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Subject: search.filters.subject.510501 Biological physics

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    Structure-rheology relationships of protein-polysaccharide complexes at oil/water interfaces : 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
    (Massey University, 2021) Ramamirtham, Sashikumar
    The complexation of proteins with polysaccharides to form bio-complexes is being utilized in a variety of applications including food formulations, microencapsulation, protein separation and bioactive deliveries. Understanding the impact of these biomolecules on each other with discernment will not only improve our existing usages but also aid in devising newer applications. The duo of beta -lactoglobulin (beta -lg), a surface active globular whey protein, and pectin, a plant-derived polysaccharide, is the model protein-polysaccharide system of this study. Beta -lg and pectin have been reported to undergo complexation driven by electrostatic attraction leading to contrasting interfacial rheological properties depending on the fine structures of the polysaccharide. The aim of this thesis is to understand the role of fine structures of the polysaccharide in protein adsorption and the interfacial film formation. Given that beta -lg is the interfacially active molecule in this study, assemblies of beta -lg at dodecane/water interfaces at pHs 3 and 4, and at different conditions of ionic strength, salt type and temperature were studied. These parameters were tuned to vary the relative amounts of two native species, namely, monomer and its smallest aggregate, the dimer, while the interface was monitored using rheology and tensiometry. Unfolding of beta -lg dimers at the interface triggers the formation of disulfide linkages between the free thiol groups located at cys121 of the monomers. In this way, it is demonstrated here for the first time that beta -lg dimers are the smallest elastic network building unit of the protein. A higher concentration of dimers increases the final interfacial elastic strength of the network. The lack of the elastic film forming ability of beta -lg monomer is attributed to the absence of multiple free thiol groups. Moreover, beta -lg monomer exhibited minimal reduction in interfacial tension akin to a pure buffer solution. This fundamental relation between the quaternary structure of beta -lg and its subsequent interfacial network suggests a possible interfacial role in its biological function. Besides, these results will also be used as control for assessing the behaviors of beta -lg/polysaccharide complexes. In the next phase of this study, transient interfacial rheology of pre-mixed solutions of beta -lg and polysaccharides with different lengths and charge densities at pHs 3 and 4 are presented. It was found that, while the interfacial activity of beta -lg/pectin complexes is dictated by the amount of charge on the polysaccharide, the kinetics of the complexed beta -lg’s adsorption and its subsequent interfacial film formation is largely controlled by the contiguity of the charges on the polysaccharide molecule. Using subphase injection techniques, it is further shown that the structure of the beta -lg in the protein/polysaccharide complex prior to adsorption is the major contributor to the lag time duration before the onset of an elastic film formation. This is exemplified by the contrasting behaviors of beta -lg/pectin complexes with high polysaccharide charge density as compared to beta -lg/pectin complexes with low polysaccharide charge density, where the latter can be used as a one shot delivery system to obtain reinforced oil/water interfaces. It is further proposed that the mechanism by which a polysaccharide molecule reinforces beta -lg interfacial film is by concatenating multiple protein units and establishing cross-links in the aqueous subphase. The final phase of this study presents microrheology measurements of oil/water interfaces laden with beta -lg and beta -lg/polysaccharide complexes. Microrheology further ascertains the viscous nature of beta -lg monomer laden interfaces and the elastic nature of the interfaces with beta -lg dimers. In addition, the presence of heterogeneity in the entangled films made of beta -lg dimers in the form of confinements was also observed. A sharp transition was exhibited from an inelastic to elastic interface occurring around a surface dimer concentration of 56 ng/m2 at pH 3, 15 mM NaCl. Further, a slightly denser interface was observed for almost all the beta -lg/polysaccharide complexes at pH 4. The heterogeneity that was observed at dimeric interfaces was not seen for interfaces with beta -lg/polysaccharide complexes indicating the presence of the polysaccharide molecules beneath the interfacial film. On the whole, this thesis demonstrates the advantages of using of interfacial rheological techniques to tease out the structure-rheology relationships of biomolecules such as proteins and protein/polysaccharide complexes and thereby provide valuable insights about molecular manipulations.
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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.