Thesis]. University of California – San Francisco; 2010. Available from: http://www.escholarship.org/uc/item/1dw4d3hd
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

University of New Mexico
21.
Phillips, Genevieve Kate.
DEVELOPING FLUOROGEN ACTIVATING PROTEIN-FLUORESCENT PROTEIN FRET PAIRS FOR LIVE CELL IMAGING.
Degree: Biomedical Sciences Graduate Program, 2017, University of New Mexico
URL: https://digitalrepository.unm.edu/biom_etds/161
► Fluorogen activating proteins (FAPs) are genetically encoded tags made from single chain antibody fragments (scFv) designed to bind fluorogens with high specificity. Both the…
(more)
▼ Fluorogen activating proteins (FAPs) are genetically encoded tags made from single chain antibody fragments (scFv) designed to bind fluorogens with high specificity. Both the fluorogen and FAP can be modified to provide flexibility in properties such as affinity, membrane permeability, spectra, and quantum yield. The fluorogen Malachite Green (MG) has two excitation peaks, the maximum at 630 nm and a secondary peak at 450 nm. The emission spectra of blue-emitting fluorescence proteins, such as mCerulean (mCer), overlap with the MG secondary peak, generating a FRET pair with large Stokes shift emission. Using 405 nm excitation of mCer, we observe acceptor sensitized emission at wavelengths greater than 650 nm with no spectral crosstalk between the donor and acceptor channels. Additionally, donor only controls can be acquired for all cells as the acceptor is not present until after the addition of the fluorogen, providing intra-cellular control.
The FAP-FRET system has been characterized using proof of principle constructs: FAP-mCer-transmembrane (TM) as a positive FRET control and FAP-TM-mCer as a negative FRET control and expressed in HeLa cells. Multiple MG derivatives were compared and imaging parameters were optimized to determine the optimal FRET pair. Analysis was performed using code written in Matlab to mask the cell membrane and quantify FRET efficiencies, based on donor intensity before and after addition of fluorogen. Data from several fluorogen showed high energy transfer efficiency (~30%) with the FAP-mCer-TM construct compared to negligible FRET (~4%) for FAP-TM-mCer. Additional techniques were performed to support the FRET efficiency data, including spectral imaging and FLIM, which also reported FRET efficiency around 30% with the positive constructs and negligible FRET with the negative constructs. The FAP-FRET system is currently being used to study the kinetics of signaling proteins within the FcεRI pathway.
Advisors/Committee Members: Dr. Diane Lidke, Dr. Angela Wandinger-Ness, Dr. Heather Ward, Dr. Bill Shuttleworth.
Subjects/Keywords: Fluorogen activating protein (FAP); FRET; Fluorescent protein; Medicine and Health Sciences
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❌
APA ·
Chicago ·
MLA ·
Vancouver ·
CSE |
Export
to Zotero / EndNote / Reference
Manager
APA (6th Edition):
Phillips, G. K. (2017). DEVELOPING FLUOROGEN ACTIVATING PROTEIN-FLUORESCENT PROTEIN FRET PAIRS FOR LIVE CELL IMAGING. (Masters Thesis). University of New Mexico. Retrieved from https://digitalrepository.unm.edu/biom_etds/161
Chicago Manual of Style (16th Edition):
Phillips, Genevieve Kate. “DEVELOPING FLUOROGEN ACTIVATING PROTEIN-FLUORESCENT PROTEIN FRET PAIRS FOR LIVE CELL IMAGING.” 2017. Masters Thesis, University of New Mexico. Accessed April 11, 2021.
https://digitalrepository.unm.edu/biom_etds/161.
MLA Handbook (7th Edition):
Phillips, Genevieve Kate. “DEVELOPING FLUOROGEN ACTIVATING PROTEIN-FLUORESCENT PROTEIN FRET PAIRS FOR LIVE CELL IMAGING.” 2017. Web. 11 Apr 2021.
Vancouver:
Phillips GK. DEVELOPING FLUOROGEN ACTIVATING PROTEIN-FLUORESCENT PROTEIN FRET PAIRS FOR LIVE CELL IMAGING. [Internet] [Masters thesis]. University of New Mexico; 2017. [cited 2021 Apr 11].
Available from: https://digitalrepository.unm.edu/biom_etds/161.
Council of Science Editors:
Phillips GK. DEVELOPING FLUOROGEN ACTIVATING PROTEIN-FLUORESCENT PROTEIN FRET PAIRS FOR LIVE CELL IMAGING. [Masters Thesis]. University of New Mexico; 2017. Available from: https://digitalrepository.unm.edu/biom_etds/161

University of Edinburgh
22.
Chen, Kai.
Use of green fluorescent protein for the analysis of protein-protein and protein-DNA interactions.
