School of Chemistry - Theses

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    Toward the synthesis and analysis of selenium-containing glucocorticoid prodrugs
    Macdougall, Phoebe Eleanor. (University of Melbourne, 2007)
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    NMR studies of amyloid ab-peptide in membranes
    Lau, Tong Lay (Crystal) (University of Melbourne, 2006)
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    Development of flow methods for the determination of salivary aldehydes and cotinine for oral cancer risk assessment
    Ramdzan, Nur' Adlin ( 2015)
    Excessive tobacco and alcohol consumption have been established to be major risk factors for the development of oral cancer. Together, they exert a synergistic carcinogenic action, accounting for about 67% of all oral cancer incidences. Early detection of oral cancer can increase the options and success of medical treatments. However, the development of simple, accurate and non-invasive analytical techniques to assist with these diagnoses is in itself a great challenge. Hence, there is a widespread need for the availability of sensitive and precise salivary diagnostic methods to assess patients’ risk of oral cancer development. This thesis aims to address these problems by developing alternative analytical methods for the determination of acetaldehyde, formaldehyde and cotinine in saliva using flow-based methodologies (viz. flow injection analysis, sequential injection analysis and paper-based microfluidics). A simple gas-diffusion flow injection method has been developed for the on-line determination of salivary acetaldehyde by its colour reaction with 3-methyl-2-benzothiazolinone hydrazone (MBTH) and ferric chloride. Under the optimum working conditions the method is characterised by a sampling rate of 9 h-1, a linear concentration range of 0.50 – 15 mg L-1 (Absorbance = 5.40 × 10-2 [Acetaldehyde, mg L-1], R2 = 0.998), a relative standard deviation (RSD) of 1.90% (n=10, acetaldehyde concentration of 2.5 mg L-1), and a limit of detection (LOD) of 12.3 μg L-1. These figures are comparable to those of the conventional gas chromatography (GC) method (RSD = 1.66 % (n=10) and LOD = 93 μg L-1). In addition, saliva samples spiked with acetaldehyde have yielded excellent recoveries (96.6 – 101.9%), comparable to those obtained by GC (96.4 – 102.3%), illustrating the reliability of the proposed method. A novel automatic sample pretreatment methodology based on the use of a micro-solid phase extraction bead injection lab-on-valve (μSPE-BI-LOV) system, coupled at-line with a hydrophilic interaction liquid chromatography (HILIC) analysis, for the determination of salivary cotinine has also been developed. The SPE methodology is based on the bead injection (BI) concept in a mesofluidic lab-on-valve (LOV) flow system to automatically perform all SPE steps. A HILIC column has been chosen based on its potential for achieving higher sensitivity and better retention of polar compounds such as cotinine. Using this approach, the working concentration range has been found to be from 5 - 500 μg L-1. The corresponding μSPE-BI-LOV system has been proven to be reliable in the handling and analysis of viscous biological samples such as saliva, achieving a limit of detection and quantification of 1.5 and 3.0 μg L-1, respectively, with a sampling rate of 6 h-1. A low cost, disposable and easy to use microfluidic paper-based analytical device (μPAD) has been developed for the simple and non-invasive determination of total aldehydes in saliva samples as well. The μPAD has been fabricated by wax printing and the colorimetric reaction between aldehydes and MBTH has been used, as in the gas diffusion flow injection method. Under optimal conditions, the μPAD is characterised by a working range of 20.4 – 114 μM, limit of detection of 6.1 μM, and repeatability of less than 12.7% (n=5). There is no significant difference at the 95% confidence level between the results obtained by the μPAD and the reference method (Student’s t-test: 0.090 < 0.38). The optimized μPAD has been found to be stable for more than 41 days when stored in a freezer and in vacuum sealed zipper bags (≤ -20 ºC).
