Blumlein, Alice (2016) The Self-Organisation of Biological Soft matter Systems at Different Length-scales. PhD thesis, National University of Ireland Maynooth.

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Abstract

Spontaneous self-organisation occurs in physical, chemical and biological systems throughout the natural world when the components of an initially unstructured system arrange to form ordered structures. Research into the mechanisms underlying these systems has led to exciting developments in materials chemistry where a bottom-up approach based on directed self-organisation has the potential to yield novel materials with a wide range of technological and scientific applications. Owing to their high specificity and potency, biopharmaceutical therapeutics are often favoured over small molecule drugs. However, protein based biopharmaceuticals are prone to degradation as a result of physical and chemical instability, a process leading to devastating financial and safety outcomes. Accordingly, understanding and quantifying the adverse effects of protein degradation is imperative. One such form of degradation is protein self-organisation in the form of aggregation. For certain solution conditions, aggregated unfolded protein leads to the formation of gels. Hydrogels are a class of gel formed from hydrophilic polymer chains capable of holding large amounts of water in their three dimensional network and have numerous medical and pharmaceutical uses. Self-organisation drives gel formation. Therefore, understanding the principles of self-organisation is a prerequisite in the development of novel hydrogels with increased functionality. At longer length-scales cells self-associate to form tissues. Spheroids are self-organised entities comprised of a single-cell type. They are the archetypal model for tumours and are an ideal system to study the biophysical phenomena associated with self-organisation. Unlike tissues, when a single cell type is used to form the spheroid, compositionally identical replicates can easily be grown. Furthermore, unlike with explants, other factors including age and the biochemical environment, which have been shown to alter the mechanical characteristics of cells and tissues can be rigorously controlled. Here, the experimental techniques of the wider soft matter field are used to investigate the biophysical properties of systems that span the biologically relevant spectrum of length-scales in which soft matter contributions are important. Differential scanning calorimetry analysis was used to quantify the reversibility of unfolding following thermal denaturation of lysozyme. Solution conditions (pH, ionic strength and the presence/absence of disaccharides) were varied to systematically alter the temperature at which the protein unfolds, Tm. The enthalpies of unfolding during successive heating and cooling cycles were compared to quantify the degree of reversible unfolding that occurs following thermal denaturation. The sugars were used to evaluate whether a disaccharide induced increase in Tm affects the reversibility of thermally induced denaturation. It was shown that there was considerable overlap between the Tm values where reversible and non-reversible thermal denaturation occurred. Indeed, at the highest and lowest Tm no refolding was observed whereas at intermediate values refolding occurred. Furthermore, similar Tm values had different proportions of refolded protein. Using this novel analysis, it was possible to quantify the degree to which protein is lost to irreversible aggregation and show that an increase in the melt transition temperature does not necessarily confer an increase in reversibility. This type of analysis may be a useful tool for the biopharmaceutical and food industries to assess the stability of protein solutions. Bigels are an emerging class of tuneable soft materials composed of two discrete but interpenetrating networks, both of which contribute to the physical and mechanical properties of the material. A bigel network was formed from two proteins, BSA and gelatin. Thorough control of the solution conditions and kinetics ensured that the inter-species attraction between the two protein systems were weak compared to the intra-protein attraction, leading to bigel formation. The protein bigel was shown to have an elastic modulus four times greater than the combined elastic moduli of the parent gels. Furthermore, the elastic response was maintained over several deformation cycles and the gel is both thermo- and chemo-responsive. These gels have the potential to be used in drug delivery, for biomedical applications such as wound healing or as a biomimetic in tissue culture. Cavitation rheology was used to show that for spheroids formed from HEK293 cells the interfacial tension was dominated by cortical tension at length-scales < 30 μm. It was found that the elastic modulus could be related quantitatively to the disruption of cell-cell adhesion molecules which facilitates the formation of the cavity. A cascade of cadherin-cadherin dissociation events, totalling a disrupted surface are equivalent to 3, 8 and 117 cells was calculated for 5, 10 and 30 μm needles, respectively, was calculate. Furthermore, the process involved was shown to be largely elastic and a mechanism involving a rapid cycle of “unzipping” and “re-zipping” the cadherin bonds was proposed to account for this elasticity. Since changes in cortical tension and cell-cell adhesion are associated with the transition from healthy to malignant cells, CR may prove a useful addition to the oncologists’ toolbox.

Item Type:	Thesis (PhD)
Keywords:	Self-Organisation; Biological Soft matter Systems; Different Length-scales;
Academic Unit:	Faculty of Science and Engineering > Chemistry
Item ID:	10379
Depositing User:	IR eTheses
Date Deposited:	07 Jan 2019 11:57
URI:
Use Licence:	This item is available under a Creative Commons Attribution Non Commercial Share Alike Licence (CC BY-NC-SA). Details of this licence are available here

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