Degree: PhD, 2011, University of Edinburgh
URL: http://hdl.handle.net/1842/4886
► Restriction modification (RM) systems play a crucial role in preventing the entry of foreign DNA into the bacterial cell. The best studied Type I RM…
(more)
▼ Restriction modification (RM) systems play a crucial role in preventing the entry of foreign DNA into the bacterial cell. The best studied Type I RM system is EcoKI from Escherichia coli K12. Both bacteriophage and conjugative plasmids have developed a variety of strategies to circumvent the host RM system. One such strategy involves the production of antirestriction proteins that mimic a short segment of DNA and efficiently inhibit the RM system. The main aim of this project was to analyse the interaction of EcoKI and its cognate methylase (MTase) with the T7 antirestriction protein, known as overcome classical restriction (Ocr), and various ArdA antirestriction proteins. Currently, there is a paucity of structural data on the complex formed between the Type I system and the antirestriction proteins. The aim of this work was twofold; (i) compare the interaction of MTase with DNA and Ocr and (ii) quantify the strength of interaction between MTase and various ArdA proteins. The MTase was fused to the Green Fluorescent Protein (GFP) to facilitate determination of the orientation of interaction with DNA and Ocr. Time resolved fluorescence measurements were carried out using the GFP-MTase fusion to determine the fluorescence lifetime and anisotropy decay. These experiments were conducted using a time resolved fluorescence instrument fabricated in-house. The values determined in these experiments were then used to perform fluorescence resonance energy transfer (FRET) measurements with fluorescently labelled DNA or Ocr. These measurements gave information concerning the relative orientation of the MTase with either DNA or Ocr. The GFP-MTase fusion was also used to quantify the strength of interaction with various ArdA proteins. Previous attempts to determine the strength of interaction between MTase and ArdA proteins by employing conventional techniques have been unsuccessful. Therefore, a novel method was developed that exploits the interaction of MTase with a cation exchange medium, which can subsequently be displaced upon binding to ArdA. This method facilitated the determination, for the first time, of a set of binding affinities for the MTase and ArdA interaction.
Subjects/Keywords: 572.8; GFP; green fluorescent protein; protein-protein; DNA restriction/modification; Time-resolved fluorescence
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❌
APA ·
Chicago ·
MLA ·
Vancouver ·
CSE |
Export
to Zotero / EndNote / Reference
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APA (6th Edition):
Chen, K. (2011). Use of green fluorescent protein for the analysis of protein-protein and protein-DNA interactions. (Doctoral Dissertation). University of Edinburgh. Retrieved from http://hdl.handle.net/1842/4886
Chicago Manual of Style (16th Edition):
Chen, Kai. “Use of green fluorescent protein for the analysis of protein-protein and protein-DNA interactions.” 2011. Doctoral Dissertation, University of Edinburgh. Accessed April 11, 2021.
http://hdl.handle.net/1842/4886.
MLA Handbook (7th Edition):
Chen, Kai. “Use of green fluorescent protein for the analysis of protein-protein and protein-DNA interactions.” 2011. Web. 11 Apr 2021.
Vancouver:
Chen K. Use of green fluorescent protein for the analysis of protein-protein and protein-DNA interactions. [Internet] [Doctoral dissertation]. University of Edinburgh; 2011. [cited 2021 Apr 11].
Available from: http://hdl.handle.net/1842/4886.
Council of Science Editors:
Chen K. Use of green fluorescent protein for the analysis of protein-protein and protein-DNA interactions. [Doctoral Dissertation]. University of Edinburgh; 2011. Available from: http://hdl.handle.net/1842/4886

Georgia Tech
23.
Fellows, William Brett.
Combinatorial synthesis of new GFP- and RFP-like chromophores and their photophysical properties.
Degree: PhD, Chemistry and Biochemistry, 2014, Georgia Tech
URL: http://hdl.handle.net/1853/52318
► A new synthetic methodology for the combinatorial preparation of C-terminus-modified Green and Red Fluorescent Protein chromophores is described. This method involves the modification of the…
(more)
▼ A new synthetic methodology for the combinatorial preparation of C-terminus-modified Green and Red
Fluorescent Protein chromophores is described. This method involves the modification of the previously reported [2+3] cycloaddition reaction scheme to incorporate new R2 groups in the imidate used in the final step. This is achieved through two primary routes: (a) the imidation of nitriles using hydrochloric acid gas and (b) the O-alkylation of amides using a variant of Meerwein's Salt to provide conjugated imidates.
The preparation of
fluorescent microcrystals and nanofibers from Green
Fluorescent Protein chromophore derivatives via the reprecipitation method is also demonstrated. The properties of these microcrystals and nanofibers, especially in relation to the powder obtained from organic solvents, are also explored. Additionally, it is demonstrated that the size and shape of the microcrystals and nanofibers can be modulated with varying experimental conditions for RP.
