The aim of this research is to develop a range of new ruthenium(II) polypyridyl complexes to be analysed spectroscopically in the presence of calf thymus-DNA to determine their relative binding affinities to the polynucleotide, thus determining the best criteria for a detection tool for DNA. Reactions of selected functional groups on one ligand of ruthenium(II) precursor complexes lead to the synthesis of six benzothiazole containing complexes; [Ru(bpY)2(B16)]2+,, [Ru(bpy)2(P5)]2+, [Ru(phen)2(B16)]2+, [Ru (phenh(P5)]2+, [Ru(Me2bpY)2(B16)]2+ and [Ru(Me2bpy)2(P5)]2+ and four "control" complexes; [Ru (bpy)2(B2)]2+, [Ru(bpy)2(P2)] 2+, [Ru(phen)2(B2)]2+ and [Ru(phen)2(P2)]2+ bearing methyl groups. Investigation established a two-step interaction occurring between the complexes and polynucleotides, which have varying effects on the luminescence of the complexes, as a result samples were equilibrated for 24 hours before steady state measurements were recorded in the presence of DNA. Denaturation studies of DNA in the presence of each complex did not show a significant change in the melting temperature to free DNA. UV / Vis absorption spectrometry of the complexes showed variation in the absorption attributed to the benzothiazole functionalised ligand and each complex became more luminescent with increasing concentrations of DNA. The circular dichroism spectra of ct-DNA and increasing concentrations of each complex indicate that the double helical shape of the DNA is not affected as the complexes interact. From equilibrium dialysis studies with each complex it can be tentatively assigned that the ∆-enantiomer is associating most favourably with DNA. There are no significant alterations between the spectra of the methyl functionalised "control" complexes and those with increasing concentrations of DNA. Selecting the promising DNA detecting complex, [Ru(phen)2(B16)]2+ and repeating the spectroscopic studies with AT and GC rich oligos, showed the complex has a clear preference for AT rich DNA, highlighting that the preferred mode of binding at equilibrium is potentially within the minor groove of the DNA double helix.

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The skin secretions of amphibians are used as a chemical defence mechanism as protection against predators within their natural environments. Among the classes of compounds contained within the secretions, peptides with diverse bioactivities, such as antimicrobial and pharmacological, are generally the most abundant. Such peptides may have therapeutic potential. In particular, the antimicrobial peptides have their own advantages, when compared with conventional clinical antibiotics. Many scientists have thus been studying the biologically-active peptides of amphibian skin and attempting to assess them as candidates for future therapeutic drugs. This thesis describes analyses performed on bioactive peptides from the skins of bombinid toads and ranid frogs. However, as the skin secretions of amphibians are complex mixtures of compounds, the identification of single active compounds in the past years was complicated and the outputs were consequently of a relatively low order. In recent years, advances in detection and analytical techniques have greatly aided in the identification of single active novel compounds from such skin secretions. In this thesis, several antimicrobial peptides and one pharmacological peptide were identified from the selected amphibian species. These isolated novel peptides demonstrate that the skins/skin secretions of amphibians continue to represent a rich source of natural peptides with varied structures and distinct bioactivities.

The goal of this work is to develop a fundamental formalism able to translate crystallographic information (contact distance and angle) into an accurate evaluation of whether a given pair of atoms participates in a hydrogen-bond or a van der Waals interaction, and, if so, what is its energy. The problem was tackled by using the enormous amount of structural data stored in the Cambridge Crystallographic Database. The distance and angle geometrical parameters were chosen for characterisation of non-covalent atom-atom contacts. Such contacts were searched within ' some pre-defined ranges of distance and angle and consequently arranged into a two-dimensional matrix form of contact counts against distance and angle. This distance/angle contact distribution matrix was fitted with a custom-designed two-dimensional analytical function, whose general form is a superposition of two terms: one describing maxima of contact density at short distances and the other capturing the continuum of disordered long-distanced contacts. The proposed analytical solution allows: 1) analysis of the statistics of short-distance contacts separately from the background noise; 2) the evaluation of analytical functions for the calculation of the probability of a contact to be a short-distance contact; 3) calculation of a pairwise statistical mean force potential through which the interaction energy of a contact can be evaluated. !twas demonstrated that the statistical mean force potential correlates well with the quantum chemically calculated energies of molecular complexes, and thus can serve as a reliable and simple means to estimate the strength of a hydrogen' bond or a van der Waals contact, provided that the contact distance and angle are known.

