Chemical and Biomolecular Engineering - Theses
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ItemThe assembly and function of the Chaplin peptides( 2016)The filamentous bacterium Streptomyces coelicolor (S. coelicolor) produces small hydrophobic peptides called chaplins that play an instrumental role in the aerial growth of this microorganism. Chaplins (Chp D-H) reduce the surface tension of water and form fibrils on the surface of hyphae that facilitate the escape of hyphae into air. Temporal variation in the expression of chaplins suggests that they may have different functions in the transition from vegetative to aerial growth in S. coelicolor, likely dependent on their unique structures, however, these functions are not yet clear. The ability of chaplins to self-assemble at the interface and lower the surface tension makes them interesting candidates to use as surface active agents in food or consumer goods but their potential in this field has not been investigated. Chp E and Chp F were selected as the primary focus for this thesis. These two peptides differ in their pattern of secretion by S. coelicolor, their primary amino acid sequence and their isoelectric points (pI) (Chp E ~7, Chp F ~4). Moreover, Chp F contains two cysteine residues that are absent in Chp E. These differences make these two peptides ideal for determining whether the structure and function of the chaplin peptides differ. Also, preliminary experiments were performed on Chp H that has a similar pI and pattern of microbial secretion to Chp E. In contrast with Chp E, Chp H contains two cysteine residues in the primary amino acid sequence. This thesis has four key objectives: 1) to examine the effect of pH on the structure and self-assembly of Chp E in solution; 2) to investigate how the different structures formed by Chp E, as a function of pH, influence the interfacial properties of this peptide; 3) to determine whether Chp F and Chp E respond differently to pH, including the structure and function of the two peptides and 4) to characterise the rheological properties and thickness of the Chp E film formed at the air/water interface in comparison with β-casein and β-lactoglobulin, commonly used surface active agents in food products. The work also has a broader goal of investigating the potential of chaplin peptides as surface active agents for applications in food or consumer goods. The techniques and methods employed in this thesis include: liquid chromatography combined with tandem mass spectrometry for mass and protein identification, dynamic light scattering to measure the size of peptide in the solution, isoelectric focusing gel to determine the isoelectric point of peptides, Synchrotron radiation circular dichroism spectroscopy and circular dichroism spectroscopy to detect the secondary structure of peptide, Thioflavin T binding to assess fibril formation, sedimentation and amino acid analysis to assess conversion of peptides into fibrils, Langmuir trough experiments to develop pressure/area isotherms and measure surface activity, atomic force microscopy to measure the thickness of chaplin films assembled at the air/water interface, canal viscometry and the application of oscillating barriers studies to investigate the rheological properties of films formed at the air/water interface, transmission electron microscopy and Brewster angle microscopy for microscopic observations, Langmuir-Blodgett deposition and Langmuir-Schaefer deposition to deposit interfacial films on solid surfaces. In this thesis, molecular dynamics simulations are used to predict the three-dimensional structure of a chaplin monomer in solution as a function of pH and also to study the adsorption of chaplins at the interface. The conformation and self-assembly of Chp E in solution was found to be controlled by pH. Increasing the pH from 3.0 to 6.7, the pI of Chp E, or 10.0 promotes the self-assembly of Chp E peptides into amyloid-like fibrils that bound the dye Thioflavin T. At high pH, this assembly is rapid with no detectable lag phase and at least 93% or 97% of the Chp E peptide was converted into fibrils at the pI and pH 10.0 respectively. In contrast, molecular dynamics simulations and measurements by dynamic light scattering indicate the peptide may be present as a dimer or other loosely associated oligomer at pH 3.0, where it remains in the predominantly soluble form (> 70%). An increase in solution pH is accompanied by changes in the size and structure of Chp E; at low pH the peptide has a random coil structure with a diameter of 3.6 ± 1 nm (> 98% of the population), while at higher pH it adopts a β-sheet conformation with a diameter of 417 nm or 1110 ± 170 nm (> 99% of the population) for the pI and pH 10.0 respectively. MD simulations suggested that electrostatic repulsion between residues His6 and Lys10 in the N-terminus stabilize the Chp E peptide at low pH. The progressive reduction in electrostatic repulsion at higher pH leads to an α-helical structure at the N-terminus, which potentially acts as an intermediate on the path to fibril formation. The different structures formed by Chp E as a function of pH influence the interfacial assembly and function of this peptide as a surface active agent. Chp E monomers at pH 3.0 were found to be more effective at lowering the surface tension of water than Chp E fibrils at pH 10.0, likely due to the greater exposure of hydrophobic residues to the interface at pH 3.0. Microscopic observations showed that at pH 3.0, Chp E forms a homogenous smooth film at the air/water interface that contains ordered structures aligned in one direction. Peptides in this film have a random coil structure with no evidence of fibrils forming under this condition. In contrast, the heterogeneous film formed by Chp E at the interface at pH 10.0 is composed of fibrils oriented randomly over the film. This film is ~10 fold thicker and ~34 fold rougher compared to the film formed by Chp E at pH 3.0, when the films were transferred onto a Si surface and dried. This finding suggests that Chp E molecules could layer on top of one another at the interface at basic pH. Molecular dynamics simulations predict a higher dimerisation propensity for Chp E at pH 10.0 compared to pH 3.0. The formation of dimers, which typically occurs through hydrophobic contacts, potentially reduces the exposure of hydrophobic residues to the hydrophobic/hydrophilic interface, consistent with a lower diffusion rate to the interface simulated at pH 10.0 and lower surface activity observed experimentally for Chp E at pH 10.0 compared to at lower pH. A greater number of inter-molecular contacts were simulated between Chp E molecules at the interface at pH 10.0 compared to at pH 3.0, consistent with the compact assembly and lower area occupied by each Chp E molecule at pH 10.0, as