A new class of AIE-active GFP chromophores is reported. These chromophores contain a benzoxazole group on the phenyl ring and varying lengths of alkyl chains on the imidazolidinone nitrogen. These benzoxazole-based chromophores exhibit unique properties in the solid state not previously observed for GFP chromophore derivatives, namely, a broadening of the excitation spectrum and red-shifting of the emission, likely caused by excimer formation. The crystal structure also reveals a unique "hot-dog" stacking motif.
Additionally, some projects which require further work are discussed at the end of the thesis. These include a stress-responsive GFP-based polymer and DNA-binding fluorophores.
Advisors/Committee Members: Tolbert, Laren M. (advisor), Kelly, Wendy (committee member), Kubanek, Julia (committee member), Liotta, Charles (committee member), Collard, David M. (committee member), Solntsev, Kyril M. (committee member).
Subjects/Keywords: Green fluorescent protein; Chromophore; Synthesis; Combinatorial; Red fluorescent protein; Hot dog stacking; Solid state; Crystal structure; Aggregate induced emission; Reprecipitation
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❌
APA ·
Chicago ·
MLA ·
Vancouver ·
CSE |
Export
to Zotero / EndNote / Reference
Manager
APA (6th Edition):
Fellows, W. B. (2014). Combinatorial synthesis of new GFP- and RFP-like chromophores and their photophysical properties. (Doctoral Dissertation). Georgia Tech. Retrieved from http://hdl.handle.net/1853/52318
Chicago Manual of Style (16th Edition):
Fellows, William Brett. “Combinatorial synthesis of new GFP- and RFP-like chromophores and their photophysical properties.” 2014. Doctoral Dissertation, Georgia Tech. Accessed April 11, 2021.
http://hdl.handle.net/1853/52318.
MLA Handbook (7th Edition):
Fellows, William Brett. “Combinatorial synthesis of new GFP- and RFP-like chromophores and their photophysical properties.” 2014. Web. 11 Apr 2021.
Vancouver:
Fellows WB. Combinatorial synthesis of new GFP- and RFP-like chromophores and their photophysical properties. [Internet] [Doctoral dissertation]. Georgia Tech; 2014. [cited 2021 Apr 11].
Available from: http://hdl.handle.net/1853/52318.
Council of Science Editors:
Fellows WB. Combinatorial synthesis of new GFP- and RFP-like chromophores and their photophysical properties. [Doctoral Dissertation]. Georgia Tech; 2014. Available from: http://hdl.handle.net/1853/52318

University of Tennessee – Knoxville
24.
Rice, John Hollis.
Bioconfinement of a putatively sterile Nicotiana hybrid and development of tools for assessing gene flow.
Degree: MS, Plant Sciences, 2013, University of Tennessee – Knoxville
URL: https://trace.tennessee.edu/utk_gradthes/2487
► Production of transgenic crops in open field environments is an ongoing concern of due to the potential for gene flow. New transgenic crops, such…
(more)
▼ Production of transgenic crops in open field environments is an ongoing concern of due to the potential for gene flow. New transgenic crops, such as plant-made-pharmaceuticals may generate additional concerns about effects of adventitious transgenes. Use of a bioconfinement strategy may alleviate any consequences by preventing gene flow. The following chapters discuss previous and current research on gene flow, testing of a
Nicotiana hybrid system for bioconfinement efficiency, and development of methods for transgene detection. The candidate ‘platform plant’ that was tested is a
Nicotiana hybrid (
Nicotiana tabacum ‘TN 90’ ×
Nicotiana glauca) previously identified to be sexually sterile. To quantify gene flow from hybrids, the
mGFP5ER gene encoding for green
fluorescent protein (GFP) was inserted into the paternal lines, which were crossed to form the hybrid. The DNA content and male fertility of these lines were used to characterize GFP-tagged hybrid lines. There were no differences in DNA content but significant differences in male fertility, in which pollen germination was observed at low rates. Two field gene flow studies revealed GFP-hybrids were not totally sterile since hybrids outcrossed and were pollinated by
N. tabacum pollen, but they produced few viable seed. These results were confirmed with manual greenhouse crosses
. Biomass studies revealed that the GFP-hybrids were comparable in productivity to an
N. tabacum cultivar typically used in field production. An additional tagging strategy was created to produce the orange
fluorescent protein (OFP), tdTomato-ER, in pollen by using pollen specific promoters in addition to the whole plant GFP cassettes.
N. tabacum ‘TN 90’ and
N. glauca were transformed, bred and hybridized to generate a hybrid that produced pollen tagged with orange
fluorescent protein. Manual crosses were performed in a greenhouse and partially similar results were obtained compared with previous GFP-tagged hybrid crosses. OFP-tagged hybrid outcrossing produced totally non-viable seed and when non-transgenic
N. tabacum was supplied as a pollen donor to the OFP-tagged hybrids some non-tagged progeny were observed. The results suggest the
Nicotiana hybrid could be a productive biomanufacturing platform and could provide total bioconfinement if grown in physical isolation from
N. tabacum and
N. glauca.
Advisors/Committee Members: C. Neal Stewart Jr., Charles Kwit, Randall L. Small.
