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  • (2022) Gautam, Shreedhar
    Thesis
    Extracellular vesicles (EVs) are phospholipid membrane bound sacs (vesicles) produced from almost all types of cells. They are found in circulation and contain the cargo biomolecules such as nucleic acids, proteins, lipids, and amino acids. EVs are involved in trafficking these biomolecules between cells and as such have the role in physiological and pathological processes. EVs are heterogenous and revealing their heterogeneity is crucial to understand their explicit physiological and pathological roles. Current isolation techniques cannot sort EVs based on their biogenesis and provides average information instead of each EVs subtype. Thus, single EVs analysis was popular and many surface protein characterization techniques are developed. But there are no techniques available for internal cargo analysis of individual EVs. The overall aim was to develop a technique to analyse internal microRNA cargo content, if possible, for single EVs, if not from the minimum number of EVs. To achieve that goal, light activated electrochemistry, a technique where focused light beam was illuminated on the semiconductor surface and make it electrochemically active was used. The surface was protected against oxidation during electrochemical reactions by grafting self-assembled monolayer of 1,8-nonadiyne. Then, silicon-based surface was patterned with polymers, antibodies, and cells using the light patterns. As a result, the first milestone to prepare light-assisted patterned semiconductor surface was achieved for our overall aim of analysing content of individual EVs. The size range of EVs is 30 to 200 nm, still very low compared to 30 µm which is the best spatial resolution achieved for light activated electrochemistry using crystalline silicon. Thus, chapter 4 developed a technique to improve the spatial resolution of light activated electrochemistry using amorphous silicon. Amorphous silicon has short diffusion length of charge carriers compared to crystalline silicon due to the defect states in band gap called as localized states. So, charge carriers are frequently trapped in these localized states leading to 60 times improvement in spatial resolution to 500 nm. But even this spatial resolution was not enough to analyse individual EVs. So, microRNA content from pool of EVs were detected using the screen-printed electrodes in a high throughput manner instead of single EVs.

  • (2022) Qiao, Laicong
    Thesis
    There has been a rapid-growing market and academic enthusiasm for small wearable molecular diagnostic platforms driven by the growing demand for continuous monitoring of human health. Wearable devices need to be portable, stretchable, and ideally re-configurable to be able to work for different analytes. Such flexible physiological monitoring devices which are non-invasive or minimally-invasive represent the next frontier of biomedical diagnostics. They may make it possible to predict and prevent diseases or facilitate treatment by diagnosing diseases at the initial stages. However, there are many problems that restrict further applications of these devices. Firstly, there are a limited number of bio-materials which are highly flexible, biocompatible and have anti-fouling properties; such biomaterials are needed as substrates for wearable devices. Secondly, traditional biosensors used in wearable devices focus on the detection of physical signals (such as heartbeat) and small chemical molecules, e.g. Na+, K+. These are not sufficient to provide in depth health information which requires sensing of large molecules such as proteins, ideally in real time, which is currently challenging. This provides a motivation to develop highly sensitive wearable biosensors for the detection of large molecules in sweat. This thesis centres on the development of a bio-material based wearable device for continuous detection of crucial analytes in human sweat. To achieve this target, our first aim was to design a highly bio-compatible flexible material as a substrate for wearable devices. A tough and anti-fouling three-network hydrogel has been prepared by integrating a zwitterionic polymer network into a robust double-network hydrogel. Secondly, to fill the gap between technological development of continuous and non-invasive detection of different analytes in human sweat, a patterned sweat-based biosensor was created for the detection of key biomolecules. This sensor was produced by placing specific aptamers or enzymes on flexible working electrodes. In addition, nanotechnology methods have been applied to refine the bio-sensing interface to further increase the sensitivity of our sensors. Finally, a sample collection chip has been combined with our high sensitivity sensors to fabricate a wearable device for sweat bio-sensing purposes. Future research may involve integration of a commercially available wireless signal readout module with this wearable biosensing device. The outcomes of this work may provide new insights for the development of wearable devices for continuous measurement of a spectrum of analytes in sweat, as an important step towards point-of-care diagnostics

