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.

Performance control of distributed generation using digital estimation of signal parameters

The Queensland University of Technology (QUT) allows the presentation of a thesis for the Degree of Doctor of Philosophy in the format of published or submitted papers, where such papers have been published, accepted or submitted during the period of candidature. This thesis is composed of seven published/submitted papers, of which one has been published, three accepted for publication and the other three are under review. This project is financially supported by an Australian Research Council (ARC) Discovery Grant with the aim of proposing strategies for the performance control of Distributed Generation (DG) system with digital estimation of power system signal parameters. Distributed Generation (DG) has been recently introduced as a new concept for the generation of power and the enhancement of conventionally produced electricity. Global warming issue calls for renewable energy resources in electricity production. Distributed generation based on solar energy (photovoltaic and solar thermal), wind, biomass, mini-hydro along with use of fuel cell and micro turbine will gain substantial momentum in the near future. Technically, DG can be a viable solution for the issue of the integration of renewable or non-conventional energy resources. Basically, DG sources can be connected to local power system through power electronic devices, i.e. inverters or ac-ac converters. The interconnection of DG systems to power system as a compensator or a power source with high quality performance is the main aim of this study. Source and load unbalance, load non-linearity, interharmonic distortion, supply voltage distortion, distortion at the point of common coupling in weak source cases, source current power factor, and synchronism of generated currents or voltages are the issues of concern. The interconnection of DG sources shall be carried out by using power electronics switching devices that inject high frequency components rather than the desired current. Also, noise and harmonic distortions can impact the performance of the control strategies. To be able to mitigate the negative effect of high frequency and harmonic as well as noise distortion to achieve satisfactory performance of DG systems, new methods of signal parameter estimation have been proposed in this thesis. These methods are based on processing the digital samples of power system signals. Thus, proposing advanced techniques for the digital estimation of signal parameters and methods for the generation of DG reference currents using the estimates provided is the targeted scope of this thesis. An introduction to this research – including a description of the research problem, the literature review and an account of the research progress linking the research papers – is presented in Chapter 1. One of the main parameters of a power system signal is its frequency. Phasor Measurement (PM) technique is one of the renowned and advanced techniques used for the estimation of power system frequency. Chapter 2 focuses on an in-depth analysis conducted on the PM technique to reveal its strengths and drawbacks. The analysis will be followed by a new technique proposed to enhance the speed of the PM technique while the input signal is free of even-order harmonics. The other techniques proposed in this thesis as the novel ones will be compared with the PM technique comprehensively studied in Chapter 2. An algorithm based on the concept of Kalman filtering is proposed in Chapter 3. The algorithm is intended to estimate signal parameters like amplitude, frequency and phase angle in the online mode. The Kalman filter is modified to operate on the output signal of a Finite Impulse Response (FIR) filter designed by a plain summation. The frequency estimation unit is independent from the Kalman filter and uses the samples refined by the FIR filter. The frequency estimated is given to the Kalman filter to be used in building the transition matrices. The initial settings for the modified Kalman filter are obtained through a trial and error exercise. Another algorithm again based on the concept of Kalman filtering is proposed in Chapter 4 for the estimation of signal parameters. The Kalman filter is also modified to operate on the output signal of the same FIR filter explained above. Nevertheless, the frequency estimation unit, unlike the one proposed in Chapter 3, is not segregated and it interacts with the Kalman filter. The frequency estimated is given to the Kalman filter and other parameters such as the amplitudes and phase angles estimated by the Kalman filter is taken to the frequency estimation unit. Chapter 5 proposes another algorithm based on the concept of Kalman filtering. This time, the state parameters are obtained through matrix arrangements where the noise level is reduced on the sample vector. The purified state vector is used to obtain a new measurement vector for a basic Kalman filter applied. The Kalman filter used has similar structure to a basic Kalman filter except the initial settings are computed through an extensive math-work with regards to the matrix arrangement utilized. Chapter 6 proposes another algorithm based on the concept of Kalman filtering similar to that of Chapter 3. However, this time the initial settings required for the better performance of the modified Kalman filter are calculated instead of being guessed by trial and error exercises. The simulations results for the parameters of signal estimated are enhanced due to the correct settings applied. Moreover, an enhanced Least Error Square (LES) technique is proposed to take on the estimation when a critical transient is detected in the input signal. In fact, some large, sudden changes in the parameters of the signal at these critical transients are not very well tracked by Kalman filtering. However, the proposed LES technique is found to be much faster in tracking these changes. Therefore, an appropriate combination of the LES and modified Kalman filtering is proposed in Chapter 6. Also, this time the ability of the proposed algorithm is verified on the real data obtained from a prototype test object. Chapter 7 proposes the other algorithm based on the concept of Kalman filtering similar to those of Chapter 3 and 6. However, this time an optimal digital filter is designed instead of the simple summation FIR filter. New initial settings for the modified Kalman filter are calculated based on the coefficients of the digital filter applied. Also, the ability of the proposed algorithm is verified on the real data obtained from a prototype test object. Chapter 8 uses the estimation algorithm proposed in Chapter 7 for the interconnection scheme of a DG to power network. Robust estimates of the signal amplitudes and phase angles obtained by the estimation approach are used in the reference generation of the compensation scheme. Several simulation tests provided in this chapter show that the proposed scheme can very well handle the source and load unbalance, load non-linearity, interharmonic distortion, supply voltage distortion, and synchronism of generated currents or voltages. The purposed compensation scheme also prevents distortion in voltage at the point of common coupling in weak source cases, balances the source currents, and makes the supply side power factor a desired value.