Subjects/Keywords: bioconfinement; Nicotiana; green fluorescent protein; orange fluorescent protein; gene flow; plant molecular farming; Biotechnology; Molecular Biology; Plant Breeding and Genetics
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APA ·
Chicago ·
MLA ·
Vancouver ·
CSE |
Export
to Zotero / EndNote / Reference
Manager
APA (6th Edition):
Rice, J. H. (2013). Bioconfinement of a putatively sterile Nicotiana hybrid and development of tools for assessing gene flow. (Thesis). University of Tennessee – Knoxville. Retrieved from https://trace.tennessee.edu/utk_gradthes/2487
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Chicago Manual of Style (16th Edition):
Rice, John Hollis. “Bioconfinement of a putatively sterile Nicotiana hybrid and development of tools for assessing gene flow.” 2013. Thesis, University of Tennessee – Knoxville. Accessed April 11, 2021.
https://trace.tennessee.edu/utk_gradthes/2487.
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
MLA Handbook (7th Edition):
Rice, John Hollis. “Bioconfinement of a putatively sterile Nicotiana hybrid and development of tools for assessing gene flow.” 2013. Web. 11 Apr 2021.
Vancouver:
Rice JH. Bioconfinement of a putatively sterile Nicotiana hybrid and development of tools for assessing gene flow. [Internet] [Thesis]. University of Tennessee – Knoxville; 2013. [cited 2021 Apr 11].
Available from: https://trace.tennessee.edu/utk_gradthes/2487.
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Council of Science Editors:
Rice JH. Bioconfinement of a putatively sterile Nicotiana hybrid and development of tools for assessing gene flow. [Thesis]. University of Tennessee – Knoxville; 2013. Available from: https://trace.tennessee.edu/utk_gradthes/2487
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

Humboldt State University
25.
Cramer, Philip B.
Using transgenic Caenorhabditis elegans as a bioindicator: a useful tool for examining potential environmental hazards, or just a bunch of glowing worms?.
Degree: MS, Biology, 2012, Humboldt State University
URL: http://hdl.handle.net/2148/1260
► A total of 17 transgenic strains of Caenorhabditis elegans were evaluated for use as bioindicators for the presence of toxins in an environmental sample. Every…
(more)
▼ A total of 17 transgenic strains of Caenorhabditis elegans were evaluated for use as bioindicators for the presence of toxins in an environmental sample. Every strain used had Green
Fluorescent Protein (GFP) production driven by the promoter region of a gene known to be involved in stress response, metabolism, or development. Controlled exposures were performed on each strain using 22 environmental toxins and contaminants. Changes in GFP intensity between exposed groups and controls were considered proof of changes in that genes expression pattern as a direct result of exposure to the toxin. The goal was to construct expression profiles for each strain using the chosen toxins. The strains could then be used to evaluate environmental samples for the presence of unknown toxins with the expression profiles serving as a preliminary indicator of the amount and type of toxin present. The metallothionein-linked strain CL 2122 showed significant upregulation in the Mtl-2 gene following exposure to 10, 50, and 100 ??M cadmium, copper, and lead. This strain also exhibited significant downregulation of metallothionein expression following exposure to diazinon, hexazinone, iron (II) sulfate, paraquat, and piperonyl butoxide, which exemplifies how even a metals-specific stress response can be altered by certain toxins to yield difficult to interpret results. Initially it was thought that a panel of 6-8 strains could be formed that showed clear expression profiles to common environmental pollutants and used to examine samples that had unknown toxins. It was envisioned that the panel would have at least 2 strains that are metals-responsive, two strains that are general organic toxin-responsive, and two strains that are endocrine disruptor and/or estrogen sensitive. This proved to be unrealistic for the genes examined as no strain yielded results that were totally unambiguous. The results indicated that single-gene biomarker-based bioassays could only be used in limited situations, such as large single-toxin releases, or when identifying the exact toxin is not as important as assessing the overall safety of the sample in question.
Advisors/Committee Members: O'Gara, Bruce A..
Subjects/Keywords: Caenorhabditis; Elegans; Biomarker; Bioindicator; Transgenic; Green fluorescent protein; GFP
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❌
APA ·
Chicago ·
MLA ·
Vancouver ·
CSE |
Export
to Zotero / EndNote / Reference
Manager
APA (6th Edition):
Cramer, P. B. (2012). Using transgenic Caenorhabditis elegans as a bioindicator: a useful tool for examining potential environmental hazards, or just a bunch of glowing worms?. (Masters Thesis). Humboldt State University. Retrieved from http://hdl.handle.net/2148/1260
Chicago Manual of Style (16th Edition):
Cramer, Philip B. “Using transgenic Caenorhabditis elegans as a bioindicator: a useful tool for examining potential environmental hazards, or just a bunch of glowing worms?.” 2012. Masters Thesis, Humboldt State University. Accessed April 11, 2021.
http://hdl.handle.net/2148/1260.