indicated by the measured pressure/area isotherms. A model was presented describing the potential role of Chp E in the transition from vegetative to aerial growth during the life cycle of S. coelicolor. At acidic pH, during the vegetative growth of the microorganism, Chp E self-assembles at the air/water interface into a non-amyloidal membrane that displays significant surface activity assisting aerial growth. At a basic pH, Chp E contributes to the self-assembly of chaplins into fibrils at the surface of hyphae, increasing the surface hydrophobicity of aerial structures. This step coincides with the formation of continuous and abundant growth of aerial hyphae favoured at neutral or basic pH. During vegetative growth, Chp H with the pI of ~7 is secreted simultaneously with Chp E. At acidic pH, a lower surface activity was observed for this peptide compared to Chp E under the same condition. Therefore, Chp H may act as a polymerization unit in the assembly of chaplins into fibrils at the surface of aerial structures. Chp F was also found to form assemblies with different size, morphology and conformation as a function of pH, likely due to changes in the net charge and the distribution of charges over the peptide. Chp F peptides formed fibrillar assemblies rich in β-sheet structure with an average diameter of 337 ± 20 nm at pH 3.0 and 439 ± 7 nm at the pI of 4.2. The short assemblies formed by Chp F at pH 10.0 were smaller in size than those observed at lower pH, with the average diameter of 59 ± 6 nm; these preparations also contained a mixed structure of random coil and β-sheet. Fibril assembly was both quick and efficient, with yields > 99% at acidic pH and ~40% at basic pH. A greater Thioflavin T binding occurred at low pH compared to high pH, consistent with the efficient assembly observed at low pH. The presence of negative charges on the C-terminus of Chp F at high pH, could lead to an electrostatic repulsion between peptides, preventing the assembly of Chp F into fibrils. The reduction in electrostatic repulsion at acidic pH, due to a decrease in the net charge and change in the distribution of charges over the peptide, likely leads to a more energetically favorable association of Chp F peptides and the formation of fibrils. The differences between the primary amino acid sequences of Chp F and Chp E result in these two peptides responding in a complementary way to solution pH. In contrast with Chp F, the presence of positive charges on the N-terminus of Chp E at pH 3.0 inhibits the formation of fibrils by inducing repulsion between Chp E peptides. The formation of disulfide bonds was not found to be necessary for the formation of the β-sheet structure and assembly of Chp F fibrils in vitro at different solution pH. Similar CD spectra were obtained for reduced and non-reduced Chp F at different solution pH, suggesting the role of disulfide bond formation in Chp F fibril assembly is of minor importance. The pH-dependent assembly of Chp F into fibrils influenced the interfacial properties of this peptide. Surface activity was highest for samples at pH 10.0 that contained a higher proportion of unaggregated material and reduced level of β-sheet conformation, compared to the surface activity measured for Chp F fibrils formed at pH 3.0. Chp F assembly is expected to proceed through hydrophobic interactions, likely lowering the exposure of hydrophobic residues to the air/water interface, reducing the surface activity of Chp F fibrils at low pH. The surface activity of Chp F as a function of pH is similar to the surface activity observed for a mixture of chaplins (Chp D-H) but the trend observed in surface activity as a function of pH is the opposite of that observed for Chp E. This complementary behaviour could be useful in potential applications, where surface activity over a wide range of pH is desired. At acidic pH, Chp F formed a heterogeneous film at the air/water interface containing aggregated material punctuated by micro pores and long randomly arranged fibrils. In contrast, at basic pH, Chp F formed a homogenous film composed of short assemblies. The slightly reduced area occupied per Chp F molecule at pH 10.0 compared to pH 3.0 indicates a better packing of the Chp F within the short assemblies observed at pH 10.0, compared to the large fibrils observed at pH 3.0. The ordered structures within the film formed by Chp E at acidic pH resulted in higher dilatational elastic modulus compared to the fibrils within the films formed at basic pH. At both pH, Chp E also showed a higher dilatational elastic modulus compared to the surface active milk proteins, β-casein and β-lactoglobulin. In contrast, a higher elasticity was observed for the Chp E fibrillar film formed at pH 10.0 after a one hour period of oscillation (corresponding to 54 compression-expansion cycles at the constant frequency of 15 mHz), compared to the Chp E film formed at pH 3.0. This increase in elasticity could be related to structural changes induced by continuous oscillations, as the fibrils at pH 10.0 broke into shorter lengths leading to an increase in the area in contact with the interface. The continuous oscillations, however, had no significant effect on the size and morphology of the ordered structures within the film formed by Chp E at pH 3.0 and only a reduction in the alignment of these structures was observed by microscopy. A greater surface shear viscosity was observed for interfacial films formed by Chp E compared to the milk proteins, β-lactoglobulin and β-casein, with a smaller flow rate measured through the canal (0.007 mN/m.s for Chp E at pH 10.0 and 0.04 mN/m.s for Chp E at pH 3.0 vs. 0.05 mN/m.s for β-lag and 0.06 mN/m.s for β-cas). The Chp E film formed at pH 10.0 also showed 5.7 fold higher surface shear viscosity compared to the film at pH 3.0, suggesting these films may provide a higher resistance to coalescence and phase separation in dispersions at basic pH. These findings indicate that Chp E is capable of forming a film with favourable rheological properties and is a promising candidate to postpone coalescence and disproportionation, a key factor in foam coarsening. This peptide and possibly other chaplin peptides may therefore provide long term stability to foams and emulsions. In summary, the ability of chaplins E and F to reduce the surface tension and self-assemble at the air/water interface into an amphipathic film with favourable rheological properties suggests that these peptides are promising surface active agents that could be used in food or consumer goods. The insights into pH controlled assembly, structures formed at the interface and complementary behaviour of different chaplin peptides gained from this study also assists our understanding of the possible role of chaplin peptides in the differential development of S. coelicolor.
