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.

The embodied hybrid space : designing ubiquitous computing towards an amplification of situated real world experiences

The emergence of mobile and ubiquitous computing technology has created what is often referred to as the hybrid space – a virtual layer of digital information and interaction opportunities that sit on top of and augment the physical environment. Embodied media materialise digital information as observable and sometimes interactive parts of the physical environment. The aim of this work is to explore ways to enhance people’s situated real world experience, and to find out what the role and impact of embodied media in achieving this goal can be. The Edge, an initiative of the State Library of Queensland in Brisbane, Australia, and case study of this thesis, envisions to be a physical place for people to meet, explore, experience, learn and teach each other creative practices in various areas related to digital technology and arts. Guided by an Action Research approach, this work applies Lefebvre’s triad of space (1991) to investigate the Edge as a social space from a conceived, perceived and lived point of view. Based on its creators’ vision and goals on the conceived level, different embodied media are iteratively designed, implemented and evaluated towards shaping and amplifying the Edge’s visitor experience on the perceived and lived level.

Mean and variance of estimates of the bispectrum of a harmonic random process-an analysis including leakage effects

Analytical expressions are derived for the mean and variance, of estimates of the bispectrum of a real-time series assuming a cosinusoidal model. The effects of spectral leakage, inherent in discrete Fourier transform operation when the modes present in the signal have a nonintegral number of wavelengths in the record, are included in the analysis. A single phase-coupled triad of modes can cause the bispectrum to have a nonzero mean value over the entire region of computation owing to leakage. The variance of bispectral estimates in the presence of leakage has contributions from individual modes and from triads of phase-coupled modes. Time-domain windowing reduces the leakage. The theoretical expressions for the mean and variance of bispectral estimates are derived in terms of a function dependent on an arbitrary symmetric time-domain window applied to the record. the number of data, and the statistics of the phase coupling among triads of modes. The theoretical results are verified by numerical simulations for simple test cases and applied to laboratory data to examine phase coupling in a hypothesis testing framework

Kallikrein-related peptidase 15 (prostinogen)

The third edition of the Handbook of Proteolytic Enzymes aims to be a comprehensive reference work for the enzymes that cleave proteins and peptides, and contains over 800 chapters. Each chapter is organized into sections describing the name and history, activity and specificity, structural chemistry, preparation, biological aspects, and distinguishing features for a specific peptidase. The subject of Chapter 619 is Kallikrein-related Peptidase 15 (Prostinogen).

The use of hypnosis in labor and delivery : a preliminary study

Self-hypnosis was taught to 87 obstetric patients (HYP) and was not taught to 56 other patients (CNTRL), all delivered by the same family physician, in order to determine whether the use of self-hypnosis by low-risk obstetric patients leads to fewer technologic interventions during their deliveries or greater satisfaction of parturients with their delivery experience or both. The outcomes of the deliveries of these two groups were compared, and the HYP group was compared to 352 low-risk patients delivered by other family physicians at the same hospital (WCH). Questionnaires were mailed postpartum to 156 patients, all delivered by the same family physician, to determine satisfaction with delivery using the Labor and Delivery Satisfaction Index (LADSI). The hypnosis group showed a significant reduction in the number of epidurals (11.4% less than CNTRL and 17.9% less than WCH, p < 0.05) and the use of intravenous lines (18.5% less for both, p < 0.05). The number of episiotomies was significantly less in the HYP group compared to WCH (15.9%, p < 0.05) and 11.5% less when compared to CNTRL. The tear rate was not statistically different. Combined use of the intervention triad (epidural–forceps–episiotomy) was less for HYP than for CNTRL (15.8% less) and WCH (10.2% less, p < 0.05). More deliveries were done in the labor room with HYP than CNTRL (21%, p < 0.05). The second stage was shortened by 10 min (HYP vs CNTRL). Overall satisfaction of HYP and CNTRL patients was similar and generally favorable.

