Engineering

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Now showing 1 - 10 of 12
  • (2021) Fu, Lu
    Thesis
    Angiogenesis is a vital step of cancer growth and metastasis and has become a promising target for cancer therapy. Due to the abnormal vascularisation and poor blood supply, tumours are usually hypoxic and lack nutrients, which triggers tumour aggressiveness and hampers efficient drug delivery. The success of traditional anti-angiogenic strategies, which focuses on decreasing the vascular supply to tumours, is limited by insufficient drug delivery or tumour resistance. Thus, vascular promotion therapy based on promoting angiogenesis is emerging as a complementary cancer therapy. Heparin is a linear negatively charged polysaccharide that, in addition to its anticoagulant action, can promote angiogenesis by potentiating angiogenic growth factors. Thus, heparin is a promising bioactive for cancer drug delivery applications. Cerium oxide nanoparticles (CNPs), a cytocompatible redox biomaterial, has gained attention as it actively scavenges reactive oxygen species (ROS) in biological systems which are related to cancer aggressiveness and tumour hypoxia. In this thesis, CNPs were functionalised with heparin with different chain lengths and density, aiming to investigate their mechanisms of interaction with the vasculature as blood vessel-targeting cancer therapeutics. CNPs were synthesised via precipitation and then functionalised with different amounts of unfractionated heparin (UFH) and low molecular weight heparin (LMWH) via an organosilane linker. CNPs and heparin-CNPs were approximately 12 nm in crystal size, exhibited face-centred cubic phase structure in morphology and as determined by high-resolution transmission electron microscopy (HRTEM) and X-ray diffraction (XRD), respectively. The successful functionalisation of CNPs with heparin was qualitatively verified by attenuated total reflectance-Fourier transform infra-red spectroscopy (ATR-FTIR) and X-ray photoelectron spectroscopy (XPS). Additionally, thermogravimetric analysis (TGA) demonstrated the conjugation of heparin onto the surface of CNPs with approximately 1.5 μmol UFH, and either 1.8 μmol or 5.7 μmol LMWH conjugated per gram of CNPs. In addition, CNPs and heparin-CNPs showed a dose-dependent cytotoxicity in human umbilical vein endothelial cells (HUVECs), indicating that concentrations of heparin-CNPs above 5 μg/mL can act as anti-angiogenic agents. Heparin-CNPs at a dose of 1.5 μg/mL had limited redox activity which correlated with their limited effect on vascular endothelial growth factor receptor 2 (VEGFR2) expression, a key receptor in angiogenesis regulated via redox. However, the heparin-CNPs signalled fibroblast growth factor 2 (FGF-2) in vitro and in vivo, a key growth factor in angiogenesis, indicating their potential to support angiogenesis. In vivo analyses of the heparin-CNPs indicated that they dose-dependently promoted angiogenesis in the CAM assay at doses up to 1 mg/mL. Moreover, compared to CNPs, heparin-CNPs reduced intracellular ROS level in melanoma cells (MM200), reducing the hypoxic level of tumour cells, which indicated their potential as an anti-cancer agent. A human tumour model in the CAM was developed in this thesis to investigate the effects of nanoparticles on tumour angiogenesis and their interactions with the tumour vasculature. It was found that heparin-CNPs, compared to CNPs, dose-dependently promoted angiogenesis in the human tumour model in the CAM and exhibited a higher level of penetration into the tumour mass via light sheet microscopy and histological analysis. Overall, heparin-CNPs dose-dependently promoted angiogenesis by modulating key processes in angiogenesis and address tumour aggressiveness, showing their potential application in cancer therapeutics.

