Spelling suggestions: "subject:"artificial lipid bilayer"" "subject:"aartificial lipid bilayer""
1 |
Developing a Cell-like Substrate to Investigate the Mechanosensitivity of Cell-to-Cell JunctionsShilts, Kent D. 08 1900 (has links)
Indiana University-Purdue University Indianapolis (IUPUI) / The role of mechanical forces in the fate and function of adherent cells has been revealed to be a pivotal factor in understanding cell biology. Cells require certain physical cues to be present in their microenvironment or the cell will begin apoptosis. Mechanical signals from the environment are interpreted at the cellular level and biochemical responses are made due to the information from outside the cell, this process is known as mechanotransduction. Misinterpretation of physical cues has been indicated in many disease states, including heart disease and asthma. When a cell is bound to the ECM, proteins such as integrins are engaged at static and stable adhesion sites. These tight and static anchoring points found at the ECM exist in stark contrast to the dynamic conditions seen at intercellular junctions. Intercellular junctions, such as gap and adherens junctions, are formed between cells to act as a mechanism to relay information and exchange material. Due to the important role intercellular junctions play in processes of wound healing, epithelial-mesenchymal transition and cancer metastasis developing more sophisticated levels of understanding of these mechanisms would provide valuable insight.
Complex biological processes, including immune cell signaling and cellular ECM adhesions, have been effectively replicated in model systems. These model systems have included the use of solid supported lipid bilayers and polymeric hydrogels that display cell adhesion molecules. Studies of cellular mechanotransduction at ECM adhesion sites has also been completed with covalently functionalized polymeric substrates of adjustable elasticity. However, developing model systems that allow the accurate reproduction of properties seen at intercellular junctions, while also allowing the investigation of cellular mechanosensitivity has proven to be a difficult task. Previous work has shown that polymer-tethered lipid bilayers (PTLBs) are a viable material to allow the replication of the dynamics and adhesion seen at intercellular junctions. Although efforts have been made to produce PTLBs with different mechanical properties, there is currently not a material with sufficient tunable elastic properties for the study of cellular mechanotransduction.
To establish a system that allows the study of stiffness effects across a biologically relevant range (~0.50 – 40 kPa) while maintaining the dynamic properties seen at cell-to-cell junctions, polymer gel-tethered bilayers (PGTBs) were developed. A fabrication strategy was established to allow the incorporation of a hydrogel support with easily tunable stiffness and a tethered lipid bilayer coating, which produced a powerful platform to study the effects of stiffness at intercellular junctions. Careful attention was given to maintain the beneficial properties of membrane diffusion, and it was shown that on different linking architectures lipid bilayers could be established and diffusion was preserved. Microscopy-based FCS and FRAP methodology were utilized to measure lipid diffusion in these systems, while confocal microscopy was used to analyze cell spreading and adhesion. Three distinct architectures to link the lipid membrane to the underlying polyacrylamide hydrogel were pursued in this work, a non-covalent biotin-streptavidin system, a covalently linked design with fibronectin, and a direct covalent linkage utilizing crosslinker chemistry. In this work, it was shown that cells were able to spread and adhere on these substrates, with cell adhesion zones visualized under plated cells that demonstrate the capability of the cell to rearrange the presented linkers, while maintaining a stable material. Also confirmed is the tunability of the polymer hydrogel across a wide range of stiffness, this was shown by quantitative changes in cell spreading area in response to polymer properties.
|
2 |
DEVELOPING A CELL-LIKE SUBSTRATE TO INVESTIGATE THE MECHANOSENSITIVITY OF CELL-TO-CELL JUNCTIONSKent Douglas Shilts (9182480) 04 August 2020 (has links)
<p>The role of mechanical forces in
the fate and function of adherent cells has been revealed to be a pivotal
factor in understanding cell biology. Cells require certain physical cues to be
present in their microenvironment or the cell will begin apoptosis. Mechanical
signals from the environment are interpreted at the cellular level and
biochemical responses are made due to the information from outside the cell, this
process is known as mechanotransduction. Misinterpretation of physical cues has
been indicated in many disease states, including heart disease and asthma. When
a cell is bound to the ECM, proteins such as integrins are engaged at static
and stable adhesion sites. These tight and static anchoring points found at the
ECM exist in stark contrast to the dynamic conditions seen at intercellular
junctions. Intercellular junctions, such as gap and adherens junctions, are
formed between cells to act as a mechanism to relay information and exchange
material. Due to the important role intercellular junctions play in processes
of wound healing, epithelial-mesenchymal transition and cancer metastasis
developing more sophisticated levels of understanding of these mechanisms would
provide valuable insight.</p>
<p>Complex biological processes,
including immune cell signaling and cellular ECM adhesions, have been
effectively replicated in model systems. These model systems have included the
use of solid supported lipid bilayers and polymeric hydrogels that display cell
adhesion molecules. Studies of cellular mechanotransduction at ECM adhesion
sites has also been completed with covalently functionalized polymeric
substrates of adjustable elasticity. However, developing model systems that
allow the accurate reproduction of properties seen at intercellular junctions,
while also allowing the investigation of cellular mechanosensitivity has proven
to be a difficult task. Previous work has shown that polymer-tethered lipid
bilayers (PTLBs) are a viable material to allow the replication of the dynamics
and adhesion seen at intercellular junctions. Although efforts have been made
to produce PTLBs with different mechanical properties, there is currently not a
material with sufficient tunable elastic properties for the study of cellular
mechanotransduction.</p>
<p>To establish a system that allows
the study of stiffness effects across a biologically relevant range (~0.50 – 40
kPa) while maintaining the dynamic properties seen at cell-to-cell junctions,
polymer gel-tethered bilayers (PGTBs) were developed. A fabrication strategy
was established to allow the incorporation of a hydrogel support with easily
tunable stiffness and a tethered lipid bilayer coating, which produced a
powerful platform to study the effects of stiffness at intercellular junctions.
Careful attention was given to maintain the beneficial properties of membrane
diffusion, and it was shown that on different linking architectures lipid
bilayers could be established and diffusion was preserved. Microscopy-based FCS
and FRAP methodology were utilized to measure lipid diffusion in these systems,
while confocal microscopy was used to analyze cell spreading and adhesion.
Three distinct architectures to link the lipid membrane to the underlying
polyacrylamide hydrogel were pursued in this work, a non-covalent
biotin-streptavidin system, a covalently linked design with fibronectin, and a
direct covalent linkage utilizing crosslinker chemistry. In this work, it was
shown that cells were able to spread and adhere on these substrates, with cell
adhesion zones visualized under plated cells that demonstrate the capability of
the cell to rearrange the presented linkers, while maintaining a stable
material. Also confirmed is the tunability of the polymer hydrogel across a
wide range of stiffness, this was shown by quantitative changes in cell
spreading area in response to polymer properties.</p>
|
3 |
Single Molecule Spectroscopy Studies of Membrane Protein Dynamics and Energetics by Combined Experimental and Computational AnalysesRajapaksha, Suneth P. 23 July 2012 (has links)
No description available.
|
Page generated in 0.0536 seconds