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Dalhousie University
1.
Charette, Nicholle Jeanine.
INVOLVEMENT OF DIFFERENT RAB GTPASES IN THE TRAFFICKING OF
CXCR4 AND CCR5 HOMO- AND HETERODIMERS BETWEEN THE ENDOPLASMIC
RETICULUM AND PLASMA MEMBRANE IN HEK293 AND JURKAT CELLS.
Degree: MS, Department of Pharmacology, 2011, Dalhousie University
URL: http://hdl.handle.net/10222/14023 
► Little is known about the outward trafficking of receptor dimers from the endoplasmic reticulum to the plasma membrane, or the role that trafficking plays in…
(more)
▼ Little is known about the outward trafficking of
receptor dimers from the endoplasmic reticulum to the plasma
membrane, or the role that trafficking plays in assembly, targeting
and specificity of receptor signalling. Bimolecular fluorescence
complementation was used to follow prescribed receptor
homo/heterodimers in Jurkat cells and clarify the trafficking
itineraries those receptors follow to reach the plasma membrane.
Chemokine receptors CXCR4 and CCR5 were chosen due to their
implication in numerous pathologies including, HIV and cancer, and
their ability to form homo and hetero-oligomers. This study
demonstrates that although the individual receptors composing
heterodimeric complexes are the same as in homodimeric complexes,
the heterodimer traffics and signals independently of its
constituent homodimers. The presence of CD4 affects the trafficking
of CCR5 containing dimers but not the CXCR4 homodimer. These
observations demonstrate the importance of considering receptor
heterodimers as distinct signalling entities that should be more
carefully and individually characterized.
Advisors/Committee Members: Dr. Neale Ridgway (external-examiner), Dr. Eileen Denovan-Wright (graduate-coordinator), Dr. Christopher Sinal (thesis-reader), Dr. Denis Dupré (thesis-supervisor), Not Applicable (ethics-approval), Not Applicable (manuscripts), Yes (copyright-release).
Subjects/Keywords: G protein coupled receptor; CXCR4; CCR5; Rab GTPase; Trafficking; Dimerization
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APA (6th Edition):
Charette, N. J. (2011). INVOLVEMENT OF DIFFERENT RAB GTPASES IN THE TRAFFICKING OF
CXCR4 AND CCR5 HOMO- AND HETERODIMERS BETWEEN THE ENDOPLASMIC
RETICULUM AND PLASMA MEMBRANE IN HEK293 AND JURKAT CELLS. (Masters Thesis). Dalhousie University. Retrieved from http://hdl.handle.net/10222/14023
Chicago Manual of Style (16th Edition):
Charette, Nicholle Jeanine. “INVOLVEMENT OF DIFFERENT RAB GTPASES IN THE TRAFFICKING OF
CXCR4 AND CCR5 HOMO- AND HETERODIMERS BETWEEN THE ENDOPLASMIC
RETICULUM AND PLASMA MEMBRANE IN HEK293 AND JURKAT CELLS.” 2011. Masters Thesis, Dalhousie University. Accessed March 05, 2021.
http://hdl.handle.net/10222/14023.
MLA Handbook (7th Edition):
Charette, Nicholle Jeanine. “INVOLVEMENT OF DIFFERENT RAB GTPASES IN THE TRAFFICKING OF
CXCR4 AND CCR5 HOMO- AND HETERODIMERS BETWEEN THE ENDOPLASMIC
RETICULUM AND PLASMA MEMBRANE IN HEK293 AND JURKAT CELLS.” 2011. Web. 05 Mar 2021.
Vancouver:
Charette NJ. INVOLVEMENT OF DIFFERENT RAB GTPASES IN THE TRAFFICKING OF
CXCR4 AND CCR5 HOMO- AND HETERODIMERS BETWEEN THE ENDOPLASMIC
RETICULUM AND PLASMA MEMBRANE IN HEK293 AND JURKAT CELLS. [Internet] [Masters thesis]. Dalhousie University; 2011. [cited 2021 Mar 05].
Available from: http://hdl.handle.net/10222/14023.
