Foot contact detection for sprint training

We introduce a new algorithm to automatically identify the time and pixel location of foot contact events in high speed video of sprinters. We use this information to autonomously synchronise and overlay multiple recorded performances to provide feedback to athletes and coaches during their training sessions. The algorithm exploits the variation in speed of different parts of the body during sprinting. We use an array of foreground accumulators to identify short-term static pixels and a temporal analysis of the associated static regions to identify foot contacts. We evaluated the technique using 13 videos of three sprinters. It successfully identifed 55 of the 56 contacts, with a mean localisation error of 1.39±1.05 pixels. Some videos were also seen to produce additional, spurious contacts. We present heuristics to help identify the true contacts. © 2011 Springer-Verlag Berlin Heidelberg.

A microfluidic device for high density hydrodynamic cell trapping, growth and super-resolution imaging

Super-Resolution imaging techniques such as Fluorescent Photo-Activation Localisation Microscopy (FPALM) have created a powerful new toolkit for investigating living cells, however a simple platform for growing, trapping, holding and controlling the cells is needed before the approach can become truly widespread. We present a microfluidic device formed in polydimethylsiloxane (PDMS) with a fluidic design which traps cells in a high-density array of wells and holds them very still throughout the life cycle, using hydrodynamic forces only. The device meets or exceeds all the necessary criteria for FPALM imaging of Schizosaccharomyces pombe and is designed to remain flexible, robust and easy to use. © 2011 IEEE.

Adaptive sequential Monte Carlo approach for real-time applications

This paper presents an adaptive Sequential Monte Carlo approach for real-time applications. Sequential Monte Carlo method is employed to estimate the states of dynamic systems using weighted particles. The proposed approach reduces the run-time computation complexity by adapting the size of the particle set. Multiple processing elements on FPGAs are dynamically allocated for improved energy efficiency without violating real-time constraints. A robot localisation application is developed based on the proposed approach. Compared to a non-adaptive implementation, the dynamic energy consumption is reduced by up to 70% without affecting the quality of solutions. © 2012 IEEE.

Real-time marker prediction and CoR estimation in optical motion capture

Optical motion capture systems suffer from marker occlusions resulting in loss of useful information. This paper addresses the problem of real-time joint localisation of legged skeletons in the presence of such missing data. The data is assumed to be labelled 3d marker positions from a motion capture system. An integrated framework is presented which predicts the occluded marker positions using a Variable Turn Model within an Unscented Kalman filter. Inferred information from neighbouring markers is used as observation states; these constraints are efficient, simple, and real-time implementable. This work also takes advantage of the common case that missing markers are still visible to a single camera, by combining predictions with under-determined positions, resulting in more accurate predictions. An Inverse Kinematics technique is then applied ensuring that the bone lengths remain constant over time; the system can thereby maintain a continuous data-flow. The marker and Centre of Rotation (CoR) positions can be calculated with high accuracy even in cases where markers are occluded for a long period of time. Our methodology is tested against some of the most popular methods for marker prediction and the results confirm that our approach outperforms these methods in estimating both marker and CoR positions. © 2012 Springer-Verlag.

Heterogeneous reconfigurable system for adaptive particle filters in real-time applications

This paper presents a heterogeneous reconfigurable system for real-time applications applying particle filters. The system consists of an FPGA and a multi-threaded CPU. We propose a method to adapt the number of particles dynamically and utilise the run-time reconfigurability of the FPGA for reduced power and energy consumption. An application is developed which involves simultaneous mobile robot localisation and people tracking. It shows that the proposed adaptive particle filter can reduce up to 99% of computation time. Using run-time reconfiguration, we achieve 34% reduction in idle power and save 26-34% of system energy. Our proposed system is up to 7.39 times faster and 3.65 times more energy efficient than the Intel Xeon X5650 CPU with 12 threads, and 1.3 times faster and 2.13 times more energy efficient than an NVIDIA Tesla C2070 GPU. © 2013 Springer-Verlag.

