Dietary restriction (DR) increases lifespan robustly in diverse species. Effects of the dietary polyphenol resveratrol consistent with delayed ageing and/or extension of lifespan have been reported. The involvement in the longevity response to DR of the protein Sirt1, which may be activated by resveratrol and deacetylates a range of cellular substrates that includes histone proteins, identifies epigenetic processes as a pathway that may mediate effects of both DR and dietary resveratrol in delaying ageing and/or extending lifespan. Based on a preliminary observation, the hypothesis underlying the study is that some of the beneficial effects of resveratrol on lifespan/aging are mediated through effects on histone expression that oppose changes observed in ageing. A secondary hypothesis, based on a degree of structural similarity between resveratrol and 17β-oestradiol, was that epigenetic effects of resveratrol are mediated through the estrogen receptor (ER). The effect of resveratrol on histone protein expression was investigated in human intestinal Caco-2 cells and human MCF-7 breast cancer cells. Histone H2a, H2b, H3 and H4 expression was decreased in response to resveratrol treatment in both cell lines. In support of our hypothesis that resveratrol affects ageing through reversing ageing-associated changes in histone proteins, higher levels of H2A, H2B, and H4 expression were detected by western blotting in the small intestine of old (38 months) mice than in younger (12 months) mice. To investigate possible consequences of effects of resveratrol, including effects resulting from altered histone expression, we studied the effect of resveratrol on global gene expression in Caco-2 and MCF-7 cells to address several objectives including: (1) investigating if resveratrol has an effect similar to that of DR at the level of gene expression; (2) identifying if genes or pathways affected by resveratrol were also affected by manipulation of the expression level of Sirt1. For both cell types, the number of genes in the intersection between those affected by resveratrol and a compiled list of genes reported in other studies to respond to DR was greater than expected by chance, supporting the view that responses to resveratrol and to dietary restriction have some commonality and that resveratrol may mimic some effects of dietary restriction. We also found that there was very little overlap between genes affected by resveratrol treatment and by knockdown of Sirt1 expression in Caco-2 cells, which adds to accumulating evidence that resveratrol does not act through effects on Sirt1. To investigate if effects of resveratrol - in particular the reduction in histone protein expression - are mediated through the estrogen receptor (ER), Caco-2 and MCF-7 cells were treated with resveratrol in the presence or absence of the ER antagonist fulvestrant, then total cell lysate was analysed by western blotting. The reduction in histone protein (H2a, H2b, H3 and H4) expression was attenuated by fulvestrant, indicating that resveratrol reduced histone expression via an ER-dependent mechanism. For further investigation of effects of resveratrol on histone expression, Caco-2 cells were transfected with a promoter reporter construct comprising the histone H3 promoter upstream of the β- galactosidase reporter gene, and the effect on reporter gene expression of treatment with resveratrol in the presence and absence of the fulvestrant was measured. Resveratrol reduced reporter gene expression and this effect was attenuated by fulvestrant, demonstrating that resveratrol acts to reduce histone H3 expression at the level of transcription through an ER-mediated mechanism. To investigate if the response to resveratrol treatment is through interaction with estrogen response elements (EREs) in the histone H3 promoter we replaced three potential EREs within the histone H3 promoter region included in the promoter-reporter construct with random sequence. Caco-2 cells were then transfected with either original or mutated promoter-reporter construct and treated with resveratrol or the endogenous ER ligand 17-β estradiol in the presence and absence of fulvestrant. Resveratrol and 17-β estradiol both reduced reporter gene expression from both promoter reporter constructs and in all cases responses were attenuated by fulvestrant, indicating that effects of neither compound, although mediated through the ER, are on the specific sequences region we identified and replaced. In conclusion, these data indicate that resveratrol reduces histone expression in both intestinal and breast cancer cells through an ER-mediated mechanism acting at the level of transcription and that this effect may oppose an accumulation of histone proteins (observed in mouse small intestine) that accompanies ageing. With respect to effects on gene expression, resveratrol was found to mimic some effects of dietary restriction but appeared to act through a mechanism independent of Sirt1.

