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Identification and characterization of critical laminin-111 sequences required for polymerization and cell surface anchorage and the requirement of both activities for proper basement membrane formation and other events

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Title
Identification and characterization of critical laminin-111 sequences required for polymerization and cell surface anchorage and the requirement of both activities for proper basement membrane formation and other events
Name (type = personal)
NamePart (type = family)
Harrison
NamePart (type = given)
David A.
NamePart (type = date)
1965-
DisplayForm
David Harrison
Role
RoleTerm (authority = RULIB)
author
Name (type = personal)
NamePart (type = family)
Amenta
NamePart (type = given)
Peter S
DisplayForm
Peter S Amenta
Affiliation
Advisory Committee
Role
RoleTerm (authority = RULIB)
chair
Name (type = personal)
NamePart (type = family)
Yurchenco
NamePart (type = given)
Peter D
DisplayForm
Peter D Yurchenco
Affiliation
Advisory Committee
Role
RoleTerm (authority = RULIB)
internal member
Name (type = personal)
NamePart (type = family)
Carr
NamePart (type = given)
Chavela M
DisplayForm
Chavela M Carr
Affiliation
Advisory Committee
Role
RoleTerm (authority = RULIB)
internal member
Name (type = personal)
NamePart (type = family)
Sahota
NamePart (type = given)
Amrik
DisplayForm
Amrik Sahota
Affiliation
Advisory Committee
Role
RoleTerm (authority = RULIB)
outside member
Name (type = corporate)
NamePart
Rutgers University
Role
RoleTerm (authority = RULIB)
degree grantor
Name (type = corporate)
NamePart
Graduate School - New Brunswick
Role
RoleTerm (authority = RULIB)
school
TypeOfResource
Text
Genre (authority = marcgt)
theses
OriginInfo
DateCreated (qualifier = exact)
2012
DateOther (qualifier = exact); (type = degree)
2012-05
CopyrightDate (qualifier = exact)
2012
Place
PlaceTerm (type = code)
xx
Language
LanguageTerm (authority = ISO639-2b); (type = code)
eng
Abstract (type = abstract)
A system was developed for expression and purification of various recombinant a1 LG4-5 and heterotrimeric laminin-111 proteins. A number of combinations involving different promoters, 5’UTRs, signal sequences, epitope tags, selectable antibiotics, and purification schemes were tested in order to optimize the system. A homology model of laminin a1 LG4-5 was generated and utilized to identify candidates for mutation and expression as recombinant a1 LG4-5 proteins. The crystal structure of a1 LG4-5 was also determined. Heparin, a-dystroglycan (aDG), and sulfatide binding of the generated mutants demonstrated wide differences and dependence upon contributions from basic residues on the surface of LG4, including: RKR2721 and KRK2793 for heparin; RAR2833, KDR2860, the Asn2811 glycation moiety, and the LG4 Ca2+ ion for aDG; the LG4 Ca2+, Arg2833 of RAR2833, and Lys2766 residue of RKGRTK2770 for one sulfatide binding site, and the Arg2831 of RAR2833 for a second sulfatide site. The produced recombinant heterotrimeric laminin-111s demonstrated a requirement for the N-terminal LN domains of laminin-111’s constituent a1, b1, and g1 chains in self-polymerization. The inability of embryoid bodies, derived from laminin g1 null embryonic stem cells, to express Lm-111 and develop past formation of an outer endodermal layer of cells without laminin, was utilized via the addition of exogeneous recombinant laminin-111s, to test the various functions of the recombinant laminins and their ability to form a basement membrane and induce both differentiation of an epiblast layer and formation of a central cavity. Laminins’ containing defective polymerization or a1 LG4 anchorage failed to form a BM or undergo any further development after endodermal differentiation. Experiments with Schwann cells, C2C12 myotubes, and mouse embryonic fibroblasts demonstrated the requirement for both polymerization and a1 LG4 mediated anchorage in laminin-111 for proper BM formation, cytoskeletal attachment, and laminin induced cell signaling via Src. The results also suggest a role for laminin-111 polymerization and anchorage in providing a means for aggregation and accumulation of low affinity interactions in a complex, thereby attaining very high net affinities for binding interactions and attaining otherwise unatainable thresholds necessary for these interactions to exert their effects through manipulation of the cytoskeleton, as well as, laminin induced differentiation and cell signaling.
Subject (authority = RUETD)
Topic
Biochemistry
RelatedItem (type = host)
TitleInfo
Title
Rutgers University Electronic Theses and Dissertations
Identifier (type = RULIB)
ETD
Identifier
ETD_3840
PhysicalDescription
Form (authority = gmd)
electronic resource
InternetMediaType
application/pdf
InternetMediaType
text/xml
Extent
xxii, 433 p. : ill.
Note (type = degree)
Ph.D.
Note (type = bibliography)
Includes bibliographical references
Note (type = statement of responsibility)
by David A. Harrison
Subject (authority = ETD-LCSH)
Topic
Proteins--Affinity labeling
Subject (authority = ETD-LCSH)
Topic
Polymerization
Identifier (type = hdl)
http://hdl.rutgers.edu/1782.1/rucore10001600001.ETD.000065159
RelatedItem (type = host)
TitleInfo
Title
Graduate School - New Brunswick Electronic Theses and Dissertations
Identifier (type = local)
rucore19991600001
Location
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NjNbRU
Identifier (type = doi)
doi:10.7282/T32806J4
Genre (authority = ExL-Esploro)
ETD doctoral
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Rights

RightsDeclaration (ID = rulibRdec0006)
The author owns the copyright to this work.
RightsHolder (type = personal)
Name
FamilyName
Harrison
GivenName
David
Role
Copyright Holder
RightsEvent
Type
Permission or license
DateTime (encoding = w3cdtf); (qualifier = exact); (point = start)
2012-03-08 01:48:13
AssociatedEntity
Name
David Harrison
Role
Copyright holder
Affiliation
Rutgers University. Graduate School - New Brunswick
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Type
License
Name
Author Agreement License
Detail
I hereby grant to the Rutgers University Libraries and to my school the non-exclusive right to archive, reproduce and distribute my thesis or dissertation, in whole or in part, and/or my abstract, in whole or in part, in and from an electronic format, subject to the release date subsequently stipulated in this submittal form and approved by my school. I represent and stipulate that the thesis or dissertation and its abstract are my original work, that they do not infringe or violate any rights of others, and that I make these grants as the sole owner of the rights to my thesis or dissertation and its abstract. I represent that I have obtained written permissions, when necessary, from the owner(s) of each third party copyrighted matter to be included in my thesis or dissertation and will supply copies of such upon request by my school. I acknowledge that RU ETD and my school will not distribute my thesis or dissertation or its abstract if, in their reasonable judgment, they believe all such rights have not been secured. I acknowledge that I retain ownership rights to the copyright of my work. I also retain the right to use all or part of this thesis or dissertation in future works, such as articles or books.
Copyright
Status
Copyright protected
Availability
Status
Open
Reason
Permission or license
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