Measuring EGFR Separations on Cells with ∼10 nm Resolution via Fluorophore Localization Imaging with Photobleaching

Detecting receptor dimerisation and other forms of clustering on the cell surface depends on methods capable of determining protein-protein separations with high resolution in the ∼10-50 nm range. However, this distance range poses a significant challenge because it is too large for fluorescence resonance energy transfer and contains distances too small for all other techniques capable of high-resolution in cells. Here we have adapted the technique of fluorophore localisation imaging with photobleaching to measure inter-receptor separations in the cellular environment. Using the epidermal growth factor receptor, a key cancer target molecule, we demonstrate ∼10 nm resolution while continuously covering the range of ∼10-80 nm. By labelling the receptor on cells expressing low receptor numbers with a fluorescent antagonist we have found inter-receptor separations all the way up from 8 nm to 59 nm. Our data are consistent with epidermal growth factor receptors being able to form homo-polymers of at least 10 receptors in the absence of activating ligands. © 2013 Needham et al.

High-level scene structure using visibility and occlusion

We demonstrate a new method for extracting high-level scene information from the type of data available from simultaneous localisation and mapping systems. We model the scene with a collection of primitives (such as bounded planes), and make explicit use of both visible and occluded points in order to refine the model. Since our formulation allows for different kinds of primitives and an arbitrary number of each, we use Bayesian model evidence to compare very different models on an even footing. Additionally, by making use of Bayesian techniques we can also avoid explicitly finding the optimal assignment of map landmarks to primitives. The results show that explicit reasoning about occlusion improves model accuracy and yields models which are suitable for aiding data association. © 2011. The copyright of this document resides with its authors.

Absence of turnover and futile cycling of sucrose in leaves of Lolium temulentum L.: implications for metabolic compartmentation

Cairns, A. J., Gallagher, J. A. (2004). Absence of turnover and futile cycling of sucrose in leaves of Lolium temulentum L.: implications for metabolic compartmentation. Planta, 219 (5), 836-846. Sponsorship: BBSRC RAE2008

Estudo epidemiológico de erosão dentária em indivíduos institucionalizados em terapias de desintoxicação de álcool

Tese apresentada à Universidade Fernando Pessoa como parte dos requisitos para obtenção do grau de Doutor em Biotecnologia e Saúde, especialidade em Epidemiologia e Saúde Pública

Innovation in Ireland's 'high-technology' businesses: the roles of interaction and proximity

This thesis explores the drivers of innovation in Irish high-technology businesses and estimates, in particular, the relative importance of interaction with external businesses and other organisations as a source of knowledge for innovation at the business-level. The thesis also examines the extent to which interaction for innovation in these businesses occurs on a local or regional basis. The study uses original survey data of 184 businesses in the Chemical and Pharmaceutical, Information and Communications Technology and Engineering and Electronic Devices sectors. The study considers both product and process innovation at the level of the business and develops new measures of innovation output. For the first time in an Irish study, the incidence and frequency of interaction is measured for each of a range of agents, other group companies, suppliers, customers, competitors, academic-based researchers and innovation-supporting agencies. The geographic proximity between the business and each of the most important of each of each category of agent is measured using average one-way driving distance, which is the first time such a measure has been used in an Irish study of innovation. Utilising econometric estimation techniques, it is found that interaction with customers, suppliers and innovation-supporting agencies is positively associated with innovation in Irish high-technology businesses. Surprisingly, however, interaction with academic-based researchers is found to have a negative effect on innovation output at the business-level. While interaction generally emerges as a positive influence on business innovation, there is little evidence that this occurs at a local or regional level. Furthermore, there is little support for the presence of localisation economies for high-technology sectors, though some tentative evidence of urbanisation economies. This has important implications for Irish regional, enterprise and innovation policy, which has emphasised the development of clusters of internationally competitive businesses. The thesis brings into question the suitability of a cluster-driven network based approach to business development and competitiveness in an Irish context.

