School of Chemistry - Theses

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    Neutron Spectroscopy and Magnetic Properties of Lanthanoid(III)-dioxolene Compounds
    Dunstan, Maja Anna ( 2022)
    This thesis presents a series of studies on the electronic structure and magnetic properties of several families of lanthanoid(III)-dioxolene compounds. Compounds of the trivalent lanthanoid (Ln) ions are the current best performing single-molecule magnets (SMMs), and efforts to improve their properties include design of the coordination geometry, as well as control of spin-phonon, magnetic exchange, and intermolecular dipolar interactions. This thesis presents an exploration of the effect of Ln(III)-radical magnetic exchange coupling, as well as other solid-state effects, on the SMM behaviour of Ln(III) dioxolene compounds. Slow magnetic relaxation in Ln-SMMs can be modulated by the introduction of magnetic exchange coupling, however, quantifying the magnitude of magnetic exchange coupling in many Ln(III) systems is difficult using conventional magnetometric techniques, due to the often large orbital angular momentum contribution to the magnetic moment of Ln(III) ions. Spectroscopic techniques are therefore required to determine the magnetic exchange coupling for non Gd(III) systems. Inelastic neutron scattering (INS) is used in this work, alongside magnetometry, EPR spectroscopy, and luminescence measurements, to experimentally determine both the crystal field (CF) splitting and magnetic exchange coupling in several families of Ln(III)-dioxolene compounds. The two families of compounds [LnTp2trop] (Tp– = tris-pyrazolylborate; tropH = tropolone) and [LnTp2dbsq] (dbsqH = 3,5-di-tert-butylsemiquinone) are investigated, and a trend in the magnitude of the antiferromagnetic magnetic exchange coupling |JLn-SQ| is found, increasing from Tb to Yb in the isostructural series of compounds. For the compound [Tb(18-c-6) Br4CatNO3] (18-c-6 = 18-crown-6; Br4CatH2 = tetrabromocatecholate), INS is used to measure the magnitude of the CF splitting of the Tb(III) in a highly axial coordination environment. The one electron oxidised compounds [Ln(18-c-6)X4SQNO3] . I3 (X4SQH = tetrahalosemiquinone; X = Cl, Br) were then synthesised, and the magnetic exchange coupling was determined for the Gd(III) analogues by magnetometry, and the Nd(III) and Tb(III) congeners by INS. The implications of the magnetic exchange coupling on the slow magnetic relaxation of both the [LnTp2dbsq] and [Ln(18-c-6)X4SQNO3] . I3 families of compounds is investigated. Slow magnetic relaxation in zero-field is engendered for the Tb(III) congeners by the magnetic exchange bias. For the case of stronger magnetic exchange coupling in the Kramers systems, the Ln(III) radical exchange coupling leads to the loss of a doubly degenerate ground state and the magnetic bistability. For the [Ln(18-c-6)X4SQNO3] . I3 compounds, the axial ligand environment and magnetic exchange coupling leads to unusual slow magnetic relaxation for the Gd(III) and Eu(III) congeners. Modulation of the slow magnetic relaxation in these systems by effects other than Ln(III)- radical exchange is also observed for several of the compounds. The effect of intermolecular dipolar interactions and crystal packing on the magnetic relaxation of the SMM analogues adds to the understanding of the contributing factors on the observed relaxation of Ln(III)-SMMs.
