Browsing by Author "Williams, Martin"

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Capture probabilities in pair-wise collisions of emulsion drops - measurement and application : report submitted in fulfilment of the requirements for the Doctoral Degree (PhD Course) in the Biophysics and Soft Matter Group, School of Fundamental Sciences, Massey University, New Zealand
(Massey University, 2019) Ravindran, Sapna
This project seeks to measure and model particle interactions under different environmental conditions with a view to being able to control these interactions. The interactions of emulsion drops will be investigated using an optical tweezer set-up and the results considered in the context of measurements of the zeta potential of the emulsion. Specifically, how the zeta potential of emulsion drops changes with the physio-chemical environment (pH and ionic environment) is captured in a concise mathematical model, the effects of depletion interactions are considered, and a novel experimental procedure is developed to allow hundreds of pairwise stickiness measurements to be taken in an automated fashion. The major research questions are: 1. Is it possible to address the effects of changes in environmental conditions which are not easily quantifiable with a pragmatic capture probability or pairwise "stickiness" measured at a single particle level? 2. Can we link these pairwise measurements to induced changes in the surface properties and understand how they yield the rheological behaviour of the system?
Polysaccharide-DNA strings for single molecular polysaccharide studies : a thesis submitted in fulfillment of the requirements for the degree of Doctor of Philosophy in Biophysics and Soft Matter, School of Natural Sciences, Massey University, Palmerston North, New Zealand
(Massey University, 2022) Mohandas, Nimisha
For several decades DNA has been the workhorse of single molecule experiments, owing to its large controllable size and simplicity of end group attachment. The use of DNA handles to study DNA-protein conjugates has also previously been employed to understand the behaviour of proteins at the single molecular level. In contrast, single molecule studies of polysaccharides are not widely known. This project attempts to develop a methodology in order to facilitate single molecule polysaccharide studies with optical tweezers (OT). Homogalacturonan (HG), a polysaccharide component extracted from pectin, a key component in plant cell walls, was chosen to be the subject of this study. The proposed strategy was to utilise DNA strands as "handles" with one end attached onto HG, and the other coupled to beads, to allow for stretching of HG, and other single molecule studies. In order to attach HG between different DNA handles, the chemistry present at the reducing and non-reducing ends of the polysaccharide which can be used to form bonds with end functionalised DNA strands was the point of focus. Ultimately the DNA-polysaccharide connection was mediated by streptavidin moieties linking biotin-functionalised ends. Streptavidin is a tetrameric protein, renowned for its strong binding to biotin that has to led to multitudinous applications. By separating streptavidin species that have differing numbers of binding sites plugged, "linking hubs" with trivalent, divalent and monovalent functionality were obtained. Species identity, and the plugging process were studied with capillary electrophoresis, which in this case provides several advantages over traditional gels. Subsequently, divalent linkers were used to concatenate two biotin-terminated 5 kb pieces of double stranded DNA, and the resulting string stretched in an optical tweezers experiment, demonstrating the "plug-and-play" potential of the methodology for coupling and extending molecules for use in single molecule biophysical experiments.
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.