Cardiovascular disease is a major killer worldwide and it is becoming clear the significance of our diet in curbing the disease. Green tea is one of the most widely consumed beverages in the world and has recently attracted significant attention in the scientific community for its health benefits. Its consumption has been associated with lower incidences of coronary artery diseases in the Japanese population. This is mainly attributed to its polyphenolic constituents that include epicatechin, epicatechin gallate, epigallocatechin and epigallocatechin gallate. The aim of this research was to synthesise the four major polyphenols present in green as well as analogues. These compounds would then be tested on H9C2 cardiac myoblast cells and neonatal rat cardiomyocytes in order to further understand the structure-activity relationship as well as potentially improve the cardioprotective function of these polyphenols following oxidative stress and ischaemia/reperfusion injury focusing on the expression of STAT-1 and ERK-1/2 proteins. In H9C2 cardiac myoblast cells following the induction of oxidative stress using H2O2, EGCG, EGC and to a minor extent ECG inhibited STAT-1 activation but not ERK- 1/2 phosphorylation suggesting that although the ERK-1/2 pathway gets activated, its downstream activation of STAT-1 is inhibited by the above polyphenols. EC, on the other hand, inhibited ERK-1/2 activation which in turn cannot activate STAT-1. Quantitative assessment of viable cells showed that pretreatment with EGCG resulted in the lowest amount of non-viable cells reducing cell death by 30%. With neonatal rat cardiomyocytes following ischaemia/reperfusion injury, pretreatment with EGCG reduced the amount of non viable cells by 5% but pretreatment with acetylated EGCG at half the concentration of EGCG reduced non-viable cells by 8%. Structure-activity relationships of the green tea polyphenol analogues identified some key aspects in the structures of the polyphenols important in their cardioprotective function. Results indicated that ABD ring system is required for cardioprotective function but the presence of a third OH group in the ring may not be necessary. Substitution of ring C with benzoic and naphthoic rings improved the potency by more than 13-fold compared to EGCG with EC50 values of 1.60 and 0.77 μM respectively. Further research into these analogues could realise their potential and contribute to the understanding of the cardioprotective activities of green tea. A review on the previous synthesis approaches, isolation and biosynthesis of the green tea polyphenols is presented in Chapter 1 and also the different signalling pathways of interest in this work. An evaluation of the biological activities of the four major polyphenols is provided in Chapter 3. Experimental procedure and characterisation data are in Chapter 5.

This thesis describes the synthetic work towards the natural product Chrysophaentin F which has been extracted from the alga Chrysophaeum taylori. This bisdiarylbutene macrocycle exhibits antimicrobial properties against gram-positive bacteria, including methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus faecium (VREF) (MIC50 (MRSA) = 4.2 ± 1.3 μg/mL). Investigation of the key steps on an unchlorinated analogue allowed us to determine what strategy would be best to access the natural product. The formation of the desired core structure relied on a Chan-Lam-Evans coupling reaction, a Pd catalysed coupling reaction and a RCAM reaction to create the pivotal bonds of the complex scaffold. The investigation on a model system to form the vinyl chloride bridge has also been performed bringing insight on the possible regioselectivity of the necessary late stage hydrochlorination reaction. Finally the synthesis towards the chlorinated macrocycle was started and advanced enough to access some key intermediates which despite some unforeseen difficulties proved that the natural product is now at reach following the strategy developed for the unchlorinated analogue.

Whilst the interest in open innovation is growing, thus far few studies have emphasised its importance beyond hi-tech industries and large multinationals. This research aims to generate an understanding of open innovation adoption within the UK Bio-pharmaceutical industry, which is chosen because of the significant growth it has experienced in recent times, and is heavy reliance on R&D. The findings of this multi-level study present a holistic view of the opportunities and barriers pertaining to open innovation in Biopharmaceutical SMEs and large firms, as well as indicating that open innovation strategies assist firms’ in terms of value creation and capture. The study also illustrates that strategies cannot necessarily be regarded as explicitly open or closed innovation, as there is a significant spectrum of approaches in the space between. The study’s theoretical contribution develops the use of critical realism, a scientific reality which is not only about constant combinations of observable events but is also about individuals, entities and structures that exist and generate the events we witness and observe. In doing so, the study utilises an alternative to inductive or deductive reasoning, the retroductive reasoning. Through retroduction, the study to explain these causal powers, mechanisms, as well as the contingent relations of individuals that are responsible for the creation of a firm’s strategic considerations based on a multi-layered approach (micro-with individuals/meso-with various organisations/macro-with various government bodies and organisations). The study comprises three sets of results, focusing on CEOs, knowledge brokers and senior executives respectively. The first set of results, based on 30 interviews of CEOs in Small-to-Medium sized Enterprises (SMEs), suggests that i open innovation practices are utilised in a multi-level, but that not all SMEs adopt the open concept. This shows the reluctance of CEOs in SMEs in sharing internal information and intellectual property. The second set, obtained from interviews with 8 Knowledge Brokers in the industry, emphasised the need for an agenda when open practices are utilised and there are personal preconceptions regarding business, which are in many cases difficult to change or adjust during open approaches. The third set, obtained from a survey questionnaire with 12 executives of 10 large biopharmaceutical firms, illustrates the importance their firms place on open innovation practices, particularly in collaborating with various firms and organisations. In spite of the industry’s recent efforts, the study identifies that benefits from the adoption of open innovation are yet to be seen, as the realisation of research and development outcomes within the Biopharmaceutical sector is a long process. Furthermore, the study clarifies that open innovation is not a monopoly of large firms in the high-tech sector, but can be adopted in various other firms in different industries. Additionally, this study contributes to the expansion of multi-layered approaches, as they give a thorough view of the processes that are involved during open innovation adoption. Finally, the research addresses the lack of empirical work in open innovation and suggests methods that identify how and why particular processes are utilised when open strategies are adopted, and elucidates the space between closed and open innovation.

