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 problem of measurement indeterminacy in complex neurobiological movement systems

In the study of complex neurobiological movement systems, measurement indeterminacy has typically been overcome by imposing artificial modelling constraints to reduce the number of unknowns (e.g., reducing all muscle, bone and ligament forces crossing a joint to a single vector). However, this approach prevents human movement scientists from investigating more fully the role, functionality and ubiquity of coordinative structures or functional motor synergies. Advancements in measurement methods and analysis techniques are required if the contribution of individual component parts or degrees of freedom of these task-specific structural units is to be established, thereby effectively solving the indeterminacy problem by reducing the number of unknowns. A further benefit of establishing more of the unknowns is that human movement scientists will be able to gain greater insight into ubiquitous processes of physical self-organising that underpin the formation of coordinative structures and the confluence of organismic, environmental and task constraints that determine the exact morphology of these special-purpose devices.

Ethyl-eicosapentaenoic acid in first-episode psychosis : a 1H-MRS study

Ethyl-eicosapentaenoic acid (E-EPA) is an omega-3 fatty acid that has been used in a range of neuropsychiatric conditions with some benefits. However, its mechanism of action is unknown. Here, we investigate its effects on in vivo brain metabolism in first-episode psychosis (FEP). Proton magnetic resonance spectroscopy at 3 T was performed in the temporal lobes of 24 FEP patients before and after 12 weeks of treatment in the context of a larger double-blind, placebo-controlled E-EPA augmentation study. Treatment group effects for glutathione (F1,12=6.1, p=0.03), and a hemisphere-by-group interaction for glutamine/glutamate (F1,20=4.4, p=0.049) were found. Glutathione increased bilaterally and glutamate/glutamine increased in the left hemisphere following E-EPA administration. Improvement in negative symptoms correlated with metabolic brain changes, particularly glutathione (r=-0.57). These results suggest that E-EPA augmentation alters glutathione availability and modulates the glutamine/glutamate cycle in early psychosis, with some of the metabolic brain changes being correlated with negative symptom improvement. Larger confirmatory studies of these postulated metabolic brain effects of E-EPA are warranted.

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.

Fingerprinting of complex mixtures with the use of high performance liquid chromatography, inductively coupled plasma atomic emission spectroscopy and chemometrics

The molecular and metal profile fingerprints were obtained from a complex substance, Atractylis chinensis DC—a traditional Chinese medicine (TCM), with the use of the high performance liquid chromatography (HPLC) and inductively coupled plasma atomic emission spectroscopy (ICP-AES) techniques. This substance was used in this work as an example of a complex biological material, which has found application as a TCM. Such TCM samples are traditionally processed by the Bran, Cut, Fried and Swill methods, and were collected from five provinces in China. The data matrices obtained from the two types of analysis produced two principal component biplots, which showed that the HPLC fingerprint data were discriminated on the basis of the methods for processing the raw TCM, while the metal analysis grouped according to the geographical origin. When the two data matrices were combined into a one two-way matrix, the resulting biplot showed a clear separation on the basis of the HPLC fingerprints. Importantly, within each different grouping the objects separated according to their geographical origin, and they ranked approximately in the same order in each group. This result suggested that by using such an approach, it is possible to derive improved characterisation of the complex TCM materials on the basis of the two kinds of analytical data. In addition, two supervised pattern recognition methods, K-nearest neighbors (KNNs) method, and linear discriminant analysis (LDA), were successfully applied to the individual data matrices—thus, supporting the PCA approach.

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.

Inhibition of myeloperoxidase-mediated hypochlorous acid production by nitroxides

Tissue damage resulting from the extracellular production of HOCl (hypochlorous acid) by the MPO (myeloperoxidase)-hydrogen peroxide-chloride system of activated phagocytes is implicated as a key event in the progression of a number of human inflammatory diseases. Consequently, there is considerable interest in the development of therapeutically useful MPO inhibitors. Nitroxides are well established antioxidant compounds of low toxicity that can attenuate oxidative damage in animal models of inflammatory disease. They are believed to exert protective effects principally by acting as superoxide dismutase mimetics or radical scavengers. However, we show here that nitroxides can also potently inhibit MPO-mediated HOCl production, with the nitroxide 4-aminoTEMPO inhibiting HOCl production by MPO and by neutrophils with IC50 values of approx. 1 and 6 μM respectively. Structure–activity relationships were determined for a range of aliphatic and aromatic nitroxides, and inhibition of oxidative damage to two biologically-important protein targets (albumin and perlecan) are demonstrated. Inhibition was shown to involve one-electron oxidation of the nitroxides by the compound I form of MPO and accumulation of compound II. Haem destruction was also observed with some nitroxides. Inhibition of neutrophil HOCl production by nitroxides was antagonized by neutrophil-derived superoxide, with this attributed to superoxide-mediated reduction of compound II. This effect was marginal with 4-aminoTEMPO, probably due to the efficient superoxide dismutase-mimetic activity of this nitroxide. Overall, these data indicate that nitroxides have considerable promise as therapeutic agents for the inhibition of MPO-mediated damage in inflammatory diseases.

