A 'click approach' to the generation of novel profluorescent nitroxides

This project focused on the first application of the copper catalyzed azide alkyne cycloaddition reaction for the generation of novel profluorescent systems. Through this approach four novel profluorescent nitroxides were prepared both rapidly and in good yield from coumarin and nitroxide CuAAC coupling partners. Specifically, 7-hydroxy, 7-diethylamino, 6-bromo and unsubstituted coumarin analogues bearing an azide group in the 3-position were prepared and conjugatively joined to an alkyne isoindoline nitroxide previously reported by our group. To explore the impact of the nitroxide moiety on the fluorescence of these systems, methoxyamine analogues of the corresponding nitroxide analogues were prepared. Spectrophotometric analysis of these methoxyamine analogues revealed that the aromatic systems possessed high quantum yields. However, the quantum yield efficiency was found to be dependent on the presence of electron donating substituents in the 7-position of the coumarin motif, which enhanced the charge-transfer character of the system. Furthermore, spectrophotometric analysis of nitroxide analogues demonstrated that the triazole effectively mediated fluorophore-nitroxide communication, as evidenced by the low quantum yield values of the nitroxide analogues. These results suggest that this technique can be used to conjugatively join any azide bearing fluorescent system with the key alkyne isoindoline coupling partner allowing for the rapid generation of diverse profluorescent systems.

Novel numerical methods for time-space fractional reaction diffusion equations in two dimensions

We consider time-space fractional reaction diffusion equations in two dimensions. This equation is obtained from the standard reaction diffusion equation by replacing the first order time derivative with the Caputo fractional derivative, and the second order space derivatives with the fractional Laplacian. Using the matrix transfer technique proposed by Ilic, Liu, Turner and Anh [Fract. Calc. Appl. Anal., 9:333--349, 2006] and the numerical solution strategy used by Yang, Turner, Liu, and Ilic [SIAM J. Scientific Computing, 33:1159--1180, 2011], the solution of the time-space fractional reaction diffusion equations in two dimensions can be written in terms of a matrix function vector product $f(A)b$ at each time step, where $A$ is an approximate matrix representation of the standard Laplacian. We use the finite volume method over unstructured triangular meshes to generate the matrix $A$, which is therefore non-symmetric. However, the standard Lanczos method for approximating $f(A)b$ requires that $A$ is symmetric. We propose a simple and novel transformation in which the standard Lanczos method is still applicable to find $f(A)b$, despite the loss of symmetry. Numerical results are presented to verify the accuracy and efficiency of our newly proposed numerical solution strategy.

Development and validation of novel methods for microbial source tracking based on Escherichia coli as an indicator of water quality

Microbial pollution in water periodically affects human health in Australia, particularly in times of drought and flood. There is an increasing need for the control of waterborn microbial pathogens. Methods, allowing the determination of the origin of faecal contamination in water, are generally referred to as Microbial Source Tracking (MST). Various approaches have been evaluated as indicatorsof microbial pathogens in water samples, including detection of different microorganisms and various host-specific markers. However, until today there have been no universal MST methods that could reliably determine the source (human or animal) of faecal contamination. Therefore, the use of multiple approaches is frequently advised. MST is currently recognised as a research tool, rather than something to be included in routine practices. The main focus of this research was to develop novel and universally applicable methods to meet the demands for MST methods in routine testing of water samples. Escherichia coli was chosen initially as the object organism for our studies as, historically and globally, it is the standard indicator of microbial contamination in water. In this thesis, three approaches are described: single nucleotide polymorphism (SNP) genotyping, clustered regularly interspaced short palindromic repeats (CRISPR) screening using high resolution melt analysis (HRMA) methods and phage detection development based on CRISPR types. The advantage of the combination SNP genotyping and CRISPR genes has been discussed in this study. For the first time, a highly discriminatory single nucleotide polymorphism interrogation of E. coli population was applied to identify the host-specific cluster. Six human and one animal-specific SNP profile were revealed. SNP genotyping was successfully applied in the field investigations of the Coomera watershed, South-East Queensland, Australia. Four human profiles [11], [29], [32] and [45] and animal specific SNP profile [7] were detected in water. Two human-specific profiles [29] and [11] were found to be prevalent in the samples over a time period of years. The rainfall (24 and 72 hours), tide height and time, general land use (rural, suburban), seasons, distance from the river mouth and salinity show a lack of relashionship with the diversity of SNP profiles present in the Coomera watershed (p values > 0.05). Nevertheless, SNP genotyping method is able to identify and distinquish between human- and non-human specific E. coli isolates in water sources within one day. In some samples, only mixed profiles were detected. To further investigate host-specificity in these mixed profiles CRISPR screening protocol was developed, to be used on the set of E. coli, previously analysed for SNP profiles. CRISPR loci, which are the pattern of previous DNA coliphages attacks, were considered to be a promising tool for detecting host-specific markers in E. coli. Spacers in CRISPR loci could also reveal the dynamics of virulence in E. coli as well in other pathogens in water. Despite the fact that host-specificity was not observed in the set of E. coli analysed, CRISPR alleles were shown to be useful in detection of the geographical site of sources. HRMA allows determination of ‘different’ and ‘same’ CRISPR alleles and can be introduced in water monitoring as a cost-effective and rapid method. Overall, we show that the identified human specific SNP profiles [11], [29], [32] and [45] can be useful as marker genotypes globally for identification of human faecal contamination in water. Developed in the current study, the SNP typing approach can be used in water monitoring laboratories as an inexpensive, high-throughput and easy adapted protocol. The unique approach based on E. coli spacers for the search for unknown phage was developed to examine the host-specifity in phage sequences. Preliminary experiments on the recombinant plasmids showed the possibility of using this method for recovering phage sequences. Future studies will determine the host-specificity of DNA phage genotyping as soon as first reliable sequences can be acquired. No doubt, only implication of multiple approaches in MST will allow identification of the character of microbial contamination with higher confidence and readability.