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ItemDroplet interactions in structured fluids and charged colloidal systems( 2016)Emulsion systems are widespread in many industries but a full understanding of how the bulk properties of emulsions are influenced by the structure and components within an emulsion is still incomplete. The focus of thesis is to investigate droplet interactions with a specific focus on investigating various surface forces with unique, poorly understood, or unknown characteristics. This was achieved through both AFM experiments to directly measure the forces between drops and through microfluidic platforms to observe the behaviour and collisions of drops during bulk flow. The thesis can be separated into three main sections. The first section contains an investigation into surfactant free high concentration salt solutions. Measurements were taken using atomic force microscopy (AFM) to investigate the interactions between drop pairs. The specific focus was on a previously observed pull off phenomenon that is not expected based on the current understanding of surface and intermolecular forces. AFM measurements between two drops were taken in solutions of 50 mM NaNO3, 500 mM NaNO3, and 50 mM NaClO4. The measurements were in agreement with previous findings that the magnitude of the pull off force is primarily determined by the contact time between drops but also demonstrated the possibility that additional factors such as maximum compressive force or a force limit for very slow pull offs may also be important. The next section presents research of high concentration surfactant systems and the influence of nanocolloid shape on structural forces. Measurements were taken using atomic force microscopy (AFM) to investigate the interactions between drop-drop and particle-plate systems. Solutions of sodium dodecyl sulphate (SDS) and sodium bromide (NaBr) as well as solutions of hexadecyltrimethylammonium bromide (C16TAB) and sodium salicylate (NaSal) were used to generate micelles of varying profiles. Although the SDS and NaBr micelles were too similar in shape and too different in solution ionic strength, changes in behaviour explicitly from differing micellar profiles of the CTAB and NaSal micelles were successfully demonstrated. It was observed that the surface force behaviour was not sensitive to small changes to the micelle aspect ratio, however, once the micelles were elongated further the long range forces changed from oscillatory to that of a single attractive force well. The final section includes an inquiry into the influence of surface forces on droplet behaviour within and upon exiting a microfluidic device. Many different arrangements were tested using a variety of components including hexadecyltrimethylammonium chloride (C16TAC), sodium dodecyl sulphate (SDS), and polyvinylpyrrolidone (PVP) with the oils tetradecane, bromodecane, and perfluorooctane. One arrangement investigated the influence of close range attractive forces between droplets on droplet behaviour and breakup when flowing into an external stream. It was found that the behaviour of the drops was influenced by not only the attractive forces but also longer range repulsive forces that may prevent drops entering an attractive region. Another arrangement probed the collision between droplets with different interfacial coatings; a phenomenon not able to be investigated using bulk solutions and techniques. Overall it was found that investigating surface forces with microfluidics allows for new insights into colloidal solutions and properties.
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ItemCarbon capture using novel water-lean solvents( 2016)Global warming is attracting much research attention due to the continuous increase in global CO2 emissions. There are a number of strategies and technologies, which can be used to mitigate the CO2 emissions. One of these technologies is the absorption technology, which is not only the most mature of all, but also has the advantage of easy retrofitting to existing power plants that currently are the largest CO2 point sources. In order to achieve efficient CO2 absorption, a number of solvent systems are used, including aqueous monoethanolamine (MEA), diethanolamine (DEA), Selexol® and Rectisol®. Although MEA is the most widely used solvent to preferentially absorb CO2 from flue gases, its high regeneration cost, corrosive nature, thermal degradation and harmful byproducts have diverted the attention of researchers to find alternate solvents. More specifically, due the presence of water in MEA solvent, its regeneration cost is the highest among various solvent systems. In order to address these issues, one of the possible alternatives is the use of water-lean solvents, namely ionic liquids (ILs) and deep eutectic solvents (DESs). Ionic liquids (ILs) have shown significant potential, however their complex manufacturing procedure, toxicity, high viscosity, and expensive nature have hindered their application in the field of carbon capture and storage (CCS). In order to address most of these issues, researchers have been focussing on developing DESs, which are easy to manufacture and can be green in nature. In the present work, a modified Lydersen-Joback-Reid (LJR) method was combined with Lee Kesler’s mixing rules to estimate the critical temperature, critical pressure, critical volume, acentric factors, and normal boiling points of 39 different DESs. An independent density-based method was used to determine the accuracy of the method. Based upon the estimated critical properties, the densities of these 39 DESs were estimated and compared with the experimental values. The results showed that absolute deviations ranged from 0% to 17.4%. For DESs consisting of aliphatic precursors, the deviations ranged from 0% to 9.5%, whereas for DESs consisting of at least one aromatic precursor, the deviations ranged from 5.8% to 17.4%. The accuracy of the method decreased with an increase in the content of hydrogen-bond donors (HBD) in a DES. The method was also found to accurately take into account the variation of density with temperature. Later on, three choline chloride based DESs, namely reline, ethaline and malinine were chosen to study the CO2 absorption capacity at conditions prevalent in a post-combustion capture process. The experiments were conducted in a static vapour-liquid equilibrium (VLE) rig in the temperature range of 309 – 329 K and at pressures up to 160 kPa. Henry’s law constants of CO2 for these solvents were determined under these conditions and were found to be 43.38 MPa (for reline), 42.10 MPa (for ethaline), and 45.60 MPa (for malinine). Additionally, thermodynamic modelling using a modified Peng-Robinson (PR) equation of sate was used to correlate the experimental data. The results showed that the