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.

Functional outcome and usage of the prosthesis of lower limb amputees fitted with osseointegrated fixation

This study established that the core principle underlying categorisation of activities have the potential to provide more comprehensive outcomes than the recognition of activities because it takes into consideration activities other than directional locomotion.

Integrated versus non-integrated management and care for clients with co-occurring mental health and substance use disorders : a qualitative systematic review of randomised controlled trials

The purpose of this paper is to conduct a qualitative review of randomised controlled trials in relation to the treatment of adults with co-occurring mental health and substance use disorder (MH/SUD). In particular, integrated approaches are compared with non-integrated approaches to treatment. Ten articles were identified for inclusion in the review. The findings are equivocal with regard to the superior efficacy of integrated approaches to treatment, although the many limitations of the studies need to be considered in our understanding of this finding. Clearly, this is an extremely challenging client group to engage and maintain in intervention research, and the complexity and variability of the problems render control particularly difficult. The lack of available evidence to support the superiority of integration is discussed in relation to these challenges. Much remains to be investigated with regard to integrated management and care for people with co-occurring and MH/SUD, particularly for specific combinations of dual diagnosis and giving consideration to the level of inter-relatedness between the disorders.

Deepening our understanding about school leadership : learning from educators and non-educators

Increasingly, leadership is argued as a way forward to improve performance and practice in a variety of contexts. School leadership is no different. There is little doubt that in the current globalised world characterized by change and complexity, effective school leadership is a key requirement. The contribution of this chapter is framed around a synthesis of current research, writing and theoretical insights regarding leadership. It draws upon three bodies of writing, Firstly, it begins by distilling several key themes and trends regarding educational leadership from the current research and writing. Secondly, it reports on the findings of a current research project carried out by the authors that explored the leadership stories of ten outstanding leaders from non-educational settings in Australia. Finally, it concludes by referring to some of the paradoxes and tensions inherent in the work of school leaders. It is argued that understanding and endeavouring to reconcile these dilemmas is a pre-requisite for school leaders as they continue to operate in an environment fraught with change and complexity.

Optimisation of performance in top level athletes : an action-based coping approach : a commentary

INTRODUCTION In their target article, Yuri Hanin and Muza Hanina outlined a novel multidisciplinary approach to performance optimisation for sport psychologists called the Identification-Control-Correction (ICC) programme. According to the authors, this empirically-verified, psycho-pedagogical strategy is designed to improve the quality of coaching and consistency of performance in highly skilled athletes and involves a number of steps including: (i) identifying and increasing self-awareness of ‘optimal’ and ‘non-optimal’ movement patterns for individual athletes; (ii) learning to deliberately control the process of task execution; and iii), correcting habitual and random errors and managing radical changes of movement patterns. Although no specific examples were provided, the ICC programme has apparently been successful in enhancing the performance of Olympic-level athletes. In this commentary, we address what we consider to be some important issues arising from the target article. We specifically focus attention on the contentious topic of optimization in neurobiological movement systems, the role of constraints in shaping emergent movement patterns and the functional role of movement variability in producing stable performance outcomes. In our view, the target article and, indeed, the proposed ICC programme, would benefit from a dynamical systems theoretical backdrop rather than the cognitive scientific approach that appears to be advocated. Although Hanin and Hanina made reference to, and attempted to integrate, constructs typically associated with dynamical systems theoretical accounts of motor control and learning (e.g., Bernstein’s problem, movement variability, etc.), these ideas required more detailed elaboration, which we provide in this commentary.

