Insulin, and the structurally related insulin-like growth factors (IGFs), are peptide growth factors believed to play a role in embryonic development. In addition to factors produced by the embryo, certain maternally derived growth factors may also be important during development. These are likely to act upon the extraembryonic membranes which surround the embryo throughout gestation, before they, or their breakdown products, are transported to the developing embryo. This thesis examines the processing of both insulin and insulin-like growth factor I (IGF-I) by the visceral yolk sac, an extraembryonic membrane in the rat. Both radiolabelled and fluorescently labelled ligands have been examined in 17.5 day yolk sacs, and their cultured equivalent. It appears that both factors are digested by this tissue very rapidly, which may involve receptor-mediated pinocytosis or surface digestion. Further, the role of the IGF-I receptor during rat embryonic development has been examined using a monoclonal antibody reported to block this receptor. When this antibody was applied to rat embryos cultured from 9.5 to 11.5 days of gestation, it resulted in growth retardation plus an associated increase in morphological abnormalities. These effects were largely reversed by the addition of an excess of IGF-I to the culture medium in the presence of this antibody, while the addition of insulin or IGF-II had no effect. In conclusion, receptors mediating insulin and IGF-I uptake appear to be present on the surface of the rat visceral yolk sac. The growth inhibition seen in the presence of the antibody also implicates that IGF-I plays a role in the normal development of post-implantation rat embryos.

Frog defensive skin secretions have been studied for decades as pharmaceutical components of traditional medicines due to their wealthy possession of wide-spectrum pharmacological effects. With the emergence of antimicrobial resistance generated by over use of conventional antibiotics and the unexpected effects caused by conventional cancer treatments, peptide-based therapeutic candidates from frog skin secretions have become one of the targets for novel drug discoveries. At present, construction of cDNA libraries from different frogs for cloning skin defensive skin secretion peptides has been used in the application of novel peptide discoveries without harming frogs. The genomic studies and peptidomic studies used in parallel on the discovery of novel bioactive peptides from the skin secretion of the selected South American frogs, Phyllomedusa hypochrondrialis and Phyllomedusa sauvagei, and the selected Australian frog, Litoria caeulea, resulted in the identification of 4 novel peptides with different bioactivities. This thesis includes 6 chapters. The general introduction introduces the background information relevant to the project. The general methods describe the general experimental principles and methods in the research. The experimental chapters, from Chapter3 to Chapter 5, report the novel peptides discovered in the research. The general discussion and conclusions summarise the project research. The functional genomic studies between the South American and Australian frogs provided more clues and benefits for cloning novel bioactive peptides from their cDNA libraries, which still require more research. What is strongly believed is that the bioactive peptide discovery will give rise to more improvements in pharmaceutical treatments and clinical applications. The on-going search for novel peptides holds the key to a healthier tomorrow for us all.

