Biochemical characterization of Aprataxin, the protein deficient in Ataxia with Oculomotor Apraxia type 1

Neurodegenerative disorders are heterogenous in nature and include a range of ataxias with oculomotor apraxia, which are characterised by a wide variety of neurological and ophthalmological features. This family includes recessive and dominant disorders. A subfamily of autosomal recessive cerebellar ataxias are characterised by defects in the cellular response to DNA damage. These include the well characterised disorders Ataxia-Telangiectasia (A-T) and Ataxia-Telangiectasia Like Disorder (A-TLD) as well as the recently identified diseases Spinocerebellar ataxia with axonal neuropathy Type 1 (SCAN1), Ataxia with Oculomotor Apraxia Type 2 (AOA2), as well as the subject of this thesis, Ataxia with Oculomotor Apraxia Type 1 (AOA1). AOA1 is caused by mutations in the APTX gene, which is located at chromosomal locus 9p13. This gene codes for the 342 amino acid protein Aprataxin. Mutations in APTX cause destabilization of Aprataxin, thus AOA1 is a result of Aprataxin deficiency. Aprataxin has three functional domains, an N-terminal Forkhead Associated (FHA) phosphoprotein interaction domain, a central Histidine Triad (HIT) nucleotide hydrolase domain and a C-terminal C2H2 zinc finger. Aprataxins FHA domain has homology to FHA domain of the DNA repair protein 5’ polynucleotide kinase 3’ phosphatase (PNKP). PNKP interacts with a range of DNA repair proteins via its FHA domain and plays a critical role in processing damaged DNA termini. The presence of this domain with a nucleotide hydrolase domain and a DNA binding motif implicated that Aprataxin may be involved in DNA repair and that AOA1 may be caused by a DNA repair deficit. This was substantiated by the interaction of Aprataxin with proteins involved in the repair of both single and double strand DNA breaks (XRay Cross-Complementing 1, XRCC4 and Poly-ADP Ribose Polymerase-1) and the hypersensitivity of AOA1 patient cell lines to single and double strand break inducing agents. At the commencement of this study little was known about the in vitro and in vivo properties of Aprataxin. Initially this study focused on generation of recombinant Aprataxin proteins to facilitate examination of the in vitro properties of Aprataxin. Using recombinant Aprataxin proteins I found that Aprataxin binds to double stranded DNA. Consistent with a role for Aprataxin as a DNA repair enzyme, this binding is not sequence specific. I also report that the HIT domain of Aprataxin hydrolyses adenosine derivatives and interestingly found that this activity is competitively inhibited by DNA. This provided initial evidence that DNA binds to the HIT domain of Aprataxin. The interaction of DNA with the nucleotide hydrolase domain of Aprataxin provided initial evidence that Aprataxin may be a DNA-processing factor. Following these studies, Aprataxin was found to hydrolyse 5’adenylated DNA, which can be generated by unscheduled ligation at DNA breaks with non-standard termini. I found that cell extracts from AOA1 patients do not have DNA-adenylate hydrolase activity indicating that Aprataxin is the only DNA-adenylate hydrolase in mammalian cells. I further characterised this activity by examining the contribution of the zinc finger and FHA domains to DNA-adenylate hydrolysis by the HIT domain. I found that deletion of the zinc finger ablated the activity of the HIT domain against adenylated DNA, indicating that the zinc finger may be required for the formation of a stable enzyme-substrate complex. Deletion of the FHA domain stimulated DNA-adenylate hydrolysis, which indicated that the activity of the HIT domain may be regulated by the FHA domain. Given that the FHA domain is involved in protein-protein interactions I propose that the activity of Aprataxins HIT domain may be regulated by proteins which interact with its FHA domain. We examined this possibility by measuring the DNA-adenylate hydrolase activity of extracts from cells deficient for the Aprataxin-interacting DNA repair proteins XRCC1 and PARP-1. XRCC1 deficiency did not affect Aprataxin activity but I found that Aprataxin is destabilized in the absence of PARP-1, resulting in a deficiency of DNA-adenylate hydrolase activity in PARP-1 knockout cells. This implies a critical role for PARP-1 in the stabilization of Aprataxin. Conversely I found that PARP-1 is destabilized in the absence of Aprataxin. PARP-1 is a central player in a number of DNA repair mechanisms and this implies that not only do AOA1 cells lack Aprataxin, they may also have defects in PARP-1 dependant cellular functions. Based on this I identified a defect in a PARP-1 dependant DNA repair mechanism in AOA1 cells. Additionally, I identified elevated levels of oxidized DNA in AOA1 cells, which is indicative of a defect in Base Excision Repair (BER). I attribute this to the reduced level of the BER protein Apurinic Endonuclease 1 (APE1) I identified in Aprataxin deficient cells. This study has identified and characterised multiple DNA repair defects in AOA1 cells, indicating that Aprataxin deficiency has far-reaching cellular consequences. Consistent with the literature, I show that Aprataxin is a nuclear protein with nucleoplasmic and nucleolar distribution. Previous studies have shown that Aprataxin interacts with the nucleolar rRNA processing factor nucleolin and that AOA1 cells appear to have a mild defect in rRNA synthesis. Given the nucleolar localization of Aprataxin I examined the protein-protein interactions of Aprataxin and found that Aprataxin interacts with a number of rRNA transcription and processing factors. Based on this and the nucleolar localization of Aprataxin I proposed that Aprataxin may have an alternative role in the nucleolus. I therefore examined the transcriptional activity of Aprataxin deficient cells using nucleotide analogue incorporation. I found that AOA1 cells do not display a defect in basal levels of RNA synthesis, however they display defective transcriptional responses to DNA damage. In summary, this thesis demonstrates that Aprataxin is a DNA repair enzyme responsible for the repair of adenylated DNA termini and that it is required for stabilization of at least two other DNA repair proteins. Thus not only do AOA1 cells have no Aprataxin protein or activity, they have additional deficiencies in PolyADP Ribose Polymerase-1 and Apurinic Endonuclease 1 dependant DNA repair mechanisms. I additionally demonstrate DNA-damage inducible transcriptional defects in AOA1 cells, indicating that Aprataxin deficiency confers a broad range of cellular defects and highlighting the complexity of the cellular response to DNA damage and the multiple defects which result from Aprataxin deficiency. My detailed characterization of the cellular consequences of Aprataxin deficiency provides an important contribution to our understanding of interlinking DNA repair processes.

