Atherosclerosis, a chronic inflammatory disorder of the vasculature, is one of the major causes of cardiovascular disease and is responsible for most deaths in western societies. The disease is characterised by a number of steps that occur during the lifespan of an individual, including fatty streak formation, development of complex lesions containing a fibrous cap, and thinning and rupture of such plaques leading to thrombosis and clinical complications of this disease. Macrophages play key roles during all stages of this disease such as foam cell formation, amplification of the inflammatory response and control of plaque stability. The actions of macrophages during this disease are regulated by cytokines present in atherosclerotic lesions such as interferon-γ (IFN-γ), tumour necrosis factor-like protein 1A (TL1A) and interleukin-17 (IL-17). Statins are widely used for the primary and secondary prevention of atherosclerosis and its complications because of their ability to inhibit cholesterol biosynthesis and, thereby, plasma levels of pro-atherogenic low density lipoprotein. However, statins have actions beyond lowering cholesterol levels, the so-called pleiotropic effects, and includes acting in an anti-inflammatory manner. Unfortunately, the anti-inflammatory effects of statins are not fully understood and therefore formed the focus of studies in this thesis using a combination of human macrophage THP-1 cell line, primary cultures of human monocyte-derived macrophages and mouse RAW264.7 macrophage cell line. Simvastatin generally acted in an anti-inflammatory manner in macrophages in relation to the expression of several pro-atherogenic genes, such as monocyte chemoattractant proein-1 (MCP-1) and intercellular adhesion molecule-1 (ICAM-1),regulated by IFN-γ, TL1A or IL-17. Such an anti-inflammatory action also extended to another statin, Atorvastatin. The inhibitory action of Simvastatin on IFN-γ induced expression of MCP-1 and ICAM-1 was reversed, at least in part, by intermediates of the 3-hydroxy-3-methyl-glutaryl coenzyme A pathway such as farnesyl pyrophosphate and geranylgeranyl pyrophosphate. This suggested a potential role for monomeric G-proteins that require such intermediates for activation. In addition, Simvastatin inhibited the phosphorylation-mediated activation of signal transducer and activator of transcription-1 (STAT1), a key transcription factor in IFN-γ signalling, on tyrosine 701 and serine 727 in response to the cytokine. The effect of Simvastatin on MAP Kinase (MAPK) pathways in macrophages was also analysed. The statin attenuated the IFN-γ induced activation of p38 MAPK and extracellular signal activated kinase (ERK)-1/2. Simvastatin also affected the constituve expression of many components of the MAPK pathways (e.g. ERK-1/2) along with downstream genes involved in atherosclerosis (e.g. ATP-binding cassette transporters-A1 and-G1). The effect of Simvastatin on lipid profile of THP-1 and RAW264.7 macrophages was also investigated. Simvastatin does not affect total polar lipids and triacylglycerol. The statin also had no significant effect on fatty acid distribution into polar lipids and triacylglycerol. The studies presented in this thesis provide insights into the actions, and potential mechanisms, underlying the anti-inflammatory effects of statins on human macrophages along with their effects on lipid profiles. Such studies are essential given the widespread use of statins and a need to gain a deeper understanding of their actions.

The complexity and size of the higher animal genome and relative scarcity of DNA-binding factors with which to regulate it imply a complex and pleiotropic regulatory system. Cisregulatory modules (CRMs) are vitally important regulators of gene expression in higher animal cells, integrating external and internal information to determine an appropriate response in terms of gene expression by means of direct and indirect interactions with the transcriptional machinery. The interaction space available within systems of multiple CRMs, each containing several sites where one or more factors could be bound is huge. Current methods of investigation involve the removal of individual sites or factors and measuring the resulting effect on gene expression. The effects of investigations of this type may be masked by the functional redundancy present in some of these regulatory systems as a result of their evolutionary development. The investigation of CRM function is limited by a lack of technology to generate and analyse combinatorial mutation libraries of CRMs, where putative transcription factor binding sites are mutated in various combinations to achieve a holistic view of how the factors binding to those sites cooperate to bring about CRM function. The principle work of this thesis is the generation of such a library. This thesis presents the development of microstereolithography as a method for making microfluidic devices, both directly and indirectly. A microfluidic device was fabricated that was used to generate oligonucleotide mixtures necessary to synthesise combinatorial mutants of a CRM sequence from the muscle regulatory factor MyoD. In addition, this thesis presents the development of the optimisation algorithms and assembly processes necessary for successful sequence assembly. Furthermore, it was found that the CRM, in combination with other CRMs, is able to synergistically regulate gene expression in a position and orientation independent manner in three separate contexts. Finally, by testing a small portion of the available combinatorial mutant library it was shown that mutation of individual binding sites within of the CRM is not sufficient to show a significant change in the level of reporter gene expression.