Heritage language for Chinese Australians : negotiating 'Chineseness' and, capitalising on resources in the lived world

The ethnic identity and commitment of Heritage Language Learners play salient roles in Heritage Language learning process. The mutually constitutive effect amongst Heritage Language Learner's ethnic identity, commitment, and Heritage Language proficiency has been well documented in social psychological and poststructuralist literatures. Both social psychological and poststructural schools offer meaningful insights into particular contexts but receive critiques from other contexts. In addition, the two schools largely oppose each other. This study uses Bourdieu's sociological triad of habitus, capital, and field to reconcile the two schools through the examination of Chinese Heritage Language Learners in Australia, an idiosyncratic social, cultural, and historical context for these learners. Specifically, this study investigates how young Chinese Australian adults (18-35 in age) negotiate their 'Chineseness' and capitalise on resources through Chinese Heritage Language learning in the lived world. The study adopts an explanatory mixed methods design to combine the quantitative approach with the qualitative approach. The initial quantitative phase addresses the first research question: Is Chinese Heritage Language proficiency of young Chinese Australian adults influenced by their investment of capital, the strength of their habitus of 'Chineseness', or both? The subsequent qualitative phase addresses the second research question: How do young Chinese Australian adults understand their Chinese Heritage Language learning in relation to (potential) profits produced by this linguistic capital in given fields? The initial quantitative phase applies Structural Equation Modelling to analyse the data from an online survey with 230 respondents. Findings indicate the statistically significant positive contribution made by the habitus of 'Chineseness' and by investment of capital to Chinese Heritage Language proficiency (r = .71 and r = .86 respectively). Subsequent multiple regression analysis demonstrates that 62% of the variance of Chinese Heritage Language proficiency can be accounted for by the joint contribution of 'Chineseness' and 'capital'. The qualitative phase of the study uses multiple interviews with five participants. It reveals that Chinese Heritage Language offers meaningful benefits for participants in the forms of capital production and habitus capture or recapture. Findings from the two phases talk to each other in terms of the inherent entanglement amongst habitus of 'Chineseness', investment of capital, and Chinese Heritage Language proficiency. The study offers important contributions. Theoretically, by virtue of Bourdieu's signature concepts of habitus, capital, and field, the study provides answers to questions that both social psychological and poststructuralist theories have long been struggling to answer. Methodologically, the position of 'pluralism' talks back to Bourdieu's theory and forwards to the mixed methods design. Particularly, the study makes a methodological breakthrough: A set of instruments was developed and validated to quantify Bourdieu's key concepts of capital and habitus within certain social fields. Practically, understanding Chinese Australians' heterogeneity and the potential drivers behind Chinese Heritage Language learning contributes to the growing interest in Chinese Australians' contemporary life experiences and helps to better accommodate linguistically diverse Chinese Heritage Language Learners in Chinese language courses. In addition, this study is very timely. It resonates with the recently released Australia in the Asian Century White Paper: Chinese Australians, with sound knowledge of Chinese culture and language obtained through negotiating their 'Chineseness' and capitalising on diverse resources for learning, will help to serve Australia's economic, social, and political needs in unique ways.

Natural and engineered plasmin inhibitors : applications and design strategies

Plasmin is the primary enzyme responsible for dissolution of fibrin in the circulatory system. Plasminogen, the zymogen of plasmin is expressed ubiquitously in the human body [1], with the predominant source being the liver [2, 3]. Plasminogen is produced as an 810 amino acid protein with a 19 amino acid leader peptide, which is cleaved during secretion to produce the mature 791 amino acid one-chain zymogen. This is converted to plasmin by cleavage of the Arg561 - Val562 scissile bond [4], resulting in an active protease consisting of two disulfide linked chains. The amino-terminal heavy chain (residues Glu1-Arg561) is comprised of a plasminogen/apple/nematode (PAN) domain [5] and five kringle domains of approximately equal size [6] while the light chain (residues Val562-Asn791) contains a serine protease domain homologous to trypsin with a catalytic triad comprising His603, Asp646 and Ser741 [7]. Both plasmin and plasminogen occur in two forms, full length and a Lys77-Lys78 activated variant produced through self catalysis (Figure 1). The former exists in a tight conformation through binding of Lys50 and/or Lys62 to kringle domain 5 [8, 9] while Lys78-plasminogen assumes a more relaxed conformation rendering it more susceptible to plasmin conversion [10, 11].