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    Controlling stem cell fate via chemical inhibition of DYRK kinases
    Bellmaine, Stephanie Frances ( 2015)
    A high-throughput screen for small molecules that can enhance the self-renewal of embryonic stem (ES) cells led to the discovery of ID-8, an indole derivative that can maintain the pluripotency of ES cells in long term culture, under conditions that usually lead to spontaneous differentiation. A biotinylated derivative of ID-8 was found to bind to DYRK kinases, and DYRK1A miRNA knockdown elicited the same enhancement of ES self-renewal as ID-8 treatment. Of the five mammalian DYRK isoforms, DYRK1A is best characterised due to its overexpression in Down's Syndrome (DS), and is believed to promote neural differentiation processes, thus contributing to the neurodegenerative phenotype of DS. In this thesis we provide the first evidence that ID-8 is a potent DYRK1A inhibitor and through kinase profiling demonstrate that ID-8 is selective for DYRK1A and other closely related kinases. We investigate the effects of ID-8 on human ES cell processes, and show that it is a potent inhibitor of neural induction. A range of ID-8 analogues were synthesized and used to establish structure-activity-relationships for the ability of this class of small molecules to inhibit DYRK1A and related kinases, and neural induction of human ES cells, leading to the discovery of 28, an ID-8 analogue with more potent ability to inhibit neural differentiation. Finally, we show that structurally-unrelated DYRK1A inhibitors from the literature do not inhibit neural induction of human ES cells to the same extent as ID-8 and 28, suggesting that effective DYRK1A inhibition is not the sole criterion necessary for inhibition of neural differentiation.
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    Functional conjugated polymers: from design to devices
    Song, Aaron Zhifan ( 2015)
    Conjugated polymers are among the most promising materials for future technologies. These organic materials combine many of the properties of conventional polymers with the behaviour of inorganic semiconductors. The low-cost and solubility of many conjugated polymers make them attractive materials for use in future electronic and optoelectronic devices, such as field-effect transistors (FETs), polymer light-emitting diodes (PLEDs) and organic solar cells (OSCs). However, both design and synthesis of conjugated polymers with well-defined structures involve many intellectual and technological challenges. Although conjugated polymers of simple aromatic monomers have been studied in great detail, to date there is no sound methodology in rationalised design and synthesis of functional conjugated polymers. Therefore, controlling and fine-tuning the properties of conjugated polymers remains rather difficult to achieve, and thus limits their applications in devices. The purpose of this research is to develop a coherent strategy for the design and synthesis of functional conjugated polymers with well-controlled structures and properties, from simple aromatic molecules. The history, background and applications of conjugated polymers are reviewed before introducing the central concept in this work - modular design and synthesis of new materials. The approach adopted seeks to develop “greener” pathways to target materials, by avoiding many highly toxic solvents, highly corrosive reagents, and expensive organometallic catalysts. Some simple reactions in water or ethanol were utilised to synthesise early building blocks in large quantities, while Horner-Wadsworth-Emmons (HWE) olefination and Kumada coupling reactions were employed to construct carbon-carbon double and single bonds, respectively. A group of three conjugated polymers were first synthesised to investigate the practicality of the modular approach. These polymers were based on the alternating copolymer of phenylenevinylene (PV) with (2-ethylhexyloxy-4-methoxy)phenylenevinylene (MEHPV), or alt-co-MEH-PPV. Each polymer consists of three types of aromatic monomers in an alternating pattern. The properties of the novel polymers were compared to their parent polymers to reveal effects of inserting an additional type of monomer. Subsequent work investigated the synthesis and properties of conjugation confined MEHPPV polymers. The target polymers differ from polymers of similar type studied by other groups in both synthesis and properties. Regioregular, bifunctional oligomers of MEHPV were first synthesised, then