  • (2021) Li, Bitong
    Thesis
    In the past decade, the discovery of the CRISPR/Cas system launched a new era of genome editing and has rapidly become a universal research tool. The specific recognition between guide RNA and target nucleic acids enables CRISPR/Cas systems to exhibit high specificity compared to gene editing systems such as zinc finger or transcription activator-like effector nucleases. In this thesis, the CRISPR/Cas12a and CRISPR/Cas13a systems were designed to modify DNA or RNA associated with the HSPG2 gene which encodes perlecan, the major extracellular heparan sulphate (HS) proteoglycan in basement membranes. Perlecan has essential roles in organ development and contributes angiogenesis in pathological processes including cancer. The Cas12a and Cas13a nucleases were expressed in transformed E. coli and purified via His-tag affinity followed by size exclusion chromatography. The purified Cas12a nuclease exhibited high activity and specificity in a collateral activity assay in which the reporter sequence was only cleaved in the presence of the Cas12a protein and target ssDNA. Similarly, Cas13a nuclease exhibited high activity and specificity in a collateral activity assay with target ssRNA. A CRISPR/Cas12a gRNA was designed to target exon 2 of HSPG2 and was able to cleave amplified genomic DNA extracted from human melanoma cell line, MM200. Additionally, the collateral activity assay revealed that Cas12a nuclease dose-dependently cleaved the reporter ssDNA when used with target HSPG2 DNA. Similarly, a CRISPR/Cas13a gRNA was designed to target exon 2 of HSPG2 RNA and was able to cleave the target RNA extracted from MM200 cells and was active in the collateral activity assay when used with target HSPG2 RNA. Modification of HSPG2 nucleic acids in both MM200 cells and human umbilical vein endothelial cells (HUVECs) was also established. The CRISPR/Cas12a system resulted in up to 39 and 24 % reduction in HSPG2 gene expression in MM200 and HUVECs, respectively. Moreover, the CRISPR/Cas13a system achieved up to 69 and 99% reduction in HSPG2 RNA in MM200 and HUVECs, respectively. The HSPG2 mRNA modification in both MM200 and HUVECs resulted in decreased expression of FGF2 and VEGF-A, genes involved in the perlecan signalling pathway networks and associated with angiogenesis. The established CRISPR/Cas12a and CRISPR/Cas13a systems provide novel and efficient nucleic acid editing tools to further study the functions of perlecan in vitro and potentially in vivo. In addition, the LbCas12a or LwCas13a-based collateral cleavage assay enabled efficient and specific detection of the HSPG2 genome or transcripts, suggestive of its potential in perlecan-related disease diagnoses, such as cancer or genetic disorders.

  • (2021) Deng, Fei
    Thesis
    Cytokines are a large, diverse family of small soluble proteins (4-60 kDa) expressed by immune and nonimmune cells. They play significant roles in cellular signalling, activation, growth and differentiation, resulting in a wealth of information related to health and disease. Defects in the regulatory networks may cause fluctuation of cytokine levels and accompany in inflammation or diverse diseases, indicating that cytokine measurement could be an effective approach for characterizing the function of immune system, diagnosing and predicting disease, and evaluating the effects of treatment. However, detection of cytokines is challenging because of their low biological concentration (pg/mL), complicated interacting networks, and inhomogeneous distribution. The current methods for detection of cytokines are mainly based on immunoassay techniques. Among them, enzyme-linked immunosorbent assay (ELISA) and Meso Scale Discovery (MSD) have become the most common cytokine quantification tools due to the simple protocols and relatively high sensitivity (pg/mL). However, all these approaches were limited on multiplex monitoring of in vivo cytokines with ultralow concentration (fg/mL) and superb spatial resolution. In this project, four novel biosensors have developed for sensitive, multiplex and in vivo monitoring of cytokines, which include three published papers and two revised manuscripts. Optical fibre and stainless steel were applied as the substrates for the development of the deployable biosensors with the capability of in vivo application. Sandwich structure was designed with capture module (antibody) immobilized on the surface of substrates and detection module (antibody or aptamer) conjugated with fluorescent reporter for signal amplification and readout. We first tested the sandwich-based biosensors in in vitro experiments by assessing and optimising their performance in PBS buffer, serum, plasma, saliva, perspiration, and whole blood. After validation of in vitro work, we applied the biosensors for in vivo monitoring of cytokines in rat brain or spine cord for the study of chronic pain or the development of diseases. The findings indicated that the designed sensing devices have the potential to be used for sensitive and multiplex monitoring of analytes (such as cytokines) in clinical practice and help with the better understanding of various disease conditions.