PR equation of state correlated the data satisfactory with maximum average absolute relative deviation of 1.6%. Furthermore, the Gibbs free energy, enthalpy of dissolution, and entropy of dissolution showed that the CO2 absorption was exothermic and the entropy of the system decreased as a result of the gas absorption. Finally, guanidine carbonate, malic acid, and ethylene glycol were mixed in 2:1:9.5 (PPIL-1) and 2:1:16.3 (PPIL-2) to prepare new solvents specifically for CO2 capture applications. Later, two more variants of PPIL-1 and PPIL-2 were prepared by taking 90wt% of each and mixing it with 10wt% arginine (hereafter called PPIL-3 and PPIL-4 respectively). Water content in these solvents was varied from 0.1wt% to 20wt%. Experiments were conducted to determine the CO2 capacity within a temperature range of 303.2 – 330.2 K and pressures up to 200 kPa. Additionally, viscosity of these solvents was also determined within a temperature range of 303.2 – 330.2 K. The results showed that the Henry’s law constants of CO2 for PPIL-1, PPIL-2, and PPIL-4 were 15.96 MPa, 23.57 MPa and 1.33 MPa, respectively. Furthermore, the viscosities of PPIL-1, PPIL-2, and PPIL-4 at 318.2K were found to be 49.8 mPa.s, 27.1 mPa.s, and 117.0 mPa.s, respectively. In comparison, the viscosities of reline, ethaline, and malinine at 318.2K were found to be 278.7 mPa.s, 31.5 mPa.s, and 203.6 mPa.s, respectively. Furthermore, suitable technologies like microscale systems and microencapsulated carbon sorbents (MECS), which can use these solvents for carbon capture, are still in the developing phase and further studies on their potential suitability to incorporate DESs are needed.
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ItemImproving success rates for in meso crystallization using integral membrane proteins and membrane protein mimetics in the bicontinuous cubic phase( 2016)The novel in meso crystallization method has facilitated the structural determination of several biologically relevant integral membrane proteins using macromolecular X-ray crystallography. To date, successful crystal growth from the lipid cubic phase is the bottleneck step in this process. An improved understanding of this technique can lead to increased success rates and more 3D structures of membrane proteins being solved, which are important for rational drug design for a wide range of diseases. Chapter 1 of this thesis outlines the relevant background about in meso crystallization, the cubic phase nanostructure, membrane proteins and peptides, methods for characterization and an outline of the thesis structure. While the mechanism of in meso crystallization is still not well understood, the bulk bicontinuous cubic phase has been well studied and is thought to be essential for crystallization in meso. In order to improve success rates for in meso crystallization we therefore need to understand the effect of multiple components on the cubic phase nanostructure within the context of a crystallization trial. An extensive review of the literature on the effect of additives, such as cholesterol and phospholipids that modify the fluidity of the lipid bilayer and soluble components like PEG and ions that are present in crystallization screens on the cubic phase nanostructures is therefore included in this thesis in Chapter 2. Lyotropic liquid crystal engineering design rules were established that can be used to improve the experimental design for in meso crystallization, since they will facilitate selection of the best lipid bilayer composition, cubic phase nanostructure and crystallization screen components. Crystallization screen conditions that control the water activity and rate of water evaporation are also favorable to maximize the likelihood of obtaining large and well-ordered membrane protein crystals. The effect of integral membrane proteins and peptides on the cubic phase nanostructure is equally of importance, as protein/peptide-lipid combinations that do not retain the cubic phase may not be suitable for in meso experiments. An improved understanding of the factors controlling peptide and protein encapsulation can also help to maximize protein loading, which is important to achieve supersaturation in the system to induce crystallization. These factors were studied using a wide range of different proteins and peptides, including synthetic WALP peptides with varying hydrophilic domain length, which are membrane protein mimics (Chapter 3). The Ag43 (Chapter 4) and BamA-E (Chapter 5) proteins from Gram-negative bacteria were also studied. It was found that the hydrophilic domain size and charge were the most important factors governing the changes in the lipid mesophase nanostructure. For this reason, cubic phase water channels that are larger than the hydrophilic domain of the protein may be preferred for in meso crystallization. In addition, screening of protein charges such that the Debye length is < 1 nm may be of use in preventing charge-induced swelling and disruption of the cubic phase. Hydrophobic mismatch and the diameter of the hydrophobic domain, the specific amino acid sequence of the proteins and local charges were also found to have an effect. This suggests that a strategy of matching of the hydrophobic segment of the lipid to the hydrophobic protein domain should also be used. It was observed in Chapters 3 and 4 that there were significant differences in protein and peptide α-helical and β-sheet secondary structures when present in detergent micelles compared to the lipid membrane of the cubic phase. Even though these conformational changes may be small, they could affect membrane protein function. The activity of the Neisserial amphiphilic protein Lipid A PEA Transferase (NmEptA) upon encapsulation in the cubic phase was therefore studied directly in Chapter 6. Transfer of the phosphoethanolamine (PEA) headgroup of different phospholipids that were doped in the cubic phase membrane indicated the enzyme was active, even though the large hydrophilic domain must be confined within the nanoscale water channels. This reaffirms that cubic phase water channels larger than the hydrophilic domain of the protein might be favorable for encapsulation. The enzymatic reaction could also be followed using high-throughput techniques, reinforcing the prospect of using high-throughput sample preparation and analysis methods as a drug screening tool in meso. In Chapters 7 and 8, the mechanism for in meso crystallization was studied. It is shown in Chapter 7 that small-angle neutron scattering can be used to study the location of peptides within the contrast-matched cubic phase. A preferential location of membrane proteins at the flat points of the cubic phase was suggested in several modelling studies. This arrangement was suggested to be a driving force for nucleation during in meso