Distribution feeder voltage support and power factor control by distributed multiple inverters

The potential of distributed reactive power control to improve the voltage profile of radial distribution feeders has been reported in literature by few authors. However, the multiple inverters injecting or absorbing reactive power across a distribution feeder may introduce control interactions and/or voltage instability. Such controller interactions can be alleviated if the inverters are allowed to operate on voltage droop. First, the paper demonstrates that a linear shallow droop line can maintain the steady state voltage profile close to reference, up to a certain level of loading. Then, impacts of the shallow droop line control on line losses and line power factors are examined. Finally, a piecewise linear droop line which can achieve reduced line losses and close to unity power factor at the feeder source is proposed.

Temporal tracking of mineralization and transcriptional developments of shell formation during the early life history of pearl oyster Pinctada maxima

Molluscan larval ontogeny is a highly conserved process comprising three principal developmental stages. A characteristic unique to each of these stages is shell design, termed prodissoconch I, prodissoconch II and dissoconch. These shells vary in morphology, mineralogy and microstructure. The discrete temporal transitions in shell biomineralization between these larval stages are utilized in this study to investigate transcriptional involvement in several distinct biomineralization events. Scanning electron microscopy and X-ray diffraction analysis of P. maxima larvae and juveniles collected throughout post-embryonic ontogenesis, document the mineralogy and microstructure of each shelled stage as well as establishing a timeline for transitions in biomineralization. P. maxima larval samples most representative of these biomineralization distinctions and transitions were analyzed for differential gene expression on the microarray platform PmaxArray 1.0. A number of transcripts are reported as differentially expressed in correlation to the mineralization events of P. maxima larval ontogeny. Some of those isolated are known shell matrix genes while others are novel; these are discussed in relation to potential shell formation roles. This interdisciplinary investigation has linked the shell developments of P. maxima larval ontogeny with corresponding gene expression profiles, furthering the elucidation of shell biomineralization.

Tracking employees' twists and turns : describing a mixed methods approach to assessing change in the psychological contract

The psychological contract is a key analytical device utilised by both academics and practitioners to conceptualise and explore the dynamics of the employment relationship. However, despite the recognised import of the construct, some authors suggest that its empirical investigation has fallen into a 'methodological rut' [Conway & Briner, 2005, p. 89] and is neglecting to assess key tenets of the concept, such as its temporal and dynamic nature. This paper describes the research design of a longitudinal, mixed methods study which draws upon the strengths of both qualitative and quantitative modes of inquiry in order to explore the development of, and changes in, the psychological contract. Underpinned by a critical realist philosophy, the paper seeks to offer a research design suitable for exploring the process of change not only within the psychological contract domain, but also for similar constructs in the human resource management and broader organisational behaviour fields.