Monitoring the Performance of Complex Projects from Multiple Perspectives over Multiple Time Frames

When complex projects go wrong they can go horribly wrong with severe financial consequences. We are undertaking research to develop leading performance indicators for complex projects, metrics to provide early warning of potential difficulties. The assessment of success of complex projects can be made by a range of stakeholders over different time scales, against different levels of project results: the project’s outputs at the end of the project; the project’s outcomes in the months following project completion; and the project’s impact in the years following completion. We aim to identify leading performance indicators, which may include both success criteria and success factors, and which can be measured by the project team during project delivery to forecast success as assessed by key stakeholders in the days, months and years following the project. The hope is the leading performance indicators will act as alarm bells to show if a project is diverting from plan so early corrective action can be taken. It may be that different combinations of the leading performance indicators will be appropriate depending on the nature of project complexity. In this paper we develop a new model of project success, whereby success is assessed by different stakeholders over different time frames against different levels of project results. We then relate this to measurements that can be taken during project delivery. A methodology is described to evaluate the early parts of this model. Its implications and limitations are described. This paper describes work in progress.

Structural health monitoring of bridges using acoustic emission technology and signal processing techniques

Structural health monitoring (SHM) is the term applied to the procedure of monitoring a structure’s performance, assessing its condition and carrying out appropriate retrofitting so that it performs reliably, safely and efficiently. Bridges form an important part of a nation’s infrastructure. They deteriorate due to age and changing load patterns and hence early detection of damage helps in prolonging the lives and preventing catastrophic failures. Monitoring of bridges has been traditionally done by means of visual inspection. With recent developments in sensor technology and availability of advanced computing resources, newer techniques have emerged for SHM. Acoustic emission (AE) is one such technology that is attracting attention of engineers and researchers all around the world. This paper discusses the use of AE technology in health monitoring of bridge structures, with a special focus on analysis of recorded data. AE waves are stress waves generated by mechanical deformation of material and can be recorded by means of sensors attached to the surface of the structure. Analysis of the AE signals provides vital information regarding the nature of the source of emission. Signal processing of the AE waveform data can be carried out in several ways and is predominantly based on time and frequency domains. Short time Fourier transform and wavelet analysis have proved to be superior alternatives to traditional frequency based analysis in extracting information from recorded waveform. Some of the preliminary results of the application of these analysis tools in signal processing of recorded AE data will be presented in this paper.

Prediction of psychological adjustment to multiple sclerosis

This study examined the utility of self-efficacy as a predictor of social activity and mood control in multiple sclerosis (MS). Seventy-one subjects with MS were recruited from people attending an MS centre or from a mailing list and were examined on two occasions that were two months apart. Clinic patients were more disabled than patients who completed assessments by post, but they were of higher socioeconomic status and were less dysphoric. We attempted to predict self-reported performance of mood control and social activity at two months, from self-efficacy or performance on these tasks at pretest. Demographic variables, disorder status, disability, self-esteem and depression were also allowed to compete for entry into multiple regressions. Substantial stability in mood, performance and disability was observed over the two months. In both mood control and social activity, past performance was the strongest predictor of later performance, but self-efficacy also contributed significantly to the prediction. The disability level entered a prediction of socila activity, but no other variables predicted either type of performance. A secondary analysis predicting self-esteem at two months also included self-efficacy for social activity, illustrating the contribution of perceived capability to later assessments of self-worth. The study provided support for self-efficacy as a predictor of later behavioural outcomes and self-esteem in multiple sclerosis.