crystallization. No enrichment was observed, however, for the model WALP21 and WALPS53 peptides at the flat points or most negative Gaussian curvature saddle points of the diamond cubic phase of phytanoyl monoethanolamine indicating the importance of characterizing proteins with different lengths or physicochemical characteristics. The work presented in this thesis is the first time that contrast-matching techniques for neutron scattering were used for experiments with the bicontinuous cubic phase. This study is therefore expected to provide new insights into the assembly of these complex hybrid protein-lipid materials and to assist further studies. In Chapter 8, the proposed mechanism for in meso crystallization was explored during crystallization of the single transmembrane α-helical peptide DAP12-TM. Small-angle X-ray scattering results with a micro-sized beam were found to be consistent with the proposed mechanism for in meso crystallization; crystallization occurred from the gyroid cubic mesophase via a highly oriented local lipid lamellar phase. No other direct experimental evidence for this mechanism has been published since a single study on bacteriorhodopsin in 2007. A new experimental protocol using equipment available in standard crystallography laboratories should enable further testing of the proposed mechanism using different proteins. Diffraction peaks at wide angles related to the peptide crystals were also observed, which are of potential use in locating crystals in meso, as they are often difficult to observe within the lipid cubic phase. In Chapter 9 the conclusions and future perspectives of this thesis are presented. The research includes important new insights with respect to lipid–protein interactions, the location of amphiphilic peptides when encapsulated in the bicontinuous cubic phase and the mechanism of in meso crystallization. Advances in our understanding of the relationship between the lipidic material and the encapsulated protein as a result of this work should lead to improvements in experimental design for in meso crystallization and subsequently a higher success rate for this promising technique.
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ItemThe incorporation of Curcuminoids in oat fibre extruded products( 2016)Curcuminoids are polyphenolic bioactive ingredients found in the roots of the plant Curcuma Longa. These compounds have health benefits and can be used to develop health promoting functional foods but the low water solubility of these compounds limits their bioavailability. One of the main approaches to overcome the low solubility and stability of curcuminoids in an aqueous environment is to make use of the interaction between curcuminoids and other food ingredients. Oat dietary fibre is one such food ingredient that could potentially be used as a carrier for curcuminoids. Oat fibre is known to have health benefits for humans, including lowering cholesterol serum, the risk of coronary heart disease and blood pressure. Dietary fibre has a potential to interact with polyphenolic compounds, such as the curcuminoids, by a number of mechanisms but the possible interactions between curcuminoids and oat fibre ingredients have not been studied to date. Extrusion cooking is one of the main technologies known to have a great potential for the manufacture of snack products. Bioactive components can be added to extruded snacks in order to improve their health benefits but these components can degrade during extrusion processing. The possible effect of bioactive addition on the physical properties of extruded products should be also considered. This thesis aims to investigate the potential of oat fibre ingredients as a carrier for curcuminoids. It also aims to examine the feasibility of producing a curcuminoid-enriched oat fibre-corn based extruded product, with a focus on curcuminoid stability during extrusion processing. It also aims to evaluate the effect of curcuminoid addition on the physical properties of extruded products. The thesis is divided into three main sections: 1. The first section focuses on the use of oat fibre as a potential carrier material to increase the solubility and stability of curcuminoids. Studies are carried out to understand the interaction between curcuminoids and the oat fibre ingredients. The stability of the curcuminoids in the presence of oat fibre materials during storage was also determined. 2. In section two, the effect of extrusion technology on the physico-chemical properties of oat fibre containing 28 % β-glucan are examined. The physico-chemical characteristics of oat fibre including molecular weight, soluble solids content, water absorption index, dynamic vapour sorption, thermal and pasting properties were measured after extrusion using two feed or barrel moisture contents (50 % - 60 %) and two screw speeds (200 rpm – 300 rpm) and results were compared with the properties of non-extruded oat fibre. 3. In the third section, extrusion technology was used as a delivery method for the production of a curcuminoid enriched oat fibre-corn based extrudates. This section focuses on the stability of curcuminoids, as affected by extrusion conditions, including the two levels of feed moisture content and two levels of screw speed. In addition, the effect of curcuminoids on the physical characteristics of extrudates including bulk density, expansion, hardness and colour were investigated. The spectroscopic experiments showed that both protein and β-glucan components of oat fibre are able to interact with curcuminoids. Solubility experiments indicate the curcuminoids in the supernatant fraction of a 1 % w/w oat fibre dispersion in 2 % v/v EtOH (88 μg/mL) increased by a factor of 21 compared to only 2 % v/v EtOH (4.1 μg/mL). This concentration of curcuminoids in the supernatant is also much higher than that of the reported for the solubility of curcuminoids in aqueous media (11 ng/mL, pH 5). In the presence of oat fibre materials, curcuminoids were converted from a crystalline to an amorphous state, as observed by Wide-angle X-ray powder diffraction. The amorphous state of the curcuminoids in the precipitate of (25.8 μg/mL) curcuminoids−oat fibre (1% w/w TS) dispersion with 2 % v/v EtOH resulted in higher stability for curcuminoids in the precipitate rather than supernatant of the oat fibre dispersion. These findings show the potential of oat fibre as a carrier for curcuminoids in functional foods. Extrusion of oat fibre with high levels of β-glucan under conditions of high moisture did not significantly change the molecular weight of the soluble fraction, the total soluble solids, the water absorption index and thermal properties of oat fibre. A higher specific mechanical energy was found to result in an increased specific surface area and absorption of water vapour as a surface