No description available.

The cellular mechanical environment, including the elasticity of the extracellular matrix, profoundly affects cellular mechanical and biological responses. This responsiveness depends on and may influence the inherent mechanical properties of the cell and the nucleus. In this thesis, the local and global elastic moduli of valve interstitial cells (VICs) cultured on substrates of varying stiffness were characterized using atomic force microscopy (AFM). A novel AFM technique used to directly determine nuclear elastic moduli in situ was also tested and preliminary results for VIC nuclear elasticity and isolated VIC nuclei elasticity were presented. This study confirmed that both local and global elasticity of VICs were sensitive to substrate compliance, and demonstrated that the nucleus was consistently two to four times stiffer than the cytoplasm and that isolated VIC nuclei were significantly softer than the intact nuclei in situ. It also provides practical guidelines for efficient AFM-based measurement of cell mechanical properties.

This thesis describes the development of a high-throughput approach towards the encapsulation of cancer cells in biopolymer microgels with tunable mechanical properties. In particular, this thesis is focused on: i) the high-throughput generation of biopolymer microgels with tunable mechanical properties ii) the measurement of the mechanical properties of the microgels, and iii) the high-throughput encapsulation of a cancer cell line within biopolymer gels. The microgels will be generated by (i) introducing in a microfluidic device two distinct streams of biopolymer solutions; (ii) mixing the streams; (iii) emulsifying the biopolymer and (iv) using thermosetting to transform the droplets in situ into microgels. By applying a compression force to the hydrogel microbead and measuring its deformation, the Young’s modulus and relaxation time of the microgel can be examined. The properties of cells were examined within the gels using various spectroscopic techniques such as absorption (UV-Vis) and fluorescence microscopy (fluorescent microscopy, confocal microscopy).

This thesis describes the development of a high-throughput approach towards the encapsulation of cancer cells in biopolymer microgels with tunable mechanical properties. In particular, this thesis is focused on: i) the high-throughput generation of biopolymer microgels with tunable mechanical properties ii) the measurement of the mechanical properties of the microgels, and iii) the high-throughput encapsulation of a cancer cell line within biopolymer gels. The microgels will be generated by (i) introducing in a microfluidic device two distinct streams of biopolymer solutions; (ii) mixing the streams; (iii) emulsifying the biopolymer and (iv) using thermosetting to transform the droplets in situ into microgels. By applying a compression force to the hydrogel microbead and measuring its deformation, the Young’s modulus and relaxation time of the microgel can be examined. The properties of cells were examined within the gels using various spectroscopic techniques such as absorption (UV-Vis) and fluorescence microscopy (fluorescent microscopy, confocal microscopy).

The cellular mechanical environment, including the elasticity of the extracellular matrix, profoundly affects cellular mechanical and biological responses. This responsiveness depends on and may influence the inherent mechanical properties of the cell and the nucleus. In this thesis, the local and global elastic moduli of valve interstitial cells (VICs) cultured on substrates of varying stiffness were characterized using atomic force microscopy (AFM). A novel AFM technique used to directly determine nuclear elastic moduli in situ was also tested and preliminary results for VIC nuclear elasticity and isolated VIC nuclei elasticity were presented. This study confirmed that both local and global elasticity of VICs were sensitive to substrate compliance, and demonstrated that the nucleus was consistently two to four times stiffer than the cytoplasm and that isolated VIC nuclei were significantly softer than the intact nuclei in situ. It also provides practical guidelines for efficient AFM-based measurement of cell mechanical properties.

This thesis is an investigation of methods to remediate methicillin-resistant Staphylococcus pseudintermedius (MRSP) biofilms through conventional and microfluidic-based in vitro assays. MRSP biofilm related infections are a major concern for veterinary clinicians as they may complicate remediation by the immune system or antimicrobials. Novel antimicrobials that have been found to reduce biofilm growth in other staphylococci were assessed in both mono- and combination therapy against MRSP biofilm. Quantitative assay results (p < 0.05) suggest fosfomycin alone and in combination with clarithromycin can significantly reduce biofilm formation. Morphological examination using scanning electron microscopy and atomic force microscopy further demonstrated the effectiveness of fosfomycin alone on biofilm formation on orthopaedic screws and mica sheets. Fabricated microfluidic assays were utilized to assess multiple concentrations of antimicrobial therapy against pre-formed biofilm under physiologically relevant conditions in a quick and repeatable manner. Results demonstrated the usefulness of microfluidic platforms in determining minimum biofilm eradication concentrations.

No description available.

