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This work explores the novel applications of 3D inkjet bioprinting technology in the areas of biomedical sciences and environmental sciences. The versatility of inkjet bioprinting was reviewed and explored by experimenting with silk fibroin as the biomaterial ink for fabricating millimetre-sized 3D fibroin structures and investigating them through three different projects. In the first project, the feasibility of fibroin structures for use as 3D carriers for on-carrier culture of neuronal cells was explored. 3D cell culture is a solution to the limitations, such as flattening and loss of physiological relevance of cells, observed in the conventional 2D culture. The results in this study demonstrated proliferation of cells and the ability of cells to differentiate under specific conditions while adhering to the fibroin structures. This study, thus, promises a new and robust platform for 3D culture of neuronal and other cells for applications in such techniques as guided neuronal growth, 3D cell-patterning and in vitro cell proliferation for adoptive cell transfer. In the second project, 3D fibroin structures were utilised as self-propelled motors (SPMs) for their potential use in inducing fluid flow in microwells for the enhancement of diffusionrate limited biomedical assays by rapid agitation of the analytes. The SPMs were fabricated in four different geometric shapes, propelled with two physical mechanisms and their propulsion data were analyzed to deduce the most favourable geometric shape and propulsion mechanism among the samples. It was concluded that among the four designs and two propulsion mechanisms tested, the line-shaped 1-armed surface tension driven SPMs showed the most reliable and predictable propulsion behaviour with maximum rotations throughout the observation time period and, therefore, could be used in the future for further investigations with different assays. In the third project, 3D fibroin structures were utilised as surface tension driven selfpropelled sensors for the approximate determination of surface tension of an unknown water sample and thereby indicate the level of dissolved contaminants in the sample. The results showed significant differences in the propulsion velocity decay over time or deceleration, with sensors showing propulsion for much longer periods of time in water samples containing low level of contaminants and having higher surface tension compared to those propelling in relatively more contaminated water samples which had lower surface tension. This study put forward a simple to use technique for on-the-site detection of dissolved contaminant levels in a water sample. Together, the experiments conducted and the results obtained across the three projects in this work demonstrate the flexible and multi-faceted applicability of 3D inkjet bioprinting technology in biomedical and environmental areas of research. This work will thus inspire and lay the foundations of numerous future research and investigations involving inkjet bioprinting.

Transketolase (TK) is an interesting enzyme for the biotechnology industry because it can catalyse the formation of specific carbon-carbon bonds with high stereospecificity and selectivity. These characteristics make TK interesting for the formation of high-value chemicals and pharmaceutical intermediates such as those used to synthesise antibiotics and others according to the substrates on the bioconversion. However, its application within large-scale processes is currently limited by low activity on new reactions, and poor stability at the high temperatures often used during industrial processes. One route to overcome these limitations is to use site-directed mutagenesis or directed evolution to improve the enzyme function and stability. The success of directed evolution relies upon designing a suitable screening method that can directly identify the best mutants from large numbers of variants, with the desired set of attributes. This thesis aims to develop an improved screening platform by adaptation of a previous screening method based upon colorimetric reactions. To assess and quantify TK activity towards the conversion of lithium hydroxypyruvate (Li-HPA) and propionaldehyde (PA) to (3S)-1,3-dihydroxypentan-2-one (HK), over a wide range of substrate concentrations. Moreover, several experiments were performed to establish the best conditions to grow E. coli for TK production, protein extraction methods and quantification of TK. In addition, PCR conditions were established for the development of mutagenic libraries using the MEGAWHOP. Finally, five different truncated TK variants were generated, all of them showed activity using Li-HPA, glycolaldehyde (GA) and PA as substrates. Results obtained in this project set up the basis to generate TK variants with better stability and activity, screen large numbers of variants using the high-throughput platform developed and finally it was shown that truncated versions of TK could keep its activity.

The rate limiting steps of biopharmaceutical process development are clone evaluation and process optimisation. To improve the efficiency of this step, miniature bioreactors are increasingly being used as a tool for high throughput experimentation. At industrial scale, microbial cultivations are usually performed in fed-batch mode to allow for high cell density cost-effective processes; however, many commercially available miniature bioreactors do not have an inbuilt feeding capacity. There are several challenges that need to be addressed to establish high cell density fed-batch cultivation at microscale: attaining high oxygen mass transfer rates, achieving good mixing for the duration of the culture and implementation of an industrially relevant feeding strategy requiring low volume additions. The overall aim of this project was to develop a scale-down fermentation platform suitable for the study and optimisation of high cell density cultures. The first objective of this work was to evaluate options for fed-batch cultures in a commercially available 24-well shaken microbioreactor. To achieve this, two feeding strategies were evaluated using an E. coli strain expressing a domain antibody: in situ feeding by the enzymatic release of glucose from polymeric starch, and direct feeding using a bespoke feed delivery system. In situ feeding was investigated as it is a simple option that does not require a physical method of feed delivery; cellular productivity was enhanced in comparison to batch cultures, however the glucose release was insufficient to sustain high cell density cultures representative of laboratory and pilot scale processes. To enable direct and continuous feed delivery to the microbioreactor a bespoke 3D-printed feeding system was developed that can operate at flow rates of 20μL h-1 and above, and enables up to twelve fed-batch cultures to be run in parallel. E. coli fermentations were performed on complex medium containing glycerol with direct feeding of a 23% w/v glycerol solution initiated at around 18 hours. The second objective of this project was to establish an industrially relevant feeding strategy in the microbioreactor, comparable to a laboratory scale fed-batch process. To this end, the direct feeding strategy was refined in terms of cell growth and product expression; the feed rate and concentration were modified, the DO set point was increased, and a pre-feeding hold period was implemented to allow for consumption of the inhibitory by-products generated in the batch phase. It was found that direct feeding enhanced biomass production by ~70% and product expression by ~2.4 fold in comparison to non-fed cultures. The third objective of this work was to demonstrate the applicability of the new feeding system as a tool for process optimisation experiments. The effect of IPTG concentration and post-induction temperature on product expression was performed using the both the microbioreactor feeding system and the 1L laboratory scale process. The data trends were consistent between scales; product expression was enhanced at a higher post-induction temperature, and IPTG concentration did not affect product expression over the concentration range tested. This demonstrates that the microbioreactor, is predicative of the 1L laboratory scale process terms of sensitivity to change in process conditions The fourth objective of this work was to characterise the microbioreactor in terms of oxygen transfer capability and fluid mixing. To achieve this aim, the volumetric oxygen mass transfer coefficient (kLa) and liquid phase mixing time (tm) of the microbioreactor were determined. The impact of shaking frequency, total gas flow rate and fill volume on oxygen transfer and fluid mixing were investigated and the optimum operating conditions were determined. Within the operating ranges of the miniature bioreactor system, it was found that oxygen transfer was dependant on both shaking frequency and gas flow rate, but was independent of fill volume. The oxygen mass transfer coefficient, kLa increased with both increasing shaking frequency (500-800rpm) and gas flow rate (0.1-20 mL min-1) over the range 3-101h-1; this is at the lower end of the range for conventional stirred tank reactors. It was demonstrated that the miniature bioreactor system is well mixed under the range of operating conditions evaluated. The liquid phase mixing time, tm under non-aerated conditions increased with shaking frequency and decreased with fill volume over the range 0.5-15s. The final objective this project was to demonstrate suitability of the microbioreactor as a scale-down model of an industrial fermentation process. 50L pilot scale, 1L laboratory scale, and 4mL microbioreactor fed-batch fermentations were performed under optimum conditions. The 4mL microbioreactor fed-batch process was shown to better predict the 50L pilot-scale process than the 1L laboratory-scale process based on cell growth, product expression and product quality. This could be explained by mixing and oxygen mass transfer phenomena. At 1L scale, oxygen mass transfer and fluid mixing are most efficient, meaning cell growth and productivity were the highest of the three processes. It appears that the limitations in oxygen mass transfer in the microbioreactor and fluid mixing in the 50L scale vessel, results in a comparable cellular environment, and therefore cell growth, productivity and product quality. In summary, this work has demonstrated the ability to conduct high cell density, fed-batch microbial cultures in parallel, using a shaken miniature bioreactor system. A bespoke, 3D-printed feed delivery system was developed allowing for twelve industrially-relevant microbial fed-batch cultures to be run in parallel. The microbioreactor fed-batch cultures were shown to be predictive of, a 50L pilot scale process in terms of cell growth, productivity and product quality.

The scale-up of protein precipitation processes proves to be a challenging task due to the complexity of the reactions and transport processes involved. A good understanding of the molecular processes underpinning precipitate formation and the reaction kinetics are therefore required in order to devise a scale-up strategy. The doctoral project was first set out to establish micro-mixing as an engineering tool for the scale-up of antibody precipitation from cell culture, and secondly to design a downstream process with the goal of purifying a therapeutic mAb to clinical grade levels. Studies were first conducted in batch and transferred to a continuous process, with the scale-up approach focusing on the latter. Interactions between precipitation conditions and centrifugal recovery were then examined by employing an ultra scale-down (USD) methodology to mimic large-scale centrifugation. The downstream process design was on the basis of integrating precipitation with non-affinity chromatography steps to avoid the cost of affinity chromatography. Precipitate formation in batch and continuous settings was governed by the mixing at the molecular scale, which determined the final particle properties. Based on this, the mean energy dissipation rate for a continuous precipitation process proved an effective scale-up criterion, enabling high process throughputs relative to batch operation. The strength of protein precipitates, as evaluated by exposing particles to turbulent shear in a rotating disc device, was shown to correlate with particle fractal dimensions. Despite excellent precipitate solids removal from the USD methodology, these could not be predicted by disc-stack centrifugation. Differences in hindered settling between the systems were proposed to explain this observation which suggests routes to resolve this scale-up challenge. To provide an integrated DSP solution for therapeutic mAbs processes anion exchange and mixed-mode chromatography steps subsequent to precipitation were designed. Parameter ranges were studied to identify the optimal conditions in maximising antibody yield and HCP removal. Using optimal conditions, precipitation and anion exchange demonstrated an 18-fold removal in HCPs, whilst precipitation and mixed-mode provided a 40-fold removal. For a three step process comprising the sequence precipitation, anion exchange and mixed mode, an overall HCP removal of 260-fold was seen; however such levels remain at least 38-fold higher than the typical specification of a clinical grade product. This therefore necessitates further optimisation in one or more steps.

Microalgae have great potentiality to act as reservoirs of viable bioactive compounds due to the attractive composition of high value-added compounds. Besides, lots of advantages have been reported using microalgae compared to plant-based biomolecules as it does not create food competition and has rapid growth rate. The resistant microalgae cell wall has to be overcome through effective yet simple and rapid techniques in separating desired compounds from the biomass for further applications. The conventional processes for processing biomass in the current industries is inefficient and not feasible to be applied on microalgae, at which a cost- and time-saving technique is in desperate need for the efficient production of biomolecules. This research work reports the development of sonoprocessing-assisted techniques in extracting and purifying biomolecules such as proteins and phycobiliproteins from Chlorella sp.: Chlorella vulgaris FSP-E, Chlorella sorokiniana CY1 and Spirulina sp.: Spirulina platensis, respectively. The proposed techniques in this research work are liquid biphasic system, three phase partitioning and liquid biphasic flotation integrated with the utilization of sonication waves. The protein extraction is performed using liquid biphasic flotation and three phase partitioning assisted with sonication while the phycobiliproteins extraction is conducted using liquid biphasic system and liquid biphasic flotation. A total protein recovery of 80% and 49% of separation efficiency was obtained using sonication-assisted liquid biphasic flotation while 56.57% of protein recovery and 74.59% of separation efficiency were gained using ultrasonication-assisted three phase partitioning. To investigate the scalability of the proposed techniques, the study related to up-scaling of system in extracting the biomolecules are presented and discussed as well. This is an important aspect to determine the feasibility of the proposed technique for large scale and commercializing its usage for industrial production. A scale-up study was performed using ultrasonication-assisted three phase partitioning, from total working volume of 10 mL to 150 mL, obtaining around 57% of protein recovery and 71% of separation efficiency in larger scale of system. On top of that, different types of pre-treatment methods, namely freeze-thawing, homogenisation, microwave and sonication were studied in conjunction with the multiphase separation techniques for phycobiliprotein extraction. Phycobiliproteins was recovered at 94.89% with 6.17 of purification factor using liquid biphasic system while 95.10% of recovery and 5.23 of purification factor were gained using liquid biphasic flotation. The conclusions and future work of this research work are further described in the last chapter of the thesis.