Abduction : the writing of a historical novel

This is a practice-led project consisting of a historical novel Abduction and related exegesis. The novel is a third person intimate narrative set in the mid-nineteenth century and is based on actual events and persons caught up in, or furthering, the mass dispossession of small farmers in Scotland known as the ‘Clearances’. The narrative focuses on the situation in the Outer Hebrides and northern Scotland. It is based on documented facts leading up to a controversial trial in 1850 that arose because a twenty year old woman of the period (the central protagonist, Jess Mackenzie) eloped with a young farmer to escape her parent’s pressure to marry a rival suitor, himself a powerful lawyer and ‘factor’ at the centre of many of the Clearances. The young woman’s independent ideas were ahead of her time, and the decisions she made under great pressure were crucial in some dramatic events that unfolded in Scotland and later in the colony of Victoria, to which she and her new husband emigrated soon after the trial. The exegesis is composed of two unequal parts. It briefly considers the development of the literary historical fiction genre in the nineteenth century with Walter Scott in particular, a genre found useful in representing women’s issues of the Victorian era by Victorian and contemporary authors. The exegesis also briefly considers the appropriateness of the fiction genre (as opposed to creative nonfiction) in creating the lived experience in a fact-based work. The major part of the exegesis is a detailed, reflective analysis of the problem-solving process involved in writing the novel, structured by reference to Kate Grenville’s Searching for the Secret River – a work of metawriting that explains her creative process in researching and writing historical fiction based on fact.

Sulfated fibrous ZrO2/Al2O3 core and shell nanocomposites : a novel strong acid catalyst with hierarchically macro-mesoporous nanostructure

A series of solid strong acid catalysts were synthesised from fibrous ZrO2/Al2O3 core and shell nanocomposites. In this series, the zirconium molar percentage was varied from 2 % to 50 %. The ZrO2/Al2O3 nanocomposites and their solid strong acid counterparts were characterised by a variety of techniques including 27Al magic angle spinning nuclear magnetic resonance (MAS-NMR), scanned electronic microscopy (SEM), transmission electron microscope (TEM), X-ray photoelectron spectroscopy (XPS), Nitrogen adsorption and infrared emission spectroscopy (IES). NMR results show that the interaction between zirconia species and alumina strongly correlates with pentacoordinated aluminium sites. This can also be detected by the change in binding energy of the 3d electrons of the zirconium. The acidity of the obtained solid acids was tested by using them as catalysts for the benzolyation of toluene. It was found that a sample with a 50 % zirconium molar percentage possessed the highest surface acidity equalling that of pristine sulfated zirconia despite the reduced mass of zirconia.