polymerised with 4,4’-bis(methylenediethylphosphonate)-1,1’-biphenyl (bis(MDEP)-biphenyl) to yield poly(MEHPV-oligomer-alt-co-bp) polymers. These polymers have a higher density of chromophores, and demonstrated a clear structure-property correlation in their photophysical properties. Furthermore, a simple, empirical quantitative model was developed to describe the basic absorption and emission characteristics of the polymers. Successes in developing an efficient synthesis of bifunctional MEHPV oligomers paved the paths towards conjugated polymers with increased structural complexity. Similar schemes were developed to synthesis bifunctional oligomers of 3,4-dihexylthienylenvinylene (DHTV). Using a combinatorial matrix approach, varying the combination of the MEHVP and DHTV oligomers modules produced 16 conjugated polymers with well-defined structures. Basic photophysical properties of these polymers were studied in some detail and a number of simple quantitative, empirical models were discovered from data analysis, which described their absorption and emission characteristics to a high degree of accuracy. Photovoltaic devices were fabricated with selected polymers as the active layer in blends with PC60BM and an efficiency of 2.0% was obtained. The device performance was superior compared to devices utilising MEH-PPV or PTV. The combined combinatorial-modular approach was then used to synthesise another type of copolymers of interest, those of MEHPV and regioregular 3-hexylthiophene (3HT) oligomers. MEH-PPV and P3HT were chosen as they are among the most intensely investigated conjugated polymers. An efficient, palladium free synthesise of 3HT oligomers was developed, and the bifunctional modules were then combined with MEHPV modules to produce 12 alternating copolymers of MEHPV and 3HT oligomers with well-defined structures. The absorption and emission characteristics showed the limitations of the empirical model developed in the other synthesised polymers, demonstrating the complex role of polymer chain morphology in solutions and the solid state polymer photophysics. At Finally, 4 polymers consisting of DHTV and 3HT oligomers were synthesised and their basic photophysical properties were determined.
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    Enhanced synthetic methods and stabilization of insulin-like peptides
    Karas, John Andrew ( 2015)
    Basal-bolus insulin therapy is essential for maintaining tight glycemic control in patients with type 1 diabetes. Despite recent advances, there are issues with its long-term heat stability, which particularly affects patients living in poorer tropical regions. Hence there is a need to develop more thermally stable analogues of insulin. Some degradation products are caused by disulfide shuffling which leads to oligomerization. This could be avoided by substituting the disulfide bonds with non-reducible cystine isosteres such as cystathionine, but incorporation of non-natural mimetics in peptides generally requires chemical synthesis. All current insulin therapeutics on the market are produced biosynthetically due to the large mass of material required and there are currently no chemical methods available that rival these yields. A more efficient chemical synthesis is required to make a modified analogue viable. Described herein are three key accomplishments which attempt to address the issues raised above. Firstly, an improved method for forming disulfide bonds in peptides utilizing a photocleavable thiol protecting group in combination with an “activated” thiol is reported. The efficacy of this new approach was established in simpler model systems and then applied to insulin, whereby higher yields were achieved compared to existing synthetic methods. Secondly, a proof-of-concept study was designed such that lactam bridges were incorporated into insulin-like peptide 3 as replacements for the intermolecular disulfide bonds. The insights gained from this study helped formulate new approaches for incorporating non-natural mimetics in insulin. Finally, replacement of the A6 – A11 disulfide bond with cystathionine in insulin was achieved via the use of orthogonally-protected monomeric building blocks. This new analogue was synthesized in excellent yield and has been fully characterized. It was found to have native binding activity for both insulin receptors and a tertiary structure similar to human insulin. Its serum stability was maintained and it exhibited improved thermal stability, which could lead to an enhanced therapeutic.
