Automation, artificial intelligence, and autonomous machines are already having a significant impact across many industries and these technologies have huge potential for advancing chemical research. This thesis focuses on two complex physicochemical systems, oil-in-water droplets and chemical gardens, and how smart-automation can be a powerful tool for their research. Smart-automation is herein defined as ‘physically implemented platforms that undertake experiments autonomously as directed by an algorithm, rather than undertaking operations or experiments as directed by a human scientist’. Overall, this thesis illustrates how smart-automation is suitable for exploration, optimisation, discovery, and developing deeper understanding of oil-in-water droplet systems. It is proposed that the approach used in this work is also suitable for other systems studied within chemical and materials sciences, including chemical gardens. Initially, three new approaches for the analysis of oil-in-water droplet systems were developed, building on traditional analytical chemistry techniques and previous work exploring oil-in-water droplet systems using smart-automation.1 These analytical methods are orthogonal and complementary: a machine learning approach predicts bulk properties and correlates these to droplet behaviours; 1H NMR spectroscopy is used to measure the state of the system at a given time; and the use of pH indicators allows the visualization of spatiotemporal variations within the system. The machine learning approach enabled the prediction of droplet behaviour directly from the droplet formulation for the first time. This included the prediction of more instances of a rare cooperative ‘swarming’ droplet behaviour. Subsequently, a new smart-automated platform was developed that expands the exploration of oil-in-water droplet systems to include the variation of both the aqueous and oil phases. Thus, the aqueous and oil phase formulations could be optimised for a property of interest; here droplet movement is the targeted property. In doing so, both new and extreme droplet behaviours were identified. The newly described droplet behaviours are termed swarming, fusion, pulsing, and sorting. Next is reported a new platform for oil-in-water droplet experiments used to compare different formulation exploration methods. In undertaking this work, a previously unknown delicate temperature sensitivity of droplet behaviours was identified; this would not have happened without the use of smart-automation. This was studied in detail for one formulation which led to the identification of six phases of droplet motion and the preparation of a time-temperature phase diagram. Analytical methods including 1H NMR spectroscopy, DLS, and further droplet experiments enabled the proposal of mechanisms driving these phases of motion. Overall, this section of the work illustrates how smart-automation and the expert scientist can work together effectively. Following this, new avenues for reactive droplet constituents were explored and a formulation resulting in reliable droplet fusion was identified. Diels-Alder and carbonyl chemistry were then investigated as suitable reacting chemical systems to use in oil-in-water droplets. Amide and imine formation were both successfully realised in oil-inwater droplets with their macroscale effects on droplet behaviour visible to the naked eye. Finally, a wholly polyoxometalate based chemical garden system was identified and studied for the first time. This work bridges a significant difference between polyoxometalate based and classic chemical gardens as it has the same charge distribution as classic chemical gardens. Six growth regimes were identified and similarities and differences between this and previous systems are discussed. To finish, a smart-automated platform for exploring mixed chemical gardens is discussed and a prototype presented.

This thesis describes efforts towards size reduction of a human tumour necrosis factor alpha (hTNFα)-targeting monoclonal antibody (mAb) infliximab (Remicade®) by means of construction of medium-sized antibody mimics, denoted as synthetic antibodies. The synthetic antibodies constructed by Cu(I) catalyzed azide-alkyne cycloaddition of complementarity determining region (CDR) mimics onto semi-orthogonally protected CTV scaffold derivatives have less than 4% of the molecular weight of the original monoclonal antibody infliximab (144 kDa). The introductory Chapter 1 describes different approaches of targeting hTNFα with small, medium, and large molecules. Furthermore, it explains research undertaken in the Liskamp group which served as a basis for the research reported in this thesis. Chapter 2 describes the development of a synthetic route for the synthesis of semi-orthogonally protected CTV scaffold derivatives. The synthetic route was applied in the synthesis of four semi-orthogonally protected CTV scaffold derivatives with different aqueous solubility-modifying spacer. Chapter 3 focuses on the selection of CDR sequences for mimicry of the mAb infliximab. It further describes their synthesis, cyclisation, and sequential attachment to the CTV scaffold derivatives towards synthesis of synthetic antibodies. Chapter 4 describes an evaluation of the capability of the prepared synthetic antibodies to mimic mAb infliximab. The evaluation was attempted by means of an MTT cell cytotoxicity assay, by affinity determination using isothermal titration calorimetry (ITC), and by surface plasmon resonance (SPR). Chapter 5 focuses on the expression of a recombinant hTNFα using an E. coli expression system. Furthermore, the characterisation of the expressed hTNFα, which was used in the ITC and SPR experiments, is described.