Evaluation of novel Streptococcus pyogenes vaccine candidates incorporating multiple conserved sequences from the C-repeat region of the M-protein

A major challenge for Streptococcus pyogenes vaccine development is the identification of epitopes that confer protection from infection by multiple S. pyogenes M-types. Here we have identified and characterised the distribution of common variant sequences from individual repeat units of the C-repeat region (CRR) of M-proteins representing 77 different M-types. Three polyvalent fusion vaccine candidates (SV1, SV2 and SV3) incorporating the most common variants were subsequently expressed and purified, and demonstrated to be alpha-helical by Circular Dichroism (CD), a secondary conformational characteristic of the CRR in the M-protein. Antibodies raised against each of these constructs recognise M-proteins that vary in their CRR, and bind to the surface of multiple S. pyogenes isolates. Antibodies raised against SV1, containing five variant sequences, also kill heterologous S. pyogenes isolates in in vitro bactericidal assays. Further structural characterisation of this construct demonstrated the conformation of SV1 was stable at different pHs, and thermal unfolding of SV1 a reversible process. Our findings demonstrate that linkage of multiple variant sequences into a single recombinant construct overcomes the need to embed the variant sequences in foreign helix promoting flanking sequences for conformational stability, and demonstrates the viability of the polyvalent candidates as global S. pyogenes vaccine candidates.

A novel duplication polymorphism in the FANCA promoter and its association with breast and ovarian cancer

The FANCA gene is one of the genes in which mutations lead to Fanconi anaemia, a rare autosomal recessive disorder characterised by congenital abnormalities, bone marrow failure, and predisposition to malignancy. FANCA is also a potential breast and ovarian cancer susceptibility gene. A novel allele was identified which has a tandem duplication of a 13 base pair sequence in the promoter region. Methods: We screened germline DNA from 352 breast cancer patients, 390 ovarian cancer patients and 256 normal controls to determine if the presence of either of these two alleles was associated with an increased risk of breast or ovarian cancer. Results: The duplication allele had a frequency of 0.34 in the normal controls. There was a nonsignificant decrease in the frequency of the duplication allele in breast cancer patients. The frequency of the duplication allele was significantly decreased in ovarian cancer patients. However, when malignant and benign tumours were considered separately, the decrease was only significant in benign tumours. Conclusion: The allele with the tandem duplication does not appear to modify breast cancer risk but may act as a low penetrance protective allele for ovarian cancer.

Development of novel vaccine strategies to prevent genital tract chlamydial infections

Chlamydia trachomatis is the most prevalent bacterial sexually transmitted infection in the developed world and the leading cause of preventable blindness worldwide. As reported by the World Health Organization in 2001, there are approximately 92 million new infections detected annually, costing health systems billions of dollars to treat not only the acute infection, but also to treat infection-associated sequelae. The majority of genital infections are asymptomatic, with 50-70% going undetected. Genital tract infections can be easily treated with antibiotics when detected. Lack of treatment can lead to the development of pelvic inflammatory disease, ectopic pregnancies and tubal factor infertility in women and epididymitis and prostatitis in men. With infection rates on the continual rise and the large number of infections going undetected, there is a need to develop an efficacious vaccine which prevents not only infection, but also the development of infection-associated pathology. Before a vaccine can be developed and administered, the pathogenesis of chlamydial infections needs to be fully understood. This includes the kinetics of ascending infection and the effects of inoculating dose on ascension and development of pathology. The first aim in this study was to examine these factors in a murine model. Female BALB/c mice were infected intravaginally with varying doses of C. muridarum, the mouse variant of human C. trachomatis, and the ascension of infection along the reproductive tract and the time-course of infection-associated pathology development, including inflammatory cell infiltration, pyosalpinx and hydrosalpinx, were determined. It was found that while the inoculating dose did affect the rate and degree of infection, it did not affect any of the pathological parameters examined. This highlighted that the sexual transmission dose may have minimal effect on the development of reproductive sequelae. The results of the first section enabled further studies presented here to use an optimal inoculating dose that would ascend the reproductive tract and cause pathology development, so that vaccine efficacy could be determined. There has been a large amount of research into the development of an efficacious vaccine against genital tract chlamydial infections, with little success. However, there have been no studies examining the effects of the timing of vaccination, including the effects of vaccination during an active genital infection, or after clearance of a previous infection. These are important factors that need to be examined, as it is not yet known whether immunization will enhance not only the individual's immune response, but also pathology development. It is also unknown whether any enhancement of the immune responses will cause the Chlamydia to enter a dormant, persistent state, and possibly further enhance any pathology development. The second section of this study aimed to determine if vaccination during an active genital tract infection, or after clearance of a primary infection, enhanced the murine immune responses and whether any enhanced or reduced pathology occurred. Naïve, actively infected, or previously infected animals were immunized intranasally or transcutaneously with the adjuvants cholera toxin and CpG-ODN in combination with either the major outer membrane protein (MOMP) of C. muridarum, or MOMP and ribonucleotide reductase small chain protein (NrdB) of C. muridarum. It was found that the systemic immune responses in actively or previously infected mice were altered in comparison to animals immunized naïve with the same combinations, however mucosal antibodies were not enhanced. It was also found that there was no difference in pathology development between any of the groups. This suggests that immunization of individuals who may have an asymptomatic infection, or may have been previously exposed to a genital infection, may not benefit from vaccination in terms of enhanced immune responses against re-exposure. The final section of this study aimed to determine if the vaccination regimes mentioned above caused in vivo persistence of C. muridarum in the upper reproductive tracts of mice. As there has been no characterization of C. muridarum persistence in vitro, either ultrastructurally or via transcriptome analysis, this was the first aim of this section. Once it had been shown that C. muridarum could be induced into a persistent state, the gene transcriptional profiles of the selected persistent marker genes were used to determine if persistent infections were indeed present in the upper reproductive tracts of the mice. We found that intranasal immunization during an active infection induced persistent infections in the oviducts, but not the uterine horns, and that intranasal immunization after clearance of infection, caused persistent infections in both the uterine horns and the oviducts of the mice. This is a significant finding, not only because it is the first time that C. muridarum persistence has been characterized in vitro, but also due to the fact that there is minimal characterization of in vivo persistence of any chlamydial species. It is possible that the induction of persistent infections in the reproductive tract might enhance the development of pathology and thereby enhance the risk of infertility, factors that need to be prevented by vaccination, not enhanced. Overall, this study has shown that the inoculating dose does not affect pathology development in the female reproductive tract of infected mice, but does alter the degree and rate of ascending infection. It has also been shown that intranasal immunization during an active genital infection, or after clearance of one, induces persistent infections in the uterine horns and oviducts of mice. This suggests that potential vaccine candidates will need to have these factors closely examined before progressing to clinical trials. This is significant, because if the same situation occurs in humans, a vaccine administered to an asymptomatic, or previously exposed individual may not afford any extra protection and may in fact enhance the risk of development of infection-associated sequelae. This suggests that a vaccine may serve the community better if administered before the commencement of sexual activity.