Evaluation of multiple sewage-associated bacteroides PCR markers for sewage pollution tracking

The host specificity of the five published sewage-associated Bacteroides markers (i.e., HF183, BacHum, HuBac, BacH and Human-Bac) was evaluated in Southeast Queensland, Australia by testing fecal DNA samples (n = 186) from 11 animal species including human fecal samples collected via influent to a sewage treatment plant (STP). All human fecal samples (n = 50) were positive for all five markers indicating 100% sensitivity of these markers. The overall specificity of the HF183 markers to differentiate between humans and animals was 99%. The specificities of the BacHum and BacH markers were > 94%, suggesting that these markers are suitable for sewage pollution in environmental waters in Australia. The BacHum (i.e., 63% specificity) and Human-Bac (i.e., 79% specificity) markers performed poorly in distinguishing between the sources of human and animal fecal samples. It is recommended that the specificity of the sewage-associated markers must be rigorously tested prior to its application to identify the sources of fecal pollution in environmental waters.

Queensland Emergency Medical System : a structural and organizational model for the emergency medical system in Australia

Objective: The emergency medical system (EMS) can be defined as a comprehensive, coordinated and integrated system of care for patients suffering acute illness and injury. The aim of the present paper is to describe the evolution of the Queensland Emergency Medical System (QEMS) and to recommend a strategic national approach to EMS development. Methods: Following the formation of the Queensland Ambulance Service in 1991, a state EMS committee was formed. This committee led the development and approval of the cross portfolio QEMS policy framework that has resulted in dynamic policy development, system monitoring and evaluation. This framework is led by the Queensland Emergency Medical Services Advisory Committee. Results: There has been considerable progress in the development of all aspects of the EMS in Queensland. These developments have derived from the improved coordination and leadership that QEMS provides and has resulted in widespread satisfaction by both patients and stakeholders. Conclusions: The strategic approach outlined in the present paper offers a model for EMS arrangements throughout Australia. We propose that the Council of Australian Governments should require each state and Territory to maintain an EMS committee. These state EMS committees should have a broad portfolio of responsibilities. They should provide leadership and direction to the development of the EMS and ensure coordination and quality of outcomes. A national EMS committee with broad representation and broad scope should be established to coordinate the national development of Australia's EMS.

A new day breaks for the Aussie film industry

The Australian film industry is evolving. The days when government film agencies handed out millions of taxpayers' dollars for filmmakers to produce "Australian stories" with little regard to commercial returns are limited. If the Australian film industry is to reach mainstream audiences – and increase its relevance – then filmmakers need to take greater notice of genre movies and the possibilities they create within the financial restraints of the local industry. The $20 million Aussie vampire movie, Daybreakers, is a prototype for how this can be achieved.

Structural health monitoring of bridges using acoustic emission technology

Bridges are an important part of society's infrastructure and reliable methods are necessary to monitor them and ensure their safety and efficiency. Bridges deteriorate with age and early detection of damage helps in prolonging the lives and prevent catastrophic failures. Most bridges still in used today were built decades ago and are now subjected to changes in load patterns, which can cause localized distress and if not corrected can result in bridge failure. In the past, monitoring of structures was usually done by means of visual inspection and tapping of the structures using a small hammer. Recent advancements of sensors and information technologies have resulted in new ways of monitoring the performance of structures. This paper briefly describes the current technologies used in bridge structures condition monitoring with its prime focus in the application of acoustic emission (AE) technology in the monitoring of bridge structures and its challenges.

Resilience in chronic disease : the relationships among risk factors, protective factors, adaptive outcomes, and the level of resilience in adults with diabetes

