An Investigation and Application of Biology and Bioinformatics for Activity Recognition

Activity recognition in a smart home context is inherently difficult due to the variable nature of human activities and tracking artifacts introduced by video-based tracking systems. This thesis addresses the activity recognition problem via introducing a biologically-inspired chemotactic approach and bioinformatics-inspired sequence alignment techniques to recognise spatial activities. The approaches are demonstrated in real world conditions to improve robustness and recognise activities in the presence of innate activity variability and tracking noise

Exploring the role of metals and senescence in cutaneous wound repair

Acute wound healing involves a tightly regulated cascade of cellular signalling and functional events. Deterioration at any stage of this sophisticated system can lead to healing impairment and chronic, non-healing wounds. Chronic wounds, which are prevalent in the elderly and diabetic, are a global socioeconomic burden and remain a major area of clinical unmet need. Improved understanding of the cellular and molecular aetiology of chronic wounds is essential to develop new therapies. The aim of this work was to explore the role of cellular senescence and the metallome in governing normal and pathological wound repair. Novel data presented in this thesis show increased senescence in both aged and diabetic wounds, while biologically-important metals, such as calcium, were reduced. Transcriptional profiling of wounds strongly linked the transcriptome, metallome and senescence. A direct role for senescence in pathological healing was mechanistically demonstrated in vitro, ex vivo and in vivo. Crucially, pharmacological inhibition of the explicated senescence receptor, Cxcr2, accelerated diabetic wound healing in vivo. Collectively, these data reveal a hitherto unappreciated role for Cxcr2 in mediating cellular senescence during pathological skin repair. Global profiling of the wound metallome highlighted significant changes in wound iron levels during late-stage healing. In vitro studies uncovered a new role for iron in mediating extracellular matrix deposition during wound remodelling, while reduced iron levels in diabetic wounds correlated with impaired ECM deposition. In summary, temporospatial metallome profiling identified multiple defects in metal-linked cellular processes in the pathological wound environment. Taken together, the research platform delivered in this work will provide an unprecedented opportunity to further interrogate transcriptional and functional relationships between cellular senescence and the metallome in wound repair. Indeed, this research may underpin the development of novel, efficacious metal-targeted therapies for chronic healing wounds in the future

Do differentiated macrophages display distinct metabolic profiles reflecting their different functions?

Macrophages are key players in both regulatory and inflammatory immune responses. They are implicated in the pathogenesis of rheumatoid arthritis (RA) where they accumulate in the synovium and produce pro-inflammatory cytokines including TNFα and IL-6. The rheumatoid synovium is metabolically distinctive, with low oxygen perfusion and high concentrations of lactate and reactive oxygen species (ROS). Macrophages are known to respond to metabolic signals, therefore we wanted to explore whether metabolic phenotypes of differentiated macrophages could play a role in the persistence of RA. We used an in vitro model of pro-inflammatory “classically activated” and “alternatively activated” macrophages to study macrophage behaviour using metabolomic and transcriptomic techniques. Differentiation with GMCSF and M-CSF produced macrophages with distinctive profiles. GM-CSF macrophages were metabolically active, metabolising glucose, glutamine and fatty acids, while M-CSF macrophages utilised fatty acid β-oxidation alone. Activation of macrophages with LPS, LPS+IFNγ or IL-4 produced metabolic changes, however, differences between MCSF groups were modest. LPS activation of GM-CSF macrophages drove both depletion of intracellular metabolites and transcriptional downregulation. In contrast, IL-4 activation of M-CSF macrophages was metabolically activating. We propose that the metabolic adaptability of GM-CSF macrophages may put them at an energetic advantage in the hypoxic, ROS-enriched rheumatoid synovium

Design and Development of an Automated Mobile Manipulator for Industrial Applications

