No description available.

Estrone sulfatase (STS) has been shown to play a major role in the biosynthesis of estrogens [in particular estrone (E 1) which subsequently leads to the biosynthesis of estradiol (E2)] and therefore the initiation and progression of hormone-dependent breast cancer in post menopausal women. Recently, a range of thiosemicarbazone-based compounds have been reported as inhibitors of STS, however, due to the mode of action of these inhibitors, no detailed structure-activity relationship (SAR) was offered. In an effort to investigate the SAR within the thiosemicarbazone-based compounds, the current project considered the synthesis and biochemical evaluation of a range of novel thiosemicarbazone-based compounds. The target compounds were synthesised through a reaction between a carbonyl containing starting material (benzaldehyde- or acetophenone-based starting material) with N-cyclohexylhydrazinecarbothioamide using ethanol as the reaction solvent. When benzaldehyde was used for the reaction to produce benzaldehyde N-cyclohexylthiosemicarbazone-based compounds, the target compounds were obtained in good to excellent yield -the target compounds were obtained in a range of 60% [for 1-(4-methylphenyl) ethan-1-one Ncyclohexylthiosemicarbazone (218)] to 92% [for benzaldehyde Ncyclohexylthiosemicarbazone (201)]. However, when acetophenone-based starting material was used, for example 1-phenylethan-1-one Ncyclohexylthiosemicarbazone- based compounds, the target inhibitors were obtained in poor yield (30% or lower)- it was postulated that steric hindrance was the major factor in the reduced yield, indeed, in the most hindered case no product was obtained. Attempts to increase the yield, involving the use of a higher boiling point solvent such as toluene resulted in the production of a by-product, namely, N,N -dicyclohexylhyd razine-1 ,2 -d icarbothioam ide(200). The biochemical evaluation (using STS from rat liver) of a small range of the thiosemicarbazone-based compounds showed that these compounds are weak inhibitors of STS in comparison to the sulfamate-based standard compounds, namely COUMATE and EMATE, however, the evaluation of these compounds suggested that hydrogen bond interaction between the inhibitor and the binding site was important in the inhibition of STS. A range of sulfonate-based potential inhibitors were also synthesised using a multistep synthetic route with the initial synthesis of a range of 4-hydroxyphenyl ketone-based compounds. The phenyl ring was then derivatised involving the synthesis of the 3,5-dibrominated derivatives which were subsequently derivatised to both the methanesulfonate- or the trifluoromethanesulfonatederivatives. These compounds were produced without any major difficulties (except for the dibromination step where the larger alkyl chain containing compounds could not be synthesised in sufficient quantity to proceed to the sulfonate derivatives). The sulfonate-based compounds were not evaluated against STS due to the relocation of the research group, however, they are currently undergoing biochemical evaluation at the University of the West of Scotland.

The role of reactive oxygen species and proteinases involved in the destruction of the extra-cellular matrix, microbial infection and inflammation have been shown as key factors influencing the skin ageing process. Many herbal extracts and natural compounds have shown promising activity in neutralising free radicals, preventing oxidative stress and acting as inhibitors of proteinases involved in cellular degradation and this evidence provides the platform for this study. The main aim of this study was to develop a panel of assays which would enable the broad screening of a large variety of herbal extracts, individual product ingredients and whole products for their antimicrobial, antioxidant, and antiageing activities. Over 100 herbal extracts from 37 different plant families were screened at several concentrations in 5 main bioassays. Antioxidant activity of each extract was assessed utilising 3 assays. The gallic acid equivalent assay to assess phenolic content, the trolox equivalent antioxidant capacity assay to demonstrate and rank anti-radical activity, and the superoxide dismutase mimetic activity assay to assess for catalytic antioxidant activity. For antiageing evaluation, two assays incorporating 2 ageing associated enzymes; collagenase and elastase were employed. Thin-layer chromatography was used to demonstrate antioxidant activity for extracts not soluble in the three antioxidant assays and, for similar reasons, a new TLC method was developed to demonstrate anti-collagenase activity for commercial products and problematic extracts. A white tea (WT) extract demonstrated the most desirable activity in all assays. Other extracts which performed particularly well include green tea (GT), witch hazel (WH), rose (R), rooibos tea (RB) and mahonia. Several whole commercial products containing WT and WH also demonstrated promising activities. Cell-based assays involving the effects of the 5 extracts and 2 products above and their activity against H[sub]20[sub]2 induced oxidative stress on human dermal fibroblasts were then implemented. The WT again demonstrated powerful protective effects on the cells and prevented the secretion of the inflammatory cytokine interleukin-8. These results demonstrate that plant extracts have the potential to act as antioxidant, anti ageing and anti-inflammatory ingredients in formulations. The factors involved in skin-ageing are also significant contributors in several inflammatory diseases therefore further investigation of active extracts may prove beneficial for future treatments.