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    Development of novel polymer inclusion membranes by casting and electrospinning and study of their extraction properties for thiocyanate
    O'Bryan, Yukie ( 2015-08-11)
    Polymer inclusion membranes (PIMs) are a type of liquid membranes in which a liquid extractant is immobilised in a polymer matrix. Separation processes using PIMs have gained increasing interest as an alternative to solvent extraction. A typical PIM consists of a base-polymer and an extractant (carrier) and it may also contain a plasticiser and/or a modifier. Most PIMs that have been developed so far have used either poly(vinyl chloride) (PVC) or cellulose triacetate (CTA) as the base-polymer and the use of other polymers as base-polymers has been limited. This thesis describes the development of PIMs using non-conventional base-polymers and the study of their extraction properties for thiocyanate. Also, fibrous materials in which a liquid extractant (Aliquat 336) was immobilised in a polymer matrix were prepared by electrospinning. These materials were tested for their suitability as packing materials for online preconcentration columns incorporated in a flow injection system for the determination of thiocyanate. PIMs containing poly(vinylidene fluoride-co-hexafluoropropylene) (PVDF-HFP) as the base-polymer and Aliquat 336 as the extractant were prepared by solvent casting using tetrahydrofuran as the solvent. It was found that membranes containing between 20 and 40% Aliquat 336 were capable of extracting thiocyanate. The PVDF-HFP-based PIMs showed significantly higher extraction and back-extraction rates than the corresponding conventional PVC-based PIMs. Also, these PIMs exhibited initial flux values about an order of magnitude higher than those of previously reported Aliquat 336/PVC PIMs. However, slow leaching of the extractant was observed over repeated use which resulted in membrane performance loss. PIMs were prepared based on a matrix of PVDF-HFP and crosslinked poly(ethylene glycol) dimethacylate (PEG-DMA). The presence of PEG-DMA significantly improved the membranes’ thiocyanate flux values and reusability. These PIMs could be prepared by solvent casting followed by photocrosslinking. The PIM that contained 40% Aliquat 336 in 5:5 PVDF-HFP:PEG-DMA had the highest transport rate among the PIMs studied and showed good reusability for 5 repeated experiments. Surface modification of a PVDF-HFP-based PIM was performed in an attempt to create a layer of positively charged functional groups in order to minimise extractant leaching. PIMs were treated with water or air plasma to introduce the OH group on the surface, followed by the reaction of the OH groups with a silane reagent containing a quaternary ammonium group to form covalent bonds. The presence of some quaternary ammonium groups on the PIM surface was confirmed by XPS after the reaction, however, these PIMs did not show improvement in stability. Copolymers of methyl methacrynate (MMA) and n-decyl methacrylate (DMA) with different MMA:DMA ratios were synthesised and tested for their suitability as basepolymers for PIMs containing Aliquat 336. The copolymer containing 3:1 MMA:DMA was found to be compatible with Aliquat 336 and the thiocyanate extraction rate of this PIM was comparable to that of the corresponding conventional PVC-based PIM. An electrospun fibre mat containing PVDF-HFP and Aliquat 336 was prepared and evaluated for its suitability as the column packing material for preconcentration of thiocyanate in a flow injection system. The column prepared did not provide successful enrichment of thiocyanate. This was likely to be because the pore size of the fibre mat was too small and the liquid flow in the preconcentration column was not uniform under the low pressures used in the flow injection system. The results suggested that loose fibres should serve better for this application. In response to this, a PIM wool was prepared from polystyrene and Aliquat 336 by electrospinning. This fibrous material was successfully used as packing material in a tubular preconcentration column in a flow injection system for the determination of This flow injection system is characterised by a linear calibration range between 0.02 and 1.0 mg/L with a correlation coefficient of 0.999 of the calibration curve and a preconcentration factor of 20.