Drugs that are preferentially absorbed through the stomach or the small intestine have a narrow time window for absorption since passage through this region of the gastrointestinal tract is rapid. A drug delivery system that can adhere to the gastric epithelium will substantially slow down drug transit and help overcome this problem. To achieve this, this study proposes the novel use of a glycan-binding adhesion protein from Helicobacter pylori, BabA, to create targeted drug delivery vectors that can mimic the attachment of this bacterium to the gastric epithelium. In this work, a recombinant form of BabA was expressed in the periplasmic space of Escherichia coli; it was found that after the incorporation of a C-terminal hexa-lysine tag, the expression and purification of this protein was significantly improved to amounts that enabled its subsequent characterisation and application. Recombinant BabA retained the highly selective glycan-binding properties of H. pylori and next, its crystal structure was solved in the absence and presence of Lewisb – a glycan well studied for its role in serving as a receptor for BabA. The structural models revealed that Lewisb binding occurred through a network of hydrogen bonds within a single, shallow binding pocket at the tip of a β-unit in BabA. Binding studies then confirmed that this site was also responsible for the recognition of other glycan receptors. Using this insight, recombinant BabA was conjugated to model drug delivery vectors via a linkage that favoured exposure of its glycan-binding β-unit; the binding properties of BabA successfully translated to these model BabA-vectors. The research presented in this thesis lays a strong foundation for future work to assess the in vitro and in vivo efficacy of biomimetic BabA drug carriers.

Ganoderma lucidum also known as lingzhi or mushroom of immortality in East Asia countries, has been known for over 2000 years. Ganoderic acids (GA), which are important secondary metabolites of this mushroom, are highly oxygenated lanostane-type entities with extraordinary pharmacological properties. GA are biosynthesised from lanosterol by a still unknown mechanism. The synthesis of thirteen GA analogues and the biological evaluation of ten of them against prostate cell-lines is described in this thesis. Analogues with different functionalities at C-3, C-7, C-8, C-9, C-11, C-23, C-24, C-25 and C-26 of the lanostane frame have been synthesised. As functional groups, alcohols, acetates, ketones, carboxylic acids and single and conjugated double bonds have been introduced. For their preparation, aldehyde 89 was formed from commercial lanosterol in up to 47% overall yield over a 3 or 4 step sequence. Wittig olefination, Reformatsky reaction and direct oxidation of aldehyde 89 provided the precursors from which the analogues were achieved. Analogues were prepared after a total of six to eight steps in 7-44% overall yield. GA and their analogues inhibited the cell viability of prostate cell-lines. However, they were less effective in the inhibition than Taxotere® (docetaxel). Besides, by comparing their IC50 values and thus, building a structure activity relationship (SAR) analysis, the most important positions for activity were determined. Interestingly, some of the ganoderic acids, GA-H (24), and analogues 3-ketone 108 and 7,11-diketone 146, were less toxic for the normal prostate cells than for the pre-malignant ones, conferring on them prophylactic activity. It is believed that ketones at C-3, C-7 and C-11 could be responsible for this behaviour. Ganoderma lucidum is rare in nature. However, due to its great demand, it is now being farmed. For their cultivation sawdust, logs or cork is used. New substrates, otherwise disposed as waste, are being investigated to produce high quality mushrooms, among them oil palm fibres such as mesocarp or empty fruit bunch fibres. Therefore, to unveil new cultivation methods, the triterpene composition of the mushroom grown at different stages, with different substrates or conditions has been assessed and their biological activity evaluated against prostate cancer cell-lines. Cultivating the mushroom in mesocarp or empty fruit bunch fibres produced similar results to those of sawdust. However, poor ventilated conditions reduced their biological activity against prostate cancer cell-lines. Likewise, it was established that triterpene composition varied during different stages and for different target body parts, their biological activity also varied with age, increasing as the mushroom aged during the 2- to 8-week period studied.