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    Photochemistry and Electronic Spectroscopy of Gas-Phase Ions
    Buntine, Jack Thomas ( 2022)
    This dissertation focuses on investigations of gas-phase molecular and cluster ions -- deprotonated dithienylethene molecular photoswitches and positively charged carbon clusters -- using electronic action spectroscopy. Dithienylethene (DTE) photoswitches are members of the diarylethene (DAE) class of molecules that undergo rapid, reversible photoisomerisation between two isomeric forms in response to light, and have been incorporated into functionalised materials as light-triggered switches. Carbon clusters are structurally diverse molecules that exist as several different conformational isomers and allotropes, and are astrophysically important species. By measuring gas-phase electronic spectra for these species, information concerning their electronic and molecular structures can be obtained in an environment where potential sources of complications are absent. The experiments described in this thesis utilise ion mobility spectrometry (IMS) to separate isomeric forms of gas-phase ions prior to spectroscopic interrogation. DTE photoswitches undergo 6$\pi$-electrocyclisation (ring closing) and cycloreversion (ring opening) reactions upon exposure to light in the ultraviolet (UV) and visible spectral regions, respectively, making them ideal photoswitches for a range of applications. The experiments described in this thesis are concerned with the photoisomerisation of \textit{meta}- (\textit{m}) and \textit{para}- (\textit{p}) substituted DTE carboxylate anions using a tandem ion mobility mass spectrometer coupled with laser excitation. Density functional theory calculations were used to aid in the interpretation of the gas-phase photoisomerisation action (PISA) spectra. Gas-phase PISA spectra for \textit{p}-DTE$^-$ and \textit{m}-DTE$^-$ were obtained by measuring the wavelength-dependent photoisomerisation yields over the 300--700\,nm range. For \textit{p}-DTE$^-$, the PISA spectrum for 6$\pi$-electrocyclisation has an onset at $\approx$360\,nm and likely reaches a maximum at a wavelength just shorter than 300\,nm. For the ring-closed isomer, the cycloreversion PISA spectrum exhibits a band in the 450-700\,nm range with a maximum response at $\approx$615\,nm and a smaller band in the 320-415\,nm range peaking at $\approx$365\,nm. For \textit{m}-DTE$^-$, 6$\pi$-electrocyclisation of the ring-open isomer was not observed, presumably due to this isomer adopting a structure in the gas phase that is not suitable for cyclisation. This emphasises the importance of the carboxylate substitution position on the gas-phase photochemistry of DTE photoswitches. For the ring-closed isomer, the cycloreversion PISA spectrum shows an intense band spanning the 430-680\,nm range peaking at $\approx$590\,nm and a second band of comparable intensity in the 330-420\,nm range with a maximum at 355\,nm. Furthermore, the gas-phase investigations show no evidence for the formation of the cyclic byproduct, the main cause of fatigue for DAE-based photoswitches in solution, when either \textit{p}-DTE$^-$ or \textit{m}-DTE$^-$ were exposed to UV light. The PISA spectra for the DTE anions should help to develop more robust and efficient DTE-based photoswitches for use in a wide variety of functional materials. The work reported in the rest of this thesis involves the spectroscopic characterisation of isomer- and mass-selected cluster ions produced by laser ablation. This thesis describes an instrument that has been designed and built for obtaining electronic spectra of positively charged carbon clusters produced by laser ablation of graphite that have been selected according to both their isomeric form and their mass-to-charge ratio. Prior to the work presented here, there have been no previous isomer-specific spectroscopic investigations of carbon clusters. Gas-phase electronic spectra were measured for monocyclic C$_{2n}^+$ (\textit{n}=6--18) clusters over the 400--2000\,nm range by photodissociating C$_n^+$-(N$_2$)$_m$ complexes in a cryogenically cooled, three-dimensional quadrupole ion trap (QIT). The most striking feature of the spectral series is the linear shift of the wavelength for the origin band transitions to longer wavelength with the number of carbon atoms, indicating a common structural motif supporting the assignment of the electronic spectra to monocyclic structures. The spectra also exhibit a series of weaker transitions that lie to higher energy from the origin bands that are probably vibronic progressions in the ring deformation vibrational modes. The bands for the C$_{4k}^+$ cluster series (C$_{12}^+$, C$_{16}^+$, C$_{20}^+$, C$_{24}^+$, C$_{28}^+$, C$_{32}^+$ and C$_{36}^+$) are relatively broad (FWHM$>$100\,cm$^{-1}$), presumably due to rapid non-radiative deactivation from the excited state, whereas those for the C$_{4k+2}^+$ clusters (C$_{14}^+$, C$_{18}^+$, C$_{22}^+$, C$_{26}^+$ and C$_{30}^+$) are much narrower (FWHM$\approx$10--20\,cm$^{-1}$), consistent with slower non-radiative deactivation. The C$_{2n}^+$ ($n$=6--14) clusters are possibly connected to the diffuse interstellar bands (DIBs) as their electronic absorptions lie in the visible wavelength region where most DIBs are found. The electronic spectra of C$_{n}^+$ clusters should provide benchmark data for the development of quantum chemical methods for modelling the electronic structure of these clusters.