Teacher Reporting Questionnaire : Western Australia Non-Government Schools

This instrument was used in the project named Teachers Reporting Child Sexual Abuse: Towards Evidence-based Reform of Law, Policy and Practice (ARC DP0664847)

Patterns of fitting cosmetically tinted contact lenses

Cosmetically tinted soft contact lenses are an attractive option for contact lens wearers. Data that we have gathered from annual contact lens fitting surveys demonstrate that those wearing tinted lenses are more likely to be female (4.6% of all soft lenses fitted vs. 1.6% for males; p < 0.0001) and younger (27 11 years vs. 33 13 years for those wearing non-tinted lenses; p < 0.0001). Tinted lenses tend to be worn more on a part-time basis and are replaced less frequently than non-tinted lenses. The decline in fitting tinted lenses over the past 12 years may be due to (a) the current limited availability of tinted lenses in silicone hydrogel materials and daily disposable replacement frequencies, which together represent a significant majority (78%) of new soft lenses fits today, (b) growing concerns among lens wearers and practitioners relating to the risks of complications associated with the wearing of tinted lenses, and (c) reduced promotion of such lenses by the contact lens industry.

Social networking : investigating the features of Facebook application

Research on social networking sites like Facebook is emerging but sparse. The exploratory study investigates the value users derive from self-described ‘cool’ Facebook applications, and explores the features that either encourage or discourage users to recommend application to their friends. Thus the concepts of value and cool are explored in a social networking setting. Our qualitative data shows that consumers derive a combination of functional value along with either social or emotional value from the applications. Female Facebook users indicated self-expression as important, while mates then to use Facebook application to socially compete. Three broad categories emerged for application features; symmetrical features can both encourage or discourage recommendation, asymmetrical features one encourage or discourage but not both, and polar features where different levels of the same feature encourage or discourage. Recommending or not recommending an application tends to be the result of a combination of features rather than one feature in isolation.

Writing together, learning together : the value and effectiveness of a research writing group for doctoral students

The high level of scholarly writing required for a doctoral thesis is a challenge for many research students. However, formal academic writing training is not a core component of many doctoral programs. Informal writing groups for doctoral students may be one method of contributing to the improvement of scholarly writing. In this paper, we report on a writing group that was initiated by an experienced writer and higher degree research supervisor to support and improve her doctoral students’ writing capabilities. Over time, this group developed a workable model to suit their varying needs and circumstances. The model comprised group sessions, an email group, and individual writing. Here, we use a narrative approach to explore the effectiveness and value of our research writing group model in improving scholarly writing. The data consisted of doctoral students’ reflections to stimulus questions about their writing progress and experiences. The stimulus questions sought to probe individual concerns about their own writing, what they had learned in the research writing group, the benefits of the group, and the disadvantages and challenges to participation. These reflections were analysed using thematic analysis. Following this analysis, the supervisor provided her perspective on the key themes that emerged. Results revealed that, through the writing group, members learned technical elements (e.g., paragraph structure), non-technical elements (e.g., working within limited timeframes), conceptual elements (e.g., constructing a cohesive arguments), collaborative writing processes, and how to edit and respond to feedback. In addition to improved writing quality, other benefits were opportunities for shared writing experiences, peer support, and increased confidence and motivation. The writing group provides a unique social learning environment with opportunities for: professional dialogue about writing, peer learning and review, and developing a supportive peer network. Thus our research writing group has proved an effective avenue for building doctoral students’ capability in scholarly writing. The proposed model for a research writing group could be applicable to any context, regardless of the type and location of the university, university faculty, doctoral program structure, or number of postgraduate students. It could also be used within a group of students with diverse research abilities, needs, topics and methodologies. However, it requires a group facilitator with sufficient expertise in scholarly writing and experience in doctoral supervision who can both engage the group in planned writing activities and also capitalise on fruitful lines of discussion related to students’ concerns as they arise. The research writing group is not intended to replace traditional supervision processes nor existing training. However it has clear benefits for improving scholarly writing in doctoral research programs particularly in an era of rapidly increasing student load.

Higher Order Thinking through ICT: What do middle years teachers think really matters?