monolayer. The viscoelastic properties of oat fibre were also maintained after extrusion. This study indicates that extrusion processing is a promising technology to produce extruded products based on oat fibre where the functional properties and potential health benefits of oat fibre are preserved. The physical properties of oat fibre based extrudates containing curcuminoids were significantly affected by feed moisture content, whereas the effect of screw speed and curcuminoid addition was not significant. Higher feed moisture resulted in darker extruded snacks with higher bulk density, hardness, 90 % retention of curcuminoids after extrusion and drying but lower expansion. Curcuminoids were also stable in dried extruded products during 80 days of storage at 25 °C. These studies provided information for the selection of process variables for extrusion. The supporting compositional evidence will help with the introduction of curcuminoid-enriched extruded snacks as a new product category in the functional food market. In conclusion, this study showed that both protein and β- glucan components of oat fibre are able to interact with curcuminoids and increase the solubility of curcuminoids in an aqueous solution of 2 % v/v EtOH. It is possible that curcuminoids also interact with proteins and dietary fibres in the precipitated fraction. These findings illustrate the potential for the curcumin carrying capacity of oat fibre to be capitalized upon in the fortification of food with curcuminoids. The application of extrusion processing to modify the functional properties of oat fibre, showed that current extrusion conditions can be used to produce extruded products from a commercially available oat fibre preparation with high β-glucan content where the properties of the extrudates were largely preserved and the health benefits are expected to be similar with non-extruded oat fibre. The ability to achieve high retention of curcuminoids (~ 90 %) at feed moisture content of 35% and screw speed of 200 rpm or 300 rpm during extrusion combined with the stability of the curcuminoids in the dried extruded snack shows the potential to improve the health benefits of oat fibre extruded products by the incorporation of curcuminoids.
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ItemBlocking endocytosis: a novel cancer treatment( 2016)This thesis studied the modified GO as a potential therapeutic-tracking conjugate. The imaging/tracing properties and toxicity effects of the proposed system was investigated. This system has shown encouraging results for cancer therapy in a selectively toxic way considering the observed cytotoxic and synergistic effects with the conventional chemotherapeutic drugs.
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ItemStructurally nanoengineered peptide polymers for combating multidrug-resistant bacteria( 2016)Antimicrobial resistance has been named as one of the clinical ‘super-challenges’ of the 21st century. With the rise and prevalence of multi-drug resistant (MDR) ‘superbugs’, such as methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Enterococcus (VRE), and more recently, the ‘ESKAPE’ pathogens, a return to the pre-antibiotic era is rapidly becoming a reality in many parts of the world. Infections caused by MDR pathogens are a major burden to modern healthcare, as the available treatment options are drastically reduced, leading to increased treatment costs, and high morbidity and mortality rates. However, this growing epidemic of infections caused by MDR pathogens has not been accompanied by an increase in the discovery of novel antimicrobials. In fact, it has been reported that aside from a few narrow spectrum drugs and teixobactin, no new chemical class of antibiotics has appeared in the last 40 years. The challenge remains to develop antimicrobial agents with new mechanisms of action that can overcome acquired resistance without contributing to resistance development. Over the past few years, our work in drug and gene delivery has demonstrated the potential of star peptide polymers as therapeutic agents, with significant advantages over linear peptides. Building on our prior knowledge, this thesis explores the possibility of using macromolecular engineering techniques to design and develop star peptide polymers that could function as novel antimicrobial agents capable of combating antibiotic-resistant bacteria. Inspired by antimicrobial peptides (AMPs) that form part of the innate immune response in multicellular organisms, 16-and 32-arm star peptide polymers were synthesized, with arms composed of cationic and hydrophobic amino acid moieties co-polymerized in a random fashion. The star polymers were found to demonstrate superior efficacy against clinically-relevant Gram-negative bacteria, including MDR species, compared to their linear ‘one arm’ equivalent and several well-known AMPs, while possessing high therapeutic indices. The lack of any observable bacterial resistance development against the star peptide polymers was attributed to their unique, multi-modal antimicrobial mechanism, which differs from that of antibiotics and AMPs. Based on these attributes, the star peptide polymers were classified as a new class of antimicrobial agents, referred to as ‘Structurally Nanoengineered Antimicrobial Peptide Polymers’ (SNAPPs). The subsequent part of this thesis focused on developing further understanding on the structural design and bio-nano interactions of SNAPPs. Through a structure-activity relationship study, the effects of the star arm (co)polymer structure and overall macromolecular architecture on antimicrobial activity and biocompatibility were investigated. Further, the behavior of SNAPPs in physiologically-relevant settings, such as in a bloodstream-mimicking environment, was probed in terms of their antimicrobial efficacy and mode of action. Lastly, this thesis also demonstrated the ability of SNAPPs to synergize with different classes of antibiotics, potentially offering a method to ‘revive’ antibiotics that have already been deemed ineffective. Collectively, the discovery and development of SNAPPs, as presented in this thesis, represents a breakthrough in the fight against infections caused by antibiotic-resistant bacteria and, hence, a significant advancement in the field of antimicrobial research. This thesis also provides fundamental understanding on the properties and performance of SNAPPs, which will be useful for the optimization of next-generation SNAPPs with enhanced antimicrobial performance and minimal toxic side-effects in vivo. It is thus with hope that this thesis will not only serve as a platform for the development of SNAPPs for actual clinical use, but also act as a stimulus for other researchers in the pursuit of innovative and more effective treatment methods against ‘superbugs’.