In order to develop novel antibacterial therapies that combine anti-adhesion, anti-quorum sensing and the delivery of conventional antibiotics, the effects of polymers on bacterial aggregation and quorum sensing (QS) were studied. QS is a term used to describe method by which bacteria use chemical signal molecules to modulate pre-infection behaviour such as surface attachment. Polymers that can interfere with bacterial adhesion or the signal molecules used for QS are therefore a potential means to control bacterial population responses. In this thesis, the ability of the cationic polymers poly (N-[3-(dimethylamino) propyl] methacrylamide) (p(DMAPMAm), P1) and poly (N-dopamine methacrylamide-co-N-[3-(dimethylamino) propyl] methacrylamide) (p(DMAm-co-DMAPMAm), P2) to cluster a range of bacteria, such as Staphylococcus aureus(Gram-positive), Vibrio harveyi, Escherichia coli and Pseudomonas aeruginosa(Gram-negative) under conditions of varying pH and polymer concentration was investigated. It was identified that clustering ability was strongly dependent on the balance between charge and hydrophobicity. The results also suggested that catechol moieties might have a positive effect on adhesive properties. Moreover, the potency of polymers against QS of Vibrio harveyi was assayed via testing bioluminescence. P1 which was able to bind to the surface of bacteria through electrostatic interactions enhanced the expression of QS and P2 which could bind to both the bacteria and QS signals showed the ability to both enhance and reduce light production. Furthermore, polymeric vesicles made of copolymers containing poly (3,4-dihydroxy-L-phenylalanine methacrylamide) (p(L-DMAm)) which displayed similar dual affinity compared toP2 were prepared and their ability to modulate QS responses in Vibrio harveyi was demonstrated. All the vesicles showed higher potency in quenching bioluminescence than their linear polymer analogues. To explore the feasibility of using self-assembled polymers for anti-microbial drug delivery, silver loaded DOPG lipid vesicles were made and were found to interfere with QSwhile reducing bacterial viability when the concentration of Ag+ was above the MIC (0.1 μg/mL). The results overall suggested that combined antimicrobial therapies might be possible using polymers and both QS and cytostatic or cytotoxic agents.

Amphipathic, non-ionic surfactants are widely used in pharmaceutical and food industries to enhance product features; as pharmaceutical excipients they achieve increased cell membrane permeability and consequently can improve the oral absorption of drugs across the intestinal epithelial barrier. The use of non-ionic surfactants has grown rapidly, and is predicted to increase, however, the mechanism(s) surrounding the induction of surfactant toxicity is not well established and, consequently, the potential risks of surfactant exposure are not well understood. This work studies the concentration- and time-dependent succession of events that occur during and following exposure of an intestinal epithelial cell model to a ‘typical’ non-ionic surfactant – Solutol HS15. The resulted gathered demonstrate that prior to a significant increase in membrane permeability to a model drug (FITC-dextran 4kDa), non-ionic surfactant, at concentrations above its critical micellar concentration (CMC), produced almost immediate redox and mitochondrial effects manifested as an increased NADH pool, increased ROS levels, and hyperpolarisation of the mitochondrial membrane potential. Apoptosis was triggered early in this initial phase, and relied on mitochondrial hyperpolarisation as a crucial step leading to subsequent depolarisation and caspase-3/7 activation. Inhibition of mitochondrial hyperpolarisation prolonged cell survival, delayed the onset of metabolic reduction by the mitochondrial, and inhibited caspase activation. The apoptotic cell death pathway appears to be triggered prior to the emergence of substantial membrane damage by the surfactant: loss of plasma membrane integrity, nuclear membrane permeabilisation, and perturbations in calcium homeostasis - indicators of a necrotic process. It is proposed that the rapid cellular response is triggered via rapid surfactant-induced increases in plasma membrane fluidity; a phenomenon akin to the membrane-regulated stress response following membrane fluidisation by heat shock, and consequently cell death events. Furthermore, work performed on differentiated Caco-2 monolayers, alongside culture models replicating the basement membrane and paracrine signalling, demonstrate surfactant toxicity is reduced. Toxicity in vivo is therefore predicted to be less than measured on the standard model.