Although manuka honey has been shown to inhibit Pseudomonas aeruginosa, an opportunistic pathogen implicated in cutaneous wound infections, the mechanisms of action are not yet defined. The purpose of this study, therefore, was to investigate the inhibitory effects of manuka honey on P. aeruginosa. Initially, a bactericidal mode of action was confirmed using suspension cultures of P. aeruginosa. Laser scanning confocal microscopy, atomic force microscopy and hydrophobicity assays identified physiological changes in manuka honey treated cells, such as reduced surface hydrophobicity, membrane blebbing, cell lysis and the release of extracellular material. The effects of manuka honey on intracellular proteins and the expression of cell envelope components was explored using two dimensional gel electrophoresis and qPCR, respectively. Expression of the gene encoding the structural anchor protein, OprF, was significantly suppressed in manuka honey treated cells. This resulted in a marked reduction in cell envelope stability with a reduced capacity to resist osmotic stress. The effect of manuka honey on P. aeruginosa surface associated functions (motility and biofilm formation/integrity) was also investigated. Transmission electron microscopy and motility assays identified significant de-flagellation and reduced motility of P. aeruginosa cells following exposure to sub-inhibitory concentrations of manuka honey. qPCR of various genes within the flagella regulon, identified suppression of genes encoding master regulators FleQ and FliA. Biofilm cells, inherently more tolerant to antimicrobial agents, required a higher concentration of manuka honey to inhibit biomass and bioactivity than planktonic cells. Reductions in the number of biofilm cells treated with manuka honey and dressings coated/impregnated with manuka honey were monitored using an in vitro cutaneous wound model. qPCR of treated biofilm cells suggested that similar mechanisms to those observed in planktonic cells occurred. These in vitro studies confirm that manuka honey inhibits both planktonic and sessile (biofilm) P. aeruginosa cells through the differential regulation of key genes required for cell envelope stability and motility.

This thesis describes the development of a linked 1000th scale mimic of four distinct unit operations, including precipitation and centrifugation. These operations are utilised during the production of an anti-snake venom Fab fragment. A new approach has been used to define the important engineering parameters that impacted on the recovery process the effect of these was verified by experimental work. The mimic requires only millilitre quantities of material to predict changes in the physical and biological properties of key components, such as particle size and antibody activity. The potential impact of several process changes on the characteristics of the feed stream and on purification steps further downstream has been assessed, which is not possible without a linked system. Microfiltration has been examined as a possible alternative to the current centrifugal method of recovering the antibody particles. A small-scale stirred cell device was used to carry out a number of operations in a single piece of equipment these included separation, concentration and buffer exchange. An overall increase in the yield of 10% was observed. This was attributed to the ability of microfiltration to reduce material losses by integrating a number of operations. A preliminary investigation has been conducted into the possibility of utilising Protein G affinity chromatography in place of the current four-step precipitation and centrifugation recovery operation. A mathematical model capable of predicting the size and shape of the breakthrough curves has been developed the predicted curves were tested by comparison with breakthrough curves produced under different experimental conditions. The initial results demonstrated the feasibility of utilising affinity chromatography as a one step recovery process. However the predicted breakthrough curves varied from the experimental curves suggesting that the mathematical model requires refinement.

The antibiotic compound pleuromutilin, produced by Clitopilus passeckerianus, has antibiotic activity against some clinically important bacteria, including MRSA. However yield from the native producer is low and there is no derivative compound available with oral or intravenal bioavailability. The identification of the pleuromutilin gene cluster in Clitopilus passeckerianus allows for molecular biology techniques to be used to attempt to resolve these issues. This investigation has sought to help ameliorate the problems with pleuromutilin via three experimental approaches. Gene silencing in Cl. passeckeriqnus was used to attempt to generate intermediate compounds from the pleuromutilin pathway. Although several strains transformed with an antisense Acyltransferase (ATF) produced no pleuromutilin, no other. intermediatesaccumulated. Chemical analysis revealed the accumulation of a novel sesquiterpene compound, pilobarbatriol. QRT+PCR was used to determine the impact of ATF silencing on the transcription of other genes in the cluster. This approach revealed that the initial two genes in the pathway, the Geranylgeranyl diphosphate synthase (GGS) and the Cyclase, were not transcriptionally active in ATF silenced lines preventing, accumulation of intermediate compounds. This approach demonstrated the viability of antisense silencing in Cl. passeckerianus and its potential for metabolite engineering. Heterologous expression in Coprinopsis cinerea was then attempted with the transfer of the pleuromutilin biosynthetic cluster in a genomic configuration. Co. cinerea transformants were generated with the entire gene cluster; however bioassay analysis revealed no antibiotic activity. RTPCR determined that all but two genes, Cyclase and p450-3, were being transcribed in Co. cinerea. Promoter-GFP analysis showed that the Cyclase and p450-3 promoter regions were active in Co. cinerea and therefore not preventing transcription. Transformation with cDNA versions of the two genes led to the transcription of p450-3. The successful expression of six of seven genes in Co . . cinerea may suggest that this approach would perhaps be effective for gene clusters smaller than that of the pleuromutilin biosynthetic gene cluster but not the pleuromutilin gene cluster itself. Expression of genes from the pleuromutilin gene cluster was subsequently attempted in the ascomycete fungus Aspergillus oryzae. On transformation with a GGS expression plasmid, a compound with similar characteristics to GGPP was found to be accumulating. Subsequently A. oryzae was transformed with both the GGS and Cyclase expression constructs and transcripts were detected for both genes. Chemical characterisation revealed the accumulation of compounds in transformed strains which were absent in controls suggesting that these two genes are generating products in A. oryzae, demonstrating this approach is appropriate for the heterologous production of secondary metabolites.