This work reports the various aspects of the application of atomic force microscopy (AFM), for the characterization of organic semiconductors and DNA-based arrays, for organic electronics and biological applications. On these soft surfaces, the amplitude modulation AFM mode was chosen. This choice is argued by a study of dissipative processes, performed on a particular sample, a DNA chip. We showed the influence of experimental parameters on the topographic and phase image quality. By calculating the dissipative energy, it was shown that the dissipation on the DNA chip was mainly induced by a viscoelastic tip-sample interaction.The AFM study of the "thickness-driven" pentacene growth was made to link the morphology to the nature of the substrate and to the electrical performance of created pentacene-based Organic Field Effect Transistor (OFET). Deposited on two polymer substrates, parylene and benzocyclobutene (BCB), pentacene has been characterized for nanoscale film thicknesses between 6 and 60nm. It has been shown that the larger grains were created for a deposited thickness of 30nm. Spectroscopic AFM mode was used as an alternative to the method of contact angles, to measure local surface energy. Decrease of surface energy is characteristic of a more ordered surface and was measured for a thickness of 30 nm of pentacene deposited on both substrates. Models of statistical analysis of spectral images, based on the Power Spectrum Distribution (PSD) have been used to explain the morphology of pentacene films. In addition, these models have provided a comprehensive description not only of the accessible surface of the sample, but also of its internal structural properties. Highlighted in the models, the critical thickness of 30 nm corresponds to a transition from the orthorhombic phase to the triclinic phase for pentacene molecules deposited on parylene. Similarly, a polymorphic transition occurs on the BCB. On OFETs, based on pentacene on BCB, the largest mobility of 3.1x10-2 cm²/Vs corresponds to the pentacene layer of 30nm, that shows a better ordering of the orthorhombic molecular packing in comparison with the triclinic packing.The molecular arrangement of X and Y structures based on DNA was observed, by AFM, in air and in two buffer solutions of Tris and HEPES on a mica substrate. It was shown that the treatment of the mica by Ni2 + ions increases the strength of the DNA/substrate interaction and reduces the diffusivity of the molecules. In air, wired macromolecules containing one double-stranded structure are observed on untreated mica and macromolecules with a 2D geometry on pretreated mica. Onto a non-treated, the greater thermal motion of weakly bounded to mica DNA molecules leads to the rupture of intermolecular bonding and the forming structures are more simple and not branched. The organization is different in solutions of Tris and HEPES. In the Tris solution, containing Mg2+ cations, the arrangement leads to a well-organized 2D architecture. In the HEPES solution, containing Ni2+ cations, the ionic strength is 10 times lower, this leads to a breaking of the bonds previously formed between DNA and mica. However, DNA molecules are near each other due to a partial substitution of already adsorbed Mg2 + cations by Ni 2 + cations of higher affinity with the mica. These results show that the two liquids promote a 2D assembly. In air, the networks are not stable and the few observed ones remain in a dendritic structure on the surface of pretreated mica and as a linear macromolecule on the untreated mica.

No description available.

<p>The role of articular cartilage in diarthrodial joints is primarily mechanical as the tissue provides a nearly frictionless, load-bearing surface that supports and distributes forces generated during joint loading. Embedded within the extensive cartilage extracellular matrix (ECM), chondrocytes are surrounded by a narrow, distinct pericellular matrix (PCM) that is thought to regulate the biomechanical microenvironment of the cell and influence chondrocyte metabolism, cartilage homeostasis, and overall joint health. While previous studies of PCM mechanical properties required physical extraction of the cell and PCM from the tissue, atomic force microscopy (AFM) provides a means for high resolution microindentation testing that can be used to measure local mechanical properties in situ. This dissertation develops and applies AFM microindentation techniques to 1) evaluate the microscale elastic properties of the cartilage PCM and ECM in situ and 2) correlate site-specific biochemical composition with biomechanical properties of the PCM and ECM. </p><p>An AFM-based stiffness mapping technique was experimentally validated and applied to cartilage sections to evaluate ECM and PCM properties in situ with minimal disruption of native matrix integration. As expected, PCM elastic moduli were significantly less than ECM moduli, uniform with depth, and mechanically isotropic. ECM moduli exhibited distinct depth-dependent anisotropy and unexpectedly, were found to decrease with depth from the articular surface. Both the PCM and ECM exhibited alterations in microscale moduli and their spatial distributions when evaluated in cartilage presenting early degenerative changes associated with osteoarthritis (OA) as compared to healthy tissue. </p><p>The ability to correlate site-specific biochemical composition with local biomechanical properties provides a more complete characterization of the chondrocyte microenvironment. To this end, we developed novel immunofluorescence (IF)-guided AFM stiffness mapping and demonstrated that PCM mechanical properties correlate with the presence of type VI collagen. Extending this technique by using dual IF, we presented new evidence for a defining role of perlecan in the PCM, showing that interior regions of the PCM rich in perlecan and type VI collagen exhibit lower elastic moduli than peripheral PCM and ECM regions lacking perlecan. Furthermore, lower moduli at the PCM interior were significantly influenced by the presence of heparan sulfate. IF-guided AFM stiffness mapping was combined with enzymatic digestion to demonstrate that the micromechanical properties of the PCM exhibit high resistance to specific enzymatic digestion of aggrecan and aggrecan-associated glycosaminoglycans but are vulnerable to proteolytic degradation by leukocyte elastase. </p><p>Overall, this research generates new insights into the complex structural, compositional, and functional relationships between the cartilage ECM and PCM and provides the tools and framework for further studies to continue to investigate their importance in regulating chondrocyte physiology in health and disease.</p> / Dissertation