MLA Handbook (7th Edition):
Cramer, Philip B. “Using transgenic Caenorhabditis elegans as a bioindicator: a useful tool for examining potential environmental hazards, or just a bunch of glowing worms?.” 2012. Web. 11 Apr 2021.
Vancouver:
Cramer PB. Using transgenic Caenorhabditis elegans as a bioindicator: a useful tool for examining potential environmental hazards, or just a bunch of glowing worms?. [Internet] [Masters thesis]. Humboldt State University; 2012. [cited 2021 Apr 11].
Available from: http://hdl.handle.net/2148/1260.
Council of Science Editors:
Cramer PB. Using transgenic Caenorhabditis elegans as a bioindicator: a useful tool for examining potential environmental hazards, or just a bunch of glowing worms?. [Masters Thesis]. Humboldt State University; 2012. Available from: http://hdl.handle.net/2148/1260

University of California – Riverside
26.
Fan, Yichong.
Expanding the Current Imaging Toolkit With Novel Fluorescent Protein Based Biosensors.
Degree: Environmental Toxicology, 2017, University of California – Riverside
URL: http://www.escholarship.org/uc/item/627146dj
► The objective of my Ph.D. study is to develop novel genetically encoded fluorescent biosensors to image and dissect biological signaling pathways in the context of…
(more)
▼ The objective of my Ph.D. study is to develop novel genetically encoded fluorescent biosensors to image and dissect biological signaling pathways in the context of live cells. I utilized protein engineering techniques to convert fluorescent proteins into fluorescent biosensors that can actively respond to specific, spatiotemporally organized cellular changes.In this thesis, we expanded the fluorescent protein toolkit by engineering one of the first red fluorescent probes⎯rxRFP1⎯for sensing general redox states in the live cells. To further extend the usage of this sensor in various subcellular domains, such as mitochondria, endoplasmic reticulum, and the cell nucleus, we developed a group of rxRFP1 mutants showing different midpoint redox potentials for studying compartmentalized redox dynamics under various pathophysiological conditions. We also developed the first genetically encoded fluorescent biosensor for thioredoxin (Trx) redox by engineering a redox relay between the active-site cysteines of human Trx1 and rxRFP1. We utilized the resultant biosensor⎯TrxRFP1⎯to selectively monitor the perturbations of Trx redox in various mammalian cell lines. We further combined TrxRFP1 with a green fluorescent Grx1-roGFP2 biosensor to simultaneously monitor the dynamics of the two major cellular antioxidant systems, Trx and glutathione, in live cells in response to chemically and physiologically relevant stimuli. We exploit another strategy which introduces reactive functional groups into circular permutated fluorescent proteins (cpFPs) using a genetic code expansion technology. Through a powerful directed protein evolution process, we were able to modulate the reactivity and chemoselectivity of an introduced p-boronophenylalanine (pBoF) in a cpRFP scaffold, resulting in fluorescent probes selectively responsive to hydrogen peroxide (H2O2) and peroxynitrite (ONOO—). Furthermore, by using boronic acid and short peptides as synergistic recognition motifs, we were able to engineer a series of reversible probes for nucleotides and carbohydrates showing surprisingly high specificity and large dynamic ranges. We have successfully utilized this new family of fluorescent probes to visualize various cellular activities.
Subjects/Keywords: Toxicology; biological processes; biosensor; fluorescent protein; imaging; redox signaling
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❌
APA ·
Chicago ·
MLA ·
Vancouver ·
CSE |
Export
to Zotero / EndNote / Reference
Manager
APA (6th Edition):
Fan, Y. (2017). Expanding the Current Imaging Toolkit With Novel Fluorescent Protein Based Biosensors. (Thesis). University of California – Riverside. Retrieved from http://www.escholarship.org/uc/item/627146dj
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Chicago Manual of Style (16th Edition):
Fan, Yichong. “Expanding the Current Imaging Toolkit With Novel Fluorescent Protein Based Biosensors.” 2017. Thesis, University of California – Riverside. Accessed April 11, 2021.
http://www.escholarship.org/uc/item/627146dj.
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
MLA Handbook (7th Edition):
Fan, Yichong. “Expanding the Current Imaging Toolkit With Novel Fluorescent Protein Based Biosensors.” 2017. Web. 11 Apr 2021.
Vancouver:
Fan Y. Expanding the Current Imaging Toolkit With Novel Fluorescent Protein Based Biosensors. [Internet] [Thesis]. University of California – Riverside; 2017. [cited 2021 Apr 11].
Available from: http://www.escholarship.org/uc/item/627146dj.
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Council of Science Editors:
Fan Y. Expanding the Current Imaging Toolkit With Novel Fluorescent Protein Based Biosensors. [Thesis]. University of California – Riverside; 2017. Available from: http://www.escholarship.org/uc/item/627146dj
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

University of Alberta
27.