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ItemEngineering biomacromolecule-based particles with tunable functionality in biological systems( 2016)Particles with tailored physicochemical properties have numerous applications in diagnosis, therapy, and management of human diseases. In this context, elucidation of the interactions between biological systems and nanoengineered materials has emerged as an important research discipline, with the ultimate aim of controlling the interactions to achieve desired physiological responses. Biomacromolecules, such as peptides, proteins and polysaccharides, have diverse physiological functions, such as target recognition, signaling, and catalysis, which remain a challenge to mimic by synthetic methods. Biological systems precisely control synthesis, assembly, and disassembly of the biomacromolecules to guide physiological events. Therefore, controlled assembly of biomacromolecules into nano- and microscale particles may offer a promising platform to study bio-nano interactions, and ultimately to engineer functional materials for biomedical applications. However, previous studies have primarily been limited to particles assembled from biomacromolecules with little function. In this thesis, a robust strategy of assembling functional biomacromolecules into particles is developed, through the use of porous particles as sacrificial templates and reversible chemistry integrated into the biomacromolecular network. The advantages of this strategy include simplicity, versatility, tunability of particle morphology, triggered disassembly, and bioactivity that can be triggered in certain biological conditions. Three types of biomacromolecule-based particles were engineered: (1) peptide nanoparticles with proapoptotic activity (Chapter 3), (2) protein particles with pH-triggered recovery of enzymatic activity (Chapter 4), and (3) polysaccharide-based particles that can be targeted to tumour associated macrophages and Escherichia Coli (Chapter 5). These systems are used to demonstrate how the functionality of the particles in biological systems can be tuned using a chemistry and materials science-based approach.
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ItemMagnetic manipulation of cells for soft tissue engineering( 2016)Engineering adipose tissue for healing defects caused by injury or trauma is a very important aspect of tissue engineering. These problems may affect the quality of patient`s lives cosmetically or mentally other than the functional impairment. However, challenges can arise in the application of scaffolds in tissue engineering due to issues including inflammatory reactions caused by biomaterials or their degradation products and also foreign body reactions. In addition, the rate and extent of cell migration and vascularisation may not be adequate, and cell-cell interactions in co-culture may be lacking, especially in large constructs. Moreover, it is known that mechanical stimulation of cells can affect their morphology and behaviour. But, using regular methods of imparting force to adipose tissue may cause problems such as tissue damage and infection, as well as difficulties in measurement of the exact forces applied to cells. To address these issues, scaffold-free tissue engineering approaches are very attractive. One such concept is the use of magnetic particles (MPs) to label and thereby manipulate cells by application of an external magnetic field (MF). In this thesis, 3T3-L1 preadipocyte and 3T3 fibroblast cells, as two of the main components of adipose tissue, were labelled with magnetic micro-particles to investigate the potential of using magnetic manipulation in 2D and 3D culture for soft tissue engineering. Large size of particles can be used to reduce the possibility of engulfment by cells and thereby reduce the adverse effects on the cells’ function compared to nanoparticles. Also, tunability and possibility of applying higher magnitudes of force to cells can be achieved. We investigated the effect of MF in patterning the cells in 2D culture as well as generation of 3D cell constructs. Pre-adipocyte cells were cultured in different patterns by changing the shape of the magnets. Also, their co-culture with fibroblast cells could be shaped to layer-by-layer or core-shell structures using different types of magnets. Rapid construction of uniform and stable 3D cell spheroids from 3T3-L1 pre-adipocytes was observed in presence of MF which had higher density and more symmetric structure compared to the centrifuge method as the standard method. These spheroids could be fused together to produce larger and more complicated structures for future tissue engineering applications. Also, co-culturing with 3T3 fibroblasts could convert the spheroids to hybrid multi-cellular aggregates. Moreover, MF was utilized for mechanical stimulation of labelled cells with MPs in 2D and 3D culture. Two dimensional results showed that using the mechanical stretch did not change the proliferation of fibroblast and pre-adipocyte cells, significantly. However, following the compression concept by placing the magnets under the plates led to a reduction in their proliferation which was more evident in samples with uncoated MPs compared to RGD-coated particles. Also, differentiation of pre-adipocytes towards the mature adipocytes was not affected significantly when MPs and MF were utilized. In 3D culture, while spheroids supported the differentiation and proliferation of pre-adipocytes in 10 days, presence of MF did not cause a significant difference likely due to the low MF gradient and small resulting magnetic force. For migration studies, high density area of spheroids was influenced and the migration towards the magnets was enhanced. In conclusion, the results of this thesis showed how micro magnetic particles and magnetic field can be used for production 3D spheroids as well as mechanical stimulation of cells in 2D and 3D culture which represents a promising approach towards the success of soft tissue engineering.