Polybrominated diphenyl ether (PBDE-47) and hexabromocyclododecane (HBCD) are brominated flame retardants (BFRs) commonly used in a wide range of consumer products. They bioaccumulate and persist in the environment, and have been detected in humans and wildlife. Their ability to pass the blood-brain barrier (BBB) and to accumulate in the brain has raised concern about the potential of BFRs to cause neurotoxicity. Functional genomics was used to investigate the modes of action of PBDE-47 and HBCD in two neuronal cell models namely mouse neuroblastoma (N2A) and neuroblastoma x spinal cord (NSC-19). It was established that PBDE-47 and HBCD reduce cell viability, increase lactate dehydrogenase (LDH) leakage and cause apoptosis as indicated by increased caspase-3 activity at low micromolar concentrations (1 - 4μM). A pre-incubation with the omega-3 fatty acid, docosahexaenoic acid (DHA), mitigated the toxic effect of HBCD in the N2A cell line, with a significant decrease in the LDH leakage. Transcriptome profiling revealed that exposure to PBDE-47 and HBCD affects expression of genes with overlapping functionalities. Both toxicants regulated genes related to calcium homeostasis, endoplasmic reticulum stress and lipid metabolism. Genes involved in thyroid hormone signalling, neurodegenerative diseases and nervous system development were also preferentially regulated. The fatty acid DHA altered the expression of genes that were also regulated by PBDE-47 or HBCD, and HBCD modulated DHA-induced gene expression. It was also established that HBCD exposure affect cellular zinc homeostasis, increasing the level of intracellular weakly bound Zn2+. This effect was ameliorated by the antioxidant NAC, suggesting that the [Zn2+] increase could have been caused by oxidative stress with release of Zn2+ from zinc-binding proteins. This result indicates for the first time that zinc signalling is a potential target of POP toxicity. In conclusion, we established that PBDE-47 and HBCD have toxicity effects on cells of neuronal origin starting at a concentration of 1μM and that exposure to either of these BFRs causes regulation of genes related to many cellular functions, several of which have been observed as BFR phenotypes in animal studies. It was also shown that zinc is a POP toxicity target and that cellular effects of BFRs are modulated by DHA.

The ubiquitous second messenger molecule, cyclic adenosine monophosphate (cAMP), regulates a multitude of cellular functions, with Protein Kinase (PK)A being the major effector of its actions. There is increasing evidence to suggest that phosphodiesterases (PDEs), a class of enzymes that selectively hydrolyses cyclic nucleotides, play a key role in determining cAMP signalling specificity to a number of PKA substrates in the heart. Phospholemman (PLM) is a phosphoprotein that is physically and functionally coupled to the cardiac Na/K-ATPase (NKA). The latter provides the primary route of Na extrusion from mammalian cardiac myocytes, thereby contributing to the maintenance of transmembrane ion gradients and the indirect control of myocardial contractility. The work described in this thesis investigated whether individual cardiac PDE subtype(s) are responsible for modulating cAMP signalling to PLM and the NKA in adult rat ventricular myocytes (ARVMs). The use of selective inhibitors for PDE subtypes 2-4 in biochemical assays identified PDE4 as the predominant PDE involved in modulating PLM phosphorylation at Ser68, the consensus PKA site, in the presence of the β-AR agonist isoprenaline hydrochloride (ISO). The augmentation of PLM-Ser68 phosphorylation was accompanied by a corresponding increase in intracellular cAMP accumulation, indicating that PDE4 selectively modulates downstream PKA activity via cAMP hydrolysis. In contrast, ISO-mediated PDE2 inhibition was found to induce the greatest increase in NKA activity, suggesting that upon neurohormonal stimulation, PDE2 may modulate NKA function via a mechanism independent of PKA phosphorylation of PLM. As PDE2 activity comprises a relatively low proportion of total cardiac PDE activity in the rat heart, our findings suggest that the actions of PDE2 may be compartmentalised. A recombinant adenovirus encoding a novel fluorescence resonance energy transfer (FRET)-based cAMP sensor, PLM-Epac1-camps, was generated to enable direct monitoring of cAMP levels in the vicinity of PLM/NKA in ARVMs. Using fluorescence confocal microscopy, PLM-Epac1- camps was observed to be predominantly targeted to the perinuclear regions of the myocytes. However, preliminary ratiometric FRET measurements using a dual-photometer setup demonstrated a robust response to intracellular cAMP elevation with forskolin. Collectively, our findings suggest that further characterisation of this methodology could provide insights into whether PDE2 activity locally regulates cAMP signalling to the NKA in a compartmentalised manner during β-AR stimulation.