Council of Science Editors:
Charette NJ. INVOLVEMENT OF DIFFERENT RAB GTPASES IN THE TRAFFICKING OF
CXCR4 AND CCR5 HOMO- AND HETERODIMERS BETWEEN THE ENDOPLASMIC
RETICULUM AND PLASMA MEMBRANE IN HEK293 AND JURKAT CELLS. [Masters Thesis]. Dalhousie University; 2011. Available from: http://hdl.handle.net/10222/14023
Dalhousie University
2.
Crawford, Michelle Audrey.
Investigating Tom1 as a Candidate Regulator of Ptch1.
Degree: MS, Department of Anatomy & Neurobiology, 2012, Dalhousie University
URL: http://hdl.handle.net/10222/15842
► Sonic hedgehog (Shh) is a signaling molecule that is involved in patterning the embryo and regulates adult stem cell homeostasis. Patched1 (Ptch1) is the receptor…
(more)
▼ Sonic hedgehog (Shh) is a signaling molecule that is
involved in patterning the embryo and regulates adult stem cell
homeostasis. Patched1 (Ptch1) is the receptor for Shh and upon
binding to Shh is endocytosed, allowing downstream signaling to
occur. Ptch1 is critical to the cellular response to Shh because it
is both a negative regulator of the Shh signaling pathway and a
transcriptional target of the pathway. Therefore, the regulation of
Ptch1 levels will directly affect the ability of cells to respond
to Shh. Understanding this process requires the characterization of
novel Ptch1-interacting proteins that regulate Ptch1 levels in the
cell. This thesis investigated a role for the adapter protein Tom1
as a putative Ptch1-interacting protein involved in regulating
Ptch1 levels through endocytic cycling. It was found that Tom1
overexpression did not regulate the patterning of vertebrate
nervous system, but did play a role the sub-cellular localization
of Ptch1.
Advisors/Committee Members: Dr. Neale Ridgway (external-examiner), Dr. Kauze Semba (graduate-coordinator), Dr. James Fawcett (thesis-reader), Dr. Victor Rafuse (thesis-reader), Dr. Angelo Iulianella (thesis-supervisor), Not Applicable (ethics-approval), Not Applicable (manuscripts), Not Applicable (copyright-release).
Subjects/Keywords: Developmental biology; Neuroscience; Sonic hedgehog; Patched1; Tom1
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APA (6th Edition):
Crawford, M. A. (2012). Investigating Tom1 as a Candidate Regulator of Ptch1. (Masters Thesis). Dalhousie University. Retrieved from http://hdl.handle.net/10222/15842
Chicago Manual of Style (16th Edition):
Crawford, Michelle Audrey. “Investigating Tom1 as a Candidate Regulator of Ptch1.” 2012. Masters Thesis, Dalhousie University. Accessed March 05, 2021.
http://hdl.handle.net/10222/15842.
MLA Handbook (7th Edition):
Crawford, Michelle Audrey. “Investigating Tom1 as a Candidate Regulator of Ptch1.” 2012. Web. 05 Mar 2021.
Vancouver:
Crawford MA. Investigating Tom1 as a Candidate Regulator of Ptch1. [Internet] [Masters thesis]. Dalhousie University; 2012. [cited 2021 Mar 05].
Available from: http://hdl.handle.net/10222/15842.
Council of Science Editors:
Crawford MA. Investigating Tom1 as a Candidate Regulator of Ptch1. [Masters Thesis]. Dalhousie University; 2012. Available from: http://hdl.handle.net/10222/15842
Dalhousie University
3.
Liu, Xinwei.
The study on oxysterol-binding protein (OSBP) and related
protein 9 (ORP9) ligands: sterols and phosphatidylinositol
4-phosphate.