There is a common belief that mammalian cell gene mutation assays are prone to false positives, thus questioning the relevance of these tests in regulatory screening paradigms and the mechanisms responsible for these uninterruptable results. False positives can lead to unnecessary animal testing and delays in the development of efficacious new medicines. The initial aim of this thesis was to put into perspective the rate of positives and firstly to consider the extent off target aneugenicity (chromosome loss or gain) may contribute to this rate. Secondly the contribution of topoisomerase II poisoning and its relationship to genotoxicity was considered. Topoisomerase II maintains DNA topology by inducing transient breaks in one strand so a second strand can pass. Chemicals that interact with the enzyme (topoisomerase II poisons; e.g. the antibiotic gemifloxacin and the chemotherapeutin etoposide), yield topoisomerase II bound DNA cleavage complexes, making breaks permanent, leading to mutation or cell death. Structurally, topoisomerase II poisons are diverse, so their genotoxicity is difficult to predict. To estimate the incidence of positives seen in pharmaceutical research, a retrospective review of data from 10 years of mouse lymphoma assays (MLA) conducted at AstraZeneca was undertaken. This showed that the rate of unexplainable positives was only 5%, vindicating the use of the test in screening paradigms. Consideration was then made of what mechanisms might contribute to this 5%. Aneugenicity was considered but it was shown that the MLA was a poor screen, failing to identify 7 known anuegens. To gain a better understanding of the relationship between topoisomerase II and genotoxicity, assays to assess enzyme poisoning were examined, including the ability of the cell free decatenation assay to predict the results of the in vitro micronucleus test. However, even when combined with an estimate of cellular uptake the predictivity was low. Assays to investigate topoisomerase II poison / DNA cleavage complexes in vivo were then investigated. The TARDIS and ICE assays both use antibodies to target topoisomerase II bound in the complex. TARDIS was found to be insensitive, failing to identify cleavage complex formation with gemifloxacin. Using the ICE assay, cleavage complex formation was seen for etoposide (0.1 μmol/L; FITC intensity 3.53 ± 0.79) and gemifloxacin (100 μmol/L; FITC intensity 3.37 ± 0.86), but not at equivalent concentrations to those inducing micronuclei (MN) (0.03 μmol/L etoposide; MN/1000 6 ± 2.6 and 10 μmol/L gemifloxacin; MN/1000 5 ± 3.4), thus questioning assay sensitivity, or suggesting a role for other mechanisms of genotoxicity e.g. reactive oxygen species (ROS). The hOGG Comet assay showed that neither etoposide nor gemifloxacin induced ROS related genotoxicity. Improvements to the sensitivity of the cleavage complex assays were made by preparation of mouse specific antibodies, but TARDIS was still unable to identify gemifloxacin. This work also suggested that when developing antibodies for DNA bound topoisomerase II, the n-terminus of the enzyme should be targeted. Over the last 4 years, emerging data linked the topoisomerase IIβ isoform to genotoxicity. As research within this thesis had investigated the activity of topoisomerase IIα, this may have explained the difficulty encountered equating topoisomerase II poisoning to genotoxicity. Following siRNA knockdown of topoisomerase IIα, the genotoxicity of etoposide and gemifloxacin was investigated. It was shown that for 0.3 μmol/L etoposide, topoisomerase IIα knockdown of 42% (± 2%) was associated with a reduction in micronuclei of 49% (± 9.7%). For 30 μmol/L gemifloxacin, topoisomerase IIα knockdown of 37% (± 9.5%) was associated with a reduction in micronuclei of 48% (± 0.2%). This was the first time such direct relationships had been demonstrated between the alpha isoform and genotoxicity. In conclusion, the predictivity of the MLA was confirmed but it was clear the assay is not a suitable screen for aneugenicity. The relative sensitivity of assays to measure topoisomerase II poisoning was shown and linked to genotoxicity. Whilst it was not possible to demonstrate cleavage complex formation at concentrations below which genotoxicity was seen, this was likely due to the insensitivity of the assays used rather than topoisomerase II poisons having other genotoxic mechanisms. For the first time the link between topoisomerase IIα and genotoxicity was confirmed and use of knockdown cells holds real promise as a tool for investigating off target topoisomerase II poisoning.

Vitamin D is a prohormone produced in the skin following ultraviolet B (UVB) radiation exposure. It can also be obtained from natural sources such as oily fish, as well as from fortified foods or supplements. The main physiological role of vitamin 0 is in the maintenance of skeletal health. However, there is also emerging evidence that vitamin 0 may also be associated with several non-skeletal health outcomes including; type 2 diabetes mellitus (T2DM), cardiovascular disease (CVD) and decreased pregnancy complications. Suboptimal vitamin 0 status is a common global issue. The accurate measurement of 250HD is hindered by epimers. Epimers are isomers which only differ in the configuration at one carbon atom. Subsequently, they have the same molecular weight, and can overlap chromatographically with vitamin 0 metabolites or internal standard peaks and give false estimates of true 250HD levels. In the present study, an ultra-performance liquid chromatography tandem mass spectrometry (UPLC/MS-MS) assay was developed, which was capable of assessing 250HD and 3-epi-250HD concentrations in human serum/plasma. The UPLC/MS-MS assay was used to measure 250HD and 3-epi-250HD levels in two cohorts; the first cohort consisted of pregnant women with type one diabetes and pregnant controls, while the other was a nested case-control cohort from the Prospective Epidemiological Study of Myocardial Infarction (PRIME) study, which consisted of men who had suffered a CVD event and controls. These results were then used to determine potential relationships between 250HD and 3-epi-250HD concentrations and; gestational and pregnancy outcomes, BMI, cardiovascular disease risk, as well as seasonal effects.

No description available.