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    Towards Total Synthesis of Icumazole A
    Salimimarand, Mina ( 2022)
    Detailed is work towards the enantioselective synthesis of the NADH oxidase inhibitor icumazole A. Key reactions include an organocatalysed asymmetric self-aldol reaction of propionaldehyde towards the synthesis of the oxazole containing fragment. An enantioselective synthesis of the Diels-Alder precursor of the isochromanone fragment has been performed. Key steps included an enzyme catalysed enantioselective desymmetrisation which was then substituted with epoxide formation and ozonolysis. Further work towards the intramolecular [4+2] cycloaddition/aromatisation sequence to give aryl bromide followed by elaboration of product to the desired isochromanone fragment through borylation and obtaining the aldehyde constituted a formal total synthesis of noricumazole B.
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    Experimental and Numerical Investigations of Chemical and Physical Effects by Oscillating Bubbles
    MONDAL, JOYDIP ( 2022)
    Microbubbles, along with ultrasound waves (frequency greater than 20 kHz), play an essential role in biomedical and engineering applications, e.g. the medical imaging of internal organs, permeabilization of tissues, food/dairy processing, and nanoparticle generation. For example, tissue-permeabilization is made possible when oscillating bubbles in the presence of sound waves produce recirculation flows in the surrounding fluid (commonly known as microstreaming). Alongside, bubble-collapse generates high temperature and pressure that can help break down molecules into free radicals, triggering chemical reactions. But, there is a dearth of information and understanding of the relationship between these physical and chemical effects with applied power and frequency of the ultrasound, when the amount of dissolved gas is less and surplus gas intrusion from outside is checked. This study addresses some of these issues through different experimental and numerical techniques. An experimental sonoreactor setup is fabricated to connect to a degassing setup and different frequency plates, thereby allowing the measurement of radical generation during bubble oscillation. Sonochemiluminescence and iodide dosimetry tests are conducted in the water in this setup without overheating or gas-intrusion. Again, the same liquid is transferred without any agitation to another in-house fabricated glass cell connected to a frequency transducer. This arrangement facilitates the levitation of a single bubble registering stable oscillation, thereby generating flow structures in the adjacent liquid. These are captured using particle image velocimetry (PIV) for different cases of applied power. In addition, computational fluid dynamics is performed to closely resolve these flow features and explore these phenomena over a broader applied power and frequency parameter space. It has been observed from our chemical investigations in water that at low dissolved gas conditions, limited radical generation occurs that is governed by acoustic cavitation (not by "ordering effect") irrespective of applied frequency. Our results suggest the absence of undesirable chemical reactions under low gas conditions. The concept of "cavitation-free" radical generation in liquids is also clarified. The PIV experiments for a single bubble reveal that periodic shape evolution of the bubble takes place beyond an acoustic pressure amplitude, known as threshold for onset of surface or shape mode oscillation. This results in a mean motion in the adjacent liquid leading to unique flow patterns characteristic to microstreaming. Through CFD models, attempts have been made to understand these flow patterns exhibited by the formation of a certain number of vortices at the bubble interface due to the subharmonic behaviour of the bubble, where the radial amplitude varies periodically. Additionally, different significant modes of bubble oscillation are identified for various combinations of applied power and resting radii of the bubble. The modal decomposition of each such significant mode provides further insight into the zonal harmonics of bubble oscillation. This phenomenon is simulated in inert and homogenous liquid nitrogen (LN2), where nitrogen vapour bubbles oscillate at relatively higher surface modes in the presence of ultrasound without any harmful reactions and simultaneously generate shear stresses. All these make controlled oscillating bubbles suitable for ultrasound-aided cryosurgical operations to remove cancerous tissues efficiently. Furthermore, a numerical investigation on the interaction of multiple bubbles in LN2 has been performed to gain insight into the erosive potential of bubbles on solids.