Clinical efficacy and safety of zoledronic acid in osteoporosis and Paget's disease

Osteoporosis and Paget’s bone disease are the most common diseases of the bone. In addition to glucocorticoid treatment, there are many other secondary causes of osteoporosis. Bisphosphonates are used to treat these bone conditions. Zoledronic acid is the most potent bisphosphonate at inhibiting bone resorption. In osteoporosis, zoledronic acid increases bone mineral density for at least 1 year following a single intravenous administration. The efficacy and safety of zoledronic acid in the treatment of osteoporosis and Paget’s bone disease are reviewed. This article also covers the studies of the effects of zoledronic acid in the bone loss associated with the secondary osteoporosis.

Anti-cancer activity of zoledronic acid in breast cancer

Background: Zoledronic acid is used to prevent the bone loss associated with antioestrogen treatments in subjects with breast cancer. Preclinical studies suggest that zoledronic acid may have anticancer activity in its own right. This anticancer possibility with zoledronic acid has not been investigated extensively in clinical trials. Objectives/methods: This evaluation is of a large clinical trial that investigated the effect of zoledronic acid on cancer outcomes in premenopausal women with breast cancer. Results: The trial showed that after 4 years, 94.0% of subjects who were treated with zoledronic acid were disease-free compared with 90.8% of those not treated with zoledronic acid. Recurrence survival was a secondary end point; this occurred in 94.0% with, and 90.9% without, zoledronic acid treatment. Conclusions: Zoledronic acid does have anticancer activity in premenopausal women with cancer.

Clinical efficacy and safety of zoledronic acid in prostate and breast cancer

The anti-estrogen treatment for hormone-sensitive breast cancer and the androgen deprivation therapy for prostate cancer can lead to the development of osteoporosis and bone fractures. Metastases associated with prostate and breast cancer can also occur in bone. Bisphosphonates are used in these types of bone dysfunction. Zoledronic acid is the most potent bisphosphonate. In osteoporosis, zoledronic acid inhibits bone reabsorption and increases bone mineral density for at least a year after intravenous administration. The efficacy and safety of zoledronic acid in osteoporosis secondary to hormone-sensitive cancers (prostate and breast), and in the bone metastases associated with these cancers are reviewed.

Performing with grid music systems

Grid music systems provide discrete geometric methods for simplified music-making by providing spatialised input to construct patterned music on a 2D matrix layout. While they are conceptually simple, grid systems may be layered to enable complex and satisfying musical results. Grid music systems have been applied to a range of systems from small portable devices up to larger systems. In this paper we will discuss the use of grid music systems in general and present an overview of the HarmonyGrid system we have developed as a new interactive performance system. We discuss a range of issues related to the design and use of larger-scale grid- based interactive performance systems such as the HarmonyGrid.

Androgen levels increase by intratumoral de novo steroidogenesis during progression of castration-resistant prostate cancer

Although systemic androgen deprivation prolongs life in advanced prostate cancer, remissions are temporary because patients almost uniformly progress to a state of a castration-resistant prostate cancer (CRPC) as indicated by recurring PSA. This complex process of progression does not seem to be stochastic as the timing and phenotype are highly predictable, including the observation that most androgen-regulated genes are reactivated despite castrate levels of serum androgens. Recent evidence indicates that intraprostatic levels of androgens remain moderately high following systemic androgen deprivation therapy, whereas the androgen receptor (AR) remains functional, and silencing the AR expression following castration suppresses tumor growth and blocks the expression of genes known to be regulated by androgens. From these observations, we hypothesized that CRPC progression is not independent of androgen-driven activity and that androgens may be synthesized de novo in CRPC tumors leading to AR activation. Using the LNCaP xenograft model, we showed that tumor androgens increase during CRPC progression in correlation to PSA up-regulation. We show here that all enzymes necessary for androgen synthesis are expressed in prostate cancer tumors and some seem to be up-regulated during CRPC progression. Using an ex vivo radiotracing assays coupled to high-performance liquid chromatography-radiometric/mass spectrometry detection, we show that tumor explants isolated from CRPC progression are capable of de novo conversion of [(14)C]acetic acid to dihydrotestosterone and uptake of [(3)H]progesterone allows detection of the production of six other steroids upstream of dihydrotestosterone. This evidence suggests that de novo androgen synthesis may be a driving mechanism leading to CRPC progression following castration.

Near infrared spectroscopy of stearic acid adsorbed on montmorillonite

The adsorption of stearic acid on both sodium montmorillonites and calcium montmorillonites has been studied by near infrared spectroscopy complimented with infrared spectroscopy. Upon adsorption of stearic acid on Ca-Mt additional near infrared bands are observed at 8236 cm-1 and is assigned to an interaction of stearic acid with the water of hydration. Upon adsorption of the stearic acid on Na-Mt, the NIR bands are now observed at 5671, 5778, 5848 and 5912 cm-1 and are assigned to the overtone and combination bands of the CH fundamentals. Additional bands at 4177, 4250, 4324, 4337, 4689 and 4809 cm-1 are attributed to CH combination bands resulting from the adsorption of the stearic acid. Stearic acid is used as a model molecule for adsorption studies. The application of near infrared spectroscopy to the study of this adsorption proved most useful.