  • (2022) Li, Zihao
    Thesis
    Therapeutic proteins have long been considered difficult to mimic synthetically. While the chemistry to make very complex polymers is generally available, the tools to efficiently screen for the effect of a polymer’s structure on its biological activity have yet to be demonstrated. In this thesis, a high throughput platform was developed for the synthesis of multivalent polymer scaffolds and applied to design synthetic mimics of the chemotherapeutic protein, tumor necrosis factor related apoptosis inducing ligand (TRAIL), which triggers apoptosis by receptor clustering. The platform makes use of a simple dual-wavelength, two-step polymerise & click approach to prepare star-shaped polymer-peptide conjugates. Polymerisations were performed in open well plates at 565 nm using an oxygen tolerant porphyrin-catalysed photoinduced electron/energy transfer-reversible addition-fragmentation chain-transfer (PET-RAFT) process. Subsequent UV irradiation results in deprotection of the polymerisation friendly cyclopropenone-masked dibenzocyclooctyne (cp-DIBAC) group at the α-chain end and the click conjugation of the desired peptide. Using this approach, the valency and position of ligands on a polymer scaffold can be precisely controlled, in a high throughput manner, without purification. Leveraging this approach, libraries of star shaped polymers which present exactly one receptor binding peptide at the end of each arm were prepared and screened for their ability to bind to the target death receptor (DR5), and trigger apoptosis through receptor clustering. Structure-activity relationships generated on a colon cancer line (COLO205) led to the identification of ~ 10 kDa trivalent structures as the most promising leads, which showed IC50 values of ~ 2 µM. Elevated levels of caspase-8 were used to confirm the mechanism of cell death. The scaffold design was then iterated by introduction of hydrophobic blocks into the centre of the star polymer, which resulted in improved spatial control over peptide presentation in solution. This led to around 30-fold improvement in IC50 (75 nM). These results demonstrate the potential for high throughput methods in designing polymer mimics of complex therapeutic proteins, and offer promising leads in the development of better TRAIL-like agents, which are long expected as novel chemotherapies for cancer treatment.

  • (2022) Pointing, Lewis
    Thesis
    Wastewater processing conditions in manufacturing environments often involve the three key factors for optimum bacterial growth - water, ideal temperature, and a constant food source. Bacteria are problematic because they can reduce product yield by consuming product and metabolise it into organic acids which lower the process pH, requiring large amounts of chemicals to control. At a casestudy wastewater treatment plant, a site-wide analysis of the impacts of chemical sanitation methods had not been conducted and the efficacy of these chemicals had not been established. To understand the impacts of current sanitation practices, standard microbiological plating techniques combined with HPLC testing to measure lactic acid as a proxy for microbial activity were used. Nitrogensource determination and solids analysis were used extensively to provide a comprehensive picture of the stream properties throughout the plant. I show that current microbial control methods are ineffective for significantly limiting microbial growth in the water treatment plant. The most important factors impacting this are the concentration of nitrogen-sources followed by total organic solids at chemical dosing sites, which react more rapidly with oxidative sanitisers than bacteria do. These findings indicate that chemical sanitisers would be more effective if dosed in locations with minimal concentrations of nitrogen-sources and organic solids. In practice, this is difficult to achieve in an existing plant without significant capital expenditure and so investigation of alternative, nonchemical methods of sanitation in combination with more effective use of chemical methods is recommended.