Activation of the G-protein coupled receptor (GPCR) chemokine receptor 5 (CCR5) has been shown to result in activation of the Gαi family of heterotrimeric G-proteins. However, little is currently known about the temporal characteristics of G-protein heterotrimer activation by CCR5. Furthermore, this simplistic model does not account for the range of changes CCR5 activation brings about. Recent evidence suggests that GPCRs may be able to signal through numerous permutations of heterotrimer. In order to assess G-protein activation and interaction with CCR5 functional stably transfected cell lines were created expressing Gαi2 fused to ECFP and Gβ1 fused to EYFP. The interaction of these constructs was confirmed by measuring fluorescent resonance energy transfer (FRET). Stably transfected cells exhibited a FRET ratio of 2.63% (±0.345%, n=3) over that of control cells. Following CCR5 stimulation with CCL3, Gαi2β1γ activation could be monitored in real time, in whole cell populations, on a fluorescent plate reader by monitoring FRET emissions. This assay system represents a novel approach to measuring G-protein activation which can be used as a foundation to build more powerful FRET based assays. Measurement of G-protein interactions was further invested by BRET based studies. Transfection of dominant negative and constitutively active G-proteins alongside siRNA knockdown of G-proteins revealed that CCR5 is capable of signalling through other members of the Gαi family, with strikingly similar efficacy and potency to CCL3 stimulation. Dual knockdown of Gαi subunits and Gαq resulted in much attenuated calcium release following CCL3 stimulation, providing evidence that CCR5 may functionally couple to several types of G-proteins. Findings also support the theory that GPCRs can participate in domain swapping in order to rescue function. Treatment with gallein resulted in higher resting cytosolic cAMP but did not prevent CCR5 mediated inhibition of cAMP production. Gallein treatment also resulted in significant increases in calcium release following CCR5 activation, highlighting Gβγ as a potential target for modulating CCR5 signalling events. Data herein emphasize the complexity of GPCR signalling, and also provide a foundation for exploring GPCR signalling using fluorescence resonance energy transfer methods in the future.

The expression of cartilage-degrading metalloproteinases (MMPs or ADAMTSs) is regulated in part via changes in acetylation mediated by histone acetyltransferases and histone deacetylases (HDACs). Classical HDACs can be divided into class I (HDAC1, 2, 3 and 8), class II (HDAC4, 5, 6, 7, 9 and 10) and Class IV (HDAC11). Broad spectrum HDAC inhibitors (HDACi) block cytokine-induction of key proteases in SW1353 chondrosarcoma cells and primary human articular chondrocytes (HACs), resulting in chondroprotection. This study aimed to elucidate the role of HDACs in chondroprotection using selective chemical HDACi and siRNA technology. Trichostatin A (TSA) (broad spectrum) and valproic acid (VPA) (class I selective at <1mM) repressed all cytokine-induced metalloproteinase genes in SW1353 cells, and MMP13 expression in HACs. MS-275 (class I selective) failed to repress cytokine-induced MMP1 and MMP13 expression in SW1353 cells, but repressed MMP13 expression in HACs. Tubacin (HDAC6 specific) decreased cytokine-induced MMP1 and MMP13 in SW1353 cells. All inhibitors prevented cytokine-induced degradation of bovine nasal cartilage, where MS-275 was also able to repress cytokine-induced MMP expression, including MMP1 and MMP13. A profile of HDAC expression showed that the majority have reduced expression in OA cartilage compared to normal. TSA increased HDAC3 expression and decreased HDAC7 expression in SW1353 cells, suggesting that HDACi may both inhibit HDAC catalytic activity, and regulate HDAC expression. Knockdown of individual HDACs in SW1353 cells using siRNA showed that inhibition of all HDACs, except HDAC1, caused a repression of basal and IL-1α-induced MMP13 expression, with HDAC1 knockdown potentiating IL-1α-induced MMP13 expression. In HACs, HDAC3 or HDAC8 knockdown resulted in reduced basal and IL-1α-induced MMP13 expression, HDAC1 or HDAC11 knockdown potentiated both of these and HDAC5 or HDAC6 knockdown potentiated only IL-1α-induced MMP13 expression. Problems with non-targeting control siRNAs made interpretation of experiments difficult. HDACs therefore play a key role in metalloproteinase expression, with inhibition of class I HDACs, or separately HDAC6, capable of altering metalloproteinase expression to confer chondroprotection.