Wu, Jiahui.
Development of red fluorescent protein-based calcium ion and
glutamate indicators.
Degree: PhD, Department of Chemistry, 2014, University of Alberta
URL: https://era.library.ualberta.ca/files/c9s1616409
► The discovery and subsequent applications of fluorescent proteins (FPs) launched a new era for live cell fluorescence imaging. The design and developments of FP-based indicators…
(more)
▼ The discovery and subsequent applications of
fluorescent proteins (FPs) launched a new era for live cell
fluorescence imaging. The design and developments of FP-based
indicators have further solidified the versatility of FPs and
rendered them as indispensable tools in life science research now
more than ever. Despite the tremendous developments and efforts
invested in the field of FP-based indicators, there remain numerous
opportunities in engineering indicators with improved or novel
properties for studying biological processes in vivo. In this
thesis we describe our efforts in developing a series of FP-based
calcium ion (Ca2+) and glutamate indicators with various colors and
useful spectral properties as versatile tools for interrogating
cell signaling in cell biology. In this thesis, we first describe
our efforts in employing protein engineering to expand the color
palette of genetically encoded Ca2+ indicators to include
intensiometric orange, improved red and ratiometric red fluorescent
variants. We demonstrate these new indicators' utility by
performing Ca2+ imaging in cultured human cell lines, slice culture
of developing mouse neocortex, organotypic hippocampal slice
cultures and the visual system of albino tadpoles. Using our
intensiometric red Ca2+ indicators, R-GECO1 and R-GECO1.2, as
templates, we further engineered a series of low affinity R-GECOs
with dissociation constants (Kds) ranging from 12 µM to more than
540 µM. We demonstrate that these indicators can be used to image
cell compartments with high Ca2+ concentration or with a broad
range of Ca2+ change, such as the endoplasmic reticulum (ER) and
mitochondria. We also demonstrate these new red Ca2+ indicators
with low affinities can be used to monitor ER and mitochondrial
Ca2+ in combination with a green fluorescent protein (GFP)-based
reporter. We also report in this thesis the development,
optimization and characterization of the first red fluorescent
protein (RFP)-based glutamate indicator, GltR1. We demonstrate
GltR1 can detect glutamate changes on the surface of cultured human
cells, as well as the glutamate dynamics during spontaneous
activities of dissociated rat hippocampal neurons.
Subjects/Keywords: fluorescent protein; GECO; biosensor; glutamate indicator; fluorescence Ca2+ imaging
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❌
APA ·
Chicago ·
MLA ·
Vancouver ·
CSE |
Export
to Zotero / EndNote / Reference
Manager
APA (6th Edition):
Wu, J. (2014). Development of red fluorescent protein-based calcium ion and
glutamate indicators. (Doctoral Dissertation). University of Alberta. Retrieved from https://era.library.ualberta.ca/files/c9s1616409
Chicago Manual of Style (16th Edition):
Wu, Jiahui. “Development of red fluorescent protein-based calcium ion and
glutamate indicators.” 2014. Doctoral Dissertation, University of Alberta. Accessed April 11, 2021.
https://era.library.ualberta.ca/files/c9s1616409.
MLA Handbook (7th Edition):
Wu, Jiahui. “Development of red fluorescent protein-based calcium ion and
glutamate indicators.” 2014. Web. 11 Apr 2021.
Vancouver:
Wu J. Development of red fluorescent protein-based calcium ion and
glutamate indicators. [Internet] [Doctoral dissertation]. University of Alberta; 2014. [cited 2021 Apr 11].
Available from: https://era.library.ualberta.ca/files/c9s1616409.
Council of Science Editors:
Wu J. Development of red fluorescent protein-based calcium ion and
glutamate indicators. [Doctoral Dissertation]. University of Alberta; 2014. Available from: https://era.library.ualberta.ca/files/c9s1616409

University of Georgia
28.
Majsztrik, John Christopher.
Subnuclear localization and interaction of selected Aux/IAA and ARF proteins in vivo.
Degree: 2014, University of Georgia
URL: http://hdl.handle.net/10724/21898
► The plant hormone auxin has been studied for many years and is instrumental for plant growth and development. Auxin regulates gene transcription of at least…
(more)
▼ The plant hormone auxin has been studied for many years and is instrumental for plant growth and development. Auxin regulates gene transcription of at least five families including the Aux/IAA family. Aux/IAA and ARF proteins are known to
interact through conserved domains in both the Aux/IAA and ARF families. This research is the first to report individual nuclear and sub-nuclear localization patterns of the Arabidopsis ARF and Aux/IAA protein in vivo, focusing on ARF1 and IAA17. Several
co-localizations of proteins from the same and different families exhibited protein recruitment to structures and locations individual proteins did not localize to when expressed alone. This work strongly supports the putative interactions between ARF
and Aux/IAA proteins in vivo. Northern Blot and semi-quantitative RT PCR showed that ARF1 and IAA17 were co-expressed in tissues sampled. The role of putative nuclear localization signals was investigated by localization of deletion constructs of IAA17
and ARF1.