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ItemSynthetic polypeptides for biomedical and bioactive applications( 2016)Synthetic polypeptides are bioinspired mimics of natural polypeptides, readily prepared through controlled synthetic polymerization processes. Their use has offered chemists and biologists around the world the ability to precisely control the synthesis, scale-up, modification and engineering of polypeptides with properties similar to those seen in the natural world. The plethora of possible functionality gives synthetic polypeptides an array of properties shown to be highly useful in the biomedical and antimicrobial (bioactive) fields. Despite this, significant deficiencies surrounding the application of synthetic polypeptide materials in these fields remain. This thesis reports on the fabrication and testing of novel synthetic polypeptide and synthetic polypeptide-based materials to address deficiencies related to the application of synthetic polypeptides in nanoparticle drug delivery, cellular scaffolds for tissue engineering and antimicrobial materials for water treatment. For drug delivery, investigations into the use of synthetic polypeptide-based nanoparticles for cisplatin delivery have traditionally focused on micelle assemblies. Different synthetic polypeptide-based self-assemblies such as vesicles offers the prospect of introducing a new biocompatible and biodegradable architecture for cisplatin delivery. In this study, the preparation of novel cancer-targeting synthetic polypeptide-based vesicles for cisplatin drug delivery is described. The vesicles were prepared through a novel drug-induced self-assembly process. Folic acid was conjugated to the vesicle corona to form an active targeting drug delivery system. In vitro studies on these targeted vesicles showed significantly higher cellular binding/uptake and dose-dependent cytotoxicity toward cancerous cells (HeLa) compared to non-cancerous cells (NIH-3T3). Next, preliminary studies into the preparation of aptamer (advanced targeting ligands composed of single strand nucleotides) targeted synthetic poly(L-glutamic acid)-based drug delivery systems was investigated. Poly(L-glutamic acid) or PLG, has been utilized in a range of synthetic polypeptide-based drug delivery systems owing to its biocompatibility and favorable enzymatic biodegradability profiles. Whilst aptamers have been used as advanced targeting ligands in a wide range of polymeric nanoparticle drug delivery systems, they have yet to be investigated in delivery systems composed of PLG. Conjugation of a model single stranded DNA (ssDNA) aptamer to poly(ethylene glycol)-b-poly(L-glutamic acid) (PEG-b-PLG) block copolymers, common synthetic PLG-based nanoparticle precursors, was achieved through thiol-maleimide coupling chemistries and the conjugates successfully isolated through preparative gel electrophoresis. The DNA-polymer conjugation and isolation protocols established in this work offer potential use in future studies employing aptamer-targeting of PLG-based delivery systems. For tissue engineering, the biocompatible, biodegradable and cell adhesive properties of synthetic polypeptides makes them useful materials for the fabrication of 3D polymeric hydrogels with macroporous morphologies ideally suited for cell in-growth. Traditionally, synthetic polypeptides have been used as partial components of these gel networks, and often require side-chain modifications to allow for cross-linking to take place, thus hindering the effective study of these materials as cellular scaffolds. In this study, macroporous hydrogels composed entirely of synthetic polypeptides were prepared through direct cross-linking of a single poly(L-glutamic acid)-b-poly(L-lysine) (PLG-b-PLL) polypeptide component under cryogelation conditions. Tuning the relative ratios of the amino acid constituents could result in cryogels with very different pore structures, swelling, and mechanical properties, suitable for a range of soft tissue engineering applications. These cryogels were shown to be enzymatically biodegradable and demonstrated excellent biocompatibility, cell attachment and cell proliferation profiles with mammalian fibroblast (NIH-3T3) cells. The inherent antimicrobial (bioactive) properties of peptides are utilized in this study through the preparation of synthetic polypeptide-based cryogels with inherent antimicrobial (bioactive) properties, for potential water purification applications. Traditionally, the effective bioactive properties of antimicrobial cryogels come from the incorporation of known antimicrobial agents to the gel structure rather than from the polymer itself. The leaching of these toxic agents is commonly reported in these systems, leading to potential toxicity issues. Cryogels composed of a polycationic poly(L-lysine) and hydrophobic poly(D,L-valine) copolymer were prepared with the gels displaying high swelling, and inherent antimicrobial activity against E. coli after brief 1 h exposure, with no toxic leaching. Compared to a conventional ‘nanoporous’ hydrogel, the cryogel macropores and their integrity were found to be crucial for bactericidal activity where they allow for effective uptake of bacteria into the gels, and provide a confined environment and increased surface area for contact of the bacteria with the antimicrobial polymer walls. The materials prepared in this thesis and the study of their properties, demonstrate an advancement in the scientific understanding and applicability of synthetic polypeptides in the relevant biomedical and bioactive fields.