The regulatory light chain (RLC) of myosin is a component of the lever arm of the myosin motor in muscle thick filaments. Phosphorylation of RLC by myosin light chain kinase (MLCK) modulates the force and speed of muscle contraction, but the mechanisms of RLC‐mediated regulation in striated muscles are less well understood than those of Ca2+‐dependent regulation via troponin in the thin filament. To help elucidate the role of RLC in muscle regulation, its orientation in the native environment of cardiac muscle cells was measured. Pairs of cysteine residues were genetically introduced into the N‐ and C‐terminal lobe of the human cardiac RLC. Each pair of cysteines was crosslinked with a bifunctional‐rhodamine (BSR). The pure BSR‐RLC conjugates were exchanged into demembranated trabeculae from rat ventricle, and the orientation of the BSR fluorescence dipole determined by polarized fluorescence. The orientations of the N‐lobe were similar to those determined from chicken gizzard RLC probes exchanged into skeletal muscle fibers indicating a conserved domain orientation. The orientation of the RLC C ‐ lobe was similar in relaxation, active isometric contraction and rigor, suggesting that either the orientation of the RLC is relatively insensitive to strong binding of myosin heads to actin, or that only a small fraction of myosin heads are strongly bound to the thin filament in both active isometric contraction and rigor. Bending between the two RLC lobes occurs in ‐ situ and may have a functional significance in cardiac muscle contraction and regulation. Expressed and purified catalytic subunit of human cardiac MLCK efficiently mono ‐ phosphorylates cardiac RLC on serine 15 in a calcium/calmodulin dependent manner. Exchange of in ‐ vitro phosphorylated BSR‐RLCs into demembranated trabeculae to replace 10‐15% of native RLC showed that the orientation of phosphorylated RLC C ‐ lobe is similar to that of unphosphorylated RLCs.

I Four electrophoretic variants of chloramphenicol acetyltransferase (E.C.2.3.1.99) have been purified to homogeneity from cell-free extracts of staphylococci by affinity chromatography. All four enzymes show similar Km values for the substrates, acetyl Coenzyme A and D, threo chloramphenicol. Amino acid analyses and tryptic peptide maps of the four enzymes are similar. II the N-terminal sequences of seven chloramphenicol acetyltransferase variants from both Gram positive and Gram negative bacteria have been determined by the method of Edman degradation of proteins covalently attached to solid phase supports. More than 90 percent of the primary sequence of one of the staphylococcal variants has been determined. III Kinetic studies and the results of chemical modification experiments have implicated the importance of a histidine residue in the mechanism of catalysis, and a unique histidine residue in the native enzyme has been found to react with iodoacetamide with consequent formation of 3-amidocarboxymethylhistidine. There is no evidence of a covalent acyl-enzyme intermediate in the catalytic process. IV The use of secondary structure prediction methods has allowed the comparison of both primary and predicted secondary structures of the N-termini of ten chloramphenicol acetyltransferase variants. The results of this study are consistent with the view that the chloramphenicol acetyltransferase enzymes have evolved from a common ancestral protein and, although their primary sequences differ considerably in some cases, their secondary structures and catalytic mechanism are likely to be similar.