Subjects/Keywords: Auxin Response Factor (ARF); Aux/IAA; Fluorescent protein; Nuclear localization
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APA ·
Chicago ·
MLA ·
Vancouver ·
CSE |
Export
to Zotero / EndNote / Reference
Manager
APA (6th Edition):
Majsztrik, J. C. (2014). Subnuclear localization and interaction of selected Aux/IAA and ARF proteins in vivo. (Thesis). University of Georgia. Retrieved from http://hdl.handle.net/10724/21898
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Chicago Manual of Style (16th Edition):
Majsztrik, John Christopher. “Subnuclear localization and interaction of selected Aux/IAA and ARF proteins in vivo.” 2014. Thesis, University of Georgia. Accessed April 11, 2021.
http://hdl.handle.net/10724/21898.
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
MLA Handbook (7th Edition):
Majsztrik, John Christopher. “Subnuclear localization and interaction of selected Aux/IAA and ARF proteins in vivo.” 2014. Web. 11 Apr 2021.
Vancouver:
Majsztrik JC. Subnuclear localization and interaction of selected Aux/IAA and ARF proteins in vivo. [Internet] [Thesis]. University of Georgia; 2014. [cited 2021 Apr 11].
Available from: http://hdl.handle.net/10724/21898.
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Council of Science Editors:
Majsztrik JC. Subnuclear localization and interaction of selected Aux/IAA and ARF proteins in vivo. [Thesis]. University of Georgia; 2014. Available from: http://hdl.handle.net/10724/21898
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

McMaster University
29.
Kuryllo, Kacper.
Detecting RNA Regulatory Interactions in Bacterial Cells.
Degree: PhD, 2014, McMaster University
URL: http://hdl.handle.net/11375/16296
► Non-coding RNAs are involved in the regulation of most major cellular process in Escherichia coli. With current technologies, many of these molecules have been identified;…
(more)
▼ Non-coding RNAs are involved in the regulation of most major cellular process in Escherichia coli. With current technologies, many of these molecules have been identified; however, the full scope of their regulatory interactions is still unknown. None of the techniques currently in use employ the regulatory effect of the RNAs, which is the major unifying attribute of these molecules, for their identification. This thesis presents projects involving the design of a dual-reporter plasmid and screening method in the discovery and characterization of RNA regulatory interactions
The first project details the engineering of the dual reporter plasmid. This vector utilizes one fluorescent protein to detect regulatory events and a second to normalize for off-target effects. The second project utilizes this tool in the discovery and characterization of novel regulatory responses. This is accomplished by screening a library of intergenic regions for regulatory responses against a collection of metabolite. Interesting interactions involving nitrogen abundance, iron and uracil are identified and further examined.
Finally, this thesis examines how this technology can be further expanded for the study of RNA regulatory functions. The use of the screening method for the detection of regulatory events caused by alternative minimal media composition and the potential for the dual reporter plasmid to aid in the study of riboswitches are investigated.
Thesis
Doctor of Science (PhD)
Advisors/Committee Members: Li, Yingfu, Brown, Eric, Elliot, Marie, Biochemistry and Biomedical Sciences.
Subjects/Keywords: RNA; gene regulation; sRNA; riboswitch; biosensor; fluorescent protein; GFP; Escherichia coli
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❌
APA ·
Chicago ·
MLA ·
Vancouver ·
CSE |
Export
to Zotero / EndNote / Reference
Manager
APA (6th Edition):
Kuryllo, K. (2014). Detecting RNA Regulatory Interactions in Bacterial Cells. (Doctoral Dissertation). McMaster University. Retrieved from http://hdl.handle.net/11375/16296
Chicago Manual of Style (16th Edition):
Kuryllo, Kacper. “Detecting RNA Regulatory Interactions in Bacterial Cells.” 2014. Doctoral Dissertation, McMaster University. Accessed April 11, 2021.
http://hdl.handle.net/11375/16296.
MLA Handbook (7th Edition):
Kuryllo, Kacper. “Detecting RNA Regulatory Interactions in Bacterial Cells.” 2014. Web. 11 Apr 2021.
Vancouver:
Kuryllo K. Detecting RNA Regulatory Interactions in Bacterial Cells. [Internet] [Doctoral dissertation]. McMaster University; 2014. [cited 2021 Apr 11].
Available from: http://hdl.handle.net/11375/16296.
Council of Science Editors:
Kuryllo K. Detecting RNA Regulatory Interactions in Bacterial Cells. [Doctoral Dissertation]. McMaster University; 2014. Available from: http://hdl.handle.net/11375/16296
30.
Drufva, Erin.
