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.

Damage assessment of 3D reinforced concrete frame under external explosion loading

Multi-storey buildings are highly vulnerable to terrorist bombing attacks in various parts of the world. Large numbers of casualties and extensive property damage result not only from blast overpressure, but also from the failing of structural components. Understanding the blast response and damage consequences of reinforced concrete (RC) building frames is therefore important when assessing multi-storey buildings designed to resist normal gravity loads. However, limited research has been conducted to identify the blast response and damage of RC frames in order to assess the vulnerability of entire buildings. This paper discusses the blast response and evaluation of damage of three-dimension (3D) RC rigid frame under potential blast loads scenarios. The explicit finite element modelling and analysis under time history blast pressure loads were carried out by LS DYNA code. Complete 3D RC frame was developed with relevant reinforcement details and material models with strain rate effect. Idealised triangular blast pressures calculated from standard manuals are applied on the front face of the model in the present investigation. The analysis results show the blast response, as displacements and material yielding of the structural elements in the RC frame. The level of damage is evaluated and classified according to the selected load case scenarios. Residual load carrying capacities are evaluated and level of damage was presented by the defined damage indices. This information is necessary to determine the vulnerability of existing multi-storey buildings with RC frames and to identify the level of damage under typical external explosion environments. It also provides basic guidance to the design of new buildings to resist blast loads.

Infrastructure Sustainability : Damage Assessment in Multiple-Girder Composite Bridges Using Vibration Characteristics

Assessing the structural health state of urban infrastructure is crucial in terms of infrastructure sustainability. This chapter uses dynamic computer simulation techniques to apply a procedure using vibration-based methods for damage assessment in multiple-girder composite bridges. In addition to changes in natural frequencies, this multi-criteria procedure incorporates two methods, namely, the modal flexibility and the modal strain energy method. Using the numerically simulated modal data obtained through finite element analysis software, algorithms based on modal flexibility and modal strain energy change, before and after damage, are obtained and used as the indices for the assessment of structural health state. The feasibility and capability of the approach is demonstrated through numerical studies of a proposed structure with six damage scenarios. It is concluded that the modal strain energy method is capable of application to multiple-girder composite bridges, as evidenced through the example treated in this chapter.

Automation of an underground mining vehicle using reactive navigation and opportunistic localization

This paper describes the implementation of an autonomous navigation system onto a 30 tonne Load-Haul-Dump truck. The control architecture is based on a robust reactive wall-following behaviour. To make it purposeful we provide driving hints derived from an approximate nodal-map. For most of the time, the vehicle is driven with weak localization (odometry). This need only be improved at intersections where decisions must be made - a technique we refer to as opportunistic localization. The truck has achieved full-speed autonomous operation at an artificial test mine, and subsequently, at a operational underground mine.

Robust outdoor visual localization using a three-dimensional-edge map

Visual localization systems that are practical for autonomous vehicles in outdoor industrial applications must perform reliably in a wide range of conditions. Changing outdoor conditions cause difficulty by drastically altering the information available in the camera images. To confront the problem, we have developed a visual localization system that uses a surveyed three-dimensional (3D)-edge map of permanent structures in the environment. The map has the invariant properties necessary to achieve long-term robust operation. Previous 3D-edge map localization systems usually maintain a single pose hypothesis, making it difficult to initialize without an accurate prior pose estimate and also making them susceptible to misalignment with unmapped edges detected in the camera image. A multihypothesis particle filter is employed here to perform the initialization procedure with significant uncertainty in the vehicle's initial pose. A novel observation function for the particle filter is developed and evaluated against two existing functions. The new function is shown to further improve the abilities of the particle filter to converge given a very coarse estimate of the vehicle's initial pose. An intelligent exposure control algorithm is also developed that improves the quality of the pertinent information in the image. Results gathered over an entire sunny day and also during rainy weather illustrate that the localization system can operate in a wide range of outdoor conditions. The conclusion is that an invariant map, a robust multihypothesis localization algorithm, and an intelligent exposure control algorithm all combine to enable reliable visual localization through challenging outdoor conditions.

A modified particle filter for simultaneous robot localization and landmark tracking in an indoor environment

This paper presents the implementation of a modified particle filter for vision-based simultaneous localization and mapping of an autonomous robot in a structured indoor environment. Through this method, artificial landmarks such as multi-coloured cylinders can be tracked with a camera mounted on the robot, and the position of the robot can be estimated at the same time. Experimental results in simulation and in real environments show that this approach has advantages over the extended Kalman filter with ambiguous data association and various levels of odometric noise.