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    Interactions of a lytic peptide with supported lipid bilayers investigated using surface-selective techniques
    Rapson, Andrew Cyrus ( 2015)
    The development of antimicrobial resistance, which is the ability of microorganisms to prevent cell death by antimicrobial agents (i.e. antibiotics, antivirals), is an increasing problem. Traditional antimicrobial drugs target aspects of microorganisms responsible for infectious diseases cell structure than can be altered, greatly reducing their cell-killing efficiency. To combat this, research has been performed into the application of naturally-occurring antimicrobial peptides, sourced from venoms or host-defence systems of plants and animals. These peptides display broad-spectrum cell-killing activity by directly attacking the lipid matrix of cell membranes, thereby bypassing the means through which cells develop resistance. In this study, a proline-substituted, fluorescently-labelled analogue of the antimicrobial peptide melittin (a major component of venom from the European honey bee) was designed and synthesised. The, analogue, named melittin P14K-Alexa 430 (MK14-A430), displayed twice the membrane-lytic activity of native melittin through means of a lipid vesicle dye-release assay. The interactions of MK14-A430 with lipid membranes consisting of 1,2-dipalmitoyl-sn-glycero-3-phoshatidylcholine (DPPC) were investigated using circular dichroism and surface-selective analytical techniques. The structure and orientation of MK14-A430 were studied by circular dichroism and oriented circular dichroism. In contrast to native melittin, MK14-A430 displayed high helicity under solution conditions mimicking physiological conditions. This helicity was retained in small unilamellar vesicles, except at low peptide density in ordered DPPC vesicles. In oriented lipid multibilayers, MK14-A430 inserted completely into a transmembrane orientation in fluid-phase DPPC, while only a fraction inserted in ordered-phase bilayers (up to an observed maximum of 25%). The interaction mechanism of MK14-A430 with supported lipid bilayers (SLBs) was studied using quartz crystal microbalance with dissipation monitoring. MK14-A430 initially associated with all SLBs in a surface-aligned state, after which different behaviour was observed depending on the lipid phase. Similar to the oriented circular dichroism experiments, MK14-A430 inserted completely into a transmembrane orientation in fluid-phase DPPC, while a significant fraction in a membrane surface-aligned state was observed for both ordered-phase SLBs. The predominating orientation of MK14-A430 could be reversibly changed with the lipid phase: transmembrane above the phase transition temperature and surface-aligned below. No mass removal indicative of detergent-like membrane solubilisation was observed. The location and mobility of MK14-A430 was assessed through fluorescence of the AlexaFluor 430 label by time resolved, evanescent wave-induced fluorescence spectroscopy and anisotropy. MK14-A430 penetrated the lipid headgroup structure in pure, ordered-phase DPPC membranes but was located near the headgroup-water when cholesterol was included. MK14-A430 formed lytic pores in DPPC SLBs, and an increase in pore formation with incubation time was observed through an increase in polarity and mobility of the probe. When associated with the DPPC-Chol SLB, the probe displayed polarity and mobility that indicated a population distributed near the lipid headgroup-water interface with MK14-A430 arranged predominantly in a surface-aligned state. The information from this study showed that the lytic activity MK14-A430 occurred through a pore-forming mechanism. The lipid headgroup environment experienced by the fluorescent label where MK14-A430 displayed pore information indicated that pore formation was best described by the toroidal pore model.
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    Dipolar dispersion forces in liquids: mechanisms of hydrophobic and Hofmeister effects
    Besford, Quinn Alexander ( 2015)
    This thesis gives a study into dipolar dispersion interactions in liquids. A new mechanism for long-ranged dipolar dispersion forces, based on dipole pair correlation functions in solution, and the resulting free energy, is derived and shown to provide closer agreement with experiment than previous theories of dipolar dispersion forces.The new dipolar dispersion interaction is called φ-dispersion. The action of φ-dispersion between thermally rotating waters' produces a long-ranged attraction between hydrophobic surfaces immersed in water. Comparing to experiments for the long-ranged attraction, φ-dispersion is found to explain most of the observed force. Inclusion of φ-dispersion into the Derjaguin-Landau-Verwey-Overbeek theory improves the theory's accuracy for the prediction of attractive forces in aqueous solutions. The level of agreement with experiment cannot be found with Lifshitz theory. At high ionic strengths φ-dispersion is strongly screened, consistent with recent experiments which cannot be explained by Lifshitz theory. The surface tension of water primarily stems from lost φ-dispersion interactions, and accounts for almost all of the interfacial tension with n-alkanes. Simple models based on φ-dispersion are given for the prediction of contact angles and the hydrophobic collapse of polymers. The Hofmeister effect, whereby different ions make solutes more or less hydrophobic, is investigated by simulations. A new free energy resulting from ion-water correlations is found, the strength of which spans -17.6 kJ/mol for cesium to -175.2 kJ/mol for calcium. A Hofmeister series exists in the magnitude of this free energy, resulting from a change in water's structure due to each ion, and its impact on pair correlations. Most crucially, the calculations for the hydrophobic and Hofmeister effects are based on the same mechanism; a change in pair correlation functions due to a solutes presence. This thesis therefore demonstrates a mechanistic link between the hydrophobic and Hofmeister effects.