This commentary reports on work published between 2005 and 2015 forming a record of a varied career building technical competence alongside strategic skills in the analytical chemistry and molecular biology of food. The unifying theme is practice based problem solving in the scientific regulation and enforcement of food safety and authenticity. The work demonstrates advances in sound, forensically robust, measurement science addressing problems arising from food additives, food authenticity and food allergens. In particular the mature discipline that underpins the regulation and enforcement of food additives is shown to be needed for the management of food allergens. The background to food regulation and enforcement is described alongside technical appeals in the official food control system to develop societally meaningful food surveillance, supported by a sustainable UK based official food control infrastructure (Public Analyst service) at the interface between science and the law. For food additives, publication of previously un-collated results informs regulatory practice and demonstrates the value of scientific collaboration between both jurisdictions on the island of Ireland. A definitive strategy is reported for the chemical analysis and risk assessment of ‘jelly mini-cups’ in which gel forming additives have caused choking fatalities and solutions to problems in the analysis of two illegal toxic additives, morpholine and dimethyl yellow are described. For food allergens the portfolio includes the first study to assess in quantitative terms the level of risk to peanut allergic consumers in take-away catering, leading to better training and similar work on coeliac disease and the availability of ‘gluten-free’ food. Systematic assessment of food allergen analysis and a programme of analytical improvement to support allergen risk assessment and risk management are discussed. A narrative account of an allergen related food sabotage incident and the subsequent Crown Court case and previously uncollated reports of court-sanctions for allergen noncompliances, severe incidents and deaths make key policy and practice recommendations for improvement in these areas. Page 7 of 162 In the study of food authenticity a critical review describes the nitrogen content of important species in the food supply chain as a proxy in the quantitative estimation of high value flesh foods in compound products. An exemplar study follows determining previously unavailable nitrogen factor data for farmed salmon and salmon frame mince. A critical survey of the up skilling of the UK Official Food Control System in DNA food authenticity techniques and major historical and contemporary reviews of food fraud (butter and horsemeat) support substantial policy and analytical recommendations. Many threats to our food supply can be assessed and managed only with the assistance of measurement science. Integrating elements of chemico-legal practice including lessons learned from ‘referee analyses’ and metrology in chemistry this commentary concludes with a synthesis describing major changes in the UK scientific food control system stemming from the author’s involvement in the ‘Elliott Review’ and recommendations for an international programme of work on food allergen analysis with interconnected learning for the benefit of the analytical and regulatory communities and society at large.