With the advent of industrial biotechnology, we can use microbes to produce myriad valuable compounds with a range of useful applications. These may be drugs, flavourings preservatives or many others. With contemporary synthetic biology approaches we are able to bring together enzymes from a variety of different genomic contexts and build pathways that don't yet exist in nature. This naturally proposes an increase in complexity in pathway design and function, that will require more complex methods to regulate and control. One way to achieve this regulation is using magnetic fields. Physical stimuli like this hold many advantages over the chemical induction methods used contemporarily. Every cell could be reached simultaneously using physical stimuli, promoting metabolic synchronicity. There is also no risk of some cells sequestering chemical inducers, leaving some cells experiencing greater levels of induction, and others none at all. A magnetic stimulus could penetrate an entire culture and stimulate each cell simultaneously and repeatedly. Magnetic nanoparticles when placed in oscillating magnetic fields have their magnetic dipoles forcibly switched to align with oscillations. At high frequency magnetic oscillations, the MNP loses some energy as heat. By conjugating magnetic nanoparticles with temperature sensitive proteins it may be possible to produce stimulus of specific genetic elements with hitherto unseen precision. To achieve this, microbes must be equipped with two things - a magnetic "aerial" module which can receive the magnetic stimulus, and genetically encoded heat-sensitive apparatus that responds to changes in temperature induced by the magnetic field. This thesis has surveyed the potential for Pd nanoparticles to perform the role of a magnetic aerial, and heterologous gene expression with genes from magnetotactic bacterial genomes to produce magnetic material de novo. This strand of work illuminated the potential for a model contrary to how Pd-NPs are made in E. coli. This being that instead of microbial hydrogenases directly nucleating production of a nanoparticle in the active site of the enzyme, hydrogen produced by microbial hydrogenases reacts with soluble Pd(II) precipitating Pd nanoparticles. Work performed in parallel has provided a novel mechanism to produce magnetic material de novo using heterologous expression of the mms6 gene from Magnetospirulum gryphiswaldense. This work has also used contemporary synthetic biology approaches to produce genetic programs that respond to temperature changes, using the genes tlpA, mogR and gmaR, with mogR and gmaR being new vi additions to the pantheon of genetic elements used in synthetic gene circuits. This thesis contains designs, characterisation and optmisation of functional novel genetic circuits which utilise mogR and gmaR in the pMOGMAR-20K plasmid. Using genetic elements characterised in this work in concert, the apparatus to begin producing novel magnetoreceptive mechanisms in E. coli is now available.

Cyanobacteria produce a variety of metabolites with diverse functions and bioactive properties that have potential uses in industrial biotechnology. Metabolomic profiles closely relate to the physiology of an organism and can be used to investigate any alteration in metabolism and production of industrially relevant metabolites. Little work has been conducted on metabolomic time-course profiles within cyanobacteria with the majority of research on targeted metabolite level changes during abiotic stress such as ultraviolet (UV) radiation. In this thesis the metabolite profile analysis of the less well investigated cyanobacterium; Chlorogloeopsis fritschii (C. fritschii) PCC 6912 was undertaken. The main aim was to evaluate changes in low molecular weight metabolite levels during standard growth and UV exposure using gas chromatography-mass spectrometry (GC-MS). The focus was on identifying biologically relevant metabolites with roles in cyanobacterial metabolism associated with growth phases and adaptation to UV stress. Extracts were assessed using in vitro assays for extraction of potential anti-inflammatory and antibacterial activity from the cyanobacteria with complementary metabolomic analysis utilised for identification of potential active metabolites. Results demonstrated a significant reduction in intracellular metabolites involved in carbon and nitrogen metabolism during UV-B exposure with a higher proportion of metabolites increasing in levels during UV-A exposure. Pre-treatment of C. fritschii to low dose UV prior to high dose UV had a lesser effect on metabolism indicating stress tolerance and adaptation to UV stress. Metabolite levels were seen to reflect the changing growth phases over 120 days during standard conditions with overall similarity in metabolite levels between axenic and xenic cultures over 28 days. Finally, extracts showed promising anti-inflammatory and antibacterial activity with identification of potential active metabolites with relevance to personal care products. This is the first GC-MS based metabolite profiling of C. fritschii during standard growth and UV exposure. This builds on the experimental data and knowledge-base already available for cyanobacteria, including C. fritschii, for their potential use in industrial biotechnology.

Bacteria are ubiquitous in the environment and can adhere onto abiotic or biotic surfaces to form biofilms. These three-dimensional (3D) communities of sessile cells are encased in a matrix of extracellular polymeric substances (EPS). Bacterial biofilms can be detrimental to human health, causing infections and diseases. Notably, bacterial biofilms are robust structures and are difficult to treat via traditional antibiotic therapy. The EPS matrix acts as a barrier to agents trying to access the interior of the biofilm, subsequently triggering the development of antibiotic resistance, which has been shown for both Staphylococcus epidermidis and Pseudomonas aeruginosa. Physical strategies, in particular the use of rationally surface design, have gained interests and present us with an effective approach to prevent bacterial adherence and biofilm growth without the requirement for antimicrobials. In this study, we aim to develop biomaterial surfaces via surface modifications that can control bacterial growth, as well as investigate the bacterial-material interactions on these surfaces. We firstly designed and fabricated nano-pillar structured surfaces via electron-beam lithography and polymer moulding technique. The results showed that rod-shaped Pseudomonas aeruginosa can align within the pillars if the space is comparable to the bacteria size; and the extended bacterial growth showed that fibrous network was formed and can help to connect isolated bacterial clusters within the pillars thereby aid in the continuous biofilm growth. Therefore, biomimetic hierarchical structured surfaces were fabricated based on the natural rose-petal via the same method of replicating nano-pillars. The key results showed that hierarchical structures are more effective in delaying biofilm growth of Staphylococcus epidermidis and Pseudomonas aeruginosa compared to the unitary structure. The nano-folds across the hemispherical micro-papillae restrict initial attachment of bacterial cells and delay the direct contacts of cells via cell alignment, and the hemispherical micro-papillae arrays isolate bacterial clusters and inhibit the formation of a fibrous network. Finally, we made two kinds of slippery surfaces via infusing the silicone oil. These slippery surfaces showed superior anti-wetting properties and exhibited excellent "self-cleaning" effects. Additionally, either slippery surface can prevent around 90% of bacterial biofilm growth of Staphylococcus epidermidis and Pseudomonas aeruginosa after 6 days, as compared with the unmodified control PDMS surfaces. This study detailed investigated the different bacterial responses when making contacts with artificial biomaterial surfaces. Multiply imaging techniques such as fluorescent microscopy, scanning electron microscopy and wettability analysis were adopted in this study, will instruct researchers to reveal the physic-chemical interactions of bacteria and materials. Particularly, the anti-biofilm surface design in this study will give insights to develop a more effective way for controlling robust biofilm growth, thereby paving a high way for preventing infection or fouling problems in either medical or industry contexts.

The traditional “one-size-fits-all” mass production model commonly used in biologics manufacturing is insufficient to accommodate the advent of personalised medicines and the necessity of on-demand production. The design and validation of novel manufacturing platforms is necessary for on-demand and personalised medicines production. To address this, an E. coli-based cell-free protein synthesis (CFPS) manufacturing platform was developed and applied to self-assembling particles for vaccine and gene therapy production. This in-house CFPS system consistently produces over 400 μg/mL superfolder green fluorescent protein (sfGFP) in 4 hours. A three-step process development strategy that can be completed in under 48 hours was designed and then validated with two products. Using this strategy, sfGFP production was improved by 38% and hepatitis B core antigen (HBcAg) production by 190%. The CFPS system was then used to produce self-assembling products and iterate upon their construct design. Two tandem-core HBcAg virus-like particles (VLPs), called VLP3 and VLP1, that have been modified to display influenza antigens as universal influenza vaccine candidates were produced and assembled. Using a minimal plasmid backbone designed for CFPS improved titres by 1.8 times over the original VLP1 construct and 1.4 times over the original VLP3 construct. Titres were further increased to over 100 μg/mL for VLP3 when the linkers around the influenza inserts were shortened, although improvements in particle quality were not seen. Further, any constructs with the C-terminal arginine-rich region removed resulted in asymmetric particles of poor quality. Additionally, the three capsid proteins of the adeno-associated virus were produced, which have been shown to form particles in vitro and can be used for the delivery of genetic material, potentially as a gene therapy treatment. Taken together this shows the potential for CFPS systems in the on-demand manufacture of self-assembling vaccine and gene therapy products.