  • (2021) Fetanat Fard Haghighi, Masoud
    Thesis
    Heart failure (HF) is one of the most prevalent life-threatening cardiovascular diseases, affecting more than 23 million worldwide. Although heart transplantation is the gold standard treatment for end-stage HF patients, the number of donor hearts is significantly less than the demand. Mechanical circulatory support for a patient with a failing left ventricle can be achieved by implanting a left ventricular assist device (LVAD) by pumping blood from the left ventricle to the aorta. Currently, clinicians set the LVAD speed at a fixed value, which can lead to different hazardous events. A physiological control system (PCS), which automatically adjusts pump speed can mitigate the hazardous events and improve a patient’s mobility, lifespan and quality of life. However, there are two main reasons that the current PCSs are not used commercially. Firstly, previously developed PCSs have been evaluated in specific conditions for only single-patient scenarios. Secondly, previously developed PCSs require implanted pressure or flow sensors. Therefore, the aim of this thesis was to design novel methods for estimation of preload and sensorless PCS for LVADs than can accommodate interpatient and intrapatient variations (IAIV), by way of three objectives. The first objective was to design a PCS for an implantable heart pump that accommodates IAIV. A novel model free adaptive control (MFAC) system was developed that maintained the preload in the normal range of 3 to 15 mmHg for different patient conditions. The second objective was to design a sensorless PCS for LVADs across different patient conditions, by combining a preload estimator using a deep learning method and the MFAC. The third objective was to design an improved non-invasive preload estimator, based on deep learning methods using LVAD flow waveforms recorded clinically. The proposed preload estimator was extremely accurate with a correlation coefficient of 0.97 and root mean squared error of 0.84 mmHg. The proposed sensorless PSC works similarly to the preload-based PCS using measured preload to prevent suction and congestion. This study shows that the LVADs can respond appropriately to changing patient states and physiological demands without the need for additional implanted sensors.

  • (2021) Rinaudo, Carlo
    Thesis
    The vestibulo-ocular reflex (VOR) is the main retinal-image stabilizing mechanism during rapid (i.e. high-frequency content) head movements. When the peripheral vestibular system is impaired, i.e. vestibular hypofunction, through disease, trauma or the aging process, the VOR response is reduced resulting in retinal image slip. Subsequently, along with other vestibular conditions, the image of the world are blurred during head movements. In these conditions, vestibular information is less accurate and reliable, introducing perceptual uncertainty due to the sensory mismatch, leading to compromised balance, gait and ability to perform daily activities. Vestibular rehabilitation therapy (VRT) is a set of prescribed exercises that help improve function and reduce symptoms. The main pillar of VRT are gaze-stabilization exercises (GSE) which aim to improve the VOR response (by a process we call adaptation) or compensate for vestibular hypofunction by improving other related (lower-frequency operating) systems via substitution or habituation. To date, despite their acceptance in clinical guidelines, little evidence exists showing GSE improves the high-frequency VOR response using quantitative measures. Recently, the incremental VOR adaptation (IVA) technique has shown promising results, with marked VOR adaptation after as little as 15 minutes of training. However, these preliminary studies mostly looked at healthy subjects. The few IVA patient studies had small numbers, minimal follow up, and were not compared to conventional GSE or evaluated for clinical effect. In the following thesis, I sought to determine GSE training parameters for optimal VOR adaptation, as well as perform studies to compare short and long-term clinical outcomes after conventional GSE versus IVA training in patients with isolated vestibular hypofunction.