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    Structural and functional characterisation of bacterial proteins that act in the utilisation of arsenic oxyanions for respiration
    Poddar, Nilakhi ( 2022)
    Arsenic is a toxic metalloid present as a contaminant in drinking water, affecting more than 140 million people worldwide. Ingestion of arsenic as the soluble oxyanions arsenite and arsenate, is linked to neurological, reproductive and respiratory disorders, cancer and diabetes mellitus. Although toxic to human health, arsenite and arsenate can be utilised by some prokaryotes as sources of energy from the environment. Such prokaryotes are Pseudorhizobium banfieldiae sp. str. NT-26 (NT-26) and Chrysiogenes arsenatis (C.arenatis) respire using these oxyanions by the actions of arsenite oxidase (AioAB) and arsenate reductase enzymes (ArrAB), respectively. The focus of the present work details structural and functional characterisation of proteins that allow these bacteria to use arsenic oxyanions for respiration. This includes characterisation of periplasmic binding proteins, ArrX and AioX that bind to arsenic oxyanions, the interaction of these proteins with the sensor histidine kinase, AioS and the respiratory arsenite oxidase enzyme, AioAB, and its interaction with its electron acceptor, cytochrome c552. The substrate specificity of periplasmic binding proteins, AioX and ArrX to arsenite and arsenate, from NT-26 and C.arsenatis, respectively were investigated. The aims of this study were to establish how both these proteins distinguish between arsenite and arsenate, and to determine the structure of ArrX protein in an apo- and arsenate-bound state. The X-ray crystal structure of ArrX with arsenate was determined to 1.74 Angstrom resolution. Structural comparison of the AioX and the ArrX proteins and isothermal titration calorimetry (ITC) analyses of mutant proteins identified a conserved Cys residue in their substrate binding sites that play a key role in the discrimination between arsenite and arsenate for both proteins. Structural analyses also revealed that the nature of neighbouring residues (Gly in AioX and Thr in ArrX) may provide varied structural flexibilities that contribute to the differential interaction of the conserved Cys residue to arsenic oxyanions. The biophysical characterisation of the interaction between the AioX protein and its sensor histidine kinase AioS was performed to investigate the processes that control the expression of the AioAB enzyme in NT-26. Size exclusion chromatography (SEC) and analytical ultracentrifugation (AUC) experiments revealed that AioX and AioS proteins can form a stable heterodimer complex in the absence of arsenite. These findings also revealed that the oligomeric state of the complex does not change in the presence of arsenite. A loop in the AioX protein, which was proposed to be involved in the interaction with AioS was shown experimentally not to directly participate in the interface between the two proteins. Structural and functional characterisation of the interaction between the AioAB enzyme with its electron acceptor cytochrome c552 was carried out to investigate the structural basis of the electron transfer process that underpins the respiration of Pseudorhizobium banfieldiae sp. str. NT-26. The crystal structure of the AioAB/cytc552 was determined to 2.25 Angstrom resolution. The structure showed two AioA2B2/(cytc552)2 complexes per asymmetric unit. Three of the four AioAB/cytc552 complexes revealed that the cytc552 molecule docks in a cleft at the interface of between the AioA and AioB subunits. The positioning of the cytc552 proteins in these complexes revealed an edge-edge distance of 7.5 Angstrom between the heme of cytc552 and the Rieske 2Fe-2S cluster in the AioB subunit, which is considered an ideal distance for fast electron transfer between proteins. The interface of the AioAB and the cytc552 protein also shows electrostatic interactions and is stabilised by two salt bridges, a classic example of transient complex formation for fast electron transfer. Additionally, the structure also highlights the unique positioning of one of the four cytc552 proteins that sit at a long distance from the redox cofactors of AioAB subunits and presumably aid in crystallisation. Enzyme kinetics analysis also revealed the AioAB/cytc552 system to be catalytically very efficient.
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    Development and characterisation of micro polymer inclusion beads (µPIBs) for the separation of rare earth elements
    Croft, Charles Frederick ( 2022)
    The global demand for rare earth elements (REEs) is predicted to significantly increase in the near future. This is due to expanding production of green technologies heavily dependent on the incorporation of REEs, such as electric vehicles (EVs) and wind turbines. However, REEs are currently supplied by mining REE minerals, which require high intensity industrial processing and releases REEs into the environment. As a result, our demand for REEs is causing a significant environmental impact. Therefore, there is a considerable interest in the development of more sustainable extraction technologies and their application to alternative sources of REEs, namely recycling from end-of-life (EOL) electronics. This thesis details the development of a new polymer-based extraction material, denominated as micro polymer inclusion beads (uPIBs), and its application to the separation of REEs. The thesis covers the development and optimisation of uPIBs composed of the base polymers poly(vinyl chloride) (PVC) or poly(vinylidene fluoride-co-hexafluoropropylene) (PVDF-HFP) which