Degree: MS, Department of Biochemistry & Molecular
Biology, 2014, Dalhousie University
URL: http://hdl.handle.net/10222/54063
► The oxysterol-binding protein OSBP-gene family is composed of 12 members with a common C-terminal sterol-binding domain (SBD). OSBP and ORP9 are members of the family…
(more)
▼ The oxysterol-binding protein OSBP-gene family is
composed of 12 members with a common C-terminal sterol-binding
domain (SBD). OSBP and ORP9 are members of the family that are
localized to the endoplasmic reticulum (ER) and Golgi apparatus by
their FFAT (two phenylalanines in an acidic tract) motif and PH
(pleckstrin homology) domain, respectively. These two proteins are
implicated in sterol transfer between these organelles. Here we
utilized Frster resonance energy transfer (FRET) between
cholestatrienol (CTL), a fluorescent cholesterol analog, and
dansyl-PE (DNS-PE) to demonstrate that both full-length ORP9L and
truncated ORP9S rapidly extract sterols from liposomes. In vitro,
OSBP, ORP9L and ORP9S specifically extracted phosphatidylinositol
4-phosphate (PI(4)P) from liposomes, suggesting PI(4)P is also a
ligand for these proteins. Overexpression of ORP9L or ORP9S
attenuated PI(4)P immunofluorescence detection in CHO cells. These
results indicate that OSBP and ORP9 may transport sterol and PI(4)P
in cells, possibly in opposite directions.
Advisors/Committee Members: N/A (external-examiner), Dr. John Archibald (graduate-coordinator), Dr. Roy Duncan, Dr. Roger McLeod, Dr. Catherine Too (thesis-reader), Dr. Neale Ridgway (thesis-supervisor), Not Applicable (ethics-approval), Not Applicable (manuscripts), Not Applicable (copyright-release).
Subjects/Keywords: Cholesterol; cholestatrienol; phosphatidylinositol
4-phosphate; oxysterol-binding protein (OSBP); oxysterol-binding
protein-related protein 9 (ORP9); sterol transport; Frster
resonance energy transfer (FRET)
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APA (6th Edition):
Liu, X. (2014). The study on oxysterol-binding protein (OSBP) and related
protein 9 (ORP9) ligands: sterols and phosphatidylinositol
4-phosphate. (Masters Thesis). Dalhousie University. Retrieved from http://hdl.handle.net/10222/54063
Chicago Manual of Style (16th Edition):
Liu, Xinwei. “The study on oxysterol-binding protein (OSBP) and related
protein 9 (ORP9) ligands: sterols and phosphatidylinositol
4-phosphate.” 2014. Masters Thesis, Dalhousie University. Accessed March 05, 2021.
http://hdl.handle.net/10222/54063.
MLA Handbook (7th Edition):
Liu, Xinwei. “The study on oxysterol-binding protein (OSBP) and related
protein 9 (ORP9) ligands: sterols and phosphatidylinositol
4-phosphate.” 2014. Web. 05 Mar 2021.
Vancouver:
Liu X. The study on oxysterol-binding protein (OSBP) and related
protein 9 (ORP9) ligands: sterols and phosphatidylinositol
4-phosphate. [Internet] [Masters thesis]. Dalhousie University; 2014. [cited 2021 Mar 05].
Available from: http://hdl.handle.net/10222/54063.
Council of Science Editors:
Liu X. The study on oxysterol-binding protein (OSBP) and related
protein 9 (ORP9) ligands: sterols and phosphatidylinositol
4-phosphate. [Masters Thesis]. Dalhousie University; 2014. Available from: http://hdl.handle.net/10222/54063
Dalhousie University
4.
Arsenault, Daniel.
The role of the CDP-choline pathway in the anoikis resitance
of Ras transformed intestinal epithelial cells.
Degree: MS, Department of Biochemistry & Molecular
Biology, 2012, Dalhousie University
URL: http://hdl.handle.net/10222/15710
► Phosphatidylcholine (PC) is an essential component of biological membranes and is synthesized by the CDP-choline pathway under the control of the rate-limiting enzyme CTP:phosphocholine cytidylyltransferase-alpha…
(more)
▼ Phosphatidylcholine (PC) is an essential component of
biological membranes and is synthesized by the CDP-choline pathway
under the control of the rate-limiting enzyme CTP:phosphocholine
cytidylyltransferase-alpha (CCT?). Ras transformed cells have
increased lipid synthesis; the aim of this study was to determine
if upregulation of CCT? was part of this transformed phenotype. Rat
intestinal epithelial cell lines (IEC) and three oncogenic H-ras
expressing IEC (IEC-Ras) were used to investigate the role of CCT?
and phosphatidylcholine (PC) synthesis in resistance to detachment
dependant apoptosis, termed anoikis. IEC-Ras have increased CCT?
expression within the nucleus. Reduction of CCT? expression with
lentiviral short hairpin RNA sensitized IEC-Ras to anoikis and
decreased PC degradation, but did not change PC synthesis. Thus, in
addition to CCT? being involved in anoikis-resistance in IEC-Ras
these data indicate the possibility that it may also have
nuclear-specific functions.