This thesis presents the modeling, control and coordination of an automated mobile manipulator. A mobile manipulator in this investigation consists of a robotic manipulator and a mobile platform resulting in a hybrid mechanism that includes a mobile platform for locomotion and a manipulator arm for manipulation. The structural complexity of a mobile manipulator is the main challenging issue because it includes several problems like adapting a manipulator and a redundancy mobile platform at non-holonomic constraints. The objective of the thesis is to fabricate an automated mobile manipulator and develop control algorithms that effectively coordinate the arm manipulation and mobility of mobile platform. The research work starts with deriving the motion equations of mobile manipulators. The derivation introduced here makes use of motion equations of robot manipulators and mobile platforms separately, and then integrated them as one entity. The kinematic analysis is performed in two ways namely forward & inverse kinematics. The motion analysis is performed for various WMPs such as, Omnidirectional WMP, Differential three WMP, Three wheeled omni-steer WMP, Tricycle WMP and Two steer WMP. From the obtained motion analysis results, Differential three WMP is chosen as the mobile platform for the developed mobile manipulator. Later motion analysis is carried out for 4-axis articulated arm. Danvit-Hartenberg representation is implemented to perform forward kinematic analysis. Because of this representation, one can easily understand the kinematic equation for a robotic arm. From the obtained arm equation, Inverse kinematic model for the 4-axis robotic manipulator is developed. Motion planning of an intelligent mobile robot is one of the most vital issues in the field of robotics, which includes the generation of optimal collision free trajectories within its work space and finally reaches its target position. For solving this problem, two evolutionary algorithms namely Particle Swarm Optimization (PSO) and Artificial Immune System (AIS) are introduced to move the mobile platform in intelligent manner. The developed algorithms are effective in avoiding obstacles, trap situations and generating optimal paths within its unknown environments. Once the robot reaches its goal (within the work space of the manipulator), the manipulator will generate its trajectories according to task assigned by the user. Simulation analyses are performed using MATLAB-2010 in order to validate the feasibility of the developed methodologies in various unknown environments. Additionally, experiments are carried out on an automated mobile manipulator. ATmega16 Microcontrollers are used to enable the entire robot system movement in desired trajectories by means of robot interface application program. The control program is developed in robot software (Keil) to control the mobile manipulator servomotors via a serial connection through a personal computer. To support the proposed control algorithms both simulation and experimental results are presented. Moreover, validation of the developed methodologies has been made with the ER-400 mobile platform

Development of sensitive proteomic approaches for protein tyrosine phosphorylation detection.