  • (2021) Lau, Kieran
    Thesis
    Cardiovascular disease is the leading cause of death globally, mainly due to blood vessel occlusions resulting in tissue necrosis and ischaemic strokes. When pharmacological intervention and native vessel transplantation are not viable, synthetic vascular conduits have been used. However, they are prone to failure at small diameters (<6 mm), largely due to mechanical and biological mismatch with the native vessel. Bombyx mori silk fibroin (silk) is a mechanically tuneable, cytocompatible natural polymer that can be manufactured into vascular grafts with mechanical properties matching those of native vessels. However, silk lacks cell binding domains and requires biofunctionalisation to modulate vascular cell interactions. The limited functional groups available for covalent crosslinking chemistries has prompted exploration of an alternative biofunctionalisation strategy. This thesis aims to evaluate plasma immersion ion implantation (PIII) as a novel technique for biofunctionalisation of silk and to assess the potential of immobilised recombinantly expressed domain V (rDV) of the human basement membrane proteoglycan perlecan in modulating vascular interactions toward graft applications. PIII is a one-step, gas plasma-based surface treatment that induces strong attachment of biomolecules onto polymer surfaces in the absence of chemical crosslinkers. PIII-treated silk films (PIII-silk) were evaluated in terms of physical and chemical properties, rDV immobilisation, in vitro vascular cell interactions, ex vivo blood interactions, and in vivo immune responses. PIII treatment affected the surface chemistry and mechanical properties of silk films, but did not alter the bulk mechanical properties, degradation, or immune responses to implanted silk biomaterials. PIII-silk support covalent immobilisation of rDV (rDV-PIII-silk) with significantly more rDV on PIII-silk compared to physisorption or carbodiimide-based covalent immobilisation methods. rDV-PIII-silk retained its bioactivity supporting endothelial cell adhesion, spreading, proliferation and cell-cell junction formation, while limiting smooth muscle cell and platelet adhesion. Furthermore, rDV-PIII-silk was found to be non-thrombogenic in the presence of whole blood in both static and dynamic flow models. Overall, the thesis results show the potential of rDV-PIII-silk as a small-diameter vascular graft material and warrant further investigation to assess endothelialisation and blood interaction in vivo.

  • (2021) Sy, Luke Wicent
    Thesis
    Human pose estimation involves tracking the position and orientation (i.e., pose) of body segments, and estimating joint kinematics. It finds application in robotics, virtual reality, animation, and healthcare. Recent miniaturisation of inertial measurement units (IMUs) has paved the path towards inertial motion capture systems (MCS) suitable for use in unstructured environments. However, commercial inertial MCS attach one-sensor-per-body-segment (OSPS) which can be too cumbersome for daily use. A reduced-sensor-count configuration, where IMUs are placed on a subset of body segments, can improve user comfort while also reducing setup time and system cost. This work aims to develop pose estimation algorithms that track lower body motion using as few sensors as possible, towards developing a comfortable MCS for daily routine use. Such a tool can facilitate interactive rehabilitation, performance improvement, and the study of movement disorder progression to potentially enable predictive diagnostics. This thesis presents pose estimation algorithms that utilise biomechanical constraints, additional distance measurements, and balance assumptions to infer information lost from using less sensors. Specifically, it presents a novel use of Lie group representation of pose alongside a constrained extended Kalman filter for estimating pelvis, thigh, shank, and foot kinematics using only two or three IMUs. The algorithms iteratively use linear kinematic equations to predict the next state, leverage indirect observations and assumptions (e.g., pelvis height, zero-velocity update, flat-floor assumption, inter-IMU distance), and enforce biomechanical constraints (e.g., constant body segment length, hinged knee joints, range of motion). The algorithm was comprehensively evaluated on nine healthy subjects who performed free walking, jogging, and other random movements within a 4x4 m2 room using benchmark optical and inertial (i.e., OSPS) MCS. In contrast to existing benchmark datasets, both direct kinematics (e.g., Vicon plug-in gait commonly used in gait analysis) and inverse kinematics (used in robotics and musculoskeletal modelling) pose reconstruction, along with the corresponding measurements, are shared publicly. The mean position root-mean-square error relative to the mid-pelvis origin was 5.3±1.0 cm, while the sagittal knee and hip joint angle correlation coefficients were 0.85±0.05 and 0.89±0.05 indicating promising performance for joint kinematics in the sagittal plane.