Advisors/Committee Members: N/A (external-examiner), Dr. John Archibald (graduate-coordinator), Dr. Graham Dellaire (thesis-reader), Dr. Paul Liu (thesis-reader), Dr. Catherine Too (thesis-reader), Dr. Neale Ridgway (thesis-supervisor), Received (ethics-approval), Not Applicable (manuscripts), Not Applicable (copyright-release).
Subjects/Keywords: H-ras; Anoikis; Phosphatidylcholine; Cancer
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APA (6th Edition):
Arsenault, D. (2012). The role of the CDP-choline pathway in the anoikis resitance
of Ras transformed intestinal epithelial cells. (Masters Thesis). Dalhousie University. Retrieved from http://hdl.handle.net/10222/15710
Chicago Manual of Style (16th Edition):
Arsenault, Daniel. “The role of the CDP-choline pathway in the anoikis resitance
of Ras transformed intestinal epithelial cells.” 2012. Masters Thesis, Dalhousie University. Accessed March 05, 2021.
http://hdl.handle.net/10222/15710.
MLA Handbook (7th Edition):
Arsenault, Daniel. “The role of the CDP-choline pathway in the anoikis resitance
of Ras transformed intestinal epithelial cells.” 2012. Web. 05 Mar 2021.
Vancouver:
Arsenault D. The role of the CDP-choline pathway in the anoikis resitance
of Ras transformed intestinal epithelial cells. [Internet] [Masters thesis]. Dalhousie University; 2012. [cited 2021 Mar 05].
Available from: http://hdl.handle.net/10222/15710.
Council of Science Editors:
Arsenault D. The role of the CDP-choline pathway in the anoikis resitance
of Ras transformed intestinal epithelial cells. [Masters Thesis]. Dalhousie University; 2012. Available from: http://hdl.handle.net/10222/15710
Dalhousie University
5.
Garant, Katy.
Oncolytic Reovirus Induces Intracellular Redistribution of
Ras to Promote Apoptosis and Progeny Virus Release.
Degree: PhD, Department of Microbiology &
Immunology, 2014, Dalhousie University
URL: http://hdl.handle.net/10222/54002
► Ras is a dynamic protein capable of interacting with plasma membrane microdomains and intracellular membranes. Its activity, as controlled by post-translational modifications, is essential in…
(more)
▼ Ras is a dynamic protein capable of interacting with
plasma membrane microdomains and intracellular membranes. Its
activity, as controlled by post-translational modifications, is
essential in regulating cell growth, differentiation, and death.
Consequently, mutations that result in constitutive Ras activity
lead to cell transformation, and are associated with over 30% of
all human cancers. Reovirus is a naturally oncolytic virus that
preferentially replicates in Ras-transformed cells and is currently
undergoing clinical trials as a cancer therapeutic. It was
previously demonstrated that Ras transformation promotes uncoating
of the parental virion during entry, production of infectious viral
progeny, and virus release through apoptosis; however, the
mechanism behind the latter is not well understood. This study set
out to determine whether reovirus alters the intracellular location
of oncogenic Ras to induce apoptosis of H-RasV12-transformed
fibroblasts. Here, I show that reovirus decreases palmitoylation
levels of H-RasV12 and causes accumulation of the oncogenic protein
in the Golgi body through Golgi fragmentation. With the Golgi body
being the site of Ras palmitoylation, treatment of target cells
with the palmitoylation inhibitor, 2-bromopalmitate (2BP), prompts
a greater accumulation of H-RasV12 in the Golgi body, as well as a
dose-dependent increase in reovirus release and spread. Use of 2BP
on cell lines expressing H, N, or K-Ras oncogenic isoforms causes
an increase in reovirus release that correlates with the
palmitoylation status of the respective Ras protein. Alternatively,
tethering H-RasV12 to the plasma membrane, and preventing its
movement back to the Golgi, allows for efficient virus production,
but results in basal levels of reovirus-induced cell death.