Melanoma cell invasiveness is regulated by miR-211 suppression of the BRN2 transcription factor

To identify microRNAs potentially involved in melanomagenesis, we compared microRNA expression profiles between melanoma cell lines and cultured melanocytes. The most differentially expressed microRNA between the normal and tumor cell lines was miR-211. We focused on this pigment-cell-enriched miRNA as it is derived from the microphthalmia-associated transcription factor (MITF)-regulated gene, TRPM1 (melastatin). We find that miR-211 expression is greatly decreased in melanoma cells and melanoblasts compared to melanocytes. Bioinformatic analysis identified a large number of potential targets of miR-211, including POU3F2 (BRN2). Inhibition of miR-211 in normal melanocytes resulted in increased BRN2 protein, indicating that endogenous miR-211 represses BRN2 in differentiated cells. Over-expression of miR-211 in melanoma cell lines changed the invasive potential of the cells in vitro through directly targeting BRN2 translation. We propose a model for the apparent non-overlapping expression levels of BRN2 and MITF in melanoma, mediated by miR-211 expression.

Characterisation of the TaNF-Y family of transcription factors in wheat

Light plays a unique role for plants as it is both a source of energy for growth and a signal for development. Light captured by the pigments in the light harvesting complexes is used to drive the synthesis of the chemical energy required for carbon assimilation. The light perceived by photoreceptors activates effectors, such as transcription factors (TFs), which modulate the expression of light-responsive genes. Recently, it has been speculated that increasing the photosynthetic rate could further improve the yield potential of three carbon (C3) crops such as wheat. However, little is currently known about the transcriptional regulation of photosynthesis genes, particularly in crop species. Nuclear factor Y (NF-Y) TF is a functionally diverse regulator of growth and development in the model plant species, with demonstrated roles in embryo development, stress response, flowering time and chloroplast biogenesis. Furthermore, a light-responsive NF-Y binding site (CCAAT-box) is present in the promoter of a spinach photosynthesis gene. As photosynthesis genes are co-regulated by light and co-regulated genes typically have similar regulatory elements in their promoters, it seems likely that other photosynthesis genes would also have light-responsive CCAAT-boxes. This provided the impetus to investigate the NF-Y TF in bread wheat. This thesis is focussed on wheat NF-Y members that have roles in light-mediated gene regulation with an emphasis on their involvement in the regulation of photosynthesis genes. NF-Y is a heterotrimeric complex, comprised of the three subunits NF-YA, NF-YB and NF-YC. Unlike the mammalian and yeast counterparts, each of the three subunits is encoded by multiple genes in Arabidopsis. The initial step taken in this study was the identification of the wheat NF-Y family (Chapter 3). A search of the current wheat nucleotide sequence databases identified 37 NF-Y genes (10 NF-YA, 11 NF-YB, 14 NF-YC & 2 Dr1). Phylogenetic analysis revealed that each of the three wheat NF-Y (TaNF-Y) subunit families could be divided into 4-5 clades based on their conserved core regions. Outside of the core regions, eleven motifs were identified to be conserved between Arabidopsis, rice and wheat NF-Y subunit members. The expression profiles of TaNF-Y genes were constructed using quantitative real-time polymerase chain reaction (RT-PCR). Some TaNF-Y subunit members had little variation in their transcript levels among the organs, while others displayed organ-predominant expression profiles, including those expressed mainly in the photosynthetic organs. To investigate their potential role in light-mediated gene regulation, the light responsiveness of the TaNF-Y genes were examined (Chapters 4 and 5). Two TaNF-YB and five TaNF-YC members were markedly upregulated by light in both the wheat leaves and seedling shoots. To identify the potential target genes of the light-upregulated NF-Y subunit members, a gene expression correlation analysis was conducted using publically available Affymetrix Wheat Genome Array datasets. This analysis revealed that the transcript expression levels of TaNF-YB3 and TaNF-YC11 were significantly correlated with those of photosynthesis genes. These correlated express profiles were also observed in the quantitative RT-PCR dataset from wheat plants grown under light and dark conditions. Sequence analysis of the promoters of these wheat photosynthesis genes revealed that they were enriched with potential NF-Y binding sites (CCAAT-box). The potential role of TaNF-YB3 in the regulation of photosynthetic genes was further investigated using a transgenic approach (Chapter 5). Transgenic wheat lines constitutively expressing TaNF-YB3 were found to have significantly increased expression levels of photosynthesis genes, including those encoding light harvesting chlorophyll a/b-binding proteins, photosystem I reaction centre subunits, a chloroplast ATP synthase subunit and glutamyl-tRNA reductase (GluTR). GluTR is a rate-limiting enzyme in the chlorophyll biosynthesis pathway. In association with the increased expression of the photosynthesis genes, the transgenic lines had a higher leaf chlorophyll content, increased photosynthetic rate and had a more rapid early growth rate compared to the wild-type wheat. In addition to its role in the regulation of photosynthesis genes, TaNF-YB3 overexpression lines flower on average 2-days earlier than the wild-type (Chapter 6). Quantitative RT-PCR analysis showed that there was a 13-fold increase in the expression level of the floral integrator, TaFT. The transcript levels of other downstream genes (TaFT2 and TaVRN1) were also increased in the transgenic lines. Furthermore, the transcript levels of TaNF-YB3 were significantly correlated with those of constans (CO), constans-like (COL) and timing of chlorophyll a/b-binding (CAB) expression 1 [TOC1; (CCT)] domain-containing proteins known to be involved in the regulation of flowering time. To summarise the key findings of this study, 37 NF-Y genes were identified in the crop species wheat. An in depth analysis of TaNF-Y gene expression profiles revealed that the potential role of some light-upregulated members was in the regulation of photosynthetic genes. The involvement of TaNF-YB3 in the regulation of photosynthesis genes was supported by data obtained from transgenic wheat lines with increased constitutive expression of TaNF-YB3. The overexpression of TaNF-YB3 in the transgenic lines revealed this NF-YB member is also involved in the fine-tuning of flowering time. These data suggest that the NF-Y TF plays an important role in light-mediated gene regulation in wheat.