The efficacy of antibiotics is being challenged by the emergence of bacteria resistant to antibiotics (AR), both in natural and clinical settings. Antibiotics and associated AR can be transmitted and dispersed via environmental bacteria, however AR might also be conferred in situ, without antibiotic pressure. For example, release of metal-bearing wastes to natural environments can proliferate AR. An important case is the influence of zinc (Zn) contamination on environmental AR, which increased tetracycline and quinolone resistance in wastewater isolates. However, how Zn might influence AR to therapeutically critical carbapenem antibiotics, including meropenem is unknown, which fuelled this study. Here the percentage of total isolates resistant to Zn, meropenem and-or both, were compared and assessed in varied microbial communities from natural environments and bioreactors. Overall, Zn levels, and Zn and meropenem resistant isolates correlated in all settings. For example, the abundance of combined meropenem plus Zn resistant isolates was significantly higher in high Zn (South Tyne) versus low Zn (North Tyne) sediments, and correlated with soluble and total Zn levels (p-value < 0.010 and p-value < 0.050, respectively), implying that acquired Zn resistance might confer meropenem resistance to isolates. In parallel, batch reactors seeded with North and South Tyne sediments, and amended with 2.00 mg/L (low) and 100 mg/L (high) Zn (2 x 2 design), showed increased relative percent meropenem resistant isolates in reactors with high (South Tyne: 21.0%; North Tyne: 31.0%) versus low Zn (South Tyne: 17.0%; North Tyne: 14.0%), whereas, sediment source (South vs North Tyne) was not important. Further, sediment soluble Zn levels significantly correlated with meropenem resistant isolates in all reactors, suggesting that the observed meropenem resistance was a possible “side effect” of cellular defence against Zn toxicity. Similar results were seen in Zn and meropenem-amended rotating tubular reactors treating domestic wastewater. Reactors dosed with 2.00 mg/L meropenem plus 100 mg/L Zn showed combined resistance in 51.0% of reactor effluent isolates, whereas only 24.0% displayed combined resistance with amendments of 2.00 mg/L meropenem and 20.0 mg/L Zn. Overall, elevated Zn levels significantly increased Zn, meropenem and combined resistance in isolates from sediments, batch reactors and tubular reactors. Therefore, one can conclude Zn levels impact meropenem resistance, although evidence suggest meropenem resistance is most apparent when Zn is present and resistance can be lost when Zn is removed, suggesting cross resistance mechanisms.

The Amaryllidaceae alkaloids e.g. galanthamine (Gal), lycorine, narciclasine and pretazettine are noted for their pharmaceutical properties. The biosynthesis of alkaloids by plants using in vitro systems has been considered as a tool for drug discovery and production since total chemical synthesis is not economic. The biosynthetic pathways, especially for Gal, are starting to be understood, but still far from complete. This study focused on understanding biosynthesis in whole plants and developing cell culture systems that could be optimised for alkaloid production. Metabolite profiling and knowledge about probable genes involved in secondary metabolism and their regulation in different tissues will provide insight into the Narcissus plant biology related to alkaloid production. In vitro cultures of N. pseudonarcissus cv. Carlton were initiated from twin-scale explants, cultured on Murashige and Skoog agar medium (MS) fortified with different concentrations of growth regulators. Callus was obtained mainly from MS medium containing high concentration of auxin, while media with low auxin and MS basal medium gave bulblets with both white and green shoots. Regenerated bulblets developed from callus developed in both high and low auxin MS media. These tissue culture derived materials and field-grown samples were analysed using GC-MS for Gal content. The highest amount of Gal was obtained from basal plate tissue followed by bulb, leaves, and in vitro tissues. A trace amount of Gal was found in callus along with possibly other alkaloids. An NMR-based metabolomic analysis showed that the relative concentrations of compounds involved in phenylalanine and tyrosine metabolism, the initial stage biosynthetic pathways that yield the Amaryllidaceae alkaloids, were higher in field samples than in vitro samples. These results support the GC-MS findings of high Gal production in field samples. Initial RT-PCR analysis using gene specific transcripts possibly involved in Gal biosynthesis i.e. P450s. PAL, TYDC and OMT showed their expression in in vitro as well in field tissues, which is intriguing. A transcriptome analysis was performed comparing expression in the highest (basal plate) versus lowest (callus) Gal containing tissues. The transcriptome analysis results were in accordance with the previous findings, as it was observed that the transcripts involved in the initial biosynthetic pathways leading to precursors (tyrosine, phenylalanine) were up regulated in callus while the transcripts involved in later pathways leading to alkaloids were up regulated in basal plate.

Polypeptide Nucleic Acids, PNAs, are analogues of DNA and have the potential to bind to DNA by base-pairing and hence act as therapeutic agents. Amino acids carrying heterocycles in their side-chains are valid targets as natural products and as components of these potential therapeutic agents (PNAs) for use in living organisms. The aim of this investigation was to synthesise a range of heterocyclic amino acids, that could be used in the formation of PNAs. The proteinogenic amino acids, serine and cysteine and the unnatural amino acids, homocysteine, 2,3-diaminopropionic acid and 2,4-diaminobutyric acid, have been used in the formation of said heterocyclic amino acids via a C-X bond (where X=C, S, O or N) in a linker chain. It was decided to approach the synthesis of heterocyclic amino acids by way of a linker approach, joining the ready-formed heterocycle with an amino acid. Once the amino acids had been suitably protected several different methods were attempted in order to form heterocyclic amino acids. To form a carbon-carbon (X=C) bond in the linker chain, radical and organocuprate conjugate addition reactions and hydroboration and metathesis coupling were attempted. The formation of a linker containing a carbon-heteroatom bond (X=S, O or N) was investigated using a substitution approach.

No description available.

No description available.