Spleen tyrosine kinase (Syk) plays a critical role in regulation of immune and inflammatory responses. The important role of Syk in inflammatory signalling cascades has led to the development of therapeutic agents designed to knock down Syk gene as novel therapeutic agents for allergic diseases (1). This thesis investigated the role of siRNA in Syk silencing using the newly developed RBL reporter systems as an in vitro model to study degranulation. Lack of an efficient and safe non-viral delivery systems has hindered the progress of siRNA into clinics. The ideal properties of the siRNA-nanoparticles for efficient delivery would require the following properties, 1. Safe with low toxic effects to the cells (non-toxic polymers and nanoparticle components); 2. Compact and well-condensed nanoparticles (thiol crosslinking and PEG); 3. Small size (20–200 nm) for easy cellular internalization (thiol crosslinking and PEG); 4. Less interaction with the environment to enable access to the target cells (PEG); 5. Neutral surface charged and sterically stable siRNA polyplexes with bio-reducible characteristics essentially required for in vivo delivery (Thiol-modified siRNA and PEG). Hence, low-toxicity linear cationic polyamidoamine (PAA) consisting of a mixture of PEGylated copolymer (CP) and non-PEGylated homopolymer (HP) were used for the formulation of the PAA-siRNA nanoparticles. This system has previously been shown to assemble into small sterically stabilised particles with reducible crosslinks. A cross-linked system of HP and HP-CP blends showed complete incorporation of thiolated siRNA during gel electrophoresis, and produced particle size less than that of non-thiolated siRNA as determined using a dynamic light scattering technique. A degranulation inhibition assay revealed that these cross-linked polyplexes prepared using thiol-modified siRNA efficiently inhibited release of fluorescent granules (NPY-mRFP) from the genetically modified RBL-2H3 NPY-mRFP cell line. There was no cytotoxicity associated with this delivery system. By using HP/SH-siRNA nanoparticle, a remarkable dose-dependent Syk gene silencing (~80%) was observed on mRNA level using RT-qPCR. Finally, the HP/SH-siRNA polyplexes produced also showed a significant reduction in Syk protein levels using Western blotting. From the data obtained it is concluded that cross-linked siRNA polyplexes prepared with thiolated siRNA are safe, effective and easy to prepare for future siRNA in vivo gene silencing applications to treat allergic diseases.

The most successful antimicrobial agents in clinical use are of microbial origin and of these the greatest variety has been found in the genus Streptomyces. However, the resistance of the pathogenic microbes to the commonly used antibiotics is increasing as a result of the wide-spread and long-term use of these antibiotics. Therefore, understanding the strategies that bacteria use to become resistant is of crucial need. Streptomycetes are Gram positive bacteria, commonly found in soil and are known antibiotic-producers. The focus of this thesis was to underpin the mechanism of resistance to penicillin G in isolated strains of streptomycetes that exhibit elevated resistance to penicillin G and to characterise these organisms. Moreover, to investigate the interaction between penicillin G and PBPs in Streptomyces strains and investigate the relationship between growth rate and penicillin G resistance in Streptomyces in vitro. Ninety six Streptomycetes were isolated and characterized. Morphological examination and the16s rRNA sequences of these strains indicated that strains belong to the species Streptomyces. The MICs and MBCs for penicillin G for the isolated Streptomyces strains were measured by plate culture. Some strains showed growth up to 400 μg/ml with penicillin G, which indicate that the strains were highly resistant against penicillin G. Some strains were unable to grow at penicillin concentrations above 200μg/ml. Also, The MICs of penicillin G for isolated Streptomyces strains were measured using a novel OxoPlates® system in 96-well culture format employing Mueller-Hinton broth culture. The MICs of all strains ranged from 1-100 μg /ml. Results indicate that the sensitivity of Streptomyces strains of penicillin G is not directly related to β-lactamase production in the panel of isolates examined. There was no correlation between the MICs of penicillin G and the growth rate in these isolates. Likewise, there was no association between the position of beta-lactamase producing and non-beta-lactamase producing strains on the phylogenetic tree and their beta-lactamase xii activity. Beta-lactamase producing and non-producing strains refers to the same ancestral origin clade. Additionally, the comparative analysis of 16S rRNA gene sequence and phylogenetic relationship of strain (W43) revealed that the isolate clustered with (W76) Streptomyces lividans strain YLA0. Bocillin (a penicillin binding protein stain) staining in β-lactamase producing strains showed staining throughout the mycelia whereas in non β-lactamase producing strains staining only occurred in certain parts of the mycelia. Bocillin also revealed that in spores PBPs were located on both poles of the spores. Streptomyces coelicolor has the ability to grow at high concentrations of penicillin G up to 640 μg/ml in continuous culture. It also has the capacity to grow at very low amounts of dissolved oxygen in continuous culture. Significantly, there was a correlation between the growth rate of S. coelicolor and the resistant to penicillin G. S. Coelicolor was more sensitive to penicillin G at a high dilution rate. Furthermore, our strategy of using the Bug-Lab for monitoring the progress of S. Coelicolor 1147 in continuous culture, even at low concentrations of cells in real time was successful.

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