The modification of natural nucleic acids or synthesis of novel DNA mimics can facilitate new structure, function and properties. In particular, the use of modified nucleic acids for applications in biosensing has become a popular field of study given the desire for rapid and reliable theranostic devices. The aim of the projects detailed in this thesis was to study a range of DNA modifications, with a view to gaining an enhanced understanding of their effects on DNA structure, but also on their ability to act as sensing platforms for the extraction of important biological information stored within DNA targets. The four projects discussed include: organometallic mimics of DNA based on ferrocene (FcNA) and corresponding FcNA-DNA conjugates and their effect on structure; FcNA-DNA conjugates that bind mercury; redox-active macrocycles incorporated into DNA as SNP sensors and DNA labeled virus particles that probe the presence of pathogens via a bionanoparticle supported PCR reaction which can be monitored by Linear Dichroism spectroscopy.

GCN4 is a yeast transcriptional factor, which binds sequence selectively to DNA as a homodimer. Two domains are required to achieve this, an N-terminal basic domain, which interacts with the target site directly, and the C-terminal leucine zipper responsible for dimerisation. The latter can be replaced by chemically linking together two basic domains, to retain similar DNA binding. This work investigates using a ferrocene complex as the dimerization unit due to its flexibility and its characteristic electrochemical redox properties. A strategy was proposed which took advantage of coupling to the thiol side chains of two cysteine residues, and detailed studies on dimerising two equivalents of Cys, or the tripeptide glutathione, were performed. Design of five peptides based on GCN4, which were synthesized and dimerized with a ferrocene linker molecule, to combine the attractive redox electrochemistry of the stable ferrocene and the DNA binding ability of GCN4 dimerized basic domain. Molecular dynamic models were produced for four of the ferrocene peptide dimer complexes bound to target DNA over 10 ns. Their DNA binding ability was investigated by circular dichroism, and electrochemistry was studied in the absence and presence of non-specific and target DNA respectively.

Work described in this thesis shows the design, synthesis, DNA binding activity and gold nanoparticle interaction of functionalised triple stranded supramolecular helicates. DNA structures and the way molecules recognise and bind to them are reviewed, with specific emphasis on supramolecular compounds. Supramolecular helicates are discussed in detail with consideration of how they may be used as anticancer agents. Nanoparticles, specifically gold nanoparticles are studied, with specific reference to how they are able to enhance anticancer properties of drug molecules and how they may be used to develop potent anticancer therapies. With the formation of three novel supramolecular iron helicates and the incorporation of supramolecular chemistry and nanotechnology, through binding such complexes to the surface of gold nanoparticles, foundations of work in this area are discussed, showing promising results for future research.

Single nucleotide polymorphisms (SNPs) are single base variations in DNA which give genetic variation. However, SNPs can also be linked to the development of certain diseases. Modified oligonucleotides used to probe biological changes and processes have become an important focus of scientific research. Fluorescent tagging of DNA can be used to sense SNPs in DNA targets through differences in emission intensity on the formation of a duplex. An anthracene-tagged DNA probe developed by Tucker \(et\) \(al\). is able to discriminate between a fully complementary DNA target sequence and one with a single base difference. This thesis describes how SNP sensing with anthracene-tagged DNA has been extended to SNPs in RNA targets and sequences associated with Alzheimer's disease. Finally, a new dual fluorophore DNA probe was designed for SNP sensing via FRET.