Biologically inspired visual landmark processing for simultaneous localization and mapping

This paper illustrates a method for finding useful visual landmarks for performing simultaneous localization and mapping (SLAM). The method is based loosely on biological principles, using layers of filtering and pooling to create learned templates that correspond to different views of the environment. Rather than using a set of landmarks and reporting range and bearing to the landmark, this system maps views to poses. The challenge is to produce a system that produces the same view for small changes in robot pose, but provides different views for larger changes in pose. The method has been developed to interface with the RatSLAM system, a biologically inspired method of SLAM. The paper describes the method of learning and recalling visual landmarks in detail, and shows the performance of the visual system in real robot tests.

Improved correlation-based modal strain energy method for global damage detection of truss bridge structures

The previous investigations have shown that the modal strain energy correlation method, MSEC, could successfully identify the damage of truss bridge structures. However, it has to incorporate the sensitivity matrix to estimate damage and is not reliable in certain damage detection cases. This paper presents an improved MSEC method where the prediction of modal strain energy change vector is differently obtained by running the eigensolutions on-line in optimisation iterations. The particular trail damage treatment group maximising the fitness function close to unity is identified as the detected damage location. This improvement is then compared with the original MSEC method along with other typical correlation-based methods on the finite element model of a simple truss bridge. The contributions to damage detection accuracy of each considered mode is also weighed and discussed. The iterative searching process is operated by using genetic algorithm. The results demonstrate that the improved MSEC method suffices the demand in detecting the damage of truss bridge structures, even when noised measurement is considered.

Wide-angle visual feature matching for outdoor localization

Wide-angle images exhibit significant distortion for which existing scale-space detectors such as the scale-invariant feature transform (SIFT) are inappropriate. The required scale-space images for feature detection are correctly obtained through the convolution of the image, mapped to the sphere, with the spherical Gaussian. A new visual key-point detector, based on this principle, is developed and several computational approaches to the convolution are investigated in both the spatial and frequency domain. In particular, a close approximation is developed that has comparable computation time to conventional SIFT but with improved matching performance. Results are presented for monocular wide-angle outdoor image sequences obtained using fisheye and equiangular catadioptric cameras. We evaluate the overall matching performance (recall versus 1-precision) of these methods compared to conventional SIFT. We also demonstrate the use of the technique for variable frame-rate visual odometry and its application to place recognition.

Reactive navigation and opportunistic localization for autonomous underground mining vehicles

This paper describes an autonomous navigation system for a large underground mining vehicle. The control architecture is based on a robust reactive wall-following behaviour. To make it purposeful we provide driving hints derived from an approximate nodal-map. For most of the time, the vehicle is driven with weak localization (odometry). This need only be improved at intersections where decisions must be made – a technique we refer to as opportunistic localization. The paper briefly reviews absolute and relative navigation strategies, and describes an implementation of a reactive navigation system on a 30 tonne Load-Haul-Dump truck. This truck has achieved full-speed autonomous operation at an artificial test mine, and subsequently, at a operational underground mine.

Experiments with underwater robot localization and tracking

This paper describes a novel experiment in which two very different methods of underwater robot localization are compared. The first method is based on a geometric approach in which a mobile node moves within a field of static nodes, and all nodes are capable of estimating the range to their neighbours acoustically. The second method uses visual odometry, from stereo cameras, by integrating scaled optical flow. The fundamental algorithmic principles of each localization technique is described. We also present experimental results comparing acoustic localization with GPS for surface operation, and a comparison of acoustic and visual methods for underwater operation.

Further results with localization and mapping using range from radio

In this paper, we present recent results with using range from radio for mobile robot localization. In previous work we have shown how range readings from radio tags placed in the environment can be used to localize a robot. We have extended previous work to consider robustness. Specifically, we are interested in the case where range readings are very noisy and available intermittently. Also, we consider the case where the location of the radio tags is not known at all ahead of time and must be solved for simultaneously along with the position of the moving robot. We present results from a mobile robot that is equipped with GPS for ground truth, operating over several km.

Localization and navigation assisted by cooperating networked sensors and robots

In this paper we discuss how a network of sensors and robots can cooperate to solve important robotics problems such as localization and navigation. We use a robot to localize sensor nodes, and we then use these localized nodes to navigate robots and humans through the sensorized space. We explore these novel ideas with results from two large-scale sensor network and robot experiments involving 50 motes, two types of flying robot: an autonomous helicopter and a large indoor cable array robot, and a human-network interface. We present the distributed algorithms for localization, geographic routing, path definition and incremental navigation. We also describe how a human can be guided using a simple hand-held device that interfaces to this same environmental infrastructure.