Campylobacter jejuni is a foodborne pathogen recognised as the leading cause of human bacterial gastroenteritis. Undercooked poultry products and contaminated water are considered as the most important sources of infection. Antimicrobial therapy is warranted only for immunocompromised patients and, although most people recover from this disease, others may develop rare neurodegenerative disorders such as Guillain-Barre Syndrome (GBS). The latter affects the nerves of the body leading to paralysis and requires extensive medical treatment. The wide use of antibiotics in medicine and in animal husbandry has led to an increased incidence of antibiotic resistance in Campylobacter over the last decade. Investigation of the molecular mechanisms of antibiotic resistance is considered important to control the spreading of resistant bacteria. CmeABC RND-type multidrug efflux (MDR) pump and the tetO gene found on pTet plasmids mediate tetracycline resistance in Campylobacter. CmeABC MDR pump consists of three components: an outer membrane protein CmeA, an inner membrane drug transporter CmeB and a periplasmic protein CmeC. Even though C. jejuni strains G1 and 11168H do not contain the pTet plasmid, the former was shown to be more resistant to tetracycline (Tet). Comparison of the genome of the G1 strain with that of the reference strain, 11168H, revealed a remarkable difference between the nucleotide sequences of their cmeB genes. In addition, it was observed that the transfer of the pTet plasmid from C. jejuni 81-176 to the G1 strain increased the level of Tet resistance above that of the former strain carrying this plasmid. This finding suggests that CmeB of strain G1 has a higher capacity to excrete this drug than its analogue in C. jejuni strains 81-176 and 11168H and thus, the former strain could be considered as an efflux pump variant with increased resistance to antibiotics. In this study we demonstrate that contribution of MDR pumps to antibiotic resistance might be dependent on the sequence variation of CmeB. Although antibiotic resistance is the main function of MDR pumps, these pumps may have other physiological roles, such as in virulence. An important mechanism of bacterial pathogenesis is the survival of Campylobacter inside environmental hosts. As a host of pathogenic microorganisms, the protozoan Acanthamoeba is a good model for the investigation of bacterial survival in the environment and the molecular mechanisms of pathogenicity. The endosymbiotic relationship between this eukaryotic organism and microbial pathogens may contribute to persistence and spreading of the latter in the environment, which has significant implications for human health. Although some studies suggest that Acanthamoeba supports Campylobacter survival in the environment, the type of interaction between these microorganisms needs to be elucidated. Also, the bacterial factors involved in this interaction remain unknown. Using a modified gentamicin protection assay it was found that C. jejuni 81-176 is able to survive and multiply inside this eukaryotic host. Thus, since these microorganisms can co-exist in the same environments (e.g. in poultry farms) the risk of infection with this foodborne pathogen is elevated. It is also reported that the CmeABC MDR pump is beneficial for the intracellular survival and multiplication of C. jejuni within A. polyphaga. However, this MDR pump was found to be dispensable for C. jejuni biofilm formation, motility and oxidative stress. Moreover, it was observed that capsule production is also required for the interaction between C. jejuni and with amoebae. Due to their role in antibiotic resistance and virulence of C. jejuni, MDR pumps could be considered as good targets for the development of antibacterial drugs against this pathogen. During the course of this study, a new chimeric C. jejuni strain was created due to horizontal gene transfer between two different strains, 81-176 and G1, which were growing together. This finding emphasises how easily Campylobacter can exchange its genetic material and thus adapt to the surrounding environment.

In comparison with the vast amount of literature available on the trypanosomes of mammals, relatively little is understood of those of lower vertebrates, including those present in the blood of fishes. Previous studies on fish trypanosomes have been concerned mainly with their life cycles and morphology, but this project presents data on their ultrastructure, polyamine and thiol metabolism and their responses to polyamine biosynthesis inhibitors. ‘Trypanosoma granulosum’ from the European eel and ‘Trypanosoma danilewskyi’ from the crucian carp were isolated from the blood of their respective hosts and successfully cultured ‘in vitro’ in an undefined diphasic medium. Several semi-defined and fully-defined media were then tested for their suitability to sustain growth of the parasites. A modified version of SDM-79 successfully provided the growth and, conditions necessary for morphological, ultrastructural and biochemical studies of both parasites. The ultrastructure of bloodstream ‘T. granulosum’ highlighted similarities with some stercorarian trypanosomes. In diphasic medium and modified SDM-79 the bloodstream parasites transformed into forms similar to those reportedly found in their leech vector. Mainly trypomastigote forms were present in modified SDM-79, although epimastigotes, not found in any blood smears were also present. Ultrastructurally, cultured ‘T. granulosum’ was similar to other members' of the genus ‘Trypanosoma’. Biochemical analyses of the parasites using high performance liquid chromatography, enzyme assays and radiolabelling studies revealed that their polyamine and thiol metabolisms were unusual amongst trypanosomatids for example the African trypanosomes and ‘Leishmania’ spp. Low levels of intracellular free polyamines, a rapid high affinity saturable uptake system for putrescine, the presence of glutathione as the major intracellular thiol, and the presence of the newly discovered thiol homotrypanothione, suggested that both ‘T. granulosum’ and ‘T. danilewskyi’ were similar to ‘Trypanosoma cruzi’ in their polyamine and thiol metabolism. Known inhibitors of polyamine metabolism Dl-[alpha]-difluoromethylornithine (DFMO), methylglyoxal-bis-(guanylhydrazone) (MGBG) and diminazene aceturate (Berenil) were all shown to inhibit growth and to cause drastic alterations to the morphology and ultrastructure of ‘T. granulosum’ (and ‘T. danilewskyi’). These anti-parasitic actions did not appear to result from polyamine depletion.