Background: There are innumerable diabetes studies that have investigated associations between risk factors, protective factors, and health outcomes; however, these individual predictors are part of a complex network of interacting forces. Moreover, there is little awareness about resilience or its importance in chronic disease in adulthood, especially diabetes. Thus, this is the first study to: (1) extensively investigate the relationships among a host of predictors and multiple adaptive outcomes; and (2) conceptualise a resilience model among people with diabetes. Methods: This cross-sectional study was divided into two research studies. Study One was to translate two diabetes-specific instruments (Problem Areas In Diabetes, PAID; Diabetes Coping Measure, DCM) into a Chinese version and to examine their psychometric properties for use in Study Two in a convenience sample of 205 outpatients with type 2 diabetes. In Study Two, an integrated theoretical model is developed and evaluated using the structural equation modelling (SEM) technique. A self-administered questionnaire was completed by 345 people with type 2 diabetes from the endocrine outpatient departments of three hospitals in Taiwan. Results: Confirmatory factor analyses confirmed a one-factor structure of the PAID-C which was similar to the original version of the PAID. Strong content validity of the PAID-C was demonstrated. The PAID-C was associated with HbA1c and diabetes self-care behaviours, confirming satisfactory criterion validity. There was a moderate relationship between the PAID-C and the Perceived Stress Scale, supporting satisfactory convergent validity. The PAID-C also demonstrated satisfactory stability and high internal consistency. A four-factor structure and strong content validity of the DCM-C was confirmed. Criterion validity demonstrated that the DCM-C was significantly associated with HbA1c and diabetes self-care behaviours. There was a statistical correlation between the DCM-C and the Revised Ways of Coping Checklist, suggesting satisfactory convergent validity. Test-retest reliability demonstrated satisfactory stability of the DCM-C. The total scale of the DCM-C showed adequate internal consistency. Age, duration of diabetes, diabetes symptoms, diabetes distress, physical activity, coping strategies, and social support were the most consistent factors associated with adaptive outcomes in adults with diabetes. Resilience was positively associated with coping strategies, social support, health-related quality of life, and diabetes self-care behaviours. Results of the structural equation modelling revealed protective factors had a significant direct effect on adaptive outcomes; however, the construct of risk factors was not significantly related to adaptive outcomes. Moreover, resilience can moderate the relationships among protective factors and adaptive outcomes, but there were no interaction effects of risk factors and resilience on adaptive outcomes. Conclusion: This study contributes to an understanding of how risk factors and protective factors work together to influence adaptive outcomes in blood sugar control, health-related quality of life, and diabetes self-care behaviours. Additionally, resilience is a positive personality characteristic and may be importantly involved in the adjustment process among people living with type 2 diabetes.

Statistical analysis and reduction of multiple access interference in MC-CDMA systems

Multicarrier code division multiple access (MC-CDMA) is a very promising candidate for the multiple access scheme in fourth generation wireless communi- cation systems. During asynchronous transmission, multiple access interference (MAI) is a major challenge for MC-CDMA systems and significantly affects their performance. The main objectives of this thesis are to analyze the MAI in asyn- chronous MC-CDMA, and to develop robust techniques to reduce the MAI effect. Focus is first on the statistical analysis of MAI in asynchronous MC-CDMA. A new statistical model of MAI is developed. In the new model, the derivation of MAI can be applied to different distributions of timing offset, and the MAI power is modelled as a Gamma distributed random variable. By applying the new statistical model of MAI, a new computer simulation model is proposed. This model is based on the modelling of a multiuser system as a single user system followed by an additive noise component representing the MAI, which enables the new simulation model to significantly reduce the computation load during computer simulations. MAI reduction using slow frequency hopping (SFH) technique is the topic of the second part of the thesis. Two subsystems are considered. The first sub- system involves subcarrier frequency hopping as a group, which is referred to as GSFH/MC-CDMA. In the second subsystem, the condition of group hopping is dropped, resulting in a more general system, namely individual subcarrier frequency hopping MC-CDMA (ISFH/MC-CDMA). This research found that with the introduction of SFH, both of GSFH/MC-CDMA and ISFH/MC-CDMA sys- tems generate less MAI power than the basic MC-CDMA system during asyn- chronous transmission. Because of this, both SFH systems are shown to outper- form MC-CDMA in terms of BER. This improvement, however, is at the expense of spectral widening. In the third part of this thesis, base station polarization diversity, as another MAI reduction technique, is introduced to asynchronous MC-CDMA. The com- bined system is referred to as Pol/MC-CDMA. In this part a new optimum com- bining technique namely maximal signal-to-MAI ratio combining (MSMAIRC) is proposed to combine the signals in two base station antennas. With the applica- tion of MSMAIRC and in the absents of additive white Gaussian noise (AWGN), the resulting signal-to-MAI ratio (SMAIR) is not only maximized but also in- dependent of cross polarization discrimination (XPD) and antenna angle. In the case when AWGN is present, the performance of MSMAIRC is still affected by the XPD and antenna angle, but to a much lesser degree than the traditional maximal ratio combining (MRC). Furthermore, this research found that the BER performance for Pol/MC-CDMA can be further improved by changing the angle between the two receiving antennas. Hence the optimum antenna angles for both MSMAIRC and MRC are derived and their effects on the BER performance are compared. With the derived optimum antenna angle, the Pol/MC-CDMA system is able to obtain the lowest BER for a given XPD.