Characterisation of the non-muscle α-actinins

Actinins are cytoskeleton proteins that cross-link actin filaments. Evolution of the actinin family resulted in the formation of Ca++-insensitive muscle isoforms (actinin-2 and- 3) and Ca++-sensitive non-muscle isoforms (actinin-1 and -4) with regard to their actin-binding function. Despite high sequence similarity, unique properties have been ascribed to actinin-4 compared with actinin-1. Actinin-4 is the predominant isoform reported to be associated with the cancer phenotype. Actinin-4, but not actinin-1, is essential for normal glomerular function in the kidney and and is able to translocate to the nucleus to regulate transcription. To understand the molecular basis for such isoform-specific functions I have comprehensively compared these proteins in terms of localisation, migration, alternative splicing, actin-binding properties, heterodimer formation and molecular interactions for the first time. This work characterises a number of commercially available actinin antibodies and in doing so, identifies actinin-1, -2 and -4 isoform-specific antibodies that enabled studies of actinin expression and localisation. This work identifies the actinin rod domain as the predominant domain that influences actinin localisation however localisation is likely to be effected by the entire actinin protein. si-RNA- mediated knockdown of actinin-1 and -4 did not affect migration in a number of cell lines highlighting that migration may only require a fraction of total non-muscle actinin levels. This work finds that the Ca++-insensitive variant of actinin-4 is expressed only in the nervous system and thus cannot be regarded as a smooth muscle isoform, as is the case for the Ca++-insensitive variant of actinin-1. This work also identifies a previously unreported exon 19a+19b expressing variant of actinin-4 in human skeletal muscle. This work finds that alternative splice variants of actinin-1 and -4 are co-expressed in a number of tissues, in particular the brain. In contrast to healthy brain, glioblastoma cells express Ca++-sensitive variants of both actinin-1 and -4. Actin-binding properties of actinin-1 and -4 are similar and are unlikely to explain isoform-specific functions. Surprisingly, this work reveals that actinin-1/-4 heterodimers, rather than homodimers, are the most abundant form of actinin in many cancer cell lines. Taken together this data suggests that actinin-1 and -4 cannot be viewed as distinct entities from each other but rather as proteins that can exist in both homodimeric and heterodimeric forms. Finally, this work employs yeast two-hybrid and proteomic approaches to identify actinin-interacting proteins. In doing so, this work identifies a number of putative actinin-4 specific interacting partners that may help to explain some of the unique functions attributed the actinin-4. The observation of alternative splice variants of actinin-1 and -4 combined with the observed potential of these proteins to form homodimers and heterodimers suggests that homodimers and heterodimers with novel actin-binding properties and interaction networks may exist. The ability to behave in this manner may have functional implications. This may be of importance considering that these proteins are central to such processes as cell migration and adhesion. This significantly alters our view of the non-muscle actinins.

Investigating the function of PINK1 in cancer development and pathogenesis

PTEN‐induced kinase 1 (PINK1) was identified initially in cancer cells as a gene up‐regulated by overexpression of the central tumour suppressor, PTEN. Loss‐of‐function mutations in PINK1 were discovered subsequently to cause autosomal recessive Parkinsonʹs disease (ARPD). Despite much research focusing on the proposed mechanism(s) through which loss of PINKI function causes neurodegeneration, few studies have focused on a direct role for this serine/threonine kinase in cancer biology. The focus of this thesis was to examine a direct role for PINK1 function in tumourigenesis. Initial studies showed that loss of PINK1 reduces tumour‐associated phenotypes including cell growth, colony formation and invasiveness, in several cell types in vitro, indicating a pro‐tumourigenic role for PINK1 in cancer. Furthermore, results revealed for the first time that PINK1 deletion, examined in mouse embryonic fibroblasts (MEFS) from PINK1 knock‐out animals, causes cell cycle defects, whereby cells arrest at in cytokinesis, giving rise to a highly significant increase in the number of multinucleated cells. This results in several key changes in the expression profile of cell cycle associated protein. In addition, PINK1‐deficient MEFs were found to resist cell cycle exit, with a proportion of cells remaining in proliferative phases upon removal of serum. The ability of cells to progress through mitosis conferred by PINK1 expression was independent of its kinase activity, while the cell cycle exit following serum withdrawal was kinase dependent. Investigations into the mechanism through which loss of PINK1 function gives rise to cell cycle defects revealed that dynamin related protein 1 (Drp1)‐mediated mitochondrial fission is enhanced in PINK1‐ deficient MEFs, and that increased expression of Drp1 on mitochondria and activation of Drp1 is highly significant in PINK1‐deficient multinucleated cells. Deregulated and increased levels and activation of mitochondrial fission via Drp1 was shown to be a major feature of cell cycle defects caused by PINK1 deletion, both during progression through G2/M and cell cycle exit following serum removal. Altered PINK1 localisation was also observed during progression of mitosis, and upon serum deprivation. Thus, PINK1 dissociated from the mitochondria during the mitotic phases and localised to mitochondria upon serum withdrawal. During serum withdrawal deletion of PINK1 disabled the ability of MEFs to increase mitochondrial membrane potential (ΔΨm), and increase autophagy. This was co‐incident with increased mitochondrial fission, and increased localisation of Drp1 to mitochondria following serum deprivation. Together, this indicates an inability of PINK1‐negative cells to respond protectively to this stress‐induced state, primarily via impaired mitochondrial function. In contrast, PINK1 overexpression was found to protect cells from DNA damage following treatment with oxidants. In addition, deletion of PINK1 blocked the ability of cells to re‐enter the cell cycle in response to insulin‐like growth factor‐1 (IGF‐1), a major cancer promoting agonistwhich acts primarily via PI3‐kinase/Akt activation. Furthermore, PINK1 mRNA expression was significantly increased following serum deprivation of MCF‐7 cells, and this was rendered more significant upon additional inhibition of PI3‐kinase. Conversely, IGF‐1 activation of PI3‐kinase/Akt causes a time‐dependent and significant reduction of PINK1 mRNA expression that was PI3‐kinase dependent. Together these results indicate that PINK1 expression is necessary for IGF‐1 signalling and is regulated reciprocally in the absence and presence of IGF‐1, via PI3‐kinase/Akt, a signalling system which has major tumour‐promoting capacity in cancer cell biology. The results of this thesis indicate PINK1 is a candidate tumour-promoting gene which has a significant function in the regulation of the cell cycle, and growth factor responses, at key cell cycle checkpoints, namely, during progression through G2/M and during exit of the cell cycle following removal of serum. Furthermore, the results reveal that the regulation of mitochondrial fission and Drp1 function is mechanistically important in the regulation of cell cycle control by PINK1. As deregulation of the cell cycle is linked to both tumourigenesis and neurodegeneration, the findings of this thesis are of importance not just for understanding cancer biology, but also in the context of PINK1‐associated neurodegeneration.