The health benefits of tea, one of the most consumed beverages in the world, are largely attributed to phenolic constituents and their antioxidant properties. Teas have been shown to neutralise free radicals, prevent oxidative stress, and act as inhibitors of proteinases involved in cellular degradation, which has led to the platform for this study. The aim of this study was to investigate the antioxidant activities of phenolics and other components of major types of tea (white, green and black) using various assays, for screening components of these three types, for their antioxidant activity and for their anti-inflammatory abilities. A new functional High Performance Thin Layer Chromatography approach was adapted and employed to (i) separate and identify antioxidant components of tea, (ii) compare the antioxidant profiles of major types of tea, and (iii) screen the antioxidant activities of identified compounds in tea via treatment with redox-active metal ions. Twelve phenolic compounds were identified which were grouped according to their reactivity towards redox-active metal ions. In addition, non-phenolic constituents were identified as antioxidants and assigned as chlorophyll analogues by comparison to reference compounds. A further key finding was that the coutner ion to the metal salt could play a marked role in antioxidant assays used with chloride, but not sulfate, enhancing antioxidant depletion. The HPTLC observations were supported by HPLC profiling to compare the constituents of tea types and phenolic loss upon challenge with oxidants. The predominant phenolics were (-)-EGCG, caffeine, (-)-ECG and quercetin - the latter two being the most active against oxidant challenge. Metal ion analysis by ICP-MS revealed the teas contained several redox-active metal ions and black tea had significantly higher levels of redox-active Mn ions. The antioxidant enzyme activity (catalase and superoxide dismutase) of the teas and selected compounds was also investigated, a variety of activity across the tea samples and the selected compounds with regards to their Catalase activity and SOD inhibitory/activity capabilities was observed. Chlorophyll showed the highest catalase activity, and black and green teas showed the highest superoxide dismutase activity. The effect of tea extracts and selected compounds on immortalised human keratinocyte cells (HaCaT) and human cervical carcinoma cells (CaSki), and their abilities to guard against oxidative damage in cell lines was also investigated. White tea (Wt), green tea (Gt), Black tea (Bt), EC, ECG, EGCG, quercetin and chlorophyll a and b showed powerful antioxidant activities in HaCaT cells both H[sub]2O[sub]2- and paraquat-induced cell death and for CaSki cells H[sub]2O[sub]2- induced cell death. These promising data demonstrate that teas and the individual components of tea have the potential to act as antioxidants and anti-inflamatory agents both in vitro and in vivo, and therefore further investigation of their bioactive effects may prove beneficial.

No description available.