DEVELOPMENT OF PROTEIN DISPLAY SYSTEMS AND GENETIC TOOLS FOR SPORE-FORMING BACTERIA.
Degree: PhD, 2018, University of New Hampshire
URL: https://scholars.unh.edu/dissertation/2398
► One major area of synthetic biology is to engineer microbial cells and subcellular systems for diverse applications including biosynthesis, biocatalysis, therapeutics, drug delivery, and…
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▼ One major area of synthetic biology is to engineer microbial cells and subcellular systems for diverse applications including biosynthesis, biocatalysis, therapeutics, drug delivery, and bioremediation. For most applications, robust cellular systems are preferred for longer activity half-life and resistance to harsh environments. Two projects related to robust cellular systems involving Gram-positive bacteria are presented in this work. One is to develop thermostable genetic reporters for Geobacilli species and the other is to display an enzyme on the Bacillus subtilis spore surface to enhance its robustness and present an alternative to purified enzymes for industrial applications.
Bacillus subtilis and Geobacillus thermoglucosidans are gram-positive, spore-forming bacteria. They secrete many proteins used industrially for the production of paper, food, textiles, chemicals, medicine, and cosmetics. Since G. thermoglucosidans is thermostable with an optimal growth temperature of 60ºC, its secreted proteins are also thermostable which proves advantageous for a variety of industrial applications. Additionally, a strain of G. thermoglucosidans has been used for the production of ethanol from biomass. Unfortunately the inner workings of G. thermoglucosidans are still poorly understood and a genetic toolkit is necessary to better discover how to improve them via genetic engineering for industrial use. Important components of this toolkit are genetic reporters which allow for the analysis of gene expression in G. thermoglucosidans.
Fluorescent proteins are commonly used reporters for other bacterial species due to their easily observed and readily measured signal, however no thermostable
fluorescent proteins have been shown to be functional in Geobacillus. Seven different
fluorescent proteins including mCherry, Venus, GFP, sfGFP, GFPmut3, mCherry (Gt), and Venus (Gt) were tested for stability and functionality in Geobacillus thermoglucosidans. Venus (Gt) and mCherry (Gt) were codon optimized for this bacterium with the goal of increasing expression level and thus improving the fluorescence signal. The fluorescence intensity of each
fluorescent protein expressed in G. thermoglucosidans was measured after several hours of bacterial growth at 50ºC and 60ºC. Venus, mCherry, Venus (Gt), mCherry (Gt), and sfGFP all had signal when expressed in G. thermoglucosidans at 50ºC and sfGFP had signal at 60ºC. Therefore,
fluorescent reporter proteins in three different colors were found to be functional in G. thermoglucosidans. This will further genetic engineering of the species for thermostable
protein production, bioremediation, and biofuel production.
Bacillus subtilis is Generally Regarded as Safe (GRAS) by the FDA and amenable toward genetic manipulation. Thus it has been engineered for the production of many heterologous proteins. Oftentimes, proteins secreted by bacteria are purified for industrial use. However,
protein purification is expensive and time-consuming and long-term storage of purified proteins…
Advisors/Committee Members: Kang Wu, Kang Wu, Russell Carr.
Subjects/Keywords: Bacillus; fluorescent protein; Geobacillus; immobilize; laccase; spore; Microbiology; Biology; Chemical engineering
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APA (6th Edition):
Drufva, E. (2018). DEVELOPMENT OF PROTEIN DISPLAY SYSTEMS AND GENETIC TOOLS FOR SPORE-FORMING BACTERIA. (Doctoral Dissertation). University of New Hampshire. Retrieved from https://scholars.unh.edu/dissertation/2398
Chicago Manual of Style (16th Edition):
Drufva, Erin. “DEVELOPMENT OF PROTEIN DISPLAY SYSTEMS AND GENETIC TOOLS FOR SPORE-FORMING BACTERIA.” 2018. Doctoral Dissertation, University of New Hampshire. Accessed April 11, 2021.
https://scholars.unh.edu/dissertation/2398.
MLA Handbook (7th Edition):
Drufva, Erin. “DEVELOPMENT OF PROTEIN DISPLAY SYSTEMS AND GENETIC TOOLS FOR SPORE-FORMING BACTERIA.” 2018. Web. 11 Apr 2021.
Vancouver:
Drufva E. DEVELOPMENT OF PROTEIN DISPLAY SYSTEMS AND GENETIC TOOLS FOR SPORE-FORMING BACTERIA. [Internet] [Doctoral dissertation]. University of New Hampshire; 2018. [cited 2021 Apr 11].
Available from: https://scholars.unh.edu/dissertation/2398.
Council of Science Editors:
Drufva E. DEVELOPMENT OF PROTEIN DISPLAY SYSTEMS AND GENETIC TOOLS FOR SPORE-FORMING BACTERIA. [Doctoral Dissertation]. University of New Hampshire; 2018. Available from: https://scholars.unh.edu/dissertation/2398
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