The study presented in this thesis provides details regarding the synthesis and characterization of different redox active molecules which can be applied to form the active layer of photovoltaic devices. For example in chapter two, star-shaped thiophene based molecules are described and their electronic and optical properties have been investigated. In chapter three, oligomers featuring bipyridinium units with different thienyl moieties have been successfully synthesized. Preliminary electropolymerization studies are achieved. In chapter four, two different series of powerful push-pull systems containing dimethyaniline DMA moieties as a strong donating group and TCNE or TCNQ as electron accepting groups have been prepared. These series feature quinone and oligothiophene units as supporting acceptor and donor unit. In chapter five, two different series of powerful organic dyes that could improve the efficiency of DSSCs by modulating the absorption of light towards the near-IR region are achieved. The influence of π-conjugated spacers on the optical and physical properties of synthesized dyes has been investigated. Preliminary DSSCs have been fabricated from some of these systems and their properties have been compared to dye N719.

During the course of this PhD, methodology for the synthesis of a series of novel, highly fluorescent pyridine-derived α-amino acids was developed. Enone-derived α-amino acids were subjected to an inverse electron demand hetero-Diels-Alder cycloaddition and aromatisation reaction, which led to a twelve-membered library of pyridine analogues. The optical properties of these compounds were analysed, with several exhibiting interesting fluorescent characteristics. One of the analogues was incorporated into a cell penetrating pentapeptide via solid phase peptide synthesis. The resulting hexapeptide was incubated with human fibroblast cells and fluorescence microscopy was used to show accumulation of the peptide in the cells. The total syntheses of piperidine containing natural products, spruce alkaloid and (+)-241D were also examined during this PhD. A short nine-step linear sequence was developed giving spruce alkaloid and (+)-241D in 21% and 19% overall yield, respectively. A base mediated 6-endo-trig cyclisation was employed as the key-step followed by stereoselective ketone reduction to complete the total syntheses. The scope of the cyclisation and reduction was examined with a range of enone side-chains resulting in a small library of novel 4-hydroxy-2,6-disubstituted piperidines.