Mass produced chemicals have revolutionised the way in which humans live and has led to both a significant increase in quality of life and a concomitant change in the way we pollute our habitats. Such chemicals include pharmaceuticals, plasticisers, perfluorinated compounds and metabolites which enter rivers via sources such as sewage treatment works (STW) effluent outfalls. The presence and distribution of these contaminants is of concern due to their increasing use and ability of some to biologically affect non-target aquatic organisms at trace (ng/L) levels. This work investigated the fate and distribution of selected pharmaceuticals, plasticisers, illict drugs, perfluorinated compunds (PFCs) and metabolites are investigated in three rivers of southern England. Specific objectives included establishing spatial distribution patterns of selected contaminants along the entire course of rivers receiving inputs from multiple STWs, their partition between bound (to suspended particulate material) and dissolved phases of river water, accumulation in sediment, bioaccumulation in primary producers including plants and biofilm/ periphyton, bioaccumulation in benthic aquatic organisms, adsorption of studied contaminants to polyethlene microparticles (similar to those used in cosmetic products) both in the lab and in rivers, and adsorption to 'natural' replacements for polyethylene microparticles (MPs). Spatial distribution analysis showed that selected contaminants are ubiquitous in the studied rivers with trace levels of plasticisers and perfluorinated compounds identified in river headwaters and beyond. Within the rivers studied in this work, pharmaceuticals were shown to be exclusively introduced by STW effluent and persisted exclusively in the dissolved phase of river water through the studies areas. Plasticisers and PFCs however were additionally introduced via runoff from streets, at times at higher concentrations than in SFWs effluent outfall and bound to suspended particulate material. Only PFCs and plasticisers were found to be bioaccumulative or very bioaccumulative in aquatic plants, biofilm. periphton and benthic organisms, and accumulated in sediment. Illicit drugs were only found in STW effluent and downstream river flow, almost exclusively in the dissolved phase of collected water. Laboratory assessment of adsorption to polyethylene MPs showed adsorption largely occurred via a linear isotherm and only for PFCs. However, trace levels of pharmaceuticals and an illicit drug urinary metabolite were extracted from polyethylene MPs deployed downstream from STW effluent outfalls in three rivers. Investigation of a 'natural' alternative to polyethyle MPs (walnut husk Mps) showed adsorption to these replacement particles occurred to a greater amount with more of the studied contaminants than to polyethylene MPs. This work significantly contributes to the knowledge of organis contaminant occurrence, bioaccumulation, fate and distribution in the aquatic environment. Future research should focus on elucidating the fate and distribution of conugated pharmaceuticals and metabolites in the aquatic environment, establishing organism-specific contaminant bioaccumulation guideline, the effect of microparticle fragmentation on contaminant adsorption, identification of adsorption-free MPs for use in cosmetic products and developing study designs to incorporate environmental epigenetics with toxicology and chemistry in the aquatic environment.

Owing to an increase in the number of doping cases with performance enhanc-ing drugs (PEDs), mainly anabolic androgenic steroids (AASs), and the limitations of urinalysis and self-reports, there is an ever increasing need to develop new methods for detecting doping. This thesis reports the development and application of a series of nov¬el analytical methods for the detection of frequently used AASs in hair and other matri¬ces using liquid-chromatography tandem mass-spectrometry (LC-MS/MS). Assays capable of detecting 0.5 pg/mg stanozolol and 3.0 pg/mg nandralane (with 20 mg hair) were developed. Hair samples from 180 subjects (108 males, 72 fe-males) were screened using ELISA, which revealed 16 subjects to be positive for stanozolol and 3 for nandrolone. LC-MS/MS analysis confirmed that only 11 subjects were positive for stanozolol (5.0 pg/mg to 86.3 pg/mg) and just 1 for nandralane (14.0 pg/mg), thus showing the inaccuracy of using ELISA screening alone. The analytical fmdings were successfully employed to verify self-reported drug use data. An assay capable of detecting 0.1 pg/mg testosterone (T) and 0.25 pg/mg epitestosterone (E) (with 50 mg hair) was developed. Seventy-five hair samples were collected from healthy volunteers (49 males, 26 females), with the natural levels of T 0.7-11.81 pg/mg and 0.33-6.05 pg/mg and the natural levels ofE 0.63-8.27 pg/mg and 0.52-3.88 pg/mg, in males and in females respectively. This thesis reports for the first time the TÆ ratio in hair, 0.5-3.37 in males and 0.56-1.81 in females. Assays capable of detecting 0.125 pg/mg stanozolol/0.25 pg/mg 3'-hydroxystano-zolol (with 50 mg rat hair) and 0.063 ng/mL stanozolol/0.125 ng/mL 3'-hydroxystanozolol (with 100 uL of rat urine or serum) were developed. Diclofenac was found to significantly reduce the urinary excretion of 3'-hydroxystanozolol, but did not influence the concentration of both compounds in hair. This is the first in vivo study to report this effect. Assays capable of detecting 0.25 pg/mL stanozolol and 0.5 pg/mL of 3'-hydroxystanozolol in 5 mL aqueous matrices were developed in order to investigate en-vironmental contamination. Three out of six samples from the River Danube, collected from December '09 to November '10, were found to contain stanozolol, (upto 1.82 pg/mL). In contrast, stanozolol was detected only once (1.19 pg/mL) in tap water from Budapest city.