Improving posture and decreasing pain with novel shoe insoles

A single subject longevity study is presented as a case study for the Medical Device Partnering Program (MDPP). The MDPP supports the development of cutting-edge medical devices and assistive technologies, through unique collaborations between researchers, industry, clinical end-users and government. The study aimed to identify what effect the innersole has on specific muscles that may influence stability and whether the innersole had any influence on gait. Three tests were conducted; a standard gait test, dynamic balance test and a standing balance test. Results from the kinematic analysis showed reduced variability in post testing results when compared to pre testing results. Reductions in muscle activation levels were also found across all tests. Further testing with a larger sample size is required to determine if these effects are due to the innersole.

A novel method for the synthesis of monodisperse gold-coated silica nanoparticles

Monodisperse silica nanoparticles were synthesised by the well-known Stober protocol, then dispersed in acetonitrile (ACN) and subsequently added to a bisacetonitrile gold(I) coordination complex ([Au(MeCN)2]?) in ACN. The silica hydroxyl groups were deprotonated in the presence of ACN, generating a formal negative charge on the siloxy groups. This allowed the [Au(MeCN)2]? complex to undergo ligand exchange with the silica nanoparticles and form a surface coordination complex with reduction to metallic gold (Au0) proceeding by an inner sphere mechanism. The residual [Au(MeCN)2]? complex was allowed to react with water, disproportionating into Au0 and Au(III), respectively, with the Au0 adding to the reduced gold already bound on the silica surface. The so-formed metallic gold seed surface was found to be suitable for the conventional reduction of Au(III) to Au0 by ascorbic acid (ASC). This process generated a thin and uniform gold coating on the silica nanoparticles. The silica NPs batches synthesised were in a size range from 45 to 460 nm. Of these silica NP batches, the size range from 400 to 480 nm were used for the gold-coating experiments.

Discovering novel knowledge using granule mining

This paper presents an extended granule mining based methodology, to effectively describe the relationships between granules not only by traditional support and confidence, but by diversity and condition diversity as well. Diversity measures how diverse of a granule associated with the other granules, it provides a kind of novel knowledge in databases. We also provide an algorithm to implement the proposed methodology. The experiments conducted to characterize a real network traffic data collection show that the proposed concepts and algorithm are promising.

A novel CDVM based high-voltage converter using low power solid-state switches and a tuned resonant circuit designed for pulsed power applications

A novel concept of producing high dc voltage for pulsed-power applications is proposed in this paper. The topology consists of an LC resonant circuit supplied through a tuned alternating waveform that is produced by an inverter. The control scheme is based on the detection of variations in the resonant frequency and adjustment of the switching signal patterns for the inverter to produce a square waveform with exactly the same frequencies. Therefore the capacitor voltage oscillates divergently with an increasing amplitude. A simple one-stage capacitor-diode voltage multiplier (CDVM) connected to the resonant capacitor then rectifies the alternating voltage and gives a dc level equal to twice the input voltage amplitude. The produced high voltage appears then in the form of high-voltage pulses across the load. A basic model is simulated by Simulink platform of MATLAB and the results are included in the paper.

