Browsing by Author "Williams, Martin (Bill)"

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Biophysical investigations of cells focusing on the utility of optical tweezers : a thesis presented in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Physics at Biophysics and Soft Matter Group, School of Fundamental Science, Massey University, New Zealand
(Massey University, 2021) Pradhan, Susav
The aim of this thesis was to explore the utility of different biophysical techniques, particularly optical tweezers (OT), in the investigation of the mechanical properties and interactions of biological samples. Specifically, MCF7 cells and their extracted nuclei were investigated mechanically, while the adhesion property of selected bacteria to the milk fat globule was also used as an exemplar. Biological cells have the ability to actively respond to external mechanical forces exerted by the microenvironment. The cellular response can be viscous, elastic, or viscoelastic in nature depending on the nature of the applied forces and the mechanical stresses applied. Changes in the mechanical properties of cells and nuclei have emerged as a prominent hallmark of many human diseases, particularly in neurodegenerative and metastatic diseases. In this thesis, to understand the application of these techniques to biological systems better, bulk rheology and microrheology studies were first performed on a model viscoelatic fluid (PEO). Particularly, the passive and active microrheology of this model viscoelastic material was characterized using optical tweezers and video particle tracking to develop the prerequisite experimental and analytical methods. Using the experimental knowledge gained from applying optical tweezers to standard materials, a mechanistic approach was developed in order to better understand how the mechanical properties of MCF7 cells change when the amount of heterochromatin protein (HP1a) present inside the nuclei was reduced. (HP1a) is an architectural protein that establishes and maintains heterochromatin, ensuring genome fidelity and nuclear integrity. While the mechanical effects of changes in the relative amount of euchromatin and heterochromatin brought about by inhibiting chromatin modifying enzymes have been studied previously, here we measure how the material properties of the cells are modified following the knockdown HP1a. Indentation experiments using optical tweezers revealed that the knockdown cells have apparent Young’s modului significantly lower than control cells. Similarly, tether experiments performed using optical tweezers revealed that the membrane tensions of knockdown cells were lower than those of control cells. This assay led to further work on studying the mechanical properties of nuclei extracted from MCF7 cells. A combination of atomic force microscopy, optical tweezers, and techniques based on micropipette aspiration was used to characterize the mechanical properties of nuclei extracted from HP1a knockdown or matched control cells. Similar to the previous finding on cells, local indentation performed using atomic force microscopy and optical tweezers found that the knockdown nuclei have apparent Young’s modului significantly lower than control nuclei. In contrast, results from pipette-based techniques in the spirit of microaspiration, where the whole nuclei were deformed and aspirated into a conical pipette, showed considerably less variation between HP1a knockdown and control, consistent with previous studies reporting that it is predominantly the lamins in the nuclear envelope that determine the mechanical response to large whole-cell deformations. The differences in chromatin organisation observed by various microscopy techniques between the MCF7 control and HP1a knock-down nuclei correlated well with the results of our measured mechanical responses and our hypotheses regarding their origin. Finally, not just the mechanical properties of the cells but also their interactions (an interaction between the milk fat globule membrane and two bacterial strains - Lactobacillius fermentum strains - 1487 and 1485) was explored as a side project by probing with optical tweezers. The difference in bacterial cell surface properties of these two strains and its effects on intestinal epithelial barrier integrity has already been studied. This study focuses on measuring the adhesion force between membrane and bacteria using optical tweezers. The results suggested that L. fermentus AGR1487 strongly interacts with MFGM compared to AGR1485. All in all, this thesis demonstrates how biophysical techniques can provide valuable insights into understanding biological systems.
The disordered- and ordered-state structures of κ-carrageenan : an X-ray scattering, molecular dynamics, and density-functional theory study : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Physics at Massey University, Palmerston North, New Zealand
(Massey University, 2022) Westberry, Benjamin
κ-carrageenan is a biopolymer extracted from marine algae. It exists in aqueous solution, at high temperatures and/or low salt concentrations as a ‘disordered-state’, and at low temperatures and in the presence of certain salts as an ‘ordered-state’. The transition between disordered- and ordered-states involves molecular structural changes, which are essential to its interesting viscoelastic properties that are routinely exploited in a plethora of applications. Despite this, the molecular conformations of the disordered- and ordered-states, as well as the details of the transitional pathway connecting them, remain a source of contention. While decades of research have amassed a vast trove of information on the disorder-order transition, an atomistic understanding of the structure in solution has remained elusive. This study takes advantages of recent advances in computational capabilities in order to simulate κ-carrageenan solutions on length scales of ∼10 nm over μs time scales, and thus develop atomistic models of the disordered- and ordered-states. Both models are used to calculate wide-angle X-ray scattering profiles, and these are subsequently validated by comparison to data obtained at a synchrotron facility. The models will be further explored using density functional theory to calculate their expected optical rotation behaviour, which finds that the formation of double-helices from single chains is able to explain the increase in optical rotation measured experimentally when transitioning from the disordered to ordered-state. Structural analysis of both experimentally-verified models find the disordered-state to have a significant amount of residual helical secondary-structure, whereas the ordered-state is mostly double-helical. Crucially, simulations show that the ordered-state arises spontaneously from the so-called disordered-state at a rate dependant on salt concentration, without prior uni-molecular changes. The findings of this research are the most detailed model of the disorder-order transition to-date, and demonstrate that the existing paradigm of a ‘coil-to-helix’ transition is in need of revision.