EUELC project : a multi-centre, multipurpose study to investigate early stage NSCLC, and to establish a biobank for ongoing collaboration

The European Early Lung Cancer (EUELC) project aims to determine if specific genetic alterations occurring in lung carcinogenesis are detectable in the respiratory epithelium. In order to pursue this objective, nonsmall cell lung cancer (NSCLC) patients with a very high risk of developing progressive lung cancer were recruited from 12 centres in eight European countries: France, Germany, southern Ireland, Italy, the Netherlands, Poland, Spain and the UK. In addition, NSCLC patients were followed up every 6 months for 36 months. A European Bronchial Tissue Bank was set up at the University of Liverpool (Liverpool, UK) to optimise the use of biological specimens. The molecular - pathological investigations were subdivided into specific work packages that were delivered by EUELC Partners. The work packages encompassed mutational analysis, genetic instability, methylation profiling, expression profiling utilising immunohistochemistry and chip-based technologies, as well as in-depth analysis of FHIT and RARβ genes, the telomerase catalytic subunit hTERT and genotyping of susceptibility genes in specific pathways. The EUELC project engendered a tremendous collaborative effort, and it enabled the EUELC Partners to establish protocols for assessing molecular biomarkers in early lung cancer with the view to using such biomarkers for early diagnosis and as intermediate end-points in future chemopreventive programmes. Copyright©ERS Journals Ltd 2009.

Understanding the implications of digital interactions on the design of public urban spaces

This study investigated the impact of digital networked social interactions on the design of public urban spaces. Urban informatics, social media, ubiquitous computing, and mobile technology offer great potential to urban planning, as tools of communication, community engagement, and placemaking. The study considers the function of public spaces as 'third places,' that is, social places that are familiar, comfortable, social and meaningful for everyday life outside the home and work. Libraries were chosen as the study's focus. The study produced findings and insights at the intersection of urban planning (place), cultural geography and urban sociology (people), and information communication technology (technology) – the triad of urban informatics.

Exploring a biomimicry based approach and key barriers to evolving water infrastructure systems

Access to clean water is essential for human life and a critical issue facing much of modern society, especially as a result of the 21st Century triad of challenges – population growth, resource scarcity and pollution – which contribute to the rising complexity of providing adequate access to this essential resource for large parts of society. As such, there is now an increasing need for innovative solutions to source, treat and distribute water to cities across the globe. This position paper explores biomimicry – emulating natural form, function, process and systems – as an alternative and sustainable design approach to traditional water infrastructure systems. The key barriers to innovations such as biomimicry are summarised, indicating that regulatory and economic grounds are some of the major hindrances to integrating alternative design approaches in the water sector in developed countries. This paper examines some of the benefits of moving past these barriers to develop sustainable, efficient and resilient solutions that provide adequate access to water in the face of contemporary challenges.

Production and isolation of ligated metal(IV)-oxo ions by tandem mass spectrometry

High valent metal(IV)-oxo species, \[M(=O)(Melm)(n)(OAc)](+) (M = Mn-Ni, MeIm = 1-methylimidazole, n = 1-2), which are relevant to biology and oxidative catalysis, were produced and isolated in gas-phase reactions of the metal(II) precursor ions \[M(MeIm)(n)(OAc)](+) (M = Mn-Zn, n = 1-3) with ozone. The precursor ions \[M(MeIm)(OAc)](+) and \[M(MeIm)(2)(OAc)](+) were generated via collision-induced dissociation of the corresponding \[M(MeIm)(3)(OAc)](+) ion. The dependence of ozone reactivity on metal and coordination number is discussed. Copyright (C) 2010 John Wiley & Sons, Ltd.

Rendering to Caesar and God : St Paul, the natural law tradition and the authority of law

The question of the authority of law has occupied and vexed the literature and philosophy of law for centuries. Law is something that characteristically implies obedience, but the precise nature of law’s authority remains contentious. The return to the writings of the Apostle Paul in contemporary philosophy, theology and jurisprudence begs attention in relation to the authority of law, and so this article will consider his analysis and critique of law with a focus on his Epistle to the Romans. It argues that Paul’s conception of the authority of law is explained on the basis that the law is from God, it externally sanctions obedience by virtue of the civil authorities, and it convicts internally in conscience. This triad is justified by the law of love (‘‘love your neighbor as yourself’’), and will be explained in relation to the natural law tradition as well as converse ideas in positivism. Hence, considering the reasoning of Paul in relation to traditional jurisprudential themes and the law of love provides a useful alternative analysis and basis for further investigation regarding the authority of law and the need for its obedience.