The middle years of schooling has emerged as an important focus in Australian education. Student disengagement and alienation, the negative effects of non-completion of the senior years of schooling and underachievement have raised concerns about the quality of education during the middle years. For many schools, reshaping the middle years has involved incorporating Information and Communication Technologies (ICT) to motivate students. However, simultaneously there is a need to ensure that programs are academically rigorous. There is little doubt that there are potential benefits to integrating ICT into programs for middle years’ students. However, little is known about how middle years’ teachers perceive higher order thinking, which is a component of academic rigour. This paper investigates the question of What are teachers’ perceptions of higher order thinking in an ICT environment? The study is underpinned by socio-cultural theory which is based on the belief that learning occurs through social interaction and that individuals are shaped by the social and cultural tools and instruments they engage with. This investigation used a collective case study design. Two methods were used for data collection. These methods are semi-structured interviews with individual teachers and a class and a focus group discussion with teachers. Findings indicate that teachers hold various perceptions of higher order thinking that lead to productive approaches to integrating ICT in middle years’ classrooms. The paper highlights that there may be a continuum of perceptions of higher order thinking with ICT. This continuum may inform professional developers who are guiding and supporting teachers to integrate ICT into middle years’ classrooms.

Ghrelin gene-related peptides : multifunctional endocrine/autocrine modulators in health and disease

Ghrelin is a multi-functional peptide hormone which affects various processes including growth hormone and insulin release, appetite regulation, gut motility, metabolism and cancer cell proliferation. Ghrelin is produced in the stomach and in other normal and pathological cell types. It may act as an endocrine or autocrine/paracrine factor. The ghrelin gene encodes a precursor protein, preproghrelin, from which ghrelin and other potentially active peptides are derived by alternative mRNA splicing and/or proteolytic processing. The metabolic role of the peptide obestatin, derived from the preproghrelin C-terminal region, is controversial. However, it has direct effects on cancer cell proliferation. The regulation of ghrelin expression and the mechanisms through which the peptide products arise are unclear. We have recently re-examined the organisation of the ghrelin gene and identified several novel exons and transcripts. One transcript, which lacks the ghrelin-coding region of preproghrelin, contains the coding sequence of obestatin. Furthermore, we have identified an overlapping gene on the antisense strand of ghrelin, GHRLOS, which generates transcripts that may function as non-coding regulatory RNAs or code for novel, short bioactive peptides. The identification of these novel ghrelin-gene related transcripts and peptides raises critical questions regarding their physiological function and their role in obesity, diabetes and cancer.

Prevention of postnatal distress or depression : an evaluation of an intervention at preparation for parenthood classes.

Determined the effectiveness of a psychosocial intervention, provided to expectant couples in routine antenatal classes, on the postpartum psychosocial adjustment of women and men. Preparation for Parenthood programs were randomly allocated to one of three conditions: usual service ('control'), experimental ('empathy'), or non-specific control ('baby-play'). The latter condition controlled for the non-specific effects of the intervention, these being: the provision of an extra class; asking couples to consider the early postpartum weeks; and receiving booster information after the antenatal class, and again shortly after the birth. Women and men were categorised into three levels of self-esteem, as measured antenatally: low, medium and high. 268 participants were recruited antenatally. Interview data and self-report information was collected from 202 of these women at 6 weeks postpartum, and 180 women at 6 months postpartum. The intervention consisted of a session focusing on psychosocial issues related to becoming first-time parents. Participants discussed possible postpartum concerns in separate gender groups for part of the session, and then discussed these issues with their partners

