Cell-extracellular matrix and cell-cell adhesion are essential for biological processes such as cell motility, signaling, proliferation, cytoskeletal organization and gene expression. For this reason, extensive effort has been devoted in the past few decades to measure cell adhesion as well as identify key molecules involved. This thesis focuses on two outstanding problems in this area, namely, how to quantitatively characterize the adhesion between neural cells and the substrate and how to model the turnover of adhesions in the intriguing phenomenon of stretch-induced cell realignment. First of all, using a combined atomic force (AFM) and total internal reflection fluorescence microscope (TIRFM) system a novel method was developed to systematically and quantitatively examine the adhesion between neurite branches and the extracellular matrix. Specifically, a tipless AFM cantilever was used to penetrate between a well-developed neurite and the functionalized substrate and then gradually peel the neurite from the surface. At the same time, a laser TIRFM was utilized to monitor the activities of different adhesion molecules during the detaching process. This approach provides a solution to the long-standing problem of how to quantitatively measure neuron-extracellular matrix interactions while, simultaneously, identify the roles of various adhesion proteins in the process. Besides heathy neurons, testes have also been conducted on cells affected by the Alzheimer's disease (AD) where the influence of such disease on the mechanical response of neural cells was demonstrated. Secondly, to better understand the observed peeling response of the neurite, as well as extract key information from it, finite element (FEM) simulation was carried out using ABAQUS. It was shown that a good fit between the simulation results and experimental data can be achieved by representing the adhesion between two surfaces with simple cohesive interactions. In particular, it was found that the apparent adhesion energy density, a quantity of central interest in cell adhesion studies, is in the range of 0.2-0.8 mj/m^2. Last but not the least, a mechanochemical modeling framework was developed to investigate the mechanism of cell reorientation induced by cyclic stretching on the substrate. It was shown that the final alignment of cells reflects the competition between stress fiber assembly or disassembly, focal adhesion growth or disruption, substrate stiffening and whole-cell rotation. Predictions from the model are consistent with a variety of experimental observations, suggesting that the main physics of this intriguing phenomenon may have been well captured. / published_or_final_version / Mechanical Engineering / Doctoral / Doctor of Philosophy

Diss. (sammanfattning) Stockholm : Karol. inst. / Härtill 7 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2000. / Härtill 5 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst. / Härtill 4 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2001. / Härtill 4 uppsatser.

Thesis (Ph.D.)--University of Delaware, 2009. / Principal faculty advisors: Babatunde Ogunnaike, Dept. of Chemical Engineering, and Ulhas P. Naik, Dept. of Biological Sciences. Includes bibliographical references.

Ubiquitylation is a versatile post-translational modification that participates in regulation of protein stability, via proteasomal and lysosomal degradative pathways, regulation of membrane protein internalisation and other trafficking events, and regulating the biological activity of some proteins independent of degradation. The diverse functions of ubiquitylation as a post- translational protein modification allow speculation that regulation of protein ubiquitylation status may be of crucial importance during the dynamic process of development. A screen of known, and suspected, ubiquitin pathway enzymes was designed to test this hypothesis. Whole mount in situ hybridisation was conducted on early post implantation mouse embryos to determine expression patterns of the ubiquitin pathway enzyme targets. This screen was not pursued in depth due to difficulties in resolving doubts regarding the sensitivity of the method and the validity of weak ubiquitous staining patterns. The FAM deubiquitylating enzyme is a known developmentally regulated ubiquitin pathway enzyme, and although believed to antagonise the conjugation of ubiquitin to specific substrates its cell biology remains poorly characterised. In different cellular contexts FAM has been reported to localise to points of cell-cell contact or to endosomes, and circumstantial evidence suggests a role in regulating trafficking of a cell-cell adhesion complex (Murray et al., 2004; Taya et al., 1999; Taya et al., 1998). It was sought to further investigate the role of FAM in cell-cell adhesion in the well characterized polarized epithelial cell line, MDCKII, by creating clonal MDCKII cell lines that overexpress FAM. These cell lines were to be analysed for alterations in cell-cell adhesive properties and the biochemistry of proposed FAM substrates, which include the cell adhesion molecules β-catenin, E-cadherin and AF-6. MDCKII cell lines expressing exogenous V5-tagged murine FAM were successfully isolated, but failed to show changes in cell-cell adhesive properties. Generation of an antibody that reliably recognised both the canine and murine FAM protein demonstrated that the FAM-V5 expressing cell lines did not have increased total FAM protein. Other approaches taken to facilitate the study of FAM include attempts to express GFP-FAM fusion proteins and to generate an inducible FAM overexpressing cell line. Further alternative approaches are discussed. / Thesis (Ph.D.)--School of Molecular and Biomedical Science, 2005.

No description available.

Ubiquitylation is a versatile post-translational modification that participates in regulation of protein stability, via proteasomal and lysosomal degradative pathways, regulation of membrane protein internalisation and other trafficking events, and regulating the biological activity of some proteins independent of degradation. The diverse functions of ubiquitylation as a post- translational protein modification allow speculation that regulation of protein ubiquitylation status may be of crucial importance during the dynamic process of development. A screen of known, and suspected, ubiquitin pathway enzymes was designed to test this hypothesis. Whole mount in situ hybridisation was conducted on early post implantation mouse embryos to determine expression patterns of the ubiquitin pathway enzyme targets. This screen was not pursued in depth due to difficulties in resolving doubts regarding the sensitivity of the method and the validity of weak ubiquitous staining patterns. The FAM deubiquitylating enzyme is a known developmentally regulated ubiquitin pathway enzyme, and although believed to antagonise the conjugation of ubiquitin to specific substrates its cell biology remains poorly characterised. In different cellular contexts FAM has been reported to localise to points of cell-cell contact or to endosomes, and circumstantial evidence suggests a role in regulating trafficking of a cell-cell adhesion complex (Murray et al., 2004; Taya et al., 1999; Taya et al., 1998). It was sought to further investigate the role of FAM in cell-cell adhesion in the well characterized polarized epithelial cell line, MDCKII, by creating clonal MDCKII cell lines that overexpress FAM. These cell lines were to be analysed for alterations in cell-cell adhesive properties and the biochemistry of proposed FAM substrates, which include the cell adhesion molecules β-catenin, E-cadherin and AF-6. MDCKII cell lines expressing exogenous V5-tagged murine FAM were successfully isolated, but failed to show changes in cell-cell adhesive properties. Generation of an antibody that reliably recognised both the canine and murine FAM protein demonstrated that the FAM-V5 expressing cell lines did not have increased total FAM protein. Other approaches taken to facilitate the study of FAM include attempts to express GFP-FAM fusion proteins and to generate an inducible FAM overexpressing cell line. Further alternative approaches are discussed. / Thesis (Ph.D.)--School of Molecular and Biomedical Science, 2005.

No description available.