There are strong incentives for a transition of the current fossil fuel-based economy towards a bio-based one, stemming from the unsustainable nature of fossil fuels, climate change and difficult disposal of non-biodegradable products. As a result, there is increasing interest in waste valorisation, especially of agricultural waste, as this could provide a low-cost feedstock with potential for upgrading to valuable products, and/or contains high-value naturally occurring bioactive compounds. Waste valorisation could also address the problems of purposefully grown biomass feedstocks and the associated negative socio-economic impacts, such as high food prices and deforestation. Additionally, integration of multiple product valorisation processes via a biorefining scheme could lead to more viable economics from product diversification, and may also provide additional economic and environmental benefits via process integration and waste reduction. Rapeseed meal (RSM) is a potential candidate for valorisation due to its high content of potentially valuable phenolics, proteins and polysaccharides. This thesis details the progress made towards RSM valorisation within an integrated biorefinery context, with particular focus on the more sustainable aspects of the scheme, specifically green solvent extraction of RSM phenolics, and the production of biodegradable polymers from RSM polysaccharides. This thesis is presented in a journal format. The experimental phase of the PhD is roughly separated into four stages as follows: Stage I: preliminary work; Stage II: assessment of phenolic extraction and viability of integrated processing; Stage III: optimisation of protein extraction and acid hydrolysis, and in depth investigation into the use of RSM-derived acid hydrolysate for PHA synthesis; Stage IV: Optimisation of enzymatic hydrolysis of untreated and pre-treated RSM for use in PHA synthesis. The work conducted in Stages II, III and IV have been peer-reviewed and published in the Journal of Cleaner production (Wongsirichot et al., 2019), Waste Management (Wongsirichot et al., 2020b) and ACS Sustainable Chemistry and Engineering (Wongsirichot et al., 2020a), respectively. RSM phenolics are of interest due to their antioxidant activities, which could lead to high-value applications such as health supplements in the food industry, or as preservative in the chemical industry. Additionally, the antimicrobial activity of RSM phenolics means that their removal could lead to improved fermentation of other residual fractions. This PhD investigated the extraction of RSM phenolics using novel green deep eutectic solvents (DES). Thermodynamic simulations using the Conductor like Screening Model for Real Solvents (COSMO-RS) shown that DES extractions had greater hydrogen-bonding interaction between the sinapic acid and the DES compared to methanol which has been traditionally used in RSM phenolic extraction. This results in greater yields with DES compared to the methanol. Although this was subjected to the influence of mass transfer, leading to lower yields in the highly viscous glucose-based DES. The highest sinapic acid yield at 91.5 % was achieved using aqueous choline chloride: glycerol (1:1), which outperformed aqueous methanol (68 % yield) at the same conditions. RSM proteins are of interest to the food industry, due to their abundance, and the fact that RSM amino acids are complementary to the amino acid profiles recommended for human consumption. Beyond this, the results found in this thesis demonstrate that the extraction of RSM proteins was crucial for expanding the use of the residual RSM polysaccharides as a fermentation feedstock; as high residual proteins in the polysaccharide fraction hinder its use in nitrogen-limited applications, particularly the synthesis of polyhydroxyalkanoates (PHA) biopolymers. During the PhD, optimisation studies of protein extraction, acid hydrolysis and enzymatic hydrolysis were conducted to determine the most suitable condition for protein extraction and fermentation media synthesis. Maximum protein yield from alkali extraction was obtained (68.8 %) at 60 °C and 100 minutes. The use of consecutive extraction allowed for the creation of post-extraction RSM with high carbon and low nitrogen content. It was also demonstrated that without an integrated scheme to first extract the protein, neither acid or enzymatic hydrolysis of the RSM could produce media with a sufficiently high glucose and low nitrogen concentration for significant PHA accumulation. Both acid and enzymatic hydrolysis of the post-protein extraction RSM were assessed. Results demonstrated the difficulty in producing the high C:N ratio required for PHA synthesis with traditional diluted acid hydrolysis, especially after scale-up. An alternative scheme which uses oxygen-limitation lead to increased PHA accumulation compared to the fermentation with no nutrient limitation. However, the final PHA accumulation in RSM media could still be further improved. Finally, using a combination of post protein-extraction RSM and optimised enzymatic hydrolysis, suitable concentration of glucose and nitrogen in the media was produced and successful PHA accumulation achieved, at both shake flask and bioreactor scales with PHA accumulations of 9.34 and 7.11 % cell dried weight, respectively. During the PhD project, more traditionally studied aspects of RSM valorisation were greatly improved upon through optimisation and scale up of the protein extraction, as well as the application of green solvents and scale up of phenolic extraction. The less explored RSM polysaccharides were successfully upgraded via fermentation to produce sustainable biopolymers. Most importantly, the integrated biorefining scheme combining the valorisation of all three fractions is demonstrated to not only be viable but essential for expanding the possible scope of holistic RSM valorisation. This thesis not only provides a solid foundation for future development and implementation of RSM-based biorefining, but the insights gained during the PhD could also be applied to the valorisation of other wastes containing multiple valorisable fractions.

This thesis concentrates on one of the least studied mechanical properties of cuticle - toughness. In particular, it investigates how cuticle microstructure impacts crack propagation, and the toughening mechanisms cuticle possesses to protect the vulnerable soft tissues contained within the exoskeleton. I use time-lapse 3D nCT imaging with in situ mechanical tests to investigate toughness and damage progression in arthropod cuticle. In addition, I develop new methodologies of standardised sample preparation and hydration preservation to perform quantitative analysis of fresh locust tibiae and beetle elytra. The results obtained in this thesis have shown that it is possible with these techniques to analyse toughening in fresh and dry cuticles, by qualitative visualisation of the interaction between microstructure and crack propagation and quantitative measurement of toughness values from standardised test samples. It was shown that microstructure is responsible for the numerous extrinsic toughening mechanisms present in cuticle, of which many were previously unreported, and that hydration is responsible for improving the effectiveness and frequency of their occurrence. Furthermore, it was found that the exocuticle contributes little to toughness and that the difference in angle between the crack direction and the fibre orientations of a lamina directly affected the toughening capability of that lamina. To summarise, this thesis displays how a combined approach using state-of-the-art 3D imaging, in situ mechanical testing, sample preparation and hydration preservation techniques can provide us with new insights in how arthropod cuticle shape, microstructure, composition and mechanical properties interact.

Streptomyces clavuligerus is the producer of clavulanic acid; a β-lactamase inhibitor that isused in combination with the antibiotic amoxicillin to treat many bacterial infections. The unique genome of S. clavuligerus includes a chromosome, and four linear plasmids pSCL1, pSCL2, pSCL3 and the megaplasmid pSCL4, which is the largest linear plasmid ever identified at 1.8 Mb in size. In order to study the genomic architecture of S. clavuligerus strains, we established an optimised method of pulsed-field gel electrophoresis, which allowed visualisation of the three giant linear plasmids pSCL2, pSCL3 and pSCL4, as well as chromosomal fragments in order to establish the physical map of the S. clavuligerus genome. Furthermore, to establish a foundation on which the genomic analyses of industrial strains of this species could be carried out, we closed the genome sequence of the S. clavuligerus type strain DSM 738, using a combination of short-read Illumina sequencing and long-read PacBio technology. Additionally, the telomeres of the five replicons were purified by a self ligation method, and identification of the sequences confirmed that the five replicons carry archetypal telomeres, with the chromosome and megaplasmid sharing identical telomeric sequences. Streptomycetes telomeres are maintained by the terminal proteins Tap and Tpg, however, while copies of the genes encoding these proteins were identified in pSCL4, pSCL3 and pSCL2, the chromosome carries none, making these the only known essential elements missing from the chromosome. Moreover, in order to investigate the essentiality of the megaplasmid in S. clavuligerus, we cured pSCL4 by targeting parB with CRISPR/Cas9. This caused deletion of the chromosomal ends and circularisation, confirming that pSCL4 Tap-Tpg are required for maintaining the chromosome telomeres, and cannot be complemented by the proteins encoded on the other two plasmids. In addition, pSCL4-free strains were confirmed to be non-sporulating as well as presenting a phenotype of short and thin hyphae, in addition to higher branching frequency. Some mutant strains also exhibited reduced clavulanic acid production which confirmed that the loss of pSCL4 has important effects onmorphological development and secondary metabolism of S. clavuligerus. Overall, with this study we have closed the genome sequence of S. clavuligerus type strain and optimised a method for rapid screening of the S. clavuligerus mutant strains that will greatly benefit the industry for development of production strains. Additionally, we have confirmed that pSCL4 is a dispensable plasmid, but is yet necessary for maintaining optimum fitness of this bacterium.

The ever-growing need to characterise manufacturability is a key driver for a therapeutic’s success. Early information can reduce costs massively and speed up a product’s delivery to market. Current monoclonal antibody (mAb) methods are limited by the amount of material required and the time it takes to gain valuable information. The formation of aggregates poses a significant hindrance to biopharmaceutical companies. This project aims to develop a novel assay sequence to forecast the manufacturability of a mAb by predicting its propensity to aggregate from low populations. The assay will form part of a biophysical screening process in order to de-risk lead candidates. A panel of six mAb candidates were characterised using currently employed techniques: SEC, DLS and AUC. The limitations were evaluated for each technique. An extensive literature review was conducted to select novel methods to explore as potential replacements for the current methods. After ranking each potential method based on several criteria, fluorescence-based methods were chosen to investigate further. Several fluorescence techniques were evaluated: fluorescence intensity (FLI), red-edge excitation shift (REES) and time-correlated single photon counting (TCSPC). After evaluating each technique, a 14-day time-course study, where samples were isothermally held at elevated temperatures, was carried out to mimic the current lead panel screening. Spearman’s ranking was used to compare the novel techniques to SEC. The earliest time-point at which each technique could detect aggregation of mAbs, formed the basis for a 4-hour time-course study. REES and TCSPC were successful in detecting the aggregation propensity of mAbs. Each technique’s ability to detect concentration-dependent aggregation was also evaluated. TCSPC was successful in identifying the aggregation-prone mAbs from their time-decays, and the impact of different buffers. Finally, a novel assay, using a combination of TCSPC (emission at 330 nm and 395 nm) and REES, was proposed as a method to obtain fast information (<1 minute) on mAbs from low volumes (< 20 μL).

A deeper understanding of the nanoscale and mesoscale structure of chromatographic adsorbents and the distribution of proteins within the media, is critical to a mechanistic understanding of separation processes using these materials. Characterisation of the media’s architecture at this scale and protein adsorption within, is challenging using conventional techniques. In this study, a novel resin characterisation technique that enables in-situ measurement of the structure of the adsorbed protein layer within the resin, under typical chromatographic conditions was examined. A quartz flow-through cell was designed and fabricated for use with Small-Angle Neutron Scattering, in order to measure the structures of a silica based protein A chromatography resin during the monoclonal antibody sorption process. We were able to examine the pore-to-pore ( ̃133nm) and pore-size ( ̃63 nm) correlations of the resin and the in-plane adsorbed antibody molecules ( ̃4.2nm) correlation at different protein loadings and washing buffers, in real time using a contrast matching approach. The effect of different washing buffers was also investigated. When 0.03M sodium phosphate with 1M urea and 10% isopropanol buffer, pH8, was introduced, it disrupted the system’s order by causing partial unfolding of the adsorbed antibody, as evidenced by a loss of the in-plane protein correlation. Neutron Reflectivity experiments were undertaken on a planar surface to enhance the understanding of how adsorption processes might impact the stability of the antibody molecules. A 56Å antibody size and 13.7% increase in layer thickness when urea was introduced was observed. The effect of temperature and antibody incubation times on the chromatography system were also explored. The methodology developed here offers new ways to investigate the nanoscale structure and ligand immobilisation within chromatography resins. Which allows for a deeper understanding of the in-situ behaviour of adsorbed proteins within the media under different mobile phase conditions within a sample environment replicating that of a chromatography column.

Recent improvements in volumetric antibody productivity (often in excess of 5 g/L) have been achieved by advances in cell lines and upstream processing, but often lead to harvest material becoming more difficult to recover. These intensified upstream operations require a renewed prioritisation of the integration of upstream and downstream process development to ensure product purification issues are taken into consideration, to avoid extensive and expensive clearance strategies downstream. Here, it was demonstrated that changes to upstream process parameters at the bioreactor stage of monoclonal antibody production affect product quantity and quality. Culture pH, temperature and seed density setpoints leading to high titre are commonly also linked to higher post-protein A HCP levels, reduced monomer percentages and increased percentages of undesirable glycan structures. To predict post-protein A product quality, several potential indicators that can be measured in harvest material (prior to using expensive purification resources) were explored, including culture viability and osmolality, revealing unexpectedly that culture viability could not be used for such a purpose, but that osmolality has the potential to be used as a product quality indicator. The impact of culture duration on product quality was also investigated and it was shown that as cultivation progressed and antibody titre increased, product quality declined, in one case due to post-protein A HCP levels increasing by 75% from day 14 to day 17 of culture. HCP identification by mass spectrometry was applied to this system to provide insights into cellular behaviour and HCP carryover during protein A purification. It showed increases in several classes of post-protein A HCPs (e.g. stress response proteins) as the culture progressed, particularly on days 15 and 17 of culture which were associated with significant increases in total HCP levels. This provides a new level of insight into HCPs that are retained during mAb purification which may be used to aide process development strategies.