Furthermore, treatment of H-RasV12 cells with the FKBP12 inhibitor,
FK506, which is known to retain Ras at the plasma membrane, reduces
virus-related death and plaque size. Analysis of Ras downstream
signalling reveals that cells expressing cycling H-RasV12 have
elevated levels of phosphorylated JNK, and that Ras retained at the
Golgi body by 2BP increases activation of the MEKK1/MKK4/JNK
signalling pathway to promote cell death. Collectively, the data
suggests that reovirus induces Golgi fragmentation of target cells,
and the subsequent accumulation of oncogenic Ras in the Golgi body
initiates apoptotic signalling events required for virus
release.
Advisors/Committee Members: Dr. Samuel Rabkin (external-examiner), Dr. Brent Johnston (graduate-coordinator), Dr. Roy Duncan (thesis-reader), Dr. Chris Richardson (thesis-reader), Dr. Neale Ridgway (thesis-reader), Dr. Kirill Rosen (thesis-reader), Dr. Patrick Lee (thesis-supervisor), Not Applicable (ethics-approval), Not Applicable (manuscripts), Not Applicable (copyright-release).
Subjects/Keywords: Reovirus; Ras; Cancer Biology; Oncolytic Virus; Palmitoylation; Apoptosis
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APA (6th Edition):
Garant, K. (2014). Oncolytic Reovirus Induces Intracellular Redistribution of
Ras to Promote Apoptosis and Progeny Virus Release. (Doctoral Dissertation). Dalhousie University. Retrieved from http://hdl.handle.net/10222/54002
Chicago Manual of Style (16th Edition):
Garant, Katy. “Oncolytic Reovirus Induces Intracellular Redistribution of
Ras to Promote Apoptosis and Progeny Virus Release.” 2014. Doctoral Dissertation, Dalhousie University. Accessed March 05, 2021.
http://hdl.handle.net/10222/54002.
MLA Handbook (7th Edition):
Garant, Katy. “Oncolytic Reovirus Induces Intracellular Redistribution of
Ras to Promote Apoptosis and Progeny Virus Release.” 2014. Web. 05 Mar 2021.
Vancouver:
Garant K. Oncolytic Reovirus Induces Intracellular Redistribution of
Ras to Promote Apoptosis and Progeny Virus Release. [Internet] [Doctoral dissertation]. Dalhousie University; 2014. [cited 2021 Mar 05].
Available from: http://hdl.handle.net/10222/54002.
Council of Science Editors:
Garant K. Oncolytic Reovirus Induces Intracellular Redistribution of
Ras to Promote Apoptosis and Progeny Virus Release. [Doctoral Dissertation]. Dalhousie University; 2014. Available from: http://hdl.handle.net/10222/54002
6.
Reddy, Tyler.
Structure, Flexibility, And Overall Motion Of Transmembrane
Peptides Studied By NMR Spectroscopy And Molecular Dynamics
Simulations.
Degree: PhD, Department of Biochemistry & Molecular
Biology, 2012, Dalhousie University
URL: http://hdl.handle.net/10222/15719
► Nuclear magnetic resonance (NMR) spectroscopy was used to determine the structure of transmembrane (TM) segment IX of the Na+/H+ exchanger isoform 1 (NHE1) in dodecylphosphocholine…
(more)
▼ Nuclear magnetic resonance (NMR) spectroscopy was used
to determine the structure of transmembrane (TM) segment IX of the
Na+/H+ exchanger isoform 1 (NHE1) in dodecylphosphocholine
micelles. Studying isolated TM segments in this fashion constitutes
a well-established "divide and conquer" approach to the study of
membrane proteins, which are often extremely difficult to produce,
purify, and reconstitute in full-length polytopic form. A similar
approach was combined with NMR spin relaxation experiments to
determine the peptide backbone flexibility of NHE1 TM VII. The
combined NMR structural and dynamics studies are consistent with an
important role for TM segment flexibility in the function of NHE1,
a protein involved in apoptosis and myocardial disease. The study
of the rhomboid protease system is also described from two
perspectives: 1) I attempted to produce several TM constructs of
the substrate spitz or a related construct and the production and
purification are described in detail; and 2) I present
coarse-grained molecular dynamics simulation results for the E.