Improving cyclodiode - a risk factor analysis of complications

PURPOSE: The purpose of this study is to identify risk factors for developing complications following treatment of refractory glaucoma with transscleral diode laser cyclophotocoagulation (cyclodiode), to improve the safety profile of this treatment modality. METHOD: A retrospective analysis of 72 eyes from 70 patients who were treated with cyclodiode. RESULTS: The mean pre-treatment IOP was 37.0 mmHg (SD 11.0), with a mean post-treatment reduction in intraocular pressure (IOP) of 19.8 mmHg, and a mean IOP at last follow-up of 17.1 mmHg (SD 9.7). Mean total power delivered during treatment was 156.8 Joules (SD 82.7) over a mean of 1.3 treatments (SD 0.6). Sixteen eyes (22.2% of patients) developed complications from the treatment, with the most common being hypotony, occurring in 6 patients, including 4 with neovascular glaucoma. A higher pre-treatment IOP and higher mean total power delivery also were associated with higher complications. CONCLUSIONS: Cyclodiode is an effective treatment option for glaucoma that is refractory to other treatment options. By identifying risk factors for potential complications, cyclodiode can be modified accordingly for each patient to improve safety and efficacy.

Cabergoline Decreases Alcohol Drinking and Seeking Behaviors Via Glial Cell Line-Derived Neurotrophic Factor