Syntaxin 6 and Vti1b form a novel SNARE complex, which is upregulated in activated macrophages to facilitate exocytosis of TNF.

A key function of activated macrophages is to secrete proinflammatory cytokines such as TNF; however, the intracellular pathway and machinery responsible for cytokine trafficking and secretion is largely undefined. Here we show that individual SNARE proteins involved in vesicle docking and fusion are regulated at both gene and protein expression upon stimulation with the bacterial cell wall component lipopolysaccharide. Focusing on two intracellular SNARE proteins, Vti1b and syntaxin 6 (Stx6), we show that they are up-regulated in conjunction with increasing cytokine secretion in activated macrophages and that their levels are selectively titrated to accommodate the volume and timing of post-Golgi cytokine trafficking. In macrophages, Vti1b and syntaxin 6 are localized on intracellular membranes and are present on isolated Golgi membranes and on Golgi-derived TNF� vesicles budded in vitro. By immunoprecipitation, we find that Vti1b and syntaxin 6 interact to form a novel intracellular Q-SNARE complex. Functional studies using overexpression of full-length and truncated proteins show that both Vti1b and syntaxin 6 function and have rate-limiting roles in TNF� trafficking and secretion. This study shows how macrophages have uniquely adapted a novel Golgi-associated SNARE complex to accommodate their requirement for increased cytokine secretion.

Sourdough & other stories : a story told in parts (a mosaic novel and exegesis)

The mosaic novel - with its independent 'story-tiles' linking together to form a complete narrative - has the potential to act as a reflection on the periodic resurfacing of unconscious memories in the conscious lives of fictional characters. This project is an exploration of the mosaic text as a fictional analogue of involuntary memory. These concepts are investigated as they appear in traditional fairy tales and engaged with in this thesis's creative component, Sourdough and Other Stories (approximately 80,000 words), a mosaic novel comprising sixteen interconnected 'story-tiles'. Traditional fairy tales are non-reflective and conducive to forgetting (i.e. anti-memory); fairy tale characters are frequently portrayed as psychologically two-dimensional, in that there is no examination of the mental and emotional distress caused when children are stolen/ abandoned/ lost and when adults are exiled. Sourdough and Other Stories is a creative examination of, and attempted to remedy, this lack of psychological depth. This creative work is at once something more than a short story collection, and something that is not a traditional novel, but instead a culmination of two modes of writing. It employs the fairy tale form to explore James' 'thorns in the spirit' (1898, p.199) in fiction; the anxiety caused by separation from familial and community groups. The exegesis, A Story Told in Parts - Sourdough and Other Stories is a critical essay (approximately 20,000 words in length), a companion piece to the mosaic novel, which analyses how my research question proceeded from my creative work, and considers the theoretical underpinnings of the creative work and how it enacts the research question: 'Can a writer use the structural possibilities of the mosaic text to create a fictional work that is an analogue of an involuntary memory?' The cumulative effect of the creative and exegetical works should be that of a dialogue between the two components - each text informing the other and providing alternate but complementary lenses with which to view the research question.

Isolation and characterisation of novel microsatellite and mitochondrial DNA markers for the Eastern Water Dragon (Physignathus lesueurii)

Habitat fragmentation as a result of urbanisation is a growing problem for native lizard species. The Eastern Water Dragon (Physignathus lesueurii) is a social arboreal agamid lizard, native to Australia. This species represents an ideal model species to investigate the effect of urbanisation because of their prominent abundance in the urban landscape. Here we describe the isolation and characterisation of a novel set of 74 di-, tri-, and tetramicrosatellites from which 18 were selected and optimised into two multiplexes. The 18 microsatellites generated a total 148 alleles across the two populations. The number of alleles per locus varied from 2 to 18 alleles and measures of Ho and He varied from 0.395 to 0.877 and from 0.441 to 0.880, respectively. We also present primers for four novel mitochondrial DNA (mtDNA) markers. The combined length of the four mtDNA marker pairs was 2,528 bp which included 15 nucleotides changes. In comparison to threatened species, which are generally characterised by small population sizes, the Eastern Water Dragon represents an ideal model species to investigate the effect of urbanisation on their behavioural ecology and connectivity patterns among populations.