Metal-Organic Frameworks (MOFs) are multidimensional crystallised porous materials built from inorganic nodes separated by organic linkers. Their permanent porosity and their large surface area has brought a widespread interest to these structures for the last twenty years regarding their potential applications such as gas storage, catalysis, sensing and drug delivery. The predictable topologies of MOFs arising from the isoreticular principle have opened the way to functionalisation through rational design and the study of their structure-property relationships. In keeping with that reasoning, this thesis focuses on the introduction of new functionalities into MOFs through careful design of functionalised linkers, and, the work reported in this thesis can be divided in two different subjects. The first is the synthesis of novel functionalised amide containing dicarboxylic linkers for the synthesis of zirconium MOFs. The second subject describes the utilisation of a new hybrid manganese Anderson polyoxometalate as a new kind of functionalised spacer for the so-called POMOF (Polyoxometalate Metal-Organic Framework) materials. Following a general introduction on MOFs describing these materials in terms of structure, synthesis, properties and functionalisation in Chapter 1, Chapter 2 reports the synthesis of amide containing dicarboxylic linkers possessing intramolecular hydrogen bonds and their introduction into UiO-66 type frameworks through both direct synthesis and Solvent-Assisted Linker Exchange procedures. The presence of pendant nucleophilic groups such as amines and hydroxyl units on the MOFs’ organic struts, as well as amide moieties, have shown to increase the CO2 uptake capabilities of porous structures though favourable interactions between consequently created hydrogen bonds and the substrate. Most of these weak interactions generally take place between catenated frameworks and the synthesis of intramolecular hydrogen bond containing linkers is a path to a more rational and controlled way of introducing this kind of functionalisation into a MOF and therefore a path to a higher control over the MOF properties. The synthesis of MOFs containing these new linkers has, however, proven to be more delicate than expected in the first place, and so the unsuccessful initial direct synthesis attempts have directed the thesis to an alternative synthetic route through Solvent-Assisted Linker Exchange of the linkers of a pre-synthesised MOF with the new linkers. The principle and the results as well as the study of a limited incorporation rate in UiO-66 like frameworks alongside stability issues that arised from the presence of the amide containing linkers are described. Following that study, the treatment of the surface of the borosilicate glassware in which the MOF syntheses take place with a chlorinated organopolysiloxane coating has afforded the direct synthesis of single crystals of the desired zirconium MOFs. The loss of their structural integrity upon solvent loss seem to confirm what was observed in the SALE materials, that the amide linkers bring some instability to the usually very stable zirconium MOFs. Two of the amide containing linkers described in Chapter 2 were designed to have metal coordination pockets though a picolinamide moiety. Metalation of linkers containing coordination pockets in MOFs is one controlled way of creating well-defined catalytic centres. In Chapter 3, the coordination properties of these sparsely described binding units are investigated by the synthesis of molecular coordination complexes with the protected version of the linkers (protected carboxylic acid groups by esters that avoid unwanted complexation) and a range of transition metals. The confirmation of the coordinative properties of the picolinamide units by the synthesis of coordination complexes as single crystals was followed by a study of the geometrical deformation of the organic struts induced by the metalation and their potential structural compatibility with UiO-66 like frameworks. This first study was followed by the post-synthetic metalation of the zirconium MOFs described in Chapter 2. The catalytic properties of a palladium functionalised MOF toward Heck couplings were then investigated, and the scope for utilising these systems as heterogeneous catalysts discussed. Chapter 4 differs from the previous chapters by the nature of the investigated functionalised spacers and the resulting coordination networks. Pyridine-functionalised hybrid manganese Anderson POMs were studied as a new class of pre-functionalised linkers for coordination polymers. To situate the work in a larger perspective toward the interest of that approach, a specific introduction on polyoxometalates is present as the beginning of the chapter. This introduction describes the POMs’ nature, chemistry, properties as well as the previously described approaches with which they have been introduced in porous framework so far. The synthesis of six novel coordination polymers as single-crystals as well as their structural description and characterisations are discussed. Some synthetic guidelines are proposed for directing the self-assembly of coordination polymers of this type in future.

It is predicted that ocean acidification (OA) threatens coral reefs worldwide, by lowering seawater pH which in turn compromises essential metabolic processes such as carbonate genesis of corals. Inshore waters however, experience different spatial and temporal carbonate chemistry variability, raising questions over the future impact of OA within these habitats. It also remains unclear whether local biogeochemical conditions of some marine habitats can buffer, or provide a refuge against OA. The thesis systematically examines the response (ecological abundance, distribution, recruitment, and metabolic expenditure) of corals that have expanded their niche into variable pH habitats, to assess both the potential impact of OA and whether any habitats may act as a refuge against its effects by: (i) establishing robust methods to measure the local carbonate chemistry and the metabolic activity of corals in situ, (ii) characterising the natural carbonate chemistry variability over different temporal and spatial scales, and evaluating the biological versus abiotic control of non-reef habitats, (iii) quantifying the metabolic expenditure of corals living within non-reef habitats and assessing whether there are similarities in the physiological responses of corals existing in different regions to ascertain commonalities, and finally (iv) testing the impact of future predicted changes in temperature and pH on the physiological responses of corals from different variability habitats. The thesis demonstrates that across bioregion sites non-reef habitats exist that have highly variable carbonate chemistry but still house corals. These non-reef habitats have very different carbonate chemistry, influencing both their own susceptibility to future OA and their potential services (buffering versus pre-conditioning) for local coral populations. Future studies can expand on this work by assessing the molecular differences of corals found within these highly-variable habitats to explore further the potential of adaptation and/or acclimatisation of coral species to low pH.