Within this thesis, the use of anthracene to perform various applications within biomolecules is assessed. Anthracene displays two interesting photo properties which make it an appealing molecule for incorporation; fluorescence and photodimerisation. The former is utilised to develop a single nucleotide polymorphism detection assay which is shown to allow for determination of the base present in a complementary strand of DNA. In addition to this, the photodimerisation properties of anthracene are used within a protein for the first time. This is utilised to develop a photoswitched binding protein, allowing for control of DNA binding of the protein. Further to this, the photodimerisation properties are utilised within oligonucleotides to achieve structural control of a G-quadruplex as well as photo-triggered release of single stranded DNA.

The partitioning of low-copy number broad host range plasmids depend on a centromere binding protein (CBP) that binds to a centromere-like site on plasmid. For RK2 plasmid, the CBP is a 358 residue, multi-domain protein, KorB. KorB contains an N-terminal domain (NTD), a central DNA-binding domain (DBD), and a C-terminal dimerisation (CTD) domain and the protein binds to the O\(_B\) site on the plasmid as a dimer. Structures of central DBD and CTD have been elucidated whilst limited information is available about the N-terminal of the KorB. In this study, NTD KorB protein was expressed and purified. Size exclusion chromatography and analytical ultracentrifugation data confirm that the NTD KorB behaves as a monomer in solution. Using solution-state NMR spectroscopy data, majority of the backbone and side-chain resonances for the NTD KorB are assigned and an ensemble structure of the protein is calculated. The flexibility of the NTD KorB is studied with coarse-grain Molecular Dynamics simulation package, AWSEM. The N-terminal of the NTD KorB is mostly unstructured whilst two α-helices towards the C-terminal of the protein exhibit limited motion. Two C-terminal KorB deletion mutants capable of binding the OB DNA (CΔ100 KorB and NΔ31CΔ100 KorB) were purified and characterised using circular dichroism (CD) and mass spectrometry. DNA-binding properties of these two deletion mutants are compared to the KorB wild-type (WT) using circular dichroism, fluorescence anisotropy and microscale thermophoresis measurements, indicating weaker binding of the deletion mutants with respect to the KorB WT to its centromere-like site (O\(_B\)). Considering the current state of structural information about the KorB and homologous proteins, a DNA partitioning model for the KorB is proposed.

The structure, occurrence and biological relevance of G-quadruplex DNA structures has been reviewed, along with a review of several notable G-quadruplex binding compounds published in the literature to date. The synthetic route towards two G-quadruplex DNA binders previously developed within the Hannon group has been modified and improved. Electrospray ionisation mass spectrometry studies have been carried out to evaluate nucleotide binding. The in vitro biological activities of these compounds have been validated against the human ovarian carcinoma cell line A2780 via MTT and comet assays, flow cytometry and inductively coupled plasma mass spectrometry. Both compounds and the corresponding metal-free ligand exhibited higher drug efficiencies than cisplatin against A2780 cells. Both compounds display mild genotoxicity and induce G2/M phase cell cycle arrest. The overall cellular uptake and nuclear localisation demonstrated by both complexes exceeds that of cisplatin. A new class of palladium and platinum(II) complexes have been synthesised from methylthio-substituted terpyridine ligands. In addition to assessing their stability in solution via UV-Vis spectroscopy, initial DNA binding studies with both duplex and quadruplex-forming sequences of DNA have been carried out via circular dichroism and gel electrophoresis. The design and synthesis of alternative ligand systems proffering a range of desirable characteristics to aid future ligand and complex development has been investigated.

DNA sequencing has vastly opened up the world of molecular biology, leading to new areas of interest, especially in medical research. Unfortunately the methods of DNA sequencing have only ever seen gradual improvements, as Sanger sequencing is still very much the norm despite its high cost and slow speed. Nanopores present an exciting opportunity for DNA sequencing, however, despite the concept being presented in 1996 several problems have prevented the creation of a publicly available sequencing device. The two main focuses of research into nanopores so far have been improving the resolution between bases and the slowing down of DNA translocation through the pore so modern ammeters can read the sequence accurately. The simulation work presented in this thesis largely focuses on the energetics associated with DNA translocation. This is performed in several parts; an investigation into the probability of pore entry, study into the free energy of translocation for two proteins in addition to solvent contribution to this free energy, finally a theoretical project was undertaken to investigate bottom up nanopore design.