The challenge of relinquishing the use of fossil fuels, and the move to a more sustainable energy model, depends on our ability to effectively and economically capture and store renewable forms of energy. Electro- and photocatalytic techniques present an attractive route towards the respective conversion of renewable electricity and direct sunlight into chemical energy. Carbon nitride (CNx), a polymeric semiconductor, has recently received much attention, stemming from its successful application as a heterogeneous photocatalyst for solar water splitting and visible light mediated organic transformations, in addition to its nontoxic properties and facile, low-cost synthesis. However, these reactions have mostly been confined to batch reactors, and the advantages offered by continuous flow chemistry vis-à-vis improved light transmission, compatibility with multiphasic systems, and catalyst recyclability, have not been fully explored for this class of materials. In the first part of this thesis, different design strategies are proposed and assembled, in order to carry out CNx-based photocatalysis under continuous flow. An investigation into the aerobic oxidation of a variety of organic substrates was made, and a comparison between the performance of batch and flow CNx photoreactors was conducted. Design of the initial flow prototype involved some computational fluid dynamics analysis and was based on a thin channel device concept. The next iteration was centred around the use of a packed column photoreactor and was tailored towards triphasic flow chemistry. The second part of the thesis is focused on the development of an anodic system for the electrochemical oxidation of alcohol substrates. It is incentivised by the need to replace the oxygen evolution reaction (OER) within a conventional CO2 reduction electrolyser, on account of the high energy penalty of the OER and the low commercial value of O2. A novel hybrid anode was fabricated, featuring a silatrane-modified TEMPO catalyst which was covalently immobilised on a mesoporous indium tin oxide scaffold. The performance of the assembled anode was first optimised towards the oxidation of representative biomass substrates, and then integrated with a precious-metal-free CO2 reduction electrocatalyst, for coupled alcohol oxidation and CO2-to-syngas conversion. The system, comprised only of earth-abundant materials, demonstrates the ability to produce chemical feedstocks from sustainable resources, such as biomass-derived alcohols, CO2, and renewable electricity.

The mid-infrared (MIR) spectral range is of special interest for establishing optical chemical sensor technologies by allowing specific molecular identification and quantification, whether the sample is in a liquid, gas or solid form, in addition to providing highly sensitive, rapid, reagent-free and non-destructive detection. This thesis explores four different liquid- and gas- sensing applications and methods using MIR spectroscopy by integrating it with other technologies, such as microfluidics and fibre-optics. Firstly, fibre-optic integrated microfluidic devices were developed and tested for con- tinuous fluid monitoring. These showed good sensing capabilities for online, continuous and real-time liquid sensing in hard-to-reach locations. Next, this thesis presents the establishment and clinical testing of a novel method for continuous monitoring of the brain chemistry of traumatically brain-injured patients by MIR transmission spectroscopy. Here, the outlet of a cerebral microdialysis catheter is cou- pled to a micro flow-cell and the flowing microdialysate is continuously analysed. Clinical studies were carried out and showed the capability of this system for performing continuous patient monitoring over several hours. With further optimisation, the implementation of this system could lead to improved patient outcome. This thesis also presents a novel method and system based on MIR fibre-optic evanescent- wave spectroscopy, which enables enhanced detection of volatile organic compounds (VOCs). Here, a nanoporous silicon cladding was used to reversibly concentrate molecules close to the fibre surface, thus enhancing VOC detection. A significant increase in sen- sitivity was seen compared to that of an uncoated fibre and successful detection of three different VOCs, both independently and in binary mixtures, was achieved. Finally, this thesis introduces a simple and relatively low-cost fibre-optic sensor for in-line, real-time bioprocess monitoring. The sensor was successfully able to monitor varying concentrations of product (sophorolipids) in fermentation broth and was able to distinguish between the two types of generated product (acidic and lactonic sophorolipids). The work presented in this thesis showed that MIR-integrated sensors have great potential to provide novel and/or enhanced sensing solutions in a wide range of applications, including medical, industrial and environmental.

Cofactor NADH and NADPH regeneration is a crucial topic in the area of biotechnology, as many redox enzymes such as alcohol dehydrogenases, imine reductases, and P450 monooxgenases require cofactor to deliver their redox equivalent. However, as the cofactors are particularly expensive, it is not economically feasible to supply stoichiometric cofactor when these enzymes are used in chemical synthesis. Instead a number of cofactor regeneration systems have been developed to tackle this problem. Traditional regeneration systems for the reduced cofactors suffers from drawbacks, as most of them require carbon based sacrificial co-substrate reductant and produce a by-product which is likely to be burnt as waste, hindering the downstream product purification process and lowering atom economy. The Vincent group has successfully developed a modular H2 driven NADH regeneration system, in which hydrogenase, NAD+-reductase, and the cofactor dependent enzyme are co-immobilised on carbon particles. However, several enzymes require exclusively NADPH as their redox equivalent. Therefore, this thesis focuses on the development of a new H2 driven NADPH regeneration system and coupling it with NADPH dependent enzymes to demonstrate its feasibility. To achieve a new NADPH regeneration, the NAD+-reductase was targeted and several variants are produced. The most robust variant was then selected to be incorporated into the new NADPH regeneration system. The new system was then coupled with NADPH dependent enzymes imine reductase and P450 monooxygenase to demonstrate its feasibility. Another approach to achieve NADPH regeneration is to introduce a fourth enzyme, soluble transhydrogenase into the NADH regeneration system. The soluble transhyrogenase was produced, isolated and partly characterised, but not yet incorporated to the regeneration system. This thesis also discusses the further development for a more robust and easy accessible H2 driven NADH regeneration system, as the NAD+-reductase currently used is not easy to produce, and not very efficient. A promising candidate to replace the NAD+-reductase currently used in the system is the NAD+-reductase part of respiratory Complex I subunit NuoF. The subunit was successfully isolated and characterised. However, it suffers from stability problem. Therefore, it is not ideal yet to be incorporated into the NADH regeneration system.

Active matter is the study of many-body systems driven out of equilibrium at a local level. Typical examples are found in biology and span many length scales, such as flocks of birds or tissues of cells. This thesis focuses on non-momentum conserving (dry), polar systems, whose hydrodynamics are generically described by the Toner-Tu equations. These systems exhibit a variety of emergent behaviour, such as collective motion and phase separation, which often only emerge at high densities. By adapting the standard lattice Boltzmann method for fluid mechanics, we develop a new method for simulating dry, polar active fluids. In particular this method is easy to implement and effective at high densities. Through a Chapman-Enskog style expansion, we confirm that the corresponding macroscopic equations are the Toner-Tu equations, and connect the system parameters with the coefficients of the equations. We demonstrate the functionality and adaptability of our method by recreating two different phenomena: motility-induced phase separation and collective motion. Furthermore, by incorporating contact inhibition of locomotion effects into the collective motion model, we uncover two new first order phase transitions and a potentially new critical transition. We interpret these transitions through a stability analysis. In addition, we perform a stability analysis on an open interface of a fluid that obeys an incompressible version of the Toner-Tu equations. We find that collective motion stabilises the interface, but the interface is unstable when starting from a stationary state. This has implications for both wound healing and the results of our simulations.

Aspergillus fumigatus can produce in vitro an extracellular hydrophobic matrix with biofilm features under static growth conditions. Microbial quorum sensing (QS) system regulates genetic competence and biofilm formation. Notwithstanding, triclosan, as a synthetic antimicrobial, disrupts QS signal in some bacteria, yeasts, and dermatophytes through blocking the biosynthesis of amino acid and fatty acids in the microbes. In this study, triclosan quorum quenching role as well as the effects of microbial quorum quenchers, furanone, farnesol and tyrosol were investigated against A. fumigatus for the first time. Amphotericin B (AMB), a common antifungal agent used against A. fumigatus infection, carries adverse effects against human health. So, triclosan’s antifungal effect in combination with AMB was also studied. Microbial cells’ detrimental attachment and biofilm formation on the indwelling medical implants, healthcare and hospital facilities, prompts interests to introduce feasible and cost-effective methods for eliminating/reducing microbial attachment to the surfaces. Using water contact angle (WCA) measurements, hydrophobicity of some hydrophilic surfaces (glass, acrylic, HDPE, nylon 6 and UPVC), and hydrophobic surfaces (PTFE and silicone) was measured following coating them with the mentioned antimicrobial agents. A. fumigatus conidia were exposed to the agents and the subsequent changes in the conidial viability, biofilm biomass production, total and EPS-related protein/ polysaccharide/ and nucleic acid were studied. The effect of AMB was weaker than the other agents on mitigating the biofilm formation. eDNA release was observed from A. fumigatus mycelia treated with triclosan, tyrosol and farnesol as compared with untreated control group. This eDNA release appeared to be due to necrosis occurrence in the treated samples verified by gel electrophoresis and protein quantity analysis. As a novel finding, the necrosis induced by triclosan followed by apoptosis induced by AMB resulted in a synergistic interaction to reduce the conidia viability even at the sub-MIC doses of triclosan and AMB. Confocal microscopy of the A. fumigatus exposed to triclosan-AMB combination confirmed the synergistic interaction between them against the fungus biofilm. Assaying for hydrophobicity of the conidia cell wall revealed a change from hydrophobic to hydrophilic property. ags3 down-regulation approved with real-time PCR assay showed triclosan’s quorum sensing inhibitory role against A. fumigatus. This was through inhibiting conidia aggregation, and hence prohibiting initiation of quorum sensing signalling pathway. Through SDS-PAGE assay it was established that the conidia cell wall hydrophobic rodlet layer was absent in farnesol-treated sample. This study revealed some of the physio-chemical properties that are involved in A. fumigatus conidia attachment to the surfaces. This work for the first time established that hydrophobic surfaces, PTFE and silicone, coated with farnesol showed hydrophilicity, while AMB changed UPVC surface charge from hydrophilic to hydrophobic. Taken together, besides influencing the conidia cell wall hydrophobicity, farnesol and AMB can also be used to coat the surfaces in clinical healthcare settings to diminish hydrophobic/hydrophilic microbial cells attachment to the surfaces.

The aim of the studies described in this thesis was to develop a set of single shot biosensors, for low concentration measures (less the 1 mM) of glucose, sucrose and fructose. In addition, the development of a user friendly potentiostat instrumental system to display and save the data acquired, via the use of an android tablet. Chapter one describes an introduction and background to screen printed biosensors. Including the electrochemical techniques used and a basic explanation of screen printing. A brief discussion of the sugars, that will be measured in this thesis. Chapter two describes the development of a simple, low cost chronoamperometric assay, for the measurement of fructose, using a graphite-nanoparticle modified screen-printed electrode (SPCE-G-COOH). Cyclic voltammetry showed that the response of the SPCE-G-COOH enhanced the sensitivity and precision, towards the enzymatically generated ferrocyanide species, over a plain SPCE; therefore the former was employed in subsequent studies. Calibration studies were carried out using chronoamperometry and tested using a commercial fruit juice. Chapter three continues from chapter two, using studies to explore the conversion of the fructose bioassay into a fructose biosensor. More analysis of the interference of ascorbic acid (vitamin C) and a method of removing the interference signals via linear subtraction, using a secondary base electrode. Chapter four explains measuring glucose, using a unique mediator, and measuring the amperometric signal, in the reduction phase. The chapter describes the use of multiple enzymes and the optimisation of them. This section then demonstrates the modification of the glucose biosensor to a sucrose biosensor. This was achieved by the inclusion of an invertase enzyme. The studies of the glucose and sucrose biosensor were then compared against a commercially available system. Chapter five investigates a possible potentiostat system, linked to an android based tablet. The intention was to produce a simple and user-friendly system to measure and record the concentrations found. Chapter six comprises future work to be carried out and ideas. These suggestions are to improve the system allowing it to be more user-friendly and to remove some of the human errors that could be introduced into the system.