The ability to adapt to changing environments is essential for survival. Bacteria have developed sophisticated means by which they sense and respond to stresses imposed by changes in the environment. Escherichia coli (E. coli) have served as a model organism for studies in molecular genetics and physiology since the 1960s. I have undertaken this study and address three outstanding questions. Firstly, the involvement of morphogene bolA and RNA polymerase sigma factor (rpoS) in biofilm formation. Secondly, the effect on respiratory activity of E. coli in presence and absence of these two genes and thirdly, the adherence pattern and formation of biofilm by E. coli on stainless steel, polypropylene and silicon surfaces under various stress-induced conditions. Bacterial biofilms are structural assemblages of microbial cells that encase themselves in a protective self produced matrix and irreversibly attach to surface. Their intense resistance to antibiotic and various environmental stresses has implicated them as playing a possible role in the pathogenesis of many chronic diseases. Although, the role of rpoS and bolA genes in long term stationary phase growth conditions and their response to it is now well-known, their objective presence and importance in short term response to different environmental cues which may lead to biofilm formation remains unknown. The rpoS gene encodes a stationary phase specific sigma factor of RNA polymerase and is a key regulator of E. coli stationary phase responses. It has been experiential under laboratory conditions that gene expression is induced by stressful environmental conditions and certain metabolic intermediates. Various stress environments were employed both in planktonic and biofilm phases to examine the sudden response of rpoS against different environmental conditions. However, it was observed that sudden rpoS response varies from stress to stress conditions. The gene bolA has been shown to trigger the formation of round cells when over expressed in stationary phase. From this research, it is concluded that bolA is not only confined to stationary phase, it also involves in biofilm formation under stress environments and essential for normal cell morphology. It also plays a major role in respiration and attachment of E. coli under diverse environmental stress surroundings. The main objective of this study was to understand the impact of heat, cold, acid and hydrogen peroxide on E. coli K-12 MG1655 and its stress response in presence and absence of rpoS and bolA genes. E. coli cells were exposed to sub-lethal levels of each stress for 15 minutes in both planktonic and biofilm phases and post-stress response i.e. gene expression level was evaluated. A real-time reverse transcription polymerase chain reaction (RT-PCR) assay, using the Applied Biosystems 7500™ real-time cycler, was developed for the purpose of this investigation of rpoS and bolA genes transcription. The assay was used specifically to quantify rpoS and bolA mRNA levels; however the method can readily be applied to the study of other E. coli genes. The method was uniquely applied to the investigation of these two genes throughout the growth cycle of E. coli in planktonic and biofilm phase in LB broth, in order to ascertain the patterns of expression for these genes. Scanning electron microscopy (SEM) was used for direct examining the cell attachment and biofilm formation on various surfaces under different stress conditions. In summary, this thesis embodies research investigating the role of rpoS and bolA genes in E. coli K-12 MG1655 biofilm formation and provides further evidence, that bacterial biofilms play a major role in resistance to various environmental cues.