coli rhomboid ecGlpG and a spitz TM construct. Spitz appears to
preferentially associate with rhomboid near TMs 1 and 3 rather than
the proposed substrate gate at TM 5. The two proteins primarily
interact at the termini of helices rather than within the
hydrocarbon core of the bilayer. Finally, I present a detailed
analysis of coarse-grained molecular dynamics simulations of the
fibroblast growth factor receptor 3 TM domain dimerization.
Specifically, algorithms are described for analyzing critical
features of wild-type and G380R mutant constructs. The G380R
mutation is the cause of achondroplasia, the most common form of
human dwarfism. The results suggest that the proximity of a residue
to the dimer interface may impact the severity of the mutant
phenotype. Strikingly, heterodimer and mutant homodimer constructs
exhibit a secondary dimer interface which may explain the increased
signaling activity previously reported for the G380R mutation – the
helices may rotate with the introduction of G380R. The unifying
theme of this work is the 'study of membrane proteins' using
complementary techniques from structural biology and computational
biochemistry.
Advisors/Committee Members: Dr. Ayyalusamy Ramamoorthy (external-examiner), Dr. John Archibald (graduate-coordinator), Dr. Neale Ridgway (thesis-reader), Dr. Christian Blouin (thesis-reader), Dr. Raymond Syvitski (thesis-reader), Dr. Jan K. Rainey (thesis-supervisor), Not Applicable (ethics-approval), Not Applicable (manuscripts), Not Applicable (copyright-release).
Subjects/Keywords: Nuclear Magnetic Resonance Spectroscopy (NMR); Membrane Proteins; Coarse-grained molecular dynamics simulations; NMR spin relaxation experiments; Structural Biology
…Dalhousie University provided assistance for parts of my project: Bruce Stewart, Caitlin
Reid… …Neurobiology Department at Dalhousie University.
I have been supported by an NSERC Canada Graduate… …Dalhousie
University. I also acknowledge support from the Department of Biochemistry &
Molecular… …Biology at Dalhousie University, and the SBCB and Department of Biochemistry at the University…
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APA ·
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MLA ·
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CSE |
Export
to Zotero / EndNote / Reference
Manager
APA (6th Edition):
Reddy, T. (2012). Structure, Flexibility, And Overall Motion Of Transmembrane
Peptides Studied By NMR Spectroscopy And Molecular Dynamics
Simulations. (Doctoral Dissertation). Dalhousie University. Retrieved from http://hdl.handle.net/10222/15719
Chicago Manual of Style (16th Edition):
Reddy, Tyler. “Structure, Flexibility, And Overall Motion Of Transmembrane
Peptides Studied By NMR Spectroscopy And Molecular Dynamics
Simulations.” 2012. Doctoral Dissertation, Dalhousie University. Accessed March 05, 2021.
http://hdl.handle.net/10222/15719.
MLA Handbook (7th Edition):
Reddy, Tyler. “Structure, Flexibility, And Overall Motion Of Transmembrane
Peptides Studied By NMR Spectroscopy And Molecular Dynamics
Simulations.” 2012. Web. 05 Mar 2021.
Vancouver:
Reddy T. Structure, Flexibility, And Overall Motion Of Transmembrane
Peptides Studied By NMR Spectroscopy And Molecular Dynamics
Simulations. [Internet] [Doctoral dissertation]. Dalhousie University; 2012. [cited 2021 Mar 05].
Available from: http://hdl.handle.net/10222/15719.
Council of Science Editors:
Reddy T. Structure, Flexibility, And Overall Motion Of Transmembrane
Peptides Studied By NMR Spectroscopy And Molecular Dynamics
Simulations. [Doctoral Dissertation]. Dalhousie University; 2012. Available from: http://hdl.handle.net/10222/15719
. 
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