Alzheimer's disease (AD) and Parkinson's diseases (PD) are neurodegenerative diseases that affect ~ 60 million people worldwide. Both diseases are linked to the misfolding of proteins from their native conformational state into β-sheeted amyloid fibrils. In AD the implicated proteins are amyloid-β and tau, and for PD the implicated protein is α-synuclein (aSyn). The motivation for this work is to develop and use physical techniques to better understand the role of amyloid proteins in neurodegenerative diseases. Two techniques used in amyloid research are fluorescence microscopy, to map the protein location and aggregation state, and electrophysiology, to examine the effect of the proteins on neurons. To enable these techniques to be combined, a transparent graphene microelectrode array (MEA) was designed, fabricated and characterised. The active electrode site was graphene since it is electrically conductive, optically transparent and biocompatible. The graphene MEA was characterised using Raman spectroscopy to check the graphene quality, and electrochemical impedance spectroscopy (EIS) to probe the electrode-electrolyte interface. The graphene MEAs enabled voltage trace recordings from cultured neurons to be combined with widefield, confocal fluorescence and fluorescence lifetime imaging microscopy (FLIM). Combining fluorescence imaging and electrophysiology will allow amyloid aggregation to be correlated with neuronal firing patterns. Another physical technique used was Fourier transform infrared spectroscopy (FTIR). A script was written to estimate the protein secondary structure content, and used to investigate polymorphism in the monomeric amyloid protein aSyn.

Every year millions of lives are saved by vaccination and millions more could be saved with more efficient vaccination coverage. Thermostability of vaccines is one of the major reasons why many children who need vaccines most do not get all the shots they need. Vaccines need to be kept below 8°C throughout manufacturing, delivering and shipping which requires a cold-chain. This involves carrying vaccines in a cool box for days even weeks to small villages or islands with no electricity or any other infrastructure. One of the grand challenges in global health is to produce vaccines that do not require refrigeration. Here, a biologically inspired vaccine stabilisation method is proposed. The method is based on the fact that proteins entrapped in the fossil avian eggshell crystals are well preserved up to thousands of years in Africa where high-temperature vaccine stability is needed. The persistence of the ancient intra-crystalline proteins suggests that protein incorporation into the inorganic host could retard protein degradation for long periods of time without refrigeration. In the present work, biomimetic protein incorporation in calcium carbonate was investigated to produce intra-crystalline heat-stable vaccines. Intra-crystalline protein persistence in the fossil record has been shown for ostrich eggshell proteins and the most durable proteins are known to belong to the C-type lectin-like protein family. It is thought that at least one C-type lectin-like protein is found in the eggshells of every species. The most well known C-type lectin-like protein is the OC-17 from chicken eggshell. OC-17 and other C-type lectins have the highest concentration in eggshells compared to other eggshell proteins which suggests that they could lead understanding of efficient protein incorporation in calcium carbonate crystals. Apart from ostrich and chicken, C-type lectins from other species have not been studied in detail. For this reason, eggshells from 14 species were studied in the present work. The aim of the investigation is to quantify the effect of the organic matter on the eggshells which could allow inferring protein incorporation efficiency of different species. The mechanical properties of the eggshells were studied with nanoindentation. This allows probing the differences in the elastic modulus and hardness of eggshells which are affected by the intra-crystalline protein content. It was found that the elastic modulus differs among species, which is lowest at around 10 GPa for Bali myna and highest at around 60 GPa for ostrich. Similarly, the hardness changes from around 1 GPa for Bali myna to around 3 GPa for rhea. The chemical analysis was conducted with IR spectroscopy. The deviations in the absorption peaks of eggshells compared to pure calcium carbonate allows probing the extent of the amorphous structure of the eggshells. In addition, the comparison of the full-width at half maximum values of vibration modes in each spectrum provides information of the crystal order of the eggshells. Comparison of mechanical and chemical analyses of different species offers insights on the protein content of different eggshells which could lead identification of the most efficient C-type lectins in terms of protein incorporation ability. Because of the importance of eggshell C-type lectins for the vaccine preservation method proposed in the present work, the OC-17 was studied here in detail. First, a purification method was developed to extract OC-17 from chicken eggshell. Liquid chromatography was used to extract OC-17, OC-23 and lysozyme. Then, lysozyme was removed from solution using a lysozyme binding protein. In addition, OC-17 was cloned to synthesize a recombinant C-type lectin-like eggshell protein in bacteria for the first time. Growth conditions were optimized and OC-17 expression was verified with anti-Histag antibody. Lastly, the model protein incorporation into synthetic calcium carbonate was studied. Because the incorporated and reconstituted protein structure is of utmost importance, the secondary and tertiary protein structures of incorporated proteins were analyzed with circular dichroism and intrinsic tryptophan fluorescence spectroscopy. The effect of both the reconstitution and incorporation were studied independently as intra-crystalline proteins could denature during the interaction with the reconstitution buffer or during the incorporation process. It was shown that the model proteins BSA, lysozyme and diphtheria anti-toxin could be reconstituted using EDTA without structural change. The incorporation was found efficient for BSA only with an efficiency of %84 in terms of total amount of protein incorporated into the crystals. The secondary structure of BSA was shown to be stable during reconstitution and incorporation. A structural change in the tertiary structure was observed in BSA. Possible improvements to incorporate a target protein as a fusion construct using the OC-17 or using the ostrich intra-crystalline peptides as ’incorporation tags’ were discussed in order to move towards a real-world application of biomimetic vaccine stabilisation.

Synthetic biology applies engineering principles to make progress in the study of complex biological phenomena. The aim is to develop understanding through the praxis of construction and design. The computational branch of this endeavour explicitly brings the tools of abstraction and modularity to bear. This thesis pursues two distinct lines of inquiry concerning the application of computational tools in the setting of synthetic biology. One thread traces a narrative through multi-paradigm computational simulations, interpretation of results, and quantification of biological order. The other develops computational infrastructure for describing, simulating and discovering, synthetic genetic circuits. The emergence of structure in biological organisms, morphogenesis, is critically important for understanding both normal and pathological development of tissues. Here, we focus on epithelial tissues because models of two dimensional cellular monolayers are computationally tractable. We use a vertex model that consists of a potential energy minimisation process interwoven with topological changes in the graph structure of the tissue. To make this interweaving precise, we define a language for propagators from which an unambiguous description of the simulation methodology can be constructed. The vertex model is then used to reproduce laboratory results of patterning in engineered mammalian cells. The assertion that the claim of reproduction is justified is based on a novel measure of structure on coloured graphs which we call path entropy. This measure is then extended to the setting of continuous regions and used to quantify the development of structure in house mouse (Mus musculus) embryos using three dimensional segmented anatomical models. While it is recognised that DNA can be considered a powerful computational environment, it is far from obvious how to program with nucleic acids. Using rule-based modelling of modular biological parts, we develop a method for discovering synthetic genetic programs that meet a specification provided by the user. This method rests on the concept of annotation as applied to rule-based programs. We begin with annotating rules and proceed to generating entire rule-based programs from annotations themselves. Building on those tools we describe an evolutionary algorithm for discovering genetic circuits from specifications provided in terms of probability distributions. This strategy provides a dual benefit: using stochastic simulation captures circuit behaviour at low copy numbers as well as complex properties such as oscillations, and using standard biological parts produces results that are implementable in the laboratory.

Four types of complex polymeric nanoparticles have been developed and investigated for their use in biomedical applications such as drug delivery, pH sensing and self-propulsion. Each type of nanoparticle was synthesised using a combination of controlled radical polymerisation, polymer self-assembly, polymer phase separation and gold nanoparticle preparation and conjugation. Polymers were characterised using gel permeation chromatography (GPC) and nuclear magnetic resonance (NMR) spectroscopy and synthesis was optimised to produce monomodal, low dispersity polymers. Assembled nanoparticles were characterised using a range of advanced techniques, including dynamic light scattering (DLS) and transmission electron microscopy (TEM). Patchy micelles were prepared from the aqueous assembly of pH responsive (poly (oligo(ethylene glycol)methyl ether methacrylate)-b-(2-(diisopropyl amino)ethyl methacrylate)-b-(2-(methacryloyloxy)ethyl phosphorylcholine) (POEGMA-PDPA-PMPC) ABC block copolymers and characterised using selective phosphotungstic acid staining, which revealed phase separation in the corona of the micelles. The micelles were confirmed to be biocompatible and exhibited different drug encapsulation and release properties compared to homogenous micelles. Triblock copolymer coated spherical gold nanoparticles were prepared and found to exhibit low protein fouling, good biocompatibility and triggered doxorubicin release. Cell studies revealed they were more effective at killing cancer cells than doxorubicin controls. Triblock copolymer coated anisotropic gold nanoparticles (nanotriangles and nanohelices), were prepared successfully despite challenges relating to the instability of the structures, and the use of these nanoparticles for pH sensing was investigated. Phase separation of various homopolymers on the surface of gold nanoparticles was investigated and characterised with selectively stained TEM. Enzymes were successfully conjugated and self-diffusiophoresis behaviour of these nanoparticles was investigated.

Stem cell balance of proliferation, differentiation and self-renewal, is regulated by the microenvironment in which they reside, termed the stem cell niche (Schofield, 1978). Niche microenvironments provide physical and functional regulatory cues that control fundamental cell intrinsic and extrinsic mechanisms. In adults, the process of haematopoiesis is sustained by a population of haematopoietic stem cells (HSCs) that are found primarily in the bone marrow (BM) (Jagannathan-Bogdan and Zon, 2013). The BM niche also houses populations of mesenchymal stromal and perivascular cells (MSPCs), that are themselves regulated by the niche, and are fundamental cellular constituents in HSC regulation (Pinho and Frenette, 2019). Both HSCs and MSPCs hold enormous clinical potential. HSCs have the ability to reconstitute the entire blood and immune system (Jagannathan-Bogdan and Zon, 2013), whereas MSPCs contain immunosuppression capacity and have the ability to regenerate damaged and diseased tissue (Caplan, 1991; Uccelli et al., 2008). However, there are still important hurdles that must be overcome before the potential of these cells are fully realised. The regenerative capacity of these stem cells is quickly lost upon ex vivo culture, meaning achieving clinically relevant numbers of cells is challenging (Dalby et al., 2018; Zon, 2008). Although BM MSPCs (such as nestin+ MSPCs) contain HSC support activity, their ability to maintain HSCs ex vivo is only modest due to loss of expression of these niche factors in culture (Kunisaki et al., 2013; Nakahara et al., 2019). The absence of sustained self-renewal or maintenance of the stem cell phenotype could be related to the lack of integration of biophysical and biochemical cues required for stem cell regulation, provided by the native BM niche microenvironment in vivo. This has led to a focus on biomaterial and engineering strategies that aim to recapitulate BM niche properties in vitro (Müller et al., 2014). It is envisaged that bioengineered artificial niches will offer protocols for ex vivo expansion and maintenance of HSCs without the need for high risk protocols (e.g. genetic manipulations (Nakahara et al., 2019)), but also platforms on which to study the fundamental mechanisms that control self-renewal in both HSCs and MSPCs in the niche. In this thesis, biomaterial strategies were employed to mimic aspects of the BM niche microenvironment. First retention of HSC support activity in a population of MSPCs was investigated and the metabolic mechanisms that may support this phenotype were probed. The ability of the system to support HSC maintenance in vitro was then assessed. Poly(ethyl acrylate) (PEA) is a polymer that causes spontaneous unfolding of the extracellular matrix protein (ECM) fibronectin (FN). These physiological-like networks expose key binding sites on the FN molecule, which can be harnessed for cell adhesion and growth factor tethering (Cheng et al., 2018; Llopis-hernández et al., 2016). Noting that low-stiffness matrices support nestin expression in MSPCs (Engler et al., 2006), a key niche marker (Kunisaki et al., 2013; Pinho et al., 2013), low-stiffness collagen hydrogels were introduced into the system. We were able to use this system to promote a population of nestin+ MSPCs that express key HSC support factors and were able to maintain a population of HSCs in vitro. The nestin+ MSPCs utilise hypoxic-like metabolic mechanisms in response to low-stiffness, that may be important in retaining this BM niche-like phenotype in long term in vitro culture.