No description available.

No description available.

The focus of this thesis was to develop a two-tier delivery system suitable for DNA delivery in vivo. Firstly, 4 peptides were investigated to determine their ability to overcome the intracellular and extracellular barriers which inhibit the expression of 'naked' DNA when administered in vivo. The RALA peptide was identified as the most efficient DNA delivery vehicle, eliciting greater gene expression in vitro and in vivo following intradermal injection compared to the delivery of 'naked' DNA. The RALA delivery vehicle was also significantly less toxic than the current commercially available gold-standard transfection agent. Subsequently, a microneedle platform for delivery of these RALA/DNA complexes w.as investigated. Three polymer matrices were examinee, PMVE/MA, PVA and PVP and scrutinised for suitability as the structural polymer for the fabrication of the microneedle arrays through analysis of their compatibility with the bioactive RALAIDNA complexes, cellular toxicity and mechanical strength. It was determined that the PVP polymer was the most suitable for microneedle fabrication and as such, research then focused on the determination of the stability of PVP microneedle arrays loaded with RALAIDNA nanoparticles, optimising cargo loading and utilising them in vivo for the delivery of RALAIDNA nanoparticles. The combination of these two technologies proved to be a successful method of eliciting gene expression in vivo. Microneedle administration of RALAIDNA nanoparticles encoding the luciferase protein generated levels of gene expression superior to that observed with the delivery of 'naked' DNA. Moreover, it was proven that the delivery of an antigen-encoding DNA via this method was capable of generating an antigen-specific CD8+ T-cell response. Thus, the nanoparticle/microneedle delivery system developed in this research has the vast potential to revolutionise the field of DNA vaccination.

Iron is vital for all living creatures but becomes toxic when it exceeds the levels catered for by their natural cellular buffering mechanisms, causing free radical formation via the Fenton reaction and ultimately, therefore, leading to oxidative stress. In such situations, for example in repeatedly transfused patients suffering from β-thalassaemia or sickle cell anaemia, iron chelation therapy is required. Desferioxamine (DFO), the most widely used therapeutic chelator, is a hexadentate ligand possessing a very high affinity for Fe3+, but it is not orally active. 3-hydroxypyridin-4-ones (HPOs), the more recently introduced synthetic alternatives, have also been shown to be useful therapeutic chelators, demonstrating the essential affinity and selectivity for Fe3+ along with good oral activity. Deferiprone, a typical HPO, has emerged as a prominent therapeutic, able to remove accumulated excess iron from the heart and mitochondria. However, because Deferiprone has some drawbacks of relatively high metabolic instability and a side effect of lowering white blood cell count for a small number of patients, the search continues for other such synthetic chelators with improved properties. In the work reported here, various computational studies have been performed to aid in the rational design of Fe3+ chelators, with their physicochemical properties (pKa, Fe3+ affinity, hydration and membrane permeability) predicted by means of quantitative structure property relationship (QSPR) methods, quantum mechanical (QM) calculations, and molecular dynamic (MD) simulations. The pKa and Fe3+ affinity were also studied experimentally with a novel approach devised for dealing with ligands possessing substituents with hydrogen bond donor and/or acceptor groups. The pKa values predicted using QSPR and QM static calculations (Gaussian 09) were found to differ very significantly from the experimentally determined values. When data from the QM static calculations were combined with a regression model, however, the pKa predictions were significantly improved, with the predicted values then within ± 0.2 log units of the experimental values, and computing times of the order of 1 day per molecule. These calculations also allowed the determination of possible deprotonation sequences for the predicted compounds. Further pKa predictions were made by means of QM MD calculations, using Car-Parrinello molecular dynamics (CP-MD). These simulations were found to yield pKa values within ±0.3 log units of the experimental values but involved much longer computing times (of the order of 20 days per molecule). In addition, however, the CP-MD simulations also provided valuable insights into the atomistic details of the proton transfer mechanism and the solvation structure and dynamics at all stages of the reaction. For the three HPOs studied, it was observed that proton transfer takes place along a chain of three H20 molecules, although direct hydrogen bonds were observed to form transiently. The (Fe3+) log /C, predictions for the HPOs were made using an entirely novel QM-based methodology (and without knowledge of the chelator pKa values), yielding log K-\ values within ± 0.32 log units of the experimental values. For the preparation of membrane permeability study, novel Chemistry at HARvard Molecular Mechanics (CHARMM) force fields specifically for use in HPO simulations were developed. These new force fields were validated using Large-scale Atomic/Molecular Massively Parallel Simulator (LAMMPS) MD simulations of the chelators’ behaviour in aqueous solution.