immobilise between their entangled chains the extractant di-(2-ethyl hexyl)phosphoric acid (D2EHPA). The uPIBs were fabricated via a recently developed phase inversion microfluidic method, which was modified to allow effective fabrication of the PVC and PVDF-HFP-based uPIBs containing up to 60 wt% D2EHPA. These modifications included acidification of the fabrication solutions to 0.1 M sulfuric acid, and increasing desolvation solution salinity from 5 to 15 wt% NaCl. The uPIBs demonstrated superior extraction performance over their polymer inclusion membrane (PIM) counterparts, attributed to the higher surface area exposed to the feed and receiving solutions. This thesis also covers the uPIBs characterisation via thermogravimetric analysis (TGA), which was used to confirm the uPIB’s D2EHPA content, using PIMs with equivalent composition as reference standards. Negligible D2EHPA leaching occurred from both the PVC and PVDF-HFP-based uPIBs under the fabrication conditions applied here. Characterisation of PVC-based uPIBs using an isothermal step highlighted the presence of Na+, extracted during the fabrication process, which was successfully removed through a 1 M sulfuric acid washing step. The removal of Na+ was found to significantly improve the extraction performance of the uPIBs and would not have been identified if not for the application of the isothermal TGA method developed here. The performance of the newly developed uPIBs (60 wt% D2EHPA, 40 wt% PVC) was characterised for the on-line separation of REEs by packing a column with uPIBs. La3+ and Gd3+ were initially used as model REE ions and could be separated by selective extraction or back-extraction. The column separation method was then applied successfully to the recovery of the REE ions Nd3+ and Dy3+ from EOL rare earth permanent magnet (REPM), which was digested in 2 M sulfuric acid. Upon decreasing the acidity of the digest to 0.03 M sulfuric acid, Nd3+ and Dy3+ were selectively extracted after the introduction of ascorbic acid at a 3:1 ratio with Fe to reduce Fe3+ to Fe2+. Nd3+ and Dy3+ could then be selectively back-extracted from the uPIB-column using 0.3 and 2.0 M sulfuric acid solutions, respectively. On the basis of the results presented in this thesis it can be concluded that the column separation method based on the newly developed uPIBs has shown promising potential as a future sustainable method for recycling REEs from digested EOL electronics.
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    Sonosynthesis of Functional Micro/Nano-structures using Biomolecules
    Zhu, Haiyan ( 2022)
    Malnutrition and access to affordable health services are some of the world’s most urgent problems. The development of nutrient and drug delivery systems by using sub-micro particles as carriers has attained much attention for improving the nutritional value of food and the efficacy of diagnostic/therapeutic treatments. Common methods for synthesizing biofunctional particles usually require many reagents and involve multiple steps. In this regard, a novel and advanced approach for material synthesis needs to be developed and investigated to address those limitations. Ultrasonic techniques have emerged as one-pot and eco-friendly methods for the synthesis of organic and inorganic materials. It has been found that acoustic cavitation derived from sound waves can induce emulsification of liquids to form microcapsules and promote chemical modifications of biomolecules. Protein-shelled microcapsules have been synthesized by using low-frequency ultrasound and reducing agents for food-based applications. However, the direct use of food ingredients for the microencapsulation of microcapsules without resorting to additional external agents was never explored and needs to be investigated further for food-based applications. On another note, drug carriers are usually prepared in nanoscale to enhance interaction with cell membranes for achieving efficient therapeutic treatment. Conventional strategies for preparing drug loaded nanoparticles require matrix materials as carriers, resulting in low drug loading capacity and safety issues. Therefore, synthesis of nanodrugs solely made of antibiotic molecules is a better method for development of drug delivery platforms. Many molecules bearing aromatic groups have been successfully sono-assembled into nanoparticles by high-frequency ultrasound, but they are mainly used as drug carriers. Transforming drug molecules into carrier-free nanodrug has not been widely investigated. As such, I intend to expand new research towards other drug molecules with aromatic moieties. In this regard, my Ph.D. project aims to sono-chemically synthesize various micro and nano structures from biomolecules by tuning the frequency/power of ultrasound without the usage of external reagents. The size of the obtained bio-functional structures is controllable, and their compositions are suitable for use in specific applications such as : i) nutrients delivery in food industries; ii) drug delivery for biomedical applications. The fundamental concepts of sono-chemistry for material synthesis, along with biomolecules (proteins, nutrients and antibiotics) based micro/nano structures and their applications are discussed in Chapter 1. Chapter 2 provides an overview of microencapsulation techniques for food industries and fabrication of nanoparticles for antibiotics delivery. In particular, methodology, formulating materials, current challenges, limitations and innovation are discussed. In Chapter 3, the materials, equipment and methodologies involved in the reactions used in this thesis are thoroughly