Microbial communities refer to a number of microorganisms living together. To-date, a number of naturally occurring microbial communities are used for engineering purposes, e.g. anaerobic digestion, and the construction of artificial communities opens the door to wider applications. In microbial communities, the ecological interactions between species (e.g. competition, predation and mutualism) play important roles. The purpose of this thesis is to explore (1) the implications of such interactions in a bioreactor in terms of productivity and stability, and (2) the inference of such interactions particularly in a complex microbial community in order to form a basis for further manipulation such as optimisation and control. This work firstly studies the cases involving different microbial species at the same trophic level, with a particular focus on the potential of overyielding (the collective yield of multiple microalgae species, in terms of overall biomass abundance or a specific biomass component, is greater than that of any single species grown in the same system) in the poly-culture of microalgae arising from the complementary use of a key resource, namely light over the spectrum range of photosynthetically active radiation. Using a mathematical model, it is demonstrated that complementarity of light absorption alone does not guarantee overyielding; other factors such as the operational settings (e.g. loss rate of the bioreactor) and species traits (complementarity of light absorption spectra) can also affect the possibility and the degree of overyielding and even the co-existence of multiple species.

Bacteriophages are viruses able to infect and kill their bacterial host upon replication within it. They are thought to be the most abundant organism on earth, since their population exceed the one of bacteria (about 1030) by a factor of 10. In many cases, the host range where a given bacteriophage is active is so narrow that it is usually specific to one species of bacteria. In the early 1920s, phage were used to treat bacterial infections in eastern Europe and spread to western companies that began to commercialize typhoid and urinary-tract infection therapies. Soon after, because of penicillin discovery, researchers abandoned this therapy for antibiotic treatment, mostly due to phage susceptibility to the immune system and other unknown behaviours. Due to the overuse and misuse of antibiotic drugs, antibiotic resistance fast become a serious problem of global concern, currently with no real solution, which represents a threat to humanity leading to a post-antibiotic era if no counter-measures and alternative strategies are undertaken. In the last decade, bacteriophage application for the treatment of bacterial infections has re-gained huge attention. However, phages delivered in buffer solutions are suboptimal, since they are not shielded from host defences that inexorably hinder phage activity. Therefore, for effective in vivo applications, e.g. crossing the acidic environment of the stomach or infecting intracellular pathogens, phages need to be delivered to the site of infection, for example via encapsulation. Encapsulation in targeted delivery systems will help phages to reach the target with high specificity, minimizing losses, undesired adverse side effects, and limiting the likelihood of emerging "super bacteria". In the last decades, a considerable variety of stimuli-responsive micro/nanocarriers have been designed. Such systems show a transition to the supramolecular structure or in their chemical structure in response to given stimuli. A wise choice of the stimulus that triggers the variation allows releasing of their cargo in a spatial- and temporal- controlled fashion. The stimuli can be either external or internal and most commonly are thermal variation, pH, enzyme concentration, redox potential, magnetic field, ultrasound intensity and photo sensitivity. In this thesis, we study the encapsulation of bacteriophages in lipid carriers able to deliver and release the cargo in a controlled manner. Liposomes are biodegradable non-toxic micro/nano vesicles composed of a lipid bilayer, which encloses an aqueous core that offer many advantages in medicinal applications. It was demonstrated that phage encapsulation in liposomes improves stability. Indeed, when inside the aqueous core of a vesicle, the thermodynamics of the system is advantageous for phage storage. In this doctoral thesis we have assessed the encapsulation of two model phages, an Escherichia coli T3 podovirus (size ~65 nm) and a myovirus Staphylococcus aureus phage K (capsid head ~80 nm and phage tail length ~200 nm). Encapsulation in liposomes was carried out by producing the vesicles by alcohol injection method in a capillary microfluidic device, in the presence of the cargo. We generated liposomes having mean sizes between 100-300 nm and the encapsulation yield of T3 phages was 109 PFU ml-1 and for phage K was much lower at 105 PFU ml-1. The encapsulation efficiency was affected by the aggregation state of the virions. Furthermore, we discovered that phage K is able to interact with the lipid bilayer resulting in phages bound to the outer membrane of the liposomes instead of being encapsulated inside them. We utilised the encapsulated phages to determine whether 1) they are internalized by human macrophages in higher number compared to free phages and whether 2) they are able to reach the cytosol were intracellular pathogens reside. We discovered that, regardless the formulation of liposomes, encapsulated phages are intracellularized more efficiently in comparison to free phages. However, following uptake, liposomes and encapsulated phages are trafficked to the lysosome, where they are irreversibly digested. Therefore, we envisioned a different approach: a smaller antimicrobial agent than phages, namely a phage lysin, was co-encapsulated with a hemolysin able to make pores in the endosomal membrane. We observed that the co-encapsulated of the two proteins effectively escaped lysosomal degradation and defeated intracellular S. aureus. We expect this approach to be easily extended to phages and we suggest further perspectives and concrete future experiments. In this doctoral thesis, we made significant advances in the encapsulation and intracellular delivery of phages for antimicrobial treatment, and we obtain promising results that pave the way to the actual utilization of phages for medical treatment of infections, especially of antibiotic resistant ones.

The focus of this thesis is on the applications of nonlinear dynamical systems in bioengineering which are mainly used in large-scale and generally categorised into two groups: (1) dynamical systems from biology (2) dynamical systems for biology. The mathematical models describing the dynamical systems used in the above systems can be simulated with the use of powerful software such as MATLAB, however, for large-scale simulations software begins to collapse. Besides, computer-based simulations are not always suitable for interfacing with biological/physical systems where continuous monitoring with low power and area consumption might be required. To alleviate these issues, a few novel hardware techniques for both aforementioned groups are proposed and their hardware results compared and validated by software simulations. Under group (1), a compact and fully reconfigurable digital hardware model capable of mimicking 1-D, 2-D and 3-D nonlinear dynamical systems in real-time and large-scale is presented. Results, and theoretical analysis confirm that the proposed model can mimic the biological behaviour with considerably low hardware overhead and is, on average, ~83 times faster than the CPU version. The proposed model has been also fabricated in the AMS 0.35 um technology capable of emulating slow intracellular calcium dynamics. The fabricated chip occupies an area of 1.5 mm^2 and consumes 18.93 nW for each calcium unit from a power supply of 3.3 V. In addition, under the same group, a novel analog circuit supporting a systematic synthesis procedure of log-domain and strong inversion circuits capable of computing bilateral fast/slow dynamical systems is proposed. The application of the method is demonstrated by synthesising four different case studies. The validity of our approach is verified by nominal and Monte Carlo simulated results with realistic process parameters from the AMS 0.35 um technology. The resulting circuits exhibit various bifurcation phenomena, time-domain responses in good agreement with their mathematical counterparts. Under group (2), a flexible and efficient hardware classifier for biomedical time-series classification is proposed. In this classifier, throughput is traded off with hardware complexity and cost using resource sharing techniques. Results confirm that the proposed hardware can accurately classify surface EMG and heart time-series data with low area and power consumption. Most notably, our classifier reaches 1.3x higher GOPs/Slice than similar state of the art FPGA-based accelerators.

The surprising discovery of intramolecular isopeptide bonds within Gram-positive bacteria pili and surface adhesins gave rise to the development of a new type of protein conjugation method, namely the Tag/Catcher system. Among the different kinds of Tag/Catcher platforms, SpyTag/SpyCatcher is the leading system with more than 200 articles utilising the technology due to the rapid and buffer-independent nature of the reaction. In this thesis, I have developed a purification method called Spy&Go to complement the SpyTag/SpyCatcher platform. Spy&Go enables the purification of proteins using SpyTag as the affinity peptide tag. Spy&Go thereby eliminates the dependence on extraneous tags for purification of proteins intended for SpyCatcher-based protein conjugation reactions. Spy&Go was developed by engineering SpyCatcher2.1, a fast reacting variant of SpyCatcher, to become SpyDock by removing reactivity whilst maintaining tight binding to SpyTag. The purity of proteins purified through Spy&Go was comparable to the purity of proteins purified by Ni-NTA using a polyhistidine tag. Subsequently, a different route for elution of SpyTag-proteins was explored by conferring pH-sensitivity to a SpyDock variant through the introduction of histidine residues at the SpyTag003-binding interface. With the combined effects of three histidine mutations, SpySwitch was developed, which can bind SpyTag003 at pH 8.0 and release at pH 5.0. SpyTag/SpyCatcher has been used to modularly multimerise proteins for vaccination, nanomedicine and making enzymatic nanoreactors. To extend the scope of SpyCatcher-based protein multimerisation platforms, I developed a Spy oligomerisation toolbox composed of SpyCatcher002 fused to coiled coils, to multimerise SpyTag-proteins into dimer, trimer, tetramer, pentamer, hexamer and heptamer assemblies. The SpyCatcher002-oligomers enable a rapid and scalable route to multimerise different proteins into defined assemblies. An agonistic nanobody against Death Receptor 5 was multimerised through SpyCatcher002-oligomers and showed a stoichiometry-dependent effect on breast cancer cell line apoptosis upon receptor activation.

Research in systems and synthetic biology in the past decade has resulted in important advances in understanding as well as engineering cellular systems through the underpinning molecular networks. Furthermore, there is an increasing blurring of boundaries between natural and engineered cellular systems. Dissecting information processing through these molecular networks is a central aspect of this. This brings up a number of systems challenges to be tackled. In this thesis, we focus on the dissection of two features of information processing molecular networks, modularity and spatial organisation. Modularity, a basic and cross-cutting theme in engineering is a key underlying theme for both systems and synthetic biology. It is a basis for both building complex circuits in synthetic biology using bottom up approaches, and for understanding the behaviour of complex molecular networks in terms of their constituent building blocks. Spatial organisation is a hallmark of cellular systems ranging from bacteria to eukaryotes and an ingredient actively exploited in evolution. It is also a vital tool in manipulating and engineering the behaviour of molecular networks through multiple, recently developed tools focussing on compartmentalisation. In the first part of this thesis, we develop a systems engineering framework to understand the behaviour of a module within a network, accounting for different aspects of the module and the ambient network. In spatial organisation, we focus on multiple themes (i) the analysis and evaluation of compartmental models as appropriate descriptions of compartmentalised reacting systems, (ii) developing systems engineering frameworks for engineering spatially organised genetic circuits, (iii) a theoretical and systems framework for elucidating the effect of spatial organisation in molecular networks. Taken together, these studies provide systems tools and approaches for dissecting and engineering vital aspects of the complexity of cellular information processing systems.