Undifferentiated cancer stem cells (CSCs) with their unique potential of self-renewal and multi-lineage differentiation fuel tumour growth and relapse. Efficacy of glioma therapy can be considerably improved if the target is focused towards successful identification and elimination of CSCs. The aim of this research lies in defining specific and selective marker(s) to isolate glioma stem cells, to explore the differentiation state of brain tumour cells and to determine the protein profile changes that assist tumour cells to sustain stem cell-like characteristics. The three stem cell-related protein (CD133, Oct4-A and BMP3) expressions were investigated in control, hypoxic and serum-deprived U87-MG cells in order to shed light on the influence the micro-environment has in generating stem cells. Hypoxia offered a rapid state of undifferentiation as compared to serum deprivation by expressing a basal level of CD133 protein, a designated stem cell marker. Subsequent measurements of chemosensitivity and cell cycle analysis under undifferentiation conditions added to the cytotoxic potential of Taxol and showed an enhanced sensitivity of serum-deprived cells towards chemotherapeutic drugs. Moreover, proteomic analysis produced a wide dataset, depicting the changes that occur at the proteomic level in the differentiated and undifferentiated U87-MG cells. With ingenuity pathway analysis (IPA), human protein research database (HPRD) and the literature review, several proteins were proposed to be tested as potential biomarkers. They included Uracil DNA glycosylase (UDG), Phosphoglycerate kinase 1 (PGK1), Heterogeneous nuclear riboprotein K (HNRNPK) and moesin that can be used as differentiated markers for glioma cells. Vimentin, Eukaryotic translation initiation factor 4e (EIF4e), Casein kinase II alpha 1 (CSNK2A1) should be further investigated to study their precise role in gliomagenesis. Laminin binding protein associated with Integrin α6β1 and BMP2 should also be explored as a potential biomarker for isolation of glioma stem cells. This novel study envelops diverse aspects related to CSCs such as biomarkers, stem cell niche, chemoresistance, cell cycle and proteomics and also suggests the existence of two sub-types of CSCs within glioma population. It can be concluded that the finding thus obtained may be a step in the right direction in helping treat brain tumours.

The uptake of dissolved CO2 by phytoplankton in the surface ocean and its delivery to the deep ocean via the remineralisation of sinking particles, the biological pump, is an important control on the exchange of CO2 between the ocean and the atmosphere. Ocean biogeochemical models suggest that atmospheric CO2 is sensitive to changes in the depth at which the majority of particles have been remineralised in the ocean interior. However, the key mechanisms involved are not well understood. The function of the biological pump in the past and future is a large uncertainty for the carbon cycle. This thesis uses observations and modelling to further constrain our mechanistic understanding of the biological pump. Geographically Weighted Regression is applied to an updated sediment trap dataset to explore the spatial variability in statistical relationships between organic matter and CaCO3 that are the basis for the ballast hypothesis. No uniform strong relationship at smaller spatial scales and patterns consistent with surface biogeochemistry suggests ecosystem processes may be important. In response to the limited sampling of particulate uxes analysis explored whether annual average uxes could be estimated from a PO4 climatology using modelled ocean transport rates in the form of a transport matrix. The Earth System Model GENIE was used to create a synthetic dataset to test this approach, �nding signi�cant sources of uncertainty from errors in the observations, the use of modelled transport rates and the assumption that remineralisation is from particles only. The transport matrix formed a basis for a steady-state phosphorus-only model used to �nd optimal solutions of spatially varying remineralisation using a 600 member Latin Hypercube ensemble and observed [PO4]. Modelled [PO4] was predominantly sensitive to global mean remineralisation depths although some spatial variability could be constrained. This has implications for using nutrient distributions to validate mechanistic parameterisations in models.