Amphibian skin is a multi-functional organ which has adapted to function as a barrier against hostile environmental pathogens and to protect against predatory attack. Highly specialised dermal glands release a complex cocktail of bioactive compounds onto the skin; this mixture contains biogenic amines, proteins, alkaloids and a plethora of bioactive peptides. A vast array of peptides have been identified from over 500 species of amphibians to date which have been found to exert, a range of biological effects. Families of peptides derived from amphibian skin secretions which have been studied in particular detail include the bombesins and related peptides, the bradykinins and related peptides, the skin opiates and those with antimicrobial activities. This thesis presents a study of the biological activities of a novel hybrid pentapeptide derived from amphibian skin secretions.

Honey has been shown to have therapeutic properties, which include immunomodulatory and antibacterial activity in vitro and anti-inflammatory, antipyretic and wound healing properties in vivo. A complex mix of factors such as acidity, osmolality and hydrogen peroxide content contribute to antibacterial activity. Unusually manuka honey has been shown to contain methylglyoxal which is derived from nectar collected from the blossom of manuka trees and this confers high antibacterial activity. Manuka honey is used in licensed wound dressings in the UK. Its ability to inhibit staphylococci has been reported, but its efficacy with Gram negative bacteria is less well documented. Since these bacteria are difficult to control and commonly infect military wounds and burns, there is a need to investigate their susceptibility to manuka honey. The main aim of this study is to assess the antimicrobial potential of manuka honey against multi-drug resistant (MDR) Gram negative rods with the potential to infect wounds. Eighty five clinical isolates were tested in this study (30 MDR Acinetobacter and 55 extended spectrum beta-lactamases [ESBL] producing members of the Enterobacteriaceae). The minimum inhibitory concentration (MIC) of manuka honey for each isolate was determined by agar incorporation and broth dilution methods, as well as the minimum bactericidal concentration (MBC). The kinetics of inhibition of selected isolates with high MIC values was monitored by total viable counts. Also, ultrastructural changes in cell morphology were studied before and after exposure to manuka honey using scanning (SEM) and transmission electron microscopy (TEM). Electron micrographs were examined for structural changes, such as altered shape, surface abnormalities and evidence of cell division. Eight Omani honeys were assayed for their antibacterial activity using bioassay, MIC and MBC methods. Omani honeys were also analysed for their chemical and physical properties such as pH, protein, water and sugar contents, hydroxymetheylfurfural (HMF), colour and antioxidant properties. Pollen analysis was also used for identifying the flora origin of honey. All Omani honeys were found to possess peroxide activity nonetheless it exhibited a bactericidal mode of activity against all MDR and ESBLs tested. In addition honey analysis revealed unadulterated and natural honey. A study of anti-radical activity and phenolic contents demonstrated that Omani honey could be used to promote a rapid wound healing and aid its antibacterial activity. The proximity of MIC and MBC values indicates that manuka honey had a bactericidal mode of action against these isolates and this was confirmed by the time to kill curves. The SEM and TEM of images of representative isolates after treatment with manuka honey showed some physical membrane damage, septa formation and irregular shape; whereas non honey treated cells (control) did not appear to be obvious damage. In conclusion manuka honey possesses strong antibacterial activity against the antibiotic-resistant wound pathogens tested here and further investigation into cellular target sites is needed. Both manuka and selected Omani honeys have clinical potential to inhibit pathogens that commonly colonise wounds.