described. Chapter 4 is the first chapter of result and discussion section. Microcapsules made of egg white protein (EWP), as commonly available biopolymers, were first conceptualized. Oil-soluble nutrients (Vitamin A, D and E) were encapsulated into EWP to form nutrients loaded proteinaceous microcapsules by employing 20 kHz ultrasound. This work primarily points out that high availability of free thiol groups in protein solution is crucial in forming stable microcapsules with robust shells, in order to protect micronutrients from degradation against detrimental effects. In Chapter 5, another two plant-based protein isolates extracted from soybean (SPI) and corn (CPI) were also formulated to form microcapsules. This study provided further insights into the structural, chemical and surface properties of proteins for efficient ultrasonic microencapsulation of micronutrients. A double emulsion technique was further developed to co-encapsulate both oil- (vitamin A and D) and water-soluble (vitamin B, C and minerals) micronutrients. In-vitro digestion study showed that the proteinaceous microcapsules enable sustained release of micronutrients, demonstrating their potential in food fortification applications. In Chapter 6, a sono-chemical strategy for transforming antibiotic doxycycline into carrier-free nanodrugs via high-frequency ultrasound (490 kHz) is reported. This study demonstrates that doxycycline undergoes hydroxylation and dimerization processes upon sonication in an aqueous solution to ultimately self-assemble into nanoparticles. The size of obtained particles could be finely controlled by tuning the applied ultrasonic powers. The nanodrugs exhibited antioxidant properties, along with antimicrobial activity against both Gram-positive (S. aureus) and Gram-negative (E. coli) bacterial strains. These results highlight the feasibility of the ultrasound-based approach for engineering carrier-free nanodrug with multiple controlled bio-functionalities. Chapter 7 provides an overall summary of the entirety of my PhD project as well as my conclusion and thoughts on it.
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    Investigating the Mechanism of Cysteine Dioxygenase
    Shirani Sarmazeh, Zahra ( 2022)
    Non-heme enzymes utilise reactive iron-oxygen intermediates in their respective mechanisms, which are key to their reactivity. Common intermediates postulated are Fe(III)-superoxide and Fe(IV)-oxo. Among non-heme enzymes, the most well-known mammalian thiol dioxygenase is cysteine dioxygenase (CDO). CDO is responsible for the oxidation of cysteine by adding molecular oxygen to the sulfur of cysteine to form a sulfinic acid known as cysteine sulfinic acid. Many studies have proposed which intermediate occur during CDO turnover, but the mechanism is still unknown. Chapter 1 provides a comprehensive review of the mechanisms of non-heme enzymes and the iron-oxygen intermediates involved, as well as a general introduction to thiol dioxygenases. Thiol dioxygenases exhibit high specificity with their respective substrates, and catalyse very similar reactions. However, due to the challenges faced by the lack of intense absorption and direct spectroscopic evidence of oxygen-bound intermediates, the mechanism of these enzymes remains unclear. Chapter 2 describes the purification of WT CDO and specific variants of CDO. It also describes all the different techniques performed in this work. In chapter 3, the different CDOs included C93G, Y157F and H155Q CDO were used to study the mechanism of disulfide formation, a side product of the CDO reaction. The amount of disulfide formed increases when a non-native substrate is used or substitutions of catalytically important residues are made. These effects were investgated through kinetic studies, 1H-NMR and mass spectrometry. In particular, the presence of the sulfenate, which is an intermediate in the formation of disulfide was probed during turnover. The identity and quantification of sulfenate supports formation of a Fe(IV)-oxo during turnover and that proper substrate chelation appears to be critical for S-oxygenation. In chapter 4, WT CDO, C93G and Y157F CDO were used to study reactivity upon substitution of iron with the non-native metal, Co(II). This was done in the hope of stabilising new reactive intermediates. A Co(III)-superoxide was characterised by EPR and UV-Vis for Y157F and C93G CDO . In contrast, a tyrosyl radical was observed in WT and C93G CDO. In the case of WT CDO this led to an increase in crosslink between C93 and Y157, suggesting that the Co(III)-superoxide undergoes rapid H atom abstraction from Y157. Chapter 5 describes the formation of cysteic acid (CA) as a second subsequent product of the CDO reaction at high pH (9.1). Product distribution of the reaction of C93G CDO pre-bound with cysteine and CSA were quantified by HPLC-mass spectrometry. The data shows that under these conditions, cysteine is oxidised initially to CSA and then further to CA. The final chapter re-evaluates the mechanism of CDO proposed by computational studies. The thesis provides one of the first protein-based evidence that support Fe(III)-superoxide and Fe(IV)-oxo intermediates are formed during the CDO catalytic mechanism. It also provides first experimental evidence that a sulfenate is indeed formed during catalytic turnover and that a reactive Co(III)-superoxide can be detected when iron is replaced by the non-native metal cobalt. Lastly, the products of the reaction of C93G CDO with cysteine and CSA at higher pH are presented and the implications of these results to the mechanism of CDO with its native substrate cysteine are discussed.