Identification of novel innate immune mechanisms regulating inflammation in the gastrointestinal tract

The Gastro-Intestinal (GI) tract is a unique region in the body. Our innate immune system retains a fine homeostatic balance between avoiding inappropriate inflammatory responses against the myriad commensal microbes residing in the gut while also remaining active enough to prevent invasive pathogenic attack. The intestinal epithelium represents the frontline of this interface. It has long been known to act as a physical barrier preventing the lumenal bacteria of the gastro-intestinal tract from activating an inflammatory immune response in the immune cells of the underlying mucosa. However, in recent years, an appreciation has grown surrounding the role played by the intestinal epithelium in regulating innate immune responses, both in the prevention of infection and in maintaining a homeostatic environment through modulation of innate immune signalling systems. The aim of this thesis was to identify novel innate immune mechanisms regulating inflammation in the GI tract. To achieve this aim, we chose several aspects of regulatory mechanisms utilised in this region by the innate immune system. We identified several commensal strains of bacteria expressing proteins containing signalling domains used by Pattern Recognition Receptors (PRRs) of the innate immune system. Three such bacterial proteins were studied for their potentially subversive roles in host innate immune signalling as a means of regulating homeostasis in the GI tract. We also examined differential responses to PRR activation depending on their sub-cellular localisation. This was investigated based on reports that apical Toll-Like Receptor (TLR) 9 activation resulted in abrogation of inflammatory responses mediated by other TLRs in Intestinal Epithelial Cells (IECs) such as basolateral TLR4 activation. Using the well-studied invasive intra-cellular pathogen Listeria monocytogenes as a model for infection, we also used a PRR siRNA library screening technique to identify novel PRRs used by IECs in both inhibition and activation of inflammatory responses. Many of the PRRs identified in this screen were previously believed not to be expressed in IECs. Furthermore, the same study has led to the identification of the previously uncharacterised TLR10 as a functional inflammatory receptor of IECs. Further analysis revealed a similar role in macrophages where it was shown to respond to intracellular and motile pathogens such as Gram-positive L.monocytogenes and Gram negative Salmonella typhimurium. TLR10 expression in IECs was predominantly intracellular. This is likely in order to avoid inappropriate inflammatory activation through the recognition of commensal microbial antigens on the apical cell surface of IECs. Moreover, these results have revealed a more complex network of innate immune signalling mechanisms involved in both activating and inhibiting inflammatory responses in IECs than was previously believed. This contribution to our understanding of innate immune regulation in this region has several direct and indirect benefits. The identification of several novel PRRs involved in activating and inhibiting inflammation in the GI tract may be used as novel therapeutic targets in the treatment of disease; both for inducing tolerance and reducing inflammation, or indeed, as targets for adjuvant activation in the development of oral vaccines against pathogenic attack.