  • (2021) Leong, Chen Onn
    Thesis
    Acute myocardial infarction (MI) is one of the leading causes of death worldwide that commonly affects the left ventricle (LV). Following MI, the LV mechanical loading is altered and may undergo a maladaptive compensatory mechanism that progressively leads to adverse LV remodelling and then heart failure. One of the remodelling processes is the infarct extension which involves necrosis of healthy myocardium in the border zone (BZ), progressively enlarging the infarct zone (IZ) and recruiting the remote zone (RZ) into the BZ. The mechanisms underlying infarct extension remain unclear, but myocyte stretching has been suggested as the most likely cause. A recent personalized LV modelling work found that infarct extension was correlated to inadequate diastolic fibre stretch and higher infarct stiffness. However, other possible factors of infarct extension may not have been elucidated in this work due to the limited number of myocardial locations analysed at the subendocardium only. Using human patient-specific left- ventricular (LV) models established from cardiac magnetic resonance imaging (MRI) of 6 MI patients, the correlation between infarct extension and regional mechanics impairment was studied. Prior to the modelling, a 2D-4D registration-cum-segmentation framework for the delineation of LV in late gadolinium enhanced (LGE) MRI was first developed, which is a pre-requisite for infarct scar quantification and localization in patient-specific 3D LV models. This framework automatically corrects for motion artifacts in multimodal MRI scans, resolving the issue of inaccurate infarct mapping and geometry reconstruction which is typically done manually in most patient-specific modelling work. The registration framework was evaluated against cardiac MRI data from 27 MI patients and showed high accuracy and robustness in delineating LV in LGE MRI of various quality and different myocardial features. This framework allows the integration of LV data from both LGE and cine scans and to facilitate the reconstruction of accurate 3D LV and infarct geometries for subsequent computational study. In the patient-specific LV mechanical modelling, the LV mechanics were formulated using a quasi-static and nearly incompressible hyperelastic material law with transversely isotropic behaviour. The patient-specific models were incorporated with realistic fibre orientation and excitable contracting myocardium. Optimisation of passive and active material parameters were done by minimizing the myocardial wall distance between the reference and end-diastole/end-systole LV geometries. Full cardiac cycle of the LV models was then simulated and stress/strain data were extracted to determine the correlation between regional mechanics abnormality and infarct extension. The fibre stress-strain loops (FSSLs) were analysed and its abnormality was characterized using the directional regional external work (DREW) index, which measures FSSL area and loop direction. Sensitivity studies were also performed to investigate the effect of infarct stiffness on regional myocardial mechanics and potential for infarct extension. It was found that infarct extension was correlated to severely abnormal FSSL in the form of counter-clockwise loop, as indicated by negative DREW values. In regions demonstrating negative DREW values, substantial isovolumic relaxation (IVR) fibre stretching was observed. Further analysis revealed that the occurrence of severely abnormal FSSL near the RZ-BZ boundary was due to a large amount of surrounding infarcted tissue that worsen with excessively stiff IZ.

  • (2021) Mohd Addi, Mitra
    Thesis
    Concurrent stimulation in a visual prosthesis is necessary in order to deliver sufficient phospenes (perceived spots of light) for effective vision. Major issues with concurrent stimulation are the effects of inter-electrode current distribution which lead to current leakage and issues with charge recovery which determines whether balanced charges being delivered are recovered at each electrode. This thesis investigates concurrent stimulation in multi-electrode arrays of different electrode configurations and orientations using platinum electrodes immersed in physiological saline bath along with results from computational modelling. Current waveforms were recorded to determine current interactions and charge recovery in each electrode. Current interaction was found to be highest when imbalanced stimuli were delivered. Current interaction and charge recovery were found to be minimal for combined current source and sink stimulation mode, especially in the tripolar configuration of the hexagonal orientation, indicating that the combination of appropriate electrode configuration, orientation and stimulus mode will aid in current focusing and avoiding current interaction. Simplified models were developed to mimic the experimental setup and used to fit multiple experimental current waveforms, based on the stimulus currents and electrode-electrolyte properties. The models were optimized to predict the electrode-electrolyte properties of electrode arrays as well as the current interactions and voltage distributions in different electrode configurations and orientations. The resistance and capacitance of the electrode-electrolyte interface were found to decrease and increase with stimulus current, respectively. The optimized models were able to reproduce current results from the experimental recordings in terms of the dynamic waveforms , with a goodness of fit between 53 % - 90 %, where the percentage reduces in more complex model with multi-electrode. To validate that the optimized model is appropriate to present concurrent stimulation of multi-electrode array, 3D models of the multi-electrode array were constructed to mimic and reproduce the experimental results and to validate the optimized model. The model was able to predict current and voltage distribution as well as the electrode-electrolyte interface voltages and voltages at each individual electrodes which is not possible to measure experimentally. Improved version of the model with additional parameters will be useful to predict the performance of the implant in various numerical settings which may not be possible to be conducted experimentally. The new methodology developed in this thesis shows a strong link between experimental and computational modeling of concurrent stimulation in multi-electrode arrays, allowing for prediction for specific electrode design simulations and also as a platform for neural prosthesis evaluation using multi-electrode arrays.