Accurate, timely, and sensitive diagnosis is the first step in appropriately treating disease. The development of diagnostics that can be used in non-hospital and point-of-care (PoC) settings is key for democratizing access to disease diagnosis and treatment when it is most effective. The application of PoC diagnostics in early disease detection is often limited due to insufficient sensitivity for the short time frames and limited resources available in these settings. To overcome these challenges, this thesis presents the synthesis, characterization, and application of catalytic nanomaterials for signal amplification and enhanced sensitivity in PoC diagnostic platforms. The nanomaterials developed here span a broad size regime from 1.5 nm clusters of atoms to 300 nm diameter particles and have been demonstrated for use in both in vitro and in vivo biosensing platforms. The platinum and gold nanomaterials exhibited robust and efficient peroxidase-like activity in their ability to oxidize chromogenic substrates in the presence of hydrogen peroxide to generate additional colored signals that could be used for amplification of disease detection, even after exposure to harsh conditions such as elevated temperatures. The larger catalytic nanomaterials were employed as extraordinarily stable and highly amplifying labels in a simple paper-based lateral flow assay (LFA). The nanocatalyst-labeled LFA surpassed the sensitivities of both commercial and published reports to date for paper-based detection of p24, one of the earliest and most conserved biomarkers of HIV. The smaller catalytic nanoclusters were efficiently renally cleared and were deployed in a modular nanosensor platform to monitor disease-associated protease activity in vivo. Catalytic activity of cleared gold nanoclusters in collected urine provided a simple, sensitive, and rapid colorimetric urinary readout of disease state. The clinical utility of both platforms presented here was investigated through detection of acute phase HIV in clinical human plasma samples using the nanocatalyst-labeled LFA, and successful non-invasive detection of tumors in a mouse model of colorectal cancer using the protease-nanosensors with colorimetric urinary readout. This thesis demonstrates the broad applicability and versatility of catalytic nanoparticle amplification for use in disease detection. A pipeline for further development of both sensing platforms for detection of other biomolecules at the PoC is presented.

Synthetic biology is a powerful collection of tools that facilitates the design and construction of novel biological systems, with applications ranging from the development of synthetic life to the production of high value chemicals. The development of novel biological parts is a core principle of synthetic biology, as expanding the biological 'tool-kit' opens up new possibilities for the production of novel tools, pathways and compounds. Antibodies demonstrate high antigen affinity and specificity, making them ideal candidates for the development of novel synthetic biology tools. However full length antibodies require post-translational modifications that prevent their use in prokaryotic chassis, such as Escherichia coli, which are commonly used for synthetic biology applications. Single domain antibodies (sdAb), such as heavy chain variable domains (VHH) of camelid heavy chain only antibodies (HCAb), demonstrate improved stability and robustness when compared to conventional recombinant antibodies whilst retaining high antigen affinity and specificity. Furthermore, sdAb such as VHH are also capable of binding 'cryptic' epitopes on the target antigen, such as those that occur within clefts and cavities. As a result, these recombinant antibody fragments are ideally positioned for initial investigations into their use as tools for synthetic biology applications. This thesis describes the generation of, and subsequent investigations into, VHH with functionalities relating to the production of monoterpenoids in E. coli. First, a VHH phage display library was constructed and subsequently enriched against Streptomyces clavuligerus linalool synthase (bLinS). Next, Design of Experiments (DoE) was used to construct and optimise a platform that facilitated the production of VHH in the cytoplasm of E. coli, such that these recombinant antibody fragments may be utilised for synthetic biology applications. Finally, the ability of three anti-bLinS VHH to modulate the production of monoterpenoids in E. coli containing a heterologous linalool production pathway was investigated, revealing that these anti-bLinS VHH were capable of altering the flux within the pathway, thereby altering the profile of the terpenoid products obtained. As such, the platforms developed in this project, and the insights gained following their implementation, represent a foundation upon which further investigations and developments may build, so as to realise the full potential of VHH antibodies as powerful tools in the field of synthetic biology.

Photosynthesis provides the mechanisms through which sunlight powers most of our biosphere. Either through direct application or inspiration, natural solar energy conversion strategies offer mankind potential solutions to impending energy and food crises through the exploitation of free solar power. However, the overall efficiency of photosynthesis is limited by a variety of factors including the selective light spectral coverage displayed by the choice of major pigments. Intriguingly, the complementary absorption profiles of chlorophyll-based photosystems and bacteriochlorophyll-based photosystems from oxygenic or anoxygenic phototrophs provide a pathway toward enhanced light capture across the photosynthetically-useful spectrum by synthetic biology. The research described in this thesis explores the effectiveness of a range of linking strategies to assemble bacteriochlorophyll-containing reaction centers (RC) and chlorophyll-containing light harvesting complexes (LHCs) into single polychromatic photosystems. Among the all biological strategies, a SpyTag/SpyCatcher linker offered the most effective way to form macromolecular "chimeras" between the RC from photosynthetic bacterium Rhodobacter sphaeroides and LHCs from Arabidopsis thaliana. Energy transfer from LHC to RC was confirmed both in solution and on an electrode and the current output of bacterial-RC photoelectrodes was shown to benefit from light capture by LHCs. In parallel, synthetic optically-active quantum dots (QDs) were shown to act as hubs for the self-assembly of LHC/RC/QD conjugates and to act as an energy bridge to augment direct LHC to RC energy transfer. A tight-binding interface between proteins and QDs was characterized. Based on the in-depth understanding of thermodynamics of this photosystem, it was found that energy flow within the tri-component conjugates could be tuned in a manner similar to natural photosystems and to a comparable level of efficiency. The project demonstrated the power of applying synthetic biology principles to the systematic redesign of natural photosynthesis and the expansion of solar energy conversion beyond the natural boundaries of living systems.

The biopharmaceutical industry is at a crossroads, where changing industry and ethical demands means it must invest in research and development (R&D) to maintain a diverse pipeline of novel drugs, and simultaneously reduce the substantial risks and costs associated with this R&D. In the future, a complete toolkit of synthetic expression elements could be used to create a streamlined, rationalised next generation cell line development (CLD) process, maximising the benefits offered by synthetic biology. However, synthetic, predictable, titratable molecular biology tools have not yet been developed for all the desired steps of biopharmaceutical expression. In this thesis, I will present work endeavouring to expand this toolbox, by investigating synthetic control of glycoprotein expression through messenger RNA (mRNA) engineering. The tethering of various RNA-binding proteins to recombinant mRNA was tested, as a method of controlling mRNA processing, and stimulating intronless mRNA export. C1orf35 and HuR were identified as target effector genes to increase transient protein production. However, inconsistency in their effect disqualified them as effective molecular biology tools. Two families of 3’ untranslated region (UTR) RNA elements were screened for their ability to control productivity through enhancement of mRNA stability. Though triple helices failed to increase expression, a stability element was discovered to increase transient SEAP productivity by 1.28-fold, via a mechanism of extension of SEAP mRNA half-life from 0.68h to 4.04h, compared to an industry-standard vector. 5’ terminal oligo-pyrimidine (TOP) motifs were investigated for their ability to control productivity through translation initiation. Enhancement of transient titre was demonstrated with various recombinant proteins and culture conditions, chemical supplements screened for their ability to specifically affect 5’TOP activation, mechanism of titre control investigated, and 5’TOP motifs integrated with synthetic proximal and core promoters. Different 5’TOP motifs were shown to control titre of a biotherapeutic fusion protein in an industry transient production process, up to a 2.12-fold increase compared to an industry-standard vector. These synthetic elements were screened together for their modularity, demonstrating titratable control of SEAP titre, from a 0.82-fold decrease to a 5.23-fold increase compared to an industry-standard vector. Further steps were then recommended, to render these tools truly predictable, and contribute maximally towards a rationalised, next-generation CLD process.

Natural protein-based materials are exploring their new applications from the traditional uses. Structural proteins provide scaffolds, whereas functional proteins carry essential biological activities through millions of biochemical reactions. The idea of implementing functionalities into natural structures could provide manufactured protein products with a better fit for human desire. The development of synthetic biology and molecular assembly methods illustrates possibilities for the production of functional structures in-situ, which provides better connections between the functional partners with the structural scaffolds. Protein fibres are one of the natural structures which possess a unique shape and superior mechanical properties. Apart from the natural protein fibres, many disease-related peptides self-assembled into amyloid filaments and fibrous structures. Natural globular proteins could also form fibres through the directed assembly by changing their storage conditions or through fusion. Elongated polyglutamine peptides cause many neurodegenerative diseases as they assemble. In this thesis, a polyglutamine peptide (Q77) was fused with functional partners to direct the protein assembly in vitro. The role of the polyglutamine was studied during assembly and after the formation of a self-supportive fibrous product. The extensibility of traditionally size-limited fibrous materials formed by disease-related peptides was tested experimentally for the first time. The resultant fibrous product with embedded functionalities mimics the structure of silk, but the mechanical behaviour of collagen. Two structurally distinct proteins were chosen as the functional partners for Q77: a monomeric red fluorescent protein (mcRFP), which is relatively small in size and possesses a b-barrel structure, and firefly luciferase (Luc), which is a larger protein with a fragile structure consisting of two mobile domains. Both proteins have been widely used as reporters for intracellular activities with either fluorescence or bioluminescent signal. In this work, the functionalities of both proteins were investigated after Q77 fusion and after assembly towards respective fibrous products. The structural variation of these recombinant proteins resulted in the changes of their functionalities. Finally, a self-supportive fibrous ATP sensor was achieved for the first time with this dual functional protein product.

Growth factors are signalling molecules that orchestrate cell proliferation, survival and migration. As such, they have been identified as promising therapeutics to promote tissue repair. However, there is currently a lack of approaches that enable their effective delivery. A promising strategy to make growth factor-based therapies more potent is to mimic the way the extracellular matrix (ECM) coordinates the presentation of growth factors in time and space. For example, the ECM contains domains that bind growth factors with high affinity and contains enzymatically cleavable domains whose cleavage leads to a transient increase of the available growth factor. One mechanism that evolved to achieve a delayed, on-demand activation of growth factors is the traction force mediated release of the transforming growth factor-beta β (TGF-β) from the lasso-resembling Large Latent Complex (LLC). While the use of bioactive ECM-derived motifs has already extended the capabilities of growth factor delivery scaffolds, traction forces have not yet been explored as a general stimulus to trigger an on-demand release of growth factors. This thesis presents the design of synthetic, functional mimics of the LLC, termed the Traction Activated Payloads (TrAPs). TrAPs consist of an aptamer and a cell adhesive peptide. The single-stranded oligonucleotide aptamer binds a growth factor via affinity interactions, which are disrupted when the cell pulls on the complex via integrins. This thesis demonstrates the versatility of the platform by showing: (i) that TrAPs can be designed to deliver multiple different growth factors and (ii) that TrAPs retain their functionality when conjugated to both a variety of surfaces and therapeutically relevant collagen scaffolds. This thesis further demonstrates that TrAPs can be combined with various cell adhesive peptides, making this platform the first step towards the design of cell type-specific, sequential growth factor activation.

The therapeutic monoclonal antibody (mAb) market is witnessing an unprecedented growth having nearly duplicated since 2012. Faster regulatory approvals and an increasing number of biosimilars are among the most important mAb market expansion drivers. As the market grows, so does competition making timelines more aggressive. Additionally, political pressure from governments and regulatory authorities over prices of life-saving biological drugs is also increasing. As a consequence, biopharmaceutical companies need to reduce costs while being faster, particularly in having drug substance ready for clinical trials. To this aim, reducing process development times and costs is crucial. In this thesis, mathematical models integrating several cellular activities important for biomanufacturing are developed. A previously published model development framework is followed to ensure the development of predictive models. An integrated framework combining multivariate data analysis and biomarker identification techniques for cell culture understanding supports the development of predictive mathematical models for two industrially relevant cell lines, GS-NS0 and GS-CHO, capturing cell cycle, metabolism, energy production, mAb production and apoptosis. As the models herein suggested can capture population heterogeneity by describing the cell cycle and gene expression, their applications to bioreactor optimization is envisaged. To this aim, the models developed in this work can be combined with the description of product quality attributes and/or computational fluid dynamics descriptions of bioreactors. These are deemed important applications to accomplish significant reduction in process development time and costs.