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    Biological Interactions of Responsive Polymeric Nanoparticles
    Kermaniyan, Sarah Sadat ( 2022)
    Nanoparticles need to be capable of endosomal escape to facilitate the effective delivery of therapeutic cargo, particularly biological materials. While there are many mechanisms postulated, some of them remain poorly understood. In this study, pH-induced swelling and proton sponge mechanisms for endosomal escape have been investigated. In the first two projects, swellable pH-responsive nanoparticles were designed and synthesised through emulsion polymerisation. Nanoparticles exhibited pH responsive swelling when the pH decreased from pH 7.4 to lower than 6.2. The swelling pH and percentage were tuned by altering the type of crosslinker or the composition and amount of pH responsive monomers. Also, particles showed strong buffering capacity as well as the ability to associate with cells, however there was no clear evidence of endosomal escape using the calcein assay. The results have generated serious questions about the validity of both these mechanisms to explain the endosomal escape of nanoparticles. In the third project, covalently- and physically-doxorubicin (Dox)-loaded particles with the capability of pH-induced release were designed and studied. Loading capacity was tunable by introducing various level of pentafluorophenyl functional moieties into particles in covalently-Dox-loaded particles. However, physically-Dox-loaded particles showed higher cytotoxicity and lower IC50. These findings suggests that both covalently- and physically-Dox-loaded particles could be potential candidates as drug delivery systems. In the fourth project, camptothecin (CPT)-loaded disulfide particles were also designed and synthesised by the oxidation of thiol monomers. Hydrophobic CPT was loaded into particles covalently and was able to be released by thiol-disulfide exchange reaction in response to the redox environment of cells. Drug release was tuned by the change in particles compositions. Cytotoxicity of drug-loaded particles was confirmed compared to control particles (non-drug-loaded) by using Alamar blue viability assay on MCF-7 cells. The study on swellable pH-responsive polymeric nanoparticles indicates that more work needs to be done to understand the endosomal escape of cargo/particles through swelling and proton sponge effect mechanisms. The study on introducing novel methods to load drug into polymeric particles showed these particles have potential for the effective controlled release of hydrophilic/hydrophobic therapeutic cargo in physiological conditions in response to pH variation and redox increase.
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    Transport, bioaccumulation, and risk of per- and polyfluroalkyl substances (PFASs) in birds from south-east Australia
    Szabo, Drew ( 2022)
    Over six million per- and polyfluoroalkyl substances (PFASs) have been identified with a broad range of uses, including commercial and industrial products such as aqueous-film-forming foam (AFFF), mist suppressant, food contact materials, cosmetics, and therapeutic drugs. Some classes of PFASs have been termed “forever chemicals” due to the strong carbon-fluorine bond that resists metabolism and degradation. To help inform key stakeholders of the potential risks involved in the exposure to these compounds the occurrence, transport, and fate of legacy and emerging PFASs within the Southeast Australian environment, this thesis aims to describe the exposure pathways and bioaccumulation in native avian species. Wastewater treatment plants (WWTPs) are a common source of PFASs to the environment and hourly variations in PFAS concentrations could have wide ranging implications for the estimation of discharge to the environment that determine risk-based human and environmental health policies. Optimised and novel sampling and analytical methodologies were developed to measure the concentrations of PFASs in biological matrices from avian species exposed to long-range and diffuse sources of PFASs in Australia, such as WWTPs. Exposure to PFASs derived from long-range transport in marine and freshwater species results in concentrations generally considered to be low risk. Conversely, a moderately impacted surface water body in Melbourne has revealed the relatively high exposure of PFASs, including novel substances, to a resident swan population that may serve as an important model for the distribution and bioaccumulation of the chemicals in Australian species for the first time. Due to the limited information regarding the occurrence, fate, and potential impact to avian species in Southeast Australia, monitoring and hypothesis-driven research is required to assess the risk of PFASs to animals and humans that may also be exposed to similar environments.