Background: Cabergoline is an ergotamine derivative that increases the expression of glial cell line-derived neurotrophic factor (GDNF) in vitro. We recently showed that GDNF in the ventral tegmental area (VTA) reduces the motivation to consume alcohol. We therefore set out to determine whether cabergoline administration decreases alcohol-drinking and -seeking behaviors via GDNF. Methods: Reverse transcription polymerase chain reaction (RT-PCR) and Enzyme-Linked ImmunoSorbent Assay (ELISA) were used to measure GDNF levels. Western blot analysis was used for phosphorylation experiments. Operant self-administration in rats and a two-bottle choice procedure in mice were used to assess alcohol-drinking behaviors. Instrumental performance tested during extinction was used to measure alcohol-seeking behavior. The [35S]GTPγS binding assay was used to assess the expression and function of the dopamine D2 receptor (D2R). Results: We found that treatment of the dopaminergic-like cell line SH-SY5Y with cabergoline and systemic administration of cabergoline in rats resulted in an increase in GDNF level and in the activation of the GDNF pathway. Cabergoline treatment decreased alcohol-drinking and -seeking behaviors including relapse, and its action to reduce alcohol consumption was localized to the VTA. Finally, the increase in GDNF expression and the decrease in alcohol consumption by cabergoline were abolished in GDNF heterozygous knockout mice. Conclusions: Together, these findings suggest that cabergoline-mediated upregulation of the GDNF pathway attenuates alcohol-drinking behaviors and relapse. Alcohol abuse and addiction are devastating and costly problems worldwide. This study puts forward the possibility that cabergoline might be an effective treatment for these disorders. © 2009 Society of Biological Psychiatry.

A fibrocontractive mechanochemical model of dermal wound closure incorporating realistic growth factor kinetics

Fibroblasts and their activated phenotype, myofibroblasts, are the primary cell types involved in the contraction associated with dermal wound healing. Recent experimental evidence indicates that the transformation from fibroblasts to myofibroblasts involves two distinct processes: the cells are stimulated to change phenotype by the combined actions of transforming growth factor β (TGFβ) and mechanical tension. This observation indicates a need for a detailed exploration of the effect of the strong interactions between the mechanical changes and growth factors in dermal wound healing. We review the experimental findings in detail and develop a model of dermal wound healing that incorporates these phenomena. Our model includes the interactions between TGFβ and collagenase, providing a more biologically realistic form for the growth factor kinetics than those included in previous mechanochemical descriptions. A comparison is made between the model predictions and experimental data on human dermal wound healing and all the essential features are well matched.

Using strategic movement to calibrate a neural compass : a spiking network for tracking head direction in rats and robots

The head direction (HD) system in mammals contains neurons that fire to represent the direction the animal is facing in its environment. The ability of these cells to reliably track head direction even after the removal of external sensory cues implies that the HD system is calibrated to function effectively using just internal (proprioceptive and vestibular) inputs. Rat pups and other infant mammals display stereotypical warm-up movements prior to locomotion in novel environments, and similar warm-up movements are seen in adult mammals with certain brain lesion-induced motor impairments. In this study we propose that synaptic learning mechanisms, in conjunction with appropriate movement strategies based on warm-up movements, can calibrate the HD system so that it functions effectively even in darkness. To examine the link between physical embodiment and neural control, and to determine that the system is robust to real-world phenomena, we implemented the synaptic mechanisms in a spiking neural network and tested it on a mobile robot platform. Results show that the combination of the synaptic learning mechanisms and warm-up movements are able to reliably calibrate the HD system so that it accurately tracks real-world head direction, and that calibration breaks down in systematic ways if certain movements are omitted. This work confirms that targeted, embodied behaviour can be used to calibrate neural systems, demonstrates that ‘grounding’ of modeled biological processes in the real world can reveal underlying functional principles (supporting the importance of robotics to biology), and proposes a functional role for stereotypical behaviours seen in infant mammals and those animals with certain motor deficits. We conjecture that these calibration principles may extend to the calibration of other neural systems involved in motion tracking and the representation of space, such as grid cells in entorhinal cortex.

Autonomous aerial navigation and tracking of marine animals

In this paper, we describe the development of an independent and on-board visual servoing system which allows a computationally impoverished aerial vehicle to autonomously identify and track a moving surface target. Our image segmentation and target identification algorithms were developed with the specific task of monitoring whales at sea but could be adapted for other targets. Observing whales is important for many marine biology tasks and is currently performed manually from the shore or from boats. We also present hardware experiments which demonstrate the capabilities of our algorithms for object identification and tracking that enable a flying vehicle to track a moving target.