  • (2022) Yu, Tsz Tin
    Thesis
    The rapid emergence and development of antibacterial resistance is a major global threat to public health. There is an urgent need for the development of antibacterial agents with novel therapeutic strategy to tackle the increasing incidence of antibacterial resistance. In recent years, antimicrobial peptides (AMPs) and their synthetic mimics have been under the spotlight of the development of a novel class of antibiotics to combat antibiotic resistance. This research project focused on the utilisation of phenylglyoxamide and benzothiazole scaffolds in the development of antimicrobial peptidomimetics. The synthesis of phenylglyoxamide-based peptidomimetics was achieved via the ring-opening reactions of N-sulfonylisatins with primary amines followed by salt formation. Minimum inhibitory concentrations (MIC) of the peptidomimetics against different bacterial strains were determined to assess their antibacterial activity. Structure-activity relationship (SAR) studies revealed the inverse relationship between the alkylsulfonyl chain length and the bulkiness of the phenyl ring system for high antibacterial activity. The most active peptidomimetics exhibited high antibacterial activity with the lowest MIC to be 4, 16 and 63 μM against S. aureus, E. coli and P. aeruginosa, respectively. These peptidomimetics also showed significant biofilm disruption (up to 50%) and inhibition (up to 70%) against S. aureus at 2–4× MIC. In addition, terphenylglyoxamide-based peptidomimetics synthesised by the ring-opening reaction of N-acylisatins with amines and amino acid esters were evaluated for their quorum sensing inhibition (QSI) activity against P. aeruginosa MH602. The most potent peptidomimetic possessed high QSI activity of 82%, 65% and 53% at 250, 125 and 62.5 μM, respectively, with no bacterial growth inhibition. On the other hand, benzothiazole-based peptidomimetics were synthesised via the Jacobson method of cyclisation of phenylthioamides, followed by the installation of cationic groups. 2-Naphthyl and guanidinium hydrochloride as the hydrophobic and cationic groups, respectively, were important for high antibacterial activity of the peptidomimetics against both Gram-positive and Gram-negative bacteria. The most potent peptidomimetics against S. aureus, E. coli and P. aeruginosa possessed MIC values of 2, 16 and 32 μM, respectively. These active peptidomimetics inhibited 39% of S. aureus biofilm formation and disrupted 42% of preformed S. aureus biofilms at sub-MIC.

  • (2022) Gadde, Satyanarayana
    Thesis
    High-risk neuroblastoma is one of the most aggressive and treatment-refractory childhood malignancies. MYCN (v-myc avian myelocytomatosis viral related oncogene, neuroblastoma derived) is a major oncogenic driver for neuroblastoma (NB) tumorigenesis. Developing direct inhibitors of MYCN has been challenging due to several limitations. Hence, targeting MYCN-binding proteins which regulate the stability of MYCN protein is a promising alternative approach. This study is aimed at developing novel inhibitors of ubiquitin specific protease 5 (USP5), a deubiquitinating enzyme, which is known to prevent MYCN protein degradation by deubiquitination. The first results chapter describes the synthesis of novel pyrido[1,2-a]benzimidazole compounds and their cytotoxicity against MYCN amplified NB cells with high expression of USP5 protein (SK-N-BE(2)-C and Kelly cells). However, none of the tested compounds displayed better cytotoxicity than the parental compound, SE486-11. The second results chapter describes a one-pot synthesis of novel γ-carbolinone, γ-carboline and spiro[pyrrolidinone-3,3′]indoles. One of the γ-carboline compounds (42d) displayed promising cytotoxicity against NB cells (SK-N-BE(2)-C (IC50 = 1.21 μM) and Kelly (IC50 = 3.09 μM)) but showed little therapeutic selectivity when compared to normal human fibroblasts, MRC-5 cells (IC50 = 3.75μM). The synthesis and cytotoxicity of novel pyrimido[1,2-a]benzimidazoles is described in the third results chapter. The active compound, 65a displayed promising cytotoxicity against SK-N-BE(2)-C (IC50 = 0.78 μM) and Kelly (IC50 = 2.00 μM) cells with a reasonable therapeutic window compared to MRC-5 cells (IC50 = 15.0 μM). 65a bound to USP5 protein by microscale thermophoresis assay (Kd = 0.47 µM). USP5 and MYCN protein levels were decreased in NB cells by treatment with 65a. Moreover, the cytotoxicity of 65a was dependant on the expression of USP5 and MYCN proteins. 65a showed synergy in combination with HDAC inhibitors, SAHA and panobinostat. In the fourth results chapter, the synthesis of more potent pyrimido[1,2-a]benzimidazoles with di- and tri- substitutions on the pendant phenyl ring (86b (SK-N-BE(2)-C IC50 = 0.31 μM; Kelly IC50 = 0.65 μM) and 91 (SK-N-BE(2)-C IC50 = 0.03 μM; Kelly IC50 = 0.07 μM)) are described. Importantly, 86b displayed significant in vivo efficacy in TH-MYCN homozygous NB mice when treated with 60 mg/kg for three weeks. The last results chapter describes the synthesis and cytotoxicity of novel benzo[4,5]imidazo[2,1-b]thiazole and pyrido[2,3-b]indole compounds. Collectively, this thesis identifies promising novel scaffolds with great potential for further development.