Streptomyces are filamentous soil-dwelling bacteria with a complex life cycle. Elucidating the signals that regulate morphological change in microbes is of fundamental importance for biotechnology applications. In the case of Streptomyces a development switch occurs with the concomitant production of secondary metabolites, many of which have pharmaceutical properties. For the industrially used strain S. lividans this switch is dependent on the bioavailability of copper (Cu) in the environment. This thesis has explored the relationship between Cu-chaperones and a haem peroxidase, part of the sco operon, with the maturation of a Cu-containing oxidase, GlxA. In S. lividans the GlxA gene is part of the cslA/glxA operon that contains genes encoding putative enzymes involved in glycan processing. Both these gene clusters are highly conserved in streptomycetes. In Chapter 2 the characterisation of GlxA is reported. It was found to be membrane associated with a mononuclear Cu site and possess a Cys-Tyr redox cofactor capable of housing a protein radical, comparable to the fungal galactose oxidase (Gox). The tertiary structure of GlxA revealed a unique domain arrangement, atypical spectroscopic properties compared to Gox and a lack of enzymatic activity with classical Gox substrates. Generation of the ΔglxA null mutant was found to stall aerial hyphae development on solid media and dramatically change the morphology in liquid cultures. This was ascribed to the absence of the oxidation of a glycan by GlxA produced by CslA (a cellulose-synthase), required for morphogenesis on solid and liquid cultures. The molecular nature of this glycan is unknown. A number of GlxA variants were created in Chapter 3 to elucidate the proteins unique spectroscopic properties. It was found that the second coordination sphere residue, Trp288, plays a major role in tuning the electronic properties of the buried Cu site in GlxA. Its removal abolishes the Cys-Tyr radical and perturbs the spectroscopic properties such that they resemble Gox. Monoclonal antibodies were used to follow the maturation of GlxA through observing mobility differences on denaturing PAGE gels based on the presence or absence of the Cys-Tyr cross-link. X-ray crystallography provided structural insight into the maturation process. A surprising outcome of Chapter 3 was that upon removal of the crosslinking Cys121, a new protein radical is formed as opposed to the expected abolition. Chapter 4 addresses another surprising finding in that a putative haem peroxidase (DtpA), part of the sco operon, plays a role in GlxA maturation and in the Cu-dependent morphological development. DtpA is shown through enzymology and structural analysis to be a member of the dye-decolourising peroxidase (DyP) family. Crucially, it is shown that DtpA functions as a peroxidase in the presence of GlxA using the GlxA substrate, glycolaldehyde. Synthesis and modification of the CslA/GlxA glycan will inevitably require degradation during the life cycle. As part of the cslA-glxA gene cluster are two genes encoding for putative polysaccharide degrading enzymes. One of these is a putative Cu lytic polysaccharide monooxygenase, SliLPMO10E. Chapter 5 structurally characterises SliLPMO10E and also investigates the kinetics of Cu-binding. The latter brings to the attention that LPMOs are able to bind Cu in two forms at a single site before relaxing into a final substrate active form. Importantly, SliLPMO10E is found to be active only with chitin via a C1 sugar ring oxidation mechanism. This hints at the possibility that the glycan produced by CslA and modified by GlxA is chitin-like possessing N-acetyl glucosamine moieties. By combining the in vitro results from this thesis together with the in vivo results obtained through the duration of this work from collaborators at Leiden University an overall model of the Cu-dependent morphogenesis and glycan processing in the hyphal tips of S. lividans is presented. Chapters 2 through to 5 focus on events that occur under Cu limitations, i.e. homeostasis. Chapter 6 extends on previous work that characterised the CsoR regulon. The Cu sensitive operon repressor (CsoR) protein determines the set point of Cu(I) concentration in the cytosol. Under Cu stress, Cu(I) binds to CsoR and de-represses genes under its transcriptional control. Chapter 6 explores the possibility of whether CopZ-like Cuchaperones can traffic Cu(I) to the DNA-bound CsoR, resulting in the up-regulation of control systems to return the cell to homeostasis. Size-exclusion and EMSA studies showed that Cu(I) was transferred from CopZ to CsoR in a unidirectional manner. Re-analysis of previous RNA-seq data using the S. lividans genome as input, enabled for a more complete model for the CsoR regulon in S. lividans to be proposed.