Investigating the invasive and cytoprotective properties of the heme binding protein HRG-1 in cancer cells

This thesis investigates the mechanisms by which HRG-1 contributes to the invasive and cytoprotective signalling pathways in cancer cells through its effects on VATPase activity and heme transport. Plasma membrane-localised V-ATPase activity correlates with enhanced metastatic potential in cancer cells, which is attributed to extrusion of protons into the extracellular space and activation of pH-sensitive, extracellular matrix degrading-proteases. We found that HRG-1 is co-expressed with the V-ATPase at the plasma membrane of certain aggressive cancer cell types. Modulation of HRG-1 expression altered both the localisation and activity of the VATPase. We also found that HRG-1 enhances trafficking of essential transporters such as the glucose transporter (GLUT-1) in cancer cells, and increases glucose uptake, which is required for cancer cell growth, metabolism and V-ATPase assembly. Heme is potentially cytotoxic, owing to its iron moiety, and therefore the trafficking of heme is tightly controlled in cells. We hypothesised that HRG-1 is required for the transport of heme to intracellular compartments. Importantly, we found that HRG-1 interacts with the heme oxygenases that are necessary for heme catabolism. HRG-1 is also required for trafficking of both heme-bound and nonheme-bound receptors and suppression of HRG-1 results in perturbed receptor trafficking to the lysosome. Suppression of HRG-1 in HeLa cells increases toxic heme accumulation, reactive oxygen species accumulation, and DNA damage resulting in caspasedependent cell death. Mutation of essential heme binding residues in HRG-1 results in decreased heme binding to HRG-1. Interestingly, cells expressing heme-binding HRG-1 mutants exhibit decreased internalisation of the transferrin receptor compared to cells expressing wildtype HRG-1. These findings suggest that HRG- 1/heme trafficking contributes to a hitherto unappreciated aspect of receptormediated endocytosis. Overall, the findings of this thesis show that HRG-1-mediated regulation of intracellular and extracellular pH through V-ATPase activity is essential for a functioning endocytic pathway. This is critical for cells to acquire nutrients such as folate, iron and glucose and to mediate signalling in response to growth factor activation. Thus, HRG-1 facilitates enhanced metabolic activity of cancer cells to enable tumour growth and metastasis.

Helicobacter pylori: comparative genomics and structure-function analysis of the flagellum biogenesis protein HP0958

Helicobacter pylori is a gastric pathogen which infects ~50% of the global population and can lead to the development of gastritis, gastric and duodenal ulcers and carcinoma. Genome sequencing of H. pylori revealed high levels of genetic variability; this pathogen is known for its adaptability due to mechanisms including phase variation, recombination and horizontal gene transfer. Motility is essential for efficient colonisation by H. pylori. The flagellum is a complex nanomachine which has been studied in detail in E. coli and Salmonella. In H. pylori, key differences have been identified in the regulation of flagellum biogenesis, warranting further investigation. In this study, the genomes of two H. pylori strains (CCUG 17874 and P79) were sequenced and published as draft genome sequences. Comparative studies identified the potential role of restriction modification systems and the comB locus in transformation efficiency differences between these strains. Core genome analysis of 43 H. pylori strains including 17874 and P79 defined a more refined core genome for the species than previously published. Comparative analysis of the genome sequences of strains isolated from individuals suffering from H. pylori related diseases resulted in the identification of “disease-specific” genes. Structure-function analysis of the essential motility protein HP0958 was performed to elucidate its role during flagellum assembly in H. pylori. The previously reported HP0958-FliH interaction could not be substantiated in this study and appears to be a false positive. Site-directed mutagenesis confirmed that the coiled-coil domain of HP0958 is involved in the interaction with RpoN (74-284), while the Zn-finger domain is required for direct interaction with the full length flaA mRNA transcript. Complementation of a non-motile hp0958-null derivative strain of P79 with site-directed mutant alleles of hp0958 resulted in cells producing flagellar-type extrusions from non-polar positions. Thus, HP0958 may have a novel function in spatial localisation of flagella in H. pylori