The elucidation of the complex array of cell signalling cascades is imperative for a deeper understanding of cell biology in both physiological and patho-physiological states. Extensive biochemical characterisation of signalling networks has revealed the importance of post-translational modifications (PTMs), particularly phosphorylation. Signalling via protein phosphorylation occurs across homeostatic proliferative, differentiative and anti-apoptotic events. Dysregulation of the kinase signalling pathways as well as mutations in kinases involved in phosphorylation have been implicated in a number of pathologies such as cancer or immune deficiencies. While it is estimated that 50% of all proteins are phosphorylated during their lifetime, phosphorylated proteins are present in relatively low abundance compared to their non-phosphorylated counterparts. The rarity of phosphorylation, which occurs on serine, threonine and tyrosine residues, has prompted the development of sensitive approaches to improve phosphorylation characterisation. Proteomic-based strategies offer novel approaches to overcome the limitations of currently available strategies for phosphoprotein analysis. The research presented within describes the development of proteomic-based methodologies for phosphotyrosine identification, quantitation and characterisation. These methods utilise the antiphosphotyrosine. Antibody 4G10 along with other MS-compatible approaches for phosphotyrosine enrichment prior to MS analysis. Methods for more targeted phosphoprotein analyses involved coupling of 4G10 covalently to super para-magnetic beads or by affinity to super para-magnetic beads with protein G covalently attached. These 4G10-coupled beads successfully enriched tyrosine phosphopeptides derived from tryptic phosphoprotein digests for identification and characterisation of phosphopeptides using MALDI-TOF/TOF MS analysis. The limited capacity of the magnetic bead approach for analysis of more complex samples necessetated the development of a more global proteomic strategy for tyrosine phosphorylation analysis. A global strategy that provides not only qualitative pTyr information but also shows quantitative changes that occur with pTyr signalling is imperative for detailed signalling cascade analyses. The global approach presented here utilised the 4G10 Ab/bead approach as well as Hydrophilic interaction chromatography (HILIC) for the enrichment of pTyr peptides from complex samples isotopically-labelled to quantify tyrosine phosphorylation after LC-MALDI-TOF/TOF MS analysis. Aspects of this approach were modified to improve phosphopeptide detection and characterisation, including the development of a novel optimised matrix-deposition strategy for LCMALDI-TOF/TOF MS. The strategy, termed EZYprep LC, allowed the effective use of the atypical 2,5-DHB matrix with phosphoric acid to improve phosphopeptide ionisation and subsequently identify and characterise more phosphorylation sites on phosphoprotein samples compared with LC-ESI-IT-MS/MS. Another aspect of the global strategy was the development of a modified isotope protein coded label strategy (modified ICPL). The optimised ICPL approach ensured quantitative information from a larger sub-set of peptides after tryptic digest of complex samples. The improved ability to quantify using this approach was highlighted by a comparative analysis of complex cell lysates labelled using the conventional ICPL strategy and the modified ICPL strategy. The modified ICPL labelling strategy identified more proteins and provided more quantitative information that the conventional ICPL methodology. As such, the global phospho-tyrosine strategy, combined the modified ICPL labelling and 4G10 Ab/bead enrichment with peptide fractionation and MALDI-TOF/TOF MS analysis, was subsequently utilised to identify and quantify tyrosine phosphorylation occurring in insulin-stimulated insulin receptor A- and B-subtypes.Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Science, 201

The effects of neo-adjuvant chemotherapy on myeloid cells in high-grade serous ovarian cancer metastases

PhD Thesi

Urinary exosomes protein cargo as biomarkers of Autosomal Dominant Polycystic Kidney Disease (ADPKD)

ADPKD is the most common genetic renal disease and affects 1:1000 people worldwide with highly variable rates of progression to end-stage renal disease (ESRD). While 50% of patients (PKD1 mutation) will reach ESRD at an average age of 53 years, it is not possible to predict individual rates of progression. There is an unmet clinical need for reliable biomarkers of disease progression. One potential source of biomarkers is exosomes, small vesicles released via the endosomal pathway into the extracellular space and body fluids including urine, in both healthy and diseased states. Exosomes reflect their cell of origin and contain a subset of proteins and RNAs which have been shown to play a role in biological processes and provide the potential to be prognostic markers of disease and severity. Using urine collected from ADPKD patients over a 5-year period stored in 5ml aliquots, a protocol was optimised for the isolation of urinary exosomes (UEX) from small volumes. UEX yield, purity and size were validated using nanoparticle tracking analysis, transmission electron microscopy, immunoblotting and FACS of exosome membrane markers. Liquid-chromatography tandem-mass-spectrometry of UEX proteins revealed differences in expression between: i) normal healthy controls and ADPKD patients; ii) ADPKD patients at different CKD stage; iii) patients with rapid or slow disease progression regardless of renal function at clinical presentation; and, iv) patients with poor, delayed or good responses to Tolvaptan©. In vitro experiments investigated the potential role of exosomes in promoting a disease phenotype by assessing functional changes in exosome-treated normal and ADPKD renal epithelial cells. The intracellular distribution of exosomes was assessed by confocal microscopy providing insight into the uptake of exosomes into normal and ADPKD cells. Taken together the data show an important role for UEX proteins as prognostic markers of ADPKD progression and for monitoring of Tolvaptan efficacy and suggest a role for exosomes in intercellular communication in this disease