The overall aim of this thesis is to examine the encounter of the real and literature – as thematically crystallized in the contrivance of the literary real – in terms of an irreducible tension. The encounter of literature and the real (their coexistence and inseparability) is examined conjointly with the encounter (the meeting and interlinking) of Georges Bataille and Maurice Blanchot – as generated by the comparative angle that structures the thesis. The literary real addresses both the question of what kind of ‘real’ is involved and disclosed in writing (and how that might differ from reality in its more traditional sense – or more precisely from more conventional representations of reality), as well as the question of writing’s own ‘being’ (that is, the particularity of its mode of being, its peculiar reality/unreality). The thesis aims to provide a renewed (and overlooked) reading of both thinkers as situated at the crossroads of post-deconstruction (welcoming the real, experience) and anti-realism (differentiating the real from – its equation and reduction to – empirical reality and the current state of affairs). In parallel, and more broadly, the project, via Bataille and Blanchot, calls for a recasting of key terms of the literary and aesthetic tradition (such as creation and inspiration, autonomy and mimesis), but also of concepts relevant to wider current debates, such as space, inside and outside, time, experience and the event, visibility and invisibility, intimacy and distance.

Protochlorophyllide (Pchlide) is the substrate of the light-driven reaction catalysed by the protochlorophyllide oxidoreductase (POR) enzyme. This reaction is a crucial step for chlorophyll biosynthesis in plants, it plays an important role in plant development and it has been suggested to also play a photoprotective role in plant cells. The POR reaction is one of the very few enzymatic reactions activated by light. The fact that the reaction can be triggered with a single laser pulse makes it a unique model to study enzyme catalysis. A crystal structure of POR is to date unavailable and this lack has thwarted a full elucidation of the reaction mechanism as well as the determination of the protein ternary structure and the exact active state geometry. The substrate, Pchlide, has unique excited-state properties and the decay kinetics including what was presumed to be a long-lived triplet state. We have used time-resolved absorption and electron paramagnetic resonance (EPR) spectroscopy at cryogenic temperatures to provide direct evidence of the triplet state of Pchlide and the characterisation of its EPR signature. We have found that the triplet state of Pchlide reacts with solvated oxygen, and that POR-binding reduces the triplet lifetime, supporting the theory of POR playing a photoprotective role. We were able to stabilise and characterise a highly transient reaction intermediate by means of cryo-trapping as well as identifying changes in its structure, a matter where there is currently no clear consensus. By studying substrate analogues we have been able to expand current knowledge on which and how different regions of Pchlide are important for the reaction photochemistry. The work in this thesis opens up this crucial biological system to a whole new range of experimental techniques that can prove instrumental in elucidating the still unclear reaction mechanism of light-driven POR.

Protein phosphatases are regulatory enzymes with unique substrate specificity for serine/threonine/tyrosine/histidine phosphorylated proteins and phosphoinositides. They mediate critical cell processes such as cell metabolism, cell cycle, growth and differentiation. For example, phosphoinositide phosphatases act as regulatory enzymes that mediate critical cellular processes such as cell signalling, membrane trafficking, cell cycle and growth. Loss-of-function mutations in these enzymes are linked to a plethora of disease conditions. Tumour suppressor PTEN is the most frequently mutated gene in various forms of human cancer; Myotubularins are mutated in X-linked myotubular myopathy (XLMTM) and Charcot-Marie-Tooth disease type 4B (CMT4B); Synaptojanin I is mutated in early-onset progressive Parkinsonism. Engineered PTPs are therefore seen as useful tools to enhance our understanding of signalling pathways involving phosphorylation events. Here, we used a combination of structure-guided rational and computational design to alter the substrate specificity of Low Molecular Weight Protein Tyrosine Phosphatase (LMW-PTP), from tyrosine-specific to PI(3,5)P2-, PI(3)P-, PI(4)P-, and PI(5)P-specific phosphatase (Low Molecular Weight Phosphoinositide Phosphatase - LMWPIP). Four LMW-PIP mutants dephosphorylate phosphoinositides with single or broad substrate specificity. We combined Rosetta Enzyme designs containing frequently introduced mutations with molecular docking studies to assess binding affinity to the ligand substrate. This approach facilitates the design selection process by reducing the number of candidate mutants to be validated experimentally. We also designed mutants to be tested for specificity towards serine/threonine phospho-peptide. Finally, we set up a system in yeast that can be used for in vivo characterisation of LMWPTPB mutants, by cloning and confirming the expression of LMWPTP in yeast using western blot and RT-PCR. Engineered LMW-PIP mutants can be a useful tool in investigating lipid-regulated cell signalling pathways, and in the long term, could serve as biotherapeutics in enzyme replacement therapy.

The depletion of fossil fuels and the problems of greenhouse gas pollution has pushed the development of alternative sustainable biofuels including from microalgae. Microalgae can produce high abundance of lipids and carbohydrates that can be utilised as a biofuel feedstock. However, improvements are required throughout the production process for microalgae biofuel to become commercially viable. One of the major bottlenecks is low metabolite productivity. Microalgae cultivation conditions such as nutrient starvation that can boost the accumulation and storage of lipids and carbohydrates in microalgae also compromise growth. To overcome this problem, genetic engineering may offer solutions to improve the production of these macromolecules without compromising growth. For genetic engineering to be successful, a better knowledge of the molecular mechanism involved in the biosynthesis of carbon storage molecules is required. Transcription factors (TF) are of interest as potential targets for genetic manipulation since their engineering could provide stronger up-regulation of metabolic pathways. In an effort to contribute to the identification of key TFs involved in carbon storage metabolism, this study focused on the characterization of two TFs from Chlamydomonas reinhardtii. PSR1 is a TF that has previously been identified as a regulator of lipid and starch metabolism, however, the exact mechanisms of regulation are unknown. By using the electrophoretic mobility assay (EMSA) I tested the ability of PSR1 to bind to the promotor region of three genes involved in the carbon storage metabolism. PSR1 was able to bind specifically to the regulatory elements in the promotors of these three genes. These results showed the DNA binding activity of PSR1 for the first time and indicates a mechanism though which PSR1 is regulating starch and lipid metabolism in C. reinhardtii. A previously uncharacterised TF, MYB2 was predicted from genomic data to be upregulated under different stress conditions. MYB2 showed close phylogenetic relationship and structural similarity with MYB-type TFs from Arabidopsis thaliana that are regulators of various environmental stress responses. To determine if MYB2 is involved in the regulation of starch and lipid metabolism, MYB2 overexpression and knockdown lines were generated and characterised. Surprisingly, both knockdown and overexpression lines showed similar phenotypes with regard to carbon storage content. Both sets of these MYB2 modified lines showed an increase in the lipid accumulation under stress condition, particularly under nitrogen (N) limitation where there was a more than 2 fold increase in lipid content when compared to control lines. These results suggest that MYB2 is involved in the regulation of carbon storage metabolism. However, its role appears to be more complex than expected and potential mechanisms of genetic robustness and compensation in its function are proposed and discussed. These results expose the complex regulatory networks involved in the regulation of the carbon storage metabolism in microalgae. In conclusion, results from this investigation have provided important insights into the role of two TFs involved in the lipid and starch biosynthesis, which could potentially be targets for genetic engineering for the enhancement of lipid and starch production in microalgae in the future.

Staphylococcus epidermidis is an opportunistic bacteria which forms pathogenic biofilms in medical implant environment. Biofilm formation is a complex multistage process within which the initial stages of adhesion are deemed the most critical target for preventing biofilms. This research involves the characterisation of S. epidermidis (ATCC35984 and NCTC13360) by using X-ray photoelectron spectroscopy (XPS) and atomic force microscopy (AFM) on model substrates including glass, muscovite mica, silicon (111) wafer, sputter-coated titanium and sputter-coated silver, focusing on the effect of chemical properties of the material on adhesion by using surfaces with minimal roughness. AFM was used to image the surface, from which bacterial coverage can be estimated. AFM was also used to probe adhesion forces and local mechanical properties of all samples through the use of force-distance curves. AFM images were also used to estimate the bacterial coverage. XPS was used to investigate the surface chemistry from the layer thicknesses, the percentage coverage and potential composition of the overlayer. The combination of these techniques allow the relationships between the surface chemistry of the substrate and the bacteria to be correlated with changes in coverage and properties of bacterial films. Data on incubated bacterial samples were compared with those from the reference substrates, both before and after autoclaving, and from samples prepared using protein rich growth medium (tryptic soy broth) in the absence of bacteria as well as a pure bacterial pellet in an assumed non-biofilm forming state. The research indicates the potential differences between biofilm and non-biofilm former strains, with both strains being covered by an organic layer with little influence of the growth media used to incubate the bacteria. This research also shows how XPS and AFM data can be combined and applied to bacterial adhesion.

Phage display has attracted a great deal of interest in the identification of peptides specific to nanomaterials revealing distinctive binding behaviour. Though significant progress has been made in selecting and screening of biomolecule binding peptides, the accuracy of molecular recognition for inorganic materials is still challenging due to the limitations of phage display libraries and biopanning process. The study presented in this thesis is aimed at isolating mineral binding peptides by phage display and verifying them experimentally and/ or bioinformatically; exploring the role of electrostatic/ non-electrostatic interactions in the aqueous phase and the factors responsible for the adsorption or desorption of peptide or phage from the mineral surface. Firstly, silica binding peptides LPVRLDW, NDLMNRA, GQSEKHL and GASESYL have been identified using the phage display technique by varying experimental conditions including pH, detergent, washing and elution buffers to remove unique 7-mer peptide binding phages from amorphous hydrophilic silica nanoparticles via disruption of the molecular interactions between the phage attached peptides and the nanoparticles. A repanning method reported here, has experimentally reproduced the majority of the initially discovered silica binders; alongside identifying/ recovering additional peptide sequences HYIDFRW, KIAVIST and YSLKQYQ that may have been overlooked in the routine approach to biopanning. Secondly, an alternative three step elution method reported here, has eluted and recovered most target silica binders including ADIRHIK in the early panning rounds and removed the phage clones that are bound to silica by hydrophobic, hydrogen bonding and electrostatic attractions or repulsions; as opposed to one specific buffer being used for all panning rounds including elution steps in traditional biopanning experiments. Also, the phage clones that resist detection to single elution step have been eluted in the other successive elution steps, thereby recovering and improving the elution procedure for silica surfaces. In addition, these three different elution buffers have eluted phage clones that are interaction or charge specific subject to change in the elution buffer pH condition. The experimental results demonstrate that this sequential three step elution process was able to isolate tightly bound target silica binders in one or two biopanning rounds than the more typical four to five; thereby reducing biopanning rounds, cost and effort. Moreover, the bioinformatic analysis to cross check the authenticity/ quality of target binders has been reported. Furthermore, selected silica binding peptides isolated from phage display experiments were synthesized by a solid phase peptide synthesis approach and peptide-silica interactions explored in vitro, using quantitative and qualitative techniques. The fluorometric analysis of these peptides revealed that the peptide adsorption to silica surfaces would have more than one type of interactions (i.e. electrostatic/ hydrophobic/ H-bonding and Van der Waals) and could be influenced by the experimental conditions. More significantly, an increase in binding activity to negatively charged silica nanoparticles was noticed for the peptides (HYIDFRW, KIAVIST and YSLKQYQ) modified with an amide (NH2) group as opposed to a carboxyl group at the C-terminal end; driving an increase in overall charge or pI of the peptides. Insights from the studies presented may provide valuable information for designing and engineering of silica directed constructs for a range of biomedical and nanotechnological applications.