  • (2022) Chen, Xueqian
    Thesis
    How to design a biosensor with optimal performance with regards to sensitivity, specificity, response time and the ability to be multiplexed is a key issue to meet the demands of diagnostics for health cares. As modern biosensors are usually constructed with bioreceptors immobilized on nanoparticles, the surface of nanoparticle has a significant impact on sensing performance. This thesis aims at investigating how to design the surface of nanoparticle to achieve better sensing performance of single molecule biosensor. Two different biosensing systems were built using DNA functionalized gold nanoparticle (AuNPs), including fluorescent biosensor and localized surface plasmon resonance (LSPR) biosensor using dark-field microscopy. To better understand how the coverage of DNA on surface of AuNPs affects sensing performance, the coverage of DNA on AuNPs surface was adjusted and effect of coverage of DNA on the sensing performance was investigated. Firstly, the impact of coverage of aptamer on the surface of AuNPs on sensing performance was investigated using fluorescence spectroscopy. The number of anti-interferon gamma (IFN-) aptamers was adjusted from an average of 9.6 to 258 per nanoparticle. The sensing strategy was based on the conformational change of anti-IFN- aptamer and fluorescence quenching property of AuNPs. The binding isotherm and binding kinetics of the interaction between AuNPs-aptamer conjugate and IFN- as a function of coverage of aptamer were investigated. It was found that AuNPs-aptamer conjugate with the highest coverage of aptamer was the most favorable in biosensors considering the limit of detection, sensitivity, and response time of detection. Secondly, a LSPR biosensor utilizing AuNPs-DNA conjugates was built to detect SARS-CoV-2 specific RNA. Two AuNPs-DNA conjugates with different size of AuNPs were adopted to form a dimer consisting with a 80 nm gold nanoparticle and a 40 nm gold nanoparticle. The binding of target RNA with dimer resulted in colour change of dimer under dark-field microscope. A calibration curve between response and different concentration of target RNA was obtained with a determined limit of detection of 2.6 fM. Lastly, the number of DNA on the satellite nanoparticle was adjusted by different diluents to optimize the sensing performance of LSPR biosensor. By reducing the amount of DNA on 40 nm gold nanoparticle, the response for the same concentration of target RNA was improved and lower limit of detection was achieved. This thesis provides insights into how to design the surface of nanoparticle and the impact of interfacial design of nanoparticle on the sensing performance of biosensor, which advances the development of single molecule biosensor from the perspective of interface of nanoparticle.

  • (2023) Mustafa, Ahmed
    Thesis
    Polyelectrolytes (PEs) and proteins can spontaneously self-assemble in aqueous solutions to form colloidal polyion complexes. These nanoparticles are widely used in many biotechnological applications such as drug/protein delivery, protein encapsulation and protein stabilisation. Although the interaction between PEs and proteins is mainly electrostatic, it is also influenced by parameters such as the density of negative and positive charges, degree of ionisation, the presence of hydrogen bonding and van der Waals forces exerted by hydrophobic groups throughout the PE and protein, as well as their overall size and concentration. The variety of forces reflects the complexity of the protein's surface where charges are often not evenly distributed, but concentrated in pockets. In order to design polymers that form strong PICs with any given protein, it is necessary to carefully tune the polymer architecture, chain length, side chain chemistry, charge distribution, and hydrophobicity. In this research project, a range of well-defined polymers with different polymeric structures and architectures, copolymerised with a small amount of fluorescent monomer based on the cyanine dye Cy5, were synthesised in low solution volumes without prior deoxygenation using Enz-RAFT polymerisation. The self-assembly of these polymers with Cy3-labelled proteins was screened in order to understand the effect of both polymer and protein structure on binding. Supported by light scattering, isothermal calorimetry (ITC) and small angle X-ray scattering data we demonstrate that a simple plate based Förster resonance energy transfer (FRET) assay can be used as a readout for the binding strength of each polymer to the protein of interest. Because both the synthetic and read-out methodologies are amenable to high throughput processing, this technique enables the rapid combinatorial design of complex polymers for protein encapsulation.