  • (2021) Kim, Ha Na
    Thesis
    Chronic wounds, such as those exhibited by diabetic ulcers are a global health issue affecting approximately 3.75 million people worldwide. Chronic wounds are slower to heal, or fail to heal, due to decreased vascularisation among other factors. Growth factors are key signalling molecules which promote wound healing, however, they experience rapid degradation by proteolytic enzymes present in the chronic wound environment. Proteoglycans (PGs) are the natural binding, protective and signalling partners for many growth factors. However, the yield of PGs from natural sources is limited due to their low abundance in tissues and cell cultures. Recombinant DNA technology and metabolic engineering offer alternative PG production methods to increase the yield of PGs and to alter the structure of their glycosaminoglycan (GAG) chains. Serglycin is an intracellular PG, which has eight GAG attachment sites. Unlike other PGs, serglycin can be decorated with heparin, heparan sulphate (HS), chondroitin sulphate (CS) and/or dermatan sulphate (DS). Through these various GAG chains, serglycin present in intracellular granules can bind and release cytokines, chemokines and growth factors, which are ideal properties for growth factor delivery and signalling applications. Thus, this thesis examined the influence of various culture microenvironments on the yield and GAG structure of recombinant serglycin produced by both adherent and suspension mammalian cells. In addition, this thesis explored the ability of recombinant serglycin to support angiogenic growth factor binding and signalling. Adherent human embryonic kidney cells expressing recombinant serglycin (HEK-SGN) cultured in shaker flasks and continuously stirred tank reactors (CSTR) produced more protein decorated with GAGs compared to culture flasks. The cells cultured in CSTR produced more HS/heparin and CS/DS chains compared to the other culture flasks. HEK-SGN cells maintained in medium containing 25 mM glucose achieved the highest yields of protein (1.5 mg/L) and GAGs (1.9 mg/L). The cells maintained in medium containing 5.5 mM glucose produced less GAG chains compared to when these cells were grown in medium containing 25 mM glucose, however, heparin was produced. Suspension HEK-293 cells expressing recombinant serglycin (HEK-S-SGN) were established in this thesis. The presence of serum in the culture medium promoted the suspension cells to adhere during culture. HEK-S-SGN cells maintained in serum free medium produced a 4.6-fold higher yield of protein decorated with HS/heparin compared to the cells maintained in serum containing medium. Recombinant serglycin supported fibroblast growth factor 2 (FGF2) binding in vitro via its HS/heparin chains, while vascular endothelial growth factor 165 (VEGF165) binding was mediated via HS/heparin and CS/DS chains as well as the protein core of serglycin. In addition, recombinant serglycin potentiated FGF2 signalling via its GAG chains and FGF receptor 1c in vitro. Furthermore, recombinant serglycin supported angiogenesis in vivo by potentiating FGF2 and VEGF165 through its HS/heparin chains in the chicken chorioallantoic membrane assay. Thus, this thesis demonstrated that the culture conditions influenced the protein yield and type of GAG chains that decorated recombinant serglycin. In addition, recombinant serglycin was found to support angiogenesis via binding and signalling angiogenic growth factors via its GAG chains. These results suggest the potential of incorporating recombinant serglycin into tissue engineering or regenerative medicine strategies for growth factor-mediated tissue repair applications.