Digital literacies: Understanding the literate practices of refugee kids in an Afterschool Media Club

Since the 2000s, teachers in an increasing number of Australian schools have been learning how to support students with refugee backgrounds. For some of these students, entry into the Australian school system is not easy. English literacy is integral to some of the challenges confronting the students. In response, educators have been developing and researching ways of engaging with the students’ language and literacy learning. Much of the focus has been on traditional print-based school literacies. In contrast, I look here at student engagement in digital literacies in an after-school media club. Several concepts from the theory of French sociologist, Pierre Bourdieu are useful for understanding the position of students of refugee background in the Australian school system. Like other conflict theories, Bourdieusian theory has sometimes been criticised as ‘pessimistic’, that is, for suggesting that schools necessarily reproduce social disadvantage. However, others have used Bourdieusian theory to analyse and critique the reproductive work of schooling for groups of students who experience educational disadvantage. I align myself with this latter tradition. Specifically, I use Bourdieu’s triad of concepts to explain aspects of the literacy education experiences of some young people of refugee background: field, capital and habitus. In particular, I look at questions of the legitimation of students’ competences as capital in literate fields within and beyond the school context. Data are drawn from an Australian Research Council-funded project, Digital Learning and Print Literacy: A design experiment for the reform of low socio-economic, culturally diverse schools (2009-14). The data analysed in this chapter include interviews and observations relating to the participation of two Congolese girls in an after school media club. Implications are drawn for teachers of literacy in culturally and linguistically diverse contexts. Consideration is made of early childhood, primary and secondary settings.

Participatory action research for civic engagement

The future of civic engagement is characterised by both technological innovation as well as new technological user practices that are fuelled by trends towards mobile, personal devices; broadband connectivity; open data; urban interfaces; and, cloud computing. These technology trends are progressing at a rapid pace, and have led global technology vendors to package and sell the ‘Smart City’ as a centralized service delivery platform predicted to optimize and enhance cities’ key performance indicators – and generate a profitable market. The top-down deployment of these large and proprietary technology platforms have helped sectors such as energy, transport, and healthcare to increase efficiencies. However, an increasing number of scholars and commentators warn of another ‘IT bubble’ emerging. Along with some city leaders, they argue that the top-down approach does not fit the governance dynamics and values of a liberal democracy when applied across sectors. A thorough understanding is required, of the socio-cultural nuances of how people work, live, play across different environments, and how they employ social media and mobile devices to interact with, engage in, and constitute public realms. Although the term ‘slacktivism’ is sometimes used to denote a watered down version of civic engagement and activism that is reduced to clicking a ‘Like’ button and signing online petitions, we believe that we are far from witnessing another Biedermeier period that saw people focus on the domestic and the non-political. There is plenty of evidence to the contrary, such as post-election violence in Kenya in 2008, the Occupy movements in New York, Hong Kong and elsewhere, the Arab Spring, Stuttgart 21, Fukushima, the Taksim Gezi Park in Istanbul, and the Vinegar Movement in Brazil in 2013. These examples of civic action shape the dynamics of governments, and in turn, call for new processes to be incorporated into governance structures. Participatory research into these new processes across the triad of people, place and technology is a significant and timely investment to foster productive, sustainable, and livable human habitats. With this chapter, we want to reframe the current debates in academia and priorities in industry and government to allow citizens and civic actors to take their rightful centerpiece place in civic movements. This calls for new participatory approaches for co-inquiry and co-design. It is an evolving process with an explicit agenda to facilitate change, and we propose participatory action research (PAR) as an indispensable component in the journey to develop new governance infrastructures and practices for civic engagement. This chapter proposes participatory action research as a useful and fitting research paradigm to guide methodological considerations surrounding the study, design, development, and evaluation of civic technologies. We do not limit our definition of civic technologies to tools specifically designed to simply enhance government and governance, such as renewing your car registration online or casting your vote electronically on election day. Rather, we are interested in civic media and technologies that foster citizen engagement in the widest sense, and particularly the participatory design of such civic technologies that strive to involve citizens in political debate and action as well as question conventional approaches to political issues (DiSalvo, 2012; Dourish, 2010; Foth et al., 2013). Following an outline of some underlying principles and assumptions behind participatory action research, especially as it applies to cities, we will critically review case studies to illustrate the application of this approach with a view to engender robust, inclusive, and dynamic societies built on the principles of engaged liberal democracy. The rationale for this approach is an alternative to smart cities in a ‘perpetual tomorrow,’ (cf. e.g. Dourish & Bell, 2011), based on many weak and strong signals of civic actions revolving around technology seen today. It seeks to emphasize and direct attention to active citizenry over passive consumerism, human actors over human factors, culture over infrastructure, and prosperity over efficiency. First, we will have a look at some fundamental issues arising from applying simplistic smart city visions to the kind of a problem a city is (cf. Jacobs, 1961). We focus on the touch points between “the city” and its civic body, the citizens. In order to provide for meaningful civic engagement, the city must provide appropriate interfaces.