  • (2023) Pelosi, Rosina
    Thesis
    The improvement of luminescent solar concentrator (LSC) efficiency is the core focus of this project. Lead sulfide oleic acid (PbS/OA) semiconductor nanocrystals otherwise known as quantum dots (QDs) are investigated as a potential luminophore in LSCs. Their broad absorption, narrow emission and good Stokes shift are of interest, however cumulative luminophore self-absorption events contribute to losses. A possible way to circumvent these losses is through Förster Resonant Energy Transfer (FRET). In this work PbS/OA QDs of two different sizes are embedded into PMMA films in 1:1 ratios. Mixtures of suitably disparate sized PbS/OA QDs in PMMA films result in photoluminescence (PL) with increased Stokes shifts. Time resolved photoluminescence (TRPL) spectroscopy results are also consistent with FRET, where there is a reduction in high energy QD lifetimes in heterogeneous QD films. PLQY synergies are demonstrated through QD FRET connection in heterogeneous QD spin coated films and improved LSC efficiency is established. This learning may be transferred across other QD species used in LSCs, that also suffer self-absorption losses. The fabrication of a luminescent solar concentrator photovoltaic (LSC/PV) prototype device with dimensions 10 × 10 × 1 cm3 is designed with a commercially available pery- lene luminophore. A power conversion efficiency (PCE) of 22.4 % with a 2.4% increase on PV alone is demonstrated and attributable to the LSC through novel experimental measurement design. Furthermore, it was shown that a parabolic patterned textured luminescent film had a threefold increase in emission over a flat film. The parabolic design within the film was able to take broadband light on one side and augment emission preferentially out the opposite side. The film performance permits up to a threefold cost benefit in real world applications, making costly green-housing films that emit in the photosynthetically active region more accessible. Finally, future directions and pathways for green technology LSCs are discussed within a New Product Development (NPD) framework.

  • (2023) Al Taief, Karrar
    Thesis
    Self-assembled short peptide hydrogels based on natural proteins have been designed to mimic natural environment of extracellular matrix (ECM) in tissue. Yet this class of hydrogels solely lacks the ability to represent the entire complexity of the ECM. To address this problem, requires novel design of synthetic materials incorporating natural biopolymers. In this work, library of peptides based on protein motifs were designed that form self-assembled hydrogel. Animal source or human source biopolymers were then mixed with these peptides to fabricate dual-functional hybrid hydrogels. The incorporation of biopolymers at a concentration much lower than the peptide concentration, drastically enhanced the mechanical property of these hybrid systems. Both animal and human biopolymers are commercially available at high cost, however, incorporating minimum concentrations of both into this novel hybrid hydrogel will reduce the need for the biopolymer in a cost-effective manner. Additionally, these hybrid hydrogel systems are readily tuned by designing or re-arranging the target peptides sequences to fulfil the required applications of these hydrogels. Another peptide carrying cell-adhesion epitope, was designed based on a key binding motif for skin cells. The peptide self-assembled into self-supporting hydrogel. While biological compatibility of this gelator with skin cells was suboptimal over a long period of time, on the other hand, in 2D cultures of human Mesenchymal Stem Cells (hMSCs) no adverse reactions were noted and the hMSCs were shown to spread over a 7 days period on top of the hydrogel formed. Remarkably, exposure of this peptide to light triggered dynamic assembly. This photo-induced modulation of peptide assembly could be harvested for future therapeutic applications.