A re-examination of the Ghrelin and Ghrelin receptor genes

The last few years have seen dramatic advances in genomics, including the discovery of a large number of non-coding and antisense transcripts. This has revolutionised our understanding of multifaceted transcript structures found within gene loci and their roles in the regulation of development, neurogenesis and other complex processes. The recent and continuing surge of knowledge has prompted researchers to reassess and further dissect gene loci. The ghrelin gene (GHRL) gives rise to preproghrelin, which in turn produces ghrelin, a 28 amino acid peptide hormone that acts via the ghrelin receptor (growth hormone secretagogue receptor/GHSR 1a). Ghrelin has many important physiological and pathophysiological roles, including the stimulation of growth hormone (GH) release, appetite regulation, and cancer development. A truncated receptor splice variant, GHSR 1b, does not bind ghrelin, but dimerises with GHSR 1a, and may act as a dominant negative receptor. The gene products of ghrelin and its receptor are frequently overexpressed in human cancer While it is well known that the ghrelin axis (ghrelin and its receptor) plays a range of important functional roles, little is known about the molecular structure and regulation of the ghrelin gene (GHRL) and ghrelin receptor gene (GHSR). This thesis reports the re-annotation of the ghrelin gene, discovery of alternative 5’ exons and transcription start sites, as well as the description of a number of novel splice variants, including isoforms with a putative signal peptide. We also describe the discovery and characterisation of a ghrelin antisense gene (GHRLOS), and the discovery and expression of a ghrelin receptor (growth hormone secretagogue receptor/GHSR) antisense gene (GHSR-OS). We have identified numerous ghrelin-derived transcripts, including variants with extended 5' untranslated regions and putative secreted obestatin and C-ghrelin transcripts. These transcripts initiate from novel first exons, exon -1, exon 0 and a 5' extended 1, with multiple transcription start sites. We used comparative genomics to identify, and RT-PCR to experimentally verify, that the proximal exon 0 and 5' extended exon 1 are transcribed in the mouse ghrelin gene, which suggests the mouse and human proximal first exon architecture is conserved. We have identified numerous novel antisense transcripts in the ghrelin locus. A candidate non-coding endogenous natural antisense gene (GHRLOS) was cloned and demonstrates very low expression levels in the stomach and high levels in the thymus, testis and brain - all major tissues of non-coding RNA expression. Next, we examined if transcription occurs in the antisense orientation to the ghrelin receptor gene, GHSR. A novel gene (GHSR-OS) on the opposite strand of intron 1 of the GHSR gene was identified and characterised using strand-specific RT-PCR and rapid amplification of cDNA ends (RACE). GHSR-OS is differentially expressed and a candidate non-coding RNA gene. In summary, this study has characterised the ghrelin and ghrelin receptor loci and demonstrated natural antisense transcripts to ghrelin and its receptor. Our preliminary work shows that the ghrelin axis generates a broad and complex transcriptional repertoire. This study provides the basis for detailed functional studies of the the ghrelin and GHSR loci and future studies will be needed to further unravel the function, diagnostic and therapeutic potential of the ghrelin axis.

Designing the Future

While there have been improvements in Australian engineering education since the 1990s, there are still strong concerns that more progress needs to be made, particularly in the areas of developing graduate competencies and in outcomes-based curricula. This paper reports on the findings from a two-day ALTC-funded forum that sought to establish a shared understanding with the 3 stakeholders (students, academics and industry) about how to achieve a design-based engineering curriculum. This paper reports on the findings from the first day’s activities and reveals that there is a shared desire for design and project-based curricula that would encourage the development of the ‘three-dimensional’ graduate: one who has technical, personal and professional and systems-thinking/design-based competence.

Functional outcome of transfemoral amputees fitted with an osseointegrated fixation : temporal gait characteristics

The purpose of this study was to characterise the functional outcome of 12 transfemoral amputees fitted with osseointegrated fixation using temporal gait characteristics. The objectives were (A) to present the cadence, duration of gait cycle, support and swing phases with an emphasis on the stride-to-stride and participant-to-participant variability, and (B) to compare these temporal variables with normative data extracted from the literature focusing on transfemoral amputees fitted with a socket and able-bodied participants. The temporal variables were extracted from the load applied on the residuum during straight level walking, which was collected at 200 Hz by a transducer. A total of 613 strides were assessed. The cadence (46±4 strides/min), the duration of the gait cycle (1.29±0.11 s), support (0.73±0.07 s, 57±3% of CG) and swing (0.56±0.07 s, 43±3% of GC) phases of the participants were 2% quicker, 3%, 6% shorter and 1% longer than transfemoral amputees using a socket as well as 11% slower, 9%, 6% and 13% longer than able-bodied, respectively. All combined, the results indicated that the fitting of an osseointegrated fixation has enabled this group of amputees to restore their locomotion with a highly functional level. Further longitudinal and cross-sectional studies would be required to confirm these outcomes. Nonetheless, the data presented can be used as benchmark for future comparisons. It can also be used as input in generic algorithms using templates of patterns of loading to recognise activities of daily living and to detect falls.